Abstract: Members of the genus Phytophthora are notorious pathogens with world-wide distribution. The most devastating species include P. infestans, P. ramorum and P. sojae. In order to develop molecular methods for routinely characterizing their populations and to gain a better insight into the organization and evolution of their genomes, we used an in silico approach to survey and compare simple sequence repeats (SSRs) in transcript sequences from these three species. We compared the occurrence, relative abundance, relative density and cross-species transferability of the SSRs in these oomycetes. The number of SSRs in oomycetes transcribed sequences is low and long SSRs are rare. The in silico transferability of SSRs among the species was analyzed for all sets generated, and primers were selected on the basis of similarity as possible candidates for transferability to other Phytophthora species. Sequences encoding putative pathogenicity factors from all three Phytophthora species were also surveyed for presence of SSRs. However, no correlation between gene function and SSR abundance was observed. The SSR survey results, and the primer pairs designed for all SSRs from the three species, were deposited in a public database. In all cases the most common SSRs were trinucleotide repeat units with low repeat numbers. A proportion (7.5%) of primers could be transferred with 90% similarity between at least two species of Phytophthora. This information represents a valuable source of molecular markers for use in population genetics, genetic mapping and strain fingerprinting studies of oomycetes, and illustrates how genomic databases can be exploited to generate data-mining filters for SSRs before experimental validation.
Abstract:Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, adistinct lineage of fungus-likeeukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on Human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world’s population. Current annual world wide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogenis challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here were port these quence of the P. infestans genome, which at ~240 mega bases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for ~74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.
Abstract:Phytophthora species are major plant pathogens infecting herbaceous and woody plants including European beech, the dominant or co-dominant tree in temperate Europe and an economically important species. For the analysis of the interaction of Phytophthora citricola with Fagus sylvatica suppression subtractive hybridization was used to isolate transcripts induced during infection and 1,149 sequences were generated. Hybridizations with driver and tester populations demonstrated differential expression in infected roots as compared to controls and verify efficient enrichment of these cDNAs during subtraction. Up regulation of selected genes during pathogenesis demonstrated using RT-PCR is consistent with these results. Pathogenesis-related proteins formed the largest group among functionally categorized transcripts. Cell wall proteins and protein kinases were also frequently found. Several transcription factors were isolated that are reactive to pathogens or wounding in other plants. The library contained a number of jasmonic acid, salicylic acid and ethylene responsive genes as well as genes directly involved in signaling pathways. Besides a mechanistic interconnection among signaling pathways another factor explainingt he activation of different pathways could be the hemibiotrophic life style of Phytophthora triggering different signals in both stages.
Abstract: The differential expression of four Phytophthora cinnamomi elicitin genes was analysed by Real Time RT-PCR. In in vitro cultures, the a cinnamomin gene showed the highest level of expression, the b-cinnamomin gene(b-cin) was the most inducible, and the HAE transcripts were in low abundance. Transcription of all the elicitins was active during the active growth of the pathogen when infecting cork oak(Quercus suber ) roots, and as host colonization progressed, the level of b-cin expression fell, while that of a-cin rose. In anantisense transgenic strain, the silencing of b-cin also negatively affected the expression of other elicitin genes in the cluster. The reduced in planta growth of the b-cin knock-out is related to the altered pattern of elicitin gene expression, supporting the idea that one of the functions of elicitins is related, directly or indirectly, with pathogenesis.
Abstract:Phytophthora parasitica is a soilborne oomycete pathogen capable of infecting a wide range of plants, including many solanaceous plants. In a first step towards large-scale gene discovery, we generated expressed sequence tags (ESTs) from a cDNA library constructed using mycelium grown in synthetic medium. A total of 3568 ESTs were assembled into 2269 contiguous sequences. Functional categorization could be performed for 65.45% of ESTs. A significant portion of the transcripts encodes proteins of common metabolic pathways. The most prominent sequences correspond to members of the elicitin family, and enzymes involved in the lipid metabolism. A number of genes potentially involved in pathogenesis were also identified, which may constitute virulence determinants.
Abstract: To identify markers for the Phytophthora resistance gene, Rps1-d,
123 F2:3 families were produced from across between Glycinemax
(L.)Merr.Tanbakuro (a Japanese traditional black soybean)and PI103091 (Rps1-d)as an experimental population. The results of virulence tests produced 33 homozygous resistant,61 segregating and 29 homozygous susceptible F2:3 families.The chi-squared test gave a goodness-of-fit for the expected ratio of 1:2:1 for resistant, segregating and susceptible traits, suggesting that the inheritance of Rps1-d is controlled by a monogenic dominant gene. Simple sequence repeat(SSR) analyses of this trait were carried out using the cultivars Tanbakuro and PI103091. Sixteen SSR primers, which produced 19 polymorphic fragments between the two parents, were identified from
41 SSR primers in MLGN. Eight SSR markers were related to Rps1-d, based on 32 of the 123 F2:3 families, consisting of 16 homozygous resistant and 16 homozygous susceptible lines. The remaining 91 families were analysed for these eight markers, and a linkage map was constructed using all 123 F2:3 families. The length of this linkage group is 44.0cM. The closest markers, Sat_186 and Satt152, are mapped at 5.7cM and 11.5cM, respectively, on either side of the Rps1-d gene.Three-way contingency table analysis indicates that dual-marker-assisted selection using these two flanking markers would be eficient.
Abstract:Phytophthora infestans is a devastating phytopathogenic oomycete that causes late blight on tomato and potato. Recent genome sequencing efforts of P. infestans and other Phytophthora species are generating vast amounts of sequence data providing opportunities to unlock the complex nature of pathogenesis. However, accurate annotation of Phytophthora genomes will be a significant challenge. Most of the information about gene structure in these species was gathered from a handful of genes resulting in significant limitations for development of abinitio gene-calling programs. In this study, we collected a total of 150 bioinformatically determined near full-length cDNA
(FLcDNA) sequences of P. infestans that were predicted to contain full open reading frame sequences. We performed detailed computational analyses of these FLcDNA sequences to obtain a snapshot of P. infestans gene structure, gauge the degree of sequence conservation between P. infestans genes and those of Phytophthora sojae and Phytophthora ramorum, and identify patterns of gene conservation between P. infestans and various eukaryotes, particularly fungi, for which genome-wide translated protein sequences are available. These analyses helped us to define the structural characteristics of P. infestans genes using a validated data set. We also determined the degree of sequence conservation within the genus Phytophthora and identified a set of fast evolving genes. Finally, we identified a set of genes that are shared between Phytophthora and fungal phytopathogens but absent in animal fungal pathogens. These results confirm that plant pathogenic oomycetes and fungi share virulence components, and suggest that eukaryotic microbial pathogens that share similar lifestyles
also share a similar set of genes independently of their phylogenetic relatedness.
Abstract: Microsatellite markers were tested to rapidly discriminate five Japanese genotypes (US-1, JP-1, JP-2, JP-3, and JP-4) of Phytophthora infestans. Collected from 1958 to 2007, 111 isolates of Japanese P. infestans were examined using a fluorescent-labeled primer and capillary electrophoresis. Microsatellite marker Pi26 generated specific products for each genotype without any differences in terms of isolation area or year for a particular genotype. The Pi26 marker is a powerful tool for obtaining information on the structure of Japanese populations of P. infestans.
Abstract: Plant pathogenic Phytophthora species are predicted to encode a large arsenal of effector proteins that target and disrupt normal host cell function, but genes regulated by effectors are difficult to distinguish from those more generally regulated by microbe associated molecular patterns (MAMPs). To help make this distinction, expression studies of host-Phytophthora interactions can be compared to reveal patterns of gene regulation indicative of effector activity. The recently sequenced tropical fruit tree, Carica papaya, contains fewer genes than Arabidopsis and is highly susceptible to the broad-host-range pathogen P. palmivora, offering a new system for comparative genomics. In this study, genes isolated from the transcriptome of C. papaya seedling roots inoculated with P. palmivora, were evaluated for pathogen-regulation. Of these genes, a predicted peroxidase, ß-1,3-glucanase, ferulate 5-hydroxylase, and hypersensitive-induced response protein were pathogen upregulated, while a second peroxidase (Cp9) and aquaporin (Cp15), both with normally high root expression, were down-regulated. Genes from other plants with similar expression responses to Phytophthora were identified from previously reported studies and evaluated for protein sequence similarity. Interestingly, as for papaya, Phytophthora also down-regulates homologs of Cp9 and Cp15 in Glycine max and Solanum tuberosum, respectively. Because peroxidases associated with defense are generally upregulated during infection and the regulation of aquaporins have been cited in cases of both plant (Opperman et al. Science 263:221–223, 1994) and animal disease (Guttman et al. Cell Microbiol 9:131–141, 2007), Cp9 and Cp15 are potential cross-species effector targets. With whole-genome C. papaya microarrays forthcoming, this study highlights papaya as a resource for comparative genomic studies of Phytophthora-plant interaction.
Abstract: Five thousand and eight hundred publicly available expressed sequence tags (ESTs) of Phytophthora sojae were electronically searched and 415 simple sequence repeats (SSRs) were identified in 369 ESTs. The average density of SSRs was one SSR per 8.9 kb of EST sequence screened. The most frequent repeats were trinucleotide repeats (50.1%) and the least frequent were tetranucleotide repeats (8.2%). Forty primer pairs were designed and tested on 5 strains of P. sojae. Thirty-three primer pairs had successful PCR amplifications. Of the 33 functional primer pairs, 28 primer pairs produced characteristic SSR bands of the expected size, and 15 primer pairs (45.5%) detected polymorphism among 5 tested strains of P. sojae. Based on the polymorphisms detected with 20 EST-SSR markers, the 5 tested strains of P. sojae were clustered into 3 groups. In this study, the SSR markers of P. sojae were developed for the first time. These markers could be useful for identification, genetic variation study, and molecular mapping of P. sojae and its relative species.
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