Function Analysis Of Phytophthora Suppressor Of RNA Silencing PAPAR2 And PAPSR2 Homologs

Oomycetes are a group of microbial eukaryotes that include important pathogens of plants and animals. The genus Phytophthora contains many notorious pathogens of crops. For example, the potato pathogen Phytophthora infestans triggered the Irish Famine in the nineteenth century and remains a serious problem worldwide, and P. sojae causes millions of dollars of losses annually in soybean. Until now, battling oomycete-related diseases has been challenging owing to a lack of understanding of pathogenesis. Bioinformatic prediction revealed about four hundred of RxLR effectors in genome of Phytophthora sojae, Compared to infection by viral or bacterial pathogens, this remarkably large number of effectors suggests complex defense/counter-defense crosstalks between host plants and Phytophthora. Although several effectors have been shown to suppress plant defense, the functions of the vast majority of effectors remain unknown.RNA silencing serves as a major defense mechanism against RNA viruses in plants, fugus and invertebrates. As a counterdefense, viral suppressors of RNA silencing （VSRs） enable efficient infection by interfering with host silencing. Small RNA-mediated post-transcriptional regulation has also been implicated in antibacterial plant defense. Furthermore, bacterial effectors were found to suppress the miRNA pathway in plants. However, whether RNA silencing regulates defense against eukaryotic pathogens remains unknown. If so, these pathogens might have evolved virulence strategies to disrupt host RNA silencing machinery. We screened about 70 P. sojae RxLR effectors that suppress RNA silencing in plants, and found two P. sojae Phytophthora Suppressors of RNA silencing, designated PsPSR1 and PsPSR2. In contrast to the broad inhibitor activity of PsPSRl on the biogenesis of both miRNAs and siRNAs, the activity of PsPSR2 specifically controls ta-siRNA accumulation through a different mechanism.In this work, we performed detailed analysis on PsPSR2 as follows.1. To understand the conservation and distribution of PsPSR2-like genes in Phytophthora. We analyzed the genome sequences of eight Phytophthora species, including P. sojae, P. capsici, P. infestans, P. parasitica, P. ramorum, P. cinnamomi var. robiniae, P. melonis, and P. litchi. Remarkably, we identified at least one PsPSR2 homolog in each of the Phytophthora genomes that were examined. Extensive genetic and expression polymorphisms have been observed in Phytophthora RxLR effectors and conserved effectors are rare. All these PsPSR2 homologs encode proteins containing the conserved N-terminal signal peptide and the RxLR-dEER motif, suggesting that they are cytoplasmic effectors. In addition, a PsPSR2-like effector gene （Hyaar₈07752） was also found in the genome of Hyaloperonospora arabidopsidis （Hpa）, which is an obligate oomycete pathogen. The deduced amino acid sequence of Hyaar 807752 only has a conserved N-terminal secretion signal and a dEER-like motif （DEDEEDER）. These data strongly suggest that PsPSR2 family of RxLR effectors is widely distributed in Phytophthora, and possibly also present in other oomycete pathogens. Phylogenetic analysis placed the PsPSR2 homologs into three major clades, which are highly congruent to the species phylogeny of Phytophthora based on Internal Transcribed Spacer （ITS） sequences, indicating that PsPSR2-like effectors belong to an evolutionarily conserved family that has diverged together with the speciation of Phytophthora. PsPSR2 was found to be highly conserved with no polymorphism in the four major races of P. sojae. PsPSR2 was found to contain 5 L-W-Y structure and most PsPSR2 homologs also have such repeat L-W-Y structure from analysis of domains. Because W-Y motif is importmant to α- helix fold, suggesting PsPSR2 may form motif repeats in order to make the protein structure much more complex to evade host recognition.2. The function analysis of PsPSR2. We wonder if there is relationship between silencing suppress activity in plants and infection process. Firstly, we found transient silencing of PsPSR2 in P. sojae led to significantly reduced virulence on soybean hypocotyls, suggesting that PsPSR2 is a virulence effector of P. sojae. We next expressed PsPSR2 in Arabidopsis and soybean which is the natural host of P. sojae, in order to check if it can facilitate infection. PsPSR2 mainly expressed late in infection and localized in nuclear and cytoplasm. We monitored the infection process of P. sojae in soybean roots using a GFP-expressing transformant of P. sojae strain P6497. Using this natural pathosystem, we showed that PsPSR2-expressing soybean roots were hypersusceptible to P. sojae. PsPSR2 was reported to specifically affects the biogenesis of ta-siRNAs, which were recently found to regulate plant nucleotide binding-leucine-rich repeat （NB-LRR） genes. However, whether PsPSR2 was able to promote Phytophthora infection in Arabidopsis could not be tested until the recent development of the Arabidopsis-P. capsici pathosystem. The enhanced susceptibility of PsPSR2-expressing Arabidopsis plants was only observed in two independent transgenic lines, i.e. PSR2-5 and PSR2-10, that also exhibited significantly reduced abundances of ta-siRNAs. On the contrary, a third PsPSR2-expressing Arabidopsis line, PSR2-18, that was not defective in ta-siRNA accumulation due to a low expression level of PsPSR2 also showed unaltered susceptibility to P. capsici, suggesting that PsPSR2 may promote infection by interfering with specific ta-siRNAs in plants. Moreover, we also observed enhanced accumulation of PR1 transcripts in wild type plants inoculated by P. capsici. Remarkably, the PR1 induction was completely abolished in the PsPSR2-expressing plants, indicating that PsPSR2 may promote Phytophthora infection by interfering with SA-dependent defense pathway. All of these data suggest that RxLR effectors PsPSR2 play a fundamental virulence function by suppressing host RNA silencing processes in various plants.3. The function analysis of PsPSR2-like effectors. As PsPSR2 is a conserved RxLR effector family that is widely distributed in Phytophthora, that make us to know if it has the conserved function. We analyzed some PsPSR2 homologs based on the bioinformatics data. PITG₁5152, a close homolog of PsPSR2 in P. infestans with one more L-W-Ymotif, was also able to strongly suppress RNA silencing activity when expressed in N. benthamiana. It almost has the same localization and expression profile with PsPSR2, so we designated it as PiPSR2. Moreover, PiPSR2-silenced transformants has no phenotypic changes with wild type, but its virulence was significantly reduced on N. benthamiana. Expressing PiPSR2 enhanced the susceptibility of N. benthamiana to Phytophthora infection, suggesting that inhibiting host RNA silencing pathways is an important virulence strategy. PsPSR2 family may lay a fundamental virulence function by it. To futher research on the detailed mechanism that PiPSR2 adopt to suppress host RNA silencing, we successfully generated PiPSR2 transgenic Arabidopsis plants as materials for future work, so that we can know the comparisons and correlations between PiPSR2 and PsPSR2.