Phytophthora Sojae Controls The Infection Process By Cooperation And Functional Interactions Within RxLR Effectors Repertoire

Phytophthora sojae is the casual agent of soybean root rot disease which is one of the most destructive diseases that harms soybean production around the world. As an aggressive hemibiotroph pathogen, P. sojae has an initial phase of biotrophic growth require living host cells to support their obligate parasitism followed by a transition to necrotrophy that may actively kill their host during the disease process, which suggesting the manipulation of cell death might be much more complicated. P.sojae deliver a variety of extracellular effectors into the plant apoplast to promote infection by modulate plant defense circuitry and enable parasitic colonization.Bioinformatic analysis of the draft genome sequences of P. sojae has identified an extraordinarily large superfamily of RxLR effector proteins (>350). The RxLR effector proteins are presumed to contribute to virulence, and the molecular mechanisms by which they act are under research in this study. In virtue of PVX-based gene expression and functional assay in Nicotiana benthamiana, high-throughput screenings are carried out to identify the virulence effect of 169 RxLR effectors which were highly expressed during infection or with the reported W-motifs. Three quarters of these effectors block the programmed cell death (PCD) induced by mouse pro-apoptosis protein Bax, while 11 effectors elicit plant PCD response. Functional assays have demonstrated that not all W-motif containing effectors could suppress BT-PCD, an systematic comparison of all W motifs was carried out. Residues at 16 positions within the W motif showed a statistically significant bias in the presence of specific amino acids. Mutations in these residues within the predicted helix of the Avh5 W-motif severely reduced the ability to suppress BT-PCD, suggesting that these residues may play a key role in suppression of BT-PCD.Since the effectors are predicted to mediate infection related with theirs expression level, we examined the transcriptional profile of the effectors using the Affymetrix SoyChip. Based on the global transcriptome data,expression of 20 predicted RXLR effector genes were induced by infection, with seven effectors termed "immediate-early"(IE) and nine termed "early effectors"(E) were identified by their expression patterns. Given the very strong expression and/or induction of Avh238 and Avh172, we modified their expression by DNA transformation, then quantitated pathogen success during the biotrophic phase. The result suggested that premature expression of Avh238/Avh172 interfered with the normal program of infection.Functional assays have demonstrated that most IE effectors could suppress the PCD triggered by Avh238, but little could suppress PCD triggered by the PAMP-INF1, suggested the hypothesis that a key function of the immediate-early effectors was to suppress plant defenses triggered by effectors expressed later in infection. E effectors such as Avh238 that were strongly induced at 12 hr of infection might be to suppress PAMP-triggered immunity. The Agrobacterium tumefaciens infiltration experiment designed to recapitulate the program of effector expression during infection using Avh238 together with Avh172. Under the "protection" of Avh172, the recognizing of plant was completely blocked, and the really function of Avh238 is disclosed. It blocks INF1 and the MAP kinase-mediated HR, the Phytophthora PAMP triggered plant immunity. It provides a very nice illustration of how cooperation among the effectors may work in planta.Alleles of Avh238 found in isolates of P6497, P7074 and P7076 showed a significant amino acid polymorphisms. The Avh238 allele found in P7076 (Avh238P7076) did not trigger PCD in N. benthamiana but could suppress the PCD triggered by INF1 and NPK1 and MKK1. Avh238P7076 could suppress the PCD triggered by both MAP kinases, suggesting that it acted at a point in the signaling pathway downstream of the two kinases. Comparing the sequence of RxLR effectors from strains P6497, P7064, P7074 and P7076, exhibit a high level of polymorphism within the species as a result of selection for alleles that do not trigger responses deleterious to the pathogen. Advanced research on Avh238 showed that it is a nuclear localization protein, hypothesis that it may target a nuclear protein and interference the plant immunity.