Identification Of MicroRNA Involved In Powdery Mildew Resistance From Chinese Wild Vitis Pesudoreticulata

Grapevine powdery mildew(Erysiphe necator Schw. [syn. Uncinula necator(Schw.) Burr.]) is one of the most severe fungi diseases which endangers the growth of grapevines and retards the development of grape industry. Plant microRNA(miRNA) is believed to play important regμlatory roles in response to biotic stress. However, by far most researches about powdery mildew resistance of grapevines focus on finding resistance genes, uncovering metabolic pathways of their translation products and their regulation on transcriptional level; studies about miRNA mediated resistance response of fruit trees like grapevine are still rare. Searching for miRNAs from grapevine involved in powdery mildew resistance and clarifying their regulation towards disease resistance can not only enrich our understanding about how grapevine and other trees react to biotic attack, but also provides new ideas for improving or breeding resistant varieties. In this research, several vvi-miRNAs involved in powdery mildew resistance were identified from Chinese wild V. pseudoreticμlata W.T.Wang‘Baihe-35-1’ by employing small RNA high-throughput sequencing, combined with bioinformatics and molecular biological technologies. The main results were as follows:1. 25703809 and 24829158 clean reads were obtained from ‘Baihe-35-1’ leaves inoculated with Erysiphe necator and the control respectively. The proportations of mapped reads to grape genome were 6.16% and 6.55%. The length of small RNAs mainly distributed between 18 and 21 nucleotides, with 21 nt and 24 nt as dominant length.2. mi RDeep-P was exployed for miRNA mining. Finally 250 miRNAs were identified, including 126 known miRNA and 124 candidates. Of these miRNAs, 20 families were conserved and 15 were non-conserved. Especially, highly expressed NewmiR2118 was identified from grape. Blast analysis showed that mi R2118 was highly conserved.3. 485 targets were predicted for 214 miRNAs with grape genome as reference. Then GO analysis showed that the molecular functions of all target genes mainly involved in molecular binding, catalysis, transcriptional regulation, transportation and transition activity; target genes were also involved in 23 biology processes, including growth and development regulation, metabolism and response to stimulus. Combined target gene function annotation and previous researches about biotic stress resistance, 49 targets involved in disease resistance were identified for 15 known vvi-miRNA families and 17 candidates4. Small RNA high throughput sequencing showed that powdery mildew stress could affect the expression profile of miRNAs from ‘Baihe-35-1’, with 131 up regulated and 119 down regulated. Besides, 68 non-redundant sequences(represented 32 miRNAs) differentially expressed, of which 18 were induced and 11 were depressed. Noticeably, there were 3 candidates whose expression obviously increased. Finally, 14 miRNAs from ‘Baihe-35-1’ involved in powdery mildew resistance were identified with 36 corresponding targets.5. The expression patterns of mi RNAs under fungi stress were also examined by semi-quantitative RT-PCR and qRT-PCR, which displayed similar expression trends for most mi RNAs tested. The expression of those depressed miRNA was mainly down regulated from 24 to 72 hours after being inoculated with Erysiphe necator. Especially, the down regulation of NewmiR2118 happened earliest and most greatly, indicating that NewmiR2118 might involved in the powdery mildew resistance in ‘Baihe-35-1’.6. In this research, Newmi R2118 was chosen for further analysis. To prove the interaction of NewmiR2118 with one of its predicted target RPP13, we adopted the method of co-transient transformation of 35S:: MIR2118 and 35S:: RPP13-GUS into tobacco leaves mediated by Agrobacterium tumefaciens. Histochemical staining showed that little GUS protein accumulated in co-transformed leaves, GUS fluorescence quantitative assay showed there were significant difference(P<0.01) between co-transformed leaves and those positive control groups, so both histochemical staining and fluorescence activity proved that NewmiR2118 could negatively regulate the expression of RPP13, and that RPP13 was a real target of NewmiR2118 from Chinese wild V. pseudoreticulata W.T.Wang‘Baihe-35-1’.