Functional Study Of Grape Pathogenesis-related Protein 4b Against Downy Mildew

Plasmopara viticola is one of the most serious oomycete diseases in grape,which seriously affects grape production around the world.Pathogenesis-related protein(PR)plays an important role in plant disease resistance,however,there are few studies on its resistance to downy mildew in grape.In the early stage of this study,transcriptome sequencing was performed on the leaves inoculated with the downy mildew of the highly resistant Chinese wild grape Vitis piasezkii ‘Liuba-8' and the susceptible European grape Vitis vinifera cv ‘Pinot Noir' in different periods.PR4 b is one of the up-regulated genes induced by downy mildew.In this study,the structural characteristics and subcellular localization of Vp PR4 b were analyzed.The ‘Thompson Seedless' Vv PR4 b knockout transgenic grape seedlings were obtained by using CRISPR/Cas9 system,and the leaf discs of transgenic seedlings were inoculated with grape downy mildew to verify the role of PR4 b in grape downy mildew infection.The cis-acting elements of the Vp PR4 b promoter were further analyzed,and the upstream regulatory factors of Vp PR4 b were initially screened by yeast one-hybrid.The main results are as follows:(1)Both Chinese wild grape Vp PR4 b and European grape Vv PR4 b genes are induced by downy mildew,and the expression patterns are similar.Vp PR4 b and Vv PR4 b contain 432 nucleotides,encoding 143 amino acids.Amino acid sequences analysis found that Vp PR4 b and Vv PR4 b both belonged to class II-like PR4,with a signal peptide at the N-terminus and a Barwin domain at the C-terminus,without Chitin binding domain,and were located on the membrane.Sequence alignment revealed that the nucleotide sequence similarity between Vp PR4 b and Vv PR4 b was 98.61%,and the amino acid sequence similarity was 97.9%.(2)The CRISPR/Cas9 gene editing system was used to mutate the ‘Thompson Seedless' Vv PR4 b gene to obtain mutant grape materials,and the resistance of the Vv PR4 b gene mutant grape material to downy mildew was verified.The sequencing results of gene editing grape material showed that deletion was the main type of mutation,and the expression of Cas9 could be detected in these transgenic lines.In this experiment,129 regenerated grape seedlings were detected.The sequencing results showed that 26 gene editing grape seedlings were successfully obtained,and the gene editing efficiency was 20.16%.The analysis of all possible off-target sites showed that the off-target rate in the gene-edited lines was zero.The inoculation experiments using the “leaf disc method” showed that Vv PR4 b knockout transgenic lines were more sensitive to grape downy mildew compared to wild-type grapes.On the fourth day after inoculation with downy mildew,the relative biomass of grape downy mildew in gene edited lines was significantly higher than that in wild-type,and the accumulation of reactive oxygen species(ROS)around the stomata in gene-edited strains was lower than that in wild-type.The results proved that Vv PR4 b played a disease-resistant function in the defense of grape against downy mildew.(3)The sequence similarity between the promoters of Chinese wild grape Vp PR4 b and European grape Vv PR4 b gene was 95.65% and contained similar cis-acting elements.The activity of the Vp PR4 b promoter was enhanced by the induction of pathogen.It was confirmed that transcription factors WRKY40 and WRKY75 could bind to the Vp PR4 b promoter by yeast one-hybrid system.