61. Mitochondrial hypovirulence in KFC9, a Cryphonectria parasitica strain obtained from nature.
Dipnath Baidyaroy, David H. Huber, Dennis W. Fulbright and Helmut Bertrand, Michigan State University.
In C. parasitica, the chestnut blight fungus, a hypovirulence phenotype can be often transmitted from strain to strain by hyphal anastomoses (cytoplasmic transmission), and is caused by infection of the fungus by dsRNA viruses. However, hypovirulent strains have been isolated from nature which lack any dsRNA viruses but are capable of rendering virulent strains hypovirulent on hyphal anastomoses. These strains also show high levels of alternative oxidase activity, indicating that they must be deficient in cytochrome-mediated respiration and hence in mitochondrial functions. KFC9 is one such strain isolated from a healing canker on a tree in Michigan. Analysis of the mitochondrial DNA (mtDNA) of KFC9 and its derivatives (virulent strains rendered hypoviruient by contact with KFC9) revealed that a particular region of the mtDNA of KFC9 was responsible for the infectious hypoviruience phenotype. That region of the mtDNA was cloned and was found to be the small subunit rRNA gene with an inserted piece of DNA just 84 bp downstream of the beginning of the gene. This peculiar DNA is 973 bp long, has no sizable open reading frames and does not have homology to any existing sequence. The molecular basis of the infectious nature of this DNA is not clear at this time.
Supported by USDA grant 95-37303-1785.
62. Inhibition of pathogenicity of the rice blast fungus by S. cerevisiae -factor.
Janna L. Beckerman, Fred Naider*, and Daniel J. Ebbole. Texas A&M Universeity. *CUNY, Staten Island.
Magnaporthe grisea is a fungal pathogen of rice that forms a specialized cell necessary for pathogenesis, the appressorium. Saccharomyces cerevisiae -factor pheromone blocked appressorium formation in M. grisea in a mating type-specific manner in vitro and protected plants from infection by a MAT1 -2 strain.
Experiments with pheromone analogs suggest that the observed activity is due to the specific
interaction of -factor with a M. grisea receptor. Culture filtrates of MAT1-1 strains contained a
putative pheromone activity that specifically inhibited appressorium formation of mating type MAT1-2
strains. These findings provide evidence that a pheromone response pathway exists in M. grisea that
can be exploited for plant protection.
63. Map-based cloning of pathogenicity determinants of Nectria haematococca MPI.
Joanna K. Bowen, Brian T. Hawthorne, Jonathan Rees-George, Rod D. Ball and Erik H.A. Rikkerink. The Horticultural and Food Research Institute of New Zealand Ltd., Mt. Albert Research Centre, Auckland, New Zealand.
Genetic analysis of pathogenicity of Nectria haematococca MPI (Fusarium solani f. sp. cucurbitae,
race 1) on unwounded hypocotyls of Cucurbita maxima cv. Crown (buttercup squash) has
demonstrated that it is controlled by a major effect virulence (MEV) gene superimposed on a
quantitative background. A population of 800 random ascospore progeny was derived from a cross
between a high pathogenicity and a low pathogenicity isolate. A colony pigmentation gene has been
found to be closely linked to the MEV gene. Bulked DNA samples from progeny exhibiting the
highest and lowest pathogenicity phenotypes are being screened to identify RAPD markers which are
linked to the MEV locus. The high pathogenicity and low pathogenicity parents have different
mini-chromosome profiles. However, the MEV gene does not appear to be located on a
mini-chromosome. A partial BAC library with an average insert size of approximately 80 kb has been
constructed from the high pathogenicity parent. A full library is under construction. The region
containing the MEV gene will mapped and identified by chromosome walking and subsequent
complementation of a low pathogenicity isolate.
64. Identification of virulence determinants of A. fumigatus using a REMI/STM approach.
J.S.Brown, A.Aufauvre-Brown, N.Tiffin, D.W.Holden, Dept. Infectous Diseases, RPMS, London UK.
Pulmonary infection due to Aspergillus fumigatus is a major cause of morbidity and mortality in immunocompromised patients. Research using A.fumigatus strains carrying disruptions in genes thought to have a role in virulence have generally failed to prove the importance of these genes in animal models infection. Recently our laboratory has developed a method, signature tagged mutagenesis (STM), which can identify avirulent mutants by screening pools of different mutants in a single animal. We have combined restriction enzyme mediated integration (REMI) with STM to screen mutant strains of A.fumigatus for loss of virulence. REMI was employed for single copy insertional mutagenesis of a clinical isolate of A.fumigatus using 96 plasmids, each carrying a different oligonucleofide tag and the hph gene conferring hygromycin resistance. Pools of 96 mutants each carrying a different tag were assembled by combining one transformant from each different REMI transformation. Groups of 5 mice were inoculated by intranasal infiltration with spores representing all mutants from a particular pool. Fungal DNA recovered from infected lungs on days 3 and 4 post-inoculation was used as a target for PCR using primers to amplify the oligonucleofide tags. The PCR product was used to probe a dot blot membrane containing the original 96 tagged plasmids. Those tags present in the inoculum but not present in the recovered DNA will identify potentially avirulent mutants that can be further characterized. Using an avirulent A.fumigatus auxotroph we have shown the feasibility of STM/REMI screening to identify avirulent mutants from an inoculum pool of 90 mutants. By screening mutant pools we expect to identify new virulence genes and improve our understanding of the pathogenesis of pulmonary aspergillosis.
65. Development of a REMI-transformation procedure for the mutational tagging of genes in Paecilomyces fumosoroseus.
Frank A. Cantone and John D. Vandenberg, USDA/ARS Plant Protection Research Unit. US Plant, Soil, & Nutrition Lab, Tower Road, Ithaca, NY 14853.
We have developed a transformation system for an entomopathogenic fungus, Paecilomyces
fumosoroseus, that relies on restriction enzyme-mediated integration, under the selection of the
herbicide, bialaphos. This procedure is being used as a tool for the mutational tagging of genes and
the creation of transformants deficient in some aspect of pathogenicity. Depending upon the enzyme
and vector used, transformation frequencies were either unaffected or increased versus the non-REMI
treatments. Restriction mapping and Southern hybridization analyses of transformant DNAs revealed
that plasmids inserted randomly throughout the genome. However, results also revealed that
integrations occurred other than by a simple ligation-repair model. From the pattern of REMI events
revealed by Southern analysis we were unable to regenerate the original restriction sites of the
plasmid with the enzyme initially used in the transformation procedure. Instead we observed that one
or both sites were destroyed during the integration of the plasmid in the genome. We are now
characterizing several pigmentation and pathogenicity mutants obtained with this transformation
procedure.
66. The interaction between fungal polygalacturonases and plant PGIPs.
Claudio Caprari Benedetto Aracri, Alessandra Devoto, Fiona Leckie, Benedetta Mattei, Laurence Nuss, Giovanni Salvi, Giulia De Lorenzo and Felice Cervone. -Dipartimento di Biologia Vegetale, Universita "La Sapienza", Roma, Italy.
Recognition of endopolygalacturonase (PG), a factor important for pathogenesis and therefore to be
maintained by fungi during evolution for successful parasitism, may be exploited by plants as a clever
strategy to establish incompatibility. Modular leucine-rich repeat proteins, structurally related to
some of the resistance gene products recently characterized, recognize and inhibit fungal PGs. These
proteins, named PGIPs have been found in the cell wall of many dicotyledonous plants and some
monocotyledonous plants. We are studying at the genetic and biochemical level the interaction
between PGs from different fungi and PGIPs of Phaseolus vulgaris L. We have introduced, by site-directed mutagenesis, different point mutations in the PG from Fusarium moniliforme and produced
mutated proteins with a range of activity between the 48% to 0% with respect to the wild type. The
interaction between the variant enzymes and PGIP of P. vulgaris has been investigated using a
biosensor based on surface plasmon resonance. All the variant enzymes were still able to interact and
bind to PGIP with association constant comparable to that of the wild type enzyme. A family of pgip
genes is present in the genome of P. vulgaris and the composition of the family is different among
different cultivars. A major objective of our laboratory is to know how many PGIP proteins exist,
under what circumstances they are expressed and their specificity. We have evidence indicating that
the members of the gene family encode PGIP proteins with distinct specificities.
67. Differential Gene Expression During Infection by the Fruit-rot Fungus Glomerella cingulata.
Sarah J Clark1, Matthew D Templeton2 & Patrick A Sullivan3. 1Department of Biochemistry, University of Otago, PO Box 56, Dunedin. 2HORT Research, Mt Albert Research Centre, Private Bag 92169, Auckland. 3Department of Biochemistry, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Glomerella cingulata (Colletotrichum gloeosporioides) is a fungal pathogen of pip and subtropical fruits. In New Zealand it is best known for causing bitter rot of apples. Penetration of the host surface involves spore germination followed by a morphological differentiation to form an appressorium. Appressorium formation is essential for infection, therefore genes expressed during appressoria formation are of interest as they may be potential targets for control of the fungus. To facilitate our studies of genes expressed during appressoria formation, apple wax is used to induce G. cingulata spores to form appressoria in vitro. In addition, cap22 (Hwang & Kolattukudy, 1995) is being used as a control marker for appressoria formation.
Differential Display PCR has been investigated as a method of identifying genes uniquely expressed during appressoria formation. Currently, we have isolated two abundant surface proteins and we plan to study the expression of these proteins during appressoria formation.
Reference: Hwang, C.S. & Kolattukudy, P.A. (1995) Mol Gen Genet 247:282-294.
68. Gregatin-deficient mutants of Phialophora gregata.
Edmund H. Crane, and Charlotte R. Bronson, Iowa State University, Ames, IA 50011.
Phialophora gregata, a deuteromycete that causes Brown Stem Rot in soybean, produces a family
of antimicrobial and phytotoxic furanones, collectively known as gregatins. These compounds have
been proposed to cause the leaf necrosis and premature defoliation characteristic of Brown Stem
Rot-infected soybeans. The role of gregatins in causing Brown Stem Rot symptoms is being tested
using REMI mutagenesis. A transformation protocol for P. gregata was developed and used to insert
pUCATPH containing hygB into wild-type P. gregata. Hygromycin-resistant transformants were
screened for inability to produce gregatins as demonstrated by loss of their ability to inhibit the
growth of Bacillus subtilis; several putative gregatin-deficient mutants have been identified. Assays
are in progress to verify the loss of gregatin production and to determine whether these mutants have
reduced virulence on soybeans.
69. Disruption of a cutinase gene (cutA) in Fusarium solani f. sp. cucurbitae race 2 does not affect virulence and tissue specificity toward cucurbits.
Ross N. Crowhurst, Stephanie J. Binnie, Joanna K. Bowen, Brian T. Hawthorne, Kim M. Plummer, Jonathan Rees-George, Erik H. A. Rikkerink and Matthew D. Templeton. Molecular Genetics Group, Horticulture and Food Research Institute of New Zealand Ltd., Mt. Albert, Auckland, New Zealand.
A 3.9-kb genomic DNA fragment from the cucurbit pathogen Fusarium solani f. sp. cucurbitae race
2 was cloned. Sequence analysis revealed an open reading frame of 690 nucleotides interrupted by
a single 51-bp intron. The nucleotide and predicted amino acid sequences showed 92 and 98%
identity respectively, to the cutA gene of the pea pathogen F. solani f. sp. pisi. A gene replacement
vector was constructed and used to generate cutA- mutants which were detected using a PCR assay.
Seventy-one cutA- mutants were identified among the 416 transformants screened. Vector integration
was assessed by Southern analysis in 23 of these mutants. PCR and Southern analysis data show the
level of homologous integration was 14%. Cutinase activity (as measured by non-specific esterase
activity) was reduced in cutA- mutants to levels not significantly different from culture medium alone.
Disruption of the cutA locus in mutants was confirmed by RNA gel blot hybridization. Neither
virulence on Cucurbita maxima 'Delica' at any of six different inoculum concentrations, nor
pathogenicity on intact fruit of four different species or cultivars of cucurbit or hypocotyl tissue of
C. maxima `Crown' was found to be affected by disruption of the cutA gene.
70. Pathogenicity determinants of Pyrenopeziza brassicae.
Katy A. Davies, Alison Ashby and Keith Johnstone, Department of Plant Sciences, Cambridge University, UK.
Pyrenopeziza brassicae is a hemibiotrophic ascomycete. It causes the disease light leaf spot of
Brassicas and is thus economically important. Circumstantial evidence suggests a role for cutinase
in penetration of the host cuticle. P. brassicae does not develop specialised infection structures
indicating that breaching of the cuticle is mechanical, and SEM studies of infected leaves show that
host entry is not via stomata. Liquid cultures induced with apple cutin show an increase in esterase
activity which is absent in uninduced cultures. The esterase activity is inhibited by ebelactones A and
B. Incubation of the culture filtrate with tritiated cutin caused the release of soluble radioactivity.
However analysis of the products using TLC suggests that they do not correspond to those of cutin
breakdown. Several approaches are being followed to try to detect a plant-inducible cutinase.
Heterologous probing and a PCR directed approach have indicated that if there is a P. brassicae
cutinase, then it lacks homology to those characterised to date. The current approach is the use of
a subtractive cDNA cibrary. A subtracted library was constructed to enrich for fungal and plant-inducible genes and which may include a cutinase gene. Differential screening has isolated several
plant-induced fungal scenes and these are being further characterised.
71. Avirulence genes cloning in the rice blast fungus Magnaporthe grisea.
Waly Dioh1, Didier Tharreau2, Marc Orbach3, Jean - Loup Notteghem2, Marc - Henri Lebrun1. 1Universite Paris Sud, France, 2CIRAD/CA, France, 3University of Arizona.
Magnaporthe grisea is an Ascomycete responsible for the major fungal disease of rice. Three
genetically independent avirulence genes, avrMedNoi-1, avrIrat7-1, avrKu86-1, were identified in a
cross between isolates Guy11 and 2/0/3. Using 77 random progeny, we constructed a partial genetic
map with 75 RFLP and 25 RAPD markers. Two avirulence genes mapped near chromosome tips
(avrMedNoi-1 and avrKu86-1). The last avirulence gene, avrIrat7-1, mapped on chromosome one
at 30 cM from avrCo39. Using bulk segregant analysis, we identified 16 RAPD markers linked to
these avirulence genes (0-15 cM), some being completely linked to avrIrat7-1 or avrKu86-1. Most
of these RAPD markers corresponded to junction fragments between M. grisea genome and known
transposons. We screened a cosmid library of an avirulent progeny, either by hybridisation with single
copy markers or by RAPD analysis of cosmid pools (repeated sequence markers). Contigs were
started from markers linked to avrIrat7-1 and avrKu86-1. One of the cosmids from avrIrat7-1 locus
confered avirulence to rice cultivar Irat7, when introduced by transformation in a virulent recipient
strain.
72. Molecular genetics of turgor generation within the appressorium of Magnaporthe grisea. Katherine Dixon, Joke de Jong, B.McCormack, N.Smimoff, J.R.Xu* and N.J.Talbot. University of Exeter, Devon, UK. *Purdue University,USA
Magnaporthe grisea , in common with many plant pathogenic fungi, uses specialised cells known as appressoria to provide an entry route into its host plant. Appressoria are specialised, dome shaped cells which facilitate the infection of plants either by enzymatic means or via the generation of internal physical pressure, forcing a penetration peg through the rice leaf surface. We have shown that during the development of appressoria, the fungus accumulates a variety of solutes to extremely high concentrations. We suggest that accumulation of these solutes, contained within a semi-permeable melanised cell produces an osmotic gradient, drawing free water into the appressorium, inflating the cell and applying a physical pressure to the penetration peg. Biochemical analysis of the contents of appressoria have revealed the most abundant solute to be glycerol. In budding yeast, the HOG pathway (High Osmolarity Glycerol Pathway) is a signal transduction pathway which allows the cell to accumulate intra-cellular glycerol in response to external osmotic stress. We reasoned that M.grisea may use elements of the HOG pathway in order to regulate glycerol accumulation within appressoria during turgor generation. In order to test this hypothesis we have identified and characterised a homologue of the mitogen activated protein kinase (MAPK) encoding gene HOG1 in M. grisea. We have constructed a gene disruption vector and are currently working towards characterising a hog- M.grisea strain.
73. Analysis of AVR1-CO39 avirulence gene locus from Magnaporthe grisea.
Mark L Farmanl,3 and Sally A Leongl,2. 1Department of Plant Pathology, and 2USDA-ARS Plant Disease Resistance Research Unit, University of Wisconsin, Madison, USA. (3 present address: Department of Plant Pathology, University of Kentucky, Lexington KY, USA).
A Magnaporthe grisea cultivar specificity gene toward rice cultivar C039 was previously mapped to
one arm of chromosome I between RFLP markers CH5-120H and 5-10-F. These RFLP markers map
11.6 cM and 17.2 cM, respectively, on either side of Avrl-CO39. Using Achilles' cleavage methods,
CH5-120H and 5-10-F were shown to be separated by 600 kb. A chromosome walk to Avr1-CO39
was initiated from these markers and over 500 kb have been covered in 20 walking steps. Cosmid
clones cosegregating with Avrl-CO39 have been identified and selected clones obtained from the walk
were shown to confer avirulence on rice variety C039 but not 51583 when introduced by
transformation in the virulent M. grisea strain Guyl1. The DNA conferrring avirulence has been
delimited to a 1.05 kb region. Four putative open reading frames were identified by DNA sequence
analysis and the precise location of the AVR1-CO39 gene is being defined by transcript analysis, site-directed deletion of ATG codons and by introduction of frameshifts into each ORF. Hybridization
studies of DNA of the virulent M. grisea strain Guy 11 indicates that approximately 20 kb of DNA
at the Avrl-CO39 locus is deleted and that a repetitive DNA species is present at the deletion
breakpoint. This deletion eliminated AVR1-CO39 from the Guy11 genome.
74. Catalase as a potential pathogenicity factor in Clavicepes purpurea.
Victoriano Gaffe1,2, Klaus B. Tenbergel, Paul Tudzynski1. 1Botanical Institute, Westf. Wilhelms-University, D-48149
Muenster, Germany; 2University of Murcia, Spain
Claviceps purpurea colonizes young ovaries of grasses andcereals, finally establishing a stable,
balanced interaction. In following up a combined biochemical, cytological and molecular genetic
approach to indentify pathogenicity factors in the system C purpurea/rye, we found that C. purpurea
- apart from cell wall degrading enzymes - secretes catalase activity in planta; this was shown by in
situ activity staining, immunogold labelling, and biochemical analysis of infected tissue and honeydew
(in comparison to axenic culture). We cloned a gene (cat1) coding for a catalase enzyme that most
probably is secreted, since it contains a signal peptide and several glycosylation sites. We could show
by RT-PCR that cat1 is expressed in planta. A potential function of this enzyme during the parasitic
cycle could be inactivation of active oxygen species generated by the host (peroxidase activity and
lignification have been demonstrated as host defense reactions confirming the presence of H2 O2).
Targeted gene disruption experiments combined with detailed cytological analyses are under way to
clarify the role of the cat1 product (or fungal catalases in general) during the interaction.
75. Isolation of genes involved in phytoalexin detoxification in Leptosphaeria maculans through differential display.
Kevin S. Gellatly1, Janet L. Taylor1 and M. Soledade Pedras2, 1NRC-Plant Biotechnology Institute, Saskatoon, SK, Canada S7N 0W9; 2Department of Chemistry, University of Saskatchewan, Saskatoon, SK, Canada S7N 5C9.
Leptoshaeria maculans is a heterothallic ascomycete that causes blackleg or stem canker disease in
oilseed rape and other brassica plants. Virulent strains of the fungus rapidly detoxify the phytoalexin
brassinin, or its analog -chlorobenzyl dithiocarbamate. We have identified the major metabolic
products of the pathway and deduced that at least three enzymes are involved. Differential display
analysis was conducted on cDNA derived from mRNA isolated from 47 h fungal cultures, with and
without exposure to 50 µg/mL -chlorobenzyl dithiocarbamate. Seventy-seven differentially amplified
cDNA fragments were isolated using twelve anchored poly(T15) primers and a mixture of twenty 9
base oligonucleotides (56 or 67% GC). Eleven of these cDNAs showed putative differential
expression after rescreening by reverse-northern dot blot analysis. The cDNA fragments were purified
from contaminants by PCR-SSCP (PCR-single-strand conformation polymorphism) and again
subjected to reverse-northern dot blot analysis. Nine cDNA fragments with sizes ranging from 250-720 base pairs were subcloned and sequenced. The results of the characterization of the clones will
be presented.
76. Isolation and characterization of 2 xylanase genes from the fungal pathogen Claviceps purpurea.
Sabine Giesbert and Paul Tudzynski. Westfalische Wilhelms Universitat, Institut fur Botanik, Schlossgarten 3, 48149 Munster.
The phytopathogenic fungus Claviceps purpurea is a common parasite on grasses and cereals. It could be shown that while colonizing the Secale cereals ovary the fungus grows intercellularly as well as intracellularly. As the primary cell walls of monocotyledonous plants contain approximately 40% arabinoxylan, xylan-degrading enzymes probably play a role during infection of Secale cereals. 2 xylanase encoding genes (xyl1 and xyl2) were isolated from a genomic library of C. purpurea using heterologous probes. Xyll exhibits great homology to family G xylanases whereas xyl2 belongs to family F. Expression of both genes in planta were studied by RT-PCR showing that both xyl1 and xyl2 are expressed during all phases of infection of Secale cereals. Secretion of xylanases during the infection could also be demonstrated by tissue printing experiments using heterologous antibodies as probes (Lepping, unpubl.). A disrupted copy of xyl1 was used to create a Xyl1 lacking mutant by transformation mediated gene replacement. Disruption of xyl1 results in reduced total xylanase activity in axenic culture, Western analyses using monoclonal antibodies raised against Trichoderma reesei Xylanase Xynl as a probe show the disappearance of a crossreacting band with a MW of 30,000 strongly indicating that this is the xyl1 gene product. In order to elucidate the role of xylanases for pathogenicity of C. purpurea the xyl1 mutant is currently being examined in pathogenicity tests anct a xyl2 as well as a xyl1/xyl2 double mutant is created.
77. Isolation of genes from Fusarium moniliforme expressed during colonization of maize.
Karoline Hahn and Eugenio Benvenuto, ENEA, Settore Biotecnologie ed Agricoltura, Via Anguillarese 301, 00060 S. Maria Galeria (Roma), Italy.
Fusarium moniliforme is an economically important pathogen of maize and sorghum, causing stem,
root and ear rot on its host plants. In order to understand the molecular processes conditioning
pathogenicity of F. moniliforme fungal genes that show an altered expression pattern during
colonization of maize stems were identified by differential display. Using 38 various primer
combinations, five F. moniliforme cDNA fragments were isolated whose corresponding genes are
exclusively expressed during fungal growth in planta as varified by Northern blot analysis. Sequence
analysis of the cDNA fragments did not reveal any similarity to known genes, probably due to the
shortness of the fragments (394 bp - 578 bp). Therefore, a cDNA libary is now screend to obtain
longer clones.
78. Molecular studies on the cell wall degrading enzymes from Botrytis cinerea II: In planta analysis of the Bcpel1 gene and the endopolygalacturonase gene family.
Arjen ten Have1, Wietse Mulder2, Wendy Oude Breuill, Jaap Visser2 and Jan A.L. van Kan1. 1Department of Phytopathology, Wageningen Agricultural University, Binnenhaven 9, 6709 PD Wageningen, The Netherlands. 2 Section of Molecular Genetics of Industrial Micro-organisms, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.
B. cinerea causes grey mould, a plant disease resulting in serious damage on several economical important crops. During pathogenesis B. cinerea secretes a number of cell wall degrading enzymes. Despite much physiological and biochemical knowledge on these enzymes, there is no clear insight in the infection process. We started a molecular genetic approach resulting in the isolation of several genes (see abstract by Mulder et al.). Here we describe experiments performed on tomato leaves as well as the disruption of one of the isolated genes. Tomato leaves were inoculated with a conidial suspension (106/ml) . At several time points during the pathogenesis, samples were taken for RNA analysis. The results show that Bcpga1 is expressed at the early stages of pathogenesis. Bcpga1 is also expressed when grown on glucose medium. Therefore we decided to disrupt this latter gene. Two Bcpga1 -null mutants were tested in pathogenicity assays. A lower amount of spreading lesions was found for the disruptants on detached tomato leaves (T = 20 C). In addition, assays perfomed at T = 4 C show that the outgrowth of lesions formed by the disruptant, was slower compared to the outgrowth of wild type lesions. Tests performed on ripe tomato fruits showed no observable difference in pathogenicity.
These data, combined with the in vitro expression data, indicate that BcPGl plays a role in the
pathogenesis of B. cinerea on tomato leaves. Experiment aimed at getting further insight in the role
of BcPGl in pathogenesis are in progress.
79. RAPD analysis of the New Zealand population of Venturia inaequalis.
Megan Hemming1, Kim Plummer2, Mike Pearson2, Jo Bowen 1, Erik Rikkerink1. 1HortResearch, Auckland; 2 University of Auckland, New Zealand.
Apple scab caused by Venturia inaequalis is the most commercially important fungal disease of apple.
Currently none of the commercial apple cultivars used in New Zealand are resistant to the disease,
and control in the orchard relies on a rigorous fungicide regime. Breeding resistance to V. inaequalis
into commercial apple cultivars is a major focus of apple breeding programmes worldwide. Seven
races of the fungus have been identified. Since the primary inoculum is derived from ascospores a
high degree of variation in the fungus would be expected. We are currently investigating this
variation at the molecular level. Monoconidial V. inaequalis isolates (39) were collected from
infected leaves and fruit of 11 different apple cultivars from the major apple growing areas in New
Zealand. RAPD analysis of these isolates revealed a high degree of variation. Only two isolates had
identical banding patterns. Indeed, isolates from the same leaf produced different RAPD patterns.
All isolates shared common major bands, some of which have also been reported by other researchers
for V. inaequalis from a Swiss orchard. There appears to be no correlation between RAPD banding
patterns and the origin of the isolates (apple cultivar or geographical location). We are investigating
whether any markers can be used to identify particular sub-groups such as races.
80. Starvation and stress of Cladosporium fulvum - relations to pathogenesis.
B.Henricot, G.Segers, A.J Clark, K.Blissett, R.P. Oliver. Carlsberg Laboratory, Gamle Carlsberg Vej 10. DK 2500, Copenhagen, Denmark.
Cladosporium fulvum is a biotrophic pathogen of tomato (Lycopersicon esculentum) which lacks specialized infection structures and does not produce obvious extra-cellular cell wall degrading enzymes or host-specific toxins. It has been demonstrated in a number of plant-fungal interactions that genes expressed during starvation may also be those which are expressed during pathogenesis. On this basis, we have used starvation induced conditions to clone genes involved in pathogenesis.
Differential screening of cDNA libraries constructed from carbon and nitrogen starved mycelia has identified 34 starvation induced clones. To test the hypothesis that mRNAs induced during starvation are also induced during infection, Northern blots of starved, non-starved fungus, uninfected and infected plants taken at different stages of infection (7, 10, 12 and 14 days after inoculation) were made. 26 of the 34 clones were not onlv starvation induced but also plant induced. Sequencing has revealed homologies with an alcohol oxidase, aldehyde dehydrogenase, alcohol dehydrogenase, an ATP translocase and hydrophobins. Gene disruption experiments are currently in progress to establish the role of each gene in pathogenicity.
A high percentage of the carbon available to the fungus is converted to mannitol. The role of this and
other polyols in pathogenic fungi remains unclear. We are investigating changes in polyol levels in
C. fulvum under stress conditions to determine whether the high level of mannitol produced by the
fungus mediates some osmotic stress associated with growth in planta, or is involved in other aspects
of pathogenesis.
81. Saponins and plant defence.
V. Hugouvieuxl, J. Wubben1, R. Melton1, L. Flegg2, R. Oliver2, M. Daniels1 and A. Osbourn1. 1Sainsbury Laboratory and 2UEA, Norwich, UK.
Saponins occur in a great many plant species, and have been implicated as pre-formed determinants of resistance to fungal attack. The importance of the saponin-detoxifying enzyme avenacinase, produced by Gaeumannomyces graminis, in determining host range has been demonstrated. At least two other phytopathogenic fungi (Septoria avenae and Septoria lycopersici) produce saponin-detoxifying enzymes which are closely related to avenacinase, suggesting that common mechanisms for saponin detoxification may be widespread. The S. avenae and S. 1ycopersici enzymes detoxify the oat leaf avenacoside and the tomato -tomatine saponins respectively, and their roles in pathogenicity are currently being tested by the generation of fungal mutants. Expression of S. lycopersici tomatinase in the biotrophic pathogen of
tomato Cladosporium fulvum, which is not known to degrade -tomatine, gives increased
pathogenicity to tomato. Interestingly, C. fulvum transformants expressing tomatinase are also able
to cause significantly more infection than control strains in incompatible interactions, suggesting that
saponins may also contribute to variety-specific resistance of tomato to C. fulvum.
82. Identification of pathogenicity related genes from entomopathogenic fungus, Metarhizium anisopliae, using differential display-RT-PCR and immunoscreening of an expression library.
Lokesh Joshi, Raymond J.St. Leger and Donald W.Roberts. Boyce Thompson Institute for Plant research at Cornell University. Tower Road, Ithaca-NY-14853, USA.
Although Metarhizium anisopliae is the best studied entomopathogenic fungus, there is limited
information on specific gene expression during host-pathogen interactions. The objective of this
study was to identify and isolate the full range of fungal genes specifically expressed during growth
on insect host cuticle. Two populations of RNA, one from the fungus grown in the presence of the
insect cuticle, the other from the fungus grown in the absence of the insect cuticle were compared
and analyzed for differences in the expression of mRNA species. Both RNA populations were
reverse transcribed and the cDNA were PCR amplified in the presence of arbitrary 5-end primers.
The resultant PCR products were differentially displayed on polyacrylamide sequencing gels.
Differentially expressed cDNAs were reamplified and sequenced. Along with differential display,
immunoscreening of an expression library was performed with antibodies raised against the total
spectrum of proteins secreted into cultures containing insect cuticle. We have identified and cloned
a very broad spectrum of genes including cuticle degrading enzymes, putative toxins and protein
kinases by employing these two techniques. Analysis of these genes is facilitating a better
understanding of the determinants of fungal pathogenicity to insects and is providing new tools for
biotechnology, e.g.; the production of a transgenic baculovirus expressing a Metarhizium anisopliae
toxin kills insects >50% faster than the wild type baculovirus.
83. Differential cloning and partial characterization of conidia germination and/or appressorium formation stage-specific genes of Magnaporthe grisea.
Takashi Kamakura, Takamasa Kochi, Syuichi Yamaguchi, Tohru Teraoka, and Isamu Yamaguchi. Microbial Toxicology Laboratory, RIKEN Institute, Wako, Saitama 351-01, Japan.
During the infection process of Magnaporthe grisea on rice plants, the conidial germ tube
differentiates a specific infection structure, an appresorium, for penetration into the host. Formation
of appressorium was observed not only on rice leaves but also on synthetic solid substrate like
polycarbonate. Approaches toward isolation of genes specifically expressed during the differentiation
stage of appressorium formation were made. A subtractive cDNA cloning strategy using combination
of biotin labeled driver DNA method and adapter-primed PCR method was efficiently condensed the
differential cDNAs those were presumably expressed during early stage of the development of
conidial germ tube and/or appressorium formation. Part of the 700 colonies containing differential
cDNA candidates were classified by cross colony hybridization and more than 50 independent clones'
partial nucleotide sequences were determined. We have further analyzed some of those cDNA clones
which showed differential expression by Northern hybridization and RT-PCR analysis. cDNA and
genomic DNA structure and presumable character of those clones will be discussed.
84. From pisatin- and nystatin-resistance in Dictyostelium to the amino acid sequence similarity between Neurospora sterol C-14 reductase and human lamin B receptor.
Durgadas P. Kasbekar & A. Prakash, CCMB, Hyderabad 500007, India.
Dictyostelium discoideum, a cellular slime mold, can acquire resistance to toxic concentrations of pisatin (a pea phytoalexin) and nystatin (a polyene antibiotic) following pretreatment with sublethal concentrations of these compounds. Additionally, pisatin-pretreatment can induce nystatin-resistance but the converse is not true. We have isolated a mutant, sup5, that blocks inducible nystatin-resistance but does not affect inducible pisatin-resistance. We are using sup5 to unravel an apparently unusual relationship between the pisatin- and nystatin-resistance mechanisms.
Mutations affecting the sterol biosynthetic enzyme, sterol C-14 reductase, block inducible pisatin-resistance in Dictyostelium. Remarkably, mutations in erg-3, the sterol C-14 reductase gene of
Neurospora crassa also confer a pisatin-sensitive phenotype. This suggests that inducible pisatin-resistance might be conserved between Dictyostelium and fungi. We cloned erg-3 by
complementation of its mutant phenotype and were surprised to find that the encoded protein shared
~40% amino acid sequence identity with the transmembrane (TM) domain of human lamin B receptor
(LBR). To determine whether the human LBR TM domain is also a sterol C-14 reductase we have
constructed recombinant genes that encode chimeric proteins comprising of Neurospora sterol C-14
reductase and human LBR TM sequences and are testing these genes for complementation of the
Neurospora erg-3 mutant phenotype.
85. Avirulence in Mycosphaerella graminicola (anamorph Septoria tritici) is controlled by a complex locus of tightly linked genes.
Gert HJ Kema, Jos GP Koeken & Els CP Verstappen, DLO-Research Institute for Plant Protection (IPO-DLO), Wageningen, The Netherlands.
Mycosphaerella graminicola is a plant pathogenic bipolar heterothallic ascomycete. We study the molecular genetic basis of specificity for wheat species and cultivars in this fungus, using induced necrosis and pycnidium formation as parameters. Isolate IP0323 is avirulent on cvs. Shafir, Veranopolis and Kavkaz whereas isolate IP094269 is virulent on these cultivars. Both isolates are virulent on cv. Taichung 29 and avirulent on cv. Kavkaz/K4500.
IP0323 and EP094269 were crossed and a random F1 progeny was recovered. In addition, 32 BC1 progenies were generated by back crossing F1 individuals to either IP0323 or IP094269. Sixty F1 and 60 BC1 isolates, from three progenies, were assayed for avirulence on the aforementioned cultivars.
As expected, all progeny isolates were virulent on cv. Taichung. None of these isolates carried virulence for cv. Kavkaz/K4500, indicating that the parental isolates carry the same avirulence factor(s) for this cultivar. Avirulence for each of the differentiating cultivars inherited as a single gene. However, these avirulences cosegregated. Thus the entire F1 andBC1 progenies showed the parental types. This suggests that the avirulences for these cultivars are tightly linked. We hypothesize the presence of a complex locus of tightly linked avirulence genes since cvs. Shafir, Veranopolis and Kavkaz are considered to carry different resistance factors. Inheritance of other markers, such as the mating type genes and RAPDS, was independent of the avirulence loci.