Late Blight Resistance Regulated By Serine/arginine Rich Protein Families In Tomato

Phytophthora infestans is one of the destructive diseases on potatoes and tomatoes,which seriously influences the quality and yield of potatoes and tomatoes.The epidemic of potato late blight caused by P.infestans lead to the Irish famine from 1845 to 1850.Until today,late blight still causes billions of economic losses per year in the worldwide,seriously threatening the world food security.The prevention and control of late blight in agricultural production mainly relies chemical pesticides.Heavy reliance on chemical prevention and control can easily lead to the frequent occurrence of chemical resistance,environmental contamination and other problems,and the long-term effective prevention and control of late blight cannot be achieved.The existing measures have not been able to prevent and control the disease effectively in a long term.As the development of research methods such as biochemistry,bioinformatics,genetics,it is becoming an urgent problem for us to deeply analyze the mechanism of interaction between phytophthora and host from the molecular level,to understand the immune mechanism of host against Phytophthora,and to provide new research ideas for the prevention and control measures of late blight diseases.Alternative splicing(AS)is an important regulation mechanism in eukaryotic gene expression.The same pre-mRNA can form a variety of different mature mRNA through alternative splicing,which generates many different proteins.Therefore,it is one of the main sources of the transcriptome diversity and proteome diversity,and one of the main regulatory mechanisms of eukaryote in response to environmental changes.SR proteins are a class of RNA binding proteins rich in arginine and serine and play an important role in the assembly of mRNA splicing complexes and the regulation of alternative splicing.In recent years,research on plant SR protein has been limited to model plants such as Arabidopsis and rice,and the function of SR protein in plant response to biological stress has not been reported.In this study,tomato was taken as the research object,and bioinformatics technology was used to found tomato SR proteins.It is expected to find the function of tomato SR proteins in the interaction between P.infestans and tomato.Systematic identification of tomato SR genes.Based on the whole genome database of Heinz tomatoes transcriptome data sequenced and other SR genes,a total of 16 genes encoding tomato SR protein were identified by Blast and other programs.Phylogenetic tree was constructed by MEGA program and systematically named,and some biological functions of tomato SR protein were preliminarily explored.The results showed that the 16 identified tomato SR proteins were divided into 6 subfamilies according to their structural characteristics and homology through multiple amino acid sequence alignment.Analysis SR protein by bioinformation,the SR protein in tomato was closely related to Arabidopsis and rice from the evolutionary tree.Protein domain analysis showed that the structure of SR protein was conservative and different.Physicochemical properties of tomato SR proteins showed that all SR proteins were hydrophilic basic proteins.The distribution of SR gene on chromosome was not pockety,we speculated that the function of SR proteins in tomato were conservative and different.Comparing the SR genes of different species,it was found that the number of genes encoded by SR protein in tomato was similar to that in other dicotyledons and monocotyledons,far higher than that other species.In order to investigate the disease resistance function of tomato SR protein,predict its promoter cis-acting elements found that SR genes were rich in pathogen responsive elements;Transcriptome sequencing showed that the expression levels of 8 SR genes were higher after inoculation with P.infestans;Alternative splicing data revealed that 14 SR genes had alternative splicing during the infection stage of P.infestans,these results suggesting that the tomato SR protein may play a role in disease resistance.SlSR30 and SlRS2Z32 regulated the immunity of tomato to P.infestans negatively.In view of the up-regulated expression of 8 tomato SR protein coding genes in the process of infection with P.infestans,the function of these genes was identified by using virus mediated transient gene silencing(VIGS)technology and transient gene expression technology of tobacco.The results showed that the transient silencing of endogenous SlSR30 and SlRS2Z32 in tomato by VIGS technology could significantly inhibit the infection of P.infestans,while the transient expression of SlSR30 and SlRS2Z32 in tomato could promote the infection of P.capsici and P.parasitica in tobacco.The above results indicated that SlSR30 and SlRS2Z32 were negative regulators of immune response in tomato.SISR30 and SIRS2Z32 participate in alternative splicing of pre-mRNA.The SR protein family is an important factor involved in the identification of alternative splicing sites.To further investigate the involvement of SlSR30 and SIRS2Z32 in the alternative splicing function of pre-mRNA,site-directed DNA mutation SlSR30/SlRS2Z32 in binding to two key amino acid sites of RNA,and the transient expression of SISR30Y46A F48A/SlRS2Z32Y46AF48A mutant on N.benthamiana leaves,it was found that the mutants lost the function of promoting infection of phytophthora,indicating that RNA binding is one of the important mechanisms of immune regulation by tomato SR protein.The alternative splicing reporting system detected that SlSR30/SlRS2Z32 affected the expression of reporter gene in different transcripts,which further proved that this protein was involved in the alternative splicing of pre-mRNA.EMSA experiment preliminarily confirmed that SlSR30/SlRS2Z32 protein could bind RNA probe.Yeast two-hybrid screened spliceosome proteins interacting with SlSR30/SlRS2Z32,including SlSR45 and SlU1-70k,suggesting that SlSR30 and SlRS2Z32 proteins may form complexes with these spliceosome proteins to regulate pre-mRNA splicing.The above results indicate that SlSR30/SlRS2Z32 forms a complex with other splicing proteins,participates in alternative splicing of pre-mRNA,and mutations of the its key sites of RNA binding,affecting the function of this protein in plant regulating immunity.