Study On Molecular Mechanisms Of Interaction Between Tomato And Cucumber Mosaic Virus

Cucumber mosaic virus (CMV) is one of the most severe viruses infecting tomato and it causes severe yield and quality reduction all over the world. In China, yield loss caused by this virus is over 15% every year. Up to now, there is no effective method available for protecting plants from CMV infection and reducing the yield loss. Due to the lack of CMV-resistant gene in plants, the traditional breeding techniques cannot meet the requirement for crop improvement. In recent years, the rapid developments of plant genetic engineering and molecular biology have provided new tools for illustrate plant and pathogen interaction.The recent development of microarray-based expression profiling methods, together with the availability of genomic and/or EST (expressed sequences tag) sequence data for some plant species, has allowed significant progress in the characterization of plant pathogensis-related responses. Recent study revealed that virus infection can interrupt the miRNA expression of plant. In this study, a tomato-CMV gene expression microarray and a plant miRNA detection microarray were designed and constructed to employ in the profiling of tomato mRNA and conserved miRNA expression across time points after the infection of CMV.Construction and application of microarray for detecting gene expression of tomato and CMV. Based on the TIGR tomato TC sequences information and CMV-phy and satellite RNA-Rs, we design probes with length at 37-45 mer and in situ synthesized on 31×128 matrix microfulidic chip by usingμParaflo^TM technology. The first synthesized chip (Design-1) contained 3,311 tomato TC probes, 242 CMV-phy probes and 14 satellite RNA-Rs probes. In order to eliminate the cross hybridization phenomenon between tomato and CMV/sat-RNA, total RNAs, extracted from pooled healthy tomato and CMV-phy plasmid transcripts samples, were dually labeled with Cy3 and Cy5 and hybridized with Chip Design-1. The results showed that 38 CMV/sat-RNA probes and 20 tomato TC probes happened the cross hybridization. Chip Design-2 was generated according the hybridization result and applied in the comprehensive analysis of the differential gene expression patterns of tomato plants at 7 dpi by CMV infection. We also analyzed the CMV expression level. The results revealed in the follows: (1) Expression analysis identified 100 genes that displayed significant expression with 52 genes were up-regulated and 48 genes were down-regulated. These genes encode products involved in cell rescue/defense/, metabolism, energy, protein folding/modification/destination, protein synthesis, cell transport, signal transduction and transcript, binding and other diverse functions. (2) In systematically infected tomato samples, the expression of CMV sense RNAs was higher than those antisense RNAs, and the tendency of expression levels of the three genomic RNAs was RNA1*, which normally were present at a much lower level. The clustering analysis of the array data revealed two major groups were found at 20 dpi upon CMV infection and 32 detected miRNAs and 17 miRNA^* were found to be differentially expressed. Our miRNA microarray results presented herein represent the most comprehensive investigation of the tomato miRNA on a genome scale thus far and provide much needed information for post-transcriptional gene regulation mechanistic studies of this economically important plant.