Oomycete plant pathogens
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Rescooped by Damitha Wickramasinghe from Plant Pathogenomics
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Plant J: Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations (...

Plant J: Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations (... | Oomycete plant pathogens | Scoop.it

RenSeq is a NB-LRR (nucleotide binding-site leucine-rich repeat) gene-targeted, Resistance gene enrichment and sequencing method that enables discovery and annotation of pathogen resistance gene family members in plant genome sequences. We successfully applied RenSeq to the sequenced potato Solanum tuberosum clone DM, and increased the number of identified NB-LRRs from 438 to 755. The majority of these identified R gene loci reside in poorly or previously unannotated regions of the genome. Sequence and positional details on the 12 chromosomes have been established for 704 NB-LRRs and can be accessed through a genome browser that we provide. We compared these NB-LRR genes and the corresponding oligonucleotide baits with the highest sequence similarity and demonstrated that ~80% sequence identity is sufficient for enrichment. Analysis of the sequenced tomato S. lycopersicum ‘Heinz 1706’ extended the NB-LRR complement to 394 loci. We further describe a methodology that applies RenSeq to rapidly identify molecular markers that co-segregate with a pathogen resistance trait of interest. In two independent segregating populations involving the wild Solanum species S. berthaultii (Rpi-ber2) and S. ruiz-ceballosii (Rpi-rzc1), we were able to apply RenSeq successfully to identify markers that co-segregate with resistance towards the late blight pathogen Phytophthora infestans. These SNP identification workflows were designed as easy-to-adapt Galaxy pipelines.


Via Kamoun Lab @ TSL
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Rescooped by Damitha Wickramasinghe from Plant Pathogenomics
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PLoS Biology: Gene Gain and Loss during Evolution of Obligate Parasitism in the White Rust Pathogen of Arabidopsis thaliana

PLoS Biology: Gene Gain and Loss during Evolution of Obligate Parasitism in the White Rust Pathogen of Arabidopsis thaliana | Oomycete plant pathogens | Scoop.it

Biotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells. Evolution to biotrophy occurred independently in fungal rusts and powdery mildews, and in oomycete white rusts and downy mildews. Biotroph evolution and molecular mechanisms of biotrophy are poorly understood. It has been proposed, but not shown, that obligate biotrophy results from (i) reduced selection for maintenance of biosynthetic pathways and (ii) gain of mechanisms to evade host recognition or suppress host defence. Here we use Illumina sequencing to define the genome, transcriptome, and gene models for the obligate biotroph oomycete and Arabidopsis parasite, Albugo laibachii. A. laibachii is a member of the Chromalveolata, which incorporates Heterokonts (containing the oomycetes), Apicomplexa (which includes human parasites like Plasmodium falciparum and Toxoplasma gondii), and four other taxa. From comparisons with other oomycete plant pathogens and other chromalveolates, we reveal independent loss of molybdenum-cofactor-requiring enzymes in downy mildews, white rusts, and the malaria parasite P. falciparum. Biotrophy also requires “effectors” to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes, and also discover and verify a novel class of effectors, the “CHXCs”, by showing effector delivery and effector functionality. Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways, and particularly reduced selection for retention of molybdopterin-requiring biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata.


Via Kamoun Lab @ TSL
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Rescooped by Damitha Wickramasinghe from Plant-Microbe Interaction
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Mol Microbiol: Chemotaxis and oospore formation in Phytophthora sojae are controlled by G-protein-coupled receptors with a phosphatidylinositol phosphate kinase domain (2013)

Mol Microbiol: Chemotaxis and oospore formation in Phytophthora sojae are controlled by G-protein-coupled receptors with a phosphatidylinositol phosphate kinase domain (2013) | Oomycete plant pathogens | Scoop.it

G-protein-coupled receptors (GPCRs) are key cellular components that mediate extracellular signals into intracellular responses. Genome mining revealed that Phytophthora spp. have over 60 GPCR genes among which a prominent class of 12 encoding novel proteins with an N-terminal GPCR domain fused to a C-terminal phosphatidylinositol phosphate kinase (PIPK) domain. This study focuses on two GPCR-PIPKs (GKs) in Phytophthora sojae. PsGK4 and PsGK5 are differentially expressed during the life cycle with the highest expression in cysts and during cyst germination, and at late infection stages. In P. sojae transformants that constitutively express RFP-tagged PsGK4 andPsGK5, the fusion proteins in hyphae reside in small, rapidly moving vesicular-like structures. Functional analysis using gene silencing showed that PsGK4-silenced transformants displayed higher levels of encystment and a reduced cyst germination rate when compared with the recipient strain. Moreover, GK4 deficiency (or reduction) resulted in severe defects in zoospore chemotaxis towards isoflavones and soybean roots. In contrast, PsGK5-silenced transformants exhibited no obvious defects in asexual development but oospore production was severely impaired. Both, PsGK4- and PsGK5-silenced transformants showed reduced pathogenicity. These results point to involvement of GKs in zoospore behaviour, chemotaxis and oospore development, and suggest that PsGK4 and PsGK5 each head independent signalling pathways.


Via Kamoun Lab @ TSL, Guogen Yang
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Scooped by Damitha Wickramasinghe
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Oomycete pathogens encode RNA silencing suppressors

Oomycete pathogens encode RNA silencing suppressors | Oomycete plant pathogens | Scoop.it

Effectors are essential virulence proteins produced by a broad range of parasites, including viruses, bacteria, fungi, oomycetes, protozoa, insects and nematodes. Upon entry into host cells, pathogen effectors manipulate specific physiological processes or signaling pathways to subvert host immunity. Most effectors, especially those of eukaryotic pathogens, remain functionally uncharacterized. Here, we show that two effectors from the oomycete plant pathogen Phytophthora sojae suppress RNA silencing in plants by inhibiting the biogenesis of small RNAs. Ectopic expression of these Phytophthora suppressors of RNA silencing enhances plant susceptibility to both a virus and Phytophthora, showing that some eukaryotic pathogens have evolved virulence proteins that target host RNA silencing processes to promote infection. These findings identify RNA silencing suppression as a common strategy used by pathogens across kingdoms to cause disease and are consistent with RNA silencing having key roles in host defense.

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Rescooped by Damitha Wickramasinghe from Plant Pathogenomics
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PLOS ONE: Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes (2013)

PLOS ONE: Comparative Genomics Reveals Insight into Virulence Strategies of Plant Pathogenic Oomycetes (2013) | Oomycete plant pathogens | Scoop.it

The kingdom Stramenopile includes diatoms, brown algae, and oomycetes. Plant pathogenic oomycetes, including Phytophthora, Pythium and downy mildew species, cause devastating diseases on a wide range of host species and have a significant impact on agriculture. Here, we report comparative analyses on the genomes of thirteen straminipilous species, including eleven plant pathogenic oomycetes, to explore common features linked to their pathogenic lifestyle. We report the sequencing, assembly, and annotation of six Pythium genomes and comparison with other stramenopiles including photosynthetic diatoms, and other plant pathogenic oomycetes such as Phytophthora species, Hyaloperonospora arabidopsidis, andPythium ultimum var. ultimum. Novel features of the oomycete genomes include an expansion of genes encoding secreted effectors and plant cell wall degrading enzymes in Phytophthoraspecies and an over-representation of genes involved in proteolytic degradation and signal transduction in Pythium species. A complete lack of classical RxLR effectors was observed in the seven surveyed Pythium genomes along with an overall reduction of pathogenesis-related gene families in H. arabidopsidis. Comparative analyses revealed fewer genes encoding enzymes involved in carbohydrate metabolism in Pythium species and H. arabidopsidis as compared to Phytophthora species, suggesting variation in virulence mechanisms within plant pathogenic oomycete species. Shared features between the oomycetes and diatoms revealed common mechanisms of intracellular signaling and transportation. Our analyses demonstrate the value of comparative genome analyses for exploring the evolution of pathogenesis and survival mechanisms in the oomycetes. The comparative analyses of seven Pythium species with the closely related oomycetes, Phytophthora species and H. arabidopsidis, and distantly related diatoms provide insight into genes that underlie virulence.


Via Kamoun Lab @ TSL
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Genome Re-Sequencing and Functional Analysis Places the Phytophthora sojae Avirulence Genes Avr1c and Avr1a in a Tandem Repeat at a Single Locus

Genome Re-Sequencing and Functional Analysis Places the Phytophthora sojae Avirulence Genes Avr1c and Avr1a in a Tandem Repeat at a Single Locus | Oomycete plant pathogens | Scoop.it
The aim of this work was to map and identify the Phytophthora sojae Avr1c gene. Progeny from a cross of P. sojae strains ACR10×P7076 were tested for virulence on plants carrying Rps1c. Results indicate that avirulence segregates as a dominant trait. We mapped the Avr1c locus by performing whole genome re-sequencing of composite libraries created from pooled samples. Sequence reads from avirulent (Pool1) and virulent (Pool2) samples were aligned to the reference genome and single nucleotide polymorphisms (SNP) were identified for each pool. High quality SNPs were filtered to select for positions where SNP frequency was close to expected values for each pool. Only three SNP positions fit all requirements, and these occurred in close proximity. Additional DNA markers were developed and scored in the F2progeny, producing a fine genetic map that places Avr1c within the Avr1a gene cluster. Transient expression of Avr1c or Avr1a triggers cell death on Rps1c plants, but Avr1c does not trigger cell death on Rps1a plants. Sequence comparisons show that the RXLR effector genesAvr1c and Avr1a are closely related paralogs. Gain of virulence on Rps1c in P. sojae strain P7076 is achieved by gene deletion, but in most other strains this is accomplished by gene silencing. This work provides practical tools for crop breeding and diagnostics, as the Rps1c gene is widely deployed in commercial soybean cultivars.
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Rescooped by Damitha Wickramasinghe from Direct but flimsy evidence for oomycete effector translocation
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RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery

RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery | Oomycete plant pathogens | Scoop.it

RXLR-dEER-GFP Fusion Proteins Isolated from E. coli Can Enter Soybean Cells in the Absence of the Pathogen.
GFP fusion proteins were expressed in E. coli, partially purified, and incubated with soybean root tips for 12 h. The root tips were then washed for 4 h and photographed under UV and white light illumination.
(A) Protein gel electrophoresis analysis of GFP fusion proteins partially purified from E. coli cells: lane 1, Arg9-GFP; lane 2, GFP fused to the N-terminal 44 amino acids of mature wild-type Avr1b protein (RXLR1+,RXLR2+-dEER-GFP); lane 3, same as lane 2 with both RXLR1 and RXLR2 mutations (RXLR1AAAA,RXLR2AAAA-dEER-GFP); lane 4, same as lane 2 except with dEER mutation (RXLR1+,RXLR2+-dEERAAAAAA-GFP). The left lane contained molecular mass markers; the sizes of the markers are shown on the left (in kD). All expressed GFP proteins fluoresce normally under UV illumination.
(B) to (F) UV (left panels) and back-lit white light (right panels) illumination of roots after incubation with the indicated GFP protein fusion. The UV photographs represent longitudinal optical sections taken using the confocal microscope as illustrated by the dashed line in the inset of (C). The GFP concentration, illumination, and exposure of the UV photographs was identical in all 10 panels shown.
(B) Buffer alone with no fusion protein.
(C) RXLR1+,RXLR2+-dEER-GFP.
(D) Arg9-GFP.
(E) RXLR1AAAA,RXLR2AAAA-dEER-GFP.
(F) RXLR1+,RXLR2+-dEERAAAAAA-GFP.
(G) and (H) Higher-magnification photographs after the root tips were gently squashed following washing, showing nuclear accumulation of GFP.
(G) RXLR1+,RXLR2+-dEER-GFP.
(H) Arg9-GFP


Via Mark Farman
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Brett Tyler's comment, April 26, 2013 6:44 PM
Mark, I'm surprised you haven't retracted these comments following our email exchanges. So I'm posting my rebuttal here. First, while leaf epidermal cells do have large vacuoles, root meristematic zone cells, and leaf mesophyll cells do NOT. This information is available in any plant cell biology text book, e.g. Biology 4th edition Ladiges et al. 2010. With regard to the vacuole, there is no evidence whatsoever for a continuous soluble phase connection between the apoplast and vacuole. This fact can also be found in the same text books. With regard to plasmolysis, Avr1bNt-GFP plasmolysis data were shown in Figure S1 panel O of Kale et al (Cell, 142(2), 284–295 2010). Similar plasmolysis data were shown for the fungal RxLR-like effector Ps87 in Figure 2 panel I of Gu et al (PLoS ONE 6(11), e27217, 2011). With regard to DAPI-validated nuclear accumulation, Fig 2 panels F-J of Tyler et al (MPMI first look, 2013) show nuclear accumulation of Avr1bNt-GFP validated with DAPI staining; Fig S1 panels Q, R, and S of Kale et al (2010) show DAPI-validated nuclear accumulation of AvrL567Nt-GFP, AvrLm6Nt-GFP and Avr2Nt-GFP respectively; Fig S2 panels B-D of Plett et al (Current Biology 21(14), 1197-12032011) show DAPI-validated nuclear localization of fluorescently labeled MiSSP7. I quote your response from our private email exchange: "Thanks for your response. It was very informative and did a lot to assuage many of my concerns. I need to digest the information a little more before I add comments to your document but I'm certainly a lot more convinced than I was previously. "
Brett Tyler's comment, April 26, 2013 6:57 PM
On a more general note, I'd like to state that this parlor game of plucking individual figures from papers, and trashing them, is not a constructive way to advance our field. Knowledge and understanding of complex problems are built step by step. It is NORMAL for publications that are released along the way to reveal many unresolved issues, some scientific and some technical. If we insist that all publications must be perfect in every way, and contain only figures that are perfect in every way, then NOTHING will get published, and progress will be greatly inhibited. Balanced, well thought out reviews (traditional pubs or blogs) that consider the advances as well as limitations of the recent literature as a whole are an appropriate and constructive vehicle to discuss where a field stands and where it needs to go next.
Mark Farman's comment, April 26, 2013 9:26 PM
Brett, At first your statement about the meristematic cells made me question whether my original criticisms of your data were valid. However, upon careful and in depth scrutiny of ALL of the published data and figures, I hold to my original criticism. Moreover, the new data raised even more serious concerns about your interpretations of the Avr1b data. I simply haven't had time to put my very long list of concerns down in writing. However, the basic issues are that: 1) NONE of the root uptake experiments for Avr1b have ever shown fluorescence at the cell periphery in a pattern consistent with cytoplasmic accumulation (your argument about meristematic cells not having a vacuole is irrelevant because most of the time, you are not looking at such cells). As an example, peripheral fluorescence is very clearly evident in root cells treated with Arg9-GFP (Dou et al 2008) and Ps87-GFP (Gu et al 2011) but this pattern was not obtained in parallel experiments with Avr1b RXLR. Even more convincing, however, were the plasmolysis data for Ps87 which clearly showed retraction of the fluorescence signals in concert with cytoplasmic shrinkage. Based on these images, I am almost convinced of the uptake of the Ps87 protein (my only reservation: missing controls to rule out autofluorescence). What I find most concerning, however, is that the very same figure presents data for Avr1b but corresponding plasmolysis data are conspicuously absent (and the pattern of fluorescence accumulation is completely different to that of Ps87). These cells are almost certainly dead, Brett - probably killed by high concentrations of Avr1b/Avr1bNt (and I believe this to be true for all the Avr1b/Avr1bNt assays). Until you present CONVINCING plasmolysis data to prove otherwise, your conclusions about Avr1b uptake into the cytoplasm will never hold water (showing "nuclear" uptake in the absence of plasmolysis data is insufficient for reasons I'll explain below). At present, the only published plasmolysis data for Avr1b (in the SUPPLEMENTARY data of Kale et al. 2010) is wholly unconvincing. I can't even tell what is going on in that image. Show me data for Avr1b that look like Gu et al 2011 Figure 4, panels F, G and I (along with controls to rule out autofluorescence), then I (and I imagine everyone else) will be satisfied that you have ruled out all of the alternative hypotheses that could explain your data (at least ones that satisfy Occum's razor).
2) Your GFP/mcherry uptake experiments have major issues:
i) you do not consider the relative quantum yields and photostabilities of GFP versus mcherry in your calculations. GFP has three times the quantum yield and twice the photostability of mcherry. That means when you see equal fluorescence intensities, mcherry is in ~6-fold molar excess over GFP.
ii) the densitometric scans across the root sections have been cherry-picked (excuse the pun) in a way that guarantees the results will support the hypothesis. On the very same root images, I can pick transects that will produce scans positively refuting it. You should be averaging the data across multiple transects laid at specific intervals along each individual root section (and then correcting the results to account for QY and photostability differences).
3) The Avr1b/Avr1bNt nuclear accumulation data are beset with major problems:
i) your Avr1bNt-GFP "nuclear" accumulation patterns are very unconvincing and have NEVER never been verified with DAPI (the example you give above is for Dylight- not GFP-labeled protein!).
ii) the Avr1b "nuclear" accumulation patterns show irregularly sized spots and patchy distribution which is VERY different to what I would expect to see and to what, in fact, was observed with Ps87 and the A9-GFP proteins. This highlights the critical need for DAPI confirmation.
iii) In Tyler et al (MPMI first look, 2013), Figure 2, panels G and J, green spots can be seen in cells that lack the expected peripheral cytoplasmic fluorescence (see Gu et al 2011). What we see, instead, is extensive fluorescence in the middle of the cell where the vacuole should be (note these are definitely NOT meristematic cells). In my lab, the only time we EVER see staining in this pattern is with dead cells that no longer plasmolyze. Thus, I can only conclude that the cells in panels G and J are dead or at least severely compromised - read "leaky"; and, in turn, I suspect that we are looking at nucleus "staining", as opposed to nuclear accumulation. Again, plasmolysis assays are needed.
iv) Finally, in your response to my critique, you stated that "Fig 2 panels F-J of Tyler et al (MPMI first look, 2013) show nuclear accumulation of Avr1bNt-GFP validated with DAPI staining" Not true! According to the figure legend, these panels show nuclear accumulation of Avr1bNt-Dylight488! Explain that to me - does Dylight488 contain a nuclear uptake signal? If so, then why wasn't it taken up into the nuclei in panels B and E. Conversely, very nice green fluorescent spots show up in the bottom right-hand corner of panels B and E but these are NOT nuclei, as evidenced by the lack of DAPI staining. Major, major inconsistencies here Brett and, yet, you're only holding on to the data you want to see.
Hopefully, you realize that I am not plucking individual figures and papers. What I see are systemic holes in your data which means that my hypothesis (purified Avr1bNt-GFP kills soybean root cells, making them permeable to the protein) holds just as much water as yours.
Rescooped by Damitha Wickramasinghe from Plant Immunity And Microbial Effectors
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Nature Communications: Transgenerational gene silencing causes gain of virulence in a plant pathogen (2013)

Nature Communications: Transgenerational gene silencing causes gain of virulence in a plant pathogen (2013) | Oomycete plant pathogens | Scoop.it

Avirulence (Avr) genes of plant pathogens encode effector proteins that trigger immunity in plants carrying appropriate resistance (R) genes. The Phytophthora sojae Avr3a gene displays allelic variation in messenger RNA transcript levels. P. sojae strains with detectable Avr3a gene transcripts are avirulent on plants carrying the R-gene Rps3a, whereas strains lacking Avr3a mRNA escape detection by Rps3a and are virulent. Here we show non-Mendelian interactions between naturally occurring Avr3a alleles that result in transgenerational gene silencing, and we identify small RNA molecules of 25 nucleotides that are abundant in gene-silenced strains but not in strains with Avr3a mRNA. This example of transgenerational gene silencing is exceptional because it is naturally occurring and results in gain of virulence in a pathogenic organism.


Via Kamoun Lab @ TSL, IPM Lab
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Rescooped by Damitha Wickramasinghe from Plant Pathogenomics
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PLoS ONE: mRNA-Seq Analysis of the Pseudoperonospora cubensis Transcriptome During Cucumber (Cucumis sativus L.) Infection (2012)

PLoS ONE: mRNA-Seq Analysis of the Pseudoperonospora cubensis Transcriptome During Cucumber (Cucumis sativus L.) Infection (2012) | Oomycete plant pathogens | Scoop.it

Pseudoperonospora cubensis, an oomycete, is the causal agent of cucurbit downy mildew, and is responsible for significant losses on cucurbit crops worldwide. While other oomycete plant pathogens have been extensively studied at the molecular level, Ps. cubensis and the molecular basis of its interaction with cucurbit hosts has not been well examined. Here, we present the first large-scale global gene expression analysis of Ps. cubensis infection of a susceptible Cucumis sativus cultivar, ‘Vlaspik’, and identification of genes with putative roles in infection, growth, and pathogenicity. Using high throughput whole transcriptome sequencing, we captured differential expression of 2383 Ps. cubensis genes in sporangia and at 1, 2, 3, 4, 6, and 8 days post-inoculation (dpi). Additionally, comparison of Ps. cubensis expression profiles with expression profiles from an infection time course of the oomycete pathogen Phytophthora infestans on Solanum tuberosum revealed similarities in expression patterns of 1,576–6,806 orthologous genes suggesting a substantial degree of overlap in molecular events in virulence between the biotrophic Ps. cubensis and the hemi-biotrophic P. infestans. Co-expression analyses identified distinct modules of Ps. cubensis genes that were representative of early, intermediate, and late infection stages. Collectively, these expression data have advanced our understanding of key molecular and genetic events in the virulence of Ps. cubensis and thus, provides a foundation for identifying mechanism(s) by which to engineer or effect resistance in the host.


Via Kamoun Lab @ TSL
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Rescooped by Damitha Wickramasinghe from Plants and Microbes
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New Phytologist: The Phytophthora parasitica RXLR effector Penetration-Specific Effector 1 favours Arabidopsis thaliana infection by interfering with auxin physiology (2013)

New Phytologist: The Phytophthora parasitica RXLR effector Penetration-Specific Effector 1 favours Arabidopsis thaliana infection by interfering with auxin physiology (2013) | Oomycete plant pathogens | Scoop.it

Pathogenic oomycetes have evolved RXLR effectors to thwart plant defense mechanisms and invade host tissues. We analysed the function of one of these effectors (Penetration-Specific Effector 1 (PSE1)) whose transcript is transiently accumulated during penetration of host roots by the oomycete Phytophthora parasitica.Expression of PSE1 protein in tobacco (Nicotiana tabacum and Nicotiana benthamiana) leaves and in Arabidopsis thaliana plants was used to assess the role of this effector in plant physiology and in interactions with pathogens. A pharmacological approach and marker lines were used to charcterize the A. thaliana phenotypes.Expression of PSE1 in A. thaliana led to developmental perturbations associated with low concentrations of auxin at the root apex. This modification of auxin content was associated with an altered distribution of the PIN4 and PIN7 auxin efflux carriers. The PSE1 protein facilitated plant infection: it suppressed plant cell death activated by Pseudomonas syringae avirulence gene AvrPto andPhytophthora cryptogea elicitin cryptogein in tobacco and exacerbated disease symptoms upon inoculation of transgenic A. thalianaplantlets with P. parasitica in an auxin-dependant manner.We propose that P. parasitica secretes the PSE1 protein during the penetration process to favour the infection by locally modulating the auxin content. These results support the hypothesis that effectors from plant pathogens may act on a limited set of targets, including hormones.


Via Kamoun Lab @ TSL
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