Towards Understanding Mechanisms of Host Reisistance to Downy Mildew Disease of Grapevine (vitis L.)

<p>NON-TECHNICAL SUMMARY:<br/>Downy mildew (DM) is the single most damaging disease of grapes worldwide and can cause deformed shoots, tendrils, inflorescences and clusters of young berries. A major outbreak of this disease can cause severe losses in yield and berry quality. The European grape (Vitis vinifera) is generally highly susceptible to downy mildew disease. Downy mildew is also a limited factor to grow bunch grapes in East United States. In order to produce this crop, a heavy fungicide-spray program is required. A promising alternative strategy that could replace fungicides is to find out the unique genes in resistant species responding to the pathogen and improve the resistance of the bunch grapes to downy mildew through transgenic methods. In this perspective, it is important to understand the molecular mechanisms of natural defense responses of resistant
grapevine. In this regard, we propose to use muscadine grape, a grape completely "immune" to DM, as the resistant source. The overall goal of this project is to improve V. vinifera grapes and their hybrids to DM disease, making the viticulture industry more profitable /sustainable while protecting the environment. Specific objectives of this project include: 1) Identify genes resistant to downy mildew in muscadine grapes; 2) Isolate the putative downy mildew resistant genes 3) Improving downy mildew resistance of bunch grapes by genetically engineer the downy mildew resistant genes obtained from muscadine grapes; 4) Improve the biotechnology teaching capacity at FAMU, and train graduate / undergraduate students on genomics, bioinformatics and biotechnology. The proposed project is highly relevant to NIFA Strategic Goal by supporting research on global food security and food safety, as
well as sustainable rural economies.<p>
APPROACH:<br/>A highly downy mildew resistant muscadine cv. ""Noble"" will be used for identification and isolation of downy mildew resistant genes. Briefly, downy mildew pathogen Plasmopara viticola will be inoculated on the young leaves of muscadine ""Noble"", and un-inoculated ""Noble"" vines are used as a control. With three independent replicates, infected leaves will be collected every 24h after the pathogen infection for 8 days. Control samples are harvested synchronously. Total RNA will be isolated from the infected and controlled leaves from 1d to 8d using the modified CTAB method, and then converted into double stranded cDNA using standard methods. Each molecule in the library should contain a gene, and some of which would be our interest for downy mildew resistance. Based on previous research, we have identified a set of 200 candidate genes that are up- or down
regulated at least 50 times after downy mildew pathogen infection. In this project, a bioinformatics strategy will be applied to identify the up- and down- regulated transcripts from the musacdine grape ""Noble"". Northern blotting and subsequent Real Time PCR will be used for the data validation /confirmation. Genes expressed over or under-20 folds after downy mildew infection will be selected for further gene function annotation and expression analysis. Focus will be on several groups of genes such as 1) defense-related regulatory proteins; 2). Genes of pathogenesis related proteins; 3). Inhibition of pathogen virulence products 4). Gene related to cell membrane / cell wall degradation and cell death; 5) Signal transduction genes. Based on the functional analysis and expressed profiling, cDNAs or full length genes with putative downy mildew resistant function will be cloned and their
functiona will be validated with various functional genomics tools. Final confirmation of the putative downy mildew resistant genes is to transfer the genes (cloned from muscadine grape) into DM susceptible V. vinifera grapes, followed by infection and resistant evaluation. In the mean time, we will also use Arabidopsis as model plant system to test the putative DM resistant genes.</p><p>
PROGRESS:<br/>2010/09 TO 2011/08<br/>OUTPUTS: A highly downy mildew resistant muscadine cv. ""Noble"" was used for identification and isolation of downy mildew resistant genes. In this fiscal year, we have inoculated downy mildew pathogen Plasmopara viticola on the young leaves of muscadine grape ""Noble"", and un-inoculated ""Noble"" vines were used as a control. Infected leaves were harvested from 2 hours and up to three days after infection. Total RNA were isolated from both infected and controlled leaves using the modified CTAB method, cDNA libraries were constructed with each of the samples. The short ends of the cDNAs were being sequenced and the sequenced-based expression analysis has been initiated. A bioinformatics analysis strategy was applied to identify the up- and down- regulated transcripts from the cDNA sequenced musacdine grape ""Noble"". In the mean time, two
putative DM resistant genes are being cloned based on previous research results. We have also been integrating the research components and platform in biotechnology teaching and capacity building in Agriculture and Food Sciences. PARTICIPANTS: Jiang Lu, Ph.D. and professor, PI. jiang.lu@famu.edu, (850)412-7395 Xia Xu, Ph.D. Research Associate, xia.xu@famu.edu, (850)412-7395 Yali Zhang, visiting professor,olivia.yl.zhang@gmail.com 4. Jiao Wu, visiting Scientist 5. Yin Yu, graduate student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.</p>