| Grape（Vitis Vinfera L）is an important economic fruit tree in China and is often severely damaged by downy mildew.To isolate and identify the disease-resistant genes by using grape’s own resources has great value to study the mechanism of grape resisting downy mildew and to improve grape varieties.The two-layer immune system of plants provides a theoretical basis for screening disease-resistant genes.The first layer is PTI（PAMP-triggered immunity）,which triggered by the PRRs（pattern recognition receptors）of plants recognizing PAMPs pathogen-associated molecular patterns（PAMPs）.The second layer is ETI（effector-triggered immunity）,which triggered through protein encoded by the resistance gene（R gene）of plants recognizing effectors secreting by pathogenic microorganism.Based on the PTI pathway and the transcriptome data from the laboratory,we screen PRRs that were differentially expressed between compatible and incompatible Plasmopara viticola infections.Then we conducted structural analysis and functional identification.According to the ETI pathway,the function of plant hormones and related genes in Systemic Acquired Resistance（SAR）pathway was analyzed,which laid a theoretical foundation for the research on grape resisting downy mildew.Results indicated that:1.In this study,12 PRRs with significantly different expressions were selected through laboratory transcriptome data.After the V.amurensis ’Shuanghong’ infecting compatible strain "ZJ-1-1" and incompatible strain "JL-7-2",we analyzed the expression pattern of PRR by RT-PCR.The results showed that the PRRs expression patterns were divided into three types under infection with compatible and incompatible P.viticola.Clear and consistent trends were observed in the expression（both up-and downregulation）of some genes.In contrast,some PRRs were upregulated after inoculation with P.viticola ’ZJ-1-1’ but downregulated after inoculation with incompatible P.viticola ’JL-7-2’.However,among the remaining genes,we observed downregulation after inoculation with ’ZJ-1-1’ and upregulation after inoculation with ’JL-7-2’.Thus,we chose the PRR which was expressed at the earliest post-inoculation time point and exhibited maximum upregulation for further functional verification of P.viticola resistance in V.amurensis’Shuanghong’.2.We cloned VaHAESA from the ’Shuanghong’ subspecies of grape based on the aforementioned transcriptome data.The VaHAESA gene is 3,066 bp long and encodes 1,021 amino acids.Further characterization of the VaHAESA protein sequence using the PFAM and SUPERFAMILY 2 databases confirmed that VaHAESA belongs to the LRR-RLK protein family,due to the presence of a leucine-rich repeat N-terminal domain（LRRNT）,two LRR8 domains,and a protein kinase C-terminal domain.Through phylogenetic tree and simulated protein crystal structure,PRR is further identified as the HAESA subfamilyof the LRR-RLK.Then we named this PRR as VaHAESA.It was found that VaHAESA localized to the cytoplasmic membrane by transiently expressed VaHAESA in N.benthamiana.The transient expression analysis showed that 3d after inoculation,grape leaves transiently transformed with the VaHAESA construct exhibited smaller infected areas than those transformed with the empty vector and untransformed V.vinifera’Thompson Seedless’.The contents of H2O2,NO,and callose in the grape leaves of the transient VaHAESA was detected to be higher than the latter two.These results show that disease resistance was improved accordingly in the transient grape leaves.The abiotic stress experiments showed that VaHAESA increased the salt tolerance and drought tolerance of grapes,but there was no obvious effect on cold resistance.3.The contents of phytohormones ABA,JA,and SA in different resistant grape varieties were determined after inoculating P.viticola,and it was found that the content of SA was most different in susceptible and resistantgrapes.We performed qRT-PCR on the key genes of the SA synthesis and SAR pathway.From these results,we can infer that the expression levels of PAL,NPR1,TGA1 and PR1 are closely connected with the grape’s resistance to downy mildew,higher the expression level of these genes,stronger the resistance of grape species.4.In the SA-mediated disease resistance pathway,NPR1,which is located downstream of SA and upstream of PR gene,has become an important regulatory factor in the SAR pathway.We used NPR1 of grapevine as bait and screened a cDNA library of grapevine after inoculation of Plasmopara viticola using yeast two-hybrid assays.The results showed that NPR1 respectively interact with TRF1 and SnRK2 in the grapevine.We further confirmed the interaction through subcellular localization and bimolecular fluorescence complementation.However,further research is needed to elucidate these processes. |
