Functional Analysis Of PvRxLR4 Effector Of Plasmopara Viticola

In grape,downy mildew is an important disease and is widespreadly distributed in cultivated area in China.Garpe downy mildew is caused by Plasmopara viticola(Berk.& MA.Curtis Berl.& De Toni)which could secrete effectors into plant cells to suppress defense responses.The sequence of the PvRxLR4 effector was predicted by the transcriptome sequencing from in vitro germinated Plasmopara viticola spores in our laboratory.PvRxLR4 was one of the effectors to induce the cell death of N.benthamiana leaves.The functional analysis of PvRxLR4,which included its deletion mutants,localization mutations and site-specific mutation,was performed,further providing a molecular basis for revealing the pathogenic mechanism of Plasmopara viticola.The key results are as follows:1.A 750 bp nucleotide sequence of PvRxLR4 effector of Plasmopara viticola was obtained by PCR amplification.The secondary structure of PvRxLR4 protein has 13 alpha helices and one beta fold.PvRxLR4 cotains a signal peptide sequence,a Rx LR motif and a glycosylation site at the N-terminus.PvRxLR4 was highly expression in early Plasmopara viticola infection stage in Vitis vinifera cv.Piont Noir leaves.2.PvRxLR4 can cause the cell death in N.benthamiana leaves by transiently expressed.The subcellular localization assasy indicated that PvRxLR4 is localized at nucleus and cytoplasm in N.benthamiana protoplast.The cytoplasmic localization and the C-terminus of PvRxLR4 have an significant founction on triggering the cell death in N.benthamiana leaves.Because of characteristics of N-terminal signal peptide,PvRxLR4 was secreted into extracellular region,thereby being nontoxic for N.benthamiana leaves.It could trigger disease resistance and defense response in N.benthamiana,and its function was also depend on cytoplasmic localization.3.Virus-mediated gene silencing(VIGS)assays demonstrated that SGT1 and MEK2 were required for the cell death triggered by PvRxLR4 in N.benthamiana.4.The relative expression of related genes including MEK2,SGT1,PAD4,PR1 b and Rboh B of transgenic tobacco plants,was analyzed by q RT-PCR in different periods of infection.The result suggested that PvRxLR4 was able to strengthen transcript levels of SGT1,SA signaling pathway related genes,and ROS related genes to improve plant disease resistance through regulating expressing levels of MEK2 in MAPK cascade.