Identification And Functional Analysis Of Proteins Interaction With VpCDPK9 And VpCDPK13 In Chinese Wild Vitis Pseudoreticulata

The powdery mildew caused by the biotrophic fungal pathogen Erysiphe necator is one of the diseases that seriously harmed cultivated grapevines worldwide.China has abundant wild grapevine germplasm resources.Thus,the integration of effective genetic resistance into grape cultivars would improve the resistance of European grapes to powdery mildew.Previous studies have found that the VpCDPK9 and VpCDPK13 were up-regulated under powdery mildew infection,but the function and mechanism of specific disease resistance are still unclear.Based on previous studies,in this study,we used Baihe-35-1 as the material to construct yeast c DNA library,and screening proteins interacted with VpCDPK9 and VpCDPK13,to further explored its molecular mechanism in the process of powdery mildew resistance.The main research results are as follows:1.The yeast c DNA library was constructed using V.pseudoreticulata accession Baihe-35-1.The capacity of the library is 2.1x10~6cfu,and distribution range of the inserted fragment length was about 400 bp～2k bp,and the recombination rate is about 100%,which indicated the library constructed successfully.The bait plasmid of p GBKT7/VpCDPK9p GBKT7/VpCDPK13,p GBKT7/VpCDPK9-KD,and p GBKT7/VpCDPK13-KD were successfully constructed.Self-activation and toxicity test showed that PGBKT7/VpCDPK9had strong self-activation activity and could not to be used to screen interaction protein.However,p GBKT7/VpCDPK13,p GBKT7/VpCDPK9-KD,and p GBKT7/VpCDPK13-KD had no self-activation and toxicity and could be used for next library screening.Co-transformation method was used to screen yeast c DNA library.We found four target proteins,namely VpJAZ5,VpMIEL1,VpAPC and VpBSL3 might interact with the kinase domain of VpCDPK9 and VpCDPK13.2.The results of Y2H combined with bimolecular fluorescence complementation(Bi FC)showed that VpCDPK13 can interact with VpJAZ5 and VpMIEL1 in both yeast cells and plant cells.Furtherly,the analysis of key domains showed that the N-terminal domains of VpJAZ5 and VpMIEL1 proteins were required for interaction with VpCDPK13.Whereas,VpCDPK9 can interact with VpJAZ5 and VpMIEL1 in yeast cell,but not in plant cells.Tissue specific results showed that expression level of VpJAZ5 was dominant in tendrils,followed by young leaves and old leaves,and the expression level in young leaves was higher than that in old leaves.Expression level of VpMIEL1 was highest in the old leaves,followed by the young leaves,and lowest in the roots and stems.q RT-PCR was used to analyze the expression patterns of VpJAZ5 and VpMIEL1 under hormone and powdery mildew treatments.The results showed that the expression level of VpJAZ5 was increased after MeJA treatment.But under SA treatment,the expression level of VpJAZ5 was lower than without treatment.VpMIEL1 transcripts accumulated highly under MeJA and SA treatments.In addition,the expression levels of VpJAZ5 and VpMIEL1 were also affected by powdery mildew,and the expression levels of VpJAZ5 and VpMIEL1 genes showed a significant upward trend in the early and later stages of powdery mildew inoculation.Therefore,it was suggested that VpJAZ5 and VpMIEL1 might be involved in powdery mildew response through MeJA and SA signaling pathways.