Posters I: Plant and Animal Fungal Pathogens

Isolates of Colletotrichum gloeosporioides from avocado and almond fruit were compared in order to determine the genetic diversity between and among the different populations. Four almond isolates exhibited very similar nuclear banding patterns compared to avocado isolates which had differential patterns. Similar results were observed with A+T-rich DNA representative of the mitochondrial genome. PCR amplification of genomic DNA using four primers accurately grouped nine almond isolates from different geographic locations uniformly. In contrast, the avocado isolates were more diverse with 7-10 different genotypes being observed. Amplification and subsequent restriction enzyme digestion of the 4-5 ITS region of ribosomal DNA failed to distinguish between C. gloeosporioides from a diverse host range. Avocado isolates produced varying lesions on avocado and almond fruits whereas the almond isolates infected at a uniform rate. Certain avocado isolates seemed to produce perithecia in culture whereas almond isolates remained asexual. This suggests that in asexually reproducing populations such as the C. gloeosporioides almond isolates little DNA variation is expected to occur as compared with the sexually reproducing avocado isolates where multiple genotypes are found.

2. Genetics of virulence and linkage mapping in Phytophthora sojae

Phytophthora sojae belongs to the Oomycetes which are characterised by gametangial meiosis, thus having a diploid somatic phase, which contrasts with the majority of the true fungi which are haploid for most of their life cycle. Until recently the genetics of virulence/avirulence in P. sojae was considered intractable due to its homothallic nature. A race l and a race 7 isolate were co- cultured in vitro and, using RAPD markers, ten hybrids were identified among 354 oospores analysed. One F1 hybrid was allowed to self fertilise and produce an F2 population of 247 individuals. Fifty-three F2 individuals were selected at random for genetic analysis. A genetic linkage map has been constructed from this cross consisting of 15 major linkage groups and ten small linkage groups using 233 RAPD markers, 30 dominant RFLP markers, 10 co-dominant RFLP markers and four avirulence genes. Segregation of virulence against soybean resistance genes Rpsla, 3a and 5 revealed that the avirulence genes Avrla, 3a and 5 were dominant to virulence. Avirulence against these three resistance genes appeared to be conditioned by one locus for Avrla and two independent, complementary dominant loci for both Avr3a and Avr5. Segregation of virulence against Rps6 was in the ratio of 1:2:1 (avirulent : mixed reaction : virulent), suggesting a semi-dominant allele at a single locus.

3. Protease and cutinase in the pathogenic interaction between Pyrenopeziza brassicae and Brassica napus

Pyrenopeziza brassicae, a haploid hemibiotroph and a member of the Ascomycotina, is the causative agent of light leaf spot disease of brassicas. Understanding the mechanism of pathogenicity by P. brassicae is of central importance in the development of alternative strategies for disease control. Extracellular enzymes are considered to facilitate penetration and growth of fungi in planta. Consequently, we are examining the roles of extracellular cutinase and protease in pathogenesis. Here we characterise an extracellular protease and describe its role as a pathogenicity determinant of P. brassicae. Purification of the protease and amino acid sequence analysis revealed N-terminal sequence homology with a cloned Aspergillus oryzae protease gene. Together with the observed inhibition of enzyme activity in vitro by ovoinhibitor, this suggests that the P. brassicae enzyme is an alkaline serine protease. Indirect evidence that expression of the extracellular protease is essential for pathogenicity by P. brassicae is as follows: (i) the protease is produced in planta; (ii) both pathogenesis (path ) and protease (prot ) traits co-segregate in mutants of P. brassicae; and (iii) complementation of both path and prot phenotypes was achieved using a single 40 kb genomic clone. One prot mutant is able to penetrate the host cuticle but shows restricted growth in planta and does not cause tissue necrosis. We have isolated several putative protease clones from a casein induced P. brassicae cDNA library which are currently being analysed. Initial studies have shown cutinase activity in cutin-induced culture filtrates of P. brassicae. We are characterising and purifying this cutinase prior to cloning its structural gene.

4. Isolation and characterization of phase specific clones of grass pathogen Claviceps purpurea

The ergot fungus Claviceps purpurea infects the florets of rye and other grasses causing a tissue replacement disease. The infected ovary is fully replaced by fungal tissue. During this process a distinct border between fungus and plant is established and the fungus switches from a conidium producing form (sphacelium) to an alkaloid producing resting structure (sclerotium). We are characterizing the interaction between C. purpurea and its host Secale cereale. We have isolated several clones carrying genes which might be higher expressed or less expressed in the plant than in axenic culture or which even might be uniquely expressed in planta. Two clones were chosen for further analysis because they seem to carry regions which are expressed in the honeydew (plant colonization, conidia producing) phase, but not in axenic culture. The clones are currently being evaluated: 1) location of transcribed regions, 2) nucleotide sequence of transcribed regions, 3) specificity for in planta transcription and 4) transcription during parasitic stages other than the honeydew phase.

5. RAPD analysis and an aspergilloma legal case

RAPD markers were used to trace the origin of a claimed work-associated case of aspergilloma caused by Aspergillus fumigatus. The counter argument was that the patient could have been infected outside the work place, and that the disability was therefore not work associated. Samples were taken from the patient's work place, from the patient's house, and from houses of neighbors and other control houses. Several decamers were used to attempt to differentiate among the various isolates obtained from the sampling. Whereas the control houses had around 3-5 strains of A. fumigatus, all different as determined by RAPD analysis, and each appearing in only one sample, the patient's house was infested with one dominant strain that appeared in nearly all the samples. This strain was indistinguishable by RAPD analysis from a strain isolated from the patient's office and the next door boiler room and other parts of his work area. The legal interpretations of these data will have to await the actual trial. The DNA was isolated by a novel non-grinding method using xanthogenates that avoids hazardous aerosols.

6. Analysis of the role of cytokinins in a hemibiotrophic plant pathogen interaction

The fungal plant pathogen, Pyrenopeziza brassicae is a haploid member of the Ascomycotina and is the cause of light leaf spot disease of Brassicas, which is one of the most important diseases of oilseed rape in the U.K. P. brassicae is a hemibiotroph, proliferating sub- cuticularly from its initial site of infection and gaining nutrition from living cells before switching to necrotrophy at the onset of sporulation. During its biotrophic phase, a relatively balanced physiological relationship exists between P. brassicae and its host. The objective of this research is to ascertain whether cytokinins of fungal origin play a role in maintaining the biotrophic phase of P. brassicae development. Cytokinin production by P. brassicae in vitro has been established and quantified by ELISA using highly specific polyclonal antibodies to zeatin riboside and isopentenyladenosine riboside. The role of cytokinins in hemibiotrophy is now being assessed by (i) screening for UV generated cytokinin deficient mutants and (ii), using PCR- derived cytokinin biosynthetic gene sequences to insertionally inactivate the corresponding fungal genes. Current progress in these areas will be reported.

7. Map-based cloning of pathogenicity determinants of Nectria haematococca

Pathogenicity of Nectria haematococca (MPI) on unwounded hypocotyls of Cucurbita maxima cv. Crown (buttercup squash) is a quantitative trait, with estimates of the number of effective factors ranging from 3 to 15, dependent on cross. To locate, and ultimately clone the genes controlling pathogenicity, a set of markers linked to the quantitative trait loci (QTLs) and a yeast artificial chromosome (YAC) library from a high pathogenicity isolate are being developed. A population of 200 random ascopsore progeny was derived from a cross between a high pathogenicity and a low pathogenicity isolate. Random amplification of polymorphic DNA (RAPD) analysis revealed 40 markers that distinguished the parents. Currently, bulked DNA samples from either high or low pathogenicity progeny are being screened to assess linkage of these markers with QTLs controlling pathogenicity. High molecular weight DNA is being prepared by preparative pulsed field gel electrophoresis to enable a YAC library to be constructed. YACs containing both marker and QTL will be identified by library screening. Sub-cloning and chromosome walking will lead to isolation of QTLs, the importance of which will be established by complementation of a low pathogenicity isolate by transformation.

8. Exploring the use of Neurospora crassa mutant erg-1 for the isolation of pisatin detoxifying genes through heterologous complementation

The fungal pea pathogen Nectria haematococca expresses a cytochrome P450 which detoxifies the pea phytoalexin pisatin by demethylation. A pisatin demethylase (pdm) encoding gene (PDA) was originally isolated from N. haematococca genomic DNA by detecting heterologous expression of the demethylase activity in Aspergillus nidulans. However, isolation of PDA genes in A. nidulans by selection for enhanced tolerance to pisatin has not been possible due to the high intrinsic pisatin tolerance of A. nidulans wildtype. Papavinasasundaram and Kasbekar (1993) have shown that ergosterol (erg) mutants of Neurospora crassa are sensitive to pisatin. In order to develop a method for the isolation of PDA genes by selection we examined the expression of a N. haematococca PDA gene in N. crassa erg-1. We found the gene to be expressed in N. crassa and pisatin demethylating erg-1 transformants showing increased tolerance to pisatin. Spores of erg- 1 pdm+ transformants can be identified in mixes with erg-1 pdm spores by means of pisatin selection. These results suggest that pdm genes can be isolated by heterologous complementation of the pisatin sensitivity of erg-1. Papavinasasundaram and Kasbekar (1993). J. Gen. Microbiol. 139:3035-3041.

9. Mutants of Cercospora nicotianae altered in their response to active-oxygen- generating photosensitizers

Fungi in the genus Cercospora produce a singlet-oxygen-generating photosensitizing toxin cercosporin which plays an important role in the ability of these fungi to parasitize plants. Cercosporin shows generalized toxicity to plants, bacteria, mice, and many fungi, but Cercospora species are resistant. This resistance has been associated with the ability of these fungi to transiently reduce and detoxify cercosporin in contact with fungal hyphae. In order to further characterize the mechanism of resistance, cercosporin-sensitive mutants of C. nicotianae were isolated. Two classes of mutants were identified. One class consisted of five mutants which were highly sensitive to cercosporin and to five other singlet-oxygen-generating photosensitizers. These mutants were unable to reduce cercosporin and could be protected against cercosporin toxicity by the reducing agents ascorbate, cysteine, and reduced glutathione. Mutants of this class were significantly less pathogenic than wild type. The second class consisted of a single mutant which was only partially inhibited by cercosporin. This mutant was unaltered in cercosporin-reducing ability and in resistance to the other photosensitizers. It was slightly decreased in pathogenicity, and could be protected against cercosporin toxicity by cysteine and reduced glutathione, but not by ascorbate. None of the mutants was altered in endogenous levels of ascorbate, cysteine, reduced glutathione, or total soluble or protein thiols. Current efforts are directed at isolating the genes involved in resistance by mutant complementation.

10. Carotenoid-minus disruption mutants of Cercospora nicotianae show no increased sensitivity to cercosporin or other photosensitizers

Numerous phytopathogenic Cercospora species synthesize the photoactivated phytotoxin cercosporin, the presence of which is correlated with the ability of these fungi to cause plant disease. Due to its mode of action cercosporin exerts a very general and broad toxicity, not only to plants, but to bacteria, most fungi and even mice. Cercosporin absorbs light energy and interacts with molecular oxygen to generate the highly toxic active oxygen species, singlet oxygen. Even though micromolar concentrations of cercosporin are fatal to most cells, Cercospora species can synthesize up to millimolar concentrations without autotoxicity. Because carotenoids are known to be the most potent biological quenchers of singlet oxygen, we evaluated their role in Cercospora resistance to cercosporin. Targeted gene disruption of a carotenoid biosynthetic gene was used to create carotenoid minus derivatives of a wild-type and of two cercosporin-sensitive strains of C. nicotianae. These carotenoid-minus disruption strains were no more sensitive to cercosporin or to five other photosensitizers than the strains from which they were derived. Pathogenicity tests also indicated inability to produce carotenoids did not otherwise

11. Cosegregation of Avr4 and Avr6 in Phytophthora sojae

Phytophthora sojae is an oomycete pathogen of soybean that causes a severe root and stem rot. To date, at least 13 host plant resistant genes (Rps) have been identified at 7 genetic loci, each conferring resistance to specific races of the pathogen. In this study, 55 F2 progeny resulting from a cross of race 2 (avirulent on Rps4 and Rps6) and race 7 (virulent on Rps4 and Rps6) were tested for their ability to cause disease on soybean plants carrying the Rps4 or the Rps6 resistance gene. Avirulence on Rps4 and Rps6 was dominant and linked, with 41 individuals avirulent on both of these genes and 14 virulent on Rps4 and Rps6. These results support the suggestion that the soybean-Phytophthora relationship is a gene-for-gene interaction, and that the presumptive Avr4 and Avr6 genes are either tightly linked or are identical.

12. Population genetic structure of Fusarium oxysporum f.sp. albedinis

Bayoud, the Fusarium wilt of date palm, first appeared in the south of Morocco and then spread in most of the moroccan palm groves and reached the western and central oasis of the Algerian Sahara. Thanks to prophylactic measures, all other date palm growing areas in the world remain free of the disease. In order to assess the population genetic structure of Fusarium oxysporum f.sp. albedinis (FOA), most of the diseased palm groves were prospected and isolates were collected from wilted palms of several varieties. 120 FOA isolates were tested for Vegetative Compatibility Group (VCG), Restriction Fragment Length Polymorphism (RFLP) and Random amplified Polymorphic DNA (RAPD). No polymorphism was observed in RFLP studies on ribosomal and mitochondrial DNA and a unique VCG was found whatever the host genotype or the geographical origin of the 120 FOA isolates. RAPD analysis separated some isolates from two Algerian oasis (Adrar and Ghardaia), but a low level of genetic diversity was found among the whole FOA population. RFLP analysis with a dispersed repetitive DNA probe allowed to detect 52 fingerprints sharing at least 80 % similarity. Such a genetic homogeneity between FOA isolates provides evidence that Fusarium oxysporum f.sp. albedinis populations originate from a unique clone which spread in Morocco and Algeria.

13. Functional analysis of the ipiO genes of the late blight pathogen Phytophthora infestans

Phytophthora infestans is the causal agent of potato late blight, one of the most devastating diseases of potato. In order to gain more insight in the molecular and cellular processes involved in pathogenicity we have isolated P. infestans genes of which the expression is specifically induced during growth of the pathogen in the host plant. From two of these in planta induced (ipi) genes, ipiO1 and ipiO2, high levels of mRNA can be detected in infected potato leaves and tubers during the first two days of the infection cycle whereas one day later the ipiO mRNA level is strongly decreased. In in vitro grown mycelium ipiO gene expression is induced by nutrient deprivation (Pieterse et al., MGG 244:269-277). The two ipiO genes are 98% homologous. The difference between the two proteins, IPI-O1 and IPI-O2, is limited to four amino acid and there is no homology with any known protein (Pieterse et al., Gene 138:67-77). Besides a putative signal peptide both proteins contain the tripeptide Arg-Gly-Asp (RGD) which functions as a "cell attachment" sequence in several mammalian proteins. Whether the RGD tripeptide present in IPI- O has a similar function is unknown. To determine whether or not IPI-O plays a role in pathogenicity, P. infestans transformants containing the ipiO1 gene in anti-sense orientation were made. Phenotypic characterization of these anti-sense transformants were presented and the possible role of IPI-O during pathogenicity of P. infestans on potato discussed.

14. Azole resistance in Candida species

Since its introduction in 1988, fluconazole has been used to treat >25 million patients, including >250,000 AIDS patients. Like the other azole antifungals, fluconazole acts by inhibiting fungal ergosterol biosynthesis. The fungistatic nature of the azole antifungals has resulted in AIDS patients receiving prolonged antifungal therapy and consequently azole-resistant Candida species are now isolated from patients with end-stage disease. These organisms are frequently cross- resistant to all of the commercially available azole antifungals. Previous studies on C. albicans, C. glabrata, and Saccharomyces showed that there are three possible mechansisms for resistance to azoles, viz: changes in the target enzyme, cytochrome P-450-dependent 14alpha-sterol demethylase; changes in delta-5-6 sterol desaturase, another enzyme in ergosterol biosynthesis; and permeability resistance. We have been investigating the mechanisms for fluconazole resistance in post-treatment fluconazole-resistant isolates of C. glabrata and C. albicans. Both of the isolates show cross- resistance to ketoconazole and itraconazole and both show permeability resistance to fluconazole. Studies with metabolic inhibitors showed that fluconazole resistance in the C. glabrata isolate was due to energy-dependent efflux of the drug from the cells. In contrast, fluconazole resistance in the C. albicans isolate appeared to be due to a block in uptake of the drug. We are currently attempting to clone the genes responsible for fluconazole resistance in these organisms.

15. Regulation of HMGR expression in the trichothecene-producing fungus Gibberella pulicaris (Fusarium sambucinum)

Trichothecenes are sesquiterpenoid toxins produced by several Fusarium species. The importance of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) in the regulation of trichothecene production was investigated in G. pulicaris R-6380. A portion of the gene encoding HMGR (Ipr1) was isolated from G. pulicaris R-6380 using anchored PCR. The isolated gene fragment specifies the C- terminal 340 amino acids of HMGR which contains the highly conserved catalytic domain of this enzyme. The G. pulicaris sequence is 76% homologous with the corresponding region of the HMGRs from S. cerevisiae. The Ipr1 gene is present in a single copy and hybridizes to an mRNA of 3.1 Kb. Transcription of Ipr1 in G. pulicaris was analyzed in both the wild-type strain R-6380 and the mutant strain BC51 (Tri5-) which is blocked in the first step of the trichothecene pathway. Under growth conditions that support trichothecene production Ipr1 mRNA levels increased 3-fold in R-6380 cultures and 6 to 7-fold in BC51 cultures over an 18 h time period. During this same time period the levels of HMGR activity also increased 2 to 3-fold in R-6380 and 5 to 6-fold in BC51. These results suggest that increases in both Ipr1 expression and HMGR activity are closely correlated with trichothecene biosynthesis in G. pulicaris, but that the observed increases in HMGR activity do not require trichothecene biosynthesis.

16. Characterization of the trichothecene pathway gene Tri7 from Fusarium sporotrichioides

The biosynthesis of trichothecene mycotoxins occurs via a pathway involving multiple isomerization, oxygenation, and esterification reactions. The clustering of trichothecene pathway genes has permitted rapid progress in the characterization of this complex sesquiterpenoid pathway. At least four trichothecene pathway genes are closely linked in Fusarium sporotrichioides. We have identified a fifth pathway gene (Tri7) located 898 bp upstream from Tri3. The Tri7 gene encodes a protein of 399 amino acids with no significant homology to sequences present in the protein databases. Comparison of the Tri7 cDNA and genomic sequences revealed the presence of a single intron of 52 bp. Hydropathy analysis predicted the presence of several membrane spanning segments indicating that TRI7 is most likely an integral membrane protein. Disruption of Tri7 resulted in an altered trichothecene production phenotype characterized by the accumulation of HT-2 toxin but does not alter the expression of other pathway enzymes. Analysis of Tri7 transcription revealed a pattern of expression during trichothecene biosynthesis identical to that of other pathway genes. Further efforts to characterize the function of TRI7 are underway.

17. Reduced virulence of a trichothecene nonproducing mutant of Gibberella zeae on field grown wheat

Wheat head scab caused by Gibberella zeae is a serious problem for wheat growers worldwide. Head scab causes both yield losses and contamination of grain with trichothecene mycotoxins that are injurious to human and animal health. Trichothecenes are also potent phytotoxins which has led to the hypothesis that they play a role in plant diseases caused by trichothecene producing fungi. . It was previously shown that disruption of the Tri5 gene encoding trichodiene synthase blocks trichothecene toxin biosynthesis and reduces the virulence of G. zeae on wheat under growth chamber conditions. In this study, conducted under permit from USDA-APHIS, the virulence of a G. zeae strain carrying a disrupted Tri5 and the virulence of its Tri5+ progenitor were examined under field conditions. Hard red spring wheat cultivars Wheaton and Butte 86 were grown in central Illinois and inoculated at anthesis by injecting individual heads with fungal conidia or with water. After one month, the heads were harvested and the grain was analyzed for yield and quality, and for kernel infection with G. zeae Tri5+ strains and the Tri5- disruptant by fungal strain isolation on a selective medium and PCR. The Tri5- disruptant was able to infect wheat kernels, but was significantly less virulent (P< .05) than the Tri5+ progenitor on both wheat cultivars as measured by kernel number, weight and germinability, and the frequency of infected kernels. These results indicate that trichothecene mycotoxins contribute to the ability of G. zeae to cause wheat head scab under field conditions.

18. Isolation of the trichodiene synthase gene (Tri5) from the macrocyclic trichothecene producing fungus Myrothecium roridum

Macrocyclic trichothecenes are antibiotic/phytotoxic compounds that are produced by both fungi and plants. Some macrocyclic trichothecenes produced by Myrothecium roridum are identical to trichothecenes found in two species of the plant genus Baccharis. Production of macrocyclic trichothecenes by M. roridum has been implicated as a possible virulence factor in plant diseases caused by this fungus, however, role of these compounds in the biology of Baccharis species is unclear. To investigate macrocyclic trichothecene biosynthesis we have isolated a gene encoding trichodiene synthase (Tri5) the first step in the trichothecene pathway from M. roridum. Cosmids carrying Tri5 were identified by probing an M. roridum cosmid library with the Tri5 gene from F. sporotrichiodes. Tri5 was subcloned as two 1.7 Kb Hind III fragments from a single cosmid clone (MRcos13). Sequence analysis revealed a coding region of 1211 bp which specifies a protein of 385 amino acids. A 58 bp intron sequence was found in the same position as the intron present in Tri5 from Fusarium species. The predicted amino acid sequence has approximately 70% homology with other trichodiene synthases. Preliminary results indicate that homologs of other trichothecene pathway genes are also present on MRcos13 suggesting that macrocyclic trichothecene pathway genes are clustered.

19. Evidence for chromosome transfer between biotypes of Colletotrichum gloeosporioides

In Australia there are two biotypes (A & B) of C. gloeosporioides which cause distinct anthracnose diseases on Stylosanthes spp. Molecular analysis has revealed that the biotypes are genetically distinct. Laboratory pairings of nitrate reductase mutants have indicated that there is no vegetative compatibility between biotypes although isolates within each biotype are compatible. These data suggest the biotypes are genetically isolated clonal lineages. The fungus has a very variable electrophoretic karyotype and this is particularly striking for small chromosomes which are easily resolved. A detailed study of the distribution of two dispensable homologous chromosomes will be presented. One of these chromosome homologues is 1.2 Mb in size and strain-specific in biotype B but a 2 Mb homologue is present in all biotype A strains studied so far. Recent field isolates have now revealed some strains carrying both 1.2 and 2 Mb chromosomes in what appears to be an exclusively biotype B background genome. Molecular analysis of this 2 Mb chromosome appears to support the notion that it is the result of a very recent transfer from biotype A. These results suggest that genetic exchange between biotypes can occur in the field. Pathogenicity tests indicate that the biotype B strain containing the 1.2 and 2 Mb chromosomes can produce chlorotic symptoms on host species only susceptible to biotype A strains. These data suggest that some pathogenicity functions are encoded on the 2 Mb chromosome. It is suggested that the presence of the 2 Mb chromosome permits survival of biotype B genotypes on hosts such as S. scabra which is the predominant species grown commercially.

20. RAPD Characterization of Alternaria alternata isolates from California tomato fields.

The genetic diversity of 69 isolates obtained from blackmold lesions of ripe tomatoes was assessed using RAPD markers. Two major groups (Gp1 and Gp2) of isolates were identified independently by principal component analysis, and by unweighted pair group method arithmetic average (UPGMA) of Jaccard and simple matching (SM) similarity coefficients. The isolates showed a high degree of homology within each major group (Jaccard similarity (73%; SM = 81%), and 34 of 137 markers were monomorphic for all of the isolates, although the difference between Gp1 and Gp2 was large (Jaccard similarity = 50%; SM = 60%). There was no association between the genetic similarity of isolates and their geographic origin, suggesting wide dispersal of isolates across California. All isolates in the survey were tested for their ability to cause stem canker disease on susceptible tomato cultivars. None of the Gp1 isolates and only three of the 14 Gp2 isolates caused stem canker symptoms. These three isolates clustered together with 11 other isolates in Gp2, indicating a high degree of similarity between these isolates and other isolates causing blackmold disease. However, none of the 137 scored bands could be used to distinguish the three stem canker isolates from the 66 other isolates included in this study. Seven additional toxin- producing isolates were analyzed with RAPDs and found to contain Gp2-specific bands, suggesting that toxin-production may be associated with phenetic group Gp2 only. The biological basis for the large difference between Gp1 and Gp2 isolates is presently unknown.

21. Identification of corn fungal pathogens using RAPD analysis

We have used RAPD analysis to develop a test which can be used to distinguish races of Northern leaf spot (Cochliobolus carbonum anamorph: Bipolaris zeicola) from each other and to specifically detect the presence of the genome of race one of this corn pathogen found in Iowa. Differentiation of several strains of Bipolaris was accomplished by using the RAPD banding patterns produced with a single 10 base primer (5'ccaccgcgcc). This primer amplifies a prominent band of approximately 550 basepairs from the genome of B. zeicola race one (HC-23). The 550 bp fragment specific to HC-23 was cloned using the TA cloning vector pCRII (Invitrogen) and sequenced. The sequence of this cloned fragment was used to design a pair of 30 base primers which specifically amplify an internal 440 base pair fragment of the cloned sequence from (HC- 23) in the presence of a large excess of genomic DNA from Zea mays and/or DNA from other fungi. These results show the feasabilty of developing a rapid field test for the presence of agronomically important pathogens infecting field corn.

22. Possible retrotransposon in the R-pathotype of Tapesia yallundae the causal agent of eyespot disease of cereals

T. yallundae, the perfect stage of Pseudocercosporella herpotrichoides, is the pathogen responsible for eyespot disease of cereals. Pathogenicity tests in conjunction with in-vitro growth characteristics have been used in the past to classify eyespot isolates into the R- and W- pathotypes. On nutrient-rich agar medium, R-types exhibit slow growth with feathery colony margins, whereas W-types grow at twice the rate of the R-types with even colony margins. During growth in-vitro, R-types often exhibit cultural instability, forming variable sectors. Reversion of sectors to the parental phenotype suggests the possible activity of a transposable element. To investigate this hypothesis, primers were constructed from homologous regions of known fungal transposons (from Cladosporium fulvum and Magnaporthe grisea) to use in PCR amplification. A fragment generated by this approach has been cloned and sequenced and further investigation is underway to establish the presence and activity of a retrotransposon.

23. Expression of peroxidase, phenylalanine-ammonia lyase, ethylene and cutinase genes in Brassica napus in response to infection by virulent and weakly virulent isolates of Leptosphaeria maculans

The relative levels of mRNAs were monitored by Northern blot hybridization of leaf tissue RNA samples, at varying intervals following infection. Variation in the level of peroxidase mRNA was observed with the use of radiolabelled Expressed Sequencing Tags (EST) clones as probes. A high level of peroxidase mRNA was induced in plants inoculated with both pathotypes. Hybridization with radiolabelled PAL gene DNA, showed no detectable PAL message in uninfected tissue, while induction was observed after infection with both isolates. Similarly, ethylene induction--also a defense-related response--was followed in infected leaf tissue. In addition, the status of cutinase gene expression was assessed during infection. Cutinase mRNA was induced when the plant was infected with both isolates. With the avirulent strain as the inoculum, at 24 h post-infection the cutinase mRNA level was high, registering a steady decline thereafter. The opposite trend was witnessed with the use of the virulent strain as inoculum, i.e. low levels at 24 h and a steady increase thereafter.

24. A virus-resistant mutant of Cryphonectria parasitica

NB58F is a virus-resistant mutant derived from a sector of an infected isolate of Cryphonectria parasitica, the chestnut blight fungus. Morphologically, NB58F is distinguishable from both infected and non-infected isolates of C. parasitica. Mating, RFLP, and fingerprint studies have demonstrated that the NB58F morphology and virus resistance are inherited together as nuclear traits. Molecular karyotype analysis has shown that a small (~3 mB) chromosome is present in NB58F and not in the wild-type isolate. The NB58F morphology and virus resistance were inherited with the 3 mB chromosome in ascospore progeny. Although NB58F has not been stably infected with any cytoplasmic virus, it supports stable infection with the mitochondrial virus from strain NB631. Short term infection of NB58F with full-length transcripts of the cytoplasmic virus CHV1-713 was successful, demonstrating that cytoplasmic dsRNA replication can occur in NB58F. However, virus-infected transfectants were not stable and virus was lost upon frequent sectoring. Together, these results suggest that the NB58F mutation may affect movement of cytoplasmic viruses while not affecting mitochondrial movement.

25. Reversion of a disrupted toxin biosynthetic gene restores high virulence in Gibberella zeae

Gibberella zeae is an important pathogen of cereal crops and produces trichothecene mycotoxins, including deoxynivalenol (DON). Previously, to determine whether trichothecenes contribute to the virulence of G. zeae, we generated DON nonproducing mutants of the fungus by transformation mediated disruption of the gene (Tri5) encoding the first enzyme in the trichothecene biosynthetic pathway. The virulence of Tri5- transformants, in which gene replacement had occurred, was reduced on some host plants relative to the Tri5+ strain from which they were derived. To confirm that the reduced virulence of Tri5- transformants was specifically due to the loss of Tri5 function and not some unrelated effect of transformation, we generated revertants of G. zeae by selfing a transformant that had undergone additive gene disruption. According to PCR analysis, three out of 160 single ascospore progeny had undergone reversion from Tri5- to Tri5+. However, only one of the three revertants produced DON. The DON producing revertant exhibited wild type levels of virulence on wheat seedlings (cv. Wheaton) while all the DON nonproducing progeny examined, including the two DON- revertants, had the same reduced levels of virulence as the parental Tri5- transformant. These data indicate that the reduced virulence of the DON nonproducing transformants resulted from disruption of Tri5 and provide further evidence that trichothecene production contributes to the virulence of G. zeae.

26. RAPD-bulked segregant analysis based mapping of a Gibberella fujikuroi gene involved fumonisin biosynthesis

The maize pathogen Gibberella fujikuroi mating population A (Fusarium moniliforme) produces fumonisins, a family of secondary metabolites that are toxic to a number of plant and animal species. A locus, designated fum1, that confers the ability to produce fumonisins was previously identified from a cross of fumonisin producing (fum1+) and nonproducing (fum1 ) strains of G. fujikuroi (Xu and Leslie, 1993 FG Newsletter 40A:25). We are using RAPD combined with Bulked Segregant Analysis to map and eventually clone fum1. Bulked DNA consisted of pools of DNA from 25 fumonisin producing or fumonisin nonproducing ascospore progeny. To date, 260 RAPD primers have been screened. We have identified two primers, OPA16 and OPH3, that amplify a fragment from the fum1 parent and fum1 pooled DNA, but not from the fum1+ parent or pool. RAPD analysis with DNA from individual progeny indicated that the markers amplified by OPA16 and OPH3 are located on opposite sides of fum1 at distances of 14.4 and 13.1 centimorgans, respectively.

27. The interaction between a non-pathogenic mutant of Glomerella magna and plant hosts

Biochemical and microscopic analyses have been performed on anthracnose resistant and susceptible plants infected with the wild type (L2.5) and a non- pathogenic mutant (path-1) of G. magna. The temporal and spatial expression of phenylalanine ammonia lyase and peroxidase enzyme activities were monitored in plants inoculated with either L2.5, path-1, or colonized for 48 hours with path-1 followed by inoculation with L2.5 (cross protected). Cross protection against L2.5 by path-1 was not systemic and appeared to be tissue specific. The growth of L2.5 and path-1 in anthracnose susceptible and resistant plant tissues was observed by differential staining and microscopic assessment. The production of appressoria in plant tissues was also monitored microscopically. G. magna specific PCR primers were also used to monitor the growth of these fungi through plant tissues. The mRNA differential display system was used to identify transcript differences between L2.5 and path-1.

28. Isolation of pathogenicity and virulence genes of Cladosporium fulvum

Cladosporium fulvum is a hemibiotroph pathogen of tomato which causes leaf mould on its host plant. A C. fulvum isolate (Cf4) was mutagenized by transformation with a plasmid carrying a hygromycin resistance gene (pAN7-1). Individual tranformants were isolated and used to inoculate tomato seedlings and transformants that failed to form the normal conidiophores on the leaves 2 weeks after inoculation were selected for further analysis. 4550 tranformants were screened in this way and 15 mutants were found. Eight of these mutants do not differ in any obvious way from wild type C. fulvum when grown on artificial media. Two classes of phenotypes can be described: (I) pathogenicity mutants, i.e. those which do not show complete the infection cycle on the tomato leaf (3 mutants) and (II) virulence mutants i.e. transformants which do eventually form conidiophores on the tomato leaves but do so with a noticeable delay and/or cause evidently reduced symptoms on the plant (5 mutants). In the poster, we report on progress made in characterising some of these mutants and in cloning DNA from regions flanking the plasmid insertion sites. Our immediate aim is to establish whether, in any case, the phenotype is determined by insertion of the plasmid in the target gene.

29. Plant flavonoids and isoflavonoid phytoalexins stimulate germination of Fusarium solani spores independent of nutritional stimulation

Flavonoids exuded by plant roots act as important signals for most rhizobial bacteria to initiate an interaction with legumes leading to the formation of nitrogen-fixing root nodules. We show here that flavonoids, including stress-related isoflavonoids, induce spore (macroconidia) germination in Fusarium solani f.sp. pisi, (isolates of N. haematococca MPVI) a soil-borne fungal pathogen of pea. The action of specific flavonoids in stimulating germination paralleled the specificity of nod gene induction in the bacterial pea symbiont, Rhizobium leguminosarum bv. viciae. A similar parallel was observed between F. solani f.sp. phaseoli, a bean pathogen, and the bean symbiont R. leguminosarum bv. phaseoli. The flavonoid-responsive germination appears to utilize a signal pathway different than nutrient-responsive germination because the former is prevented by inhibitors of cAMP-dependent protein kinase (PKA), while the latter is not. Germination of macroconidia in root exudates was significantly inhibited by the PKA inhibitor, indicating that flavonoids may be as or more important than nutrients as a stimulatory signal in root exudates. Thus, flavonoids in root exudates may play two roles in legumes, initiating interaction with beneficial bacterial symbionts but also inducing F. solani to emerge from dormancy and initiate a pathogenic interaction with its host plant.

30. Characterisation of the pathogenicity gene MPG1 from the rice blast fungus Magnaporthe grisea

Magnaporthe grisea infects its host by producing a specialized cell known as an appressorium. This cell works by adhering tightly to the leaf surface and generating high internal turgor which is translated into the mechanical force necessary to break the underlying plant cuticle. Recently, we identified a gene known as MPG1 which appears to play an important role in the elaboration of appressoria. The gene was identified as a fungal transcript produced abundantly in planta. Temporal analysis revealed that MPG1 was highly expressed as soon as 18h after inoculation of rice seedlings and was also expressed during disease symptom expression 72- 96h later. A directed gene replacement showed that MPG1 is required for efficient appressorial development and mpg1-mutants therefore showed a reduced pathogenicity phenotype. MPG1 appears to encode a hydrophobin-like protein with homology to the Sc3, Sc1 and Sc4 genes from Schizophyllum commune, the rodA gene from Aspergillus nidulans and the eas gene from N. crassa. Consistent with this, M. grisea mpg1 mutants show an 'easily wettable' phenotype showing that cell surface hydrophobicity of aerial hyphae is reduced. Hydrophobins are unusual proteins which are known to be produced during aerial growth of fungi. Hydrophobins appear to undergo self-assembly into high molecular weight amphipathic complexes when they reach interfaces between liquids and air. Such physical characteristics would predict a number of potential roles for MPG1p in appressorial development and pathogenesis. It is, for example, conceivable that MPG1p acts either as a structural component of the appressorium, or as an adhesion protein. Its secretion and potential incorporation into cell wall complexes may therefore be a rate limiting step in the transduction of the inductive signals required for appressorial morphogenesis. In order to test these hypotheses a number of strategies have been adopted to purify and characterise MPG1p and to determine its precise role in the pathogenesis of M. grisea. Progress in these areas was discussed.

31. Evidence of a horizontal transfer event between phytopathogenic fungi in the genus Leptosphaeria and its correlation to host-range expansion

Phoma lingam (teleomorph: Leptosphaeria maculans) and Phoma wasabiae are closely related species that cause similar disease symptoms on different plants in the crucifer family. All the examined isolates of Phoma lingam that are aggressive to Brassica species possess a repetitive element, LMR1. With one exception, no isolates of the related Phoma wasabiae, that is aggressive to the crucifer Wasabi japonica, have been found to contain LMR1. The apparent horizontal transfer of the element to this isolate correlates with the expansion of its host-range to include Brassica juncea. The structure of LMR1 will be described and models will be presented for how it could become an extrachromosomal element, thus facilitating transfer. In addition, our hypotheses about how this transfer may induce host-range expansion are presented.

32. Cercosporin efflux and partial auto-resistance are encoded by the LE6 gene

The fungal pathogen of soybean Cercospora kikuchii produces the polyketide phytotoxin cercosporin, an essential pathogenicity factor for the development of disease. Light is required for the induction of cercosporin synthesis. We have used light induction to isolate light-enhanced cDNAs by a subtractive hybridization technique. One of these cDNAs, LE6, shows enhanced transcript accumulation 20 fold higher in light and is correlated with the accumulation of cercosporin in culture. The nucleotide sequence of this cDNA contains a putative open reading frame of 1818 base pairs that encodes a predominatly hydrophobic, cysteine rich peptide of 606 amino acids with a molecular weight of 65,424 and an isoelectric point of 5.08. Kyte-Doolittle analysis of the peptide indicates that LE6 contains six potential alpha helical regions, three of which stretch long enough to span the plasma membrane. Gene disruption of LE6 results in dramatically reduced cercosporin production in the light, loss of pathogenicity on soybean, diminished transcript accumulation of another light-enhanced cDNA, an altered pigment accumulation profile, and substantial loss of auto-resistance to cercosporin. An amino acid sequence homology search has identified two regions of homology common to efflux-mediated drug resistance determinants in the amino half of the protein, suggesting that LE6 belongs to the subgroup of the major facilitator protein superfamily that catalyzes efflux-mediated drug resistance.

33. Physical, genetic, and molecular characterization ofthe TOX2 locus, a complex locus controlling pathogenicity in Cochliobolus carbonum

The TOX2 locus of C. carbonum race 1 encodes the biosynthetic activities required for the production of HC-toxin. HC-toxin, a cyclic tetrapeptide, is a host-selective toxin which is the key molecule involved in the pathogenicity of the fungus on specific genotypes of maize. In all wildrace 1 strains tested, there are two copies of the TOX2 locus. The gene encoding the major HC-toxin biosynthetic activity, HTS1, has been cloned and characterized. HTS1 encodes an enzyme, HTS, which is involved in non-ribosomal peptide bond formation between the amino acids in HC-toxin. Several other genes (TOXA, TOXC, TOXD) unique to race 1 strains have also been cloned and are currently being analyzed. The TOXA gene encodes a putative integral membrane HC-toxin efflux protein. The TOXC gene contains an open reading frame with amino acid sequence similarity to yeast fatty acid synthases and may be involved in the biosynthesis of the novel HC-toxin amino acid, AEO. The TOXD gene is a Tox+ unique gene of unknown function. In addition, there is evidence of Tox+ unique regulatory gene(s). The organization of the genes within the TOX2 locus was analyzed using contour-clamped homogeneous electric field (CHEF) gel electrophoresis and sexual crosses.

34. Comparison of three xylanase genes in the plant pathogenic fungus Cochliobolus carbonum

Three endo-xylanases genes have been cloned from C. carbonum. Xyl1 is the major endo- xylanase and accounts for 85%-94% of the xylanase activity when grown in culture as well as the major protein peak when culture filtrate is fractionated by HPLC cation exchange. A degenerate oligonucleotide had been generated from direct internal amino acid sequence of Xyl1, and this oligo was used to clone XYL1 and XYL3. XYL2 was cloned from a cDNA library prepared from fungus grown on corn cell walls using XYL1 as a heterologous probe. XYL2 and XYL3 have a 62% and 42% identity respectively to XYL1 at the amino acid level. In addition, all three of these xylanases are similiar to xylanases from other organisms. The expression of the three xylanases is currently being examined. XYL3 expression has not been seen nor a cDNA clone been found in the cDNA library prepared from fungus grown on corn cell walls. Transformation-mediated gene disruption of XYL1 was already conducted and pathogenicity tests of the mutant indicate that XYL1 is not required for pathogenicity. Transformation-mediated gene disruption of XYL2 and XYL3 is currently in progress. The single endoxylanase mutants will be crossed with each other to obtain multiple disruption mutants and these will be tested for pathogenicity on maize.

35. Genetics of gibberellin biosynthesis by Gibberella fujikuroi

The rice pathogen Gibberella fujikuroi is well known as a producer of large amounts of gibberellins which induce the superelongation ("bakanae") disease on infected rice seedlings. By GC MS it could be demonstrated that the ability to produce gibberellins is widespread among fungi, but only rice pathogenic isolates of the species Gibberella fujikuroi (mating population C) seem to be deregulated and produce these phytohormones in high amounts. The genetics of gibberellin biosynthesis and the relationship to the biosynthetic pathway in higher plants are not well understood until now. Therefore, we have started to isolate the genes of the isoprenoid pathway as well as specific gibberellin genes. So far, the genes for HMG-CoA-reductase, the geranyl-geranylpyrophosphate and farnesylpyrophosphate synthetases were isolated by screening of genomic and expression libraries of this fungus with heterologous probes or with PCR- fragments synthesized on the basis of sequence homologies to other fungal prenyltransferases. Sequence comparison with analogous fungal or plant genes showed that the genes of this pathway are very conserved.

36. Victorin-deficient REMI mutants of Cochliobolus victoriae demonstrate a requirement for victorin in pathogenesis

C. victoriae is a pathogen of oats and produces victorin C, a cyclized pentapeptide which specifically affects oats containing the Vb allele; all other oat genotypes are resistant to both the toxin and the fungus. We have used Restriction Enzyme Mediated Integration (REMI) to mutate genes involved in victorin C biosynthesis. Eight mutants altered in victorin C production were isolated after screening approximately 620 hygromycin B-resistant transformants by automated HPLC analysis of crude culture fluids. One is a null mutant, several are leaky (< 0.5-5% of wild type), and two are overproducers of victorin C (3-4 X that of wild type). The null mutant produces no detectable toxin and is unable to cause disease symptoms on oats in leaf uptake, root inhibition, and whole plant assays. The leaky mutants cause symptoms on whole plants but much more slowly than wild type. The null mutation is untagged, as determined by insertion point rescue and gene disruption experiments. Similar analyses are being conducted on the other mutants with altered victorin production. To identify genes encoding a particular enzyme involved in victorin biosynthesis, we have prepared degenerate primers and isolated PCR products with sequence similarity to known cyclic peptide synthetases. These products will be used to investigate enzyme functions by gene disruption methods.

37. Molecular cloning of the gene for the host-specific toxin produced by Pyrenophora tritici-repentis

We are investigating the key events that regulate specificity in the disease, tan spot of wheat, caused by the fungus Pyrenophora tritici-repentis. The host specific toxin(s) produced by P. tritici-repentis is a protein, which provides a straight forward experimental approach to the molecular cloning of the gene. Analysis of purified toxin by mass spectroscopy indicated a molecular weight of 13.2 kD. Polyclonal and monoclonal antibodies were raised against purified toxin. Western analyses indicated that the antibodies reacted with and are specific to a 13.2 kD band associated with toxic activity. Time course studies comparing toxin production and toxic activity in culture filtrates were performed. Poly (A+) RNA was prepared from mycelia from time periods for the maximum rate of toxin production. In vitro translations of isolated total mRNA were performed and the relative abundance of the Tox message evaluated by indirect immunoprecipitations of total translation products with anti-Tox antibody. Results indicated the presence of a single, ca. 20kD translation product that reacted with anti-Tox antibody. Immunoprecipitation analyses suggest the presence of a precursor form of the toxin as expected for a secreted protein. A lambda cDNA library was prepared and screened with anti-Tox antibody. Antibody-positive recombinants were identified at a high frequency that reacted with both the monoclonal and polyclonal antibodies. We have sub-cloned a ca. 750 bp cDNA from an antibody positive recombinant and are currently conducting nucleotide sequence analysis.

38. Pathogenicity related gene expression in the entomopathogenic fungus Beauveria bassiana

The Deuteromycete fungus, Beauveria bassiana along with other fungi, is being developed as a potential biological control agent for insect pests. The pathogenicity of an entomopathogenic fungus is largely determined by its ability to penetrate the host cuticle, overcome the host defense responses and propagate inside the host body. We have mainly been working on the invasion processes by the fungus which include the coordinated secretion of cuticle-degrading enzymes and the differentiation of penetration structures. In our laboratory, we have cloned genes which are induced when the mycelia is grown in nutrient-deprived media containing a host (cockroach) cuticle. These genes encode an array of enzymes and a putative toxin, indicating strategies by which the fungus may penetrate host barriers and subdue the insect.

39. Sexual and asexual mechanisms influence genetic variation in Pythium ultimum

We are interested in studying genetic, morphological, and pathogenic variation in Pythium ultimum, a homothallic soil-borne oomycete. P. ultimum isolates are capable of outcrossing under lab conditions. Genetic analysis of isolates belonging to the Pythium taxonomic group HS (self- sterile isolates with cultural characteristics similar to P. ultimum) demonstrated that these isolates can cross with homothallic P. ultimum isolates. The group HS isolates may represent heterothallic variants of P. ultimum. F2 segregation ratios and karyotypes detected by PFG electrophoresis are consistant with diploidy for several homothallic and group HS isolates. Diploid populations segregating for sporulation rate and sensitivity to inhibitory chemicals have been established for genetic analysis. Exceptions to diploidy also exist in P. ultimum. For example, three alleles were detected in isolate Pu18 with RFLP marker PG123. Variation in allele number, karyotype, growth rate, and pathogenicity was detected in subcultures generated from single hyphal tips and single sporangia of Pu18. These results suggest that polyploidy and heterokaryosis may contribute to non-Mendelian allele numbers and that asexual mechanisms can contribute to variation. We are in the process of extending our observations to field populations. Using co-dominant PCR markers and RFLP markers we have found that field isolates are often heterozygous at unlinked loci and genetically distinct individuals are frequently isolated from the same lesion. These results support the occurrence of outcrossing in nature.

40. Location of pisatin demethylase genes on diverse dispensable chromosomes in the phytopathogenic fungus Nectria haematococca

Conventional genetic analysis has identified six genes (PDA) in the ascomycete Nectria haematococca that encode for the ability to demethylate the pea phytoalexin pisatin. It has been observed that the phenotype associated with PDA genes is occasionally lost during crosses. Miao, et. al. (Science 254:1773) showed that the loss of Pda in isolates carrying PDA6-1 was due to loss of a 1.6 Mb dispensable chromosome carrying PDA6-1. Progeny from crosses involving two independently segregating genes, PDA1 and PDA5, also exhibited loss of Pda. Southern analysis confirmed that loss of Pda was due to loss of one or both genes. CHEF analyses showed that PDA1 is on a 1.5 Mb chromosome that is absent in PDA1 progeny. PDA5 is found on a 4.9 Mb chromosome in the original Pda+ parent from these crosses but on polymorphic chromosomes, in the range of 3.5 to 5.7 Mb, in progeny from this parent. Analyses of crosses between PDA5 carrying parents showed loss of PDA5. All these PDA5 progeny had an aberrant colony morphology which suggested that not all DNA lost along with PDA5 is dispensable. Electrophoretic karyotyping of field isolates and their progeny has shown that chromosomal bands carrying different PDA genes are absent in Pda progeny. Southern analysis of CHEF gels using DNA from a PDA1-bearing chromosome as a probe, has shown that most of the PDA chromosomes thus far tested cross-hybridize with this chromosome, one notable exception being the PDA5 chromosome. There is no hybridization of the PDA1 chromosome with the chromosomes of Pda isolates, indicating that all or part of these PDA-bearing chromosomes are dispensable.

41. Transformation of Botrytis cinerea: system and applications

Botrytis cinerea, the causal agent of grey mould on various fruits, is an economically important pathogen of grape. Different hydrolytic enzymes secreted by B. cinerea have been characterized, among them, pectin methylesterase (PME, Reignault et al., 1994) might be involved in its pathogenicity. The control of B. cinerea by fungicides is not satisfactory as this fungus is variable genetically and resistant strains have appeared all over the world. A transformation system is an essential tool for developing new weapons against this fungus. Using a plasmid carrying the hygromycin phosphotransferase gene (hph) of E. coli (Hamada et al., 1994), we have obtained transformants expressing different levels of resistance to hygromycin. The hph gene was integrated into B. cinerea genome, often as tandem arrays and in different locations and was expressed at variable levels. Further investigations of these transformants are needed to understand variable gene expression as well as to analyse the segregation of integrated copies. Transformation will be helpful to clone fungicide resistance genes by complementation and to generate specific mutants by gene disruption (for exemple for PME). Developing transformation as a tool of mutagenesis will help to understand the pathogenicity of B. cinerea. We are improving transformation using REMI (restriction enzyme-mediated insertion). Hamada, Reignault, Bompeix, Boccara (1994) Current Genetics 26:251-255. Reignault, Mercier, Bompeix, Boccara (1994). Microbiology in press

42. Structural analysis of melanin biosynthetic genes of Colletotrichum lagenarium

Melanin biosynthesis of Colletotrichum lagenarium is essential for appressorial penetration. Melanin deficient albino mutants form colorless appressoria and can not penetrate host cucumber leaves and also cellulose membranes, a model substrate for host leaves. The melanin is considered to confer structural rigidity to appressorial walls needed for focusing the turgor forces in the vertical direction to facilitate penetration from pore formed at the basal area of appressorium. Three types of mutants defective in different steps in melanin biosynthetic pathway have been isolated. Albino mutant 79215 (Pks ) was defective in the polyketide synthesis, mutant 9201Y (Scd ) was defective in the dehydration of scytalone to 1,3,8-trihydroxynaphthalene and mutant 9141 (Thr ) was defective in the reduction of 1,3,8-trihydroxynaphthalene to vermelone. The three melanin biosynthetic genes, PKS1, SCD1 and THR1 were cloned and the structural analysis of these genes was made by resolving DNA sequence and their transcriptional structure. The predicted amino acids encoded by PKS1 and THR1 were 2187 and 241, respectively. PKS1 has significant homology with polyketide synthase gene, particularly Aspergillus nidulans wA involved in conidial pigmentation, and THR1 shares high homology with Ver1 gene involved in aflatoxin biosynthesis of Aspergillus parasiticus and the polyhydroxynaphthalene reductase gene of Magnaporthe grisea. The three genes were not clustered in a cosmid level.

43. Genetic improvement and the molecular basis of fungal pathogenesis

Entomopathogenic fungi represent an untapped reservoir of pesticidal genes for the production of advanced engineered pestcides; an important consideration given that the lack of "useful" pesticidal genes for transfer has been a major constraint in the implementation of biotechnology in crop protection We are assembling a bank of pathogenicity related genes from Metarhizium anisopliae and Beauveria bassiana which could be used to transform other fungi, bacteria, or viruses to create novel combinations of insect specificity, or to produce transgenic plants with improved resistance to insect pests.To perform these studies, specific vectors are being constructed which facilitate strain construction to enhance virulence using constitutive and regulatory promoter regions for expression of homologous and heterologous genes. The potential for this approach has been demonstrated by transferring the gene for the Prl protease from M. anisopliae to Aschersonia aleyrodis, which consequently became a pathogen of late instar whitefly. We have developed a direct strategy for engineering enhanced virulence in M. anisopliae by constitutive expression of some of the many, normally inducible anti-insect proteins. Our initial candidates for this approach have been genes encoding cuticle-degrading enzymes and toxins, since the active agents are encoded by single genes and have been shown to be active in vitro against insects. Constitutive expression of Prl was obtained by transforming M. anisopliae with cDNA for Prl behind the Neurospora crassa cross pathway control promoter. Transgenic strains continued to produce Prl in the haemocoel of Manduca sexta caterpillars following penetration of the cuticle causing extensive melanization in the body cavity and cessation of feeding 30-40 hr earlier than controls infected with wild type. These studies provide the first conclusive demonstration of the utility of heterologous gene expression in molecular analysis of the insect- fungus interaction, and for strain construction of improved mycoinsecticides.

44. New mutations involved in T-toxin production by Cochliobolus heterostrophus are unlinked to the Tox1 locus

Crosses between race T (which produces T-toxin) and race O (which does not produce T- toxin) of C. heterostrophus segregate 1: 1 for parental types, thus defining a gene (Tox1) controlllng toxin biosynthesis and high virulence to the host plant, corn. Twelve Tox1 mutants, generated previously, all map at Tox1, suggesting that this locus contains all the informatlon needed for T-toxin productlon. Tox1 is actually two loci, each on a different chromosome; both are linked to the breakpoint of a reciprocal translocation. Here we report identification of two additional loci unlinked to Tox1 which appear to be involved in T-toxin production. The two loci were revealed by REMI mutagenesis; mutants produce halos smaller than wild type in a microbial toxin assay. When mutants are crossed with race T, only parental progeny segregate (1 : 1); all Tox progeny contain the REMI plasmid, indicatlng each mutation is tagged. When crossed with race O, progeny segregate 25% wild type halo, 25% mutant halo and 50% no halo, indicating that the mutations do not map at Tox1. This conclusion is confirmed by crosses to any induced mutant which maps at Tox1, in which progeny segregate 25% wild type halo, 25% no halo, and 50% parental halos. When the two mutants are crossed to each other, proqeny segregate 25% wild type halo, 50% parental type and 25% double mutant (less leaky than either parental mutant), indicating the two mutations are unlinked to each other. The transforming plasmid with flanking DNA was recovered and retransformed into wild type race T; the original mutant phenotype was restored.

45. Phylogenetic relationship among PDA genes

DNA sequence for seven Nectria haematococca MP VI PDA genes which encode the enzymatic detoxification of the phytoalexin antibiotic pisatin is compared using a phylogenetic analysis technique. This analysis demonstrates that there are distinct lineages of PDA genes. PDA genes which encode a highly active pisatin demethylase phenotype (PDAH) share the closest relationship and have all been associated with virulence on pea. The gene PDA5 is the most divergent among the PDAH genes, consistent with its unique origin: this gene was found in an isolate from mulberry in Japan while all of the other highly active PDA genes are from isolates obtained from pea in the USA. Straney and VanEtten have shown that there is a region of DNA upstream from the translation start site of PDA 1 that is involved in the activation of PDA genes following exposure to pisatin (MPMI 7:256-266). While this sequence, called the pisatin responsive element, is found in all of the PDAH genes, this region is interrupted by a small insertion in all of the genes conferring the low activity pisatin demethylation phenotype. DNA sequence is also presented for homologs of the Nectria PDA gene in Fusarium oxysporum f. sp. pisi. Comparing all of these PDA genes with other fungal P450 gene sequences demonstrates that this is a unique family of genes in phytopathogens of pea.

46. The TOXA gene of Cochliobolus carbonum encodes a putative HC-toxin pump

The TOXA gene of C. carbonum is tightly linked to the HTS1 gene which encodes the multifunctional HC-toxin synthetase, HTS. Both genes are found only in toxin-producing strains of C. carbonum and TOXA message is expressed at very low levels in the cell. The TOXA gene has an open reading frame which encodes a putative protein of 58kD. Hydropathy plots of the TOXA amino acid sequence suggest that the protein is an integral membrane protein with 10-13 membrane spanning domains. The amino acid sequence is similar to bacterial and fungal proteins known to be small molecule efflux pumps, and we propose that the TOXA gene product is an HC-toxin pump. There are two linked copies of the TOXA gene in C. carbonum; deletion of either copy has no measurable effect on the cell's ability to produce HC-toxin or infect maize. However, despite many attempts, it has not been possible to construct strains with both copies of the TOXA gene disrupted. This suggests that TOXA mutants are lethal in an HC-toxin producing background due to the intracellular accumulation of HC-toxin.

47. Neutral versus selective evolution of proteins: data from the human pathogen Coccidioides immitis (Ascomycota, Onygenales)

The question of whether evolution is mainly due to selection or random drift can be addressed using DNA sequence data from protein coding loci and comparing the ratio of replacement to synonymous substitutions within and between species. A higher ratio of replacement to synonymous substitutions between species than within indicates that species divergence represents adaptation to different environments (positive selection changing amino acids), while equal ratios within and between species suggest speciation by a random accumulation of mutations (neutral model). We are studying evolution at a variety of protein-coding genes in the ascomycete fungus Coccidioides immitis, a human pathogen causing valley fever, and its closest known relatives, the nonpathogenic Uncinocarpus reesii and Auxarthron zuffianum. We have obtained data for CHSl, coding for chitin synthase, an enzyme located on the cell membrane and participating in cell wall synthesis, and for a gene coding for 4 hydroxy-phenylpyruvate dioxygenase (4HPPD), a protein identified as a human T-cell antigen of C. immitis, found on the cell membrane and cell walls of conidia and spherules, the infective and proliferative stages of the disease in humans.

48. Molecular markers and sex in the human pathogen Coccidioides immitis

Microorganisms are not easily studied in nature and even determining whether a species has sex can be difficult or impossible by direct observation. Coccidioides immitis, a dimorphic fungal pathogen and cause of a recent epidemic of "Valley fever" in California, is typical of many eukaryotic microbes in that mating and meiosis have yet to be reported, but it is not clear if this is because sex is absent, or just cryptic. To find out, we have undertaken a molecular population genetic study to test for the clonal structure expected if sex is truly absent. Molecular markers with nucleotide level resolution were found by a novel approach combining PCR amplification with arbitrary primer pairs, screening for single strand conformation polymorphisms (SSCPs), and direct DNA sequencing. Both population genetic and phylogenetic analyses indicate that C. immitis is almost completely recombining, with little evidence of clonal structure. These results suggest that sex is a regular part of the C. immitis life cycle and illustrate the utility of SSCP and sequencing with arbitrary primer pairs (SWAPP) in molecular population genetics.

49. Possible regulation of pathogenicity genes by nitrogen catabolite expression in Magnaporthe grisea

Carbon and nitrogen catabolite repression have recently been implicated in regulation of genes required for pathogenesis in both plant pathogenic bacteria and fungi. We recently demonstrated that Mpg1, a Magnaporthe grisea gene that expressed in planta and involved in pathogenicity is induced by nitrogen and carbon starvation. Thus we examined the role of nitrogen catabolite repression in the regulation of genes involved in pathogenesis. We investigated whether mutations at the major transcriptional activator, Nut-1 (for Nitrogen utilization) would render M. grisea non-pathogenic. Three hundred and eighty chlorate resistant mutants were isolated and classified according to their defects in nitrogen metabolism. Five putative nut-1 mutants were identified, and two of these mutants, Cl60 and Cl105 have reduced pathogenicity and toxin production. In addition to the commonly observed ammonium-dependent growth, they have also lost the ability to regulate Mpg1 during nitrogen and carbon starvation. Genetic analysis of Cl60 and Cl105 demonstrates that these mutations define two unlinked genes. Surprisingly, neither gene is allelic to Nut-1. We named Cl60 and Cl105 as Npr1-1 and Npr1-2 (for Nitrogen and pathogenicity regulation) respectively. We hypothesize that Npr1-1 and Npr1-2 may act as regulators that integrate nutritional starvation and pathogenesis related pathways. To begin to test this hypothesis, cloning of these genes by complementation is underway.

50. SAR gene expression in maize

Systemic acquired resistance (SAR), induced by biotic or chemical activation offers broad host resistance to a variety of pathogens (Ross, 1961; Kuc, 1982; Metraux et al., 1991). In response to some pathogens many plants form necrotic lesions on their leaves. Lesion mimic mutants form lesions in the absence of pathogens, stress, or wounding (Walbot et al., 1983). Arabidopsis lesion mimic mutants simulate the disease response by expression of histochemical and molecular markers (Dietrich et al. 1994), formation of secondary metabolites, deposition of callose in and around necrotic lesions, and SAR gene expression. To examine if maize lesion mimics possess these characteristics, plants were grown to maturity and tissue harvested before and after lesion formation. RNA was extracted and analyzed for SAR gene expression by northern analysis. Chemical inducers of resitance also induces SAR genes in maize similar to what has been reported in other systems (Ward et al. 1992, Uknes et al 1992, 1993). References: Alexander et al., PNAS 90: 7327-7331 (1993); Dietrich et al., Cell, in press (1995); Kuc, BioScience 32:854-860 (1982); Metraux et al., in Advances in Molecular Genetics of Plant-Microbe Interactions, Vol. 1, H. Hennecke and DPS Verma, eds. pp. 432-439 (1991); Ross, Virology 14: 329-339 (1961a); Ross, Virology 14:340-358 (1961b); Uknes et al., The Plant Cell 4:645-656 (1992); Uknes et al., The Plant Cell 5:159-169 (1992); Walbot et al., in Genetic Engineering of Plants, T. Kosuge and C. Meredeth, eds., pp. 431-442 (1983); and Ward et al., The Plant Cell 3:1085-1094 (1991).

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