Characterization of Ve genes in Verticillium infected tomato (Solanum lycopersicum).

摘要

The Ve locus in tomato (Solanum lycopersicum) is implicated in resistance to Verticillium spp. Studies of the Ve locus in tomato have identified two inverted genes (Ve1 and Ve2) that independently confer resistance to the same pathogen when introduced in transgenic potato (Kawchuk et al., 2001). However, little was known about the structural or regulatory features that differentiate the defensive response in susceptible and resistant plants nor was the molecular mechanism of Ve gene-mediated resistance apparent. To address these questions, Ve genes from two tomato near-isolines of the cultivar Craigella were cloned for DNA sequence analyses. The comparison revealed a deletion at position 1221 of the nucleotide sequence in the Ve1 gene that would result in a severely truncated Ve1 protein; only single amino acid changes that do not correlate with the phenotype were predicted in the Ve2 protein. More substantial DNA differences were observed in the intergenic region, including a 36 by deletion upstream of Ve2 gene in the susceptible isoline. To characterize the transcription of the Ve genes during Verticillium infection, mRNA levels of different organs were assayed using quantitative RT-PCR, based on a homologous truncated internal control template. The assays indicated that levels of Ve2 gene expression in stems of the resistant plants at 5 days post infection were two fold higher than levels in the susceptible variety. Furthermore, levels of Ve1 gene expression in the resistant near-isoline were up to seven fold higher, consistent with this gene playing a more preponderant role in the defensive response. As a step to testing, independently, the role of each Ve gene in vivo, two different RNAi plasmid constructs, controlled by an ethanol inducible promoter, were introduced into Craigella resistant tomato plants to silence each gene. Plants containing each type of transgene were regenerated and the expression assayed. The significance of all the observations with respect to Verticillium resistance is discussed.