Abstract: The Avr1b locus is required for avirulence of the oomycete pathogen Phytophthora sojae on soybeans carrying resistance gene Rps1b. One of the Avr genes of the locus (Avr1b-1) was shown to encode an elicitor. We have analyzed the spatial and temporal expression patterns of Avr1b-1 in comparison to defense-related genes induced in soybean. Avr1b-1 expression was detectable mainly in close proximity to the site of infection, in wound-inoculated hypocotyls as well as in roots infected
with zoospores. Usually, in compatible interactions, higher expression levels of
Avr1b-1 were observed in roots when compared with incompatible P. sojae–soybean interactions, whereas neither the timing nor the amount of transcript accumulation of defense-related genes showed cultivar-specific differences. In contrast, the P.sojNIP gene encoding a proposed virulence factor was expressed only during the necrotrophic phase in the compatible interaction.
Abstract: Summary Members of the oomycete genus Phytophthora cause some of the most devastating plant diseases in the world and are arguably the most destructive pathogens of dicot plants. Phytophthora research has entered the genomics era. Current genomic resources include expressed sequence tags from a variety of developmental and infection stages, as well as sequences of selected regions of Phytophthora genomes. Genomics promise to impact upon our understanding of the molecular basis of infection by Phytophthora, for example, by facilitating the isolation of genes encoding effector molecules with a role in virulence and avirulence. Based on prevalent models of plant–pathogen coevolution, some of these effectors, notably those with avirulence functions, are predicted to exhibit significant sequence variation within populations of the pathogen. This and other features were used to identify candidate avirulence genes from sequence databases. Here, we describe a strategy that combines data mining with intraspecific comparative genomics and functional analyses for the identification of novel avirulence genes from Phytophthora. This approach provides a rapid and efficient alternative to classical positional cloning strategies for identifying avirulence genes that match known resistance genes. In addition, this approach has the potential to uncover ‘orphan’ avirulence genes for which corresponding resistance genes have not previously been characterized.
Abstract: The activation of programmed cell death in the host during plant–pathogen interactions is an important component of the plant disease resistance mecha-
nism. In this study we show that activation of programmed cell death in microorganisms also regulates plant–pathogen interactions. We found that a form of vacuolar cell death is induced in the oomycete Phytophthora parasitica
– the agent that causes black shank disease in Nicotiana tabacum– by extracellular stimuli from resistant tobacco. The single-celled zoospores underwent cell death characterized by dynamic membrane rearrangements, cell shrinkage,formation of numerous large vacuoles in the cytoplasm and degradation of cytoplasmic components before plasma membrane disruption. Phytophthora
cell death required protein synthesis but not caspase activation, and was associated with the production of intracellular reactive oxygen species. This characterization of plant-mediated cell death signalling in pathogens will enhance our understanding of the biological processes regulating plant–pathogen interactions, and improve our ability to control crop diseases.
Abstract: Potato cultivars were evaluated for their resistance responses to aggressive strains of Phytophthora infestans in field and laboratory experiments. Analysis of variance revealed differential cultivar-by-isolate interactions for both foliar and tuber blight resistance. Differential responses occur as revealed by specific susceptibilities of cultivars to certain pathogen genotypes and changing rank order. In general, severity of late blight epidemics as observed in the haulms did not correlate well with foliar blight resistance ratings as presented in the National List of Recommended Potato Varieties. No significant correlation was found between tuber blight incidence under field conditions and the tuber blight rating in the National List. Also, there was no relation between the field and laboratory tuber blight resistance assessments. A significant association was demonstrated between late blight infection in the foliage and tuber blight incidence under field conditions. The presence of differential interaction, independent of R-gene-based resistance, indicates some adaptation of P. infestans to partial resistance and consequently adverse effects on the stability and durability of partial resistance to potato late blight.
Abstract: Root and stem rot disease of soybean is caused by the oomycete Phytophthora sojae. The avirulence (Avr) genes of P. sojae control race-cultivar compatibility. In this study, we identify the P. sojae Avr3c gene and show that it encodes a predicted RXLR effector protein of 220 amino acids. Sequence and transcriptional data were compared for predicted RXLR effectors occurring in the vicinity of Avr4/6, as genetic linkage of Avr3c and Avr4/6 was previously suggested. Mapping of DNA markers in a F2 population was performed to determine whether selected RXLR effector genes co-segregate with the Avr3c phenotype. The results pointed to one RXLR candidate gene as likely to encode Avr3c. This was verified by testing selected genes by a co-bombardment assay on soybean plants with Rps3c, thus demonstrating functionality and confirming the identity of Avr3c. The Avr3c gene together with eight other predicted genes are part of a repetitive segment of 33.7 kb Three near-identical copies of this segment occur in a tandem array. In P. sojae strain P6497, two identical copies of Avr3c occur within the repeated segments whereas the third copy of this RXLR effector has diverged in sequence. The Avr3c gene is expressed during the early stages of infection in all P. sojae strains examined. Virulent alleles of Avr3c that differ in amino acid sequence were identified in other strains of P. sojae. Gain of virulence was acquired through mutation and subsequent sequence exchanges between the two copies of Avr3c. The results illustrate the importance of segmental duplications and RXLR effector evolution in the control of race-cultivar compatibility in the P. sojae and soybean interaction.
Abstract: The hrmA gene from Pseudomonas syringae pv. syringae has previously been shown to confer avirulence on the virulent bacterium Pseudomonas syringae pv. tabaci in all examined tobacco cultivars. We expressed this gene in tobacco(Nicotiana tabacum cv. KY14) plants under the control of the tobacco Δ0.3 TobRB7 promoter, which is induced upon nematode infection in tobacco roots. A basal level of hrmA expression in leaves of transgenic plants activated the expression of pathogenesis-related genes, and the transgenic plants exhibited high levels of resistance to multiple pathogens: tobacco vein mottling virus, tobacco etch virus, black shank fungus Phytophthora parasitica [Phytophthora nicotianae var. parasitica] isolate 62, and wild fire bacterium Pseudomonas syringae pv. tabaci. However, the hrmA transgenic plants were not significantly more resistant to root-knot nematodes. Our results suggest a potential use of controlled low-level expression of bacterial avr genes, such as hrmA, in plants to generate broad-spectrum resistance to bacterial, fungal and viral pathogens.
Abstract: The oomycete Phytophthora parasitica is a soilborne pathogen infecting numerous plants. The infection process includes an initial biotrophic stage, followed by a necrotrophic stage. The aim here was to identify genes that are involved in the late stages of infection. Using the host tomato and a transformed strain of P. parasitica expressing the green fluorescent protein (GFP), the various infection steps from recognition of the host to the colonization of plant tissues were studied. This late stage was selected to generate 4000 ESTs (expressed sequence tags), among which approx. 80% were from the pathogen. Comparison with an EST data set created previously from in vitro growth of P. parasitica allowed the identification of several genes, the expression of which might be regulated during late stages of infection. Changes in gene expression of several candidate genes predicted from in silico analysis were validated by quantitative RT–PCR experiments. These results give insights into the molecular bases of the necrotrophic stage of an oomycete pathogen.
Abstract: The importance of segmental duplications and copy number variants as a source of genetic and phenotypic variation is gaining greater appreciation, in a variety of organisms. Now, we have identified the Phytophthora sojae avirulence genes Avr1a and Avr3a and demonstrate how each of these Avr genes display copy number variation in different strains of P. sojae. The Avr1a locus is a tandem array of four near-identical copies of a 5.2 kb DNA segment. Two copies encoding Avr1a are deleted in some P. sojae strains, causing changes in virulence. In other P. sojae strains, differences in transcription of Avr1a result in gain of virulence. For Avr3a, there are four copies or one copy of this gene, depending on the P. sojae strain. In P.sojae strains with multiple copies of Avr3a, this gene occurs within a 10.8 kb segmental duplication that includes four other genes. Transcriptional differences of the Avr3a gene among P. sojae strains cause changes in virulence. To determine the extent of duplication within the superfamily of secreted proteins that includes Avr1a and Avr3a, predicted RXLR effector genes from the P. sojae and the P. ramorum genomes were compared by counting trace file matches from whole genome shotgun sequences. The results indicate that multiple, near-identical copies of RXLR effector genes are prevalent in oomycete genomes. We propose that multiple copies of particular RXLR effectors may contribute to pathogen fitness. However, recognition of these effectors by plant immune systems results in selection for pathogen strains with deleted or transcriptionally silenced gene copies.
Abstract: The interaction between soybean and the phytopathogenic oomycete Phytophthora sojae is controlled by host resistance (Rps) genes and pathogen avirulence (Avr) genes. We have mapped the Avr1a locus in F2 populations derived from four different P. sojae races. Four RAPD and nine AFLP markers linked to Avr1a were initially identified. Nine markers were used to compare genetic linkage maps of the Avr1a locus in two distinct F2 populations. Distorted segregation ratios favoring homozygous genotypes were noted in both crosses. Segregation analysis of all the markers in one F2 population of 90 progeny generated a map of 113.2 cM encompassing Avr1a, with one marker cosegregating with the gene. The cosegregating DNA marker was used to isolate P. sojaeBAC clones and construct a physical map covering 170 kb, from which additional DNA markers were developed. Three markers occurring within the BAC contig were mapped in an enlarged population of 486 F2 progeny. Avr1a was localized to a 114-kb interval, and an average physical to genetic distance ratio of 391 kb/cM was calculated for this region. This work provides a basis for the positional cloning of Avr1a.
Author(s): Dou. D Kale. S. D Wang. X Chen. Y Wang. Q Xia. W Jiang. R. H. Y Arredondo. F. D Anderson. R. G Thakur. P. B McDowell. J. M Wang. Y Tyler. B. M
Abstract: The sequenced genomes of oomycete plant pathogens contain large superfamilies of effector proteins containing the protein translocation motif RXLR-dEER. However, the contributions of these effectors to pathogenicity remain poorly understood. Here, we show that the Phytophthora sojae effector protein Avr1b can contribute positively to virulence and can suppress programmed cell death (PCD) triggered by the mouse BAX protein in yeast, soybean (Glycine max), and Nicotiana
benthamiana cells. We identify three conserved motifs (K, W, and Y) in the C terminus of the Avr1b protein and show that mutations in the conserved residues of the W and Y motifs reduce or abolish the ability of Avr1b to suppress PCD and also abolish the avirulence interaction of Avr1b with the Rps1b resistance gene in soybean. W and Y motifs are present in at least half of the identified oomycete RXLR-dEER effector candidates, and we show that three of these candidates also suppress PCD in soybean. Together, these results indicate that theWand Ymotifs are critical for the interaction of Avr1b with host plant target proteins and support the hypothesis that thesemotifs are critical for the functions of the very large number of predicted oomycete effectors that contain them.
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