Update on Pathogenicity Factors

61. Identification of pea pathogenicity (PEP) genes on dispensable chromosome in Nectria haematococca.

X. Liu^l Y. Han², C. C. Wasmann^l H. C. Kistler² and H. D. VanEtten^l. ¹Department of Plant Pathology, Univ. of Arizona, Tucson,

²PIant Molecular and Cellular Biology Program, Plant Pathology Department, Univ. of Florida, Gainesville.

Previous research suggests that genes (PDA) for detoxifying the pea phytoalexin pisatin and other pea pathogenicity (PEP) genes are located on dispensable chromosomes in N. haematococca. Recent work indicates that the cosmid clone 55-D-8, derived from the 1.6-Mb dispensable chromosome of N. haematococca, contains PEP genes as well as the disrupted PDA1 locus. Portions of cosmid 55-D-8 have been used as hybridization probes to screen a cDNA library constructed from mRNA isolated from infected pea tissue collected two days after inoculation. Four transcripts were identified in this screening. No homologs have been found in sequence databases for the four transcripts. A 5.3 kb XhoI/NotI fragment encoding cDNAl and cDNA2 is capable of converting a dispensable chromosome-deficient, Pda^-, nonpathogenic isolate 94-6-1 to pathogenicity on pea. Furthermore, in preliminary results, a 2.9 kb NotI/BglII fragment encoding only the cDNAl transcript is sufficient to convert 94-6-1 to pathogenicity. These results indicate that genes affecting the virulence of N. haematococca on pea (PDA1 and one or more PEP genes) are linked on one of the dispensable chromosomes of this broad host range fungus.

62. A multifunctional peptide synthetase involved in pathogenesis of Cochliobolus heterostrophus to corn.

Shun-Wen Lu, O.C. Yoder and B. G. Turgeon, Cornell Univ., Ithaca NY

Peptide synthetases are large multifunctional enzymes responsible for the non-ribosomal synthesis of a structurally diverse family of bioactive peptides, including antibiotice, toxins and immunosuppressants. We have partially cloned a gene (CPS1) from the corn pathogen C. heterostrophus that appears to encode a cyclic peptide synthetase required for pathogenesis. A cps1^- mutant, identified in a screen of REMI transformants, appears morphologically and developmentally identical to wild type but is 60% less virulent to corn. Genetic analysis of the mutant, as well as site specificdisruption of the gene in wild type, confirmed that a single tagged mutation was responsible for the mutant phenotype. Sequencing of DNA flanking the REMI vector insertion site and translation reveals an open reading frame (>4.5 kb) with similarity to SafB, of the multifunctional enzymes catalyzing the biosynthesis of the cyclic peptide antibiotic safamycin Mx1 produced by the bacterium Myxococcus xanthus. A second ORF (1.1 kb; TEA1), encoding a thioesterase (proposed function: termination of non-ribosomal peptide synthesis), was identified 5' of CPS1. These results suggest that the two genes are part of a cluster controlling biosynthesis of a peptide (as yet unidentified) required for fungal pathogenesis.

63. Identification and mapping of Les2, a gene controlling lesion size in Cochliobolus heterostrophus.

Li Ma, Edward J. Braun and Charlotte R. Bronson, Department of Plant Pathology, Iowa State University, Ames, Iowa 50011-1020

A mutant strain of Cochliobolus heterostrophus that produces significantly smaller disease lesions on corn leaves than does wild-type was obtained by ultraviolet light mutagenesis. In progeny of crosses between the mutant and a near-isogenic wild-type strain, all ten of ten complete tetrads segregated 4:4 (mutant pathogenicity:wild-type pathogenicity), and random spores segregated 47:45, suggesting a single mutated locus. Intercrosses between this mutant and six other lesion-deficient mutants of C. heterostrophus indicated that the locus had not been identified previously. The locus has been named Les2 because of its lesion-deficient phenotype. The mutant retains normal growth on media, and normal abilities to germinate, attach, form appressoria and penetrate corn leaves, which suggests that Les2 is needed for lesion production only after the fungus enters the corn leaves. Les2 is now being mapped by using AFLP (amplified fragment length polymorphism); tetrad analysis indicates that AFLP is a highly reliable and reproducible method for detection of molecular markers in this fungus. Using bulked-segregant analysis, eighteen AFLP markers have been identified which may be closely-linked to Les2.

64. Isolation of the cultivar specificity gene AVR1-MARA of Magnaporthe grisea.

M. Alejandra Mandel, Uvini P. Gunawardena, Travis M. Harper and Marc J. Orbach. Department of Plant Pathology, University of Arizona, Tucson, AZ.

AVR1-MARA is a stable avirulence gene of Magnaporthe grisea that elicits a resistant response in the rice cultivar Maratelli. To address the question of how this gene acts and why it is maintained in the genome, we are trying to clone AVR1-MARA using a map-based approach. We initiated a chromosome walk from molecular marker 12B5 located on chromosome 2b in the RFLP map of Sweigard et al (1). We have isolated the virulent locus avrl-MARA and portions of the avirulent locus (2). However, parts of the AVR1-MARA locus appears to be unclonable in E. coli, as these sequences are not present in any of the lambda, cosmid and BAC libraries that we constructed. The AVR1-MARA locus spans approximately 55 kb as defined by recombination breakpoints. There are two regions of 14 kb and 25 kb present in the the avirulent strain that are absent in the virulent strain.

To address whether the gene is essential, and to localize it further, we constructed 3 plasmids to disrupt parts or all of the locus by transformation-mediated gene disruption. We have deleted the entire locus in the aviruient strain 4224-7-8, which rendered the transformed strain virulent on rice cultivar Maratelli. We are currently using the partial locus disruption plasmids, to determine which half of the locus contains AVR1-MARA. We have also used UV mutagenesis to isolate virulent mutants and to increase the virulence of a low virulence mutant we obtained. These results, as well as our latest efforts in the cloning of the AVR1-MARA gene, will be presented.

1.Sweigard, J. A., B. Valent, M. J. Orbach, A. M. Walter, A. Rafalski, and F.G. Chumley. 1993. Genetic map of the rice blast fungus Magnaporthe grisea. in: Genetic Maps, 6th Ed. CSHL Press. Cold Spring Harbor, NY.

2. Mandel, M.A., V.W. Crouch, T.M. Harper, and M.J. Orbach. 1997. Physical mapping of the Magnaporthe grisea AVR1-MARA gene reveals the virulent allele contains two deletions. (accepted Molecular PlantMicrobe Interactions)

65. Molecular studies on the cell wall degrading enzymes from Botrytis cinerea I: Characterisation of

the endopolygalacturonase gene family.

Wietse Mulder*, Arjen ten Have⁺, Jan A.L. van Kan⁺ en Jaap Visser*. * Section MGIM, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands. ⁺Department of Phytopathology, Wageningen Agricultural University, Binnenhaven 9, 6709 PD Wageningen, The Netherlands.

In order to develop an effective control strategy for the plant pathogenic fungus Botrytis cinerea, insight is required in the mechanisms of infection employed by the fungus and the roles played by various factors. Several reports have clearly shown that pectinolytic enzymes, excreted by the fungus, play an important role in the infection process by degrading the plant cell wall. Therefore, we started a molecular genetic study in order to identify and characterise these enzymes and to elucidate their roles in the process of pathogenicity.

Genes encoding pectinolytic enzymes were isolated via heterologous hybridisation, using DNA-probes derived from several Aspergilli. Six members of a endopolygalacturonase gene family were isolated. Bcpga1 and Bcpga2 code for enzymes with an alkaline pI and are highly homologous to the PGs of the related fungus Sclerotinia sclerotiorum. The other four pga genes code for enzymes with an acidic pI. Interestingly, Bcpga3 and Bcpga6 show significant homology to the PG encoding gene of the root pathogenic fungus Fusarium moniliforme. In addition, we characterised the Bcpel1 gene, encoding a pectin lyase, which displays significant homology to pelA of A. niger. Recently, we identified Lambda phages hybridising to Aspergilli genes coding for pectin methyl esterase, exopolygalacturonase and pectate lyase. Cloning and characterisation of these genes is in progress. Regulation of expression of the isolated genes is studied in time course experiments using various growth conditions in flask cultures. Obtained results will be presented and possible roles of pectinolytic enzymes in pathogenicity will be discussed.

66. Suicide-Substrate Selection of Genes that Regulate Plant Cell Wall Breakdown in Cochliobolus sativus.

Rolf A. Prade¹ ; Bauman J.¹, Allen A.R.¹, and Andrew J. Mort². Departments of Microbiology & Molecular Genetics¹

and Biochemistry & Molecular Biology². Oklahoma State University, Stillwater, OK 74078.

The fungus Cochliobolus sativus causes plant disease on cultivated barley and related crops. Infection occurs through production of differentiated cell-types initiated by vegetative propagating hyphae. Cells, specialized in invasion, produce a complex set of enzymes that depolymerize plant cell walls during the penetration process and require multiple activities that overlap functionally. We are interested in identifiing genes involved in the induction phase of these enzymatic systems. To address the problems associated with redundant substrate-enzyme interactions, we tested a novel suicide selection approach to reveal pectn-degrading induction loci. The idea is simple: Lethal-substrates are synthesized by covalently linking fungicide molecules to substrate fragments; wild-type strains degrade the substrate releasing the fungicide and die. Mutants that fail to degrade lethal-substrates survive, probably because they fail to recognize the substrate, are unable to produce inducer molecules via positional isomerization, properly transduce the activation signal or activate transcription of gene expression. Suicide substrates prepared by covalently bonding hygromycin B to pectin (HY-Pectin) or xylan (HY-xylan) fragments function as predicted, they need to be enzymatically degraded before they are able to arrest vegetative growth. Moreover, a significant number of mutants that survive HY-pectin suicide selection have been isolated and one class is unable to assimilate other polysaccharides (i.e., cellulose and xylan) as well.

67. New approaches to identify pathogenicity genes of Botrytis cinerea.

Theo W. Prins, Lia Sibbel and Jan A.L. van Kan. Dept of Phytopathology, Wageningen Agricultural University, The Netherlands.

By definition, a prerequisite for pathogenicity factors is, that they are expressed during penetration and invasion of the host plant. Examples of such factors could be genes coding for extracellular hydrolases (cutinase, pectolytic enzymes), toxins or other, as yet unidentified, genes. We are studying the B. cinerea (grey mould)-tomato interaction. The aim is to isolate fungal genes expressed during infection. This is carried out in a non-biassed approach using two methods. The role of isolated genes in pathogenicity needs to be evaluated by disruption. For a Differential Screening, poly(A)⁺ RNA was isolated from B. cinerea grown in vitro and a tomato-B. cinerea interaction, reverse transcribed into cDNA in the presence of -³²P dATP and hybridised to duplicate filters of a genomic library of B. cinerea. Since the fungal poly(A)⁺ RNA in the interaction sample only represents 3%, the in vitro probe was compensated. One phage which hybridized differentially after the first and second screening was characterised in detail. The gene appeared to encode polyubiquitin. The structure of the gene is similar to other ubiquitin genes and consists of four repeats in a head-to-tail arrangement with intervening sequences. By DDRT-PCR, the fungal expression in planta was compared with the expression of B. cinerea in vitro, uninfected tomato and P. infestans- and TNV-infected tomato as controls. Five B. cinerea cDNAs were isolated and sequenced but no homology was found in the database. Results will be presented on further characterisation of polyubiquitin and the DDRT-PCR fragments.

68. Molecular and biochemical analyses of saponin detoxiefication by the phytopathogenic fungus Botrytis cinerea.

Thomas Quidde and Paul Tudzynski, Inst. f Botanik, Westfalische Wilhelms Universitat Muenster, Schlossgarten 3, 48149 Munster, Germany

Botrytis cinerea is the causal agent of "grey mould" diseases of many economically important fruits, vegetables and flowers. Our main interest are the mechanisms of virulence and pathogenesis during the interaction ptocess between B. cinerea and its hosts. Since the detoxification of saponins - preformed fungi-toxic compounds involved in plant defense against pathogens - has been shown to determine host-specificity in the Gaeumannomyces-Avena pathosystem, we started to investigate the importance of saponin-detoxification in the interaction of B. cinerea with its hosts, especially tomato.

B. cinerea has been described to detoxify -tomatine - a saponin from tomato - by deglycosylation. A tomatinase deficient field isolate showed enhanced sensitivity towards -tomatine in in-vitro assays and reduced virulence on tomato leaves. By screening a genomic library of B. cinerea using the Septoria tomatinase cDNA we were able to clone a genomic fragment carrying an ORF with signficant homology to family 3 glycosidases on both DNA and amino acid level. Targeted gene replacement showed the loss of avenacinase activity in three transformants. Detoxification of other structurally related saponins is not affected, suggesting a high substrate specificity of the enzyme. The potential role of this avenacinase-like enzyme in pathogenicity will be discussed.

69. Analysis of pectinase genes in Cochliobolus carbonum.

John S. Scott-Craig(1), Nyerhovwo J. Tonukari(1), Felice Cervone(2), Guilia De Lorenzo(2) and Jonathan D. Walton(1). (1)MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824 U.S.A. (2)Dipartimento di Biologia Vegetale, Universita di Roma "La Sapienza", Rome, Italy.

The maize pathogen Cochliobolus carbonum secretes a large number of plant-cell-wall-degrading enzymes when grown with maize cells walls as the sole carbon source. Although pectin constitutes less than ten percent of the maize cell wall, at least three pectin-degrading enzymes are produced by the fungus. Genes encoding an endo-polygalacturonase (PGN1), an exo-polygalacturonase (PGX1) and a pectin methylesterase (PME1) have been cloned and used to create strains containing deletions of each gene. These deletions, singly and in combination, are being examined for their effects on the growth of the fungus in vitro on pectin and on the ability of the fungus to infect maize.

70. The effects on pathogenicity of tomato of heterologous expression of a ₂-tomatinase gene in Nectria haematococca MPVI and disruption of this gene in Colletotrichum coccodes and Septoria lycopersici.

Robert W. Sandrock* and Hans VanEtten. University of Arizona, Tucson, * present address, Cornell University.

The presence of the antimicrobial compound -tomatine has been proposed to function as a chemical barrier in tomato tissue to potential pathogens. However, most successful pathogens of tomato are tolerant of this glycoalkaloid. A gene encoding a P-1,2-D glucosidase, called ₂-tomatinase, which detoxifies - tomatine to ₂-tomatine, has been cloned from the fungal tomato pathogen Septoria lycopersici. In this study, a portion of a ₂-tomatinase homologue that possesses 90% similarity to the S. lycopersici ₂-tomatinase amino acid sequence was cloned from the tomato pathogen Colletotrichum coccodes. Unlike S. lycopersici, C. coccodes degrades -tomatine to the aglycone tomatidine. Transformation-mediated gene disruption was utilized to create ₂-tomatinase-deficient mutants of both these pathogens in order to evaluate the importance of this enzyme in pathogenicity. Gene disruption of the C. coccodes ₂-tomatinase homologue resulted in a loss of ₂-tomatinase activity but these mutants retained their tolerance to -tomatine and their abilities to degrade -tomatine to tomatidine. The gene-disrupted mutants also were still able to parasitize green tomato fruit, an organ containing high levels of -tomatine. Gene disruption of the S. lycopersici ₂-tomatinase gene resulted in both a loss of ₂-tomatinase activity and a loss of tolerance to -tomatine. Preliminary pathogenicity tests with this mutant were inconclusive. Expression of the S. lycopersici gene in the pea pathogen N. haematococca MPVI, a fungus that lacks ₂-tomatinase activity, increased its tolerance to -tomatine in vitro and its ability to form lesions on green tomato fruit.

71. Gene expression of the blackleg fungus Leptosphaeria maculans in the presence of alkenyl glucosinolates.

Adrienne Sexton and Barbara Howlett, Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville 3052 VIC. Australia.

Blackleg caused by the ascomycete Leptosphaeria maculans is the most economically important disease of oilseed Brassicas worldwide. Generally Indian mustard (B. juncea) is resistant to the blackleg fungus and consequently has been used as a source of resistance in canola (B. napus) breeding strategies. Indian mustard has high levels of alkenyl glucosinolates, sulphur-containing compounds that give mustard a pungent taste. Upon wounding of the plant, these compounds are cleaved by the plant enzyme myrosinase into glucose and gases such as isothiocyanates, which are toxic to many organisms including L. maculans.

Recently blackleg isolates that can attack some Indian mustard varieties have been found in Australia. These isolates may be able to tolerate or detoxify hydrolysis products of glucosinolates in the plant. We using differential display to identlfv genes expressed by L. maculans isolates in the presence of glucosinolates. Such genes may be involved in detoxification of glucosinolates or evasion of their effects. A 1 kb mRNA is expressed only in the presence of 2-propenyl glucosinolate (5 and 10 ug/ml) and myrosinase (5 ug/ml). This message has a high degree of'sequence similarity to a yeast 5S ribosomal DNA binding protein, which is transcribed at high levels as a response to growth in high concentrations of glucose. Other differentially expressed genes of L. maculans are being sought.

72. Conversion of the soybean pathogen Colletotrichum destructivum to a pathogen of alfalfa by introduction of the MAK1 locus of Nectria haematococca.

Scott Soby and Hans VanEtten, Department of Plant Pathology, University of Arizona, Tucson, AZ 85721.

The soybean pathogen Colletotrichum destructivum (Glomerella glycines) can be isolated from anthracnose lesions caused by the alfalfa pathogen Colletotrichum trifollii, but is not known to be an important or aggressive pathogen of alfalfa. An examination of its ability to detoxify the major phytoalexin produced by alfalfa, medicarpin, shows that C. destructivum is able to slowly detoxify medicarpin by 6a hydroxylation, whereas C. trifollii and other alfalfa pathogens are able to metabolize medicarpin rapidly and by more than one pathway. In an attempt to determine whether the ability to detoxify medicarpin is important in the ability to cause disease in alfalfa we transformed an isolate of C. destructivum with the Nectria haematococca MAK1 gene. The MAK1 gene encodes the ability to detoxify medicarpin and the closely related compound maackiain by la hydroxylation. MAK1 was transformed into C. destructivum as either a genomic or cDNA clone and in both cases under control of its native promoter. Transformants of C. destructivum containing genomic copies of MAK1 metabolize maackiain at 150 times the wild type rate, and those with the cDNA clone at 50 times wild type. Virulence tests in planta show an increase in pathogenicity of the transformants as measured by the number and extent of lesions, and by an increase in the amount of stem damping-off. These results are consistent with the hypothesis that detoxification of phytoalexins is an important function in the virulence of fungal pathogens.

73. Withdrawn

74. PTH11, a gene required for surface recognition by Magnaporthe grisea.

Sweigard, J., Carroll, A., Howard, R., and Valent, B. DuPont Experimental Station, Wilmington, DE 19880.

PTHII was identified twice in a mutant hunt based on restriction enzyme mediated insertional (REMI) mutagenesis. The integration events leading to both mutants were more complex than typical REMI insertions. pth11-1 contains a deletion of greater than 10 kb at the vector insertion site. pthll-2 resulted from the apparent in vivo ligation of mitochondrial DNA with the integrating vector, followed by integration into PTH11. pth11^- mutants form few appressoria both on hydrophobic surfaces in vitro and on plant surfaces and therefore the mutant causes very few disease lesions. These mutants form wild-type levels of appressoria on other surfaces, including cellulose acetate. Therefore we conclude that PTH11 is not required for appressoria formation per se, but rather for surface recognition that leads to appressoria formation. PTH11 encodes a 68 kD protein and lacks significant homology to any known protein. Hydropathy plots of Pthllp suggest seven transmembrane domains and a long hydrophilic C-terminal tail. pth11^- mutants can be rescued by cAMP and IBMX suggesting that PTH11 is upstream of a cAMP-dependent pathway required for appressoria formation.

75. Cloning, molecular characterization and disruption of chitin synthease genes in Wangiella.

Paul J. Szaniszlo, University of Texas, Austin

Polymerase chain reaction technologies were used to clone four chitin synthase (CHS) structural genes from the phaeohyphomycotic agent W. dermatitidis, which were named WdCHS1, WdCHS2, WdCHS3 and WdCHS4. Molecular characterizations showed that WdC-HSl and WdCHS2 are most similar to CHS2 and CHS1 respectively of S. cerevisiae, whereas WdCHS4 is a homolog of the CAL1/CSD2 gene that encodes the Chs3 isozyme in that species. In contrast, WdCHS3 has no homolog in S. cerevisiae. RT-PCR experiments confirmed that all four WdCHS genes are transcribed during logarithmic yeast reproductive growth at both 25 C and 37 C. However, northern analyses of total RNA from cells cultured under a variety of conditions showed that only WdCHS3 is overexpressed to any extent and on when cells are subjected to stress conditions, including shifts of cells from 20- 25 C to 37 C. This result suggests that WdCHS3 overexpression is virulence factor that enriches the cell walls of W. dermatitidis with chitin during infection. Support for this hypothesis has been obtained by disrupting WdCHS3 in the wild-type background, showing that the mutant strain grow normally at 25 C and 37 C, but has reduced activity associated with it membranes, and has reduced virulence when tested in mouse models. Studies are in progress to evaluate the virulence of the three other single, three double and even one triple wdchs disruption strains we have derived.

76. Characterization of a gene for export of the toxic compound, sirodesmin, from the phytopathogenic fungus Leptosphaeria maculans.

Janet Condie, Janet L. Taylor, Michelle Van Driel, and M. Soledade Pedras*. NRC Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada; * Dept of Chemistry, Univ of Saskatchewan, Saskatoon, SK S7N 5C9.

Leptosphaeria maculans is a heterothallic ascomycete that causes blackleg disease in many cruciferous plants. The fungus produces several non-selective toxins that contain a epipolythiodioxopiperazine ring and are called sirodesmins. A report of a yeast gene, STS1, whose overexpression conferred resistance to the related toxin sporidesmin inspired us to search for a similar gene in L. maculans. The STS1 gene is a member of the highly conserved family of translocases known as ABC transporters. Proteins of this family are frequently involved in antibiotic export and are characterized by the presence of two ATP binding cassettes, containing Walker A and Walker B motifs, and 12 transmembrane helices. In the belief that a similar protein may be involved in the export of sirodesmin from L. maculans, we used degenerate oligonucleotides encoding amino acids from Walker A & B to amplify genomic DNA from the fungus. Two PCR products were identified with high homology to ABC transporters and we have characterized the genes from which they were derived. We have transformed L. maculans with a construct for disruption of one of the genes and obtained a high percentage of transformants that apparently no longer secrete sirodesmins into the medium. Our results will be presented.

77. The biological role of the hydrophobin cerato-ulmin in the life history of Ophiostoma ulmi and 0. novo-ulmi.

B. Temple, W. E. Hintz, L. Bernier, and P. A. Horgen; Universities of Toronto, Victoria and Laval .

The causal agents of Dutch elm disease, Ophiostoma ulmi (Buisman) Nannf. and O. novo-ulmi (Brasier, 1991) have been responsible for close to 80% elm mortality in the Northern hemisphere. One of most readily noticeable differences between the aggressive O. novo-ulmi and the less aggressive O. ulmi is the differential production of a protein called cerato-ulmin (CU), which is synthesized in large amounts by O. novo-ulmi. This small protein belongs to a class of molecules known as hydrophobins and is able to cause wilting in elm seedlings exposed to CU in vitro. Our, results suggested that CU is not a major wilt toxin. Over-expression of a copy of the cu gene from an aggressive O. novo-ulmi isolate in a nonaggressive isolate of 0. ulmi did not alter the virulence of the fungus on 3 year old elm seedlings. The enhanced production of CU in the cu overexpressing transformant resulted in a dramatic phenotype change compared to the wild type. The role of CU in parasitic fitness is evaluated. Our data indicate that CU can be considered a parasitic fitness factor that may be a mediating factor in adhesion of infectious propagules to the bark beetle vector, or functions to increase resistance of infectious propagules to environmental stress.

78. Expression of the Host-Selective Toxic Protein, ToxA, in Escherichia coli.

Robert P. Tuori and Lynda M. Ciuffetti. Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97330.

ToxA is a 13.2 kD host-selective toxic protein produced by isolates of the wheat pathogen Pyrenophora tritici-repentis. It has been shown to be a primary determinant of pathogenicty of this fungus on susceptible wheat cultivars. The ToxA gene encodes two polypeptide domains, an N-terminal anionic domain of unknown function and destiny, and a C-terminal cationic domain which is the mature, secreted ToxA protein. In order to initate a study of the processing of the ToxA proprotein, the expression of various constructs of the gene was attempted in E. coli. Three constructs were initially designed and expressed in the Promega PinPoint system: 1, The N-domain, which encodes a predicted 4.5 kD anionic peptide (N-FP); 2, The ToxA or C-domain (C-FP); And 3, the entire ORF minus the signal peptide region, which encodes a 17.5 kD protein (NC-FP). Cloning into the multiple cloning site of the PinPoint vector results in the construst of interest being expressed as a fusion protein attached to an N-terminal 14 kD biotinylated protein tag. E. coli expressing C-FP and NC-FP both produce abundant amounts of the appropriate sized fusion protein, however, much of this appears to be insoluble. E. coli expressing N-FP produce scant amounts of fusion protein with cultures multipling very slowly after induction. In all three cases it has been difficult to achieve efficient proteolytic cleavage of the fusion protein with factor Xa protease. Currently, the NC-FP construct is being expressed in a His-tag vector with a TEV protease recognition site separating the tag and toxin sequences.

79. Genetics of resistance and virulence in the Hordeum vulgare:Cochliobolus sativus pathosystem.

Majda Valjavec-Gratian and Brian Steffenson, Department of Plant Pathology, North Dakota State University, Fargo, ND 58105.

The perfect stage of Cochliobolus sativus, a heterothallic ascomycete, is rarely found in nature, but it can be reliably produced in culture. The objective of this study was to examine the genetics of host specific virulence in C. sativus and to complement it with an examination of host specific resistance in barley. A cross was made between fungal isolate ND90Pr (which exhibits high virulence on barley genotype Bowman and low virulence on genotype ND 5883) and ND93-1 (which exhibits low virulence on both genotypes). For hybridization, barley seed was used as a substrate. Seed, amended with conidia of both isolates was incubated in petri plates on Sach's nutrient agar. One hundred and three ascospores were obtained from the cross, and the virulence of each single ascospore culture was evaluated on the barley genotypes Bowman and ND 5883. The fungal progeny could be clearly differentiated into two phenotypic groups based on their infection responses on Bowman. One group exhibited the high virulence character of isolate ND90Pr and the other the low virulence character of isolate ND93-1. The ascospore population segregated 48:55 for low virulence:high virulence on Bowman, indicating the presence of a single virulence gene in isolate ND90Pr. Barley progeny segregated approximately 3:1 for resistance:susceptibility to isolate ND90Pr, indicating that ND 5883 carries a single gene for resistance. These data demonstrate that both virulence in the pathogen and resistance in the host are under monogenic control in this specific host genotype:fungal isolate combination.

80. The role of arginine biosynthetic enzymes in the pathogenicity of Venturia inaequalis.

Sally P Wither and Keith Johnstone, Department of Plant Sciences, University of Cambridge, UK.

Venturia inaequalis is the cause of apple scab and is the major pathogen of apples world-wide. The aim of this study is to establish at the molecular level whether arginine biosynthetic enzymes are required for pathogenicity. An existing, argininosuccinate lyase (ASL) V. inaequalis mutant (1) was chosen for further study.

The ASL mutant was shown to be non-pathocyenic on apple seedling. An enzyme assay has been established for ASL and used to compare the enzyme activity of the wild type and mutant. The phenotype is also beina verified by western blot analysis using, chick ASL antibodies.

Prgaress in this work and in the construction and screening, of a cDNA library for the ASL gene will be reported,

(1) Boone D.M., Stauffer M.A. and Keitt G.W. ( 1956) Am J Bot 43: 199-204.

81. Application of differential display RT-PCR to analyse fungal gene expression in a plant-pathogen interaction.

Jan A.L. van Kan, Ernesto P. Benito & Theo W. Prins, Dept. of Phytopathology, Wageningen Agricultural University, PO Box 8025, 6700 EE Wageningen, the Netherlands.

Establishment of a plant-pathogen interaction involves differential gene expression in both organisms. In order to isolate Botrytis cinerea genes whose expression is induced during an interaction with tomato, a comparative analysis of its expression pattern in planta with its expression pattern during in vitro culture was performed by differential display of mRNA. Fungal genes induced in planta were distinguished from plant defense genes induced in response to the pathogen, by including in the comparison expression patterns of healthy tomato plants and of tomato plants infected with two different pathogens, (Phytophthora infestans or Tobacco Necrosis Virus). Using a limited set of primer combinations three B. cinerea cDNAs, Bipi2, Bipi5 and Bipi47, were isolated. Northern blot hybridization showed that Bipi2 and Bipi5 mRNAs accumulated at detectable levels only at late time points during the interaction. Bipi47 detected two different mRNAs, both corresponding to genes expressed at a high level during the entire interaction. None of the cDNAs displayed homology to sequences in the database. These results show that the differential display procedure possesses enough sensitivity to be applied to the detection of fungal genes during early stages of a plant-pathogen interaction. Some of the genes isolated by this method may be involved in pathogenicity of B. cinerea.

82. Cutinase A of Botrytis cinerea is not essential for infection of tomato fruits and gerbera flowers. Jan van Kan, L. Wagemakers, J.W. van 't Klooster, D. Dees, C.J.B. van der Vlugt-Bergmans. Dept. of Phytopathology, Wageningen Agricultural University, PO Box 8025, 6700 EE Wageningen, the Netherlands.

Cutinase was proposed to play an early role in the infection of host tissues by Botrytis cinerea. The enzyme was purified, characterized and amino acid sequences were used to design primers for PCR based gene cloning from strain SAS56. Expression of the cutinase (cutA) gene during the infection of gerbera flowers and tomato fruits was studied, using a cutA promoter-GUS construct, transformed into B. cinerea. During germination and penetration of host tissue, fungal structures demonstrated high GUS activity, indicating that the cutA promoter is active in planta. During in vitro growth on water agar, GUS staining was observed, indicating that substrate induction is not essential. The addition of a cutin monomer to the agar resulted in a much more intense GUS staining. In vitro, cutinase expression is subject to catabolite repression. To study the necessity of cutinase A for penetration of host tissue by B. cinerea, cutA-deficient mutants were constructed by means of gene disruption. Pathogenicity of these mutants was tested on tomato fruits and gerbera flowers. The ability of the mutants to penetrate and cause infection was not altered. Infection structures produced by the mutant did not differ morphologically from the wild type. We conclude that cutinase A is not an essential pathogenicity factor for B. cinerea.

83. Reduced pathogenicity strain of Colletotrichum gloeosporioides obtained by REMI.

Nir Yakoby, Amos Dinoor and Dov Prusky, Department of Postharvest Science. Volcani Center Bet Dagan 50250 Israel.

Colletotrichum gloeosporioides is the main postharvest pathogen attacking avocado and other subtropical fruits. In the past it was found that a mutant of C. magna strain, lacking the capability of secreting pectate lyase (PL) was non pathogenic on avocado fruit. Furthermore, antibodies developed against PL of C. gloeosporioides were able to inhibit symptom development by the pathogen on avocado fruits. Both experiments suggested the importance of pectate lyase during symptom development on fruits. We have used restriction enzyme mediated integration (REMI) to obtain tagged mutants with reduced pathogenicity as a possible tool to demonstrate the importance of pectate lyase during Colletotrichum pathogenicity. The hph gene was subcloned into pGEM-7Z and following restriction with HindIII was transformed into C. gloeosporioides protoplasts. Transformants were screened for pathogenicity in 24h assay, by flesh inoculation and on long term assay by peel inoculation. From 300 screened transformants one reduced pathogenic strain was found. The presence of C. gloeosporioides pel gene in the mutant was determined by Southern blots analysis. Further analysis of the mutant will be discussed in order to demonstrate the involvement of PL during pathogenicity of C. gloeosporioides in avocado fruits.

84. Ecm1: a locus affecting extracellular matrix production and lesion size in Cochliobolus heterostrophus.

Hong Zhu, Edward J. Braun, Jennifer L. Perry and Charlotte R. Bronson, Department of Plant Pathology, Iowa State University, Ames, Iowa 50011-1020.

A mutant of Cochliobolus heterostrophus lacking the outer layer of extracellular matrix around its germ tubes was obtained by mutagenizing protoplasts. The mutant not only lacks the outer matrix, but also produces much smaller lesions on corn leaves than non-mutant strains; mutant lesions average 1. 9 mm² compared to 6.6 mm² for non-mutant lesions. Genetic analysis demonstrated that the failure to produce the outer matrix cosegregates with the reduced lesion size, which suggests that the two traits are controlled by the same locus. This locus has been named Ecm1 (extracellular matrix deficient). The mutant retains normal growth on media and normal abilities to germinate, attach, form appressoria, and penetrate corn leaves. This indicates that the outer matrix is not necessary for infection prior to entrance of the fungus into the leaf and that the pathogenicity defect occurs after penetration. Seven markers linked to Ecml were found by analysis of amplified fragment length polymorphisms (AFLP). Ecml maps to chromosome 4; the closest marker to Ecml is 5 cM, which is estimated to be about 115 kb. These results should enable the cloning of Ecml by mapbased methods.