Identification Of Candidate Effectors Of Puccinia Helianthi

Sunflower rust,caused by Puccinia helianthi,often imposes tremendous threat to the sunflower production.During the interaction between rust and host plants,effectors,as important weapons secreted by rust,regulate the physiological process of the host and interfere with the immune function of the host.Finally,effectors invade the host tissues to obtain nutrients for propagation and reproduction.This study was based on the screening of effectors of P.helianthi in the previous study.Four proteins with more effectors characteristics were selected for bioinformatics analysis and cloning target genes.The plant transient expression vector was constructed for tobacco transient expression,and the effectors inducing or inhibiting cell death were screened and used for subcellular localization and expression pattern analysis.The main findings are as follows:1.Bioinformatics analysis of candidate effectors.With several bioinformatics softwares,including Signal P4.1,TMHMM S2.0,Pfam,big-PI Predictor,Prot Param,Sopma,Netphos 3.1 Server and others,the structural characteristics of candidate effectors were analyzed.The results showed that these four candidate effector proteins were predicted to contain signal peptide region,no transmembrane domain,no nuclear localization signal,no Pfam,no anchoring site.2.Identification of candidate effectors in inducing cell death or inhibiting cell death.The PGR107-target gene expression vector with signal peptide and no-signal peptide were constructed,and the transient expression was carried out in tobacco.It was found that P1,P2,P3,P4,P1^?SP,P2^?SP,P3^?SP,P4^?SPcouldn't induce hypersensitive reactions in tobacco cell.Candidate effectors were inoculated into tobacco leaves,after 24h,the BAX was co-expressed at the same site,the results indicated that the effectors P2 and P2^?SPcould inhibit cell death induced by BAX in tobacco leaves.3.The subcellular localization of effector.The P2 was constructed into the expression vector part-CAM-e GFP.The results showed that P2 mainly located in the cell membrane and nucleus.4.Expression pattern of P2 q RT-PCR was used to analyze the expression level of P2 in sunflower infected by rust.The results showed that compared with urediospore,the expression level of P2 was up-regulated at 24h?72h after infection,reaching a peak at24h,and the expression level was increased by about 14 times.