An Exploration Of Cellular Mechanisms Of The Interaction Between Erysiphe Necator And Wild Chinese Vitis And Functional Analysis Of Vpcdpk From Vitis Pseudoreticulata

The most economically important disease of cultivated grapevines worldwide is powdery mildew caused by the biotrophic fungal pathogen Erysiphe necator.To integrate effective genetic resistance into cultivated grapevines,numerous disease resistance screens of diverse Vitis germplasm have been conducted to identify powdery mildew resistance.Here,a new powdery mildew isolate that is infectious on grapevines was identified,designated Erysiphe necator NAFU1(En.NAFU1).Furthermore,controlled inoculations of En.NAFU1 were performed using detached leaves from 57 wild Chinese grapevine accessions to quickly evaluate powdery mildew resistance based on trypan blue staining of leaf sections.Furthermore we undertaken a survey about the histological resistance mechanisms of wild Chinese grapevines against E.necator.Calcium-dependent protein kinases(CDPKs)play vital roles in plant growth and development,biotic and abiotic stress responses,and hormone signaling.In this study,we performed a genome-wide analysis of the 12 X grape genome(Vitis vinifera)and identified nineteen CDPK genes.Furthermore,the predicted full-length coding sequences of grape VpCDPK genes were amplified by high-fidelity Taq HS-mediated PCR from cDNA of the Chinese wild grapevine V.pseudoreticulata accession Baihe-35-1leaves.Then we investigated the resistant function of VpCDPKs in Arabidopsis plants.This research obtained main results as follows:1.A new grapevine powdery mildew(PM)isolate,NAFU1,was isolated and characterized.The isolate of Erysiphe necator NAFU1 was obtained from a heavily infected powdery mildew V.vinifera cv.Rizamat plant in a vineyard.Three classical methods were compared for the maintenance of En.NAFU1.NAFU1 was maintenance on detached grapevine leaves by contact with infected leaves.2.Controlled inoculations of En.NAFU1 were performed using detached leaves from 57 wild Chinese grapevine accessions to quickly evaluate powdery mildew resistance based on trypan blue staining of leaf sections.Among the screened accessions inoculated with En.NAFU1,22.8% were resistant,33.3% were moderately resistant,and 43.9% were susceptible.The results were basically the same with previous natural epidemics in the field.3.We rooted the wild Chinese grapevine accessions,including the resistant Baihe-35-1,Baishui-40 and the susceptible Lingye,and susceptible V.vinifera cv.Thompson Seedless as control,by cutting in individual pots for the further study the resistant function of wild Chinese grapevines.In the trypan blue-stained leaf sections of ‘Baihe-35-1' and ‘Baishui-40'at 20 dpi,the rare post-invasion interaction with En.NAFU1 was accompanied by a large amount of PCD,but PCD was observed to a lesser extent in‘Lingye' and was absent in infected areas of ‘Thompson Seedless' plants.A more precise characterization at the ultrastructural level under transmission electron microscopy observations uncovered that in the case of ‘Baihe-35-1',haustoria appeared to be collapsed and formed hemispherical protuberances resembling papilla at the sites of attempted fungal penetration.However,in all cases,penetration of En.NAFU1 in the epidermis leads to the formation of haustoria in the‘Thompson Seedless' appeared well rounded and multilobed.4.To detect the accumulation of H2O2 from the four grapevines leaves post inoculation En.NAFU1,the infected leaves at 7 dpi was stained with DAB.The staining depth and stained shades were much stronger and larger in the infected leaves of ‘Baihe-35-1' and‘Baishui-40' than in the leaves of ‘Lingye' and ‘Thompson Seedless'.H2O2 accumulation in the ‘Baihe-35-1' and ‘Baishui-40' leaves was higher than in the leaves of ‘Lingye' and‘Thompson Seedless'.To assess the variation of SA levels during PM infection in the four grapevines,we measured the SA content in NAFU1-infected leaf tissues of wild Chinese grapevines accessions ‘Baihe-35-1',‘Baishui-40',‘Lingye',and V.vinifera cv.Thompson Seedless at 0dpi,1dpi,3dpi,5dpi and 7 dpi.The SA levels in NAFU1-inoculated leaf tissues of ‘Baihe-35-1' and ‘Baishui-40' are clearly higher than the content of Thompson Seedless.Total RNA of grape leaves was extracted and real-time PCR was employed to analyze the expression patterns of NPR1?PAD4?EDS1?PR1?PR2?RBOH?ARFA genes known to be involved in defense in the four grapevines.5.In this study,we performed a genome-wide analysis of the 12 X grape genome(Vitis vinifera)and identified nineteen CDPK genes.We cloned three VpCDPKs(VpCDPK2 ?VpCDPK9 ? VpCDPK19)from Chinese wild Vitis pseudoreticulata accession Baihe-35-1leaves.Then we constructed expression vectors of VpCDPKs to assessed the subcellular localizations of the encoded VpCDPKs by transient expression assays in Arabidopsis protoplasts,and obtained VpCDPKs over-expression transgenic Arabidopsis plants.The results indicated that VpCDPK2-GFP and VpCDPK19-GFP localized in the nucleus and cell membrane.Unlike the other CDPKs,VpCDPK9 localized to four places including some kind of plastids,biomembrane system,most likely on the endoplasmic reticulum(ER),as well as on vesicles and the nucleus.6.The phenotype analysis of VpCDPKs transgenic Arabidopsis showed that there are spontaneous cell death of VpCDPK9 transgenic Arabidopsis.What's more,VpCDPK9 transgenic Arabidopsis generated cell death in the leaves after inoculated powdery mildew by trypan blue staining indicated that VpCDPK9 transgenic Arabidopsis were resistent to powdery mildew.