Discovery of key genes for White Rot resistance in Vitis davidii based on WGCNA

Abstract:【Objective】White-rot [Coniella diplodiella (Speg.) Petrk & Sydow.] of grapes is one of the most dangerous diseases, and different grapevine germplasm differs in their resistance to white- rot. In the previous study, the disease- resistant germplasm of Vitis davidii 0941 was screened, which was a wild type of grape in China with the characteristics of heat and humidity tolerance and good disease resistance. In this study, we screened the key genes of resistance to white rot in V. davidii by transcriptome sequencing and WGCNA.【Methods】The disease-resistant V. davidii 0941 (Vd) and the diseasesusceptible V. vinifera‘Manicure Finger’(Vv) were used as test materials to study the differences in gene expression between disease-susceptible and disease-resistant germplasm resources under pathogen induction conditions by inoculation indoors at three time points of 0 h, 24 h and 48 h. The disease resistance genes were screened and validated using qRT- PCR.【Results】According to the p≤ 0.05 and |Log2Fold Change| ≥ 2, the 5266, 4725 and 4653 differentially expressed genes were screened 0 h, 24 h, and 48 h with the resistant cultivar (Vd) and susceptible cultivar (Vv). Among them, the numbers of upregulated genes were 1737, 1788 and 1727, and the numbers of down-regulated genes were 3529, 2937 and 2926, respectively. Further, these differential genes were enriched by GO (Gene Ontology) andKEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. Some defense responses were observed, such as transmembrane transport, some plant hormone signal transduction and other functions or pathways. In cellular components, the membranes, cells, cell parts, etc. had a high degree of enrichment, and in biological processes, the defense reactions, transmembrane transport, metabolic processes, cellular processes, etc. had a high degree of enrichment. In molecular functions, there was a high degree of enrichment in transcriptional regulatory activity and catalytic activity, and the results indicated that these functions played an important role in the defense response of V. davidii and white rot pathogens. By analyzing KEGG analysis, it showed that the up- regulated differential genes were mainly enriched in flavonoid biosynthesis, flavonoid and flavonol biosynthesis, phenylpropane biosynthesis and stilbene, diarylheptanoid and gingerol biosynthesis, α-linolenic acid metabolism, linoleic acid metabolism, and other metabolic pathways, while the down-regulated genes were mainly enriched in the plant-pathogen interaction, flavonoid biosynthesis, phenylpropane biosynthesis, plant hormone signal transduction and mitogen-activated protein kinase (MAPK). In signaling pathways, there were ABC transport, phenylalanine metabolism, amino sugar, and nucleotide sugar metabolism, diterpene biosynthesis, DNA replication, and other pathways. In addition, those genes and acids were also found, icluding phenylalanine ammonia- lyase gene PAL (Vitvi00g01431 and Vitvi16g01503), tetracoumarate CoA ligase gene 4CL (Vitvi16g00139), chalcone synthase CHS (Vitvi16g00990 and Vitvi16g01480), shikimic acid, quinic acid and hydroxycinnamic acid. The expression levels of five or seven genes of acyltransferase HCT (Vitvi11g00735) and flavonol synthase FLS (Vitvi08g01575) were much higher in the resistant cultivar Vd. The expression levels of astragalus synthase STS (Vitvi16g01465 and Vitvi16g01466) showed an upward trend in both grape and after pathogen induction, and the expression level in Vv was higher than that in Vd. In addition, In the process of grapevine induction by white rot pathogen, important defenserelated signal pathways such as auxin, cytokinin, gibberellin, abscisic acid, brassinosteroid, jasmonic acid, and salicylic acid were enriched in plant hormone signal transduction pathway. The expression of AP2/ERF (Vitvi05g01073) was 60 times higher in the Vv than that in the Vd 24 hours after inoculation, and 35 times higher in the Vv than that in the Vd 48 hours after inoculation. The expression of MYB (Vitvi02g01019), which was involved in the secondary metabolic pathway of phenyl propane in plants, was 100 times higher in Vd than in Vv 24 hours after inoculation, and 40 times higher in Vd than in Vv 48 hours after inoculation. According to the correlation analysis between the trend of gene expression modules and resistance traits, Pearson correlation coefficient (r>0.5) and significance p value (p<0.05) were used as screening conditions for MEyellow, MEblack and MEgreen. MEblack module was positively correlated with disease resistance at Vd0h, Vd24h and Vd48h, and MEyellow module was positively correlated with disease resistance at Vd48h. KEGG enrichment analysis of differentially expressed genes in the two disease-resistant related modules showed that the enrichment pathways mainly focused on flavonoid biosynthesis, MAPK signaling pathway, phenylpropane biosynthesis, and plant interaction. The top 150 genes with the highest connectivity in MEyellow and MEblack modules were selected as the core hub genes of the module, among which there were 13 candidate transcription factors. In the MEblcak module, 4 transcription factors (MYB, 2; EIN and TCP, 1) were associated with plant defense responses, among which MYB4-Like (Vitvi17g00232) had the highest number of DEGs with a total of 104; and these genes were mainly associated with plant metabolic reactions, such as phenylalanine biosynthesis, tryptophan metabolism, glycolysis, ubiquitin-mediated proteolysis, etc. In the MEyellow module, 9 transcription factors (MYB, 3; ERF, 3; bHLH, 2; bZIP, 1) were associated with plant defense responses, and the number of DEGs associated with MYB108 (Vitvi04g00076) was 134, the functionalannotation of these genes was found to be related to a variety of plant hormones, such as auxin, ethylene, salicylic acid and jasmonic acid, as well as redox reactions and flavonoid biosynthesis. In order to clarify the regulatory network between the disease resistances of different grape varieties, the WGCNA (Weighted Gene Co-expression Network Analyses) analysis was carried out to screen out some key modules and identify them through hub genes. The interaction network relationship among genes in the module was analyzed to identify the hub genes. Two flavonoid metabolism-related genes, two flavonoid metabolism- related genes and two hub genes were screened out by differential gene expression analysis, GO enrichment analysis, KEGG pathway analysis and WGCNA analysis, and their expression was verified by the fluorescence quantitative analysis.【Conclusion】Differentially expressed genes in response to white rot infestation were obtained from V. davidii and V. vinifera‘Manicure Finger’, and were significantly enriched in defence response, transmembrane transport and some phytohormone signalling pathways, and six candidate genes were screened for disease resistance, including F3'5'H, EIN and MYB, etc.