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Adhesion properties and cell surface characteristics of the entomopathogenic fungus Beauveria bassiana

University of Florida Institutional Repository
Permanent Link: http://ufdc.ufl.edu/UFE0011221/00001

Material Information

Title: Adhesion properties and cell surface characteristics of the entomopathogenic fungus Beauveria bassiana : a link between morphology and virulence
Physical Description: Mixed Material
Language: English
Creator: Holder, Diane J. ( Dissertant )
Keyhani, Nematolah ( Thesis advisor )
Kima, Peter ( Reviewer )
Boucias, Drion ( Reviewer )
Rollings, Jeff ( Reviewer )
Maupin, Julie ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2005
Copyright Date: 2005

Subjects

Subjects / Keywords: Microbiology and Cell Science thesis, Ph.D
Dissertations, Academic -- UF -- Microbiology and Cell Science

Notes

Abstract: The entomopathogen 'Beauveria bassiana' produces three distinct in vitro, single cell propagules: aerial conidia, blastospores and submerged conidia. Atomic force microscopy (AFM) was used to visualize the surface of aerial conidia and confirmed the presence of a rodlet layer that was absent from the surface of both blastospores, which were smooth, and submerged conidia, which appeared coarse. Interfacial free energies of interaction and hydrophobicity indicies, derived from contact angle data, and hydrocarbon partitioning revealed differential properties of the three propagules regarding cell surface hydrophobicity ranging from strongly hydrophobic (aerial conidia) to hydrophilic (blastospores). Adhesion studies with fluorescently labeled cells suggested that 1) aerial conidia bound better to hydrophobic surfaces, 2) blastospores bound better to hydrophilic surfaces, and 3) submerged conidia bound equally well to both types of surfaces. The effective surface charge (zeta potential) of the three single cell propagules was predominantly positive at low pH (pH 3-4) decreasing to negative values at higher pH values (pH 6-8). Aerial conidial surface charge varied the most with respect to pH (+22 mV to - 47 mV), while submerged conidia varied moderately (+7 mV to - 13.4 mV) and the blastospores showed minor variation (+ 3.2 mV to - 4.65 mV). The gene for a beauverial hydrophobin (bhd1) was identified from a suppression-subtracted library generated from cells grown in the presence of insect cuticle as opposed to glucose. Real-time, reverse transcriptase PCR of two 'Beauveria bassiana' specific hydrophobins and a hydrophobin like protein (bhd1, bhd2 and bsn) showed that bhd1 mRNA levels were relatively high in most cell types analyzed, with the highest abundance in submerged conidia. Transcript for bhd2 was detected primarily in aerial conidia and submerged conidia, whereas transcript for bsn was not detected under the conditions tested. A SDS insoluble/TFA soluble constituent of the cell wall of 'B. bassiana' conidia was identified as bhd2 by peptide mass fingerprinting.
Subject: Beauveria, blastospores, conidia, entomopathogen, hydrophobicity, submerged
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 117 pages.
General Note: Includes vita.
Thesis: Thesis (Ph.D.)--University of Florida, 2005.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0011221:00001

Permanent Link: http://ufdc.ufl.edu/UFE0011221/00001

Material Information

Title: Adhesion properties and cell surface characteristics of the entomopathogenic fungus Beauveria bassiana : a link between morphology and virulence
Physical Description: Mixed Material
Language: English
Creator: Holder, Diane J. ( Dissertant )
Keyhani, Nematolah ( Thesis advisor )
Kima, Peter ( Reviewer )
Boucias, Drion ( Reviewer )
Rollings, Jeff ( Reviewer )
Maupin, Julie ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2005
Copyright Date: 2005

Subjects

Subjects / Keywords: Microbiology and Cell Science thesis, Ph.D
Dissertations, Academic -- UF -- Microbiology and Cell Science

Notes

Abstract: The entomopathogen 'Beauveria bassiana' produces three distinct in vitro, single cell propagules: aerial conidia, blastospores and submerged conidia. Atomic force microscopy (AFM) was used to visualize the surface of aerial conidia and confirmed the presence of a rodlet layer that was absent from the surface of both blastospores, which were smooth, and submerged conidia, which appeared coarse. Interfacial free energies of interaction and hydrophobicity indicies, derived from contact angle data, and hydrocarbon partitioning revealed differential properties of the three propagules regarding cell surface hydrophobicity ranging from strongly hydrophobic (aerial conidia) to hydrophilic (blastospores). Adhesion studies with fluorescently labeled cells suggested that 1) aerial conidia bound better to hydrophobic surfaces, 2) blastospores bound better to hydrophilic surfaces, and 3) submerged conidia bound equally well to both types of surfaces. The effective surface charge (zeta potential) of the three single cell propagules was predominantly positive at low pH (pH 3-4) decreasing to negative values at higher pH values (pH 6-8). Aerial conidial surface charge varied the most with respect to pH (+22 mV to - 47 mV), while submerged conidia varied moderately (+7 mV to - 13.4 mV) and the blastospores showed minor variation (+ 3.2 mV to - 4.65 mV). The gene for a beauverial hydrophobin (bhd1) was identified from a suppression-subtracted library generated from cells grown in the presence of insect cuticle as opposed to glucose. Real-time, reverse transcriptase PCR of two 'Beauveria bassiana' specific hydrophobins and a hydrophobin like protein (bhd1, bhd2 and bsn) showed that bhd1 mRNA levels were relatively high in most cell types analyzed, with the highest abundance in submerged conidia. Transcript for bhd2 was detected primarily in aerial conidia and submerged conidia, whereas transcript for bsn was not detected under the conditions tested. A SDS insoluble/TFA soluble constituent of the cell wall of 'B. bassiana' conidia was identified as bhd2 by peptide mass fingerprinting.
Subject: Beauveria, blastospores, conidia, entomopathogen, hydrophobicity, submerged
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 117 pages.
General Note: Includes vita.
Thesis: Thesis (Ph.D.)--University of Florida, 2005.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0011221:00001


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ADHESION PROPERTIES AND CELL SURFACE CHARACTERISTICS OF THE ENTOMOPATHOGENIC FUNGUS Beauveria bassiana: A LINK BETWEEN MORPHOLOGY AND VIRULENCE By DIANE J. HOLDER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Diane J. Holder

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This document is dedicated to my parents, my sister and her children

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iv ACKNOWLEDGMENTS I would like to thank my family for al ways being there for me, and my major advisor, Dr. Nemat Keyhani, without whom th is would all have been impossible. I would also like to thank all the members of Dr. Keyhanis lab (past and present) for the help, support and friendship that I will always treasure. I also woul d like to recognize the other members of the department that help ed me with both hands on and theoretical applications, especially Waultraud Dunn, D onna Williams, Dr. Henry Aldridge, Louise Monroe, members of Dr. James Prestons la b, Dr. Keelnatham Shammungams lab, and Dr. Julie Maupins lab. I would also like to acknowledge the students, staff and professors in PERC, for their help and the use of their equipment. Special thanks goes to Brett Kirklan d, Dr. Eunmin Cho, and the myriad of undergraduates who made this work possible. I thank all the members of my committee, Dr. Nemat Keyhani, Dr. Peter Kima, Dr. Dri on Boucias, Dr. Jeff Rollings and Dr. Julie Maupin, who helped guide me through this wo rk, generously giving of time, equipment and advice. I cannot finish w ithout thanking the rest of the staff, students and faculty of the Department of Microbiology and Cell Science, for the simple day-to-day things that made this project move forward as smoothly as possible and eventually come to an end. And last but not least, thanks go to Hamlet, for just being you.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... xi CHAPTER 1 LITERATURE REVIEW.............................................................................................1 Beauveria bassiana .......................................................................................................2 Biological Control........................................................................................................7 Fungal Biocontrol Agents......................................................................................7 Beauveria bassiana as a Biocontrol Agent...........................................................7 Factors Affecting Adhesion..........................................................................................8 Specific Binding....................................................................................................9 Nonspecific Binding..............................................................................................9 Steric, Bridging and Depletion forces.................................................................10 Hydrophobic Interactions....................................................................................11 Specific Molecules or Structures Involved in Fungal Adhesion.........................12 Molecular Biology......................................................................................................14 Methods Used to Study Global Differe ntial Gene Expression in Fungi.............14 Differential screening of cDNA libraries.....................................................15 Real Time RT-PCR.............................................................................................17 Objectives...................................................................................................................18 2 ADHESION OF THE ENTOMOPAT HOGENIC FUNGUS BEAUVERIA BASSIANA TO SUBSTRATA..................................................................................20 Introduction.................................................................................................................20 Materials and Methods...............................................................................................21 Cultivation of Fungi............................................................................................21 FITC-labeling of B. bassiana Cells.....................................................................22 Adhesion Assay...................................................................................................22 Enzyme Treatments.............................................................................................23 Effect of pH Influence on Attachment................................................................24

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vi Competition Assays.............................................................................................24 Contact Angle Determination..............................................................................24 Results........................................................................................................................ .25 Discussion...................................................................................................................34 3 SURFACE CHARACTERISTICS OF THE ENTOMOPATHOGEN BEAUVERIA BASSIANA ............................................................................................38 Introduction.................................................................................................................38 Materials and Methods...............................................................................................43 Atomic Force Microscopy...................................................................................44 Zeta Potential.......................................................................................................44 MATH Assay.......................................................................................................44 Contact Angle Determination..............................................................................45 Results........................................................................................................................ .45 Atomic Force Microscopy...................................................................................45 Zeta Potential.......................................................................................................46 MATH Assay.......................................................................................................46 Contact Angles....................................................................................................48 Discussion...................................................................................................................49 4 SUPPRESSIVE SUBTRACTION HYBRIDIZATION ANALYSIS OF BEAUVERIA BASSIANA GROWN ON INSECT CUTICLE................................55 Introduction.................................................................................................................55 Virulence Factors.................................................................................................55 Adherence and Colonization factors...................................................................56 Toxins..................................................................................................................57 Molecules Involved in Evading Host Defences..................................................58 Siderophores........................................................................................................58 Molecules Involved in Toxin Transport..............................................................58 Suppression Subtrac tive Hybridization...............................................................59 Materials and methods................................................................................................60 Cultivation of Fungi............................................................................................60 Cells grown in the presence of gl ucose (source of driver RNA)..................60 Cells grown on insect cuticl e (source of tester RNA)..................................60 RNA Isolation......................................................................................................61 Supression Subtractive Hybridization.................................................................61 Results........................................................................................................................ .61 Discussion...................................................................................................................65 5 MOLECULAR ANALYSIS OF TWO BEAUVERIA BASSIANA HYDROPHOBINS AND A HYDROP HOBIN LIKE PROTEIN.............................70 Introduction.................................................................................................................70 Materials and Methods...............................................................................................73 Cultivation of Fungi............................................................................................73

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vii RNA Isolation......................................................................................................74 Rodlet Layer Extraction......................................................................................74 Mass Peptide Spectrometry (Peptide Fingerprinting).........................................75 Reverse Transcriptase RT-PCR...........................................................................76 Results........................................................................................................................ .77 Identification of a B. bassiana Cell Wall Hydrophobin......................................77 Reverse Transcriptase RT-PCR...........................................................................77 Discussion...................................................................................................................80 6 GENERAL DISCUSSION.........................................................................................83 Statement of Hypotheses............................................................................................83 Is There a Measurable Difference in The Cell Surface Characteristics of The Single Cell Propagules of B. bassiana? .................................................................83 Visual Differences...............................................................................................83 Differences in Hydrophobicity............................................................................83 Differences in Effective Surface Charge.............................................................84 Cell Wall Proteins................................................................................................85 Conclusions.........................................................................................................86 Are there Differences in the Binding Pr operties of These Propagules, Which Can Be Related to the Cell Surface Characteristics?....................................................86 Adhesion Profiles of Aerial Conidia, Submerged conidia and Blastospores......86 Conclusion...........................................................................................................86 APPENDIX AGROBACTERIUM MEDI ATED TRANSFORMATION OF BEAUVERIA BASSIANA........................................................................................88 Introduction.................................................................................................................88 Materials and Methods...............................................................................................89 Fungal Cultures...................................................................................................89 Agrobacterium tumefaciens Cultivation..............................................................90 Transformation Procedure...................................................................................90 Discussion...................................................................................................................93 LIST OF REFERENCES...................................................................................................96 BIOGRAPHICAL SKETCH...........................................................................................105

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viii LIST OF TABLES Table page 1-1 Classification B. bassiana ..........................................................................................3 2-1 Effects of various competitors and chemicals on B. bassiana cell-type adhesion...31 2-2 Effect of pH on B. bassiana cell-type adhesion.......................................................32 2-3 Effects of various enzymatic treatments on B. bassiana cell-type adhesion...........33 3-1 Contact angle values for the three ce ll types for water, bromonapthalene and glycerol with calculated interfacial and polar free energy values............................49 3-2 Advancing and receding water contact angle data with cal culated surface energy values for three B. bassiana single cell propagules.................................................50 4-1 Blast hits of sequence fragments from the SSH library to vi rulence factors and allergens...................................................................................................................63 5-1 Primer sequences and product size for the reverse transcriptase RT-PCR..............76 5-2 mRNA abundance of bhd1, bhd2 in Beauveria bassiana single cell propagules....78 A-1 Putative transformants obtained from Agrobacterium tumefaciens mediated transformation of B. bassiana with selection markers for hygromycin B ( hph ) and neomycin ( neo ) resistance.................................................................................93

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ix LIST OF FIGURES Figure page 1-1 The life-cycle of a typi cal hyphomycete entomopathogen........................................6 2-1 Bright field and fluorescent microscopy of FITC-labeled B. bassiana aerial conidia, blastospores and submerged conidia..........................................................25 2-2 Fluorescent intensity of FITC labeled aerial coni dia, blastospores, and submerged conidia as a function of cell number......................................................27 2-3 Adhesion of aerial conidia to glass and silinated glass surfaces..............................27 2-4 Adhesion of blastospores to glass and silinated glass surfaces................................28 2-5 Adhesion of submerged conidia to glass and silinated glass...................................28 2-6 Saturation point of binding sites for B. bassiana aerial conidia, blastospores, and submerged conidia to microtiter plates....................................................................29 2-7 Quantitative adhesion and influence of washing on adhesion of aerial conidia, blastospores, and submerged conidi a to silinated F-200 (hydrophobic), F-200 (weakly polar), and F-600 (hydrophilic) microtiter plates.......................................30 3-1 Contact angles ( are formed at the interface between the liquid, solid and gas boundaries of a droplet on th e surface of interest....................................................40 3-2 Atomic force micrographs of B. bassiana conidia, submerged conidia and blastospores..............................................................................................................46 3-3 Zeta potential values for the thr ee spore types as a function of pH.........................47 3-4 Microbical adhesion to hydro carbon.and hydrophobicity indices for B. bassiana aerial conidia, blastospores and submerged conidia................................................47 4-1 Relative numbers of gene fragments re presenting functional groups present in the subtracted library................................................................................................62 4-2 B. bassiana H1 hydrophobin genomic sequence.....................................................66 4-3 Alignment of H1 and H2 with homologous hydrophobins......................................67

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x 4-4 Bootstrapped (1000) phylogenetic tree of class I and class II hydrophobins...........68 5-1 SDS PAGE (10% polyacrylamide, BisTris) Gel of SDS soluble/TFA insoluble cell wall proteins......................................................................................................77 5-2 Mass spectroscopy data showing the ma ss of individual amino acids in one of the two main fragments from the 12 KDa trifluoroacetic acid soluble/sodium dodecyl sulfate insoluble B. bassiana cell wall protein...........................................79 5-3 Mass peptide fingerprinting results for tw o identifiable fragments of the 12 KDa trifluoroacetic acid soluble, sodium dodecyl sulfate insoluble B. bassiana cell wall protein...............................................................................................................80 5-4 Comparison of Bhd1 and Bhd2 and ot her hydrophobins consensus spacing for Class I and Class II hydrophobins............................................................................81

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xi Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ADHESION PROPERTIES AND CELL SURFACE CHARACTERISTICS OF THE ENTOMOPATHOGENIC FUNGUS Beauveria bassiana: A LINK BETWEEN MORPHOLOGY AND VIRULENCE By Diane J. Holder August 2005 Chair: Nemat O. Keyhani Major Department: Microbiology and Cell Science The entomopathogen Beauveria bassiana produces three distinct in vitro, single cell propagules: aerial conidia, blastospores and submerged conidia. Atomic force microscopy (AFM) was used to visualize the surface of aerial conidia and confirmed the presence of a rodlet layer that was absent from the surface of both blastospores, which were smooth, and submerged conidia, which app eared coarse. Interfac ial free energies of interaction and hydrophobicity indicies, de rived from contact angle data, and hydrocarbon partitioning revealed differential properties of the three propagules regarding cell surfac e hydrophobicity ranging from str ongly hydrophobic (aerial conidia) to hydrophilic (blastospores). Adhesion st udies with fluorescently labeled cells suggested that 1) aerial conidia bound be tter to hydrophobic surfaces 2) blastospores bound better to hydrophilic surfaces, and 3) s ubmerged conidia bound equally well to both types of surfaces.

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xii The effective surface charge (zeta potential) of the three single cell propagules was predominantly positive at low pH (pH 3-4) decreasing to negative values at higher pH values (pH 6-8). Aerial conidial surface char ge varied the most with respect to pH (+22 mV to 47 mV), while submerged conidia va ried moderately (+7 mV to 13.4 mV) and the blastospores showed minor variation (+ 3.2 mV to 4.65 mV). The gene for a beauverial hydrophobin ( bhd1 ) was identified from a suppr ession-subtracted library generated from cells grown in the presence of in sect cuticle as opposed to glucose. Realtime, reverse transcriptase PCR of two Beauveria bassiana specific hydrophobins and a hydrophobin like protein ( bhd1, bhd2 and bsn ) showed that bhd1 mRNA levels were relatively high in most cell types analyze d, with the highest abundance in submerged conidia. Transcript for bhd2 was detected primarily in aerial conidia and submerged conidia, whereas transcript for bsn was not detected under the conditions tested. A SDS insoluble/TFA soluble constituent of the cell wall of B. bassiana conidia was identified as bhd2 by peptide mass fingerprinting.

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1 CHAPTER 1 LITERATURE REVIEW The earliest reports of a f ungal entomopathogen, possibly the organism that would come to be known as Beauveria bassiana (Balsamo) Vuillemin, came from China, as far back as 2700 BC (79). It was not until 1835 that Agostino Bassi demonstrated that Calcino, or White Muscardine, a disease th at was devastating th e Italian silkworm industry, was contagious and caused by a pa rasitic fungus (63). Balsamo Crivelli officially named the organism Botrytis paradoxica eventually changing the name to Botrytis bassiana to honor the man who first described it. In 1912, Vuillemin, determined that ther e were enough features peculiar to Botrytis bassiana to assign it to the new genus Beauveria (19). There now are multiple species in the genus Beauveria Vuill. some of the most important ones are: B. bassiana, B. brongniartii, and B. alba B. bassiana and B. brogniartii well known entomopathogens with a wide host range, includi ng arthropods other than insect s, are now being used as biological control agents to control a variety of crop damaging insects. B. alba is mainly isolated as an indoor contaminant and displa ys the lowest pathogeni city of these three beauveria species (1). Due to the practical a pplications of fungal entomopathogens as biological control agents, the biology (and to a lesser extent the molecular biology) of these fungi has been the s ubject of much research. Major efforts have been targeted towards isolation and character ization of strains with high virulence, improved cost effectiveness, and to technologies that could be applied to other economically important Ascomycetes One of the most important steps in

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2 the host-pathogen interaction is the initial attachme nt of the fungus to the host cuticle. Modifying the formulation of commercial produ cts, or of the fungus itself, namely to improve targeting and attachment to the hos t cuticle, may lead to improvements in infection rates, and host mortality, and hen ce the effectiveness of the biocontrol. This dissertation examines the physiol ogical and molecular determinants of B. bassiana attachment. New techniques to ev aluate these adhesion profiles were developed, and a protein implicated in the adhesion process were isolated and characterized. Beauveria bassiana All fungal phyla include speci es that are able to re produce either sexually or asexually. The production of multiple spore types increases the ch ances of survival during adverse environmental conditions (1). These spore types can be produced in response to environmental conditions, as well as at different times in the lifecycle and can have different dispersal mechanisms. In 2002, Huang et al. (47) identified Cordyceps bassiana as the ascomycote teleomorph of B. bassiana. However, the organism is most frequently described and identified in the anamorph stage and assi gned to the Deuteromycota. Taxonomical identification within the Deuteromycota relie s heavily on physical ch aracteristics such as shape, size and color, as well as the manner in which the asexual spores, or conidia, are produced. It is a common, and often useful, practic e to use separate nomenclatures for different stages of the same species (Tab le 1-1), because most Ascomycetes produce different spore types specific to the particular stage in the life cycle in which they find themselves and in some species the sexual stag e may occur as infrequently as once a year

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3 (1). Predominant stages tend to be the mo st commonly described (t he descriptions are usually based morphological char acteristics such as spore ty pes); as a result connections between different lifecycle stages within the same fungus are not always immediately apparent. Fungi for which a sexual stage ha s not yet been identif ied are considered second (deutero) class, imperfect fungi, and are currently assigned to the artificially constructed phylum/class. Deuter omycota/Deuteromycetes (1). Correct nomenclature involves assigning the fungus a holomorphic name once the sexual and asexual stages have been officially linked and the organism is considered complete. The whole organism, with all life cycles, is the holomorph; the sexual stage, the teleomorph, and the asexual stage, the anamorph Table 1-1. Classification B. bassiana Holomorph1 Anamorph Kingdom Fungi Phylum Ascomycotina Subphylum Pezizomycotina Deuteromycota Class Sordariomycetes Hyphomycetes/Deuteromycetes Subclass Hypocreomycetidae Order Hypocreales Moniliales Family Clavicipitaceae Genus Cordyceps Beauveria Species bassiana bassiana 1 National Center for Biotechnology Information at the National Institutes of Health, Bethesda, MD Species within the genus Beauveria are typically differentiated from other fungi by morphological characteristics. They are filamentous fungi that produce colorless (hyaline) aerial conidia from conidiogenous cells fr eely on the mycelia. This characteristic places them within the moniliaceous (having hyaline conidia) Hyphomycetes (19). Aerial c onidia are initially produced as terminal swellings formed on the neck of the conidiophore. The next c onidium grows laterally, half way up the first neck of the conidiophore, in another dire ction, and is pushed upwards by sympodial

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4 growth (19). The resulting denticulate rachis, with denticles equally wide as the rachis, is characteristic of Beauveria spp. B. bassiana colonies grow relatively slowly and can appear powdery or wooly, with colors ranging from white to yellow, and occasionally pinkish. Aerial hyphae are septate, smooth, hyaline, and about 2 m wide. Submerged hyphae are similarly structured, but larger (1.5 3m) (19). Conidiogenous cells, which arise from short swollen stalk cells, are often found in dense clus ters or whorls. They consist of a globose base and the characteristic denticulate r achis. The aerial conidia are hyaline, smooth, relatively thin walled and vary from being oval to spherical depending on the species, and occasionally by cultural conditions (19, 47). Typical of hyphomycete entomopathogens, B. bassiana invades through the host cuticle, although as with other hyphomycetes, entry through the digestive tract is also possible. The initial and crucial steps in the infection process ar e attachment to, and penetration of, the host cuticle. Arthropod cuti cles are complex structures, which in the case of insects are composed of two main la yers the epicuticle and the procuticle. The epicuticle, a thin layer which overlays the procuticle, lacks chitin, but is composed of sklerotinized proteins overl aid by a waxy layer containing fatty acids, sterols, and lipids. The bulk of the cuticle, the procuticle, consists of chitin embedded in a protein matrix (15, 40). Fungal entomopathogens use mechanical pressure and a mixture of enzymes to penetrate and dissolv e the insect cuticle (15). Although several entomopathogens use swellings at the tip of the germ tube (appressoria) to generate mechanical pressure and increase attachment to the insect cuticle, such structures are rarely observed in B. bassiana However, the battery of enzymes including proteases,

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5 and chitinases produced by this entomopathogen are similar in nature to those produced by other hyphomycete entomopathogens such as Metharhizium ansiopliae (6, 77) Once the fungal hyphae reach the hemocoel, thin walled, yeast like, hyphal-bodies, or blastospores, are generated and dispersed th roughout the host (40). Host death appears to result from a number of factors including production of toxins by the fungus, physical obstruction of the circulatory system, i nvasion of organs, and nutrient depletion. Upon host death, the parasite switches from yeast-like to hyphal growth invading all the tissues of the host body, while atte mpting to reduce or eliminate competing organisms with a variety of antimicrobial metabolites. The mummified corpse can remain in the environment unchanged for months, but under favorable conditions the hyphae emerge from within the corpse, spor ulate and the resulting aerial conidia are dispersed via air or water (40). Beauveria sp. produce a number of metabolites some of which have cytotoxic effects alexopoulos (1). These metabo lites include beauvericin, bassianolide, beauveriolides, bassianin, te nellin, and oosporein. Bea uvericin and bassioanolide are ionophores that differ in specificity for cati ons. Beauvericin, a hexadepsipeptide, has antimicrobial activity against both gram-negative and gram-positive bacteria, is toxic to brine shrimp with a LD50 of 2.8 g ml-1 water, but has no demonstrated insecticidal effects (81). Bassianolide, a cyclo-octadepsipeptide, also has antimicrobial effects and is lethal to silkworm larvae at a concentration of 13 ppm (81). Although beauveriolides are st ructurally related to beauvericin and bassioanolide, they are not as well characteri zed, and their antimicrobial or insecticidal potential have yet to be described (81). Nama tame et al. (2004) have recent ly shown that beauveriolides

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6 have an inhibitory effect on lipid drop form ation in mouse erythrocytes and as a result could be marketed as anti-cholesterol drugs. According to their data, beauveriolides have few cytotoxic effects on mouse cells at levels up to 100 mg-1day-1. The pigments, bassianin, tenellin, and oospor ein are toxic to erythrocyte membrane ATPases (50). Oosporein is also a denaturi ng agent and a potent antibiotic specific to gram-positive organisms. The toxicity of these pigments towards insect host cells has not been well defined (81). Saprophyticcycle Parasiticcycle Germination(2) Hyphalproliferation(3) Spore dispersal(1) Sporulation(7) Hyphalproliferation throughthehostcuticle (4) Switchfromhyphalgrowth to yeastlikegrowth (5) Hyphalgrowthout throughthecuticle (6) Epicutcle Procuticle Heomocel Blastospores Conidia Hypha Fig. 1-1. The life-cycle of a typical hyphomycete entomopathogen. Aerial conidia disperse and land on diverse substrata (1 ). If the conditions are suitable, the condia will germinate (2) and the hypahe will proliferate (3). If there is not a suitable host, once the nutrients are used up, the hyphae will generate conidiogenous cells, produce aerial conidia and the cycle will repeat (7). If there is a suitable host, the hyphae w ill proliferate over the surface of the cuticle (3) until a suitable entry poin t is found. The hyphae will then digest their way through the layers of the cuticle (4 ) and enter the hemocoel. Within the hemocoel, growth switches from hypha l to yeast-like (5), and yeast-like hyphal bodies (blastospores) proliferate throughout the host. When the host dies, the switch occurs again and th e resulting hyphae grow out through the insect cuticle (6), upon emerging from the host the hyphae produce conidiogenous cells, and the cycle repeats (7).

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7 Biological Control Fungal Biocontrol Agents Fungi are important in limiting insect populations in nature and over 700 entomopathogenic fungal species, predominan tly hyphomycetes have been described. The effective impact on host populations depend s on host range and the ability to attack specific life stages. These attributes vary gr eatly by species and in some cases by strain (81). A number of commercial mycoin secticidal preparations containing Verticillium lecanii, Metarhizium anisopliae, Beauveria bassiana, B. brongniartii, M. flavoridiahave, and Paecilomyces fumosoroseus have been developed and used with some success in Holland, Australia, Brazil, Germany, Fran ce, the former USSR, the former Czechoslovakia, Switzerland, Austria, Australi a, and the UK (81). The targets of these formulations are diverse, with whiteflie s, aphids, coffee berry and corn borers, grasshoppers, locusts, colorado beetles and cock chafers as the most targeted organisms. Some commercial mycoinsecticides have been developed in the US, however several factors have hampered widespread use including complex large-scale production problems and potential loss of viability if precise production and st orage conditions are not met (89). Commercial mycoinsecticidal efficiency is sensitive to environmental conditions, especially low humid ity levels. Even in soil applications, where exposure to ultraviolet radiation and desiccating c onditions are limited, competition with soil microbes can limit the efficiency of fungal based insecticides (89). Beauveria bassiana as a Biocontrol Agent Commercial insecticidal formulations based on B. bassiana have been developed targeting diverse organisms including beetle larvae, plant hoppers on rice, grasshoppers, whiteflies, and locusts. As with other commercial mycoinsecticides, despite the

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8 advantages, including wide host ranges, low environmental persistence (desirable in application practices), mass pr oduction, and a wider range of ta rget hosts not effectively targeted by other biocontrol agents (viruses bacteria, nematodes etc.), there are still a number of obstacles to widespread applications. The highest success of commerci al mycopesticides, including B. bassiana based products, is in third world countries where lower production costs and fewer governmental regulations make the products more cost effective (37). In the US Companies such as Mycotech and Troy bios ciences have developed and are marketing Mycotrol and Naturalis which are B. bassiana based formulations. Information relating to how different beauveria cell types initiate the infectious processes, combined with the development of efficient methodologies to transform, genetically manipulate, and characterize fila mentous fungi at a molecular level, will significantly affect the development of impr oved mycoinsecticides which in turn could lead to increased use of these products. Factors Affecting Adhesion Intermolecular forces between biological molecules involve complex interactions. In addition to the intermolecular effects of bi ological structures such as membranes, cell wall structures, or the entire organism acting in a non-localized, non-specific manner, interactions derived from the influence ex erted by proteins, lipids, carbohydrates, and other molecules that can have localized receptor-ligand binding in addition to electrostatic and hydrophobi c/hydrophilic interactions also need to be taken into account. The parameters that influence adhesion inte ract with each other in non-linear manners and can involve competing, synergistic or hi ghly interdependent dyna mic interactions.

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9 Van der Waals forces, permanent dia pole-diapole interactions, H-bonding, hydration forces, hydrophobic inte ractions and entropy-driven forces mediate van der Waals interactions. Some of these forces are short ranged and are primarily involved in adhesion and binding, whereas longrange forces are involved in colloidal aggregation. For descriptive purposes biological intermolecu lar reactions will be separated into two main categories, non-specific and specific binding, although some processes may involve both types of interactions intermolecular (59). Specific Binding Biospecific interactions are a subclass of highly complementary, non-covalent bonds. They are characterized by the close ch emical and geometric fit between ligand and receptor binding pocket and entail relati vely strong binding energies. These high bond energies (1015 m-1 for biotin/avidin) (95) are highly dependent on very small distances between the ligand and receptor. Specific binding is dependent on distance and acts primarily when molecules are very close to each other. The molecules are brought into close proximity by the action of long-range forces, which guide the ligand into the specific receptor. Similar forces govern both specific and non-specifi c biological interactions. Specific forces tend to be attractive, relatively small and heterogene ous, whereas non-specific forces are usually repulsive, influence large areas, and th e interacting surfaces tend to be more homogeneous intermolecular (59). Nonspecific Binding Non-specific interactions important for bi ological intermolecular forces include electrostatic interactions, ster ic interactions, and hydrophobic interactions. Electrostatic

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10 interactions can be further subdivided in to van der Waals interactions, permanent diapoles, and hydrogen bonding. Electrostatic interactions ar e repulsive or attractive interactions that occur between ions and charged surfaces. Van der Waal s, diapole-diapole, and hydrogen bonds are relatively weak attractive forces between temporary or permanent diapoles. At one point, the definition of van der Waals forces en compassed all intermolecular forces, but presently only London (dispersive) forces ar e included. London for ces are transitory forces generated when electr on clouds of different molecule s oscillate in unison creating attractive forces between the molecules. Thes e forces are transitor y, aligned with each other and are dependent on the size of the mol ecule, the larger the molecule the stronger the force. The diapoles in London forces are deri ved from non-polar molecules and are induced and temporary in contrast intera ctions between compounds with permanent diapoles (polar molecules) lead to increas ed adhesion and cohesion. This explains the observed higher boiling points fo r polar liquids, as compared to non-polar liquids. If hydrogen atoms are attached to small electron egative groups such as O, N, or F, the diapoles generated are bigger and the resul ting interactions (Hbonds) are proportionally stronger. Steric, Bridging and Depletion forces Flexible polymer-like groups are present of the surfaces of many microbes; these include polysaccharides, tethered ligands, or lipids, which mediate many biological interactions. The interactions mediated by these groups can be repulsive if polymer-like groups are tethered to the surface, coiled or do not interact strongly with each other, the bulkier the groups, the stronger the repulsion. These polymers can initiate adhesion to

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11 other surfaces via attractive fo rces if the ends and/or the whole length of the tethered polymers are linked to functional groups, as the guest molecule approaches they can initiate the binding process. When polymers (such as polyethylene glycol PEG) are free in solution they can also mediate adhesion of biological surfaces by resulting in depletion forces arising from the osmotic pressure between the bulk solutio n, and the zone betw een the two surfaces devoid of polymer (depletion zone). Water is driven out of the depl etion zone and into the bulk solution thus forcing the two surfaces together. This is the principle behind using PEG in cell fusions and transformations (59). Hydrophobic Interactions Hydrophobic interactions are c onsidered to be the most important interactions of molecules dissolved in highly polar liquids (87). It is considered the predominant interaction mediating microbial binding to biosurfaces and Boucias et al. (1998) noted that it probably was the pre dominant interaction mediating fungal adhesion to insect cuticle. The actual mechanisms behind hydr ophobic interactions ar e still not fully understood, however, if a non-polar liquid is dissolved in polar liquid droplets of the former will rapidly aggregate. This gives the appearance that non-polar molecules fear water, hence the name water fe aring (hydrophobic) substances. Hydrophobic interactions can be best de scribed as the exclusion of hydrophobic molecules from water resulting in the squeezi ng together effect of these surfaces. Hydrophobic forces are relativ ely strong and long ranged, more so than other nonspecific intermolecular forces by a factor of 5-10 times (59). The driving force behind hydrophobic inte ractions is the high free energy of cohesion of water. According to Van Oss, Lifs hitz-van der Waals (LW) forces have little

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12 or no effect on hydrophobic interactions, b ecause hydrophobic materials have apolar (LW) surface tension components ( w LW) that are close to that of water ( w LW of water @ 20 0C = 21.8 mJ m-2, the i LW of completely apolar mate rials range from 18 to 33 mJ/m2). This results in LW free energies of attraction ( Giwi LW) at close range between water and the material, of 0 to .3 mJ/m-2. The Lewis acid-base surface energy components ( Giwi AB) are much larger (up to mJ/m-2). Because the total interfacial energy is equal to the combined values of the LW a nd AB components, the contribution from LW tends to be very low (87). Hydrophobic compounds do have an affinity for water with Giw values of to 55 mJ/m2, but this affinity is not strong enough to overcome the high cohesive energy of water. Hydrophilic substances, on the other hand, have higher affinities for water ( Giw values of 113 to 143 mJ/M2). Hydrophilic interactions are characterized by a net repulsion between similar hydrophili c surfaces in water. This repulsion can occur only if the surfaces have a stronger adhesion to wa ter than the polar free energy of cohesion of water ( Gw = 102 mJ/M2) (87). Specific Molecules or Structures Involved in Fungal Adhesion The forces regulating sporecuticle initial in teractions between entomopathogenic fungi and hosts are preexis ting involving various combin ations of specific and nonspecific interactions. Specific interactions can involve: (1) the recognition of specific carbohydrate groups present on the insect cuticle by lectins (carbohydrate binding proteins), (2) protein-lipid binding, (3) prot ein-protein binding, and (4) other receptorligand binding. Non-specific interacti ons are mediated by hydrophobic and/or electrostatic interactions.

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13 Freeze etched carbon replicas of the aerial conidial surfaces visualized with electron microscopy (EM) (52, 68, 96), and mo re recently Atomic Force Microscopy (AFM) (Kirkland & Keyhani, unpublished da ta), (27), have shown that hydrophobic aerial conidia from various fungi possess a well organized, uncoat ed, outer layer of hydrophobic rodlets. These rodl ets are predominantly composed of hydrophobins small, secreted, hydrophobic proteins ch aracterized by eight conserve d cysteine residues and the ability to self-assemble into amphipathic me mbranes. These proteins are ubiquitous in ascomycete and basidiomycete fungi, but have not yet been found in other phylogenetic groups. They are involved in a variety of functions including spore coat protection, escape of aerial structures from wa ter, virulence, and signaling (90). Most other fungal cell types, including hydrophilic conidiospores, either lack hydrophobin layers, coat the hydroph obin layer with mucilage (obscuring visualization), or possess very disorganized, uncoated rodl et layers (increasi ng organization, being correlated with increasing hydrophobicity) (96). For some hydrophilic fungi, preexisting muc ilaginous coats or mucus released at the time of cuticle contact mediate attach ment to hosts. Fungi that produce motile hydrophilic spores promote attachment by s ecreting adhesion vesi cles from pseudopodia when in contact with the insect cuticle; ot hers release mucilage from germ tubes, or appressoria, in order to consolidate adhesion (91). Understanding the mechanisms that underlie aerial conidial adhesion to insect cuticles and other surfaces is im portant, because this is the in itial and thus a crucial step in pathogenesis. Aside from aerial conidia, B. bassiana produces at leas t two other single cell propagules that have the potential to initiate infection. Knowledge concerning the

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14 biochemical and biophysical mechanisms that mediate the adhesive properties of aerial conidia and other specialized cells can be a basis for ma king rational decisions on cell formulations optimized towards specific targ ets. This information is important in understanding the biol ogy and ecology of B. bassiana, and can have an impact on practical considerations for the use of B. bassiana as a biological control agent. Molecular Biology Methods Used to Study Global Differen tial Gene Expression in Fungi Increasingly the information about the su rface properties of fungi, including factors affecting pathogenicity and virulence, which could lead to technologi es that may improve the efficacy of mycoinsecticid es, will be found by analyzing the organisms at a molecular level. Understanding which genes are important for the infection process may lead to the development of commercial insecticidal produc ts specifically designe d to adhere better, with higher infection rates and/or virulence, this in turn will decrease the infective doses leading to more cost effective products. Genes important for the infectious process are carefully regulated (either up or down) when the parasite comes into contact with the host. Comparison of the concentrations of individual mRNAs in sa mples obtained from cells, which have been exposed to different conditions, or which have been genetically altered, can yield valuable information about the spatial and te mporal relationships of gene expression. Isolation of differentially e xpressed transcripts can lead to the characterization of regulatory networks and motifs (promoters and other cis elements). Comparative analysis of transcript and proteome data can provide further information about how protein levels are regulated.

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15 There are a number of methods used to analyze differential expression of multiple genes (transcriptome analysis). These techniques include array technology, serial analysis of gene expression (SAGE), techni ques based on amplified fragment length polymorphism (AFLP), differential display, reverse transcription PCR (DDRT-PCR), differential screening of cDNA libraries, a nd reverse transcriptas e, real time PCR (reverse transcriptase RT-PCR). Some of these methods are combined to improve upon the limitations of the individual techniques. Differential screening of cDNA libraries Screening cDNA libraries is the classical method of isolating differentially expressed cDNAs. Successful application of this method is limited unless the mRNA of interest comprises at least 0.05% of the total RNA in one cel l line, and less than 0.01% in the other (55). Subtractive hybridization (SH) is specifically designed to remove common expressed sequences, increasing the co ncentration of differentially expressed sequences in the probe, and th erefore the overall specificity of the procedure. SH technology involves hybridizing cDNA from treat ed sample libraries (tester cDNA) to cDNA from control sample libraries (dri ver cDNA). The DS, hybridized cDNA is removed, leaving behind SS molecules represen ting genes that are e xpressed at a higher level in the tester population. A further improvement to this technology is Subtraction Suppre ssive Hybridization (SSH); a PCR based technique that norma lizes, subtracts, and amplifies message expressed at higher levels in the tester cDNA library without the requirement of physically separating DS cDNA from SS cD NA. Unlike SH, SSH does not require multiple rounds of hybridization and the normalization and subtraction steps are combined in a single reaction (23, 36, 61, 64).

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16 cDNA from the experimental groups (tester), and from the subtracting cDNA (driver) are digested with a four base cutter Then tester cDNA is split equally into two aliquots, which are then ligated to different ad aptors. Each tester sample is mixed with excess driver, denatured and allowed to a nneal. Normalization (equalization) of sequence abundance occurs during this step b ecause high abundance sequences will selfanneal faster than low abundances reducing the percent of high abundance differentially expressed molecules in the SS cDNA pool. Th e tester populations are mixed together in the prescence of excess driver, this furthe r enriches for differentially expressed SS cDNA, but it also allows the formation of hybr id molecules with different adapters at each end (heterohybrids). The ends of the molecu les are then filled in to generate primerannealing sites. During the PCR amplification only these heterohybrids (tester1/tester2) are exponentially amplified, whereas homohybrids (tester1/tester1, tester2/tester2 and driver/driver) will not be amplified. The test er homoybrids have identical adapters with long inverted terminal repeats that form self anneal at temperatures higher than the annealing temperatures of the primers and forming panhandle structures which cannot be amplified (Fig. 1-2). Adapters cDNA molecule Fig 1-2. Panhandle structure due to annealing of identical adapters Driver/driver homohybrids have no adapter a nd cannot be amplified. Driver/tester hybrids will be amplified linearly, and none of the single stranded molecules will be amplified because they dont have adapters, or they dont have primer-annealing sites.

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17 The end result is amplified sequence fragme nts representing differentially expressed sequences, normalized with respect to sequen ce abundance in the original sample. These fragments can then be cloned into appropriate vectors for further analysis. This technique was used to identify B. bassiana genes up regulated during the initial stages of pathogenesis; it was hypothesized that if hydr ophobins were important in the infection process they might be upre gulated at this stage. Real Time RT-PCR The polymerase chain reaction (PCR) has three basic reaction rates. Initially, when reagents are not limiting, the reaction pro ceeds exponentially; eventu ally one or more reagent becomes limiting and the reaction pro ceeds in a linear manner; and finally very little new product is made and amplif ication rates reach a plateau. Using PCR to accurately quantify DNA re lies on collecting the data during the exponential stage and having a large e nough dynamic range (several orders of magnitude). Real time PCR automates this procedure by automatically generating and plotting data points from all samples during each amplification cy cle, yielding a dynamic range of about 107 fold. Data analysis, standard curve generation, and copy number calculations are also automated. Converting mRNA to cDNA prior to PCR amplification and amplifying specific genes within sample s yields information about differential abundance of mRNA species under different conditions. Real time RT-PCR products are detected fl uorescently. There are currently four basic technologies used for this purpose: Taqman (Applied Biosystems), Molecular beacons, Scorpions and SYBR Green (Mol ecular probes). All these technologies, except SYBR green, rely on Frster Resona nce Energy Transfer (FRET) to quench fluorescently labeled probes until they have hybridized with the te mplate. This quenching

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18 is accomplished by physically restraining th e fluorophore in close proximity to the quencher. Once the probe is amplified, the quencher and probe are separated by a variety of processes including release of the fluorophor and inhibition of hairpin loops present in free unhybridized probes. Because fluores cence is dependent on template-probe hybridization increases in the quantity of template results in equivalent increases in fluorescence from cycle to cycle. SYBR green is a fluorescent dye which fluor esces strongly in the presence of double stranded DNA. As with the other tec hniques, the level of fluorescence increases proportionally to the amount of PCR product. SYBR-green based technologies tend to be less expensive than probe based systems, but more sensitive to errors derived from the amplification of non-specific products or th e presence of primer dimers. Although the costs associated with fluorescent probes tend to be higher, there is the added advantage of being able to perform multiplex PCR; wher e DNA species within the same sample are amplified and differentiated by labeling the probes with differently colored fluorescent molecules. Once differential abundance of message for specific genes is observed and confirmed, the importance of these genes in the pr ocesses of interest needs to be verified. This often requires targeted gene manipulation (knockout ) of wild-type genes and characterization of the resultant mutants. Objectives The objectives of this research includ e: (1) quantification of the relative contributions of surface forces in the adhesion processes of Beauveria bassiana single cell propagules to solid surfaces ; (2) analysis of the hydropho bic and electrostatic cell surface properties of B. bassiana single cell propagules; (3) the development of a

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19 quantitative assay to measure microbial adhesion to surfaces; (4) molecular characterization of proteins involved in B. bassiana adhesion to solid surfaces; and (5) analysis of the mRNA levels of these mol ecules in different cell types and under varying growth culture conditions.

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20 CHAPTER 2 ADHESION OF THE ENTOMOPATHOGEN IC FUNGUS BEAUVERIA BASSIANA TO SUBSTRATA Introduction Under intensive study for use as a biope sticide, the entomopathogenic fungus Beauveria bassiana displays a broad host range able to target a diverse number of arthropod species. Strains of B. bassiana have been selected for control of insects and other arthropods that act as di sease vectors including mosqu itoes and ticks (14, 54); crops pests such as whiteflies, caterpillars, grasshoppers, a nd borers (12, 20, 53, 58, 100) ; and even ecologically hazardous, invading pests such as fire ants and termites (11, 17). The varied cuticles of these organisms repres ent the first barrier to the pathogen, and attachment of fungal propagules to the cuticle is the initial event in establishing mycosis. Air currents, dispersion via water droplets, as well as saprophytic growth over substrata inhabited by insects are consid ered the major routes for cont act of fungal spores with host cuticles. Upon contact, funga l cells bind to the cuticle a nd initiate a developmental program that includes the production of special ized infection structures such as germ tubes and penetrant hyphae (9, 42). If the in fection is successful, the fungus will grow across the cuticle surface, pene trating the host cuticle to inva de and proliferate within the hemolymph, ultimately resulting in the death of the host. Fungal cell attachment to cuticle may involve specific receptor-ligand and/or nonspecific hydrophobic and electros tatic mechanisms (9, 10, 25). A haploid anamorphic fungus, B. bassiana produces a number of mono-nuc leated single cell types including

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21 aerial conidia, blastospores, and submerged coni dia that can be isolated from agar plates, rich broth submerged cultures, and nutrient limited submerged cultures, respectively. Although it is well known that culture conditi ons (and hence the cell type produced) can affect successful virulence to wards targeted hosts, little is known about the adhesion process of B. bassiana cell types other than conidi a. This report describes a quantitative assay used to determine the binding quali ties and adhesion substrata preferences of B. bassiana aerial conidia, blastospor es, and submerged conidia. Materials and Methods Cultivation of Fungi Beauveria bassiana (ATCC 90517) was routinely grown on potato dextrose agar (PDA) (4). Plates were incubated at 26oC for 10-14 days and aerial conidia were harvested by flooding the plate with sterile dH2O. Conidial suspensions were filtered through a single layer of Mira-cloth (Clabioc hem, CA) and final spore concentrations were determined by direct count using a hemo cytometer. Blastospores were produced in DifcoTM Sabouraud dextrose (Becton, Dickins on and Co., MD)+ 1-2% yeast extract liquid broth cultures (SDY) usi ng conidia harvested from plat es to a final concentration of 0.5-5 x 105 conidia/ml as the inoculum. Cultu res were grown for 3-4 days at 26oC with aeration (150-200 rpm). Cultures were filt ered (2x) through glass wool to remove mycelia, and the concentration of blastospor es determined by direct count. Submerged conidia were produced in TKI broth using fructose as th e carbon source as described by Thomas et al. (1987). For all cell types, Mi ra-cloth (Clabiochem, CA) or glass wool filtered cell suspensions were harv ested by centrifugation (10,000xg, 15 min, 4oC), washed two times with sterile dH2O, and resuspended to the desired concentration as

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22 indicated (typically 107-108 cells/ml) in water for final dilution as required for the experiment. FITC-labeling of B. bassiana Cells Fluorescein isothiocyanate (100 l of 1mg/ml stock solution per ml of fungal cells) was added to washed fungal cell-types (0.5-1x108 cells/ml) resuspended in 50 mM calcium carbonate buffer, pH 9.2. The reaction wa s incubated for 20 min in the dark, after which the cells were extensively wash ed (4-5 times with volumes equal to the original volume) with TB (50 mM Tris-HCl, pH 8). Final cell pellets were resuspended in TB (pH 8) to the desired concentrati ons as indicated. Fi nal single cell propagule concentrations were checked by direct count using a hemocytometer. Adhesion Assay Two assays were used to assess adhesion to substrata. In the first (qualitative), fungal cell suspensions (100 l, 1-20 x 106 cells/ml) were spotted and incubated in slide chambers (treated and untreated glass surf aces, Lab Tech chamber slide system, Nalgene Nunc, Naperville, Il), at 25oC and 100% humidity for various periods of time. Adhesion was assessed microscopically after a 1x wash or 3x wash in TB (pH 8). Digital images were taken using a Nikon Optiphot-2 microsc ope with a digital camera. Adhesion was also assessed quantitatively using FITC-lab eled cells incubated in various black microtiter plate test substrata. Fungal cell suspensions (100 l, 1-20 x 106 cells/ml) were placed in (black) microtiter plate wells and incubated at 25oC in the dark for various periods of time. Unbound cells were remove d by aspiration of the liquid from the wells followed by up to 3 washes with 450 l TB (pH 8). Fluorescence was measured using a Spectra Max Gemini XS microplate fluorom eter (Molecular Devices Corp., Sunnydale,

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23 CA), Ex wavelength: 495 nm, Em: 530 nm, cutoff: 515 nm. For each experiment a standard curve of fluorescent in tensity versus cell number (as measured by direct count) was prepared. Typically, the fluorescent intensity was meas ured before wash (total number of cells) and after each wash. Weakly polar microt iter plates (Fluorotrac F200) and hydrophilic microtiters plates (Fluorot rac F600) from Greiner Biotech (Longwood, Fl) and were used unmodified. Hydrophobic su bstrata were prepared by addition of a thin layer of silicone using Sigmacote (S igma Corp., St. Louis, Missouri) onto glass slides or into Fluorotrac F200 microtiter plat e wells. Typically, substrata were treated up to three times with Sigmacote and the treated pl ates or slides were placed in a fume hood overnight to ensure solvent evaporation. Enzyme Treatments Aerial conidia, blastospores, a nd submerged conidia (0.5 1.0 x 107 cells/ml) were washed twice and resuspended in the manufact urers suggested enzyme reaction buffers. Stock solutions (100 l of 10 mg/ml) of amylase (S igma, A6255), cellulase (Sigma, C9422), or laminarinase (S igma, L5272) in 0.01 M KPO4, pH 6.8 for -amylase and pH 5.6 for cellulase and laminarinase, were adde d to 0.9 ml cells resuspended in the same buffer. For protease treatments, 100 l of a 10 mg/ml stock solution and 50 l of a 1 mg/ml stock solution of Protei nase K (Sigma, p6911) and Pr onase E (Stratagene, 300140) respectively, in buffer (0.1 M Tris HCl, pH 7.8, 0.5% sodium dodecyl sulfate (SDS), and 1 mM CaCl2) were added to cells resuspended in the same buffer. Glycosidase reaction mixtures were incubated for 4 hr at 250 C, and protease treatments were performed for 4 hr at 370 C. After incubation, cells were ex tensively washed in 50 mM calcium bicarbonate buffer pH 9.2 (7-8 times, 1 ml each) by centrifugation (10,000 x g, 5 min).

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24 Treated, washed cells were then FITC-labeled and used in adhesion assays as described above. Effect of pH Influence on Attachment FITC-labeled cell types (0.5 1.0 x 107 cell/ml) were washed twice and resuspended in one of the following physiologi cal buffers (0.1 M): acetate (pH 4 and 5), MES (pH 6 and 7), HEPES (pH 7 and 8) and TB (pH 8) before being used in adhesion assays. Control wells with cells suspended in TB (pH 8) were used to determine initial cell concentrations due to the pH sensitivity of fluorescence intensity measurements. Normalization due to pH effects on the FITC intensity was achieved by allowing adhered cells ( i.e. after the adhesion assay incubation and washing steps) to equilibrate in TB buffer (pH 8) until the fluorescence intensity of signal of the cells stopped increasing. Competition Assays Cells were FITC labeled, and the final cel l pellets resulting from the washing steps of the labeling reaction were suspended in TB containing 0.3 M carbohydrate (added as a competitor), 0.1% detergent (SDS, Tween 80 or CTAB), or 1 M NaCl solution. Cells were immediately used in adhesion assays. Contact Angle Determination Contact angle measurements of the surf aces used to evaluate the adhesive properties of the fungal cell types were determined using a Ram-hart Model 500 Advanced Goniometer with automated dr op dispenser and tilting plate running DropImage Advanced software (Ram-hart ). Dynamic angle measurements were determined just prior to movement of the wa ter drop. Briefly, a 10 l drop of sterile water was placed onto the surface of the substr ata to be tested. The stage and the camera were tilted at 10oC increments until the drop was on the verge of movement. The leading

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25 edge (dynamic) contact angle was determined from the last measurement taken prior to drop movement. Results A quantitative assay was developed to meas ure the kinetics of fungal cell adhesion to various substrata. Fungal cells chemica lly treated with the fluorescent reagent FITC, appeared uniformly labeled, with clear ha lo rings defining the cell wall (Fig. 2-1). Labeling of all three cell type s, aerial conidia, blastos pores, and submerged conidia displayed a linear relationship between cell number (as measured by cell counting using a hemocytometer) and fluorescence intensity (F ig. 2-2). Little va riation was observed within experiments (each point in Fig. 2-2 re presents triplicate samples); however, some variation (up to 2-fold in fluorescence inte nsity) was observed between separate FITC labeling reactions for each experiment. Ther efore, a standard curve of fluorescence intensity versus cell number, as determined by cell counting, was determined and used for each experiment. Fig. 2-1. Bright field (A, B, C) and fluorescent microscopy (D E, F) of FITC-labeled B. bassiana aerial conidia (A, D), blastospores (B, E) and submerged conidia (C, F). Bar = 5 m (A) and (B) and 10 m (C).

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26 In order to determine the effects of the la beling reaction on the a dhesive qualities of the cells, a series of preliminary qualitative experiments was performed using untreated and silinized glass slides with both unlabeled (not shown) and labeled cells (Fig. 2-1). In all instances no difference was observed using FITC-labeled or unlabeled cells (data not shown). These experiments demonstrated: (1) that aerial conidia were able to bind to hydrophobic, but not hydrophilic surfaces (Fig. 23), (2) blastospores bound uniformly to hydrophilic surfaces, but bound poorly to hydrophobi c surfaces, forming small clumps on the latter (Fig. 2-4), and (3) submerged c onidia bound equally well to both hydrophilic and hydrophobic surfaces, forming large clumps that appeared more evenly distributed over time (Fig. 2-5). These pa tterns were identical between FITC-labeled and unlabeled cells (data not shown). For the quantitative assays, three types of black polystyrene based microtitre plates with differing surface characteristics were us ed as substrata: (1) siliconized FluorotracF200, highly hydrophobic, (2) F200 untreated poly styrene surface, weakly polar, and (3) F600, treated polystyrene, hydrophilic, polar surface containing hydroxyl, carbonyl, and amino groups with a small net negative charge. Dynamic leading edge water droplet contact angle (c a) measurements of the three substrata agreed with their decreasing hydrophobicity. The silinated F-200 plates displayed a c a = 104.7o, the untreated F-200 plates a c a = 95.6o, and the F-600 plates a c a = 85.6o (contact angles for cleaned polished glass, the glass chamber slides, and silinated glass were de termined to be: 73.1, 87.4, and 109.7, respectively).

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27 Fig. 2-2. Fluorescent intensity of FITC labeled aerial conidia ( ), blastospores ( ), and submerged conidia ( ) as a function of cell number. Fig. 2-3. Adhesion of aerial conidia to gla ss (A1, A2, A3) and silinated glass (B1, B2, B3) surfaces after 5 min (A1, B1), 4 hr (A2, B2), and 24 hr (A3, B3) incubation on substrata. Bar = 30 m.

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28 Fig. 2-4. Adhesion of blastospores to glass (A1, A2, A3) and silinated glass (B1, B2, B3) surfaces after 5 min (A1, B1), 4 hr (A2, B2), and 24 hr (A3, B3) incubation on substrata. Bar = 30 m. Fig. 2-5. Adhesion of submerged conidia to glass (A1, A2, A3) and silinated glass (B1, B2, B3) surfaces after 5 min (A1, B1), 4 hr (A2, B2), and 24 hr (A3, B3) incubation on substrata. Bar = 30 m. The number of binding sites per microtiter plate well was estimated at 4-8 x 105 cells as determined by the saturation point de rived from plots of the percent cell bound as a function of cell concentration (Fig. 2-6). Th ese data indicated that the linear range of

PAGE 41

29 each cell type was similar, although the saturati on point for each cell type varied from approximately 25% of the aerial conidial cells able to bind per well, to greater than 70% of the submerged conidia bound in wells using ~5 x 105 cells/well. All subsequent experiments were performed using cell concen trations within the linear range of the attachment curve (2-5 x 105 cells/well) (Fig. 2-6). Quantitative adhesion assays were performed using aerial conidia, blastospores, and submerged conidia on hydrophobic, weakly polar, and hydrophilic surfaces with either 1 or 3 washes (Fig. 2-7). Aerial conidia bound rapidly and tightly to hydrophobic surfaces, with no loss of cell binding by up to 10 washes with buffer (data not shown). Aerial conidia bound poorly to weakly pol ar surfaces even after prolonged (24 hr) exposure to the substrata. Interestingly, th ese cells bound weakly to hydrophilic surfaces and were readily washed off i ndicating that this binding proc ess might be biphasic, with initial weak electrostatic binding. Fig. 2-6. Saturation point of binding sites for B. bassiana aerial conidia ( ), blastospores ( ), and submerged conidia (O ) to microtiter plates

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30 In contrast, blastospores bound poorly to hydrophobic surfaces with no greater than 10% of the cells bound even after 24 hr. Blas topsores bound moderately to weakly polar surfaces, with approximately 1-2 x 105 cells bound/well (30% of 5 x 105 cells used in the assay) after 4 hr incubation. Blastospor es bound more readily to hydrophilic surfaces, with up to 50% of the cells bound within 30 min. Submerged conidia displayed the broadest binding characteristics, adhering to all three surfaces, although with slightly differing kinetics. On hydrophobic, weakly pol ar, as well as hydrophilic surfaces up to 60% of the cells used bound to the substrat a within 4 hr, although in the case of the hydrophobic and weakly polar surfaces, almost half of the bound cells could be removed using three washes. Fig. 2-7. Quantitative adhesion and influence of washing on adhesion of aerial conidia, blastospores, and submerged conidi a to silinated F-200 (hydrophobic), F-200 (weakly polar), and F-600 (hydrophilic) mi crotiter plates. Dark violet bars represent a single wash; maroon ba rs represent a triple wash.

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31 Attachment of aerial conidia to hydrophobic surfaces could not be competed with any of the carbohydrate compounds tested includ ing glucose, galactose, lactose, maltose, melibiose, or trehalose and was insensitive to salt concentrations as high as 1 M NaCl (Table 2-1). The presence of N-acetylgl ucosamine appeared to promote adhesion (confirmed by microscopic analysis). The aff ect was not due to any visible growth or mucilage production, i.e. no germination/germ t ube or secretion of extracellular matrix was observed. Conidial attachment was, howev er sensitive to the presence of detergents, inhibition (80-90%) could be observed us ing 0.1% Tween-80 (non-ionic detergent), CTAB (cationic detergent), or Tr iton-X 100 (non-ionic detergent). Table 2-1. Effects of various competitors and chemicals on B. bassiana cell-type adhesion Attachment Ratioa Compound Aerial conidiab Blastosporesc Submerged conidiac Glucose 1.0 0.1 1 0.1 1 0.1 N-Acetylglucosamine 2.0 0.1 1 0.1 1 0.1 Fucose 1.0 0.2 1 0.2 1 0.1 Melibiose 1.0 0.2 1 0.2 ndd Maltose 1.0 0.2 0.1 0.1 1 0.3 Trehalose 0.8 0.1 0.9 0.1 1 0.1 1 M NaCl 1.0 0.2 0.9 0.2 1 0.2 0.2% Tween-20 0.2 0.1 0.1 0.1 nd 0.2% SDS 0.4 0.5 0.8 0.3 0.3 0.1 0.2% CTAB 0.2 0.1 0.6 0.3 1 0.2 0.2% Triton-X 100 0.1 0.1 0.3 0.1 0.6 0.1 a) Attachment ratio = (% cells bound under condition tested)/(% cells bound under control conditions) b) Cells tested on siliconized F200 (hydrophobic) microtiter plates. c) Cells tested on F600 (h ydrophilic) microtiter plates. d) nd, not determined The anionic detergent SDS also inhib ited conidial attachment to hydrophobic surfaces although a large degree of variation wa s observed. Adhesion of aerial conidia to surfaces was only slightly affected by pH w ith 30% less cells bound at pH 4.0 than at pH 7.0 (Table 2-2).

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32 A unique feature of blastos pore attachment was that a dhesion could be competed with maltose (Table 2-1). No other sugar te sted had any affect on blastospore adhesion nor did maltose affect conidial or submerge d conidial adhesion. Blastospore attachment was insensitive to salt (NaCl), SDS and CTAB, but was inhibited by Tween-20 and Triton X-100. In contrast to the other cell type s, adhesion of blastospores appeared to be pH dependent with a 50% decrease in the number of cells bound when assays were preformed at pH values of 4-5 compared to pH 7-8 (Table 2-2). Submerged conidia behaved similar to aerial coni dia except that N-acetylgluc osamine did not increase the number of cells bound and the presence of the detergents CTAB and Triton X-100 (Table 1) and changes in pH had only a minor affect on submerged conidial adhesion. Table 2-2. Effect of pH on B. bassiana cell-type adhesion Attachment Ratioa pH Aerial conidiab Blastosporesc Submerged conidiac 4 0.7 0.1 0.5 0.3 0.8 0.1 5 0.8 0.1 0.5 0.3 0.9 0.1 6 0.8 0.2 0.6 0.4 0.8 0.1 7 1.0 0.2 0.9 0.2 0.9 0.3 8 1.0 0.2 1.0 0.1 0.9 0.3 a) Attachment ratio = (% cells bound under condition tested)/(% cells bound under control conditions) b) Cells tested on siliconized F200 (hydrophobic) microtiter plates. c) Cells tested on F600 (h ydrophilic) microtiter plates. d) nd, not determined Removal of carbohydrates (maltose, glucos e or glucuronic acid) from the cell surface of aerial conidia using either -amylase or laminarinase, but not cellulase resulted in decreased conidial adhesion to hydrophobi c surfaces but had no affect on conidial adhesion to hydrophilic surfaces (Table 2-3) Treatment of blastospores with glycosidases appeared to eith er slightly promote adhesion ( -amylase and to a lesser extent cellulase treatment) to hydrophilic su rfaces or not affect adhesion (laminarinase

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33 and/or hydrophobic surface). Glycosidase treatm ent of submerged conidia resulted in a 25-50% decrease in adhesion to hydrophilic surfaces and hydrophobic surfaces. The only other remarkable difference wa s noted in the adhesion of -amylase, treated cells to hydrophobic surfaces where a large vari ation in adhesion was noted. Some differential effects were observed by protease treatment of the cell types (Table 2-3). Aerial conidia treated with Pr onase E displayed a greater than 50% loss of adhesion to hydrophobic surfaces but no loss in adhesion to hydrophilic surfaces, although a large variation wa s observed. This variation was between experiments, i.e. different cell batches treated with the enzy me and may reflect surface heterogeneity or accessibility of target substrates to the enzy me. Protease K treatment of aerial conidia did not result in appreciable changes in adhe sion. Similar treatment of blastspores with proteases had no effect or resu lted in an almost 2-fold a pparent increase in adhesion. Microscopic analysis ( i.e. visual counting) of the numb er of cells bound indicated that there did not appear to be an actual incr ease in the number of cells bound and instead protease treatment appeared to increas e the fluorescence intensity signal. Table 2-3. Effects of various enzymatic treatments on B. bassiana cell-type adhesion Attachment Ratioa Enzyme Aerial conidia hydrophobicb hydrophilicc Blastospores hydrophobic hydrophilic Submerged conidia hydrophobic hydrophilic -amylase 0.3 0.1 1.0 0.3 1.2 0.2 1.5 0.15 1.4 0.2 0.5 0.2 Cellulase 0.8 0.2 1.1 0.2 1.0 0.7 1.3 0.05 0.4 0.2 0.6 0.2 Laminarinase 0.3 0.1 0.9 0.3 0.9 0.1 1.0 0.2 0.4 0.2 0.8 0.2 Pronase E 0.4 0.3 1.2 0.3 0.9 0.1 ndd 1.1 0.2 1.1 .1 Protease K 0.9 0.2 0.8 0.1 1.0 0.1 nd d ndd 1.0 0.2 a) Attachment ratio = (% cells bound under conditi on tested)/(% cells bound under control conditions) b) Cells tested on siliconized F2 00 (hydrophobic) microtiter plates. c) Cells tested on F600 (h ydrophilic) microtiter plates. d) Apparent increase (see text for details).

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34 These were the only conditions tested in which the fluorescent signal was affected by the treatment. In all other experiments, mi croscopic analysis was in agreement with the fluorescent intensity measur ements. Finally, little to no effect was observed for the adhesion properties of protease treated submerged conidia. Discussion Conidial adhesion has been examined in a number of plant and insect pathogenic fungi (9, 65). Adhesion of entomopathogenic fu ngi appears to involve an initial binding interaction followed by a consolid ation step, resulting in firm a ttachment to the cuticle (9, 33, 34). Similarly, studies on the phytopathogenic fungus Botrytis cinerea revealed a two-stage adhesion process; immediat e adhesion occurri ng upon hydration and characterized by relatively weak attachment and a stronger delayed adhesion observed as the conidia germinate (25, 26) Immediate adhesion of B. cinerea is passive (nonmetabolic) and although no specific structur es were visible on the conidia, adhesion is characterized as dependent (in pa rt) on hydrophobic interactions. Hydrophobic interactions have also been implicated in the attachment of conidia of the insectpathogenic fungi Nomuraea rileyi Metarhizium anisopliae and B. bassiana to both host and non-host cuticle preparations (9). B. bassiana produces at least three single cell types that can be distinguished based upon morphological and adhesive characteristics. Experime nts qalitatively assessing entomopathogenic fungal adhesion to various su rfaces including insect cuticles (9, 10, 25, 26, 42) have almost exclusively addressed c onidial binding to surfaces and have not examined the adhesion properties of either bl astospores or submerge d conidia. Using a quantitative adhesion assay our results demons trate complex interactions between various cell types and substrata with differe nt surface properties. All three B. bassiana single cell

PAGE 47

35 types studied (aerial conidia, blastospores, and submerged conidia) displayed different adhesion properties that appeared to be mediated by different cell-specific mechanisms. B. bassiana aerial conidia were able to bind both hydrophobic and hydrophilic surfaces although adhesion to the latter was weak and the cells could readily be washed off. Aerial conidia binding to hydrophobic su rfaces could not be competed with any carbohydrate tested, although addition of N-acetylglucosamine, the monomeric constituent of chitin (the major carbohydr ate polymer found in arthropod cuticles) appeared to increase adhe sion. The hydrophobic nature of B. bassiana conidial spores, as well as those from other entomopathogens such as N. rileyi M. anisopliae and Paecilomyces fumsoroseus was correlated with the pr esence of an outer cell layer comprised of rodlets or fascicles as visual ized by electron microsc opy boucias (9, 10). These rodlet layers are presumably formed by assembly of specific proteins termed hydrophobins, which in turn are thought to passively mediate adhesion to hydrophobic surfaces (92, 96). Although our results are consis tent with this model, they also indicate that the interaction of aeri al conidia with hydrophobic surf aces may be more complex. Since amylase and laminarinase as well as protease treatments reduced adhesion (but displayed no discernable effects on the r odlet layer, Kirkland, Holder, & Keyhani, unpublished results), both carbohydrates on the cell surface as well as proteins may be involved in mediating adhe sion of aerial conidia. In contrast, blastospores, cells lacking a visible rodlet layer (9, 10), bound poorly to hydrophobic surfaces forming small aggregates or clumps, but displayed high binding to hydrophilic substrata. Blastospor es were also able to bind to weakly polar substrata, although a greater incubation time compared to hydrophilic substrata was required.

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36 Intriguingly, blastospore attachment could be specifically competed with maltose. No other carbohydrate tested appeared to compete with adhesion of blastospores or the other fungal cell types including trehalose the ma jor carbohydrate constituent found in insect hemolymph. In-vivo generated blastospores (distinct but similar to the rich broth produced blastospores), produced during funga l proliferation in th e insect hemolymph after penetration of the cutic le, are able to evade recogni tion by insect hemocytes and display altered membrane characteristic s (48, 49, 70). However, the physiological significance of potential maltose inhibition of adhesion of these cells is unclear. Submerged conidia displayed the broa dest binding characteristics of the B. bassiana single cell types, capable of effici ently binding hydrophobic, weakly polar, as well as hydrophilic surfaces. Spore tip or mucilage-covered appendages and adhesive knobs have been implicated to mediate coni dial adhesion of seve ral fungi (8, 45, 85). The mucosal coat of nematophagous fungi not only appears to mediate adhesion but is attractive to host insects. A wide variety of arthropod myc opathogens appear to produce exocellular mucilage during germ tube or a ppressorial formation (9). Similarly, the hydrophilic nature of conidia of the Entomophthorales is thought to be mediated by a mucilaginous coat released upon attachment to cuticle surfaces and that acts as a glue mediating attachment (30, 57). Although blasto spores and submerged conidia attached to hydrophilic surfaces, no obvious mucilaginous coat was visible in either cell type. In addidtion, scanning electron micr oscopy did not reveal any speci fic structures in conidia, blastospores, or submerged conidia of B. bassiana that are predicted to be involved in mediating adhesion (unpublished data). It is possible, however that extracellular matrix

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37 components or mucilage located between the inner and outer wall th at is not readily detectable could be involv ed in mediating adhesion. Although aerial conidia are considered easil y dispersible via ai r currents, and are more resistant to adverse environmental c onditions such as desiccation and extreme temperatures, submerged conidiation and blastospore formation may occur under a variety of environmental conditions as well as during the host-pat hogen interaction. The ability to produce multiple cell types, with differing adhesive properties, may occur in response to specific environmental conditions. This may allow fungal cells to bind to a br oad range of host targets and provide the fungus with a means to adapt to substrata condi tions (33, 34). It is unlikely, however that alteration in adhesion can account for emergence of restricted host-range B. bassiana strains, since these strains may have altere d (cuticle degrading) enzyme production or may be unable to penetrate and/or respond to surface cues of certain hosts, but may still retain the means to initiate bi nding or adhesion interactions. Indeed, there is some evidence that when entomopathogens specialize they loose structures rather than gain them, It would be interesti ng to see whether there is a ny alteration in the adhesion kinetics of general and specialized strains of B. bassiana Our data indicates that certain practical c onsiderations should be taken into account during application of B. bassiana For instance, if blastospores were to be used, formulations should probably avoid hydrophobi c solutions. Instead aqueous or polar liquids are recommended. Such formulations may prove to be more successful in the biocontrol of certain hosts as compared to aerial conidia.

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38 CHAPTER 3 SURFACE CHARACTERISTICS OF THE ENTOMOPATHOGEN BEAUVERIA BASSIANA Introduction The entomopathogenic fungus B. bassiana has a broad host range. The varied cuticles of these arthropod hosts are the first barriers to suc cessful infection. Attachment of fungal propagules to the cuti cle is the crucial initial even t in establishing mycoses. B. bassiana produces at least three distinct single ce lled propagules in response to differing growth conditions: Aerial conidia (AC), submer ged conidia (SC) and blastospores (BS). These spores are distinct in morphology, gene sis, germination char acteristics, lectin adhesion profiles and tolera nce of extreme environmen tal conditions (10, 72). Adhesion factors typically found in other organisms, including glycoproteins and carbohydrates, are present on fungal spores or in the extra-cellular matrix surrounding most fungal structures. However, the nonspecific physical prope rties important for adhesion are less well studied, especially for submerged spores (blastospores and submerged conidia). The net result of intera ctions between the surface of a solid and its surroundings is a function of the total surface free energy of the solid. According to Van Oss et al (1990), the total free energy (E) of a surface is the sum of three main components: acid/base interactions (AB), disper sive forces (LW) and electrostatic forces (EL) (Eq. 3-1) Etot = ELW + EAB + EEL (3-1)

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39 Dispersive forces, acid/base interact ions, and hydrophobic interactions can be calculated from contact angle data and by us ing Youngs equation, whereas the effective surface charge, or zeta potential, can be deri ved from the mobility () of cells in an electric field. Spores with rodlet layers or rough surfaces are more hydrophobic than smooth spores (39, 97). In our e xperiments atomic force microscopy (AFM) was used to generate high resolution images of the su rfaces of the three spore types produced by B. bassiana AFM is an example of proximity probe microscopy, where a cantilever, placed close the surface of the sample, is scanned in a raster (back and forth in an ordered manner) pattern to measure physical features su ch as height, elasticity, or hardness. A laser directed towards the cantilever is defl ected, and the deflections are recorded on a positron-sensitive photodiode. As the probe follows the contours of the sample, it is attracted, or repulsed by it. Th e instrument measures the force of these interactions, and renders the output graphically. The resoluti on of the instrument, which depends on the size of the probe, ranges from 10 pm to 125 m; the vertical limit of the sample is 5 m. In addition to high-resolution cell surface imaging, AFM can also generate data about surface biophysical propert ies. Two basic types of images are generated, height and deflection. The former gives accurate information about cell surface roughness and size, while the latter generates images with higher resolution for view ing ultra-structural details. In addition to these images, AFM can be used to generate force curves that can be used to measure local physical proper ties such as adhesion and elasticity. AFM images can be correlated to electros tatic, or hydrophobic characteristics of the different spore types. The hydrophobic char acter of the spores can be determined

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40 indirectly, by observing how well the spores at tach to different su rfaces, or directly, by quantitatively measuring hydrophobi c indicies. Past research has shown that different methodologies used to evaluate cell surface hydrophobicity do not always correlate well with each other, and can yield statistically significant differences for similar cells even from a single strain (2, 29, 46) Phase exclusion assays, specifically micr obial adhesion to hydrocarbons (MATH) and contact angles, have been used in a number of biological systems to obtain quantifiable hydrophobic indices. The advantages of this system include ease of use, reasonable reproducibility, extens ive use in the literature, a nd no requirement for costly equipment. However other factors, including electrostatic interacti ons, temperature, and pH, can affect the results of these experiments. Contact angle (CA) da ta is affected, to a lesser degree than MATH assays by similar factors and by the requirement, when using specific equipment that the cells remain flat, and are dry. The advantage of CA data is the multitude of information that can be generated from one set of data. To obtain contact angle measurements, a drop of liquid is deposited onto a given solid an image of the drop is pr ojected onto a screen and a tangent is drawn close to the drops surface, at the liquid/s olid/gas interphase. The angle at the solid/liquid/gas interface ( is called the contact angle (CA) (Fig. 3-1). Fig. 3-1. Contact angles ( are formed at the interface between the liquid, solid and gas boundaries of a droplet on th e surface of interest. Solid Liquid Gas

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41 The contact angle is a measure of how well a liquid wets a surface. An angle of 00 indicates complete wetti ng, while an angle of 1800 indicates absolute non-wetting. More generally when contact angles are < 900 the liquid is able to wet the surface to some degree, and the surface is hydrophilic. Angles > 900 indicate that the surface is hydrophobic. At 900 the surface energy of the solid is equal to the cohesive energy of water. The surface energy parameters of the solid can be determined if multiple, fully characterized liquids are used to obtain contact angles. According to van Oss et al. (1990), this information can be used to obtain solidliquid-solid interfacial energy ( Giwi), a measure of how quickly the surfaces will aggregate in liquids, when the solute is water; this is a measure of the hydrophobic nature of the surface. The surface energy paramete rs for the solid of interest are used to calculate Giwi by applying Youngs equation (Eq. 3-2) to the contact angles of multiple, well-defined liquids present on its surface. SV SL = VL cos Other surface energy parameters, such as the work of adhesion, the energy associated with the adhesion of a solid and a liquid, also defined as the work required to separate them (Eq. 3-2). The work of cohe sion, the energy required to separate a liquid into two parts, can also be calculated fr om contact angle data using the following equations (Eq. 3-3 & 3-4). Wa = (1 + cos ) (3-3) Wc = 2 Where, Wa = work of adhesion, Wc = work of cohesion = surface tension (liqui d)/surface energy (solid)

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42 It is no longer acceptable to use a singl e, static contact angle measurement to determine surface energy properties, or wet-ability. Dynamic contact angles are measured either while the drop is actively moving (advancing and receding angles), by gradually increasing the volume of the drop, or by tilting the surface until the drop moves while continously measuring the advanced and receded angle. The advanced and receded angles are defined as the angles obtained ju st prior to movement of the droplet. The advanced/advancing angle is the angle that is formed just as the drop is about to move over a dry surface. The receded/receding angle is the angle that is formed over already wet surfaces. The advanced angle is less affect ed by hysteresis than the receded angle, so it is the one most of ten reported (31, 86, 87). Hysteresis, the difference between the advanced/advancing and the receded/receding angles, is affected by su rface homogeneities larger than approximately 10 nm and by hydrophilic and hydrophobic mi croenvironments on solid surfaces. Hydrophobic micro domains are thought to have a breaking effect on the advanced/ing angle, while hydrophilic domains appear to have a retarding effect on the receded/ing angle. Sample roughness at the microscopic le vel increases hysteresis and has an effect on hydrophobicity. Rough surfaces, that are othe rwise identical to smooth surfaces, tend to be more hydrophobic, especially if they already have hydrophobic characteristics hysteresis (56). While acid/base, dispersive and hydrophobic interactions can be calculated from contact angle data, zeta potentials are th e most effective way of determining the electrostatic contribution. The movement of ce lls in an electrical fi eld is directed by the potential at the boundary between the cell and th e ions tightly associated with it and the

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43 surrounding medium. This potentia l is known as the zeta poten tial, and is a function of the cells surface charge, absorbed layers on the surface of the cell and the medium in which the cell is suspended. It is usually the same sign as the actual charge at the surface of the cell. The zeta potential is determined by measuring the moveme nt of the cells in an electric field, or the part icle mobility (velocity under uni t field strength). There is a simple relationship between particle mob ility () and zeta potential (in water @ 25oC zeta potential = 12.85 ). Materials and Methods Cultivation of Fungal Cells B. bassiana (ATCC 90517) was routinely grown on potato dextrose agar (PDA). Plates were incubated at 26o C for 10-15 days, and aerial conidia were harvested by flooding the plate with sterile dH20. Conidial suspensions we re filtered through several layers of Mira, cloth and the final spore con centrations were determined by direct count using a hemocytometer. Blastospores and submerged conidia were grown by inoculating conidia harvested from plates to a final concentration of 0.5 to 5 x 105 conidia/ml into DifcoTM Sabouraud dextrose (Becton, Dickinson and Co., MD) with 12 % yeast extract (SDY) and TK1 broth (83), with fructo se as the carbon source, respectively. Cultures, grown for 3 to 5 days at 26oC with aeration (150-200 rpm), were filtered (2X) through glass wool to remove mycelia. Final filtered suspensi ons of the three cell types were harvested by centrifugation (10,000 x g, 15 min, 40oC), washed twice with sterile dH20, and resuspended to the desired concentrations in appropriate buffers. Precentrifugation, post-centr ifugation, and final spore concentrations were confirmed by direct cell count (106-9 cells/ml).

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44 Atomic Force Microscopy Atomic force migrographs were generated using Digital Instruments Multimode SPM Atomic force Microscope with a silica nitrate tipped cantilever. Data from the micrographs was analyzed using Nanoscope SPM v.4.4 (Digital Images) and SPM Image Magic demo v1.10 (Alexander Kryzhanosvsky, A.F. Loffe physio-technical institute, St. Petersburg, Russia). Zeta Potential Aerial conidia, blastospores and submerged conidia were washed (3X) in water. The rinsed pellets were resuspended in 1mM KCl (pH 3-10, adjusted just prior to running the samples) to a concentration of 106 cfu/ml within 1 hr of running the samples on a Zetaplus micro-electrophoret ic unit (Brookhaven Instrument s Cooperation) with native software (PALS). Each cell type was run in triplicate. Ten microphor etic readings were run for each triplicate for a total of 30 readi ngs for each sample. The readings for the triplicate runs were averaged, and the standard error of the mean was determined for each sample. MATH Assay Cell surface hydrophobicity was determined essentially as described by Thomas et al (1986). Briefly aerial conidia, blastospores and submerged conidia were washed into PUM buffer (1.0 M sodium phosphate-urea-magnesium, pH 7.1). Fungal cell suspensions were adjusted to an OD470 = 0.4 and dispensed (3 ml) into acid washed glass tubes (12 x 75 mm). Hexadecan e (300 l) was added, each tube was vortexed (3 x 30 s) and allowed to incubated at RT for 15 min before the hexadecane phase was carefully removed and discarded. Tubes were cooled to 5oC and residual solidified hexadecane was removed. The tubes were warmed to RT, and the absorbencies of the resultant cell

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45 suspensions were determined at 470 nm. The hydrophobic index (HI) was calculated using the following equation (Eq. 3-5): HI = (initial OD470 final OD470)/initial OD470 (3-5) Contact Angle Determination Contact angle measurements of the B. bassiana cell types were determined from digital images of drops obtained using a Ram-hart Instruments Co. (NJ) Model 500 Advanced goinometer (automated drop dispen ser, tilting plate, digital camera, native Dropimage Advanced software). Advancing angle measurements were determined just prior to movement of the water drop. Briefl y, a 10 l drop of solution was placed onto the surface of the substrata to be tested. The stage and the camera were tilted at 10 increments until the drop began to move. Th e leading edge (dynamic) contact angle was determined as the angle obtained ju st prior to drop movement. Interfacial energies of interaction were calculated using the Acid/Base tool of the Dropimage Advance software. Water (polar), -bromonapthalene (non-polar) and glycerol (polar) were used to determine th e free surface energy components of the solid. Results Atomic Force Microscopy Fascicle bundles (a rodlet layer) were clearly visible on B. bassiana aerial conidia, but were absent from both blastospores and submerged conidia (Fig 3-2.) The blastospore surface appeared smooth, whereas the submerged conidial surface was rough and a raised lip was apparent on most of the la tter cells. No fascic les were visible on the germ tubes of germinating aer ial conidia (data not shown).

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46 A B C Fig. 3-2. Atomic force micrographs of B. bassiana A) conidia, B) submerged conidia and C) blastospores. (bar A) 10 nm, B) 0.1 m and C) 0.5 m) Zeta Potential The surface charge of the th ree cell types decreased as the pH increased. The isoelectric point for all cell types was PI4, except 16-day-old conidi a, which was PI5. Mean zeta potential values for aerial conidi a, blastospores, and submerged conidia were obtained over a pH range from 3-9 (Fig. 32). These values were obtained for a minimum of 3 different samples; each sample was run at least 10 tim es. Aerial conidia (16 and 20-day-old combined) had a positive ze ta potential (22 2 mV) that rapidly became negative by pH 4-4.5, reaching a net ne gative surface charge of 47 4 mV at pH 6-7. Blastospores and submerged conidi a also displayed a net positive zeta potential at low pH that decreased to 4 4 mV and 13 2 mV at pH 6-7, respectively (Fig. 43). MATH Assay Hydrophobicity indices (HI) in dicated that almost 90% (HI= 0.88) of the aerial conidia partitioned into the hydrocarbon layer. Submerged conidia partitioned at an intermediate level (HI = 0.7), and at least 60% of the blastospores (HI = 0.4) partitioned preferentially in the aqueous phase. Thes e values can be interpreted as relative hydrophobicity. However, absolute determina tion of which cell t ypes are definitely hydrophobic and which are hydrophilic are not possible and the cu toff is often set arbitrarily at 70% (HI = 0.7) (66).

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47 -50 -40 -30 -20 -10 0 10 20 30 012345678910 pH Fig. 3-3. Zeta potential values for the thre e spore types as a function of pH: (x) Conidia 16 days old, () conidia, 20 days ol d, blastospores (), and submerged conidia (o ). 0 0.2 0.4 0.6 0.8 1aerial conidiablastosporesmicrocycle conidiahydrophobicity index Fig. 3-4. Microbical adhesion to hydrocarbon and hydrophobici ty indices for B. bassiana aerial conidia, blastospores and submerged conidia.

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48 Contact Angles A surface is considered wetta ble if the liquid tested forms angles smaller than 90o. Water contact angles for the conidia of all three B. bassiana phenotypes were well over 90o (Table 3-1), while that of the submer ged conidia, and the blastospores were consistently below 900. The propensity of two surfaces to adhere to each other in water is calculated by the interfacial energy of interaction (Giwi IF) between the surfaces (in this case the surfaces of two similar spores) in water. The or iginal parental 90517 a ppeared to have two distinct phenotypes, which once separated bred true (Puff, 90517p and Blue, 90517b). The Giwi IF of conidia, regardless of phe notype, was negative (90517 .8 mJ/m2, 90517p .3 mJ/m2, and 90517b .8 mJ/m2), indicating that these spores are hydrophobic; whereas the submerged c onidia and the blastospores, had Giwi IF values greater than 0 mJ/m2 (30.2 mJ/m2 to 46 mJ/m2 and for submerged conidia and 66.3 mJ/m2 to 75.2 mJ/m2 for blastospores) indicating that th ey were hydrophilic (Table 3-2). The values for acid/base compone nt of the inte rfacial energy, Giwi AB for conidial cells ranged from 23.9 mJ/m2 to 69.4 mJ/m2, are were below +102 mJ/m2 ( Gw AB)which means they would be unable to overcome the c ohesive energy of water dissolve in water. The values for blastospores and submerge d conidia were all gr eater than +102 mJ/m2; specifically, 177.4 mJ/m2 to 191.6 mJ/m2 for the blastospores and, 171.5 mJ/m2 to 212 mJ/m2 for the submerged conidia i ndicating that these cells tend to disperse in water and are therefore hydrophilic. For the Giwi AB value to be greater than 102 mJ/m2, the electron donor surface energy parameter (S +) must be much lower than 25.5 mJ/m2 (L + for water), often around zero, and the prot on acceptor surface energy parameter (L -)

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49 must be much larger than 25.5 mJ/m2 (L for water). This was found to be true for the blastospores and mycrocycle conidia, but not for the aerial conidia (Table 3-2). Discussion The surface properties of the three B. bassiana cell types, as measured by AFM, contact angles, and the MATH assay, were markedly different. Freeze-etch, carbonplatinum transmission electron microscopy has previously shown the presence of a conidial rodlet layer on the dry aeri al conidia of many hyphomycetes, including Beauveria bassiana (5, 68, 97). In this dissertati on Atomic Force Microscopy confirmed the presence of this layer on the surface of our strain of B. bassiana. Aerial conidia were shown to be highly hydrophobic, especially wh en compared to the other spore types investigated. Table 3-1. Contact angle values for the thr ee cell types for water, bromonapthalene and glycerol with calculated interfaci al and polar free energy values. Cell type mean Contact anglea Hydrophobicityb Water -Bromonapthalene Glycerol Giwi AB Giwi IF 90517 Aerial Conidia 120.3.5c 66.2.5 112.7.9 47.2 -34.8 Blastospores 23.25.8 33.6.4 59.4.8 187.4 74.4 Submerged conidia 30.6.0 56.4.8 53.6.1 167.1 46.0 90517pd Aerial Conidia 122.1 0.6 47.2.6 108.2.3 49.3 -23.3 Blastospores 33.2.0 33.6.4 61.4.4 171.2 66.3 Submergd conidia 28.3.3 29.5.4 42.3.7 152.8 36.5 90517b Aerial Conidia 119.3.7 42.6.4 122.0.4 50.0 -23.8 Blastospores 23.9.8 39.5.5 68.9.9 183.7 75.2 Submerged conidia 28.6.0 38.8 .7 40.8.7 147.3 30.2 a) Mean of advanced contact angles n = 28 diff erent samples (>2 drops were used per sample) b) AB = acid/base, IF= interfacial energy. c) Advanced/receded angles were measured just befo re the drop started to roll after the stage was tilted d) p (puff) and b (blue) two stable phenotypes derived from the parent strain (90517)

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50 Table 3-2. Advancing and receding water co ntact angle data w ith calculated surface energy values for three B. bassiana single cell propagules. Cell type Polar component s AB s LW s total + (Polar)a (Dispersive) 90517 Aerial Conidia 9.7 1.5 33.3.6 43.0.1 4.3.4 5.5.5 Blastospores 10.8.3 23.6.3 34.4.3 0.4.2 86.1.7 Submerged conidia 23.3.1 16.3.4 39.6.8 2.0.3 68.4.0 90517p Aerial Conidia 0.6 0.01 33.3.3 33.9.1 9.4.5 6.0.6 Blastospores 3.2 0.8 24.5.50 27.7.7 0.04.02 71.8.7 Submerged conidia 16.6.0 25.1.30 41.7.5 1.9.3 57.3.6 90517b Aerial Conidia 13.6.1 30.3.2 43.9.7 2.1.2 0.9.16 Blastospores 7.3 1.4 21.7.6 29.0.0 0.16.1 82.7.5 Submerged conidia 24.5.9 24.7.4 49.2.8 2.8.4 53.2.7 a) AB = acid/base, s=solid, l= liquid gamma= surface energy components. b) p (puff) and b (blue) two individual phenotypes, genetically identical to the parent strain (90517). In 1999, Jeffs et al. concluded that the hydrophilic s pores had more electronegative groups than the hydrophobic ones because amin e modified, electropos itive, polystyrene beads attached better to hydr ophilic aerial conidia than to hydrophobic aerial. Our results suggest that the most hydrophobic spores, ae rial conidia (based on zeta potential measurements) have more electronegative groups than the hydrophilic ones, but that the stronger, and thus predominant, interactio ns for these spores are hydrophobic and the electronegativity of the spor es probably has a limited on ef fect initial interactions, although surface charge may be important for binding once the aerial conidia have made contact. Blastospores, the most hydrophillic spores have fewer electronegative groups, and electrostatic interactions a ppear to be the most importa nt factors involved in surface interactions for these cells. Submerged c onidia are intermediate in both hydrophobicity

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51 and electronegativity, indicating that for thes e cells both factors are likely to play an important role in adhesion to solid surf aces, dispersion in liquids, and flocculation. Previously, surface charge measurements of fungal spores often relied on indirect techniques, or were carried out at a single pH value. Wh ile these experiments yielded valuable information about the adhesion profil es of the spores, they often did not take into account the interactions of competing and synergistic fo rces on the adhesion profiles. As a result, indirect measurements of surf ace potential should not be the only criterion used when making inferences about the actual electric potential at the cell surface of the spores. In these experiments, the electronegativ e character of aerial conidia increased steadily with pH, reaching values as low as 47 mJ/m2 at pH 8.0. St. Leger et al. (1989) observed, that during fungal entomopathogenic infections, the pH of the insect cuticle rises from 6.3 to 7.7 during the initial phases, indicating that conidi a will be moderately electronegative during the first stages of infection, but that they will become more electronegative as the infection progresses. Because hydrophobic interactions are stronge r and act over much larger distances than electrostatic forces, it is likely that the primary inte rmolecular interaction involved in the initial adhesion process is hydrophobic in nature. It is equally probable that other intermolecular interactions become relatively more important once the spore is in close proximity with the insect cutic le. The variety of intermolecu lar forces are involved in the adhesion process probably ensure that B. bassiana aerial conidia remain attached to the cuticle until the germ tube penetrates, desp ite rapidly changing micro-environmental conditions.

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52 B. bassiana spores can vary widely in hydrophobi city, between strain as well as within the same strain, depe nding on age of the cells a nd culture conditions. This phenomena has been linked to possible spec ialization of the fungus towards different strains (51). Rodlet laye rs, while important to the overall hydrophobic ity of fungal spores, are not the only cell surface featur es involved in hydrophobic interactions. Removing these layers decreases, but doe s not eliminate the hydrophobic character of Aspergillus fumigatus conidia, and has diffe rent effects on cell su rface characteristics such as surface charge and ab ility to bind to hydrocarbons on A. nidualns and A. fumigatus (39). Submerged conidia are similar in shape to aerial conidia, but are typically larger and do not appear to posses a highly organi zed rodlet layer (9). These cells (when imaged using AFM) appeared rough compared to blastospores whose surfaces appeared smooth. Previous reports have shown that if these cells do posses a rodlet layer it is highly disorganized (10, 83). Submerged conidia also had sm aller absolute zeta potential than aerial conidia at higher pH. Although the environmental condition that leads to the formation of submerged conidia has not been well characterized, it has been observed that these cells are able to initiate infection at rates equal to or higher than aerial conidia (80, 83). Submerged conidia ap pear to be less tolerant to environmental stresses than aerial conidia and may be a specialized stra tegy related to nutrient/host availability. Blastospores, the smoothest of the three single cell propagules, did not posses a rodlet layer and were the most hydrophillic of the spore types. Hyphal bodies present inside the insect during infection are similar in shape and size to these spores but appear to have much thinner cell walls than blastosp ores (71). The electro static properties of

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53 blastospores did not vary as dramatically with pH as the other spore types, although previously we have noticed that the bindi ng of these cells to hydrophilic surfaces is dependent on pH. The ability of blastospores to attach under favorable conditions increased with increasing pH until the isoelectric point was reached (around pH 4). Adhesion did not however decrease with increasing pH >4.0 as expected, considering increased electrostatic repulsion under th ese conditions. Instead, the adhesion rates continued to increase as the ambient pH increased. As not ed by Jeffs et al. (1999), this is probably to due to other intermolecular f actors stabilizing the adhesive process and minimizing the electrostatic repulsion. This may also explain why these spores have moderate electrostatic properties, comp ared with aerial conidia. Strong electronegative surface charge and weak hydrophobicity may result in increased electrostati c repulsion leading to decreased adhesion. Both the MATH assay and contact a ngle data can be used to measure hydrophobicity. However, they do not give exact ly the same information. In the MATH assay, the propensity of the spores to partiti on in one layer preferen tially over another is used to assess the hydrophobic nature of the s pores. However, this assay does not take into account other non-specific forces that may affect partitioning and as a result should be used as a direct measure of the surface hydr ophobicity of the cells. Contact angles can give more precise determinations of the cell surface hydrophobicity (CSH); however the cells are usually dry when tested and this needs be taken into account when considering the results. Given these caveats, our result s demonstrate that the three spore types are very different in terms of their surface characteristics.

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54 Previous studies had conflicting results regarding the hydrophobi city of submerged conidia. Some reports noted that they we re as hydrophobic as aerial conidia (83), but Jeffs et al. (1999), using SAT, noted that blastosp ores were less hydrophobic than aerial conidia. Our results appear to back up this last interpretatio n; however it should be noted that there appears to be great heterogeneity between the cell surface ch aracteristics of the same type of spores from different B. bassiana strains. In additi on, the hydrophobicity of the spores can vary within a strain, depending on the age of the culture and the cultural conditions. This report also notes the importance of studying cell surface ch aracteristics (CSC) of any spore that might be considered for commercial applicati ons, using a range of biochemical parameters. The rational is that the microenvironment th e spores are likely to encounter on the surface of the insect cuticle may cha nge rapidly as the infection proceeds.

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55 CHAPTER 4 SUPPRESSIVE SUBTRACTION HYBRIDI ZATION ANALYSIS OF BEAUVERIA BASSIANA GROWN ON INSECT CUTICLE Introduction Virulence Factors Deuteromycete entomopathogenic fungi usually infect their arthropod hosts through the cuticle. Infecti on is dependent on the fungus successfully adhering to and penetrating through the hosts cuticle. Once inside the hos t the fungi must avoid being destroyed by the immune response and in so me cases have a means of exiting the host and dispersing its spores into the environm ent so that the cycle can continue. The molecular factors important to the infection process determine if an organism is pathogenic and which hosts it can invade, thes e factors are collectiv ely termed virulence factors. There are a number of definitions in the literature describing virulence factors. Some of the most common of these definitions include: 1) components that when deleted specifically impair virulence, but not the ability of the organism to grow, 2) products that allow the organism to cause disease and 3) components of the pathogen that cause damage to the host. The factors involved in the pre-penetrati on and penetration step s are important for initiation of infection, but are often redundant, or they work in tandem with other virulence factors. This makes it difficult to define them as viru lence factors under the first definition because it might be difficult to show that virulence is decreased when

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56 candidate genes are deleted. In the case of adhesion factors, while they are important in initiating the infectious process, they ofte n do not cause significan t damage to the host tissue, making it difficult to define under the third definition of virulence factors requiring virulence factors to cause significant damage to host tissue. The Edward Jenner institute for vaccine research lists the following groups of virulence factors, based on definitions in current literature, as follows: 1) adherence and colonization factors (e.g. adhe sions, integrins, hydrophobins), 2) invasins (extracellular enzymes involved in breaking down host defenses including proteases and chitinases), 3) toxins (e.g. cerato-plantanin and snodprot1), 4) molecules that permit the pathogen to evade the hosts immune system, 5) side rophores, and 6) molecules involved in the transport of toxins. Adherence and Colonization factors The adhesion of entomopathogens to the in sect cuticle is mediated by specific and non-specific factors including adhesins (e.g. hydrophobins), integrins (molecules containing the tripeptide sequence arginine-g lycine-aspartic acid (RGD) recognizable by molecules on host cells, lipids, and polysaccharides (84). Invasins Entomopathogenic fungi secrete specific extracellular enzymes (invasions) during pre-penetration and penetration events. Th e activity of infection specific proteases increases in the presence of insect cu ticle and secretion is often regulated by environmental cues (pH and nutrients) that sign al to the organism that it is on the cuticle of a suitable host. Many entomopathogenic fungi secrete substilin-like proteases (e.g. Pr1), and trypsin like proteases (e.g. Pr2) in response to environmental cues including high pH and nutrient deprivation ph (78). St. Leger et al. (1998) examined pH dependent

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57 secretion of M. anisopliae proteases and noted that these enzymes (Pr1, Pr2 and metalloproteases) that are typi cally secreted in response to the presence of insect cuticle and at higher pH (pH 8) than aspartyl proteases and amino pepdidases which are not typically involved in cuticle degredation. The highest activity of chitinases is observed when the fungi are exposed to chitin and is se creted later in the infection process than infection related proteases (78). The expr ession of hydrophobins implicated in adhesion and recognition of cuticle surfaces appear s to be regulated by mechanisms and environmental cues similar to those th at regulate the serine proteases and metalloproteases (78). Toxins Information relating to to xin production during infection comes mainly from the study of plant pathogenic fungi. During the in fection process these fungi employ similar adhesion and penetration mechan isms as those observed in entomopathogenic fungi. The wheat pathogen Phaeosphaeria nodorum produces a hydrophobin-like protein (Snodprot1), which is secreted mainly during invasion. Although this protein accumulates at the site of infection and brown lesions that progressively increased in size can be observed at the site of application of drop difussates from phaeospheria nudorum infected wheat leaves, the purified snodprot1 protein ap pears to have no specific cytotoxic effects (44). Conversely, purified ex tracts of a protein (c erato-platanin) highly homologus highly produced by the tabacco pathogen Ceratocystis fimbriata the causative agent of canker stain, shown to cause necros is in tabacco leaves (69). Snodprot-like proteins are related to hydrophobins by n-terminal se quence homology, level of hydrophobicity, numerous cysteines and size.

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58 Enzymes of pathogenic fungi involved in the synthesis of small cyclic polypeptides, or polyketide synthetases, are sometimes considered virulence factors. Polyketides are secondary meta bolites produced by a variet y of organisms including, fungi, plants, insects, and mollusks (60). Polyketides important for host-pathogen interactions include antibiotics (e.g. erytr homycin A), anti-tuberculosis compounds (e.g. rifamycin B), anti-helmintic compounds (e.g. avermectin) and numerous immunosuppressant agents incl uding ripamycin polyketides. Molecules Involved in Evading Host Defences Successful entomopathogenic infections de pend on the pathogen evading the hosts immune response, and/or using this respons e to increase the spread of the organism through the organism. Aspergillus spp. have numerous virule nce factors, the ones that have been implicated with evasion of the hosts immune system include the conidial rodlet layers, conidial melanins, deto xifying systems for reactive oxygen species (catalases and supre oxide dismutases) (73). Siderophores Iron is a catalytic cofactor in many redox r eactions; as a result it is an essential nutrient for many pathogenic fungi Iron is relatively unavailable because it is typically found in the insoluble ferric form (oxides and hydroxides) and with in the host it is usually bound to carrier proteins such as tr ansferrin. Pathogenic organisms obtain iron from scavenging systems such as high affinity iron permeases, siderophores (iron specific chelators) or heam oxygenases which aquire iron from haem groups (67). Molecules Involved in Toxin Transport Shapiro-Ilan et al. (2002) examined the natural and i nduced variability of the resistance of Beauveria bassiana strains to fungicidal agents They noted that exposure

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59 to fungicides increased virule nce against insect hosts, a nd this effect was observed without concurrent exposure of the fungicide to the insect. Indicating that being exposed to fungicides, in a manner not well understood, increased the organisms virulence (76). Beauveria bassiana strains are major secretor of toxins. Transporters are required to secrete these molecules and protect the or ganism from toxins produced by the host. There are two major groups of multidrug tran sporters the major facilitator superfamily (MFS) transporters and the ATP-binding cassette (ABC) transporters (21). Suppression Subtractive Hybridization Suppression subtractive hybr idization (SSH) a technique developed by Diatchenko et al. (1996), combines normalization and suppressi on into a single procedure. Within a single subtractive cycle, SSH suppresses th e amplification of a portion of abundantly expressed sequences, normalizing the ratio of high and low abundance transcripts. Differentially expressed tran scripts are selectively amp lified without the need to physically separate single stranded ( SS) cDNA from double stra nded (DS) cDNA (22). Many proteins and enzymes involved in prepenetration, and pene tration stages are secreted in response to insect cuticle. The enzymes secret ed at this stage are more efficient in breaking down insect cuticle than similar extracellular enzymes produced by non-pathogenic fungi, or by the entomopat hogenic fungi growing saprophytically enzymes (13). It has been relatively diffi cult to correlate individual enzymes with virulence, or pathogenicity, and thus to conclu sively defined them as virulence factors. This is often due to the high redundancy of th ese enzymes, many different isoforms of the same type of enzyme (e.g. Pr1 and pr1b, multip le chitinases, etc) that appear to replace those in mutant entomopathogens lacking specific hydrolases (13).

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60 Materials and methods Cultivation of Fungi Beauveria bassiana (ATCC 90517) was routinely grown on Luria Bertani (LB) (Difco, MI) (4). Plates were incubated at 26oC for 10-14 days, and aerial conidia were harvested by flooding the plate with sterile dH2O. Conidial suspen sions were filtered through a single layer of Mira-cloth (Clabi ochem, CA) and resuspended in sterile deionized water. Final spore concentrations were determined by direct count using a hemocytometer. Czapek dox (Difco, MI) broth (24 ml) was inoculated with a loopful of the colonies on the LB agar plates and incubated at 26oC with aeration (210-230 rpm). Cells grown in the presence of gl ucose (source of driver RNA) Minimal medium with 1% (w/v) glucose (0.4 g/l KH2PO4, 1.4 g/l Na2HPO4, 0.6 g/l MgSO4.7H2O, 1.0 g/l KCl, 0.25 g/l NH4NO3, 0.01 mg/l FeSO4 and 10 g/l glucose) was inoculated with 0.1 ml of the Czapek dox (24 ml) cultures (6 days). Cultures were grown for 6 days at 26oC with aeration (210-230 rpm) and filtered through a 0.22 m filter (Corning, NY), to remove culture supernatan t. The mycelial mat was washed (2x) with water and then lyophilized. Cells grown on insect cuti cle (source of tester RNA) The insect cuticles were removed from frozen (-20oC) mole crickets (Scapteriscus vicinus). The cuticles were disinfected by so aking in them 30% ethanol (30 min), followed by 30% cholorox (2 x 15 min). The cuti cles were rinsed repeatedly with water until there was no detectable chlorine odor. Minimal medium with 1% (w/v) glucose (0.4 g/l KH2PO4, 1.4 g/l Na2HPO4, 0.6 g/l MgSO4.7H2O, 1.0 g/l KCl, 0.25 g/l NH4NO3, 0.01 mg/l FeSO4 and 10 g/l sterilized cuticle) was inoculated with 0. 1 ml of the Czapek dox (24 ml) cultures (6 days). Cultures

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61 were grown for 6 days at 26oC with aeration (210-230 rpm) and filtered through a 0.22 m filter (Corning, NY), to remove culture supernatant. The mycelial mat was washed (2x) with water and then lyophilized. RNA Isolation Fungal cells were frozen in liquid Nitr ogen, or lyophilized and stored @ -700C until used. Immediately prior to use the cells we re crushed in liquid Nitrogen and total RNA was extracted with RNAwizTM (Ambion, TX) per manufacturers instructions. Poly A RNA was enriched using magnetic beads c ovalently attached to oligo-dT tails (Dynabeads mRNA direct kit, Dynal, WI, Poly (A) Quick mRNA isolation kit, Stratagene CA, or Poly A tract mRNA isolation Systems, Promega, WI). Supression Subtractive Hybridization The PCR-Select cDNA subtraction kit (Cl ontech, CA) used as per manufactures instructions. The RNA purified from cells gr own on glucose was design ated as the driver RNA, while the RNA from cells grown on ins ect cuticle, was designated as the tester RNA. The amplified PCR product was cloned into Topo TA (Invitrogen, CA) or pGEM-T easy (Promega, WI) cloning vectors. Results Aproximately 350 clones were sequenced and analyzed, resulting in 280 nonredundant sequences. Of these, 57 % had no homology to known proteins, had weak homology (expect value < 1e-5) to known proteins, or gave hits to hypothetical, or proteins of unknown function. The remaini ng proteins were loosely grouped according to function: (i) Enzymes (10%), (ii) Miscellaneous (proteins that did not fit neatly into specific groupings (18%), (iii) signal transduc tion (6%), (iv) virulence factors (1%), (v)

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62 allergens (2%), and (vi) ribosom e/histone proteins (5%) (Fig 4-1). The proteins with weak homology to specific functional groups were listed accordingl y in figure 4-1. Signal transduction 6% Virulence factors 1% Allergens 2% Other 57% Ribosome/histone 5% Enzyme 10% possbile ribosome/histone 1% Hyopthetical proteins 1% pos. virulence factors 1% possbile signal transduction 4% possible allergens 1% possible enzymes 5% No significant homolgy 24% No Hits 6% possible misc. 15% Miscellaneous 18% Fig. 4-1. Relative numbers of gene frag ments representing functional groups present in the subtracted library, the smaller gra ph represents proteins with no homology to known proteins, strong homology to hypot hetical proteins, or proteins with unknown function or with w eak homology (expect value < 0.0001) to proteins with known functions. Among the virulence factors, two fragments were sequenced that gave strong hits (4e-7 and 2e-6) to a class I, rodlet laye r hydrophobin belonging to the Aspergillus genus (expect values for whole genes: 2e-36 A. fumigatus (rodA), 1e-34 A. nidulans (rodA) and 4e-30 Gibberella moniliformes (HYD1)). In addition to the hydrophobins, strong hits were obtained to a tetrac ycline efflux protein (1e15) an allergen (9e-5) (Table 4-1). The full expressed and genomic sequence for bhd1 was obtained (Keyhani, unpublished results) (fig. 4-2) and this information was used to design the primers for the real time RT-PCR.

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63 Table 4-1. Blast hits of sequence fragments fr om the SSH library to virulence factors and allergens. ID function e value type Known virulence factor accession # 2-4-G02.g-b1 angi_emeni antigen 1 precu rsor9.00E-05 allergen emb|CAA90537.1| 1RCA-F05.g-b1 Tertracycline efflux protein 1.0E-15 antibiotic transport x emb|CAC28808| 2-4-E02.g-b1 rodl_emeni rodlet pr otein 2.00E-06 hydrophobin x sp|P28346| 1RCA-A06.g-b1 Rodl_emeni rodlet protein precursor 4.0E-07 hydrophobin x sp|P28346| 1RCA-B06.g-b1 STARP antigen 1.0E-06 invasin x emb|CAA82996| 2-4-A05.g-b1 aldehyde dehydrogenase 7.00E-55 metabolism gb|AAF82789.1| 2-3-H05.g-b1 aldehyde dehydrogena se 1.00E-20 metabolism sp|P41751| 2-4-E09.g-b1 aldehyde dehydrogenase 5.00E-13 metabolism emb|CAA5071.1| 2-4-E08.g-b1 cyanate lyase 5.00E -06 metabolism djb|BAB73248.1| 2-3-F01.g-b1 Fatty acid synthase (EC 2.3.1.85) 2.00E-16 metabolism sp|P15368| 1RCA-E02.g-b1 fructose 1-6 bisphosphate aldolase 1.00E-07 metabolisim dbj|BAB12232.1| 2-3-B11.g-b1 fructose 1-6 bisphosphate aldolase 3.00E-19 metabolism dbj|BAB12232.1| 2-3-B03.g-b1 fructose 1-6 bisphosphate aldolase 1.00E-09 metabolism dbj|BAB12232.1| 2-3-H12.g-b1 fructose 1-6 bisphosphate aldolase 9.00E-09 metabolism dbj|BAB12232.1| 1RCA-C10.g-b1 Fructose 1-6 bisphosphate aldolase 4.00E-08 metabolism dbj|BAB12232.1| 1RCA-H06.g-b1 glucose repressible gene 1.0E-16 metabolism emb|CAA32907.1| 2-3-C12.g-b1 Glyceraldehyde-3-phosphata se 2.00E-13 metabolism gb|AAK20083| 2-4-H05.g-b1 glyceraldehyde-3-phosphate dehydrogenase 6.00E-52 metabolism sp|P54117| 2-4-G09.g-b1 glyceraldehyde-3-phosphate dehydrogenase 2.00E-14 metabolism emb|CAA37943.1| 1RCA-A03.g-b1 homocytrate synthase 4.0E-53 metabolism gb|AAF66618.1| 2-4-D01.g-b1 nitrogen metabolite rpression 9.00E-08 metabolism x emb|CAA758663.1| 1RCA-D03.g-b1 nucleoside diphosphate kinase domain 5.6E-101 metabolism pfam dom PF00334 2-3-E07.g-b1 serine biosynthesis enzyme 3.00E-17 metabolism emb|CAA86096.1| 2-4-G10.g-b1 serine biosynthesis enzyme (EC 1.1.1.95) 3.00E-30 metabolism emb|CAA86096.1| 1RCA-A07.g-b1 sphingolipid boisynthesis protein 1.0E-12 metabolism ref|NP_014814.1| 1RCA-E08.g-b1 Sterol methyltransferas e 3.00E-20 metabolism emb|BAB97289.1| 2-4-H01.g-b1 Succinate dehydrogenase (ubiquinone) 9.00E-40 metabolism gp|2801670| 1RCA-C04.g-b1 utp glucose phosphate urydyltransferase 9.0E-09 metabolism emb|CAA22857.1| 2-4-B08.g-b1 metallochaperone 7.00E-12 metal homoestasis x gi|13787053| 2-B11.g-b1 Cap20 2.00E-09 opac ity factor x gb|AAA77678.1|

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64 Table 4-1. Continued ID function e value type Known virulenc e factor accession # 1RCA-D12.g-b1 ATP synthase (EC 3. 6.1.34) 2.00E-43 other sp|P23704| 1RCA-C08.g-b1 ATP synthase (EC 3. 6.1.34) 7.00E-30 other sp|P37211| 1RCA-H10.g-b1 Canal 40S ribosomal prot ein 1.00E-68 other emb|CAA57542.1| 1RCA-G11.g-b1 chromosomal protein, non histone 8.00E-14 other emb|CAA33378.1| 2-4-C07.g-b1 Histone H2A 1.00E -32 other emb|CAA07351.1| 2-F01.g-b1 Histone H2B 1.00E-31 other gi|283352| 2-4-A08.g-b1 Histone H2B 7.00E-25 other sp|P37210| 2-4-E05.g-b1 histone H3 5.00E-19 other sp|P07041| 1RCA-E12.g-b1 Histone H4 1. 00E-18 other emb|CAA25760.1| 2-4-F11.g-b1 leucyl-tRNA synthetase (EC 6.1.1.4) 1.00E-31 other sp|P10857| 2-4-H12.g-b1 leycyl-tRNA synthetase (EC 6.1.1.4) 2.00E-30 other sp|P10857| 2-4-H06.g-b1 lukemia related prot ein 1.00E-05 other ref|NP_065882.1| 2-4-F06.g-b1 Nuclear plasma membrane ATPase (proton pump) (EC 3.6.1.35) 3.00E-34 other emb|CAB91207.1| 2-4-G06.g-b1 nuclear transport fa ctor 6.00E-19 other gb|AAK71467| 2-4-A11.g-b1 signal recognition particle receptor 6.00E-31 other ref|NP_010578.1| 2-4-D07.g-b1 Vacuolar ATP syntha se 2.00E-14 other gb|AAA03446| 2-3-F06.g-b1 zinc finger protei n 3.00E-09 other ref|NP_006620.1| 2-3-B09.g-b1 60s ribosomal protein 2.00E-29 protein metabolis m emb|CAB10813.1 2-3-C09.g-b1 ubiquitin like protein 3.00E-25 protein metabolis m gb|AAAF24230.1| 2-4-C10.g-b1 40s ribosomal protein 3.00E-13 protein synthesis emb|CAA33608| 2-3-C04.g-b1 60s ribosomal protein 4.00E-14 protein synthesis sp|Q9C0T1| 2-3-B10.g-b1 elongation factor 1 alpha 4.00E-40 protein synthesis sp|Q01765| 2-3-C11.g-b1 elongation factor 1 alpha 7.00E-35 protein synthesis gb|AAK49353.1| 2-D10.g-b1 elongation factor 2 1.00E-35 protein synthesis gb|AAK49353.1| 2-4-A12.g-b1 ribosomal protein 3.00E-58 protein synthesis emb|CAC28787.1| 2-3-D10.g-b1 ribosomal protein 5.00E-29 protein synthesis sp|P40910| 2-4-A10.g-b1 ribosomal protein 1.00E-18 protein synthesis emb|CAC18189.1| 2-3-D07.g-b1 ribosomal protein L30 3.00E-21 protein synthesis gb|AAK58056.1| 2-3-A02.g-b1 ribosomal protein L34 9.00E-14 protein synthesis gb|AAK58051| 2-4-D04.g-b1 translation elongation factor 6.00E-20 protein synthesis dbj|BAA11572.1|

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65 Table 4-1. Continued. ID function e value type Known virulence factor accession # 2-3-E04.g-b1 G protein 1.00E -40 signalling gb|AAF78478.1| 1RCA-H03.g-b1 serine/ther onine kinase 9 1.0E-05 signalling gb|ADD28798.1| 1RCA-H02.g-b1 serine/ther onine kinase 9 6.0E-05 signalling gb|ADD28798.1| 1RCA-C09.g-b1 serine/theronine protein kinase2.00E-13 signalling emb|CAA22846| 2-4-A09.g-b1 Serine/threonine protein kinase SNF1 7.00E-24 signalling gb|AAK69560| 2-A09.g-b1 Possible stress response 7.00E-13 stress response x emb|CAC22612.1| 2-4-F02.g-b1 actin 4.00E-11 structural emb|CAC28227.1| 2-D11.g-b1 hypothetical protein vesicle membrane protein 4.00E-53 structural emb|CAC18239.1| 1RCA-D04.g-b1 kinesin motor domain 7. 1E-82 structural pfam dom PFOO225 2-H08.g-b1 symbiosis related protei n 7.00E-13 symbiosis x ref|NP_193929.1| 2-3-A05.g-b1 membrane transporter MFS 2.00E-06 transporter x emb|CAC36910.1| 2-4-A02.g-b1 aquaporin 7.00E-09 transporter gb|AAC69713.1| 2-4-H07.g-b1 low/affinity zinc transport protein 1.00E-12 transporter emb|CAA62642.1| 2-F12.g-b1 hypothetical protein 4.00E-24 unknown emb|CAB88645.2| 2-4-H03.g-b1 hypothetical protei n 6.00E-30 unknown ref|NP_011435.1| 2-4-E04.g-b1 hypothetical protei n 7.00E-11 unknown emb|CAB63552.1| 2-4-G05.g-b1 hypothetical protei n 4.00E-05 unknown emb|CAA96785.1| 2-H06.g-b1 hypothetical protein 1.00E-22 unknown pir|T50065| 2-3-H11.g-b1 hypothetical protei n 2.00E-19 unknown emb|CAA95893| 2-3-C08.g-b1 hypothetical protein 1.00E-07 unknown pir|S50309| 2-3-A04.g-b1 hypothetical protei n 4.00E-07 unknown emb|CAB72282.1| Discussion Our subtracted library generated 10 pu tative virulence factors (including B. bassiana hydrophobin, bhd1, a tetracycline efflux transpor ter and two potential allergens (STARP and ASPII)), however we were unable to find a number of expected virulence factors including serine proteases and chitin ases that are secreted during the infection stage. This is probably due to the mixure of cells present in the library, many of which may not have been producing proteases, and to the lack of synchr onization or the cells with resect to the infection process.

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66 1 CTATAGGGCG AATTGGGCCC TCTAGA TGCA TGCTCGAGCG GCCGCCAGTG 51 TGATGGATAT CTGCAGAATT CGCCCTTTCT TCACATCTCT TCCTAGTGAT 101 GTCCATGCAC CAGCCTACTG GATAAGACTG CCAATGGCGA CGCAAGGCAG 151 GGCAAGGTTG ACAAGACCAC CGTAAGCCTA ATTGTATCGT TAGATCCTGA 201 ACAGCAGTCA ATTAGAGACT TGATATAACT TACGACAGAG GGAGTGTTGT 251 GGCAGCAGGC CGCACGGTTG GTGCACTCGG ACTGGAGAAG GTTGCCGACA 301 GCGAGGACTG GAGACTTGTT AGTACTTGCT CAGCGACTTT ATCGACCACT 351 TGAGGCTTAC CATTGACGGG GATCTTGGTG CAAGTACCGA GAAGGTGGGA 401 AAGAAGACCA TCAGCACCGA CAGTGGCACC GAGCAGGCCG CCGAGGCCGG 451 TTTCGGGGGA GGAGTGCTCG ATCTTGGTCG TCTTCTCGTT GCAGCAGTAG 501 ATGGACTGGT CGGCACCGCA CCTGTCGCCA GCCTGGCCGA GAGTCATCTT 551 GTGCAGATCC TTTCCACCAT GGTGGCCGGA GGGAGCAGCA GTCACAGCGG 601 CGATGAGGGT GGTGATGGCA AGAGCGAAAC GCATGATGAC GGTATTGTTT 651 GTTTGGTTGA AGGATGTGGA AGAGGTGAAG GGCGAATTCC AGCACACTGG 701 CGGCCGTTAC TAGTGGATCC GAGCTCGGTA CCAAGCTTGG CGTAATCATG 751 GTCATAGCTG TTTCCTGTGT GAAATTGTTA TCCG Fig. 4-2. B. bassiana H1 hydrophobin genomic sequence. Expressed sequence in bold. In our laboratory we have generated a series of B. bassiana stage and cell specific cDNA libraries (Cho et al personal communicatio n). From these libraries it has become apparent that many of these proteins are expressed under conditions present in our driver library, and that further analysis of subtract ed libraries of purifie d cells from different stages would give a better picture of th e expression of putativ e virulence factors. The fragments hydrophobin (bhd1) and other potential vi rulence factors found in the SSH library were sequenced, cloned a nd the full-length sequences were obtained using 5prime RACE (Keyhani, unpublished resu lts) when needed (some of the smaller genes did not require additional analysis to generate full length cDNA sequences). PSIBlast searches indicated th at the hydrophobin had the strongest homology to the RodA proteins of A. fumigatus and A. nidulans, and a weaker homology to the HYD1 and HYD2 proteins of Gibberella moniliformis (anamorph: Fusarium verticillioides). However, Bootstrapped, phylogenetic analysis (Phylip 3.6, ClustalX, and phylodendron) of class I and class II hydr ophobins deposited in the NCBI protein database, from Ascomycetes and the Basidiomycetes, places them closer to the F. verticilliodes hydrophobins (figures 4-3 & 4-4).

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67 Signal peptide M._grisea_mpg1_class MFSLKTVVLALAAAAFVQAIP------------------------APGEG M._anisopliae_ssgA_precursor MF--KALIVALAAVAA--AIPTQ----------------------QPSSN B._bassiana_H2 MLA--TTIFATLLAL-AAAAPH-----------------------GPSHG A._nidulans_rodl_emeni MKFSIAAAVVAFAAS-VAALPPAHDSQFAGNGVGNK--GNSNVKFPVPEN A._fumigatus_rodl MKFSLSAAVLAFAVS-VAALP-QHDVNAAGNGVGNK--GNANVRFPVPDD A._fumigatus_Rodbp MKF--LAVVSLLAAT-ALALP-------------NA--GVVHPTFASADK G._moniliformis_H1_Fusarium MQY--MTIVAFLAAT-VAAGP------------------QIRAY-PSIDQ G._moniliformis_H2_Fusarium MYA--YTVIAFLAAS-VAAAG--------------------NG--PSISS B._bassiana_H1 MRF-ALAITTLIAA--VTAAP-----------------SGHHGG-KDLHK A._nidulans_DEWA_EMENI__class_ MRFI-VSLLAFTAAATATALP---------------ASAAKNAKLATSAA * M._grisea_mpg1_class PSVSMAQQKCGAEKVVSCCNSKELKNSKSGAEIPID-------------M._anisopliae_ssgA_precursor ---EMN---CDSG--VYCCN--KVAQN-TGIVVPID-------------B._bassiana_H2 PSVKTG-DICGNGNTMHCCNDESVTNKLTGPSVLSDL------------A._nidulans_rodl_emeni VTVKQASDKCGDQAQLSCCN--KATYAGDTTTVDEGLLSGALSG--LIGA A._fumigatus_rodl ITVKQATEKCGDQAQLSCCN--KATYAGDVTDIDEGILAGTLKN--LIGG A._fumigatus_Rodbp YTLQQAQNKCGEHTTLSCCN--HVSKVGDTTAFNYGLLNGLLGN--AI-G._moniliformis_H1_Fusarium ITVAQANNACGNNMQVTCCN--KVTNTPAGNAVGNGAG-----------G._moniliformis_H2_Fusarium LTVQQAANSCANGQSVYCCN--KTSNKPAGNSVGDGAG-----------B._bassiana_H1 MTLGQAGDRCGADQSIYCCN--EKTTKIEHSSPETGLGG-LLGATV---A._nidulans_DEWA_EMENI__class_ FAKQAEGTTCNVG-SIACCNSPAETNNDSLLSGLLGAG--LLNGL----* : *** M._grisea_mpg1_class -----VLS----GECKNIPINILT--INQLIP--INNFCSDTVSCCSG-M._anisopliae_ssgA_precursor -----ALS----STCG----DTLK--LVTVDA--LNDKCTSQTVCCNN-B._bassiana_H2 -DLRKLLA----AECSPISVNVL---LNQLVP--IDNKCKQQSICCGE-A._nidulans_rodl_emeni GSGAEGLG--LFDQCSKLDVAVLI--G---IQDLVNQKCKQNIACCQNSP A._fumigatus_rodl GSGTEGLG--LFNQCSNVDLQIPV-IGIP-IQALVNQKCKQNIACCQNSP A._fumigatus_Rodbp -SGPEGVG--ILSGCQKISVTALI--G---VDDLLNKQCQQNVACCQDNK G._moniliformis_H1_Fusarium --ILNNLS--LFDQCSKLDVNVLA-IA----NGLLNKECQANAACCQNSG G._moniliformis_H2_Fusarium --IANGLS--LFSQCSKVDVNVIA-IA----NNLLNKECQANAACCQDSP B._bassiana_H1 --GADGLLSHLLGTCTKIPVNVLA-VG----N-LLQSECTNRAACCHNTP A._nidulans_DEWA_EMENI__class_ -SGNTGSA------CAKASLIDQLGLLALVDHTEEGPVCKNIVACCPEG* ** M._grisea_mpg1_class -EQI-GLVNIQCTPILS-----M._anisopliae_ssgA_precursor -VQQNGLVNVACTPIDV-----B._bassiana_H2 -QKLNGLVNLGCTPITVLG---A._nidulans_rodl_emeni SSADGNLIGVGLP-CVALGSILA._fumigatus_rodl SDASGSLIGLGLP-CIALGSILA._fumigatus_Rodbp SVATGGLINIATPACVALDSIIG._moniliformis_H1_Fusarium GSATGGLVNVALP-CIALSSLIG._moniliformis_H2_Fusarium GTAAAGLVNAALP-CVAISNLVB._bassiana_H1 SVAYGGLVNLALP-CVAIGSLIQ A._nidulans_DEWA_EMENI__class_ ---TTN--------CVA-----. : Fig. 4-3. Alignment of H1 and H2 with homologous hydrophobins (C lustalX) that gave significant expect values in PSI blast. Signal peptide length was determined using SignalP.

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68 0.1 Fig. 4-4. Bootstrapped (1000) phylogenetic tree of class I an d class II hydrophobins using alignments generated with Clastal X and visualized with the online tree printer Phylodendron (http://iubio.bi o.indiana.edu/tr eeapp/treeprintform.html)

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69 This is interesting because the F. vercticilliodes microconidia are hydrophilic (38) and this organism is a plant pathogen with specificity towards corn and B. bassiana, an entomopathogen with hydrophobic conidia, can in fect corn asymptomatically (88). It is possible that some of the same factors that allow F. verticilliodes to infect corn are similar to those that allow B. bassiana to colonize the same organism. It is possible that the expected genes we re present in the subtracted library. The fragments generated by the SSH procedure are quite small making it difficult to determine if the fragments did not have strong hits because there were not matches, or if they were to small to generate strong h its. Annotated information for fungal genomes lags behind that for many other organism s, and as more annotated sequences are deposited into the databases, more hits to named proteins is likely to occure.

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70 CHAPTER 5 MOLECULAR ANALYSIS OF TWO BE AUVERIA BASSIANA HYDROPHOBINS AND A HYDROPHOBIN LIKE PROTEIN Introduction Hydrophobins are small, cysteine rich, s ecreted proteins uni que to fungi. Many hydrophobins were initially identified from a bundantly transcribed mRNA present during sporulation, fruit body formation, and during pathogenic interactions with plants and animals (52). These proteins play a critical role in numerous filamentous fungal processes including; growth, conidial form ation, conidial dispersion, ae rial structure formation, and attachment to (hydrophobic) surfaces, and pathogenicity (93). Wessels et al. (1991) noted a positive correlation between Sc3 mRNA abundance and the presence of aerial hyphae and fruiting bodies in Schizophyllum commune. They estimated that by the fourth day of growth up to 8.1% of the protein synthesis activities in the dikaryon (fruiting bodies are composed of dikaryon hyphae) is directed towards the synthesis of hydrophobins. Rodlet layers have been observed on c onidial surfaces of numerous filamentous fungi including, Neuorospora crassa, Aspergillus sp., Magnaporthe grisea, Metharhizium. anisopliae and Beauveria bassiana. The presence of this rodlet layer appears to increase th e hydrophobic nature of the conidi a. The better organized, the fascicles, the more hydrophobic the spores. Hy drophilic conidia formed by fungi such as Gibberella moniliformis, Verticillium lecanii and Botrytis spp. do not appear to form a

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71 rodlet layer, and have additional structures that mediate attachment to surfaces including mucosal coats, and adhesive knobs (94, 99). Hydrophobins produced during s porulation are involved in the production of the rodlet layer that has been observed on the surface of many hydrophobic conidia. Deletion mutants lacking rodAp in A. nidulans and A. fumigatus (68) or mpg1 in M. grisea (52) generated spores lacki ng the characteristic rodlet la yer, and displaying altered adhesion characteristics. The relative importance of th e hydrophobins to rodlet layer formation and adhesion propertie s may vary with species. Paris et al. 2003 observed that both A. fumigatus and A. nidulans possess two conidial hydrophobins RodA p (both sp.) RodB (A. fumigatus) and DewAp (A. nidulans). Deleting rodAp resulted in loss of the conidial rodlet layer in A. fumigatus while deleting rodBp had no effect on rodlet layer formation. In A. nidulans similar studies showed that both rodAp and dewAp were required for conidial r odlet layer formation. Paris et al. (2003) also noted that changes in cell surfac e characteristics were more profound in the A. nidulans rodletless mutants than in the corresponding A. fumigatus mutants. Unlike A. nidulans and A. fumigatus, the rodlet layer of B. bassiana, Neurospora crassa and Magnaporthe grisea appear to posses a si ngle hydrophobin (68). In M. grisea, deleting mpg1 resulted in rodletless mutants similar to those observed for A. fumigatus and A. nidulans (52). This protein was also linked to pathogenicity by the observation that appropriate appressorium form ation, in response to external surface cues, was impaired in mpg1 mutants (3). All hydrophobins isolated to date self-a ssemble into 10 nm thick layers at hydrophobic/hydrophilic interfaces. Based on the sol ubility characteristics of the layers

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72 and hydropathy patterns of the amino acids, hydrophobins have been separated into two classes (I and II) (96). Treatments with trifl uoroacetic acid (TFA), or formic acid (FA) are often required to dissol ve class I layers which rema in insoluble in 2 % sodium dodecyl sulfate (SDS) at 1000C. In contrast, layers form ed by class II hydrophobins are more soluble and can be dissolved in 60 % ethanol and 2 % SDS. Class I hydrophobins self assemble at hydrophilic/hydrophobic interfaces, forming 5-12 rodlets that bundle toge ther into fascicles. Th e rodlets are made up of protofilaments 2.5 nm in diameter. Thus fa r, similar bundles have not been observed in the amphipathic layers formed by Class II hyd rophobins (96). Atomic resolution of the structure of HfbI and Hf bII, hydrophobins from Trichoderma reesei, revealed that these Class II hydrophobins form tetrameric supram olecules that assemble into crystalline domains (43). The rodlet layer, like the proteins from which it is composed, is amphipathic. When coating the surface of fungal structures this layer arranges itself so that the hydrophilic part of the membrane faces the cell wall of the fungus, while the hydrophobic side is exposed to the environment. In A. nidulans and other fungi with hydrophobic conidia this layer, especially when present on the su rface of conidia, is thought to be important in hydrophobic interactions between fungus and su rfaces, however in th e closely related species, A. fumigatus adhesion to hydrophobic substrates is not completely lost in mutant strains lacking the major s pore coat hydrophobins (68). There is little sequence homology between Class II hydrophobins, and even less for Cass I hydrophobins, although all hydrophobins share eight cysteines that are characteristic of these molecules. Comple mentation studies of r odlet hydrophobins from

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73 closely related species, such as A. nidulans and A fumigatus, show that they often can restore partial wild-type charac teristics, however when the f ungi that are not as closely related, as is the case with M. griseae and A. nidulans, phenotypes may not be restored unless the gene is introduced under the control of the promoter from the host organisms own hydrophobin suggesting that regulation may be more important than the hydrophobins sequence in determin ing function (52, 97, 98). In addition to being the major proteins in the rodlet layer of hydrophobic conidia, hydrophobins are also found on the surface of hyphae and fruiting bodies. They are highly surface active molecules and reduce the surface tension of water allowing the fungus to form aerial structures (62). They have also been implicated in signaling (82) and are important for mediating adhesion. Determining the mRNA levels of bhd1, bhd2 and bsn in different cell types and under different cultural conditions can be expected to yield insights into their function. In this study real-time reverse transcri ptase PCR (real-time RT-PCR), currently the most sensitive technique fo r mRNA detection was used to quantify the levels of transcripts encoding the two hydrophobins (bhd1 and bhd2) and the hydrophobin like protein (Bsn) in aerial conidia, submerge d conidia and blastospores. Materials and Methods Cultivation of Fungi Beauveria bassiana (ATCC 90517) was routinely grown on potato dextrose agar (PDA) (4). Plates were incubated at 26oC for 10-14 days, and aerial conidia were harvested by flooding the plate with sterile dH2O. Conidial suspen sions were filtered through a single layer of Mira-cloth (Clabioc hem, CA), and final spore concentrations were determined by direct count using a hemo cytometer. Blastospores were produced in

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74 DifcoTM Sabouraud dextrose (Becton, Dickins on and Co., MD) + 1-2% yeast extract liquid broth cultures (SDY) usi ng conidia harvested from plat es to a final concentration of 0.5 5 x 105 conidia/ml as the inoculum. Cultu res were grown for 3-4 days at 26oC with aeration (150-200 rpm). Cultures were fi ltered (2x) through glass wool to remove mycelia, and the concentration of blastos pores was determined by direct count. Submerged conidia were produced in TKI br oth using fructose as the carbon source as described (83). For all cell types, Mira-clo th (Clabiochem, CA) or glass wool filtered cell suspensions were harvested by centrifugation (10,000 x g, 15 min, 4oC), washed two times with sterile dH2O, and resuspended to the desi red concentration as indicated (typically 107-108 cells/ml). Cells producing oo sporein were grown in DifcoTM Sabouraud dextrose (Becton, Dickinson and Co., MD) + 1-2% yeast extract liquid broth cultures (SDY) using conidia harv ested from plates to a fina l concentration of 0.5-5 x 105 conidia/ml as the inoculum until they started producing oosporein, and the mycelium were obtained from unfiltered cultur es used to grow blastospores. RNA Isolation Fungal cells were frozen in liquid Nitr ogen, or lyophilized and stored @ -70oC until used. Immediately prior to use the cells we re crushed in liquid Nitrogen and total RNA was extracted with RNAwizTM (Ambion, TX) per manufactur ers instructions. Poly A RNA was enriched using magnetic beads c ovalently attached to oligo-dT tails (Dynabeads mRNA direct kit, Dynal, WI, Poly (A) Quick mRNA isolation kit, Stratagene CA, or Poly A tract mRNA isolation Systems, Promega, WI). Rodlet Layer Extraction The rodlet layer proteins were removed fr om the surface of the spores as described by Paris et al. (68). Briefly, aerial conidia, blas tospores, and submerged conidia were

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75 prepared as described above, resuspended in water, and sonicated at 140 W (3-mm diameter microtip, 50% duty cycle) for 2 x 10 min using a Sonifier cell disrupter B-30 (Branson Ultrasons, Rungis, France). Unly sed cells and cell debris were removed by low-speed centrifugation (10,000 g 10 min), an d the supernatant was centrifuged for 1 hr at 50,000 g. The resultant pellet was boile d in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (2% SDS, 5% -mercaptoethanol, 10% glycerol in 62 mM Tris-HCL, pH 6.8), wa shed twice with equal volumes of sample buffer then three times with equal volumes of distilled water. The final pellet was lyophilized, then treated with 100% trifluoroacetic acid (TFA ), and incubated for 10 min at room temperature. The acid was remove d under a stream of nitrogen, and dried extracts were stored at room temperature und er dry air. The sample was resuspended in water prior to analysis. Aliquots of protein sample were mixed w ith lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, CA) plus dithiothreitol (DTT) (NuPAGE reducing agent, Invitrogen, CA) separated by 10-12% BisTris NuPAGE gel electr ophoresis by a MES-SDS gel running buffer (Invitrogen, CA). Presumed protein molecular mass was estimated using SeeBlue Plus2 Pre-Stained Standards (Invitrogen, CA). Protein bands were visualized with Sypro-Ruby Red (Biorad, CA) and coomassie blue staining. Mass Peptide Spectrometry (Peptide Fingerprinting) Samples were analyzed by the Protein Core facility (ICBR) at the University of Florida. Basic procedure: Proteins separa ted by SDS gel electrophoresis were cleaved in situ with trypsin. MALDI-MS was used to obtain a mass spectrum of the peptide

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76 mixture. The resulting amino acid sequences were compared to the predicted sequences of Bhd1, Bhd2 and Bsn. Reverse Transcriptase RT-PCR Primers were designed to amplify se quences 80-275 bp in length within the Hydrophobin 1, Hydrophobin 2, Snodprot and the B. bassiana -tubulin sequences. Primers are listed in Table 4-1. Table 5-1. Primer sequences and product size for the reverse transcriptase RT-PCR Gene Product Primer Sequence 5 to 3 Hydrophobin 1 F: ATC ATG CGT TTC GCT CTT GCC ATC R: AGG TGG GAA AGA AGA CCA TCA GCA Hydrophobin 2 F: AAA TGC TTG CCA CCA CCA TCT TCG R: CTG CTG CTT GCA CTT GTT GTC GAT Snodprot F: TTC CGC GGT TAG AGT TTC TTG GGA R: ACC CTG CTT CTG ATA CTT GGG CAT Tubulin F: TCC TTC GTA CGG TGA CCT GA R: CGA GCT TGC GAA GAT CAG AG Total RNA concentration was determined with Molecular Probes (OR) Ribogreen RNA quantitation kit, per manufactur ers instructions. Plasmids containing the hyd1, hyd2 and bsn sequences were used to generate standard curves to determine the original concentration of the RNA amplified from the experimental treatments and to ensure that all genes were being amplified at the same rate. Assays were run in triplicate, in 96 well plates, a Gemini XPS system fluor escent plate reader (Molecular devices, CA) running native Soft Max Pro, software. Appropriately diluted samples (pg to ng quantities) were amplified using Biorads iScript One-Step RT-PCR kit with SYBR green, on Biorads iCycler with its native soft ware (Biorad, CA). The amplification cycle was as follows: 1 x 10 min @ 500C, 1 x 5 min @ 950C, and 45 x (10 sec @ 950 and 30 sec @ annealing temp).

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77 Results Identification of a B. bassiana Cell Wall Hydrophobin Four prominent bands (aprox. 25 KDa, 12 KDa and 4-5 KDa) were observed when SDS soluble/TFA insolu ble proteins, from B. bassiana conidial cell walls, were subjected to SDS-PAGE (Fig. 5-1). It was postul ated that the 12-14 KDa, TFA soluble/SDS insoluble protein isolated from the conidial cell wall was either Bhd1 (12KDa) or Bhd2 (14KDa). Peptide fingerprinting (peptide ma ss spectrometry) identified the sequences for two fragments of the 12-14 KDa band (Fig. 5-2 & 5-3). The sequence of fragments from the 12-13KDa band identified by mass spectros copy were compared to the sequences of Bhd1 and Bhd2 which combined covered 29. 2% of the amino acid sequence (35/116), or 30.9 % (36991/11983.9) of the mass predicted for Bhd2 (Fig. 5-3). Fig. 5-1. SDS PAGE (10% pol yacrylamide, Bis-Tris) Gel of SDS soluble/TFA insoluble cell wall proteins stained with cooma ssie blue Lane 1, protein molecular weigh standards; lane 2, aerial conidia; lane 3, blastospores; and lane 4 submerged conidia. Reverse Transcriptase RT-PCR. In addition to the sequence for Bhd1, the cDNA and genomic sequences for another B. bassiana hydrophobin (bdh2) and a B. bassiana hydrophobin like protein (Bsn) have been identified in our laboratory (E. Cho unpublished results). The bhd1 mRNA levels 188 kDa 98 kDa 63 kDa 49 kDa 38 kDa 28 kDa 17 kDa 14 kDa 6 kDa 3 kDa1 2 3 4

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78 were highest in the submerged conidia with a relative concentration (RC) of 41.77. Aerial conidia and blasto spores had much lower levels with RC 3.61 and 1.69 respectively. The relative amount of bdh2 mRNA (a cell wall SDS insoluble/TFA soluble protein) was much lower than that of bdh1 in all cell types (RC 1.69 41.77 for bdh2 and RC 0.01-1.59 for bdh1). The highest abundance of bdh2 mRNA was found in aerial conidia (RC 1.59) much lower levels we re found submerged conidia (RC 0.18) and blastospores (RC 0.01). The concentration of the tubulin mRNA was lower in conidial cells ([tub]/[total RNA] = 0.001), when compared with the other two spore types ([tub]/[total RNA] = 0.02). We were unable to demonstrate hi gh levels of expression in any of the cell types tested; we also we re unable to find significant expression of Snodprot in any of the cell t ypes analyzed (Table 5-2). Table 5-2. mRNA abundance of bhd1, bhd2 in Beauveria bassiana single cell propagules. Aerial conidia Submerged conidiaBlastospores Total RNA 89.7 ng/mla 838.6 ng/ml 649 ng/ml [tub]b ng/ml 0.09 1.46 15.96 [bhd1] ng/ml 0.33 61.02 26.97 [bhd2] ng/ml 0.15 0.26 0.20 [bhd1]/[tub]c 3.61 41.77 1.69 [bhd2]/[tub] 1.59 0.18 0.01 a) Ribogreen assay (Molecular probes, Invitrogen, CA). b) tub (tubulin gene), bhd1 (Beauveria bassiana hydrophobin 1), and bhd2 ( Beauveria bassiana hydrophobin 2). c) The concentrations were normalized using the concentra tion values obtained amplifi cation of the tubulin genes.

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79 Avg Mass: 11983.9 Seq # b y (+1) --------------L 1 114.1 1385.8 13 T 2 215.1 1272.7 12 G 3 272.2 1171.6 11 P 4 369.2 1114.6 10 S 5 456.2 1017.6 9 V 6 555.3 930.5 8 L 7 668.4 831.5 7 S 8 755.4 718.4 6 D 9 870.5 631.3 5 L 10 983.5 516.3 4 D 11 1098.6 403.2 3 L 12 1211.7 288.2 2 R 13 1367.8 175.1 1 Fig. 5-2. Mass spectroscopy data showing th e mass of individual amino acids in one of the two main fragments from the 12 KDa trifluoroacetic acid soluble/sodium dodecyl sulfate insoluble B. bassiana cell wall protein.

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80 Coverage: 13/116 = 11.2% by amino acid count, 1385.6/11983.9 = 11.6% by mass Hydrophobin sequence H2 MLATTIFATL LALAAAAPHG PSHGPSVKTG DICGNGNTMH CCNDESVTNK LTGPSVLSDL DLRKLLAAEC SPISVNVLLN QLVPIDNKCK QQSICCGEQK LNGLVNLGCT PITVLG PepStat LTGPSVLSDLDLR position: 51 63 Avg Mass: 11983.9 Coverage: 22/116 = 19.0% by amino acid count, 2313.5/11983.9 = 19.3% by mass Hydrophobin sequence H2 MLATTIFATL LALAAAAPHG PSHGPSVKTG DICGNGNTMH CCNDESVTNK LTGPSVLSDL DLRKLLAAEC SPISVNVLLN QLVPIDNKCK QQSICCGEQK LNGLVNLGCT PITVLG PepStat TGDICGNGNTMHCCNDESVTNK position: 29 50 Fig. 5-3. Mass peptide fingerprinting result s for two identifiable fragments of the 12 KDa trifluoroacetic acid soluble, sodium dodecyl sulfate insoluble B. bassiana cell wall protein confirming that th is protein is the Bhd2 hydrophobin. Discussion Fragments of the hydrophobin gene (bhd1) present in the SSH library (chapter 4) were sequenced, cloned and were ultimately found to collectively represent the fulllength sequence of the gene (Keyhani, unpubl ished results). Since then we have identified sequences for two additional proteins Bhd2 and Bsn from phage display libraries enriched for phage displaying proteins that bound to carbohydrates (E. Cho, unpublished results). Blast searches (NCBI) i ndicated that these proteins are similar (38% and 51% identity, respectively) to Mpg1 hydrophobin of Magnaporthe grisea (E value 9e-11) and the Neurospora crassa Snodprot1 toxin (E value 2e-35). Based on the number of sequences between the cyst eines, both Bhd1 and Bhd2 are class I hydrophobins (fig. 5-3) (38). Our previous results (Chapter 3) suggest that B. bassiana conidia are hydrophobic, and a rodlet layer is clearly visible by AFM on the surface (Chapter 2). Previous research

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81 (7) of the B. bassiana rodlet layer appeared to indicat e that it contains a single cell wall hydrophobin (10-14 KDa protei n), however we have identified the genes for at least two hydrophobins (Bhd1 and Bhd2) and a hydrophobin like protein (Bsn). Class I: -C-X5-8-CC-X17-39-C-X8-23-C-X5-6-CC-X6-18-C-X2-13 H1 7 27 18 5 12 7 H2 7 37 17 5 17 8 Class II: -C-X9-10-CC-X11 -C-X16 -C-X6-9-CC-X6-18-C-X2-13 Fig. 5-4. Comparison of Bhd1 and Bhd2 a nd other hydrophobins (A) consensus spacing for Class I and Class II hydrophobins as suggested by Fuchs et al. (38). At least four major bands were obser ved when TFA soluble, SDS insoluble proteins were extracted from aerial conidial cell walls (Figure 5-1). Based on reports in the literature discussing B. bassiana cell wall proteins that were insoluble in hot SDS, but soluble in TFA or FA hydrophobi n (7, 68), we selected the ba nd with a molecular weight of approximately 10-14 KDa (based on SDS pa ge anlaysis) for further analysis because we considered it the best candidate for the cell wall hydrophobin. Mass peptide spectrometry analysis of th e 10-14 KDa band, when compared to the sequence of the two hydrophobins and the hydrophobin like protein, identified the protein as Bhd2. These preliminary results i ndicate that Bhd2 is a component of the B. bassiana cell wall. Aside from aerial conidia B. bassiana produces the othe r two single cell propagules, blastospores and submerged coni dia. These cells do not appear have a visible rodlet layer and SDS insoluble, TFA soluble proteins were not isolated from purified cultures of these cells. However, real-time RT-PCR on RNA isolated from these cells indicated that tr anscripts corresponding to bhd1 and bhd2 could be detected in pure cultures of these cell ty pes, although levels of bdh2 were low in all cell types. It is likely that conidia are not metabolic ally active; as reflected by the relatively low levels of tubulin amplified by real-time RT-PCR (Table 5-2). Alternatively, the low

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82 levels of tubulin could be e xplained by low expression of tu bulin alone, with other genes being expressed at relatively higher levels. Using other hous ekeeping genes, or purifying and quantifying poly A RNA will determine which of these explanations is the most likely. There are a number of possibilitie s explaining the low levels of bhd2 RNA observed in the cell types anal yzed. It is possible that bhd2 is only produced in sporulating cultures, specif ically in cells responsib le for producing conidia (conidiophores) and was therefore not expres sed by any of the cell types tested. In contrast bhd1 was present in almost all the cultures tested at a relatively high abundance. This protein while not being recruited to the single cell propagule cell walls may instead be secreted into the medium. Similar to other hydrophobins which are not recruited to conidial cell walls it may function as a surfact ant, coat surfaces acti ng as a lubricant, or function as an adhesin directing hyphal attachment and growth. Bsn, like bhd2, is likely produced unde r specific conditions not examined in these studies. Based on homology to proteins produced by Pisolithus microcarpus and Ceratocystis fimbriata. Homologous proteins are pr oduced by these organisms are expressed by vegetative mycelium, when th e expression levels of other hydrophobins or carbon availability is low. Putative functions for the product of th is gene may include host cell toxicity (28, 69).

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83 CHAPTER 6 GENERAL DISCUSSION Statement of Hypotheses The work in this provides scientific data to examining the validity of the following hypotheses: 1) that there is a measurable difference in th e cell surface characteristics of the single cell propagules of Beauveria bassiana, and 2) there are differences in the binding properties of these propagules wh ich can be related to the cell surface characteristics. Is There a Measurable Difference in The Cell Surface Characteristics of The Single Cell Propagules of B. bassiana? Visual Differences Visual inspection of the single cell pr opagules using light or atomic force microscopy reveals that the morphology and the surfaces of B. bassiana aerial conidia, submerged conidia and blastospor es are different. Aerial coni dia are spherical, 2-4 m in diameter, hyaline and covered with a distinct rodlet layer. Submerged conidia are similar in shape to the aerial conidia but are larger (3-5 m in diameter), and while the surface appears rough, there was no visible evidence of a rodlet layer in those cells. Blastospores are rod shaped and larger than the other spor es at 8-12 m in lengt h, there is no visible rodlet layer on their surface, which is smooth (35, 83). Differences in Hydrophobicity Interfacial free energies of interaction de rived from contact angle data and percent retention of the spores in the hydrocarbon pha se (MATH) assay, both confirmed that the

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84 conidia have negative interfacial free energies of interaction indicati ng that they would be unable to overcome the high cohesive force of water, and with almost 88 % retention in the hydrocarbon layer, it is clear that these spores are hi ghly hydrophobic. Submerged conidia were less moderately hydrophilic, with slightly positive interfacial free energies of interaction and 74 % retent ion in the hydrocarbon layer. Blastospores with strongly negative interfacial free energies of inte raction and low retention (40%) in the hydrocarbon layers were the most hydrophilic. Differences in Effective Surface Charge Effective surface charge (zeta potential) of the three spores was similar in sign (positive at low pH and negative at high pH) as pH changes from 3-9. All of the spores had the same isoelectric point (pH 4), excep t for conidia that were 16-days-old, the isoelectric point for these cells was pH 5. Conidia registered the greatest magnitude of charge of the three spore types, (zeta poten tial: -29 mV, day 16 and 47 mV, day 20) at very high pH (pH 8) and at low pH (pH 3) for 16-day-old aerial c onidia (zeta potential: 22 mV. At low pH (ph 3) the zeta potentia l for all the spores, except for the 16-day-old conidia, was between 3-8 mV. The magnitude of charge for submerged conidia at high pH was intermediate with a zeta potential of -10 mV, and that for the blastospores was the lowest with a zeta potential of 5 mV. The overall change in zeta potential with respect to pH for the blastospores was 7 mV while that for the submerged conidia was 20 mV, and that of the aerial conidi a (16 and 20 days combined) was 69 mV. The conidia that were the most hydrophobic had the highest char ge, this could be indicative that: 1) electrostati c repulsion is less of a factor for the more hydrophobic cells because hydrophobic forces are stronger and act over a longer range, 2) that the surface to which aerial conidia bind to tend to be le ss electronegative at higher pH than aerial

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85 conidia (this is true for insect cuticle; many of the proteins in the insect cuticle tend to be positively charged at higher pH), whereas the surfaces that the blastospores and microccyle conidia bind to tend to be less mo re at higher pH than either of those cell types, or that 3) aerial conidia are desi gned to adhere to hydrophobic surfaces, using the surface of water as a dispersion mechanism, whereas blastospores and submerged conidia are designed to be resuspended in aqueous solutions for longer periods of time. Cell Wall Proteins Sodium dodecyl sulfate (SDS) insoluble, trifluoroacetic ac id (TFA) soluble proteins were extracted from the cell wall of conidia, but not from the cell walls of microcycleconidia, these proteins often belong to a class of sm all, cysteine rich, secreted fungal proteins called hydrophobins (99). The genes for two hydrophobins and one hydrophobin like protei n specific to B. bassiana have been identified, cloned and sequenced in our laboratory. The bhd1 sequence was identified from gene fragments obtained from a SSH library designed to fi nd virulence factors upregulated when the organism is grown in the presence of chitin, or insect cuticle. Th e other two genes were identified by E. Cho in this laboratory from phage display libraries designed to enrich for carbohydrate binding proteins. It has been not ed, but never conclu sively proven that hydrophobins poses lectin like qua lities hydrophobin (93, 99). The amino acid sequences of the two hydrohobins (Bhd1 and Bhd2) were compared to the sequences of fragments obtained by running peptid e fingerprinting on a 12 KDa SDS insoluble, TFA sol uble protein isolated from the cell wall of the conidia. The sequence of two of the fragments matched at least 29% of the se quence of Bhd2. To determine the expression patterns of all thr ee genes, real-time reverse transcriptase PCR (real time RT-PCR), was performed on the three single cell propa gules and vegetative

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86 mycelia from cells grown on chitin or in sabauroud dextrose broth (SDY) (1-2% YE). bhd1 was expressed in all cell types, bhd2 was expressed mostly in cells exposed to chitin, and submerged conidia and bsn was not expressed in any of the cell types tested. Conclusions From these results we can conclude that there are measurable differences in the surface characteristics of the three cell types with regards to morphology, surface charge, hydrophobic characteristics, and cell wall protein composition. Are there Differences in the Binding Propert ies of These Propagules, Which Can Be Related to the Cell Surface Characteristics? Adhesion Profiles of Aerial Conidi a, Submerged conidia and Blastospores Our fluorescent cell, microtitre pl ate adhesion assay determined that b. bassiana aerial conidia bound predominantly to hydrophobic surfaces (silinized polystyrene), and weakly to hydrophilic surfaces (polystyrene, factory-treated to be more hydrophilic). Submerged conidia bound well to either surface, while blastospores bound predominantly to the more hydrophilic surfaces. This correl ates well with the hydrophobic character of each spore type, however treating with amylase, laminarinase and protease could reduce the binding of aerial conidia to hydrophobic surfaces, and trea tment with maltose reduced blastospores adhesion to hydrophilic surfaces indicating that factors other than hydrophobicity are implicated in spore adhesion. Conclusion The surface characteristics of B. bassiana single cell propagules are consistent with the adhesion profiles on hydrophili c, and hydrophobic surfaces. However, it is clear that other the adhesion properties of the spores are a result of the interactions of short and long range intermolecular forces. These inte rmolecular forces are consistent with the

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87 distinct cell wall characteristics of the three cel l types, however it is probable that other features not yet identified are contributing to the adhesion characterist ics of the cell types Confirmation of the role of the B hd2 hydrophobin in the adhesion profile of B. bassiana conidia and the func tion of the other two B. bassiana specific proteins (Bhd1 and Bsn) will require generating transformants lacking Bhd2. The Agrobacterium tumefaciens mediated transformation procedure, w ith minor modifications, will be used for this purpose. Finally it should be noted that the adhesion profiles and the cell surface characteristics unique to B. bassiana must be important in th e ability of the fungus to successfully function as an entomopathogen. Although the different propagules have different surface characteristics and adhesion prof iles, all are able to successfully initiate infection, the main observable difference be tween the cells, with respect to use in mycoinsecticidal preparations, is the resistan ce of aerial conidia to environmental stresses such as UV light and desiccation spores, this difference has previously been correlated to the presence of the rodlet layer on the surface of the aerial conidia (80)

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88 APPENDIX AGROBACTERIUM MEDIATED TRAN SFORMATION OF BEAUVERIA BASSIANA Introduction Little known concerning the regulation of molecules and metabolic pathways involved in fungal pathogen-host interactions particularly for the entomopathogen. This is primarily due to the lack of efficient transformation systems for filamentous fungi. Development of efficient transformation system s for filamentous fungi have been limited to several species particularly those rela ted to human health. Successful genetic manipulation of fungi, as with any organism requires a plasmid mediated transformation system involving the entry and replication, or integration of foreign DNA (75) Once DNA has been successfully introduced into the fungal cells its fate varies depending on the host. DNA usually integrates into the chromosomes; autonomously replicating plasmids are rare in most filamentous fungi. There are a number of transformation stra tegies currently bei ng used with mixed degrees of success. The earliest, and s till widely used, tran sformation method for filamentous fungi is polyethylene glycol (PEG) mediated transformation of fungal protoplasts. More recently developed methods sometimes used in conjunction with the generation of protoplasts; include lithium acetate treatment, Agrobacterium tumefaciens mediated transformation (ATMT), electropo ration, restriction enzyme mediated transformation (REMI), and biolistic tran sformation (75). Many of these methods,

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89 simplify transformation procedures, reduce va riability, and/or increase transformation efficiencies. Agrobacterium tumefaciens mediated transformation (ATMT) is routinely used for the genetic modification, primarily of dicotyledon plant species. Agrobacterium tumefaciens causes crown gall in plants by induc ing tumor formation, and the overproduction of specific nutrients (opines) within the host cell. This is accomplished via the transfer of part of its Ti plasmid DNA to the host genome. The transferred DNA (TDNA) contains genes encoding auxins, cytoki nins and opines; the remainder of the plasmid, and part of the bacterial genome, c odes for proteins involve d in the detection of acetosyringone, a substance released by w ounded plants. Excision, transfer and integration of the T-DNA, into the host genome are not dependent on DNA present in the T-DNA region, which can be artificially modi fied without affecting the transformation process. In 1998, de Groot and Gouka demonstrated that efficient transfer of T-DNA to filamentous fungi, speci fically to the fungus Aspergillus awamori, was possible, and that transformation frequencies were increased up to 600 fold compared to traditional methodologies (18, 41). Tran sformation by the same group of other fungi, including Aspergillus niger, Fusarium venetatum and Trichoderma reesei, Colletotrichum gloeosporioides, Neurospora crassa and Agaricus bisporus (a mushroom), demonstrated that this system that could be adapted for a variety of filamentous fungi (18). Materials and Methods Fungal Cultures Beauveria bassiana (ATCC 90517) was routinely grown on potato dextrose agar (PDA) (4). Plates were incubated at 26oC for 10-14 days, and aerial conidia were

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90 harvested by flooding the plate with sterile dH2O. Conidial suspen sions were filtered through a single layer of Mira-cloth (Clabioc hem, CA), and final spore concentrations were determined by direct count using a hemocytometer. Blastospores were produced in DifcoTM Sabouraud dextrose (Becton, Dickinson and Co., MD) + 1-2% yeast extract liquid br oth cultures (SDY) using conidia harvested from plates to a final concentration of 0.5 5 x 105 conidia/ml as the inoculum. Cultures were grown for 3-4 days at 26oC with aeration (150-200 rpm) Cultures were filtered (2x) through glass wool to remove mycelia, and the concen tration of blastospores was determined by direct count. For all cell types, Mira-cloth (Clabiochem, CA) or glass wool filtered cell suspensions were harvested by cen trifugation (10,000 x g, 15 min, 4oC), washed two times with sterile dH2O, and resuspended to the desi red concentration as indicated (typically 107-108 cells/ml). Agrobacterium tumefaciens Cultivation The transformation was performed as desc ribed by Covert et al. (2001) with the following modifications. Conidia (104-7 cfu/ml) were mixed in equal amounts with an overnight Luria Bertani (LB, Difco, M I) broth (40 g/ml ampicillin and 50 g/ml kanamycin) culture of Agrobacterium tumefaciens (AGL1). The A. tumefaciens culture was in diluted to O.D. 0.15 with indu ction medium (16) supplemented with acetosyringone (IMAS) and allowed to grow fo r 4 hrs to reach an O.D. of 0.6-0.8. Transformation Procedure Then 50-200 l of the Agrobacterium and B. bassiana cultures were mixed and plated onto sterile cellophane overlaid onto IM AS plates. The plates were incubated for 3 days at 20oC and the filters were transferred to the Czapek-dox (CZD) (Difco, MI)

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91 plates supplemented with the appropriate an tibiotic (1000 g/ml hygromycin B with 350 g/ml of cefotaxime, or 750 g/ml G418 with 350 g/ml cefotaxime). The fresh plates were incubated at 200 C for 4 to 10 days until isolated colonies were visible. Sensitivity to the various antibiotics tested, was affected by the salt concentration of the medium as well as the pH It was determined that LB and the M-100 media did not have sufficient buffering capacity CZD agar gave the best results for sensitivity for all antibiotics tested, a nd buffering capacity (results not shown). Putative transformants were restreaked ont o selective media and then inoculated into SDY (1-2% YE) broth. PCR reactions were run on purified genomic DNA (GenElute Plant Genomic DNA Miniprep Kit, Sigma-Aldrich, MO) of the putative transformants, with primers designed to amplify genes (e.g. hph) present in the T-DNA, on the wild type (WT) chromosome (aspfII). Results Agrobacterium tumefaciensMediated Transformation ATMT with pPK2 resulted in at least 526 colonies able to grow on the selective media, however the background of wild type colonies (growth of colonies on control plates w/o acetosyringone) was sometimes very high. Colony PCR of the initial transformants obtained using pPK2 showed that many of the colonies in the presence of Agrobacterium (at least 66%) contained the hph gene (Fig. A-1), which was not amplified from any of the control colonies.

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92 2000 1500 1000 700 500 400 300 200 100 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 202000 1500 1000 700 500 400 300 200 100 50 A Fig. A-1. PCR analysis of putative Beauveria bassiana transformants: A) lanes 4, 7, 10, 13, and 16 positive control for Beauveria (AspfII), lanes 3, 6, 9, 12, and 15 amplification of the hph gene B) lanes 3, 5, 8, 11, 14,17 and 20 positive control for Beauveria (AspfII), lanes 4, 6, 9, 12, 16, 18, and 19 amplification of the hph gene Transformation with pPk2 derived plasmids containing the neomycin phosphotransferase gene (neor) or neor combined with the gene for green fluorescent protein gfp produced hundreds (715) of putative transformants. Many of these (198/717) failed to grow when subcultured to fresh se lective media unless the concentration of the antibiotic was reduced in half and none were confirmed to have the neor gene (Table A1). ATMT with our strain of B. bassiana, using hph as the marker, on average produced more colonies (including many back ground colonies) from blastospores than using neor ( 250 colonies/103 cells vs. 83.2 24. 8 colonies/103 cells n=4 plates) (Table A-1). Azaserine, a purine synthesis inhib itor slightly increased the number of putative transformants (gfp-neo) from an average of 16 4.24 colonies/102 cells to 32.5 17.6 colonies/102 cells (n=2 plates). Blastospores, regardless of selectivity gene, produced

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93 more putative transformants ( 250 colonies/ 104) on average, than conidia (e.g. 87.7 90.81 colonies/ 104 cells) (Table A-1). Table A-1. Putative tran sformants obtained from Agrobacterium tumefaciens mediated transformation of B. bassiana with selection markers for hygromycin B (hph) and neomycin (neo) resistance. Resistance marker Conidia 103 a Conidia 104 Conidia 105 Blastospores 102 Blastospores 103 Blastospores 104 hphb -c 8d, 10 200,200 28, 80 -,TNTC,TNTC TNTC TNTC TNTC Neor 0e 0 0 0 0 0 19 0 0 0 90f, 74f 96, 102 37g, 100g TNTC gfp-Neor 13, 19 20g, 45g 100 TNTCf a. Conidia were obtained from 4-7 day old plates, blastospores we re obtained by growing Bb in SAB dextrose broth with 2% yeast extract for 3 days. b. Beauveria bassiana strain 90517 was transformed with plasmids containing the hph gene, Neor and gfp-Neor markers. c. Not done d. Replicate 1, replicate 2 TNTC = to numerous to count e. No colonies were recovered from plates usi ng conidia and the neomycin resistance marker f. Unable to unable to subculture colonies on media with 750g/ml of G418, only on media with 350g/ml of G418. g. Beauveria was grown with Azaserine added to the media. A ll other experiments either ha d TNTC beauveria colonies on all plates, or Agrobacterium over growth Discussion An Agrobacterium tumefaciens-mediated transformation (ATMT) protocol was developed for use with B. bassiana. This procedure will eventu ally be used to conduct targeted mutagenesis of the hydrophobins (bhd1 and bhd2), the hydrophobin like protein (bsn), and other genes identified in the suppression subtrac tion hybridization library of genes upregulated in the pres ence of insect cuticle. Covert et al. (2001) described a method for the ATMT of Fusarium circinatum. The protocol and plasmid (pPK2) used in t hose experiments were obtained from Dr. S. Covert at the University of Georgia. The A. tumefaciens strain AGL1, derived from EHA101, was obtained from the American type culture collection (ATTC). The ATMT

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94 procedure described in Covert et al. (2001) was used with B. bassiana with the following modifications: 1) the selec tive medium was changed from M100 to Czapek-dox, 2) the selective marker in pPK2 is the hygromycin phosphotrans ferase gene (hph). Eventually, due to B. bassianas resistance to hygromycin at leve ls of 1000 g/ml, other selective markers were required, and the appropri ate plasmids containing genes conferring resistance to Genticin (G418), and Zeocin we re obtained, and the ti me of co-cultivation was increased from 2 days to 3 days. Other researchers have adapted ATMT for use with B. bassiana The B. bassiana strain used by Fang et al. (2003) was also highly resistant to hygromycin (up to 2 mg/ml). This resistance appears to be stra in dependent however; dos Reis et al. (2004) found their strain to be sensitive to c oncentrations of hygromycin as low as 600 g/ml transformation (24, 32). Although we successfully obtained putative transformants using the hph gene, we were unable to prevent overgrowth of b ackground colonies on c ontrol co-cultivation plates without A. tumefaciens or, plates without acet osyringone, even at high concentrations (100 g/ml) of hygromycin B. Changing the selective marker from hph to neor increased the number of putative transf ormants, and reduced the number of background colonies. However, these putative tr ansformants failed to retain resistance to the selective marker when subcultured, although colony PCR indicated that the neor gene was present in most of these colonies. Using blastospores during the co-cultivati on yielded more transformants than using conidia and this is probably due to the faster germina tion rate (Keyhani, unpublished results) of the blastospores compared to conidia. Roberts et al. (2003) reported that

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95 deleting Saccharomyces cerevisiae genes involved made the yeast supersensitive to transformation with Agrobacterium tumefaciens. They noted that the magnitude of this sensitivity was large, up to three orders of magnitude. The same group showed that plant cells were also supersensitive to transformation when treated with purine inhibitors, including azaserine (74). We used azaserine in our experiments but did not note the same supersensitivity. There was a modest increas e in the number of putative transformants obtained in the presence of azaserine, but that increase was not three orders of magnitude larger. This technique will be used for targeted mutagenesis of the hydrophobins, and other genes that were identified using the subtractive suppression hybridization procedure to find genes that were potentially upregul ated when the fungus was growing on insect cuticle.

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96 LIST OF REFERENCES 1. Alexopoulos, C. J., C. W. Mims, and M. Blackwell. 1996. Introductory mycology, Fourth ed. John Wiley & Sons, New York, NY. 2. Balebona, M. C., M. A. Morinigo, and J. J. Borrego. 2001. Hydrophobicity and adhesion to fish cells and mucus of Vibrio strains isolated from infected fish. Int. Microbiol. 4:21-26. 3. Beckerman, J. L., and D. J. Ebbole. 1996. MPG1, a gene encoding a fungal hydrophobin of Magnaporthe grisea, is involved in surface recognition. Mol. Plant. Microbe Interact. 9:450-6. 4. Beever, R. E., and E. G. Bollard. 1970. The nature of th e stimulation of fungal growth by potato extract. J. Gen. Microbiol. 60:273-279. 5. Bell-Pedersen, D., J. C. Dunlap, and J. J. Loros. 1992. The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the c onidial rodlet layer. Genes Dev. 6:2382-94. 6. Bidochka, M. J., and G. G. Khachatourians. 1990. Identification of Beauveria bassiana extracellular protease as a virulence factor in pathogenicity toward the migratory grasshopper Melanoplus sanguinipes. Journal of Invertebrate Pathology 56. 7. Bidochka, M. J., R. J. St Leger, L. Joshi, and D. W. Roberts. 1995. An inner cell wall protein (cwp1) from conidia of the entomopathogenic fungus Beauveria bassiana. Microbiology 141:1075-80. 8. Bird, A. F., and A. C. McKay. 1987. Adhesion of c onidia of the fungus Dilophosphora alopecuri to the cuticle of the nematode Anguina agrostes, the vector in annual ryegrass toxicity. Int. J. Parasitol. 17:1239-1247. 9. Boucias, D., and J. Pendland. 1991. Attachment of my copathogens to cuticle. In G. T. Cole and H. C. Hoch (ed.), The f ungal spore and disease initiation in plants and animals. Plenum Press, New York, NY. 10. Boucias, D. G., J. C. Pendland, and J. P. Latge. 1988. Nonspecific Factors Involved in Attachment of Entompathogenic Deuteromy cetes to Host Insect Cuticle. Applied and Environmental Microbiology 54:1795-1805.

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105 BIOGRAPHICAL SKETCH Dianes first school was a small British school in Rome, Ital y called the Junior English School of Rome. The school only ac commodated students up to Form 1 (age 10) so I transferred to a second school; St. Geor ges English school also located in Rome. After completing her O and A levels in 1986, she applied to the biology program at the University of Maryland at College park (UMCP). By 1991 she had completed her bachelors degree in Biology, and she c ontinued on to complete a Masters in Microbiology at UMCP (prese ntly the department of mol ecular and cell biology) under the guidance of Dr. S. Joseph. During this period she was involved in a project USDA funded project to develop a detection system for Salmonella sp. in meat products. She obtained her Masters degree in 1995, and contin ued to work at the same department for a year as a faculty assistant. She subsequen tly worked as a supervisor for the quality control laboratory at Allens Family Foods In 2001 after having successfully petitioned for a national interest waiver based on her Ma sters work allowing he r to self sponsor her permanent immigration applicati on, she decided to continue her education and applied to the doctoral program at Florida Institute of technology, in Melbourne Fl, after one year she transferred to the doctoral progra m in Microbiology and Cell Science at the University of Florida at Gainesville, Florid a where she has been working on the analysis of cell surface properties of the entomopathogenic fungus Beauveria bassiana under the guidance of Dr. N. O. Keyhani.