40. Genetic analysis and mapping of metalaxyl resistance loci in Phytophthora infestans.
Anna-Liisa Fabritius1, Samuel Roberts1, Richard Shattock2 and Howard S. Judelson1, 1University of California, Riverside USA, 2University of Wales, Bangor, UK.
Previous genetic studies have suggested that resistance by oomycetes to the phenylamide
fungicide, metalaxyl, is controlled by single loci exhibiting incomplete dominance. To study if the
same locus determines resistance in different strains of Phytophthora infestans, pedigrees in which
that phenotype was segregating were derived from resistant isolates from Mexico, The
Netherlands and the United Kingdom. The segregation of resistance in the progeny indicated that
resistance was determined primarily by one locus in each isolate, and that some of the isolates
were heterozygous and others homozygous for the resistant allele. DNA markers linked to
resistance in the Mexican and Dutch isolates were obtained by bulked segregant analysis using
random amplified polymorphic DNA (RAPD) markers, and mapped by RAPD or restriction
fragment length polymorphism (RFLP) analysis in the three pedigrees. The results suggested that
the same locus conferred resistance in the Mexican and Dutch isolates, based on the linkage of the
markers and resistance in those isolates. However, in the British isolate those DNA markers were
unlinked to the resistance gene. This implied that resistance can result from several different genes
in P. infestans, or alternatively that the same gene is always involved but that chromosomal
rearrangements exist within the species that altered its linkage relationship with the DNA markers.
41. Genetic and physical variability at the mating type loci of Phytophthora.
Howard S. Judelson, Anna-Liisa Fabritius, and Thomas A. Randall, University of California, Riverside, California, USA.
A single but highly unusual locus determines mating type in heterothallic species of the
oomyceteous genus, Phytophthora. DNA markers linked to the A1 and A2 mating types of P.
infestans and P. parasitica were isolated and used to characterize the physical and genetic nature
of mating type determination in these diploid species. Our data indicate that the A1 type is
heterozygous for two mating type alleles (M/m) while the A2 type is homozygous (m/m). In P.
infestans, the mating type locus segregates in a non-Mendelian manner consistent with its linkage
to a system of balanced lethals. However, that locus displays normal segregation in P. parasitica,
which raises the question of why and how such an unusual system evolved in P. infestans.
Physical analyses of the chromosomal interval bearing the mating type locus of P. infestans
indicated that the region was prone to duplication, transposition, deletion, and other
rearrangements which could be related to the mechanism of evolution of the apparent balanced
lethals; such aberrations were not observed for loci unlinked to mating type. In addition, genetic
studies identified several strains in which the mating type locus had become duplicated or
translocated to new regions of the genome. Chromosome walking studies are underway which
should reveal the genetic and physical nature of both the mating type loci and the factors
responsible for their unusual segregation in P. infestans.
42. Characterisation of genes encoding proteins present in large peripheral vesicles of Phytophthora cinnamomi zoospores.
JS Marshall, JM Wilkinson, T Moore and AR Hardham, Australian National University, Canberra ACT, Australia
Zoospores of Phytophthora contain several characteristic types of peripheral vesicles. One of
these, large peripheral vesicles (Lpv), has been proposed to act as a nutrient store and has been
shown to contain three immunologically-related high molecular weight proteins. We have used
antibodies directed against P. cinnamomi zoospores and cysts to isolate several cDNA clones
encoding one of the proteins present in Lpv. Northern blot analysis demonstrated the presence of
three large transcripts (9-14 kb) in mRNA isolated from hyphae which had been induced to form
sporangia. Co-ordinate accumulation of the three transcripts occurred after induction of
sporangial formation: no transcript was observed in uninduced hyphae and maximum transcript
levels were seen 4-6 h after induction. Genomic Southern blots indicated that P. cinnamomi
contains three lpv genes. Genomic clones representing two of the lpv genes were isolated and
characterized by restriction mapping and partial DNA sequencing. The genes were >99%
identical; the high degree of conservation extending at least 385 bp downstream of their
polyadenylation sites. The lpv coding regions contained a variable number (approximately 9-15)
of highly conserved 534 bp repeats, flanked by unique sequences. Variation in the number of
repeats in the lpv genes was responsible for the different sizes of the three transcripts and
proteins. Database searches using the lpv nucleotide and deduced amino acid sequences failed to
detect any similar sequences. We discuss the molecular events which may have been involved in
the evolution of the lpv genes and the nature of the products of these genes.
43. Cloning of avirulence genes in Phytophthora sojae.
Wei-Xing Shan1, Felipe Arredondo1, Helga Forster2, Michael D. Coffey2, and Brett M. Tyler1. Departments of Plant
Pathology, 1University of California, Davis, CA 95616, and 2Riverside, CA 92521.
There are 13 major resistance (Rps) genes in soybean against the oomycete pathogen P. sojae,
and more than 37 races of the pathogen. We have demonstrated that avirulence against many of
these Rps genes is controlled by single dominant genes, using crosses between three isolates of P.
sojae. We are attempting to isolate the tightly linked Avr1b and Avr1k genes using a genetics-based positional cloning strategy. Towards this goal, bulked segregant analysis was used to find
linked molecular markers. Two tightly linked Random Amplified Polymorphic DNA (RAPD)
markers were used to start chromosomal walking. A BAC (Bacterial Artificial Chromosome)
contig of 200 kb genomic region from Race 2 spanning 5 CentiMorgans was constructed.
Restriction Fragment Length Polymorphism (RFLP) markers dispersed across this region were
developed and analyzed on two F2 populations derived from crosses between Race 2 and Race 7,
and Race 2 and Race 19. These progeny segregate for Avr lb and Avrlk, respectively. A total of
110 F2 progeny of cross Race 2 X Race 7, and 48 F2 of cross Race 2 X Race 19 were
characterized. Preliminary results suggest that both Avr gene loci are within a region of 150 kb
with a possible location of the Avrlb locus within a single Notl fragment of 25 kb. Genetic
analysis also indicates that mutation of Avrlb Race 2 does not involve insertion or deletion events.
This enables isolation of functional Avr1b gene by cloning single Notl fragment from Race 7.
Progress in isolating avirulence genes from P. sojae will be reported.
44. Molecular cloning and characterization of a cDNA that is differentially expressed in steroid hormone (antheridiol)-treated A. ambisexualis mycelia.
Julie C. Silver, Evangelia Tomais, Pavel Milman, Thilaka Ponnampalam and Shelley Brunt. University of Toronto, Scarborough.
In Achlya ambisexualis strain E87, sexual differentiation resulting in the formation of gametangia
(antheridia), can be induced by adding the steroid hormone antheridiol to vegetatively growing
mycelia in culture. Differential hybridization of cDNAs constructed from RNA populations
isolated from hormone-treated and non-treated mycelia, yielded several clones. Among these was
a cDNA clone representing an RNA present at very high levels in hormone-treated mycelia but at
only basal levels in untreated (vegetative) mycelia. This cDNA clone was used to isolate a
genomic clone containing the 5' flanking region of the corresponding gene, in order to investigate
the sequence motifs present. In the event that nuclear runon's confirm that the gene is regulated by
antheridiol at the transcriptional level, the sequence information may help us identify an oomycete
steroid-hormone response element. Both the cDNA and the corresponding gene are being
sequenced. Preliminary sequence information (roughly 400bp/1.8kB) from the cDNA, was
compared with sequences in Genbank. Identities over very small regions to three very different
but nevertheless interesting proteins were found, including the Drosophila 7UP2 steroid hormone
receptor, the E.coli (mitochondrial?) DNA polymerase III chi subunit and mouse and Anabaena
P450 hydroxylases. Partial sequence information from the 5' flanking region of the gene revealed
the presence of an oomycete transcription initiation sequence (INR) (as delineated by Pieterse,
Govers, de Wit and colleagues). Similar INRs were found also in the 5' flanking regions
respectively of A. ambisexualis genes encoding the chaperone and heat shock protein Hsp90, the
mitochondrial chaperone and heat shock protein Hsp60 and the A. ambisexualis actin gene.
Further sequencing of the above and the other differentially expressed cDNAs and genes isolated,
should yield interesting information regarding the gene products involved in gametangial
differentiation and the transcription factors which regulate their expression.
45. An AFLP linkage map of the oomycete Phytophthora infestans.
Theo Van der Lee and Francine Govers. Department of Phytopathology WAU and Graduate School Experimental Plant Sciences, Binnenhaven 9, 6709 PD Wageningen, The Netherlands.
We constructed a comprehensive molecular genetic linkage map of the heterothallic oomycetous
plant pathogen Phytophthora infestans. The map is based on polymorphic DNA markers
generated by the DNA f ingerprint technique AFLP (Nucl. Acids Res. 23: 4407-4414). AFLP
fingerprints were made from single zoospore progeny and 73 Fl progeny from two field isolates of
P. infestans. The parental isolates appeared to be homokaryotic and diploid, their AFLP
fingerprints were mitotically stable and the segregation ratios of AFLP markers in the Fl progeny
were largely Mendelian. Besides 185 AFLP markers, seven RFLP markers, four avirulence genes
and the mating type locus were mapped. The markers are distributed over nine major and ten
minor linkage groups and they cover in total a distance of 861 cM which is approximately 70% of
the calculated genome size. Non-Mendelian segregation ratios were found for the mating type
locus and thirteen AFLP markers all located on the same linkage group.
46. Antisense and sense mediated gene silencing as a tool to suppress production of INF1 elicitin of Phytophthora infestans.
Pieter van West, Sophien Kainoun, Koen de Groot and Francine Govers. Department of Phytopathology, Graduate School of Experimental Plant Sciences, Wageningen Agricultural University, The Netherlands.
Most Phytophthora and Pythium species produce 10 kDa extracellular protein elicitors, generally termed eliciting. Elicitins induce a hypersensitive response in a restricted number of plants, particularly in the genus Nicotiana within the Solanaceae family. Elicitins are thought to act as avirulence factors that restrict the host-range of the pathogen by triggering plant defense responses (Kamoun et al., MPMI 6: 15-25; Yu, PNAS 92: 4088-4094).
Phytophthora infestans, the causal agent of the potato late blight disease, produces an elicitin named INF1. A cDNA clone encoding INF1 was isolated and characterized. Expression of the inf1 gene was found in mycelium grown during various conditions, whereas inf1 expression was not detected in sporangia, zoospores, cysts and germinating cysts (Kamoun et al., MPMI 10: 13-20).
Since P. infestans is a diploid organism and homologous integration of introduced DNA has not
yet been demonstrated, we adopted a gene silencing strategy to inhibit INF1 production.
Therefore, P. infestans was transformed with constructs carrying strong oomycete promoters
fused to the inf1 coding sequence in both antisense and sense orientation. Expression of the
integrated transgenes and the native inf1 gene was studied and the production of extracellular
INF1 protein was analyzed. With the phenotypic characterization of the transformants, the role of
elicitin in host specificity should be determined unambiguously.
47. Genetic analysis of chemotactic response in Phytophthora sojae.
Wang, J., Wu, R., Cheung, W., Tran, P., Morris, P., Tyler,B. Department of Plant Pathology, University of California, Davis, CA 95616
Phytophthora sojae infects roots and stems of soybean plants, primarily by means of zoospores swimming in water in the soil or on the soil. P. sojae zoospores are attracted to the isoflavonoids daidzein and genistein which are present in soybean seeds and exuded by the roots. We have examined a wide variety of compounds having some structural similarity to genistein and daidzein including isoflavonoids, flavones, chalcones, stilbenes, benzoins, benzoates, benzophenones, acetophenones and coumarins, for ability to act as attractants or inhibitors for two isolates of P. sojae, race 2 and 6. Among the 59 tested compounds 43 compounds elicited some response from at least one race, and 30 compounds have been found that elicited different responses from race 2 and race 6. Genetic crosses between the two races has identified at least 3 genes responsible for the differences in specificity.