Functional Analysis Of Rxlr Effector AVH241from Phytophthora Sojae

Soybean root rot caused by Phytophthora sojae is the most destructive disease to soybean production in the world. It is very important to find the mechanisms of the effectors secreted during P. sojae infecting soybeans. There are nearly400RxLR effectors in the genome of P. sojae. By binding PI3P outside the plant plasma membrane and RxLR effectors can enter the plant cells. Recently, some RxLR effectors were found to play virulence roles in the plant cytoplasm, suppress the plant immunity and promote pathogens infection.In our previous study, a high-throughput screening in our lab was carried out to identify the functions of the RxLR effectors of P. sojae. We found that most effectors can suppress the plants cell death, and several can induce plants cell death themselves. The most strongly expressed immediate-early effectors could suppress the cell death triggered by several early effectors, and most early effectors could suppress INF1-triggered cell death, suggesting the two classes of effectors may target different functional branches of the defense response. It is very important for P. sojae to transcript effectors exactly. These effectors can fight as a team to interrupt the plant immunity.According to the high-throughput screening result, we selected15RxLR effectors for further analysis. Transcriptional profiling by Solexa RNA sequencing, we found that nearly all the candidate effectors highly expressed during early infection stage except some not detected. It demonstrated that these effectors might play important roles during infection. When expressing effectors fused GFP protein in plant cells, we found that effeccors Avhl8, Avh94, Avh97, Avh172, Avh244, Avh247, Avh256and Avh260localized to the nuleus and cytoplasm of the plant cells; effectors Avh23, Avh52and Avh240localized to the nuclue; and effectors Avh181and Avh241lozalized to plasma membrane.According to our results, different effectors target different components of the plant cells, so the effectors might play different roles during infection. Avh241induce strongly cell death on N. benthamiana leaves. Then we identified the protein localization using different assays. Avh241can induce plant cells death not only on N. benthamiana, but also on soybean, tomato and Arabidopsis. This protein has no homologue in other species which sequence had been known. But it is very conservative in different P. sojae strains. The amino acids from59to168are essential for triggering cell death. There is a motif consist with6amino acids GAAKAK deciding the localization and cell death inducing.Because Avh241induce several plants cell death, it is recognized by plants widely. It was found to induce cell death on soybean leaves with or without Rps genes. This demonstrated that this recognition was independent of R genes we had known. Avh241induced cell death on rpml, sgt1b or rarl mutant of Arabidopsis. It demonstrated that the cell death was also independent of Rpml, SGTlb and RARl. Mitogen-activated protein kinase cascade is evolutionarily conserved signal transduction module involved in transducing extracellular signals to the nucleus for appropriate cellular adjustment. When we silenced MAPKKKa, MEK1, SIPK, NTF6, WRKY1or WRKY2of N. benthamiana, the cell death induced by Avh241was not influenced. But the cell death induced by Avh241was obviously decreased on MEK2or WIPK silenced N. benthamiana. Our results proved that the cell death triggered by Avh241did not depend on Rps protein, SGT1b or RAR1, but depended on MEK2and WIPK.Avh241was highly expressed during early infection stage. When we silenced it in P. sojae, the silent mutant showed a depressed virulence. To further prove the virulence of Avh241, we transiently expressed the protein on N. benthamiana leaves with the cell death suppressed by Avh172. Avh241could promote the infection of P. capsici to N. benthamiana. Moreover, we determined that the mutant of Avh241, Avh241-4(80-188aa) can also promote the infection of P. capsici to N. benthamiana. Avh241-4can not induce plant cell death and is not localized to the plant plasma membrane. So Avh241plays a virulence role independent of RxLR motif and membrane localization during infection.