Microarray gene expression analysis to better understand the grape / Xylella fastidiosa interaction

• Author(s): Lin, Hong;
• Abstract: The gram-negative bacterium Xylella fastidiosa is the causal agent of Pierce's disease (PD) in grape. This disease is vectored by xylem-feeding insects and continues to cause serious vineyard losses in California. PD resistant plants produced by classical or molecular breeding techniques are the ultimate solution to this disease. However, information regarding genetic control of resistance is limited and the molecular basis of resistance mechanisms to PD is unknown. To fill this latter knowledge gap, we constructed 12 tissue specific (stem, leaf and shoot) subtractive suppression hybridization cDNA libraries from highly resistant and highly susceptible sibling genotypes from a Vitis rupestris x V. arizonica segregating in response to X. fastidiosa infection. We sequenced and annotated 5,794 PD-specific transcriptional profiles. To maximize gene discovery, an in-house EST database with 33,933 ESTs available from public sources (12,593 unigene set from the V. vinifera, 10,704 accessions from V. shuttleworthii, 6,533 from V. rupestris 'A. de Serres' x V. arizonica, 2,117 of V. aestivalis, and 1,986 sequences of V. riparia) was used to generate 20,020 non-redundant Vitis transcripts. This EST set was used to design a custom 60-mer high density (382,900 probes) microarray chip. Total RNA from stem and leaf tissues was used to hybridized 36 slides (eighteen for each explant) in a two-color experiment using the monochromatic dyes Cy5 and Cy3. RNA from three time points: early (1 week), mid (6 weeks) and late (10 weeks) stages of disease development from both infected and non-infected tissues of resistant and susceptible genotypes was analyzed. For each time point, there were three slides (biological replicates) including a dye flip. Differentially expressed genes reflecting spatial and temporal responses to PD were identified. Candidate genes underlying metabolic pathways involving defense responses and pathogenicity have been analyzed and their biological function will be validated.
• Publication Date: Jul 2006
• Journal: 9Th International Conference On Grape Genetics And Breeding, Italy