fio .9.“ 1’ ‘ a.» ,5 xxx :31: V ‘ . ‘ ., ‘ .rwfiflfil . .12“. «unfit». 49).. ..r . 2%.. am ‘ . é... 512..qu . , . . . . m. . . .3: ‘ . may a. z . a. “wax. . = 1: an! 4 .1. .i. ‘a . J. J.- .«rL... . . . , km» 2 1...... . was». “410. 3:32: ,. .595 in {a . , . .. ‘ . o. '1 i. ’ . a. « . . 53:». 3 2: {.{fi . t r , . . w ». Rana? ‘.)~n Q~ l ism. §~¢me¢m¥ m . i. wwm ‘ : 3.? i) an 4U..t.tu..m unta «in! .r. 52.3 2.9. . find. :3“ :Jfim .9 . 2 n. i .19.. .51 ,3 (a. . l n , fiwm u ‘ Pi... . 4 .. 1 ‘ 325...... u... v. .5..::fi: .w..a ' a .0? .u. hibr “mm—wig lHESiS- Z 200] This is to certify that the dissertation entitled THE ROLE OF CELL WALL DEGRADINC ENZYMES IN COCHLIOBOLUS CARBONUM PATHOGENIC ITY presented by Nyerhovwo John Tonukari has been accepted towards fulfillment of the requirements for Ph.D degree in Biochemistry Major professor Date IQIZALQO MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 8/01 eJCIRC/DatoDue.p65-p.15 THE ROLE OF CELL WALL DEGRADING ENZYMES IN COCHLIOBOL US CARBONUIVI PATHOGENICITY By Nyerhovwo John Tonukari A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 2000 ABSTRACT THE ROLE OF CELL WALL DEGRADING ENZYMES IN COCHLIOBOL US CARBONW PATHOGENTCITY By Nyerhovwo John Tonukari Fungi secrete many cell wall degrading enzymes (CWDEs) for the penetration of host cell wall barrier, as well as for generation of simple molecules that can be assimilated for growth. Repression of six CWDE genes (XYLI, XYLZ, XYL3, XYL4, XYPI, and ARF 1) was observed in the maize pathogen Cochliobolus carbonum when it was grown on glucose as the sole carbon source, whereas expression was seen when the culture medium contained xylan. The Saccharomyces cerevisiae Miglp and its fungal homolog (CreAp) has been implicated in glucose (catabolite) repression. Repression by Miglp and CreAp is relieved by Snflp which is encoded by SNF 1, a gene first isolated from S. cerevisiae. An ortholog of SNF 1, ccSNF 1, was isolated from C. carbonum and ccsnfl mutants were created by targeted gene replacement. Growth of the ccsnf] mutant was reduced significantly on typical cell wall components, such as xylan, pectin, or purified maize cell walls. Extracellular CWDE activities, including B-l,3-glucanase, pectinase, and xylanase, as well as expression of several CWDE genes, were also significantly reduced in the 003an mutant. The ccsnf! mutant was strongly reduced in virulence on susceptible maize, forming fewer spreading lesions, indicating that ccSNF/ is required for biochemical processes that are important for pathogenesis by C. carbonum. The ARFI gene is not expressed in the C. carbonum ccsnfl mutant. HPLC analysis of the culture filtrate of an arfl mutant constructed by gene replacement indicated that residual a-L-arabinofiiranosidase activity remained high due to an additional two arabinofuranosidase activities. The growth and virulence of the arfl mutant were indistinguishable from the wild type. The disruption of a second arabinofuranosidase gene, ARFZ, creating an arfI/arj? double mutant, led to the disappearance of the two major arabinofuranosidase activity peaks. However, the arfI/arf? double mutant had similar virulence as the wild type C. carbonum. The remaining activities were due to a bifunctional B-xylosidase/a-L-arabinofuranosidase (Ransom and Walton, 1997; Wegener et al., 1999). Thus a major decrease in virulence may require disruption of a regulatory gene (such as ccSNFI) that activates the expression of several CWDE genes. The CreA-Snfl pathway may play an important role in the regulatory process that leads to cell wall degrading enzyme expression and ' virulence in pathogenic fitngi. For the Urhobos of West Africa. iv ACKNOWLEDGEMENTS I am sincerely gratefiJl to Dr. Jonathan Walton for the wonderihl time I spent in his laboratory and for being a very good mentor. My gratitude also to Dr. John Scott- Craig for teaching me how to do molecular biology experiments and for listening to my numerous questions. And I am grateful to my guidiance committee (Dr. P. Green, Dr. N. Raikhel, Dr. B. Burton, and Dr. G. Zeikus) for steering me in the right direction. Sridhar Venkataraman and Michael Feldbrugge taught me some important things I needed to know. Members of the Walton Lab were remarkable colleagues. I teamed up with Kerry Pedley for some important experiments. My wife (Ese) and the little boy (Gaga) brought so much joy and blessings to these memorable times. My siblings have always had confidence in me, and for that I am thankful. My appreciation also to Ike Iyioke, Steve Bakiamoh and Charles Ngowe for being great friends. And I am indebted to this great country, the United States of America, for providing me the opportunity and financial assistance to learn and to dream. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS Chapter One Enzymes and fungal pathogenicity Abstract Introduction Fuel, Enzymes and Virulence Generation of Simple Molecules Adaptation Redundancy Penetration CWDE regulation Substrate Specific Regulators Global Regulators Response of the Plant to CWDEs CWDEs as inducers of Plant defense Protease Inhibitors CWDE-Inhibitors Conclusion References Chapter Two The C ochliobolus carbonum SNFI gene is required for cell wall-degrading enzyme expression and virulence on maize Abstract Introduction Methods vi 27 28 30 33 Fungal Cultures, Media, and Grth Conditions Nucleic Acid Manipulations Functional Complementation of Yeast snfl Disruption of ccSNFI Enzyme Assays HC toxin Analysis Pathogenicity Assay Results Isolation of C. carbonum SNFI (ccSNFI) Complementation of a Yeast 3an Mutant by ccSNFl Targeted Disruption of ccSNFl Expression of CWDE Activities and mRN As in the ccsnfl Mutant Grth of the ccsnf] Mutant on Complex and Simple Carbon Sources Effect of the ccsnfl Mutation on Pathogenicity Discussion References Chapter Three Glucose Repression and Characterization of the CREA gene in the pathogenic fungus, Cochliobolus carbonum Abstract Introduction Methods Fungal cultures, media and growth conditions Nucleic acid manipulations Results Repression of C. carbonum CWDE genes in glucose substrate Expression of C. carbonum CWDE genes in different grth media Cloning of C. carbonum CREA Discussion References vii 33 33 35 35 36 38 38 38 38 45 48 50 53 56 63 68 73 74 75 77 77 77 79 79 80 83 87 94 Chapter Four Cloning and targeted mutation of Cochliobolus carbonum ct-L- Arabinofuranosidase genes ARFI and ARFZ Abstract Introduction Methods Fungal cultures, media and growth conditions Nucleic acid manipulations Construction of gene replacement vector and transformation HPLC Enzyme assays Pathogenicity test Results Cloning of ARF 1 and ARF2 Targeted Disruption of ARF l and ARF 2 Effect of the ARFI and ARF 2 mutations on enzyme activities Effect of the ARFI and ARFZ mutations on growth Effect of the ARFI and ARFZ mutations on pathogenicity Discussion References Chapter Five Conclusion and Future Directions Conclusion Suggestions for future studies Effects of SNFI mutation Regulation of Snflp Identification of Fungal Pathogenicity Genes Proteins that inactivate CWDEs Perspectives References viii 98 99 101 103 103 104 106 107 110 111 Ill 111 120 122 125 128 128 131 134 135 137 137 138 139 140 141 143 LIST OF TABLES Table l. C. carbonum extracellular enzymes capable of degrading specific maize (Zea mays) cell wall components Table 2. Proteins known to regulate cell wall degrading enzymes Table 3. Properties of $an protein kinases from several eukaryotic organisms Table 4. Sequence in 5' region of C. carbonum CWDE genes that corresponds to the consensus sequence (5'-SYGGRG-3') that CreAp binds ix 12 44 90 Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. Figure 17. LIST OF FIGURES Construction of ccsnfl Disruption Transformants Nucleotide (Genbank accession number: AF159253) and deduced amino acid sequence (Genbank accession number: AAD43 341) of C. carbonum SNFI gene Comparison of Deduced Amino Acid Sequences of C. carbonum ccSNFI and yeast SNFI Alignment of the N-terminal region of Snflp homologs from several eukaryotic organisms Copy number of SNF] gene in C. carbonum Complementation of Yeast snf] by ccSNF] Screening of ccsnfl Disruption Transformants Effect of SNFI mutation on extracellular enzyme activities Effect of SNFI mutation on extracellular enzyme activities in Tox2‘ C. carbonum strains RNA expression of Wall-Degrading Enzyme Genes in the Wild Type (WT) and in a ccsnfl Mutant (Mut) of C. carbonum Growth of C. carbonum Wild Type and a ccsnfl Mutant on Glucose, Sucrose, or Complex Carbon Sources Growth of C. carbonum Wild Type and ccsnfl Mutant on Simple Sugars Pathogenicity Assays of ccsnfl Mutants Pathogenicity Assays of Tox2+/ccsnfl Mutant HC-toxin Production by Tox2+ Wild Type (367-2A) and Tox2+lccsnf1 Mutant(T668) Infection by Wild Type and ccsnfl Mutants Thermotolerance of C. carbonum wild type and 5an mutant 37 40 42 43 46 47 49 51 52 54 55 57 58 59 61 62 64 Figure 18. Expression of C. carbonum Arabinoxylan-Degrading Enzyme Genes in Glucose and Xylan media Figure 19. Effect of carbon source on xylanase gene expression Figure 20. Nucleotide and deduced amino acid sequence of C. carbonum CREA cDNA (ccCREA) Figure 21. Comparison of Deduced Amino Acid Sequences of C. carbonum CREA and other fungal CreA proteins, and S. cerevisiae Miglp Figure 22. Copy number of CREA gene in C. carbonum Figure 23. Schematic representation of the 8an /CreA pathway of cell wall degrading enzyme regulation Figure 24. Construction of arfI Disruption Transformants Figure 25. Construction of aer Disruption Transformants Figure 26. Nucleotide and deduced amino acid sequence of C. carbonum ARFI Figure 27. Comparison of Deduced Amino Acid Sequences of C. carbonum ARFI and a-L-arabinofirranosidases of A. niger, A. sogae, S. coelicolor, and S. lividans Figure 28. Nucleotide and deduced amino acid sequence of C. carbonum ARFZ Figure 29. Comparison of Deduced Amino Acid Sequences of C. carbonum ARF 2 and a-L-arabinofiiranosidases of A. niger, E. nidulans, and T. reesei Figure 30. Copy number of ARF 1 and ARFZ in C. carbonum Figure 31. Screening of arfl and aer Disruption Transformants Figure 32. Effect of ARF 1 and ARF 2 mutations on a-L-arabinofirranosidase activity Figure 33. Cation-exchange HPLC analysis of a-L-arabinofirranosidase activity of wild-type, arfl mutant and arfl/arf? double mutant Figure 34. ct-L-Arabinofuranosidase, B-xylosidase and B-glucosidase activities in the three peaks of the wild-type culture filtrate xi 8] 82 84 85 88 92 108 109 112 115 116 118 119 121 123 124 126 Figure 35. Growth of C. carbonum wild-type, arfl mutant and aflI/arfl double mutant 127 Figure 36. Pathogenicity Assay of arfl Mutant 129 xii ARF] arf] Arfl p ARF 2 0r]? Arap ccCREA ccCreAp ccRPD3 ccsnfl ccSNFI ccSnfl p CREA CreAp CWDE(s) EX G] CPD 1. HPLC MLGI PGNI PGXI LIST OF ABBREVIATIONS ct-L-arabinofuranosidase gene (1) Cochliobolus carbonum mutant lacking fiinctional ARFI gene Protein product ofARFl gene ct-L-arabinofuranosidase gene (2) Cochliobolus carbonum mutant lacking functional ARFZ gene Protein product of ARFZ gene Cochliobolus carbonum CREA gene Protein product of Cochliobolus carbonum CREA gene Cochliobolus carbonum histone deacetylase gene Cochliobolus carbonum mutant lacking functional ccSNFI gene C ochliobolus carbonum SNFI gene Protein product of C ochliobolus carbonum SNFI gene CREA gene Protein product of CREA gene Cell wall degrading enzyme(s) Cochliobolus carbonum exo-B-l,3-glucanase gene Cochliobolus heterostrophus glyceraldehyde-3-phosphate dehydrogenase gene High-perfomance liquid chromatography Cochliobolus carbonum B -l,3- -1,4-Glucanase gene Cochliobolus carbonum endopolygalacturonase gene Cochliobolus carbonum exopolygalacturonase gene xiii snfl SNFI Snfl p XYLI XYLZ XYL3 XYL4 XYPI Mutant lacking functional SNFI gene SNFI gene Protein product of SNFI gene Cochliobolus carbonum xylanase gene (1) Cochliobolus carbonum xylanase gene (2) C och/iobolus carbonum xylanase gene (3) C ochliobolus carbonum xylanase gene (4) Cochliobolus carbonum B-xylosidase gene xiv Chapter One Enzymes and fungal pathogenicity ABSTRACT Plant pathogenic fungi secrete extracellular enzymes that are capable of degrading the cell walls of their host plants. These cell wall degrading enzymes (CWDEs) may be necessary for penetration of the cell wall barrier, as well as for generation of simple molecules that can be assimilated for growth. Most of these enzymes are substrate- inducible and their expression is controlled by both specific and global regulators. CWDE-inhibitors have been isolated fi'om plants and some evidence indicates that they are components of general resistance. INTRODUCTION When microorganisms acquire the ability to enter and grow within healthy tissues, and subsequently circumvent plant host defense mechanisms, disease develops. Recent investigations on fungal pathogenicity have focused on defining the virulence factors produced by these pathogens, as well as the methods used to penetrate their hosts. Certain pathogenic fitngi are known to produce host-specific toxins as agents of pathogenicity. In most cases, these toxins affect only a particular host plant. For example, Fusarium graminearum secretes trichothecene, a sesquiterpenoid involved in wheat head scab disease (Mirocha et al., 1994; O'Donnell et al., 2000), while pathotypes of Alternaria alternata produces several toxins specific for different plants such as AK-toxin for Japanese pear (Tanaka et al., 1999) and AAL-toxin for tomato (Abbas et al., 1995). The maize pathogen Cochliobolus carbonum makes HC-toxin, which is a critical determinant of virulence in its interaction with maize of genotype hm/hm (Ahn and Walton, 1996). Participation of these host-specific toxins in the establishment of plant diseases has been studied in the above and several other pathogenic firngi. Nevertheless, the ability of toxin-producing fungi to cause disease is dependent not only on their ability to produce a toxin, but also their fiindamental ability to penetrate and grow within plant tissues. Penetration into plant tissues and utilization of the nutrients found therein are requirements for all successful infections. Because cell wall constituents such as cellulose, xylan, pectin and proteins are typical among plants, the mechanism for penetration may be common across a variety of fungal pathogens. Most firngal pathogens and many non-pathogens produce large numbers of cell wall degrading enzymes (CWDEs) including pectinases, xylanases, cellulases, and proteases, which are capable of depolymerizing the various components of the host cell walls. Consequently, they have been studied in relation to tissue maceration, penetration, or nutrient acquisition from the cell wall polymers. In pathogenic fungi, these CWDEs may be required not only for growth but for virulence as well. The question of whether CWDEs are important or necessary for disease development has been the subject of considerable research and debate over many decades (Annis and Goodwin, 1997; Mendgen et al, 1996; Walton, 1994) FUEL, ENZYMES AND VIRULENCE Generation of simple molecules A major reason for microbial attack on plants is to obtain nutrients for growth. To achieve this, they must first overcome the physical barrier presented by the plant cell wall. Fungi utilize the food substances in their immediate vicinity to promote growth toward other areas of food availability. This continuing need to reach new food supplies for growth and reproduction may be the most significant driving force of fungal virulence. Generally, fungi secrete a wide variety of enzymes that can degrade various macromolecules to simple compounds that can be assimilated. Both the plant cell wall and the protoplasm contain nutrients that can be utilized by the growing fungus. Loosening and growing through the plant cell wall would allow the fungus access to simple sugars, amino acids, minerals and nucleotides that are abundant within the cytosol of the plant. In addition, degradation of the cell wall macromolecules would generate simpler compounds such as xylose, glucose, and amino acids that could be absorbed for growth. Adaptation It could be that a microorganism becomes pathogenic if it can adapt its extracellular enzyme secretion both in proportion and kind to the cell wall constituents of a particular plant. This would occur if the rate of inhibition or degradation of these enzymes by plant substances is less than the rate of production of the enzymes by the microorganism. Additionally, the plant cell wall repair machinery may not be able to match the speed of the cell wall loosening activities of the fungal CWDEs. Investigations by St. Leger et a1. (1997) indicate that fungi exhibit specific enzymatic adaptation to their various targets. The authors showed that plant pathogens (Verticillium albo-atrum, Verticillium dahliae, Nectria haematococca) produce high levels of enzymes capable of degrading pectic polysaccharides, cellulose, xylan and cutin, but secrete little or no chitinase and show no proteolytic activity against elastin or mucin. On the other hand insect pathogens (Verticillium lecam'i, Beauveria bassiana, Metarhizium anisopliae) degrade a broad spectrum of proteins including elastin and mucin, but produce low levels of polysaccharidases. Saprophytes (Neurospora crassa and Aspergillus nidulans) and opportunistic pathogens (Aspergillus fumigatus and Aspergillus flavus) produce the broadest spectrum of protein and polysaccharide degrading enzymes, indicative of their less specialized nutritional status. The specialized adaptation of pathogenic fungi CWDEs to their host polymers suggests a coevolved relationship between the fungi and their hosts (Cooper et al., 1988). Redundancy Fungal extracellular enzymes may have the potential to degrade the structural cell wall constituents of living plants, but proving that such interactions initiate or promote pathogenesis has been difficult. It is now well known that fungi often produce two or more distinguishable proteins with identical or similar enzymatic activity (Yao and Koller, 1995). Until a fungal genome is completely sequenced, it is impossible to determine the number of CWDE genes. Several isoforms of a particular enzyme can also occur and may be encoded by a single gene (Caprari et al., 1993). Although many other CWDE genes remain to be isolated, a search of the GENBANK database (www.ncbi.nlm.nih. gov) reveals that T richoderma reesei, C. carbonum, Magnaporthe grisea and Fusarium oxysporum have multiple xylanase genes. As documented in Table 1, C. carbonum secretes numerous enzymes to degrade the complex web of carbohydrates and glycoproteins of the maize cell wall. Functional redundancy provides a means to adapt to different conditions, and is indicative of processes with vital importance to an organism. While some CWDEs are made at all times during the life cycle of the pathogen, others may be inducible only during plant infection, making it difficult to detect them in vitro. In the wheat scab fungus F. oxysporum f.sp. lycopersici, for example, two xylanase genes (xyIZ and xyl3) are expressed in vitro during growth on oat spelt xylan or tomato vascular tissue. In contrast, RT-PCR revealed that x3213 is expressed in roots and in the lower stems of tomato plants infected by the fungus Table 1. C. carbonum extracellular enzymes capable of degrading specific maize (Zea mays) cell wall components. Cell Wall Degrading Gene(s) References Component Enzyme Arabinoxylan endo-B-1,4-xylanase XYLI, XYL2, Apel et al., XYL3, XYL4 1993; Apel- Birkhold and Walton, 1996 B-Xylosidase XYPI Wegener et al., 1999 Ransom and Walton, 1997; Arabinofilranosidase ARFI, ARFZ Tonukari et al., unpublished Pectin Endopolygalacturonase PGNI Scott-Craig et al., 1990 Exopolygalacturonase PGXI Scott-Craig et al., 1998 Pectin methylesterase PMEI Scott-Craig et al., unpublished Cellulose/ Endo- B -1,4-glucanase CEL] Sposato et al., B-LB-GIucan (Cellulase) 1995 [3 -1,3- -l,4-Glucanase MLG], MLGZ Gorlach et al., 1998 Exo-B-1,3 glucanase EXGI, EXGZ Nikolskaya et aL,l998; Schaeffer et al., 1994 Protein Protease ALP], ALPZ Murphy and Walton, 1996 throughout the whole disease cycle, whereas xyl2 is only expressed during the final stages of disease (Ruiz-Roldan et al., 1999). Other CWDEs may be encoded by cryptic genes which may be only expressed under stress or unfavorable conditions, or when the products of the normal genes are compromised. M. grisea xy12 mutants secrete three additional xylanase activities that are not expressed in the parent strain (Wu et al., 1997). Similar secretion of previously undetected pectate lyases and an endo-B-l,3-glucanase following inactivation of related genes have been reported earlier by Kelemu and Collmer (1993) in bacteria and Beffa et al. (1993) in plants, respectively. Thus it is possible that additional genes are expressed during pathogenic growth, and some CWDEs may even exhibit overlapping activities. Therefore in planta studies are required to provide a more comprehensive knowledge of the full spectrum of enzymes that are produced during fungal pathogenesis. All these factors have made the assessment of the contribution of any individual CWDE to pathogenicity difficult. Penetration Penetration of the plant surface and cell walls is a crucial event in pathogenesis. Some fungal pathogens such as the rice blast fungus M. grisea form highly melanized appressoria (swellings at the end of germ tubes) inside which a high pressure builds up, allowing the penetration of the intact host cuticle by mechanical pressure (Howard et al., 1991; Money, 1997). Nevertheless, most fungal pathogens that do not form a large melanized appressorium probably need CWDEs for penetration (Mendgen et al., 1996). Even in fimgal pathogens where the appressoria physically push through the plant cell wall, the contribution of enzymes to the softening of the wall cannot be ruled out. Depolymerization or loosening of the components of the cell walls by enzymes would facilitate the penetration and passage through this barrier. The ability to make knock-out mutants by DNA-mediated transformation now provides the means for the direct assessment of the role of CWDEs in disease development. Disruption of one or a few of the cell wall degrading enzyme genes may not result in any detectable decrease in the virulence of a fungus because of the redundancy of the enzymes. Such disruption may only eliminate a single component of the enzyme activity under investigation. Single mutations in each of 16 CWDE genes in C. carbonum have led to a reduction in specific enzyme activities in some cases but a corresponding decrease in virulence of the fungus was not observed (Apel et al., 1993; Apel-Birkhold and Walton, 1996; Gorlach et al., 1998; Murphy and Walton, 1996; Scott- Craig et al., 1990; Scott-Craig et al., 1998; Wegener et al., 1999). Multiple gene disruptions in C. carbonum have also been made including double and triple mutants (Apel-Birkhold and Walton, 1996; Scott-Craig et al., 1998) but these also had no effect in the virulence of the fungus. Despite this redundancy, however, single constitutive pectinase genes have been shown to contribute to the virulence of Aspergillus flavus on cotton bolls (Shieh et al., 1997) and Borrytis cinerea on tomato (ten Have et al., 1998). This evidence suggest that while a specific CWDE may be very important for virulence in one pathogenic fungus, others may likely require a combination of several CWDEs. CWDE REGULATION Substrate The expression of most CWDEs depends on external conditions such as substrate availability or the specific stage of disease development. The majority of CWDEs are made at low basal levels on sugars such as glucose or sucrose, but are highly expressed when the fungus is grown on the appropriate substrate. In some cases, however, the products of CWDE digestion also induce expression. Northern analysis indicates that while T richoderma reesei xyn2 is induced in the presence of xylan and xylose (a product of xylan degradation), it is virtually silent in the presence of glucose (Zeilinger et al., 1996; Mach et al., 1996). Polygalacturonase, pectate lyase and pectin lyase activities are also induced in media supplemented with galactose or galacturonic acid (Crotti et al., 1998; Scott-Craig et al, 1990). Ilmen et a1. (1997) also demonstrated that in T. reesei induction by cellulose and repression by glucose regulate cellulase expression in an actively growing fungus. However, derepression of the cellulase occurs without apparent addition of the inducer once glucose has been depleted from the medium. A similar conclusion on cellulase regulation was also obtained in Agaricus bisporus (Yague et al., 1997). Kolattukudy et a1. (1995) reported that a cutinase present in spores of F. solam' pisi releases small amounts of cutin monomers upon contact with the plant surface, which then trigger cutinase gene expression. This induction of CWDEs by cell wall components suggests a possible signaling pathway from the plant cell leading to the expression of CWDE-encoding genes inside the fitngal cell. 10 Specific Regulators Recent investigations indicate that both specific and global regulators control CWDEs (Table 2). Xlan, a transcriptional activator of the xylanolytic system, has been identified in A. niger (van Peij et al., 1998). The xlnR gene encodes a polypeptide of 875 amino acids with a zinc binuclear cluster domain similar to the zinc clusters of the GAL4 superfamily of transcription factors. A 5'-GGCTAAA-3' consensus sequence, the binding site of Xlan, is present within several xylanolytic promoters of various Aspergillus species and Penicillium chrysogenum. This sequence may be an important and conserved cis-acting element in the induction of xylanolytic genes in filamentous fungi: In contrast, the Chaetomium gracile xylanase A gene (chA) is repressed by binding of a protein designated AnRP. This protein binds specifically to a 5’TTGACAAAT-3’ element in the promoter region of the chA gene (Mimura et al., 1999). Global regulators The involvement of CreAp, the carbon catabolite repressor, in the regulation of CWDEs has been reported in several fungi (Table 2). CreAp, which is the homolog of yeast Miglp, binds to the promoter region of several genes and inhibits their expression (Dowzer and Kelly, 1991; Ronne, 1995). Deletion of this promoter region leads to an increase in the level of transcription of the regulated genes (de Graaff et al., 1994). The promoter region of the xInA gene from A. tubigensis has also been studied with respect to xylan induction and carbon catabolite repression (de Graaff et al., 1994; Orejas et al., 1999) and contains four potential CreAp target sites. de Graaff et al. (1994) suggest that 11 Table 2. Proteins known to regulate cell wall degrading enzymes. Regulatory Organism Comments Reference factor AnRP Chaetomium Binds to a xylanase gene Mimura et al., 1999 gracile (chA) promoter and represses its expression. Xlan A. niger Transcriptional activator of van Peij et al., 1998 xylanase genes. CreAp A. niger Carbon catabolite repressor de Graaff et al., 1994; B. cinerea which binds to the Orejas et al., 1999; E. nidulans promoter region of Zeilinger et al., 1996; G. fujikuroi enzymes and down- Mach et al., 1996; N. crassa regulates gene expression. Reymond-Cotton et al., S. sclerotiorum 1996 T. reesei Drysdale et al., 1993 Dowzer and Kelly, 1991 Shrofi‘et al., 1996 ccSnflp C. carbonum Relieves CreAp inhibition, Tonukari et al., 2000 thereby promoting expression of catabolite- repressible genes including CWDEs. 12 carbon catabolite repression of the xInA gene is controlled at two levels, directly by repression of xlnA gene transcription and indirectly by repression of the transcriptional activator. T reesei xynl and Sclerotinia sclerotiorum polygalacturonase pg] genes are also regulated by CreAp (Mach et al., 1996; Reymond-Cotton et al., 1996; Zeilinger et al,1996) Because the biosynthesis of most CWDEs is substrate-inducible, it may appear that these enzymes are made upon encountering plant cell walls. In contrast, most CWDE genes are repressed by glucose. Repression by the CreA complex is relieved by Snfl p, which was first isolated in S. cerevisiae (Celenza and Carlson, 1984). Snflp is a serine/threonine protein kinase (encoded by SNFI) and functions as an activator of gene expression by inactivating Miglp in yeast, and presumably, the Miglp homolog CreAp in fungi (Ostling and Ronne, 1998; Treitel et al., 1998). The yeast SUC2 gene which encodes invertase is repressed by Miglp, and yeast carrying a snfl mutation cannot grow on sucrose because they are unable to express invertase. Candida gIabrata snfl mutants also cannot use trehalose as carbon source (Petter and Kwon-Chung, 1996). Snflp is not active in the presence of glucose, and preliminary evidence suggests that it is in a dephosphorylated state under these conditions (Hardie, 1999; Sugden et al., 1999). Snflp also activates transcription in response to glucose limitation by modulating transcriptional activators such as Sip4p (Lesage et al., 1996; Vincent and Carlson, 1998). The SNF] gene is highly conserved among eukaryotes, and has been isolated from mammals, plants, and the nematode, Caenorhabditis elegans (Hardie et al., 1998). The mammalian homolog of 5an p, AMP-activated protein kinase (AMPK), phosphorylates and regulates hydroxymethylglutaryl-CoA reductase and acetyl-CoA carboxylase, both of which are 13 key regulatory enzymes of sterol synthesis and fatty acid synthesis, respectively (Hardie, 1999). AMPK is activated by high AMP and low ATP levels via a complex mechanism, which involves allosteric regulation, promotion of phosphorylation by an upstream protein kinase (AMPK kinase), and inhibition of dephosphorylation. This protein-kinase cascade is a sensitive system which is activated by cellular stresses that deplete ATP and thus acts like a cellular fuel gauge. When AMPK detects a 'low—fiJel' situation, it protects the cell by switching off ATP-consuming pathways (e.g. fatty acid synthesis and sterol synthesis) and switching on alternative pathways for ATP generation (e. g. fatty acid oxidation) (Hardie and Carling, 1997). Like Snfl p, AMPK can also regulate gene expression (Foretz et al., 1998). A tobacco Snflp homolg, NPKSp, has a predicted kinase domain whose amino acid sequence is 65% identical to that of the S. cerevisiae SNFI product. Expression of NPK5 in yeast cells allows snfl mutant cells to utilize sucrose for growth and causes constitutive expression of SUC2 in wild-type cells. NPKS-related genes are expressed in the roots, leaves, and stems of tobacco plants (Muranaka et al., 1994) Therefore, a gene disruption that abolishes Snflp activity should down-regulate CWDE expression. Mutation of the SNFI gene in C. carbonum led to a decreased expression and activities of several CWDEs including B-l,3-glucanases, pectinases and xylanases (Tonukari et al., 2000; and in this thesis). The C. carbonum snf] mutant also shows reduced virulence on maize which is consistent with the hypothesis that CWDEs are virulence factors. In S. cerevisiae, Snflp is regulated by protein phosphatase 1, which is made up of the regulatory subunit Reglp and the catalytic subunit Glc7p (Sanz et al., 2000). 14 Expression of the glucose transporter gene (HXT) in S. cerevisiae also depends on the Rgtlp transcriptional repressor and two glucose sensors in the membrane, Snf3p and Rgt2p. these sensors bind glucose and generate the intercellular signal to which Rgtlp responds (Ozcan and Johnston, 1999). Morever, mutation of two other S. cerevisiae genes, SSN3 and SSN8, suppresses the effect of a SNFI mutation. SSN3 encodes a cyclin- dependent protein kinase (cdk) homolog, and SSN8 encodes a cyclin homolog, and both are likely to function as a cdk-cyclin pair (Kuchin et al., 1995). A spinach homolog of Snfl p, protein kinase H1, is regulated by glucose-6-phosphate, and this suggests that the inhibition of Snflp may be through glucose-6-phosphate (Huang et al., 1997; Toroser et al,2000) RESPONSE OF THE PLANT TO CW DEs CW DEs as inducers of Plant Defense Although plants are exposed to many microorganisms, some of which are potential pathogens, they are naturally resistant to the vast majority. Only a few fungi are pathogenic though most are endowed with CWDEs that could serve as virulence factors. The key to pathogenicity may depend to a large extent on the degree of resistance or susceptibility of the host. Saprophytes with their wide array of digestive enzymes are unable to consume living plants suggesting that all plants may have constitutive mechanisms that resist these enzymes. In addition to the cell wall barrier, other pathways that activate a battery of defense mechanisms against potential pathogens may also exist. 15 It has been specifically shown that a 21-kDa xylanase from T. viride elicits defense responses in tobacco plants, including tissue necrosis and increased production of ' pathogenesis-related proteins (Avni et al., 1994). In tomato, a cytoplasmic protein T- SUMO (tomato small ubiquitin-related modifier) is involved in mediating the signal generated by the ethylene-inducing xylanase from T. viride that leads to induction of plant defense responses (Hanania et al., 1999). In general, resistance could involve small molecules or proteins that inhibit or promote the digestion of the fungal secreted enzymes thereby denying access to the host cell. For a pathogen to be successfirl, it must be able to circumvent or overcome these antifungal defenses. Protease Inhibitors It now common knowledge that plants produce protease inhibitors that can inactivate fungal proteins, possibly including CWDEs. A proteinase-inhibitor protein (MPI) is induced in response to fungal infection in germinating maize embryos (Cordero et al., 1994). Increase in the proteinase inhibitor mRNA is a consequence of the wound produced by the penetration and colonization of the host tissues by the pathogen. Similarly, an antifungal cysteine protease inhibitor has been identified in pearl millet (Joshi et al., 1998), while a 14-kDa trypsin inhibitor from maize inhibits both conidial germination and hyphal growth of nine plant pathogenic fungi, including Aspergillus parasiticus and Fusarium monilzforme (Chen et al., 1999). Whether protease inhibitors specifically interact with CWDEs, however, is not known at present. 16 CW DE Inhibitors The identification of CWDE inhibitors in plants provides indirect evidence that CWDEs are virulence factors. Polygalacturonase-inhibiting proteins (PGIP) are typically effective against fiingal endopolygalacturonases (Cervone et al., 1989, 1990). The PGIPs are predominantly bound to plant cell walls, and their levels increase in bean hypocotyls when primary leaves begin to develop (Salvi et a1, 1990). PGIP forms specific and reversible high affinity complexes with fungal polygalacturonases, thereby regulating the activity of the firngal enzymes (Cervone et a1, 1989). Rapid accumulation of pgip mRN A correlates with the appearance of the hypersensitive response in incompatible interactions between C. lindemuthianum and P. vulgaris, and the inhibitor transcripts accumulate to higher levels in epidermal cells proximal to the site of infection (Devoto et al., 1997). Recently, a glycosylated, basic protein that inhibits A. niger and Trichoderma viride endo-l,4-B-xylanases was identified in wheat (McLauchlan et al., 1999). This inhibitor, which is heat and protease sensitive, is a glycosylated protein with a basic isoelectric point similar to the PGIP from tomato (Stotz et al., 1994). An inhibitor of pectin methylesterase from kiwi fruit is also a glycoprotein, but has an acidic isoelectric point (Giovane et al., 1995). High-level expression of pear PGIPs in transgenic tomato fi'uits leads to increased resistance to B. cinerea (Powell et al., 1994). The PGIP inhibition of B. cinerea polygalacturonases slows the expansion of disease lesions and the associated tissue maceration in transgenic tomato plants (Powell et al., 2000). It is envisaged that more CWDE inhibitor proteins will be identified in the near fixture. l7 CONCLUSION Virtually all fungi produce a great abundance and variety of CWDEs that may be needed for softening up of the plant cell walls for penetration by firngal hyphae, as well as provision of nutrients for growth. The application of molecular technologies to the study of fungal-plant interactions offers a new and more definitive approach for examining the role played by CWDEs in disease development. Resistance to CWDEs may be part of the constitutive and/or acquired resistance that plants possesses against various pathogens. Any factor that will inhibit the production of CWDEs in firngi could lead to reduced virulence. Though it has not yet been possible to demonstrate that individual CWDEs are required for C. carbonum pathogenicity (Apel et al., 1993; Apel-Birkhold and Walton, 1996; Gorlach et al., 1998; Murphy and Walton, 1996; Scott-Craig et al., 1990; Scott- Craig et al., 1998; Wegener et al., 1999), the focus of this study was to determine whether CWDEs are globally necessary for C. carbonum pathogenicity. 18 REFRENCES Abbas, H.K., Tanaka, T., and Shier, W.T. (1995). 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Chem. 271, 25624-25629. 26 Chapter Two The Cochliobolus carbonum SNFI gene is required for cell wall-degrading enzyme expression and virulence on maize Parts of this chapter have been published: Tonukari, N.J., Scott-Craig, J.S., and Walton, J.D. (2000) The Cochliobolus carbonum SNFI gene is required for cell wall-degrading enzyme expression and virulence on maize. Plant Cell 12, 237-248. 27 ABSTRACT The production of cell wall-degrading enzymes (wall depolymerases) by plant pathogenic fungi is under catabolite (glucose) repression. In Saccharomyces cerevisiae, the SNF] gene is required for expression of catabolite-repressed genes when glucose is limiting. An ortholog of SNF 1, ccSNF 1, was isolated from the maize pathogen Cochliobolus carbonum and ccsnf] mutants of HC toxin-producing (TonI) and HC toxin nonproducing (Tox2—) strains created by targeted gene replacement. Growth in vitro of the ccsnfl mutants was reduced by 50 to 95% on complex carbon sources, such as xylan, pectin, or purified maize cell walls. Growth on simple sugars was affected depending on the sugar. Whereas growth on sucrose or glucose, fi'uctose, or sucrose was normal, growth on galactose, galacturonic acid, maltose, or xylose was somewhat reduced, and growth on arabinose was strongly reduced. HC toxin production was normal in the Tox2+ ccsnf] mutant, as were conidiation, conidial morphology, conidial germination, and in vitro appressorium formation. Activities of secreted B-l,3-glucanase, pectinase, and xylanase in culture filtrates of the Tox2+ ccsnfl mutant were reduced by 53, 24 and 65%, respectively. mRNA expression under inducing conditions of XYLI, XYLZ, XYL3, XYL4, XYPI, ARFI, MLGI, EXGI, PGNI, and PGXI, which encode secreted wall-degrading enzymes, was downregulated. The Tox2+ ccsnfl mutant was strongly reduced in virulence on susceptible maize, forming fewer spreading lesions, which, however, had normal morphology. The Ton_ ccsnfl mutant also formed fewer nonspreading lesions that also had normal morphology. The results indicate that ccSNFI is required for biochemical processes that are important for pathogenesis by C. carbonum and suggest 28 penetration as the single most important step at which ccSNFI is required. The specific biochemical processes controlled by ccSNFl probably include, but are not necessarily restricted to, an ability to degrade polymers of the plant cell wall and to take up and metabolize the sugars that are produced. 29 INTRODUCTION The plant cell wall is a major barrier to the penetration and spread of potential pathogenic organisms, and all of the major groups of cellular plant pathogens are known to make extracellular enzymes that can degrade cell wall polymers. Although the involvement of wall-degrading enzymes and their genes in penetration, ramification, plant defense induction, and symptom expression has been extensively studied, conclusive evidence for or against a role for any particular enzyme activity in any aspect of pathogenesis has been difficult to obtain (Walton, 1994). The major obstacle to addressing the fitnction of wall-degrading enzymes has been redundancy: all pathogens that have been studied in detail have multiple genes for any particular enzyme activity. Thus, most fungal strains mutated in wall-degrading enzyme genes — either by conventional (e.g., Cooper, 1987) or molecular (e.g., Scott- Craig et al., 1990) methods — retain at least some residual enzyme activity. For example, the pea pathogen Nectria haematococca (F usarium solani f sp pisi) has four functional pectate lyase genes, the maize pathogen Cochliobolus carbonum and the rice pathogen Magnaporthe grisea each have at least four xylanase genes, and the cosmopolitan pathogen Borrytis cinerea has up to five endo-polygalacturonase genes (Apel-Birkhold and Walton, 1996; Guo et al., 1996; Wu et al., 1997; ten Have et al., 1998). Even strains of fungi with multiple mutations still retain residual enzyme activity and are still pathogenic (Apel-Birkhold and Walton, 1996; Scott-Craig et al., 1998; J. S. Scott-Craig and J. D. Walton, unpublished results). Despite redundancy, single genes of a particular class have been shown, in two cases, to contribute to the virulence of pathogenic fungi. 30 Particular constitutive pectinases are virulence factors for Aspergillus flavus on cotton bolls and for B. cinerea on tomato (Shieh et al., 1997; ten Have et al., 1998). The gamut of extracellular wall-degrading enzymes produced by the ascomycete C. carbonum includes pectinases, xylanases, cellulases, mixed-linked (B-l,3-B-1,4)- glucanases, B-l,3-g1ucanases, proteases, xylosidases, arabinosidase, and undoubtedly others. None of the strains generated to date with single mutations in any of the genes encoding these enzymes have reduced virulence. Furthermore, with only a few exceptions, the mutants still grow as well as wild type on the appropriate substrate in vitro (Scott-Craig et al., 1990; Apel et al., 1993; Schaeffer et al., 1994; Sposato et al., 1995; Apel-Birkhold and Walton, 1996; Murphy and Walton, 1996; Nikolskaya et al., 1998; Gerlach et al., 1998; Scott-Craig et al., 1998; Wegener et al., 1999). An alternative approach to the individual isolation and disruption of each wall- degrading enzyme gene would be to identify genetic regulatory elements whose mutation results in the simultaneous loss or downregulation of multiple enzymes. If a mutant globally impaired in its ability to make wall-degrading enzymes were still pathogenic, it would bring into serious doubt a significant role for such enzymes in pathogenesis (Walton, 1994). In culture, the expression of most wall-degrading enzymes by most fungi, including plant pathogens, is inhibited by glucose or other simple sugars, which is a well- studied metabolic process known as catabolite or glucose repression (Ruijter and Visser, 1997). Most and perhaps all of the examined extracellular enzyme activities of C. carbonum are subject to catabolite repression (Walton and Cervone, 1990; van Hoof et al., 1991; Holden and Walton, 1992; Ransom and Walton, 1997). In yeast, release from 31 catabolite repression requires a protein kinase called Snflp. Snflp is required for the expression of glucose-repressed genes such as invertase (S UC2) when glucose is limiting. That is, glucose-repressed genes remained repressed in a snfl mutant even in the absence of glucose (Celenza and Carlson, 1984; Hardie et al., 1998; Ostling and Ronne, 1998; Treitel et al., 1998). A major function of Snflp is to phosphorylate Miglp, a DNA- binding transcriptional repressor. The ortholog of MIG] in filamentous fungi is called creA (Ronne, 1995). Phosphorylation of Miglp inhibits its binding to the promoters of the genes that it represses and promotes movement of Miglp out of the nucleus into the cytoplasm (DeVit et al., 1997). Snflp controls the response to glucose through additional mechanisms, because it also activates via phosphorylation the transcriptional activators Sip4p and Cat8p (Lesage et al., 1996; Vincent and Carlson, 1998). Orthologs of SNF] are present in many other organisms, including mammals and plants. Its counterpart in mammals is AMP-dependent protein kinase (Hardie et al., 1998). A SNFI ortholog is involved in the response of Arabidopsis to glucose (Bhalerao et al., 1999). To the best of our knowledge, the biology of SNFI genes has not been studied previously in filamentous fungi. Because SNFI is required for derepression of catabolite-repressed genes in yeast, mutation of the orthologous gene in C. carbonum might cause irreversible downregulation of catabolite-repressed wall-degrading enzymes. snf] mutants might therefore be usefiil for testing whether wall-degrading enzymes are virulence factors in pathogenic fungi. 32 METHODS Fungal Cultures, Media, and Growth Conditions The wild-type HC toxin-producing (Tox2+) isolate of Cochliobolus carbonum, designated 367-2A, was derived from isolate SBl 11 (ATCC 90305) and maintained on V8 juice agar plates (Apel et al., 1993). The wild-type HC toxin nonproducing (Tox2‘) isolate was 164R], which is a progeny of SB] 11 (Walton, 1987). The fungus was grown in liquid media or agar plates containing mineral salts, 0.2% yeast extract, and trace elements (Van Hoof et al., 1991). Carbon sources were 2% (w/v) glucose, sucrose, oat spelt xylan (Fluka), citrus pectin (Sigma P-9135), or maize cell walls (Sposato et al., 1995). For quantitation of growth on agar plates, 5 uL of a conidial suspension (104 conidia per mL) in 0.1% Tween 20 was pipetted onto the center of the plate. Plates were incubated under fluorescent lights at 21°C. Nucleic Acid Manipulations The C. carbonum genomic and cDNA libraries have been previously described (Scott- Craig et al., 1990). DNA and total RNA were extracted from lyophilized mats (Apel et al., 1993; Pitkin et al., 1996). The methods used for DNA and RNA electrophoresis, gel blotting, probe labeling, and hybridization have also been described elsewhere (Apel- Birkhold and Walton, 1996). For RNA blots, the Cochliobolus heterostrophus GPDI 33 gene, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a loading control (Van Wert and Yoder, 1992). Polymerase chain reaction (PCR) was performed in a thermocycler (MJ Research, Callahan, CA) using Taq DNA polymerase (GIBCO-BRL) and two degenerate oligonucleotide primers based on the conserved regions of SNFI genes (sense, 5'- CAYCCNCAYATHATHAA-3'; antisense, 5-TCNGGNGCNGCRTARTT-3', where Y is T or C; R is G or A; H is T, C or A; N is A, T, G, or C). Touchdown PCR (Don et al., 1991) with these primers and C. carbonum genomic DNA as template was performed under the following conditions: initial denaturation at 94°C for 3 min, followed by 45 cycles of denaturation at 94°C for 1 min, annealing for l min, and polymerization at 72°C ' for 2 min. The annealing temperature ranged from 60 to 45°C with a decrease of 1°C every three cycles. This was followed by 10 cycles of denaturation at 94°C for 1 min, annealing at 45°C for 1 min, and polymerization at 72°C for 2 min. The PCR product was cloned into the EcoRV site of pBluescript II KS+ (Stratagene, La Jolla, CA). The transcriptional start site of ccSNFI was determined using the 5’ rapid amplification of cDNA ends (RACE) system, version 2.0, following the instructions of the manufacturer (GIBCO-BRL) (Frohman et al., 1988). An oligonucleotide of sequence 5’-GCCCTCGCCCAGGGTGC-3’ was used to prime first-strand cDNA synthesis, which was then amplified by PCR using a nested primer of the sequence 5’- GCCGAGACGCTGGCTCG-3’. Automated fluorescence DNA sequencing was done at the Department of Energy- Plant Research Laboratory Sequencing Facility at Michigan State University. Sequence data were analyzed with Lasergene software (DNASTAR, Inc., Madison, Wisconsin). 34 Functional Complementation of Yeast snfl Saccharomyces cerevisiae was grown at 30°C on plates containing YPD (1% yeast extract, 2% [w/v] peptone, 2% [w/v] glucose, and 2% [w/v] agar). The ura reference strain was MG106 (MATa adeZ-I can1-100 his3-1115 leu2-3112 trpI-I ura3-1). The ccSNFI cDNA was excised from plasmid pSnfl c-3 with SacI and XbaI and cloned into pVTlOO-U, a yeast expression vector with the alcohol dehydrogenase ADHI promoter and terminator and the URA3 selectable marker. The resulting construct, pSnfl c-4, or the empty vector pVTlOO-U as a control, were used to transform yeast MCY1846 (MATa snf1A10 lys2-801 ura3-52) cells as described by Gietz and Woods (1994). Transformants were first selected on synthetic dextrose-uracil (SD-URA) medium, which contains 0.67% yeast nitrogen base without amino acids (DIFCO Laboratories, Detroit, MI), 0.062 % -Leu/-Trp/-Ura DO (dropout) supplement (Clonetech Laboratories, Palo Alto, CA), 0.01% Leu, 0.002% tryptophan, 2% (w/v) glucose, and 2% (w/v) agar at 30°C. The transformed cells were grown in liquid YPD medium overnight. The cultures were then streaked on YPS agar plates, which contain 1% yeast extract, 2% peptone, 2 % (w/v) sucrose and 2 % (w/v) agar. The plates were photographed after 2 days at 30°C. Disruption of ccSNFI The gene replacement vector was derived from a 3.3-kb SacI-EcoRV genomic fragment containing ccSNFI (Figures 1A and IE) cloned into pBluescriptII KS+. An internal 2.1- kb PstI-Sall fragment was deleted and replaced with a 1.4-kb PstI-SaII fragment 35 containing the hph gene conferring hygromycin resistance. To create convenient restriction sites, the HpaI fragment fiom plasmid CBlOO3 (Carroll et al., 1994) was cloned into the EcoRV site of pBluescriptII KS+. The replacement vector was linearized at the BssHll-Sacl sites prior to transformation of C. carbonum 367-2A (Tox2+) or 164R] (TonU protoplasts (Pitkin et al., 1996; Scott-Craig et al., 1990). Mycelium for protoplasts for transformation was obtained from germinating conidia (Apel et al., 1993). Transformants were purified by two rounds of single-spore isolation. Enzyme Assays Total xylanase, pectinase, and B-1,3-glucanase activities were assayed by release of reducing sugars from the indicated substrate (Lever, 1972). Thirty microliters of culture supernatant was assayed in a 300-uL reaction volume containing 1.0% oat spelt xylan, polygalacturonic acid, or B-1,3-glucan (laminarin), and 50 mM sodium acetate, pH 5.0, at 37°C for 30 min. A 25- or lOO-uL aliquot of the reaction mixture was mixed with 1.5 mL of a working solution of p-hydroxybenzoic acid hydrazide, the mixture heated at 100°C for 10 min, and the absorbance measured at 410 nm (Lever, 1972). Enzyme activities are based on the amount of monomers (glucose, galacturonic acid and xylose for B-1,3- Glucanase, pectinase and xylanase activities, respectively) released per uL of reaction volume. 36 A 1kb Sacl Pstl Sall EcoRV > ccSNF1mRNA B Sacl Pstl Sall EcoRV —-l-‘ _—l— hphl Figure 1. Construction of ccsnf] Disruption Transformants. (A) Map of the ccSNF1 gene. (B) An internal 2.1-kb PstI-Sall fragment of ccSNF1 was replaced with hphl. 37 HC toxin Analysis HC toxin was extracted with chloroform fi'om culture filtrates of C. carbonum grown for 14 days, fi'actionated by thin-layer chromatography on silica using a solvent system of dichloromethane2acetone (1:1 by volume), and detected using an epoxide-specific colorimetric reagent (Meeley and Walton, 1991). Pathogenicity Assay Pathogenicity was tested by spray-inoculating 18-day-old susceptible inbred maize line Pr (genotype hmI/hml) with a suspension of conidia (104 per mL) in 0.1% Tween 20. The plants were inoculated in the afternoon and covered with plastic bags overnight. The plants were grown in a greenhouse and monitored daily for eight days or until death. RESULTS Isolation of C. carbonum SNFI (ccSNF1) Two degenerate oligonucleotide primers were synthesized based on conserved regions of Snfl p-related sequences of Saccharomyces cere visiae, Candida glabrata, and tobacco NPK5 (Celenza and Carlson, 1984; Muranaka et al., 1994; Petter and Kwon-Chung, 1996). Amplification by polymerase chain reaction (PCR) with C. carbonum genomic 38 DNA as template yielded a product of the predicted size (400 bp). The PCR product was used to isolate the SNFI gene and cDNA copies from C. carbonum genomic and cDNA libraries, respectively. The C. carbonum cDNA clone of ccsnfl was cloned into pGEM7 to create plasmid pSnflc-3. The C. carbonum gene (designated ccSNF1) has two introns of 60 and 51 bp. The 3’-untranslated region is 159 bp and the 5’-untranslated region is 120 bp in length (Figure 2). The open reading frame of the product of ccSNF1, ccSnfl p, is 880 amino acids and has a molecular mass of 98 kD. ccSnflp has ~40 % overall identity with yeast Snfl p. As shown in Figure 2, the similarity is very strong at the N terminus, a region of the protein that includes the ‘activation segment,’ which is conserved in all known related protein kinases (Johnson et al., 1996; Hardie et al., 1998). This block of amino acids is 100% conserved between Snflp and ccSnfl p. The similarity between Snflp and ccSnflp is weak to nonexistent at the C terminus, with the possible exception of a few blocks of conserved amino acids that are apparent only with the introduction of many large gaps (Figures 3 and 4). ccSnflp is the largest (98 kD) of the known Snflp-related proteins. Those of various yeasts are ~70 kD, those of plants are ~56 kD, and those of Caenorhabditis elegans, Drosophila, and mammals are ~63 kD. ccSnflp also differs fi’om other proteins related to Snflp in that the PSORT program (Nakai and Kanehisa, 1992) strongly predicts localization in the nucleus (Table 3). The computed probability of ccSnflp having a nuclear localization is 78%, whereas yeast Snflp is predicted to be in the nucleus with only a 48% probability. The difference is due to the presence of three regions of basic amino acids in ccSnflp that are absent in yeast Snflp (PTKKPRA 39 CAGGCGGCCGCGAATTCACTAGTGATTCTGCACACAATTGTGCAACTTCGAGCCCTGTAC 6O CTGTGCTTCTGCACACTCCCACGGGCCGCCAACAAGCTCTCTTCCTCTCCCTCTTTCTCT 120 CCCTCTCGTTGCCTCTCCCCACCGACGCGCTCCTCTTCGCATGCTTCCGCCCTGCGTCGG 180 CACACCTGCTAACCGGATGTTACCTGTATAGTCCCCGCAGACGCAAACCGCCCTCCAGCC 240 CTACGTATCCCCGCAAAAGCTTCTGGCCAGCGCCTCCAAGCACGACCGGCGCCACCATAC 300 CACTCGCCATACAATGTCGGCCGCAATCGATAATGAGGACCTGGAGGAGCTCTCCATCTC 360 M S A A I D N E D L E E L S I S 16 CATGCCCTCGCAGCGGCGGGGCGCCGCCCAAACCAGCACAACAAAGGCCCAGGACCCCGC 420 M P S Q R R G A A Q T S T T K A Q D P A 36 CCCGCCGCCGCCTACTGCGCTCGGAACTGCGGTGCACGAAACCAAGAGCAAGGATACAAA 480 P P P P T A L G T A V H E T K S K D T K 56 GGCGAGCCAGCGTCTCGGCCAGTACACCATTGTCCGCACCCTGGGCGAGGGCTCCTTCGG 540 A. S Q R L G Q Y T I V R T L G E G S F G 76 CAAGGTCAAGCTGGCCACCCACCAGGTTAGCGGCCAAAAGGTCGCCCTCAAGATCATCAA 600 K V K L A T H Q V S G Q K V A L K I I N 96 TCGCAAGAGGCTCGTCACCAGAGATATGGCAGGCAGGATCGAGCGTGAGATTCAGTATCT 660 R K R L V T R D M A G R I E R E I Q Y L 116 GCAGCTGCTGCGCCATCCGCATATCATCAAGCTgtacgttgtttgcctgtagccgcgctt 720 Q L L R H P H I I K L 127 gcctcattttcatgctcacttttgctcccctagCTATACCGTCATAACAACGCCGACCGA 780 Y T V I T T P T E 136 AATCATCATGGTCCTCGAATACGCAGGCGGGGAATTGTTCGACTACATCGTCAACCACGG 840 I I M V L E Y A G G E L F D Y I V N H G 156 TAAACTGCAAGAGGCACAGGCTCGAAAGTTCTTCCAGCAAATTGTATGCGCTGTCGAATA 900 K L Q B A Q A R K F F Q Q I V C A V E Y 176 CTGCCATCGACACAAGATTGTCCACCGAGATCTGAAGCCCGAGAACCTCCTCCTCGACCA 960 C H R H K I V H R D L K P E N L L L D H 196 CGATAGCAATGTAAAAATTGCCGACTTTGGTCTGAGCAACATCATGACGGACGGCAACTT 1020 D S N V K I A D F G L S N I M T D G N F 216 TCTCAAGACAAGCTGTGGCAGCCCCAACTATGCTGCGCCCGAGGTCATTTCTGGCAAGTT 1080 L K T S C G S P N Y A A P B V I S G K L 236 GTACGCTGGTCCCGAAGTCGACGTCTGGAGCTGTGGTGTCATACTATACGTTTTGTTAGT 1140 Y A G P E V D V W S C G V I L Y V L L V 256 CGGCCGGCTACCCTTCGACGACGAATATATCCCGACCCTCTTTAAGAAAATTGCCGCGGG 1200 G R L P F D D E Y I P T L F K K I A A G 276 CCAGTACAGCACACCCAGCTATCTCTCACCAGGCGCCACCTCTTTGATTAGAAAAATGCT 1260 Q Y S T P S Y L S P G A T S L I R K M L 296 CATGGTCAATCCCGTACACCGCATCACCATCCCCGAGCTTCGACAAGACCCGTGGTTCAC 1320 M V N P V H R I T I P E L R Q D P W F T 316 GACAGACCTCCCAGCATACCTCGAACCGCCCGCGCAAGAGTTTTTTGACAGTGGCGCTGA 1380 T D L P A Y L E P P A Q E F F D S G A D 336 CCCCAACAAGGCCATTGATCCCAAGGCTCTTGCGCCGTTGGCCGACGCGCCTCGTGTGCA 1440 P N K A I D P K A L A P L A D A P R V Q 356 GGCGCTGCATGAAAACGTGGTGACAAAGCTTGGAAAGACAATGGGTTATGCAAAGCATGA 1500 A L H E N V V T K L G K T M G Y A K H D 376 TGTGCAAGATGCCTTGGCACGCGATGAGCCGAGTGCCATTAAAGATGCTTACCTCATTGT 1560 V Q D A L A R D E P S A I K D A Y L I V 396 Sequence continued next page. Figure 2. Nucleotide (Genbank accession number: AF 1 59253) and deduced amino acid sequence (accession number: AAD43341) of C. carbonum SNFI gene. The open reading frame encodes a protein of 880 amino acids; " transcription start site; * stop codon; # polyadenylation site. The two introns, 60 and 51 bp, are typed in italic lowercase. 40 Sequence continued from previous page. CCGAGAGAATGAGATGATGCGGGAGAACCgtaggcatgactactcttatggtattgcctg R E N E M M R E N tagctgactgtttattctagCTTTGTTAACCAACCAAGATGGTGTTCCGGTGTGGAATCA P L L T N Q D G V P V W N H CCAGTCGCCGCCTGCGCACGACAGTTATATGGAAAAGTTTAGACCACAATCATTGAATGC Q S P P A H D S Y M E K F R P Q S L N A TGTATCACGTCCGCAATTCATTCCCCCGGCGCCTTCAGACCATGAAAGAGCACGCCAAGG V S R P Q F I P P A P S D H E R A R Q G ATCCAACGCCAGCAGTCAGCTTGCAAGCATTCGCAGCCCGGTCAGCACCATAGCAATTCT S N A S S Q L A S I R S P V S T I A I L CCCGAGTAGTCTTACAGAATACCACAAGGCTTATATGAAGGGCCACCCAAGGCCCACTAA P S S L T E Y H K A Y M K G H P R P T N CAAGATTTCGGAAAGCGAGGCTCTCCCACCAACACCTGAACAGACGGAAGAACAACGGCA K I S E S E A L P P T P E Q T E E Q R Q AATATCGGCTCGAAGACTAAAACCAAATTTCCGTACAATGCCCGAAGCAGGTAGAACAAA I S A R R L K P N F R T M P E A G R T K GCCGGAGCCGATGACCAGCCTACCTACCAAGAAGCCACGTGCGACCAAGTGGCAATTCGG P E P M T S L P T K K P R A T K W Q F G TATTCGATCCAGAAACCAACCTGCAGAGGCTATGCTTGCTATATTCAAGGCATTAAAAGC I R S R N Q P A E A M L A I F K A L K A CATGGGCGCCGACTGGGAAGTACCAAAGATACGCAGAGCCGGTGGTCGTAGTGGGTCTCG M G A D W E V P K I R R A. G G R S G S R CAGCCGAAGCACCTCTCAGGCCCCAGAAGACCGCAAGTCTAAATCCAGGAATCATTCACA S R S T S Q A P E D R K S K S R N H S Q AGACTCTATCTCTTCACATTCATCAGACGAAGACCAGGGCTCGCGGAAGGGCTCGCCACG D S I S S H S S D E D Q G S R K G S P R TCGTGAGCCGCTCAGTGTACGCAACAATGGCACGAGCGAACAAGAAGCAAGAGGTCGACA R E P L S V R N N G T S E Q E A R G R Q AAAGAAGCACTACAACCATACAAATGACTGGGGCTACCACGTTCCCGAAGATCCCTGGGT K K H Y N H T N D W G Y H V P E D P W V TATCCATGCACGCTTCCTAAAGGAGGGCATGTTCCCGCCTGGTGTTGCGCATCCCTCGTC I H A. R F L K E G M F P P G V A H P S S GACGCACTCTTCACGCGTCGACCTCGCCAACGATTCTTCTGGTGCACGAAGACGGAGTTC T H S S R V D L A N D S S G A R R R S S GACTAACACCAGCACATCGAGCGCTGGCCATGGTGTCGAAGGCATGACCCCGTCCGAGCG T N T S T S S A G H G V E G M T P S E R CGCAGGGTCGGTTAGTGAGGACCATGTGAATCCCGATGAAGCAGTATACATTTACATGTC A G S V S E D H V N P D E A V Y I Y M S CATCCAACTTTACAGCATCGACCGCGACTTTTTTGTCGTCGACTTCAAATGTGCAGGCTA I Q L Y S I D R D F F V V D F K C A G Y CGAACGCCTCGTCACCAATCTTGTGCGCGAGATCAAGGCCTCAATTCCGCTTTCAGGCTC E R L V T N L V R E I K A S I P L S G S GCACCAGCCCCCACCGCATCACCAGGACGGCTGGGACGACGAGCAGGGAGTATGGCGCCG H Q P P P H H Q D G W D D E Q G V W R R ACTGGACGAAAACGAGCCACTTCCCGAAGACTTGGCCAAGAAGCTGAATGAAGGGGGCAC L D E N E P L P E D L A K K L N E G G T AGAGATTCTTCGAGAAAGAACGGAACTTGTGGGTGCGGGGCGCCAGGAAGGCGAGAAGAT E I L R E R T E L V G A G R Q E G E K I TGTCACAAGTCCGTTTCCCTTTCTCGATGTTGCAAGCACTCTTATCTTGCAATTGAGCGG V T S P F P F L D V A S T L I L Q L S G CGAGTAATTGTGTGCTTGTGTACAATCGAGGGGATGGGGAGAAAGTATGTATGTATAGAA E * AGTGGGCAAGGATGGGATAGATGCACCACCTAGATTCCGGGATTTTGCCCACTGTAAAGC AGAGAATCCGACCTCTTGATGGTCTAAATGCATTTCACTGCACTTATTCTTACCACTTTC # AGTGAAATCTGGAAATACCTCAAACTTCAAACAAATAGTGTACTCATACCGTGTCATCCA 41 1620 405 1680 419 1740 439 1800 459 1860 479 1920 499 1980 519 2040 539 2100 559 2160 579 2220 599 2280 619 2340 639 2400 659 2460 679 2520 699 2580 719 2640 739 2700 759 2760 779 2820 799 2880 819 2940 839 3000 859 3060 879 3120 880 3180 3240 3300 9’9 9’9 5”? 9’9 9‘9 5“.“ carbonum cerevisiae carbonum cerevisiae carbonum cerevisiae carbonum cerevisiae carbonum cerevisiae carbonum cerevisiae carbonum cerevisiae carbonum cerevisiae carbonum cerevisiae carbonum cerevisiae cazbonum cerevisiae carbonum cerevisiae canbanum corovisiao carbonum cerevisia- cazbonum cerevisiae 1: panama I shnmorsmmnpnppppm rm - 1. mm ------- IMannie.tannin-151114run-nutca 3 -LAD . GQYHIVRTLGEGSFGKVKLAHHO SGQKVALKIINRK IGNYEIVKTLGEGSFGKVK ‘ ‘H GQKVALKII 121 112 181 172 241 232 301 292 _ ~ . - . 361 “'1' l- 7‘ --r- -- :IKDAYLIVRENI' IRENPLLTN-- T 347 I s . us 8381- IRDAYMLIKENI' Ermmvsu- 415 - 1" anpp- usmmsf’lna IPP an - GS QgAsIHs 407 D LSQSPP FQQQSKSHQKSQHDHE :- -m FubeYEEE 475 TI-ILPSSL - - ‘ measampnnqunqmrsmmm 466 SILPTSL‘OI -- g i ----------- E ------------ sp -------------- 535 PB '-T qFGIRSRI V - 489 -- IS erwErsmss a ‘6 595 Rscsnsnsrsqmonxsxsauasqosrssassoaoqcsmsamnsvamerng 537 -------------------------------------------------------- -- 655 539 715 Raf-133 s'rssncacvzmpsamsv wfirMsIQLym- - 555 ——.- ---------------------- --I ---Lxl60%. Similarly, disruption of PGNI and PGXI together causes 3 >95% loss of extracellular polygalacturonase activity in C. carbonum, yet disruption of both genes results in only a 40-60% decrease in growth on pectin (Scott-Craig et al., 1998; J.S. Scott-Craig and J.D. Walton, unpublished results). In contrast, disruption of ccSNF1 only 65 partially down-regulates PGNI and FOX] expression and decreases total pectinase activity by only 24%, yet growth of the mutant is decreased by >50%. These comparative results are consistent with the hypothesis that ccSNF1 is also necessary for expression of enzymes needed for utilization of xylan or pectin, such as enzymes need for the transport and intracellular catabolism of xylose or galacturonic acid. This hypothesis is supported by the data shown here that indicates that ccsnfl mutants have reduced ability to grow on simple sugars, such as xylose or galacturonic acid. It is also supported by studies in other systems. Catabolism of the products of pectinase in Erwinia chrysanthemi requires at least two transport proteins and seven cytoplasmic enzymes, which are coregulated with the extracellular pectinases (Hugouvieux-Cotte-Pattat et al., 1996). Glucose, acting at least partially through SNFI, regulates the transcription of hexose transporters in yeast (Carlson, 1998). ccSNF1 is required for the expression of wall-degrading enzymes and for grth on simple sugars, but our results do not indicate which is more important for virulence of C. carbonum. Wall-degrading enzymes might be important for the actual process of penetration. Species of Cochliobolus do not require either melanization or appressoria to cause disease and, therefore, have been presumed, by default, to penetrate enzymatically and not by mechanical force (Horwitz et al., 1999). A decreased ability to produce wall- degrading enzymes would therefore be predicted to result in decreased penetration, which is what was observed for the ccsnfl mutant. Alternatively, or in addition, C. carbonum might require free sugars as a source of nutrition to sustain the metabolic activity necessary for penetration. These sugars could conceivably come from the plant leaf surface or epidermis, although the concentrations of free sugars (other than glucose or 66 sucrose, which are utilized equally well by the ccsnfl mutant and the wild type) are probably quite low in these environments. A more likely source of sugars, such as arabinose or xylose, is from the action of extracellular depolymerases on the maize cell wall. Therefore, even if the proximal cause of the decreased virulence of the ccsnfl mutant is a defect in the uptake and/or metabolism of simple sugars, the ultimate cause would still be its reduced expression of wall-degrading enzymes. Insofar as wall-degrading enzymes are important for virulence of C. carbonum, it is of interest to determine which enzymes in particular are important. Reduced virulence of the ccsnfl mutant could be due to downregulation of one enzyme, all enzymes of a particular class, or many enzymes partially. It is not possible to distinguish between these alternatives from our results, because all of the ones studied were downregulated. Previous studies have excluded many individual structural genes from making a major contribution to virulence. Of those that have not yet been directly tested, ARF1 is intriguing as a possible candidate for a solo virulence gene because (1) its expression is completely dependent on ccSNFl, (2) the ccsnfl mutant has strongly impaired growth on arabinose, (3) arabinose is a major component of maize cell walls, and (4) ct- arabinosidase has been implicated as a virulence factor in two other diseases (Howell, 1975; Rehnstrom et al., 1994). 67 REFERENCES Ahn, J.-H., and Walton, J.D. (1997). A fatty acid synthase gene required for production of the cyclic tetrapeptide HC-toxin, cyclo(D-prolyl-L-alanyl-D-alanyl-L-Z-amino- 9,10-epoxi-8-oxodecanoyl). Mol. Plant-Microbe Interact. 10, 207-214. Apel-Birkhold, P.C., and Walton, J.D. (1996). 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Plant-Microbe Interact. 10, 700-708. 72 Chapter Three Expression of cell wall degrading enzymes and characterization of the CREA gene in Cochliobolus carbonum 73 ABSTRACT The expression of four endo-1,4-B-xylanase genes (XYLI, XYLZ, XYL3, XYL4), an exo- 1,4-B-xylosidase gene (XYPI) and an ct-arabinosidase (ARF1) was studied in the presence and absence of glucose. Repression of all six genes was observed with glucose as the sole carbon source, whereas expression was seen when the culture medium contained xylan. The xylanase genes are substrate-induced, but differentially expressed. The CREA gene has been implicated in glucose repression in several fungi, and a homolog was isolated and characterized in Cochliobolus carbonum. The deduced amino acid sequences of the C. carbonum ccCREA gene was very similar to the CreA proteins of Aspergillus niger (55% identity), Gibberella fujikuroi (48% identity), Sclerotinia sclerotiorum (46% identity), and Trichoderma reesei (46% identity). It also had 22% identity with the Saccharomyces cerevisiae Miglp. The zinc finger region of the C. carbonum ccCreAp protein had between 92 and 100% identity with the corresponding zinc finger regions of the other fiingal proteins. The CreAp pathway may play an important role in the regulatory process that leads to cell wall degrading enzyme expression and therefore virulence in pathogenic fungi. 74 INTRODUCTION The full expression of cell wall degrading enzyme (CWDE) genes by fungi depends on mechanisms that are associated with the glucose regulation pathway (Tonukari et al., 2000). In many organisms, glucose represses genes whose products are used to metabolize other carbon sources. Work in yeast and filamentous fungi has revealed a mechanism for glucose repression in eukaryotes that is different from that found in bacteria (Ronne, 1995). In yeast, a CyszHisz zinc-finger-containing protein, Miglp, binds GC-boxes (GCGGGG) in promoters of several genes (Bu and Schmidt, 1998; Lundin et al., 1994) and plays a key role in mediating glucose repression. In the presence of glucose, Miglp represses the transcription of genes required for the utilization of alternative sugars (Lutfiyya and Johnston, 1996). Carbon catabolite repression is mediated in Aspergillus nidulans by a repressor, CreAp, which is a homolog of the yeast Miglp (Dowzer and Kelly, 1991). CreAp also binds to promoters (Ruijter and Visser, 1997) and prevents transcription of several genes in the presence of glucose. The CREA gene has also been isolated from other fungi including Sclerotinia sclerotiorum (Vautard et al., 1999), Trichoderma reesei, and T. harzianum (Ilmen et al., 1996). Most CreA proteins have the signature zinc finger domain of the CyszHisz type (C-x(2,4)-C-x(3)- [LIVMFYWC]-x(8)-H-x(3,5)-H) (Berg, 1988; Miller et al., 1985; Rosenfeld and Margalit, 1993). The role of the CreAp in glucose repression has been summarized by Rome (1995) and Ruijter and Visser (1997). Deletion of CREA in A. nidulans has an extremely severe effect on growth under both carbon catabolite-repressing and nonrepressing conditions (Dowzer and Kelly, 1991; 75 Shroff et al., 1997). Other authors have shown that an A. nidulans crea mutant has two- and threefold increased expression of hexokinase and fructose-6-phosphate reductase activities, respectively, while phosphofructokinase and pyruvate kinase activities decrease (van der Veen et al., 1995). Repression by CreAp is mainly relieved by the protein kinase, Snflp, which is highly conserved in fungi, plants and mammals (Celenza and Carlson, 1984; Hardie et al., 1998). We have previously reported that in the pathogenic fungus, Cochliobolus carbonum, a homolog of SNF] (the gene which encodes Snflp) is required for the expression of many cell wall degrading enzymes (Tonukari et al., 2000). Mutation of ccSNF1 in the fungus leads to varying levels of repression of cell wall degrading enzyme genes, reduced growth on complex polymers such as xylan and pectin, and also reduced virulence on its host, maize. As part of an effort to determine whether the phenomenon of glucose repression in C. carbonum depends on the CreA-Snfl pathway studied in yeast, we isolated a homolog of the CREA gene from C. carbonum and studied the relatedness of its deduced protein to other known CreA proteins. We also studied the expression of CWDE genes following the growth of the fungus in minimal medium containing different carbon sources. 76 METHODS Fungal cultures, media and growth conditions The wild type race 1 strain of C. carbonum (367-2A) used in this study was derived from strain SBl 11 (ATCC 90305) and was maintained on V8 plates (Apel et al., 1993). The fungus was grown in liquid media containing mineral salts, 0.2 % yeast extract, and trace elements (Van Hoof et al., 1991) with 2 % glucose, sucrose, xylose, oat spelt xylan (Fluka, Switzerland), pectin, cellulose, corn cell walls (Sposato et al., 1995), lyophilized corn leaves, or 5% of fresh corn leaf material as the carbon source. Nucleic acid manipulations DNA and total RNA were extracted from lyophilized mats (Pitkin et al., 1996; Apel et al., 1993). The methods used for DNA and RNA electrophoresis, blotting, probe labeling and hybridization have also been described elsewhere (Apel-Birkhold et al., 1996). The Cochliobolus heterostophus GPD] gene, encoding glyceraldehyde-3 -phosphate dehydrogenase (Van Wert and Yoder, 1992), was used as a reference. PCR was performed in a thermo cycler (MJ Research, Watertown, MA) using T aq DNA polymerase (Life Technologies, Gaithersburg, MD) and two degenerate primers based on conserved amino acid sequences of CreA homologs from Aspergillus niger, Neurospora crassa, Saccharomyces cere visiae, Sclerotinia sclerotiorum, and 77 Trichoderma reesei. Forward primer 5’ GARAARCCNCAYGCNTG 3’ is based on the amino acid sequence “EKPHAC,” and reverse primer 5’ GTRTGRTCNGGNGTNGG 3’ is based on the amino acid sequence "PTPDHT" where Y = T or C; R = G or A; I = inosine; N = A, T, G or C). The degenerate primers were used in Touchdown PCR (Don et al., 1991) to amplify a 631-bp fragment from C. carbonum genomic DNA. Touchdown PCR was performed under the following conditions: initial denaturation at 94 °C for 3 min, 45 cycles of denaturation, 94 °C for 1 min; annealing for 1 min; and polymerization at 72 °C for 2 min. The annealing temperature ranged from 60 °C to 45 °C with a decrease of 1 °C every three cycles. This was followed by 10 cycles of denaturation at 94 °C for l min; annealing at 45 °C for at for l min; and polymerization at 72 °C for 2 min. The PCR product was cloned into pGEM T-easy vector (Promega). A specific primer based on the amplified CREA sequence from the touchdown PCR (above), 5’ CTCCTTCTCCAACTACTCTCCTG 3’, was made and used with a vector-specific primer, 5’ CGTGAATGTAAGCGTGACAT 3’, to isolate a CREA cDNA from a C. carbonum cDNA library in the yeast vector, pMYR (GenBank Accession number of pMYR is AF 102577; CytoTrap XR Library Construction Kit, Stratagene). The C. carbonum cDNA library was originally constructed for the CytoTrap yeast two-hybrid screen (Stratagene), following the instructions of the manufacturer, using total mRNA from C. carbonum grown on corn cell walls. The PCR reaction was as follows: initial denaturation at 94 °C for 3 min, 35 cycles of denaturation; 94 °C for 1 min; annealing at 50 °C for 1 min; and polymerization at 72 °C for 2 min. The CREA cDNA was then cloned into the pGEM T-easy vector. 78 The transcriptional start site of the CREA cDNA was determined using the 5’ RACE (Rapid Amplification of cDNA Ends) system, version 2.0, following the instructions of the manufacturer (GIBCO-BRL) (Frohman et al., 1988). A 21-bp oligonucleotide (5’- TCATGTGTGCAAGGTTAGATC -3’) was used to prime first- strand cDNA synthesis, which was then amplified by PCR using a nested primer of the sequence 5’- GCTCCATGAGACCCGCCTGTA -3’. DNA nucleotide sequences were determined by automated fluorescence sequencing at the DOE-Plant Research Laboratory Plant Biochemistry Facility, Michigan State University. Sequence data were analyzed with the Lasergene software (DNASTAR Inc., Madison, WI). RESULTS Repression of C. carbonum CW DE genes in glucose substrate To illustrate the glucose repression mechanism in C. carbonum, the expression of cell wall degrading enzymes was analyzed by RNA gel blotting. Four endo-1,4-B-xylanase genes (XYLI, XYLZ, XYL3, XYL4) (Apel et al., 1993; Apel-Birkhold et al., 1996; Scott- Craig et al., unpublished), an exo-1,4-B-xylosidase gene (XYPI) (Ransom and Walton, 1997; Wegener et al., 1999) and an ct-L-arabinofiiranosidase gene (ARF1) (in this thesis) has been cloned fi'om C. carbonum. The expression of these genes was studied following growth of the fungus in minimal medium containing glucose or xylan, and their 79 expression was detected only when the culture medium contained xylan (Figure 18). All of these genes were repressed when the culture medium contained only glucose (Figure 18). Expression of C carbonum CWDE genes in different growth media The expression of XYL], XYLZ, XYL3, XYL4, and XYP] was also studied during growth of the fungus in minimal media containing sucrose, xylose, xylan, pectin, cellulose, purified corn cell walls, lyophilized corn leaves (ground or whole), corn leaf extracts or whole corn leaves (fresh) as the sole carbon source (Figures 19). As analyzed by RNA blotting, XYL] was expressed only when the culture medium contained xylan, cellulose, purified corn cell walls, or the various corn leaf materials. XYLZ was induced under these same conditions but only weakly by the corn leaves. XYL3 was expressed only when xylose or xylan was in the medium, and XYL4 was transcribed in the presence of xylose, xylan or corn leaf materials. XYP] expression was induced by xylose, xylan, pectin and cellulose. The presence of sucrose in the medium suppressed the expression of all five genes. 80 , It. i. #1: GPD‘I Figure 18. Expression of C. carbonum Arabinoxylan-Degrading Enzyme Genes in Glucose and Xylan media. Fungal strains were grown for 7 d in liquid still culture containing, as sole carbon source, 2 % xylan, or 2 % glucose. GPD] encodes the glyceraldehyde- 3-phosphate dehydrogenase gene. 81 12345 678910 cam-vwmw wni~~1~ 71v “avast—n “:1" . Xy]2 , ini- A“ “a a..- -117“; Xyl3 ; Vi 77... ,f Xyl4 Xyp1 .. g, .~ ._2' 1'39) 1:; $51 al.-3. a"; are... .1 Wing» GPD1 ”*fiw qua- nil-mu Figure 19. Effect of carbon source on xylanase gene expression. Fungal strains were grown for 7 d in liquid still culture containing 2% sucrose (1), 2% xylose (2), 2% xylan (3), 2% pectin (4), 2% cellulose (5), 1% corn cell walls (6), 1% lyophilized corn leaves (whole) (7), 1% lyophilized corn leaves (ground) (8), fresh corn leaf extracted with water, 1:10 (w/v) (9), or 5% fresh corn leaves (10) as sole carbon source. GPD] encodes glyceraldehyde- 3-phosphate dehydrogenase gene. 82 Cloning of C carbonum CREA Two degenerate oligonucleotide primers were designed based on conserved regions of CreAp sequences of A. niger (Drysdale et al., 1993), S. cerevisiae (Lutfiyya and Johnston, 1996), S. sclerotiorum (Vautard et al., 1999), and T. reesei (Ilmen et al., 1996). Amplification by PCR, with C. carbonum genomic DNA as template, yielded a single 631-bp DNA fragment. A gene-specific primer and a vector-specific primer were used to amplify a corresponding cDNA. The full length cDNA contains an open reading fiame encoding a predicted protein product of 430 amino acids with a molecular mass of 46.3 kD and a pI of 9.38. The 3’-untranslated region is 20 bp and the 5’-untranslated region is 212 bp in length (Figure 20). The deduced amino acid sequence of the C. carbonum CREA (called ccCREA) has 55, 48, 46, and 46 % identity with the CreA proteins of A. niger, Gibberellafiijikuroi, S. sclerotiorum and T. reesei, respectively (Figure 21). It also show 22% identity with Miglp of S. cerevisiae. Two putative DNA-binding protein zinc fingers of the CyszHisz type situated between amino acids 70-90 and 98-120 are predicted by the PROSITE program (Hofmann et al., 1999). The ccCreAp zinc finger region is 92-100% identical to that of other fungal CreA proteins. The similarity between ccCreAp and its firngal homologs can be seen across the entire protein including both the N and C termini (Figures 21). Although smaller than S. cerevisiae Migl (504 a, 55.5 kD), ccCreAp is similar in size (430 aa, 46.3 kD) to the A. niger (402 a, 43.7 kD), G filjikuroi (420 aa, 45.9kD), S. sclerotiorum (429 aa, 47 kD) and T. reesei (402 aa, 43.7 kD) CreA proteins. Like other 83 AAACTTTCCCCTTCGGCCACATGCAATCCAACTCTGCCTCGACAGGTTTCGCCAACCTGC 60 M Q 8 N S A S T G F A N L 13 TCAACCCCGAAACAGCCTCCCAGAACCAACAGCAACAACAACAACAACCCACCTCTACAC 120 L N P E T A 8 Q N Q Q Q Q Q Q Q P T 8 T 33 CAACCGCCTCCATGGCCGCTGCCACCGTCAGCCTCATGGCGCCTCTTCTCCAGAACGCCC 180 P T A. 8 M A. A A T V 8 L M A P L L Q N A 53 CGCAACAGACAGAGGAGCCTCGACAGGATCTTCCCAGGCCTTACAAGTGCCCTCTCTGCG 240 P Q Q T E E P R Q D L P R P Y K C P L C 73 ACAAGGCCTTCCACCGTCTGGAGCACCAGACTCGCCACATCCGAACCCACACTGGAGAGA 300 D K A. E H R L E H Q T R H I R T H T G E 93 AGCCACACGCCTGCACTTTCCCTGGATGCACAAAGAGATTTTCCCGCTCTGACGAACTGA 360 K P H .A C T E P G C T R R E S R 8 D E L 113 CTCGACACTCGAGGATACATAACAACCCAAACTCGCGGCGAGGCAAGGGCCAGCAACATG 420 T R H 8 R I H N N P N 3 R R G K G Q Q H 133 CTGCTGCCACGCAAGCTGCCGTTGCCGCTGTACAGGCGGGTCTCATGGAGCCTGGATCTA 480 A .A A. T Q A. A. V' A .A. V Q A G L M. E P G 8 153 ACCTTGCACACATGATGCCTCCCCCATCAAAGCCCATTTCTCGCAGCGCCCCGGGTTCTC 540 N L A. H Mi Mi P P P 8 K P I S R 8 A P G S 173 AACTAGGCTCACCCAACGTCTCGCCACCTCACTCCTTCTCCAACTACTCTCCTGGCATGA 600 Q L G 8 P N V’ 8 P P H 8 F S N Y S P G M 193 GCAACGACCTGGCCGCATACCACCAGGGCGGCTTGAGCAACAGCAGCAGCCCCAGCGGTC 660 8 N D L A A Y H Q G G L S N S 8 S P S G 213 TTGCCCGCCCCATGGATCTCCTTGCTGATGCTGCGTCAAGACTGGAGCAACGTCCTGGAC 720 L A. R P M D L L A D A A 8 R L E Q R P G 233 ACATTTCCCACTCGAGTAGACATCACCTTACGAGCGGGTACCACCCTTACGCCAACCGAC 780 H I 8 H 8 8 R H H L T 8 G Y H P Y A N R 353 TGCCAGGCCTCTCTCAATACGCCTACTCGTCGCAGCCCATGTCAAGATCGCACTCACACG 840 L P G L 8 Q Y A Y S S Q P MC 8 R 8 H 8 H 273 AGGACGACGACCCGTACTCGCACAGGATGACGAAGAAGTCGAGGCCAGGCTCACCGTCGT 900 E D D D P Y 8 H R. M T K K 8 R P G S P S 293 CGACTGCCCCGCCATCTCCAACATTTTCGCACGATTCCTGCAGTCCGACGCCAGACCACA 960 S T A P P 8 P T P 8 H D 3 C 8 P T P D H 313 CGCCTCTGGCAACCCCTGCACACTCGCCAAGATTACGGCCCCACGGCTTCAGTGATCTCC 1020 T P L A T P A H 8 P R L R P H G F S D L 333 AGCTACCACATCTACGTCATCTCAGCCTCAACCAAAACTTTGTGCCAGCACTTGCGCCCA 1080 Q L P H L R H L 8 L N Q N F V' P A L A P 353 TGGAGCCGTCGACGGAGCGTGAGCAACCGTACGTGCCAAGCCAGTCGTCGGGACTTCGTA 1140 M E P 8 T E R E Q P Y V' P S Q 8 8 G L R 373 TTGGGGACATTATTTCCAAGCCCGAAGGTGCGCAGCGCAAATTGCCGGTTCCGCAAGTAC 1200 I G D I I 8 K P E G A Q R K L P V P Q V 393 CCAAGGTTGCGGTGCAAGATCTGCTCAATGGCCCGAGCAACAGCGGCTTCTCTTCCGGTA 1260 P R ‘V A V Q D L L N G P 8 N 8 G F S 8 G 413 ATAACTCCGCAACGGCGTCATTAGCCGGCGAAGACCTCTCGAATCGCAACTAAACCCACT 1320 N N 8 A. T A 8 L A G E D L 8 N R N . 430 CTCGACTCCAACTAGCATGGCATCGGCGTTTTCGGTCTGCATCCTCTGCTTTTTTTTACT 1380 TTTTCACCTCTCTTGCATTTCGTCATTTTTTTCACGGTCAGCAAGCGATGAGATTAGACA 1440 AGGGCACACACACACACACAAAGACCTTTTTTTGTAGGGTGGAGCCTGTGGCATATTACG 1500 ATTTCCCTAATTGAAACCTGAAAGCAAAAAAAAAAAAAAA 1540 Figure 20. Nucleotide and deduced amino acid sequence of C. carbonum CREA cDNA (ccCREA). ( The open reading frame encodes a protein of 430 amino acids; .’ stop codon 84 C. cazbonum 1 A. niger 1 G. tujikuroi 1 T. zoos-1 1 S. aclorotiorum 1 S. cerevisiae 1 C. carbonum S3 ‘RQDLPRPYKCPLCDKAFHRLEHQTRHIRTHTGEKPHACQFPGCTKRFSRSDE A . niger 61 RQDLPRPYKC PLCDRAFHRLE HQTRH IRTHTGEKPHACQFPGCTKRFSRSDE G . fuj ikuroi 4 9 V'LPRPYKC PLCDKAFHRLEHQTRH IRTHTGEKPHACQFPGCSKEFSRSDE T. £00801 42 _ J LPRPYKCPLCDKAFHRLEHQTRHIRTHTGEKPHACQFPGCSKRFSRSDE S. aclorotiorum 56 ' S. coruviaiao 23 1 2 C. cazbonum 113 A. niqur 121 ; G. fujikuxoi 109 LTRHSRIHNNPNSRRGNKAEQAJ qu . ooHQMH 9- _ 1'. recur. 102 LTRHSRIl-[ENPNSRRGNKGQQO S. acloxotiorumllG - S. cerevisiae 83 C. carbonum 173 A. nigur 178 G. ftjikuroi 169 T. reesei 156 S. aclorotiorum167 S. cerevisiae 143 C. carbonum 193'4 A. night 198 G. fujikuroi 189 T. rooaoi 176 S. aclorotiorumlB? S. cerevisiae 203 C. carbonum 245 A. nigur 247 G. fujikuroi 231 T. rooaoi 218 S. aclorotiorum242 S. coroviaiao 262 Alignment continued next page. Figure 21. Comparison of Deduced Amino Acid Sequences of ccCREA, other fungal CreAps, and S. cerevisiae Miglp. Residues that are identical in at least four sequences are indicated by black shading while conserved substitutions are in gray shading. The two CyszHisz zinc-finger region (Berg, 1988; Miller et al., 1985; Rosenfeld and Margalit, 1993) are underlined (1,2). 85 Alignment continued from previous page. 9959.”? 591959?!“ 995999 599339?!“ carbonum 269 niger 270 tujikuroi 252 reesei 238 aclorotiorum263 carbonum 325 niger 325 tujikuroi 312 reesei 296 aclorotiorum319 cerevisiae 382 carbonum 376 niger 375 tujikuroi 367 reesei 349 aclorotiorum374 coroviaiao 442 carbonum niger tujikuroi reesei aclorotiorum gum“; ...... . ‘ llLSL a PALAPMEPS ILPSIRI‘ILSLEHfiPALAPMEPO, ‘ s - S o HEPSIS ...... TPALAPMEPI 1 Q nee-S- --'.0 ’PT-- 0 0 0 Q . EZMNTSNQSQNQN cerevisiae 502 END 86 CreA proteins, the deduced ccCreAp also contains a nuclear localization signal (Hicks and Raikhel, 1995), PNSRRGK, at residues 123-129. Hybridization of a ccCREA probe to C. carbonum genomic DNA digested with PstI, Sacl, SalI, or SpeI resulted in a single band in each case (Figure 22), indicating the existence of a single copy of the ccCREA gene in C. carbonum. DISCUSSION Most CWDE genes are activated only in the appropriate environment and therefore are only expressed when required (Hensel and Holden, 1996). The biosynthesis of most xylanases are induced by xylan. Monomers such as xylose, a product of xylan digestion, can also induce expression of some CWDEs. This differential expression of multiple xylanases may serve to ensure that enzymes capable of degrading xylan are present under the various conditions encountered by the fungus during growth in its host plant. The repression of CWDEs by preferred carbon sources such as glucose (Asymeric et al., 1988) is an efficient energy-conserving mechanism because the activity of enzymes that degrade xylan and other carbon sources are not required in the presence of glucose. One of the mechanisms to achieve this is repression at the transcriptional level of genes that encode enzymes involved in the catabolism of the alternative carbon sources. The involvement of a carbon catabolite repressor (CreAp) in the regulation of CWDEs has been reported in several fungi (de Graaff et al., 1994; Orejas et al., 1999; Mach et al., 87 X \ s? 9°98 90‘ 69° 14.0 4.8 2.3 ' . 0.7 Figure 22. Copy number of CREA gene in‘C. carbonum. Southern blot analysis of total genomic DNA from C. carbonum digested with restriction enzymes PstI, SacI, SaII, or SpeI. 88 1996; Reymond-Cotton et al., 1996; Zeilinger et al., 1996). CreAp, which is the homolog of yeast Miglp, binds to the promoter region of target genes in the presence of glucose and inhibits their expression (Dowzer and Kelly, 1991; Ronne, 1995). ccCreAp, like Miglp and other CreA proteins, has two CYS2H182 zinc fingers that are well conserved among all known CreA proteins. CyszHisz zinc finger-bearing proteins are a large superfamily of nucleic acid binding proteins that constitute a major subset of eukaryotic transcription factors (Berg, 1988; Miller et al., 1985; Rosenfeld and Margalit, 1993). CreAp inhibits gene transcription by binding to specific sequences (5'-SYGGRG- 3') in the promoters of these genes (Cubero and Scazzocchio, 1994; Espeso and Penalva, 1994). This consensus sequence is present in several C. carbonum CWDE genes (Table 4). In S. cerevisiae, Miglp, binds similar promoter sequences (GCGGGG) and recruits two other corepressor proteins, Tuplp and Ssn6p which inhibit the expression of genes encoding enzymes that catabolize other carbon sources (Treitel and Carlson, 1995). Ssn6p-Tuplp is a general repressor of transcription in yeast (Keleher et al., 1992; Smith and Johnson, 2000). It has been shown that CREA transcript levels are independent of the carbon source in G. fiijikuroi and Botrytis cinerea (Tudzynski et al., 2000). De Vit et al. (1997) have also found that the subcellular localization of the S. cerevisiae Miglp is regulated by glucose. Miglp is imported into the nucleus within minutes after the addition of glucose and is just as rapidly transported back to the cytoplasm when glucose is removed. Although our knowledge of catabolite repression is still very incomplete, it is possible in certain cases to propose a partial model of the way in which the different elements involved in catabolite repression may be integrated. It appears that the Snflp 89 Table 4. Sequence in 5' region of C. carbonum CWDE genes that corresponds to the consensus sequence (S'-SYGGRG-3') that CreAp binds ((Cubero and Scazzocchio, 1994; Espeso and Penalva, 1994). The consensus sequence is in either ‘+’ or ‘-’ strand, and numbering is from the ATG encoding the first amino acid. Gene Putative CreAp binding site ARF1 -111CTGGAG-106 XYP] -163 GTGGGG -168 XYL3 -453 GTGGGG ~448 XYL4 ~118 CTGGGG -123 90 in the presence of non-glucose substrate phosphorylates Miglp and probably CreAp (Ostling and Ronne, 1998; Treitel et al., 1998). The phosphorylated CreAp is unable to bind to DNA and repress CWDE gene expression, and therefore they are transcribed. Translation of the messages produces CWDE proteins that are processed and secreted to the external surface of the fungal hyphae (Figure 23). Upon encountering plant cell walls, the CWDEs degrade the constituent polymers generating simple compounds including amino acids and mono- and oligosaccharides that are assimilated by the fimgus for growth. As long as most of the absorbed molecules are not glucose, Snflp is active, CreAp is inhibited, and CWDEs are made. The Snfl/CreA regulatory pathway is the first link of a possible signaling pathway from the cell wall of the host plant to the global expression of enzymes as probable fungal virulence factors that advance the disease condition. 91 (See figure next page) Figure 23. Schematic representation of the Snfl /CreA possible pathway of cell wall degrading enzyme regulation. Snfl protein kinase in the presence of non-glucose substrate phosphorylates CreAp. The phosphorylated CreA is unable to bind DNA and repress CWDE gene expression. Translation of the message produces CWDE proteins that are processed and secreted to the external surface of the fungal hyphae. Upon encountering it, the CWDEs degrade the plant cell wall generating simple compounds including amino acid, and mono- and oligosaccharides that are assimilated by the fungus for growth. As long as most of the absorbed molecules are not glucose, Snflp is active and CreAp is inhibited and CWDEs are made. 92 Cell wall degradation products 8. 9. amino acids, mono- and H , Cell oligosaccharides H P H / Wall G CWDE-inhibitors, other defense proteins and chemicals defense 93 REFERENCES Apel, P.C., Panaccione, D.G., Holden, ER, and Walton, J.D. (1993). Cloning and targeted gene disruption of XYL] , a B-1,4-xylanase gene from the maize pathogen Cochliobolus carbonum. Mol. Plant-Microbe Interact. 6, 467-473. Apel-Birkhold, P.C., and Walton, J.D. (1996). Cloning, disruption, and expression of two endo-B-l, 4-xylanase genes, XYLZ and XYL3, from Cochliobolus carbonum. 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Carbon catabolite repression of the Aspergillus nidulans xlnA gene. Mol. Microbiol. 31, 177-184. 95 Ostling, J., and Ronne, H. (1998). Negative control of the Miglp repressor by Snfl p- dependent phosphorylation in the absence of glucose. Eur. J. Biochem. 252, 162- 168. Pitkin, J.W., Panaccione, D.G., and Walton, J.D. (1996). A putative cycle peptide efflux pump encoded by the T OXA gene of the plant pathogenic fungus Cochliobolus carbonum. Microbiology 142, 1557-1565. Ransom, RE, and Walton, J.D. (1997). Purification and characterization of extracellular B-xylosidase and a-arabinosidase from the plant pathogenic fungus Cochliobolus carbonum. Carbohydr. Res. 297, 357-364. Reymond-Cotton, P., Fraissinet-Tachet, L., and Fevre, M. (1996). Expression of the Sclerotinia sclerotiorum polygalacturonase pg] gene: possible involvement of CREA in glucose catabolite repression. Curr. Genet. 30, 240-245. Ronne, H. (1995). Glucose repression in fungi. Trends genet. 11, 12-17. Rosenfeld, R., and Margalit, H. (1993). Zinc fingers: conserved properties that can distinguish between spurious and actual DNA-binding motifs. J. Biomol. Struct. Dynll, 557-70. Ruijter, G.J.G., and Visser, J. (1997). Carbon repression in Aspergilli. FEMS Microbiol. Lett. 151, 103-114. Shroff, R.A., O'Connor, S.M., Hynes, M.J., Lockington, R.A., and Kelly, J.M. (1997). Null alleles of creA, the regulator of carbon catabolite repression in Aspergillus nidulans. Fungal Genet. Biol. 22, 28-38. Smith, R.L., and Johnson, A.D. (2000). Turning genes off by Ssn6-Tupl: a conserved system of transcriptional repression in eukaryotes. Trends Biochem. Sci. 25, 325- 330. Sposato, J P., Ahn, J-H., and Walton, J.D. (1995). Characterization and disruption of a gene in the maize pathogen Cochliobolus carbonum encoding a cellulase lacking a cellulose binding domain and hinge region. Mol. Plant-Microbe Interact 8, 602- 609. Tonukari, N.J., Scott-Craig, J.S., and Walton, J.D. (2000) The Cochliobolus carbonum SNF] gene is required for cell wall-degrading enzyme expression and virulence on maize. Plant Cell 12, 237-248. Treitel M.A., and Carlson, M. (1995). Repression by SSN6-TUP1 is directed by MIG], a repressor/activator protein. Proc. Natl. Acad. Sci. USA 92, 3132-3136. 96 Treitel, M.A., Kuchin, S., and Carlson, M. (1998). Snfl protein kinase regulates phosphorylation of the Migl repressor in Saccharomyces cerevisiae. Mol. Cell. Biol. 18, 6273-6280. Tudzynski, B., Liu, S., and Kelly, J.M. (2000). Carbon catabolite repression in plant pathogenic fungi: isolation and characterization of the Gibberella fujikuroi and Botrytis cinerea CreA genes. FEMS Microbiol. Lett. 184, 9-15. van der Veen, P., Ruijter, C.J., and Visser, J. (1995). An extreme creA mutation in Aspergillus nidulans has severe effects on D-glucose utilization. Microbiology 141, 2301-2306. Van Hoof, A., Leykam, J., Schaeffer, H.J., and Walton, J.D. (1991). A single B-1,3- glucanase secreted by the maize pathogen Cochliobolus carbonum acts by an exolytic mechanism. Physiol. Mol. Plant Pathol. 39, 259-267. Van Wert, S.L., and Yoder, O.C. (1992). Structure of the Cochliobolus heterostrophus glyceraldehyde-3-phosphate dehydrogenase gene. Curr. Genet. 22, 29-35. Vautard, G., Cotton, P., and Fevre, M. (1999). The glucose repressor CREl from Sclerotinia sclerotiorum is functionally related to CREA fi'om Aspergillus nidulans but not to the Mig proteins from Saccharomyces cerevisiae. F EBS Lett. 453, 54-58. Walton, J.D. (1994). Deconstructing the cell wall. Plant Physiol. 104, 1113-1118. Wegener, S., Ransom RE, and Walton, J.D. (1999). A unique eukaryotic B-xylosidase gene from the phytopathogenic fungus Cochliobolus carbonum. Microbiology 145, 1089-1095. Zeilinger, S., Mach, R.L., Schindler, M., Herzog, P., and Kubicek, C.P. (1996). Different inducibility of expression of the two xylanase genes xyn] and xyn2 in Trichoderma reesei. J. Biol. Chem 271, 25624-25629. 97 Chapter Four Cloning and targeted mutation of the Cochliobolus carbonum a-L- arabinofuranosidase genes ARF1 and ARF2 98 ABSTRACT The filamentous fungus Cochliobolus carbonum produces many CWDEs that can depolymerize plant cell walls. A strain of C. carbonum carrying a snf] mutation has reduced virulence and does not express ARF1, a gene encoding ct-L-arabinofuranosidase. The ARF1 gene was isolated using degenerate PCR primers based on Aspergillus niger, Aspergillus sojae, Streptomyces coelicolor, and Streptomyces Iividans et-L- arabinofuranosidases. Its encoded protein of 325 amino acid residues and 35 kD shares 64 to 66% identity with the above named arabinofuranosidase proteins. An internal fragment of ARF] was used to create arj] mutants by transformation-mediated gene deletion. Mutant strains were analyzed for growth, arabinofuranosidase activity and virulence. Residual arabinofuranosidase activity in the arf] mutant remained high, but one of the peaks of arabinofuranosidase activity, separated by cation-exchange HPLC, disappeared. A second arabinofuranosidase gene, ARFZ, encoding a protein of 503 amino acid residues was cloned from C. carbonum. It has 66 to 70% amino acid identity with a similar arabinofuranosidase present in Aspergillus niger, Emericella nidulans, Trichoderma reesei. The disruption of ARFZ in the or]! mutant, making an arfI/arfl double mutant led to the disappearance of the two major arabinofuranosidase activity peaks in culture filtrate fractionated by HPLC. The remaining activity was due to a bifunctional B-xylosidase/ct-L-arabinofiiranosidase (Ransom and Walton, 1997; Wegener et al., 1999). Grth of the arf] mutant was normal on xylan but somewhat reduced on xylose, arabinose or corn cell walls. Grth of the arfI/arfl double mutant was reduced 99 in the above sugars, but more strongly on arabinose. Both the arf] mutant and arfl/arfl double mutant had similar virulence on maize as the wild type C. carbonum. 100 INTRODUCTION The role of fungal-secreted extracellular enzymes in diseases caused by plant fungal pathogens has been postulated to include degradation of the plant cell walls to enable penetration into inter- and intracellular regions, release of nutrients that can be assimilated for growth, and triggering of plant defense responses (Walton, 1994). A number of such enzymes and their encoding genes have been isolated fiom plant pathogenic microorganisms and studied for their role in the disease process. Cochliobolus carbonum, an ascomycetous pathogen of maize, penetrates into and ramifies through intact leaves and in the process obtains nutrients for growth from the plant cell cytoplasm and walls. The disease caused by this organism, Northern leaf spot of corn, is characterized by extensive necrotization of susceptible maize tissues. The fungus synthesizes numerous extracellular enzymes that can degrade the polysaccharides of the maize cell wall (Walton, 1994). Several C. carbonum cell wall degrading enzyme genes have been cloned and characterized. C. carbonum mutants made by disruption or deletion of specific genes including polygalacturonase genes (Scott-Craig et al., 1990, 1998) and endo-l,4-B-xylanase genes (Apel et al., 1993; Apel-Birkhold et al., 1996) were unaltered in virulence on maize. A possible reason for the lack of a role of individual enzymes in the disease process is the redundancy of these enzymes, that is, the presence of multiple CWDEs with overlapping fianction. In graminaceous crops and grasses, arabinoxylan can account for up to 60 % of total cell wall carbohydrate (Carpita and Gibeaut 1993). It is composed of a B-1,4-linked D-xylopyranosyl backbone that is highly substituted, at the 0-2 and/or O-3 positions by various mono- or oligosacchaide side chains. The side chains consist largely of 101 arabinosyl, xylosyl, and/or glucuronic acid residues (Carpita and Gibeaut 1993). Arabinoxylan-degrading enzymes includes B-xylosidases, a-L-arabinofuranosidases and endo-B-1,4-D-xylanases (Sunna and Antranikian, 1997). The mRNA expression of XYL3 (an endo-l,4-B-xylanase gene) and ARF1 (an ct-L-arabinofuranosidase gene) cannot be detected in a C. carbonum ccsnfl mutant (Tonukari et al., 2000). Mutation of ccSNF1 also leads to down-regulation of other C. carbonum CWDE genes resulting in a decreased amount of enzyme production, poor growth on corn cell walls, and reduced virulence (Tonukari et al., 2000). Grth of the ccsnfl mutant is strongly impaired when D or L-arabinose is the sole carbon source (Tonukari et al., 2000). In order to determine whether XYL3 and ARF1 are required for virulence, it would be necessary to create a strain of the fungus that completely lacked these activities. However, Apel-Birkhold and Walton (1996) have previously shown by gene disruption that the XYL], XYLZ, and XYL3 genes are not required for pathogenicity in C. carbonum. The C. carbonum xyl], xylZ, and xyl3 mutants exhibits similar growth in culture and virulence on maize as the wild- type firngus. In addition to XYL], XYLZ, and XYL3, C. carbonum has at least one other endo-1,4-B-xylanase encoding gene, XYL4 (Apel et al., 1993; Apel-Birkhold and Walton, 1996; Scott-Craig et al., unpublished). Unlike xylanases, a-L-arabinofuranosidase has been implicated as a virulence factor in diseases caused by Sclerotinia fiuctigena (Howell, 1975) and Sclerotinia trifoliorum (Rehnstrom et al., 1994). Arabinofuranosidases are a class of enzymes that specifically release arabinose residues from xylan side chains (Sunna and Antranikian, 1997). These enzymes in combination with exo- and endoxylanases are important for the complete degradation of arabinoxylan to xylose and L-arabinose. Ransom and Walton 102 (1997) have described the isolation of a non-glycosylated a-L-arabinofirranosidase from C. carbonum with a molecular weight of 63 kD. In this research, we describe the isolation of the ccSnfl p-regulated C. carbonum ARF1, and a second arabinofhranosidase gene, ARF2. We also analyze the effects of their targeted mutation on growth, total arabinofuranosidase activity, and vimlence on maize. METHODS Fungal cultures, media and growth conditions The wild-type race 1 strain of C. carbonum (367-2A) was derived from strain SB] 11 (ATCC 90305) and maintained on V8 plates (Apel et al. 1993). The fungus was grown in liquid media on 1 % agar plates containing minimal salts, 0.2 % yeast extract, and trace elements (Van Hoof et al., 1991) with 1% D-xylose, D-arabinose, L-arabinose, oat spelt xylan (Fluka, Switzerland), or corn cell walls (Sposato et al., 1995) as the sole carbon source. For liquid culture, four fungal plugs (each 5 mmz) were inoculated into a l L Erlenmeyer flask containing 125 ml of medium and grown in still culture for 7 to 14 days at room temperature. For growth on the agar plates, 5 ul of a suspension of conidia (104/ml) in 0.1 % Tween 20 was pipetted onto the center of the plate. Mycelium for protoplasts preparation was obtained from germinating conidia (Apel et al., 1993). For routine enzyme production, cultures were grown on 0.8% corn cell walls plus 0.2% 811 CID SC . 103 Nucleic acid manipulations The C. carbonum genomic library has been previously described (Scott-Craig et al., 1990). DNA and total RNA were extracted fi'om lyophilized mats (Pitkin et al., 1996; Apel et al., 1993). The methods used for DNA and RNA electrophoresis, blotting, probe labeling and hybridization have also been described elsewhere (Apel-Birkhold et al., 1996). The Cochliobolus heterosiophus GPD] gene, encoding glyceraldehyde-3- phosphate dehydrogenase (Van Wert and Yoder, 1992), was used as a loading control. To isolate ARF1, PCR was performed in a thermocycler (MJ Research, Watertown, MA) using taq DNA polymerase (Life Technologies, Gaithersburg, MD) and two degenerate primers based on conserved amino acid sequence of a-L- arabinofuranosidase proteins from Aspergillus niger, Streptomyces coelicolor, and Streptomyces Iividans. Forward primer 5’ GGNGAYAAYGGIAARATITAYMG 3’ is based on the amino acid sequence “GDNGKIYR,” and reverse primer 5’ ATRTCRTYI GTCCANGTIGCNCC 3’ is based on the amino acid sequence "GATWTN/DDI" (where Y = T or C; R = G or A; I = inosine; N = A, T, G or C). The degenerate primers were used to amplify a 310-bp genomic fragment from C. carbonum DNA using the following PCR: initial denaturation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 45 sec; annealing at 50 °C for 30 sec; and polymerization at 72 °C for 1 min. This was followed by polymerization at 72 °C for 7 min. The PCR product was cloned into the pGEM T- easy vector (Promega). For ARF 2 isolation, degenerate primers were also made based on conserved amino acid sequence of arabinofuranosidases from Aspergillus niger, 104 Emericella nidulans, and Trichoderma reesei. Forward primer 5’ ARAARGCNTAYG GNGTNTT 3’ is based on the amino acid sequence “Q\KKAYGVF,” and reverse primer 5’ ADRTCNRCCATDATCCA 3’ is based on the amino acid sequence “WIMA/VDM/L” (where D = A, G or T). The degenerate primers were used to amplify a 264-bp genomic fragment from the C. carbonum cDNA library originally constructed for the CytoTrap yeast two-hybrid screen. A touchdown PCR (Don et al., 1991) with the C. carbonum cDNA library as template and the above primers was performed under the following conditions: initial denaturation at 94 °C for 3 min, 45 cycles of denaturation, 94 °C for 1 min; annealing for 1 min; and polymerization at 72 °C for 2 min. The annealing temperature ranged from 60 °C to 45 °C with a decrease of 1 0C every three cycles. This was followed by 10 cycles of denaturation at 94 °C for 1 min; annealing at 45 °C for at for 1 min; and polymerization at 72 °C for 2 min. The PCR product was cloned into pGEM T-easy vector (Promega). An ARF1 cDNA was amplified by PCR from a CytoTrap yeast two-hybrid cDNA library made from C. carbonum mRNAs. An ARF1 specific primer, 5’ TTCTTCGCC GGTGACAACGGC 3’ and the MYR 3’ primer, 5’CGTGAATGTAAGCGTGACAT, which is specific to the pMYR vector into which the cDNA library was made (GenBank Accession number: AF 102577; CytoTrap XR Library Construction Kit, Stratagene) were used in a PCR reaction (initial denaturation at 94 °C for 3 min, 35 cycles of denaturation; 94 °C for 1 min; annealing at 50 °C for l min; and polymerization at 72 °C for 2 min) to amplify an 800-bp ARF1 cDNA which was then cloned into pGEM T-easy vector. ARFZ cDNAs were amplified from the CytoTrap yeast two-hybrid cDNA library. A 1.1-kb ARFZ cDNA was amplified using an ARFZ specific primer, 5’ GGAACCGGGTACCGC 105 AATAAC 3’ and the vector pMYR primer. The PCR reaction conditions were initial denaturation at 94 0C for 3 min, 35 cycles of denaturation; 94 °C for l min; annealing at 50 °C for 1 min; and polymerization at 72 0C for 2 min. The ARFZ cDNA was cloned into pGEM T-easy vector. The transcriptional start sites of ARF1 and ARFZ were determined using the 5’ RACE (Rapid Amplification of cDNA Ends) system, version 2.0, following the instructions of the manufacturer (GIBCO-BRL) (Frohman et al. 1988). An oligonucleotide of sequence 5’ TAGCACCAGTGCTGCTGCCGG 3’ was used to prime first-strand cDNA synthesis, which was then amplified by PCR using a nested primer of the sequence 5’-TGGACA CCACCCCAGCTGTTG-3’. The initial primer for the ARFZ 5’ RACE reaction was GATGGCTCCCATGTGGCCGTT and PCR amplification primer was 5’ GTTGTTATTGCG GTACCCGGT 3’. DNA Nucleotide sequence was determined by automated fluorescence sequencing at the DOE-Plant Research Laboratory facility, Michigan State University. Sequence data were analyzed with the Lasergene sofiware (DNASTAR Inc., Madison, WI). Construction of gene replacement vector and transformation A gene replacement vector for ARF1 was constructed using a 3.8-kb Xhol genomic fi'agment cloned into pBluescript SK+/- (Stratagene). An internal 1.0-kb HindIII-EcoRI fi'agment was deleted, and replaced with a 1.4-kb Hpal fragment containing the hphl gene that confers hygromycin resistance (Sweigard et al., 1992) (Figure 23A and 238). A major portion of the coding region was thus replaced by hphl such that 0.8 to 1.5 kb of 106 colinear DNA remained on each side. The gene replacement fragment was released from vector sequences by using two restriction endonucleases, Smal and Apal to prevent recircularization prior to transformation of C. carbonum 367-2A protoplasts (Scott-Craig et al., 1990). Transformants were selected for their ability to grow in the presence of hygromycin B (Calbiochem, La Jolla, Calif). For ARF2 gene disruption, a 2.8-kb genomic fragment containing the ARFZ gene was cloned into pBluescript KS+/-, and a 1.35-kb Sall-HindIII region was deleted and replaced with a 3.0-kb StuI-HindIII sh-ble gene conferring phleomycin resistance (Figure 25A and 25B). A major portion of the coding region was thus replaced by the sh-ble gene such that 650-bp of ARFZ genomic DNA remained on one side. The sh-ble fragment was obtained from pUT 720 (CAYLA Toulouse, France). The ARFZ disruption vector was linearized at the KpnI/Spel site and used to transform protoplasts of C. carbonum arfl mutant (T708). Transformants were selected for their ability to grow in the presence of phleomycin. HPLC Seven-day old culture filtrates of C. carbonum grown in l L flasks containing 125 ml. of liquid medium with 0.2 % sucrose and 0.8 % corn cell walls were filtered through through four layers of cheesecloth and one layer of Whatman #1 filter paper. Five 107 1kb (A) Scal Hindlll EcoRI ' — D ARF1 mRNA (3) Seal d h hphl Figure 24. Construction of arfl Disruption Transformants. (A) Map of the ARF1 gene. (B) An internal 1.0-kb HindIII-EcoRI fragment of ARF1 was replaced with hphl. 108 (A) 1 kb BstXll Sall Hindlll Bgll ' ——I- ARF2 mRNA (3) H' II 8 ll BstX "10' 9| sh-ble Figure 25. Construction of arfl Disruption Transformants. (A) Map of the ARFZ gene. (B) A 1.35-kb Sail-Hindlll region was replaced with sh-ble. 109 hundred milliliters of culture filtrate was concentrated by rotary evaporation to about 50 mL and then dialyzed against 4 L of 25 mM sodium acetate, pH 5.0, for 24 h with two changes of buffer. The dialysate was then applied to a column of DEAR-cellulose to remove strongly anionic species and pigments. Material not binding to the DEAE- cellulose column was pooled and concentrated by ultrafiltration through an Amicon YM30 membrane to about 10 mL. This concentrated and clarified culture filtrate was then fractionated by cation-exchange high-performance liquid chromatography (HPLC) on a sulfoethylaspartamide cation-exchange HPLC column (The Nest Group, Southboro, Mass.) with a linear gradient of 25 mM sodium acetate (pH 5) to 25 mM sodium acetate (pH 5) plus 0.4 M KCl over 30 min. The flow rate was 1 ml/min, and l-ml fractions were collected and assayed for a-L-arabinofiiranosidase activity. Enzyme assays a-L-arabinofiiranosidase, B-glucosidase and B-xylosidase activities were determined by production of p-nitrophenol from p-nitrophenol-a-L-arabinofuranoside, p-nitrophenol-B- D-glucopyranoside and p-nitrophenol-B-D-xylopyranoside (Sigma), respectively. A mixture of 100 pl of enzyme, 200 pl of 50 mM sodium acetate, pH 5, and 100 pl 10 mM substrate was incubated for 30 min at 37°C. Sodium carbonate (600 pl of 1 M) was added and the absorbance measured at 400 nm (Ransom and Walton, 1997). 110 Pathogenicity test Eighteen days old plants of susceptible inbred maize line Pr (genotype hm/hm) were inoculated with a suspension of conidia (104/ml) in 0.1% Tween 20. After inoculation in the afternoon, the plants were covered in plastic bags overnight (approximately 12-l6h) and then grown in a greenhouse. The plants were observed daily for about seven days or until they died. RESULTS Cloning of ARF1 and ARFZ Two degenerate oligonucleotides were synthesized based on the conserved regions of a- L-arabinofuranosidases fi'om other fungi (Gielkens et al., 1997; Redenbach et al., 1996; Vincent et al., 1997) and a 310-bp fragment of ARF1 was amplified by PCR, using C. carbonum genomic DNA as template. The PCR product was used as a probe to obtain the genomic copy of the encoding gene, ARF1, which was sequenced on both strands (Figure 26). The ARF1 cDNA was amplified by PCR from a cDNA library. The transcriptional start site of ARF1 was determined by 5'-RACE. The ARF1 transcript has a 64-bp 5' untranslated region, and 161-bp 3' untranslated region. The DNA sequence of ARF1 contains an open reading flame of 978 bases predicting a protein of 325 amino acid residues with a molecular mass of 34.6 kDa. Comparison of the genomic and cDNA 111 CCGCCCGTAGGATTTCCTTCTTTATCTCTTAGGAGCTGCTCTCTTAGTGTTCGGGGGGAA ATGGCGGTTCCACTCCGTATTCTGGGCGCCCAGCACCACACCTACTTTACACTAGCTTTG CGATGTTCAAAAGCCGACGTGGAGAAAAACCCCGGATAGAGGAGAATCCACCTGCACCAC CCCACCCACCCAACCTCGCAGGAACGGGCATTGGCGGGCGAACTCCGGAAGATTTGTAAG TAGTGTTCTATCGGTGTAATAGTGGAGATGTGCCAAGCGGTGTGGGAAAAAGGTTCAAAG GTTGAATATTTGATGGATTGTTCAGAGATTGATATGGGCACTTTTTTTGAACCAGGGGCG CAATCGGCAAAAGTTACTTTTTTACGTACTGTTTAAATGTTGGTTGGGTGGTAAACAATT TGCTCCTTGGGAGACGAAAGCCGGTGTGCAAAGTAGCATTTTTTGAGCATCTTTTGTCCA GCATGCTCCTTACTCTAGACATGGGCTTGGTAGGTGGGCCGAGTTACATTGGCAACGCTC ACAACCGCCTTGGCAAGGCTAAGTACTGCGTACAATCCAAGCGCTGTGGTTACCGCTACT CTTTAATCTGTGCGGCCGAACAAATTCCGCGAGTCGATCTAGAAGCTGACCCTCCTCCCA CGTGTTTGCTGGCCACTGACAGTGAATGTTGTTTAGTGGAGTTGAGAGTGCTTAGTGGCA AAAAAGTGCCCTTGCTTATGGCCGGAGAGTGTTGCGTCAGTTATAGTTTTCATAACTGCA GATGCGCGTCCTTTGGGCGGCTCCTTACAGCGGCTCCCTGCTGTAGCACGATAATTGTGG TTTTGCAGTTTTGCGTGATTAAGGCGCAGAGCACAGTTGACGTCGGTGTAGGGACAATTT CTCGCGCCCTAGCTGTCCATGCGGCATCAATCAAGCAATGCCAAACCTCAAACCCGCTAA GTCCAAGTCTTGACTTGTGGGCGCAGCGGAGGCAGCCCTTCGACTGGTGGCACTTTTGCG CCACTGGCACCCATGCTTCTAGATTCCGGCCACCGGGTGTTTCATCGCGTGTTGACGTTT CGCTCTAGTGGAGAGACTAGCATCGGATAGGAACACTCGCGGATTCTAGTAAACACCCGG TGGGACTTTTTGGAGCAATACCGCCGAGATTGGGGCCTGGCAATCAGCGCAGCAGGCCAT GTTTCTTTGTTCGGCGCAAGAGACGCCAACAGGACTGCTTGTTAGCGCAGTGTTGGTACG TAGTCGGCCGGTCATACATCAGACGAATTGACGTCGACGTCAAAACATAGTCGCCAAGCT AGCGGGTTAGGCGAATGGGGTGCAGCACATACCTGCATTAGGATTTAAGCATGCATATAG TAGTAGATTAAGGAATTGCCCAGCCTCACTTGTTTCTGCAGTCCTACGTGCCCTTTGCCG TGCGTTCCCCGCATTACGGAGAATTGCCGTCCGTGCAGTCGCGTCAATTCCCTTGAAACA TCATGTAATTTAATATACCCACAACAACGGAACAATATGCCATACCGAGAGTAGACGCAT GATGTGGATGATCAATTTCCACGCTCAGGAGATCTAGGTTCCCGAGCCCAGTCCTGAATC GGCCAACCCTCAAGCGGTACGTGGGCAAGGCGTGATACACGGCATGCCTTCTGATTCGTA GGCCTAGACAGCATCGGCTCCAGGTATAAGTATCTTATACATGCCCTCGTTCTAATAGTC ATCCAGCTCACCAGCATCACATTCACTCATTCAACCAACCAACTACCAAACACCCCAAAA A AACCAAACATGCGTTTCGTTCCCGACCTTAGCTTCTCGGCTGCTGCCGTCGCCCTTCTGG M R F V P D L 3 P S A A A V A L L CTTCCACTGCTTCGGCCCAGAGCTGCAAGCTTCCCACCTCTTACAAGTGGACCTCCTCCG A 8 T A 8 A Q 8 C K L P T 8 Y K W T 8 S GCGCTCTTGCCCAGCCCAAGTCTGGATGGGCCAACTTGAAGGATTTCACCATCTCCAGCC G .A L A Q P K 8 G W A N L K D F T I 8 8 TTAACGGCAAGCACATTGTCTATGCTACCGACCACGACACTGGATCCAAGTACGGATCCA L N G K H I V Y A T D H D T G S K Y G S TGGCTTTCAGCCCCTTCGGTAGCTTCAGCGAGATGGCCTCTGCTTCCCAGACCGCCACCC M A P 8 P F G 8 P 8 E M .A S A 8 Q T A T CCTTCACTGCTGTTGCCCCTACCCTCTTCCGTTTCGCTCCCAAGAACATTTGGGTCTTGG P F T A. V A P T L F R F A P K N I W V L Sequence continued next page. Figure 26. Nucleotide and deduced amino acid sequence of C. carbonum ARF1 gene. The open reading frame encodes a protein of 325 amino acids; " transcription start site; * stop codon; # polyadenylation site. 112 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 17 1920 37 1980 57 2040 77 2100 97 2160 117 Sequence continued from previous page. CCTACCAGTGGGGACCTACCACCTTCTCCTACAGGACCTCCAGCGACCCCACCAACCCCA A Y Q W’ G P T T F S Y R T S 8 D P T N P ACAGCTGGGGTGGTGTCCAGACTCTCTTCTCTGGCAAGATCTCCGGCAGCAGCACTGGTG N S W G G V Q T L F 8 G K I 8 G 8 8 T G CTATTGACCAGACTGTCATTGGTGACGCCATCAACATGTACCTCTTCTTCGCCGGTGACA A I D Q T V I G D A I N M Y L F F A G D ACGGCAAGATCTACCGTAGCAGCATGCCCAAGGCCAACTTCCCTGGCAGCTTCGGCACTG N G R I Y R 8 S M P K A N P P G 8 F G T CTTCCACCGTCATCATGAGCGACAGCACCAACAACCTCTTCGAGGCTGTTCAGGTCTACA A S T V I M S D S T N N L F E A V Q V Y CCGTCAAGGGTGGCGGTTACCTCATGATCGTTGAGGCTGTTGGCTCTGGCGGCCGTTACT T 'V K G G G Y L M I V E A. V' G 8 G G R Y TCCGCTCTTTCACTGCCTCCAGCCTCAGCGGTAGCTGGACCCCCAACGCTGCCACCGAGA F R. 8 P T A 3 8 L 8 G 8 W T P N A. A T E GCAACCCCTTCGCCGGCAAGGCCAACAGCGGTGCCACCTGGACCAACGACATCTCCCACG S N P P A. G R A. N S G A T W T N D I S H GAGATCTTGTCAAGGTCACCAACGACGAGACCATGACCGTCGACCCTTGCAACCTGCAGC G D L V K V T N D E T M T V D P C N L Q TGTTGTACCAGGGCCGTGCCCCCAACTCTGGCGGCGACTACGACCGTCTCCCCTACAGGC L L Y Q G R A P N S G G D Y D R L P Y R CCGGTCTCCTTACCCTCAAGAAGTAGAGCCTTCTTTGCTCGGCTCCTGGCGAGGTACCTT P G L L T L K K * GATTGTAAATGAATTTTTGTGTGGGAGATTGTATATAGCTCAGTTTGTGGTGGAATTCGT ATATATATTATTTTGGGCCACTGGCTTTGGCTCTTTGGCATTGCGCGAGGCATCAATTGA TTCTTCAACCTATCTTTGAAAATCCATCTATCTTACCTTTTGAGTGACTTCTTGTGAAAC O ATGACTCGATGGCTTGCCTGCCTATTGCAACTCCTTGGAGAAAATCCGCCGACGCTGCTT ATGCTGGTATATCTCGCTCCTACACACTTCTAGATTCTTCCTCTCTCTAATCGCAATCTC CTGCAAGATGGCCATGCTCTTGGCAGATAACAACGGGACCGCCCTTTGCACTGGGTCCAG GCCTCGTGCAGTTCCATAAACGCCCAAGCCATACCAACAAGGGCATTCCGGTGCACTAGC CACCGCATACTCGTCGCAACCATTCATTCCGCAAAAGCAGCTGACTCGGGCTCGCCCAGC GACACTGCCTCGCGCAAATTACACGGCTGCTGGTAGCCTGCATTAAGACACAACTCTAGG CCAAGTTCCACGATGCCAACCGCCTCGCCATCATCAG 113 2220 137 2280 157 2340 177 2400 197 2460 217 2520 237 2580 257 2640 277 2700 297 2760 317 2820 325 2880 2940 3000 3060 3120 3180 3240 3300 3360 3397 sequences revealed that the ARF1 gene has no introns. The product of ARF1 (called Arflp) has a predicted signal peptide cleavage site between amino acids 23 and 24 (Nielson et al., 1997). Its deduced amino acid sequence has about 64 to 66% identity with A. niger, Aspergillus sojae, S. coelicolor, and S. lividans a-L-arabinofiJranosidases (Figure 27). A 264-bp ARF2 fragment was also amplified by PCR using degenerate oligonucleotides that were synthesized based on the conserved regions of arabinofuranosidases from Aspergillus niger (F lipphi et al., 1993), Emericella nidulans (Gielkens et al., 1999), and Trichoderma reesei (Margolles-Clark et al., 1996). The PCR product was used as a probe to obtain the genomic copy of ARF2, which was sequenced on both strands (Figure 28). The ARF2 cDNA was also amplified from a cDNA library, and the transcriptional start determined by S‘-RACE. The ARF2 DNA sequence encodes a predicted protein (Arf2p) of 503 amino acid residues with a molecular mass of 52.2 kDa. ARF2 gene has one 59-bp intron. Arf2p also has a predicted signal peptide of 21 amino acids residues. Arf2p has 66 to 70% amino acid identity with A. niger, E. nidulans, 71 reesei arabinofuranosidases (Figure 29). Hybridization of ARF1 or ARF2 probe to C. carbonum genomic DNA digested with different restriction enzymes (Hindlll, PstI, Sail, or XhoI) resulted in a single band in each case (Figure 30), indicating the existence of a single copy of C. carbonum ARF1 and ARF2. 114 Figure 27. Comparison of Deduced Amino Acid Sequences of C. carbonum ARF1 and or-L- 9???? 9???? 9???? 9???? 9???? 9???? 9???? 9???? coalicolor lividans nignr saga. caxbonum coolicolor lividnns nigor soga- carbonum coolicolor lividans nigar saga. carbonum coolicolor lividans nigor saga. carbonum coolicolor lividans nigur eagle cazbonum coclicolor lividnns niguz sagas carbonum coolicolor lividans nigh: soga- carbonum coolicolor lividans nigh: saga. carbonum MHRGSLSRGHTSAVLAAVVAALAALAALLVATTPAQAAGSGALRGAGSNRCLDVLGGSQD MHRGSL8RGQHVRGTRRRGAALAALAALLVATAPAQAAGSGALRGAGSNRCLDVLGGSQD 181 181 33 33 31 240 240 93 93 91 300 300 153 153 151 360 360 213 213 211 416 416 273 269 266 TY 'STEALAaPKs KDFTN GKHIVYASTTDTOG yes 31! STGALAEPKA - ALKD FTI SYKWTSSGALAQPKSG - . TYRWSSTG‘LAQPKSES‘A ‘ TYRW§STGHLAQPKSGWHALKDFT I GRHgVYGSTSSGS 5'3. S GKHIVYATI SAiQN- HQAAVAPTLFYFAPKNIWVLAYQWGS ‘FIYRTSSDPTDPNGWS SAgoN QAAVAPTLFYFAPIQIIWVLAYQWGSm ‘IYRTSSDPTDPNGWS QPLFTGS sns.-. srs-VAPILFYFEPKEIWVLAYQWGSermYRTsEDPwuigcwssaQfiLFTg. SASQN'ISQGu APTLFYFAPKDXWILAYQW P SFSYKTSSDPTD‘ GWSA-QPLFSG‘ SASQ-‘ p ‘VAPTLFWFAPKNIWVLAYQW - TFSYRTSSDPTNP swsg7o' LFSG' ISGSDT"IDQTLIAD t YLFFAGDNGKIYRASMPIGNFPGbFGS‘YTHIMSDTK‘ L ISGSDT 'IDQTLIAD Q LFFAGDNGKIYRASMPIGNFPG«FGSEYT IMSDTKRNL ISGSET ‘IDQTVIGDwfi LFFAGDNGKIYRQSMQII.FPGEFGSeYI ILS - L ISESDTSNIDQTVIGDmTwMYLFFAGDNGKIYRASMPIm FPGIFG . ILSDTK| L - - T IMSDS L ISGSST IDQTVIGD‘II LFFAGDNGKIYRSSMPH‘ FPGEFG SGATWTNDISHGDLVRW SGATWTNDISHGDLVRI PDQTMTVDPCNLQ PDQTMTVDPCNLQE I SGATWTDDISHGDLVRI PDQTMTVDPCNLQLLYQG HEN??? OPN GD LYQG"PNAGG§Y arabinofuranosidases of A. niger, A. sogae, S. coelicolor, and S. lividans. Residues that are identical in at least three sequences are indicated by black shading while conserved substitutions are in gray shading. 115 AGAGGAGCACAGAGGACTTGGGTTTGTCTTTTTTCTTCATATCCAAAAAAAACCATGTCT ‘ M 8 TCAAAATCGCTTCTCGTTGCGCTTGGGCTTGTAGCCACAGGTTCATTGGTGCATGCAGGG s R 8 L L V’ A L G L V A T G s L V H .A G CCTTGTGACATTTACGCCAAAGGCAACACACCATGCATTGCTGCACATGCCACCACTCGT P C D I Y A R G N T P C I A A H A T T R GCTCTGTATAATTCATACAGTGGCCCGCTCTACCAAGTCAAGCGTGGCTCAGACGGTGCC A. L Y N S Y 8 G P L Y Q V K R G 8 D G A ACAACTGACATTGCTCCACTGTCCGCCGGCGGTGTCGCCAATGCAGCTGCTCAAGACAAG T T D I A P L s A G G V’ A. N A A A Q D K TTCTGTGCCAACACGACATGTCTCATTTCCATCATCTACGACCAATCTGGCAAAGGAAAC r C A. N T T C L I 8 I I Y D Q S G R G N CACCTCACACAAGCACCACCAGGCGCTTTCAAGGGCCCAGACGTCGGCGGTTACGACAAT H L T Q A. P P G A F K G P D V G G Y D N CTGGCAAGTGCTATCGGTGCACCTGTTTCGCTAGGCGGCAAAAAGGCATATGGCGTCTTC L A. 8 A. I G A. P V S L G G R R A Y G V r ATCTCCCCTGGAACCGGGTACCGCAATAACAACGTAAAAGGCTCGGCCGTAAAAGACGAG I S P G T G Y R N N N V R G S A V K D E CCCCAAGGCATATACGCCGTCTTGGACGGTACACATTACAATGGCGGCTGCTGCTTCGAC P Q G I Y A V L D G T H Y N G G C C F D TATGGCAATGCTGAGACAAACAACCTTGACACTthaagtatatcacctaccatcaattt Y G N A E T N N L D T ttgatataagccaattgaaaacacaaaaaacagGCAACGGCCACATGGAAGCCATCTACT G N G H M E A. I Y TTGGTGACAACACCGTGTGGGGCAGCGGCGCTGGCAACGGCCCCTGGGTCATGGCTGACC P G D N T V W G s G A G N G P W V M: A. D TAGAAAACGGCCTCTTCTCCGGTGCAAACCCAAAGCAGAACACTCAAAACCCATCCGTCT L E N G L F 8 G A. N P R Q N T Q N P 8 V CAAACCGCTTCCTAACGACTGTTGTCAAGGGCAAGCCTGGCGTCTGGGCCATCCGCGCCG 8 N R r L T T V V R G R P G V W A I R A GAGACGCCACCACAGGCGGACTCTCAACATACTACAACGGCTCGCGCCCAAGTGTGTCGG G D A. T T G G L S T Y Y N G 8 R P 8 V s GCTACAACCCCATGAGCAAAGAAGGCGCCATTATCCTCGGTATCGGCGGCGACAACAGCA G Y N P M s K E G A I I L G I G G D N 8 ACGGCGCACAGGGAACCTTCACCGAGGGCGCAATGACGTTCGGGTATCCCAGCGACGCAA N G A. Q G T F T R G A. M T r G Y P S D A TCGAAAACGAAGTCCAGGCCAACCTTGTTGCGGCTGGATACTCCACTGGACGCGGATTGA I E N E V Q A N L V’ A. A G Y 8 T G R G L TGACTAGCGGACCCGCATACACTGTTGGATCCAGTGTATCTCTCAGGGCCACGACATCTG M T 8 G P A Y T V G 8 s V S L R A T T 8 Sequence continued next page. 60 120 22 180 42 240 62 300 82 360 102 420 122 480 142 540 162 600 182 660 193 720 202 780 222 840 242 900 262 960 282 1020 302 1080 322 1140 342 1200 362 Figure 28. Nucleotide and deduced amino acid sequence of C. carbonum ARF 2. The open reading frame encodes a protein of 503 amino acids; " transcription start site; * stop codon; # polyadenylation site. An introns of 59 bp is typed in italic lowercase. 116 Sequence continued from previous page. GCTACACGGACCGCTATCTCGCGCACTCCGGAGCGACAGTGAACACCCAAGTCGTATCGT G Y T D R Y L A H 8 G A T V N T Q V' V S CGTCGAGCACTGCGCTGCTCAAGCGCCAGGCGAGCTGGATTGTCCGCGCTGGCTTCACCA 8 8 8 T A L L K R Q A 8 W I V R A G P T ACAGCGAGTGCTTTGCGTTCGAATCAAAGGATACGGCTGGAAGCTTTCTCCGCCACGCAA N S I C P A r E 8 K D T A G 8 P L R R A ATTTCGTGCTGCAGGTCAATGCTAATGATGGATCCAAGGGGTTCAAGGAAGATGCGACAT N F V' L Q V' N A. N D G 3 K G P K E D A T TTTGTCCTCAGGCGGGTCTTACGGGTAAGGGTAGCTCGATTAGAACTTGGGCGTACCCGA P C P Q A G L T G R G 8 8 I R T W A Y P CGAGGTGGATTCGCCATTTTAACAATGTTGGGTATATTTCGAGCAATGGTGGTGTCAAGG T R W’ I R H P N N V G Y I 8 S N G G V K ACTTTGATAATGTCAGCTCGTTCAACGACGATATCACGTGGCTTGTTGAGAGTGCCCTGG D P D N V' 8 S I N D D I T W L V' E S A L CTTGAGGGGACCGGGTAGTATGTGGTGGAATTTTATGTAAGTATGAATGCTTCCGACGTA A * TATAAGTTGGTTTTGTCAGAATATGTTGATGAATGGTATATTCCTATGTATATATGGACG 4 GGATTGCCTCTACTATGTAAGCTGCATTTGTTAGCAAATATAAAGATACCAAAGTACTCT AGTAAACTTCTGTTAAATTTCCTCACTTACCTTGAATTTTGTAATTCATGATCGGAAGCG GGCATTGTATCATATCGCATTTGAACACGAGAAATCAACATCCAGTTCACACACACACTG CATATGAACATTGAACAAGTTTTGTTTAAGCTACTGGTACAAGTTATAGTACTGGCTATC AAGACTGCTTACAAAAAAGACCCTGACATCGCCTAGTCTATGGCTAAATCTAGGGGATAT ACACATCAACAGGGCTGGGAATAACAGAAAGAAGAAGATCCTTTTAGTCAATAAGCAGAC TGTTCAGGCGAGCCTCAGTGGCAATAACGTCCAAGGCCTGCTTGACCTCGAGGGGGGGCC CGGCACC 117 1260 382 1320 402 1380 422 1440 442 1500 462 1560 482 1620 502 1680 503 1740 1800 1860 1920 1980 2040 2100 2160 2167 9??? 9??? 9??? 9??? 99?? 99?? 99?? 99?? 99?? niger nidulans reesei carbonum nigh: nidulans rues-i carbonum nignr nidulans reesei carbonum nigar nidulans rooaci carbonum nigar nidulans reesei carbonum niger nidulans recs-i carbonum nigur nidulans reesei carbonum niger nidulans roosoi cazbonum niger nidulans roosoi carbonum 115 121 117 118 175 181 177 178 235 241 237 238 294 301 296 297 352 359 354 357 408 419 410 413 467 479 469 472 'GDTPCVAAHSTTRALYSSFS ‘LYQ _*_ALATGSLVAAGPCDIYSSGGTPCIAAHSTTRALYSSYNGPLYQ IAEGSLVAAGPCDIYSSGGIPCVAAHSTIRALFSAYQGPLYQ ~- m7 ATGSLVFAGRCDIY-«GNIRCIAAHATIRALY:SYSGPLYQ ' I QDQRCANIICLIIIIIDQSGNGNRLIQAPPGGR-Gpr TTTITPLSAGGV'w-S-QDAFCENTTCLITIIYDQSGNGNBLTQAPPGGF’GPD TTnISPLSBGEVAflAfiAQDAFCAgTTCLITIIYDQSGSGNHLTQAPPGGFgGPES ‘TT$ IAPLSAGGv-«- AQDI 1GNHLTQAPPGAFHGPD ‘FCANTTCLISIIYDQSG uGYDNLASAIGARVILNGqKAYGVFMspGIGYRNNEAIGIAIGDEpEGNYAVLDGIRyN- GGYDNLAEAIGAPVTLNGrKAYGVFVSPGTGYRNNEAEGTATGDEPEGMYAVLDGTHYNI GYDNLASAIGAPVILN 7KAYGVFVSPGIGYRNNHASGI" ”:GDMBGMYAVLDGIHY GGYDNLASAIGAPVSLEO‘KAYGVFISPGTGYRNNh - «DEPQGIYAVLDGIHY N -GYGAGNGPWIMADLENGLFSGESSDYN‘ . sfioIGNGHMEAIYFGD va 'GSGEGPWIMADLENGLFSGSSPGFN- LDTGNGHMEAIYFGDW SGYNPMSKEGAIILG ‘SGYNPMSKEGAIIL GARP-O ‘SGYNPMSKEGAIIL GDpsIsIRFVIAELKGGPNLWALRGGNAASGsLSIIY GDpsIsYRFVIAAIKG7PN7WAIRGGNAASGSLSIFYé IGGDNSNGAQGTFYE IGGDNSEAQGIFYE IGGDNSNGAQGTFYE 7 IGGDNSNGAQGTF- E - L a LiaTTSGYE aIRIIAHIGSIINIvissssssILKQQASWIvR: LR IIAEIIRYIAHSGSIVNIQWSSssnIHLKQQASWIVR LRHIISGYI7RYLAHSGATVNIQWSSssrl‘aLfiQAs I RTPGSYIRH FF DTPGSYIRHSNFALLLNANDGTKLEEEDATFCPQW GSYIRHSNFGLVLNANDGSKL DATFCEQAGINGOGS ”shes Figure 29. Comparison of Deduced Amino Acid Sequences of C. carbonum ARF 2 and or-L-arabinofuranosidases of A. niger, E. nidulans, and T. reesei. Residues that are identical in at least two sequences are indicated by black shading while conserved substitutions are in gray shading. 118 Figure 30. Copy number ofARFI (A) and ARF2 (B) in C. carbonum Southern blot analysis of total genomic DNA from C. carbonum digested with Hindlll, PstI, SaII, or Xhol. 119 Targeted Disruption of ARF1 and ARF2 Five hygromycin-resistant transformants were isolated and these were purified to nuclear homogeneity by two rounds of single-spore isolation on V8-hyg plates. All were determined by DNA gel blot analysis to have undergone gene replacement. Genomic DNA from both the mutants and the wild type were digested with SacI and probed with the deleted 1.0-kb HindHI-EcoRI ARF1 fragment. All five transformants showed no hybridization whereas there is a 2.5-kb band seen in the wild type lane (Figure 31A). To confirm the integration of the hygromycin resistance gene, a similar blot was probed with a fiagment of hphl. Only the five replacement mutants exhibited hybridization. As control, a SNF] genomic fragment was used as probe, and hybridization was observed with the wild type and the five transformants. ARF2 was disrupted in the arfl mutant in order to create a double mutant. Three phleomycin-resistant transformants were isolated. Genomic DNA from both the transformants and the wild type were digested with PstI and probed with an ARF2 fragment. All three transformants and arfl mutant showed a 1.4-kb hybridization, while two transformants have an additional 8.4 kb band. The third transformant also has an additional band, but of size 8.2 kb (Figure 31B) indicating that the resistance gene, sh- ble, integrated into the ARF 2 gene in one position for the first two transformants, and in a different position for the third transformant. To confirm the integration of the phleomycin resistance gene, a similar blot was probed with a fragment of sh-ble. Only the three transformants exhibited hybridization. As control, a SNF] genomic fragment was used as probe, and hybridization was observed with the wild type and the three transformants. 120 arfl transformants A W112 345 ‘ rr':_m- ._ _ khl 3v --_—_-._ .4. $143 . ,. 1 “: .,."-’ If ..= _' \' 1. -\' .11., .. 3 \‘~ '7. . l.» ".. I. ‘1 > ‘1‘.’ - . (5', .-. . 1" A-yv '. ‘3 ‘. arfl/arfl transformants Figure 31. Screening of arfl and arfl? Disruption Transformants. (A) DNA blotting of wild type (WT) and five arfl disruptant transformants. DNA was digested with SacI. Similar blots were probed with ARF1, hphl, or SNF] as a control. (B) The ARF2 gene was disrupted in the arfl mutant to obtain an arfl/arfl double mutantDNA from arfl mutant (am) and three arfl/arfl disruptant transformants was digested with PstI. Similar blots were probed with ARF2, sh-ble, or SNF] as a control. 121 Effect of the ARF1 and ARF2 mutations on enzyme activities In preliminary experiments, all five arfl mutant strains displayed similar phenotypes with regard to grth and enzymatic activities (data not shown) and the arfl mutant T708 (arbitrarily chosen) was used for all further experiments. Strains 367-2A (wild type) and T708 (arfl) were grown in liquid still culture with purified maize cell walls as the sole carbon source. Afier seven days of growth, a-L-arabinofiiranosidase activity was measured. Activity in the culture filtrate of the arfl mutant decreased by 3.6 i 1.76 % (Figure 32). The arfl mutant of C. carbonum had residual arabinofiJranosidase activity indicating the presence of one or more additional a-L-arabinofiiranosidases. After concentration by rotary evaporation, dialysis, and passage through an anion-exchange column to remove acidic proteins and pigments, culture filtrates of the C. carbonum arfl mutant were analyzed by cation-exchange HPLC. Two peaks of arabinofuranosidase activity were observed (Figure 33B). In the wild type C. carbonum strain, three major peaks were present (Figure 33A). From this result, it can be inferred that the ARF1 gene encodes the third arabinofuranosidase peak eluting at 29 to 31 min (Figure 33A). Each of the peaks were further analyzed to determine whether they have other activities. Fractions 24, 27 and 30 were assayed for arabinofuranosidase, B-D-xylopyranosidase and B-D- glucopyranosidase activities. While fractions 24 and 30 exhibit predominantly arabinofuranosidase activity, fraction 27 had significant B-D-xylopyranosidase activity in addition to a lesser amount of a-L-arabinofirranosidase activity. Fraction 24 had a small 122 or-L-arabinofuranosidase 0.20 - 0.15 - a 34 .5 010 1 '33 8 0.05 — 0.00 - , 1 Wild type arf1 mutant arf1/arf2 double mutant Figure 32. Effect of ARF1 and ARF2 mutations on a-L-arabinofiiranosidase activity. Arabinofuranosidase activity of C. carbonum wild type, arfl mutant and arfl/arfl double mutant after growth for 7 days in liquid cultures containing 2 % corn cell walls. 123 Arabinofuranosidase activity (U) 0.75 0.50 0.25 0.00 0.75 0.50 0.25 0.00 0.75 0.50 0.25 0.00 _ A. Wild type III II 1 10 19 28 37 B. arfl mutant 1 10 19 28 37 C. arfl/aer double mutant 11 WW 1 10 19 28 37 Retention time (min) Figure 33. Cation-exchange HPLC analysis of or-L-arabinofuranosidase activity of wild-type, arfl mutant and arfl/arfl double mutant. Culture filtrates were harvested after 7 days of growth on 0.8% corn cell walls plus 0.2% sucrose as the carbon source. Equal amounts of protein were loaded onto the HPLC column in each case. 124 amount of B-D-xylopyranosidase activity, and B-D-glucopyranosidase activity was barely detectable in all three fractions (Figure 34). Culture filtrates of the three C. carbonum arfl/arfl transformants were also analyzed by cation-exchange HPLC. Two transformants, 1 and 2 (Figure 31B), have similar HPLC profiles as the arfl mutant (not shown) indicating that the sh-ble resistance gene integrated into the C. carbonum genome ectopically. In the culture filtrate of the third transformant, the first (major) arabinofuranosidase peak in addition to the third peak disappeared (Figure 33C). From this result, it can be inferred that the ARF 2 gene encodes the major arabinofuranosidase peak eluting at 20 to 25 min (Figure 33A). Activity in the culture filtrate of the arfl/arfl double mutant decreased by 65.7 i 2.22 % (Figure 32). Effect of the ARF1 and ARF2 mutations on growth To assess if the reduced or-L-arabinofiiranosidase activity secreted by the arfl mutant and arfl/arfl double mutant had any effect on grth on cell wall components including xylose, D-arabinose, L—arabinose, xylan, or corn cell walls, the mutants and wild type were grown on agar media supplemented with the above named compounds as the sole carbon source. On D-arabinose medium, the arfl mutant has about 37% decrease in growth, but the difference is smaller (0 to 13%) than on the other media (Figure 35). Grth of the arfl/arfl double mutant was reduced 78% in the D-arabinose medium, while reduction in the other media ranged from 39 to 56%. 125 I or-L-Arabinofiiranosidase I B-Xylosidase 0'8 " El B-Glucosidase Enzyme activity (U) Fraction 24 Fraction 27 Fraction 30 Peak I Peak II Peak III Figure 34. or-L-Arabinofuranosidase, B-xylosidase and B-glucosidase activities in the three peaks of the wild-type culture filtrate. Fractions 24, 27 and 30 were from culture filtrates of wild type fractionated by cation-exchange HPLC. 126 I Wild type I arfl mutant CI arfl/arfl double mutant 30 ...... ....... Growth (cmz) N O ..... ....' ...,.~ -—- . , H. ‘-'-II | ~ . .. . H... . 01-.- 'h~ v- --...1. Iv‘ll’l' -.... “u... I ....... v o ...... u ..... . . . .. ....... a . . .1. . . ... » - .. . .- .. . .11 .- . D-Ara L-Ara Xyn ch Figure 35. Grth of C. carbonum Wild type, arfl mutant, and arfl/arfl double mutant. Fungi were grown on agar in lS-cm Petri plates. Basal salts medium was supplemented with 1% (w/v) xylose (Xyl), D-arabinose (D-Ara), L-arabinose (L-Ara), noxylan (Xyn), or corn cell walls (ch) as sole carbon source. Growth was measured after 4 days. 127 Effect of the ARF1 and ARF2 mutations on pathogenicity Maize plants were infected with C. carbonum wild-type, arfl mutant and arfl/arfl double mutant. There was practically no difference between the plants inoculated with the wild type or the mutants (Figure 36). The wild type and the mutants were indistinguishable with regard to lesion size, color, and rate of lesion formation that they caused. Thus, ARF1 and/or ARF2 do not make a significant contribution to the virulence of C. carbonum. DISCUSSION The survival of a pathogens depends on its ability to have access to and utilize nutrients in the plant. The role played by the CWDEs is of interest because of their ability to degrade the cell wall polymers of host plants to provide materials that can be used for growth of the pathogen. The impetus behind this work was the observation that C. carbonum carrying a ccsnfl mutation has reduced virulence and does not express ARF1, a gene encoding arabinofuranosidase, suggesting that this enzyme may be important in virulence. This study has shown that C. carbonum has three or-L-arabinofuranosidase activities and two are encoded by ARF1 and ARF2. These enzymes can act together or substitute with each other in removing arabinose residues from side chains of cell wall xylans. Such redundancy in enzyme activities where two or more chromatographically 128 arf1 arf1larf2 Control mutant double mutant Figure 36. Pathogenicity Assay of arfl Mutant and arfl/arfl Double Mutant. Plants of genotype hm/hm (inbred Pr) were inoculated with wild type, arfl mutant, and arfl/arfl double mutant spores. 129 separable proteins have similar enzymatic activities is common in C. carbonum and other fungi (Yao and Koller, 1995; Walton 1994). C. carbonum also secretes at least four endo- 1,4-xy1anases (Apel-Birkhold et al., 1996; Scott-Craig et al., unpublished) and three proteases (Murphy and Walton, 1996). From the position of elution compared to the major arabinofuranosidase peak, the smallest peak eluting at 26 to 28 min (Figure 29) which was also shown to have B—D-xylopyranosidase activity, corresponds to the [3- xylosidase which has been previously described by Ransom and Walton (1997) and Wegener et al. (1999). Insofar as arabinofuranosidase activity might be important in virulence, the similar pathogenicity of the arfl mutant and arfl/arfl double mutant can be attributed to the presence of the bifunctional B-xylosidase/arabinofuranosidase. The main obstacle of this and some previous research on the contribution of individual CWDEs in pathogenicity is redundancy of the enzymes. The occurrence of multiple genes that encode fiJnctionally redundant enzymes that degrade the physical barriers in the host is a common feature of fiingal pathogens that infect plants. And this is probably why the single or sometimes multiple disruption mutants of C. carbonum can still grow and be pathogenic (Apel et al., 1993; Apel-Birkhold and Walton, 1996; Gorlach et al., 1998; Murphy and Walton, 1996; Scott-Craig et al., 1990, 1998; Wegener et al., 1999). The alternative requires the disruption of a regulatory gene that promotes the expression of several CWDE genes as demonstrated in our previous experiment in which the ccSNF1 gene was knocked out resulting in reduced C. carbonum virulence on maize (Tonukari et al., 2000). 130 REFERENCES Apel, P. C, Panaccione, D. G., Holden, F. R., and Walton, J. D. (1993). Cloning and targeted gene disruption of XYLI , a B-l,4-xylanase gene from the maize pathogen Cochliobolus carbonum. Mol. Plant-Microbe Interact. 6, 467-473. Apel-Birkhold, P. C., and Walton, J. D. (1996). Cloning, disruption, and expression of two endo-B-1,4-xylanase genes, XYLZ and XYL3, from Cochliobolus carbonum. Appl. Environ. Microbiol. 62, 4129-4135. Carpita, N.C., and Gibeaut, D.M. (1993). Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 3, 1-30. Celenza, J. L., and Carlson, M. (1984). Cloning and genetic mapping of SNF 1, a gene required for expression of glucose-repressible genes in Saccharomyces cerevisiae. Mol. Cell Biol. 4, 49-53. Flipphi, M.J., van Heuvel, M., van der Veen, P., Visser, J., and de Graaff, LB. (1993) Cloning and characterization of the abjB gene coding for the major alpha- L-arabinofuranosidase (ABF B) of Aspergillus niger. Curr. Genet. 24, 525-532. Frohman, M.A., Dush, M.K., and Martin G.R. (1988). Rapid production of full-length cDNAs from rare transcripts: amplification using a single-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998-9002. Gielkens, M., Gonzalez-Candelas, L., Sanchez-Torres, P., van de Vondervoort, P., de Graaff, L., Visser, J., and Ramon, D. (1999). The ast gene encoding the major alpha-L-arabinofuranosidase of Aspergillus nidulans: nucleotide sequence, regulation and construction of a disrupted strain. Microbiology 145, 735-741. Gielkens, M.M., Visser, J. and de Graaff, L.H. (1997). Arabinoxylan degradation by fungi: characterization of the arabinoxylan-arabinofuranohydrolase encoding genes from Aspergillus niger and Aspergillus tubingensis. Curr. Genet. 31, 22-29. Gorlach, J.M., Van Der Knaap, E., and Walton, J.D. (1998). Cloning and targeted disruption of MLGI, a gene encoding two of three extracellular mixed-linked glucanases of Cochliobolus carbonum. Appl. Environ. Microbiol. 64, 385-391. Howell, H.E. (1975). Correlation of virulence with secretion in vitro of three wall- degrading enzymes in isolates of Sclerotinia fiuctigena obtained after mutagen treatment. J. Gen. Microbiol. 90, 32-40. 131 Lever, M. (1972). A new reaction for colorimetric determination of carbohydrates. Anal. Biochem. 47, 273-279. ‘ Margolles-Clark, E., Tenkanen, M., Nakari-Setala, T., and Penttila, M. (1996). Cloning of genes encoding alpha-L-arabinofuranosidase and beta-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 62, 3840-3846. Murphy, J.M., and Walton, J.D. (1996). Three extracellular proteases from Cochliobolus carbonum: cloning and targeted disruption of ALP]. Mol. Plant- Microbe Interact. 9, 290-297. Nielsen, H., Engelbrecht, J., Brunak, S., and G. von Heijne. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1-6. Pitkin, J. P., Panaccione, D. G., and Walton, J. D. (1996). A putative cycle peptide efflux pump encoded by the T OXA gene of the plant pathogenic fungus, Cochliobolus carbonum. Microbiology 142, 1557—1565. Ransom, RE, and Walton, J.D. (1997). Purification and characterization of extracellular B-xylosidase and a-arabinosidase from the plant pathogenic fungus Cochliobolus carbonum. Carbohydr. Res. 297, 357-364. Redenbach, M., Kieser, H.M., Denapaite, D., Eichner, A., Cullum, J., Kinashi, H. and Hopwood, D.A. (1996). A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome. Mol. Microbiol. 2, 77-96. Rehnstrom, A.L., Free, S.J., and Pratt, RG. (1994). Isolation, characterization and pathogenicity of Sclerotinia trifoliorum arabinofiiranosidase-deficient mutants. Physiol. Mol. Plant Pathol. 44, 199-206. Scott-Craig, J. S., Panaccione, D. G., Cervone, F., and Walton, J. D. (1990). Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize. Plant Cell 2, 1191-1200. Scott-Craig, J.S., Cheng, Y.Q., Cervone, F., De Lorenzo, G., Pitkin, J.W., and Walton, J.D. (1998). Targeted mutants of Cochliobolus carbonum lacking the two major extracellular polygalacturonases. Appl. Environ. Microbiol. 64, 1497- 1503. Sposato, J. P., Ahn, J-H., and Walton, J. D. (1995). Characterization and disruption of a gene in the maize pathogen Cochliobolus carbonum encoding a cellulase binding domain and hinge region. Mol. Plant-Microbe Interact. 8, 602-609. 132 Sunna, A., and Antranikian, G. (1997). Xylanolytic enzymes from fungi and bacteria. Crit. Rev. Biotechnol. 17, 39-67. Sweigard, J. A., Chumley, F. G., and Valent, B. (1992). Disruption of a Magnaporthe grisea cutinase gene. Mol. Gen. Genet. 232, 183-190. Tonukari, N.J., Scott-Craig, J.S. and Walton, J.D. (2000) The Cochliobolus carbonum SNF] gene is required for cell wall-degrading enzyme expression and virulence on maize. Plant Cell 12, 237-248. Van Wert, S.L., and Yoder, O.C. (1992). Structure of the Cochliobolus heterostrophus glyceraldehyde-3-phosphate dehydrogenase gene. Curr. Genet. 22, 29-35. Vincent, P., Shareck, F., Dupont,C., Morosoli, R. and Kluepfel, D. (1997). New alpha-L-arabinofiiranosidase produced by Streptomyces Iividans: cloning and DNA sequence of the abjB gene and characterization of the enzyme. Biochem. J. 322, 845-852. Walton, J. D. (1994). Deconstructing the cell wall. Plant Physiol. 104, 1113-11 18. Wegener, S., Ransom RE, and Walton, J.D. (1999). A unique eukaryotic B-xylosidase gene from the phytopathogenic fungus Cochliobolus carbonum. Microbiology 145, 1089-1095. 133 Chapter Five Conclusion and future directions 134 CONCLUSION Pathogenesis is a complex process and an understanding of the underlying biochemical mechanisms is dependent on the characterization of the fungal gene products that influence the progression of infection of the host. SUCCCSSfiJi penetration of living plant tissue by fungal pathogens is also preceded by an exchange of signals between both organisms and may depend on signaling pathways for fiingal development and virulence (Knogge, 1998). A better understanding of pathogenicity will occur when the full cascade of signaling events between and within the fungal parasite and its host plant during penetration is uncovered. The application of molecular technologies to the study of fungal-plant interactions offers a new and more definitive approach for examining the virulence process and the role played by CWDEs in disease development. The softening of the plant cell walls for penetration by fungal hyphae, as well as provision of nutrients for grth are possible roles of CWDEs in plant disease development. This work showed that fill] induction of CWDEs in C. carbonum requires a gene, ccSNF1, which is a structural and fiinctional homolog of S. cerevisiae SNF 1. The demonstration that the C. carbonum ccsnfl mutant makes less CWDEs and exhibits significantly reduced virulence strongly suggest the importance of these enzymes as virulence factors. The deficiencies in penetration of the plant surface by the ccsnfl mutant may have arisen because they are unable to secrete an adequate amount or the required threshold of CWDEs needed to degrade the cell walls of the maize epidermis. Nevertheless, partial induction of several of the CWDEs occurs in the ccsnfl mutant indicating that a SNF]-independent pathway also regulates induction. 135 Repression of xylanase genes was observed with glucose as the sole carbon source, whereas expression was seen when the culture medium contained xylan. The xylanase genes are substrate-induced, but differentially expressed. An ortholog of CREA, a gene encoding a carbon catabolite repressor, was cloned and consensus CreAp binding sites are present in C. carbonum CWDE genes. Deletion of ARF1 results in complete disappearance of a peak of ot-L- arabinofuranosidase activity in culture filtrate fractionated by HPLC. Nevertheless, the mutant had more than 96 % of wild-type levels of total a-L-arabinofuranosidase activity and grth was normal on xylan but somewhat reduced on xylose, arabinose or corn cell walls. The disruption of a second arabinofiiranosidase gene, ARF2, making an arfl/arfl double mutant, leads to the disappearance of the two major arabinofirranosidase activity peaks in culture filtrate fractionated by HPLC. Growth of the arfl/arfl double mutant was reduced in the above sugars, but more strongly on arabinose. Both the arfl mutant and arfl/arfl double mutant have similar virulence as the wild type C. carbonum but grth is reduced. The remaining activities was due to a bifunctional B-xylosidase/a-L- arabinofuranosidase (Ransom and Walton, 1997; Wegener et al., 1999) which seems adequate for virulence. A major decrease in virulence may require disruption of genes that encode more than one class of the functionally redundant barrier-degrading enzymes. Therefore, emphasis of future research will be to consider all the important genes encoding the major enzymatic activity including xylanase, pectinase, B-xylosidase, or-L- arabinofuranosidase, and glucanase. The alternative requires the disruption of a regulatory gene that promotes the expression of several CWDE genes as demonstrated in 136 our previous experiment in which the ccSNF1 gene was knocked out resulting in reduced C. carbonum virulence on maize. SUGGESTIONS FOR FUTURE STUDIES Effects of SNF] mutation The phenomenon of glucose repression is concerned with the repression of a large number of genes when glucose is an abundant carbon source. S. cerevisiae can use several different sugars for growth, but it selectively ferments glucose when less desirable carbon sources are also available (Ronne, 1995). This is achieved by glucose down-regulation of the transcription of genes involved utilization of these alternate carbon sources. In fungi, the extracellular CWDEs are prominent among the glucose- repressed genes (Hensel and Holden, 1996). It is possible that ccSnflp regulates other genes, in addition to CWDEs, that are glucose repressed. Only a handful (fewer than 25) of C. carbonum genes have been isolated, and it is therefore not possible at present to know all the genes controlled by ccSnfl p. Nevertheless, such studies can be conducted in S. cerevisiae because sequencing of all its 16 chromosomes has been completed and nearly all the genes identified (http://genome-www.stanford.edu/Saccharomyces). Analysis of the global expression of all genes in snfl mutant (compared to wild type) in the presence and absence of glucose using the microarray technology (Lee and Lee, 2000; Schena et al., 1998; Shalon et al., 137 1996) would provide valuable insights into the different genes regulated by Snflp and their possible fiinction in fungal activities. Regulation of Snflp Regulation of gene expression is an important mechanism for adaptation to the nutritional environment and there is a complex signaling network that interconnects transduction pathways from sugars and nutrient signals (Sheen et al., 1999). Because glucose down- regulates fungal cell wall degrading enzymes, it may be that the fungi use glucose not only as nutrient but also as signal molecule. The mRNA of the enzyme that hydrolyzes sucrose into glucose and fi'uctose, beta-fi'uctosidase (invertase), accumulates in plants after pathogen attack (Sturrn and Chrispeels, 1990). Sucrose is the main sugar that is transported in plants, and its hydrolysis during infection by the induced beta-fructosidase may have the effect of releasing large amounts of glucose, which, when absorbed by the fungus, would lead to repression of CWDEs. This may be a manner by which plants resist fiingal pathogens. Snfl p, which is required for CWDE expression (Tonukari et al., 2000) is not active in the presence of glucose (Hardie, 1999). Therefore, it is necessary that the specific factors that control Snflp in filamentous fiingi be identified and characterized, because any factor that inhibits Snflp or its positive effector(s) may also inhibit the expression of CWDEs. Isolation of such factors as well as other components of the C. carbonum Snfl -CreA pathway (Figure 21) could be achieved by using the yeast two-hybrid screen with either Snflp or CreAp as bait. A possible candidate is Snf4p which acts as a positive effector of the kinase activity 138 of Snfl. The yeast snf4 mutant does not express invertase and cannot grow on glucose (Celenza and Carlson, 1989). Random mutagenesis using, for example, the restriction enzyme-mediated integration (REMI) (Bolker et al., 1995; Maier and Schafer, 1999; Riggle and Kumamoto, 1998), followed by screening transformants on replica plates of glucose and corn cell walls media, could also be used to find genes in the glucose repression pathway. Transformants that grew well on glucose and poorly on corn cell walls would be candidates for further characterization of the mutated gene. Identification of Fungal Pathogenicity Genes Targeted gene disruptions and deletions have allowed the generation of specific mutants defective in various properties that have been implicated in pathogenesis. Some of these factors, such as ccSnflp (Tonukari et al., 2000), branched-chain-amino-acid transaminase (Cheng et al., 1999), alanine racemase (Cheng and Walton, 2000), fatty acid synthase (Ahn and Walton, 1997), and Topr (Ahn and Walton, 1998) have been demonstrated to be required for pathogenicity in C. carbonum. But others, like the individual CWDE genes, are not essential for C. carbonum virulence (Apel et al., 1993; Apel-Birkhold and Walton, 1996; Gorlach et al., 1998; Murphy and Walton, 1996; Scott-Craig et al., 1990, 1998; Wegener et al., 1999). Further studies in cloning and deletion of other genes predicted to be necessary for virulence in C. carbonum would further clarify our understanding of the nature of fungal pathogenicity. Complementary approaches for identification of pathogenicity genes involve the generation of random mutants with subsequent characterization of the induced mutations. These approaches do not require a 139 priori knowledge of gene function, and are likely to be of great value in the molecular dissection of phytopathogenicity. Useful mutants will include those that exhibit non- or reduced virulence, and in which the deleted gene encodes proteins or catalyzes the production of chemical substances that are secreted by the fiingi and may have targets in the plant. Proteins that inactivate CWDEs Plants defend themselves against pathogens by other mechanisms, which can be constitutive or induced (Hunt et al., 1996). Chitinases and beta-1,3-glucanases, which are induced in response to infection, have received considerable attention due to their probable antimicrobial activity through hydrolysis of the fungal cell wall components, chitin and beta-1,3-glucans (Beffa and Meins, 1996; Collinge et al., 1993; Powell et al., 2000). Enzyme inhibitors such as protease inhibitors (Joshi et al., 1998) and specific polygalacturonase inhibitors (Mahalingam et al., 1999; Stotz et al., 1993; Yao et al., 1999) that target CWDEs may be useful antifungal agents. This interplay of invading CWDEs and cellular inhibitors may play a vital role in the spread of infection and host range. Constitutive overexpression of a protein involved in plant defense is one strategy for increasing plant tolerance to fungal pathogens. Making the plant produce more of its natural protectants is one of the reigning paradigms in crop improvement (Grison et al., 1996; Shelton et al., 2000). Identification and overexpression of the CWDE inhibitor proteins in plants could provide a means for the development of plant varieties with 140 increased resistance to certain fungal pathogens. A useful approach that could be applied for the identification of CWDE-inhibitors would be screening for susceptible plants in a population of A. thaliana random mutants (Parinov and Sundaresan, 2000) using non- HC-toxin-producing strains of C. carbonum. The use of A. thaliana is suggested here because it is naturally resistant to C. carbonum, is a widely studied model plant, and a majority of its genes have been identified. This will reveal a variety of genes that make most plants naturally resistant to plant pathogens. PERSPECTIVES Although the goal of this study has been to understand the molecular mechanisms of plant fungal pathogenesis, the potential application of such knowledge to design plants that are resistant to fungi is of great importance. Moreover, as the use of chemical control of pathogenic fungi and other microorganisms become increasingly restricted, there is an urgent need to develop crop varieties with resistance to these pathogens. Achieving this goal will require identification and characterization of the genes involved in pathogenicity and host specificity so that such factors could be targeted for the development of efficient and durable disease control. To obtain a clearer picture of fungal pathogenesis, the mechanisms of susceptibility and resistance ought to be aggressively analyzed and well understood. Resistance to CWDEs may be part of the constitutive and/or acquired resistance that plants possesses against various pathogens. A broad characterization of the host defense apparatus will reveal the combination of factors that 141 give a pathogen the capacity to ignore or bypass host defenses. Hence it should be of biological interest to further explore why most plants are immune to a pathogen that can devastate other plants. 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