Host Target Screening And Functional Analysis Of Phytophthora Sojae RXLR Effector Avh5

Phytophthora sojae belongs to the Oomycota fungus species,and is a kind of eukaryotic pathogenic microorganism with great harm.Its host range is very limited,and only a few legumes such as soybean can be infected under natural conditions.Phytophthora soja will harm the roots,stems,leaves and pods of plants.The soybean rhizome rot caused by it is a devastating disease in agricultural production and causes huge losses to the agricultural economy every year.Today,Phytophthora sojae root rot occurs in every soybean producing area in the world,causing huge economic losses to the world economy every year.It is a devastating disease that threatens soybean production and even global food security.With the continuous development of economic and trade globalization,and the increasing demand for imported soybeans in China,the damage caused by Phytophthora sojae has also appeared in some parts of China,which seriously threatens the development of the soybean industry in China.Therefore,we urgently need to deeply understand the pathogenic mechanism and disaster-causing mechanism of Phytophthora sojae,provide a solid theoretical basis for breeding resistant varieties,and provide new ideas for disease prevention and control.Early studies have found that Phytophthora sojae secretes a large number of RXLR effector molecules at the initial stage of infection,and enters the host cells to suppress the plant's immune response,thereby enhancing its colonization on plants.Therefore,finding the host targets of these toxic molecules will help to further analyze the mechanism by which effector molecules interfere with the host plant's immune response,and reveal the pathogenic mechanism of P.sojae infection of the host.Previous studies found that about 60% of the RXLR effectors of Phytophthora sojae contained conserved W-motif.It is speculated that W-motif is a key region for RXLR effector molecules to inhibit programmed cell death(PCD)in plants,and the more effector molecules the more W-motif is more likely to inhibit PCD.There is only one W-motif at the C-terminus of the protein of Phytophthora sojae RXLR effector Avh5,and the structure is relatively simple,but experiments have also shown that it can significantly inhibit plant PCD,so it is the main research object.This study found that Avh5 was up-regulated in the early stages of infection,indicating that it may play an important role in the pathogenesis.By genetically manipulating the Avh5 gene of Phytophthora sojae,it was found that knockout of Avh5 in the strain of Phytophthora sojae would reduce the infectivity of soybean plants,while overexpression of Avh5 would enhance the infectivity.These results prove that Avh5 is a key effector molecule of Phytophthora sojae and can promote the pathogenic process of Phytophthora sojae.In this study,Avh5 was used as a bait protein,and yeast two-hybrid technology was used to screen host targets in soybean c DNA libraries.Through yeast interaction verification,it was found that E3 ubiquitin-protein ligase complex SLX5-SLX8(referred to as E3)in soybean can directly interact with Avh5.Pull-down,Bi FC and other methods have further proved that there is a direct interaction between Avh5 and E3.Further research found that E3 can directly interact with the C-terminus of Avh5(Avh5~C)and the W-motif region(Avh5~W),which fully indicates that E3 is the host target protein of Avh5 in soybean.E3 ubiquitin-protein ligase complex SLX5-SLX8 is a component of SUMO-targeted ubiquitin ligase(STUb L)complex SLX5 / SLX8,which mediates ubiquitination and subsequent sulfonylated proteins and contains SUMO Desulphonylation of protein-like domain degradation plays an important role in the regulation of apoptosis.It is speculated that Avh5 directly affects PCD in plants through its direct interaction.This study further found that the W-motif mutant proteins M5,M6 and E3 of Avh5 cannot interact with each other,proving that W-motif is the key structure of Avh5 and E3 interactions,and is located in four of the second ?-helix region of W-motif Amino acids play a key role.The mutant proteins M5 and M6 were not able to inhibit PCD in plants.These results indicate that W-motif may interfere with programmed cell death in plants by directly interacting with E3 protein.This study found and verified the interaction mechanism between Phytophthora sojae RXLR effector protein Avh5 and host E3 ubiquitin ligase complex SLX5 / SLX8 in plants.In order to further analyze the plant disease resistance mechanism,discover resistance breeding targets and formulate epidemic resistance The mold control strategy has laid a solid experimental foundation.