Mechanism Dissection Of Phytophthora Infestans Effector SRE4 Targets SlSR30 To Regulate Tomato Immunity

Late blight disease caused by Phytophthora infestans is a devastating disease of Solanaceae crops,which seriously threatens the quality of crops such as potato and tomato.The "Great Irish famine" was caused by Phytophthora infestans in Ireland during1845-1850,resulting in severe reduction of potato and millions of people starved to death or fled,which had a deep influence to Western society,economy and politics.The prevention and control of late blight disease has always been an important problem in agricultural production.Chemical control is mainly used at present,which not only causes environmental pollution,but also threatens human health.Therefore,it has a scientific reference value to reveal the interaction mechanism between Phytophthora infestans and hosts at the molecular level.In the process of interaction between Phytophthora infestans and host plants,a large number of effectors are transported to host cells to disturb the plant immunity,and then promote their own infection.Effectors are highly specific proteins secreted in the process of co-evolution between pathogens and plants.The molecular mechanism of effectors regulating plant immunity has long been a frontier issue in the field of plant pathology.Our previous research found that Phytophthora infestans could secrete a kind of effectors which could disturb alternative splicing of host pre-m RNA,and named SREs(splicing regulatory effectors)by using a novel alternative splicing reporter system.In this study,SRE4(Pi07569)was used as the research object.It was found that SRE4 could interact with Sl SR30,and further explored the molecular mechanism of Sl SR30 inhibiting plant immunity through a series of biochemical and genetic techniques.The contents are follows:SRE4 is a virulent effector of Phytophthora infestans.In this study,SRE4 is a conserved RXLR effector in Phytophthora sp and has three WY-motif.It was found that SRE4 is up-regulated in early stage of Phytophthora infestans infection.It was found that transient expression of SRE4 in N.benthamiana could promote the infection of Phytophthora infestans,which indicated that SRE4 plays a virulent role in the process of infection of Phytophthora infestans and promots Phytophthora infestans infection.It was found that the first or the third WY-motif played important roles in disturb host alternative splicing function and virulent function of SRE4.This result provides important clues for further study on the function of SRE4.SRE4 interacts with a splicing factor Sl SR30 in tomato.In order to further study the virulent function of SRE4,yeast two hybrid technology was used to screen the target proteins in host.It was found that SRE4 could interact with Sl SR30,a splicing factor in tomato.SRE4 can alter the subcellular localization of Sl SR30.According to the structural characteristics of SRE4 and Sl SR30,it was found that SRE4 interacts with Sl SR30 through the first WY-motif,while Sl SR30 binds SRE4 through its SR domain.In addition,this study verified the specificity of the interaction between SRE4 and Sl SR30.The results showed that SRE4 could not interact with Sl SR30 homologous proteins like Sl SR34,Sl SR34 a and Sl SR34 b in tomato.These results showed that Sl SR30 could be targeted by Phytophthora infestans for the first time,suggesting that Phytophthora infestans could inhibit plant immunity by disturb host alternative splicing.Sl SR30 is a negative regulator in plant immunity.In order to further study the role of Sl SR30 in the interaction between Phytophthora infestans and tomato,this study used our previous RNA-seq data and q RT-PCR results,and found that Sl SR30 was significantly induced in the early stage of P.infestans infection time,suggesting that Sl SR30 may play an important role in the infection of Phytophthora infestans.Transient expression of Sl SR30 in N.benthamiana and tomato could promote the infection of Phytophthora infestans.Transient silence SR30 gene in N.benthamiana and tomato respectively could significantly enhance the resistance of plants to oomycete,and also significantly enhanced the active oxygen burst induced by PAMPs,which indicated that Sl SR30 is a negative regulator of plant immunity.Sl SR30 was also knocked out in tomato by CRISPR/Cas9.It was found that the slsr30 knocked out mutant plants showed significant resistance to Phytophthora infestans,and the expression of PTI related marker genes,salicylic acid marker genes and free salicylic acid content were significantly up-regulated,which indicated that Sl SR30 can get involved in the plant PTI reaction and salicylic acid hormone signaling pathway.In this study,the mechanism of negative regulation of plant immunity by tomato splicing factor Sl SR30 was preliminarily explored.It was proved for the first time that Sl SR30 could get involved in the regulation of plant immunity,which provided a reference for improving the resistance of crops to late blight.