The Spatiotemporal Expression Of Defensive Genes Induced By Fungal Pathogen Botrytis Cinerea And Cladosporium Fulvum In Tomato

Tomato is not only an economically important crop but also a classic model for studying the immune response of plants.During long-term evolution,plants has obtained a sophisticated system to defend pathogens,which relies on tempo-spatial expression programs of defense genes.Based on the lifestyle of pathogens,there are necrotrophic and biotrophic pathogens.Different types of pathogens trigger different expression patterns of resistance genes.Botrytis cinerea(B.cinerea)and Cladosporium fulvum(C.fulvum)are of most extensively studied necrotrophic and biotrophic fungal pathogens respectively,which also caused large economic loss for tomato production.To understand the mechanism of plant immunity,it is essential to differentiate the plant defense response to necrotrophic and biotrophic pathogens.In this study,we use tomato-B.cinerea and C.fulvum systems to characterize the gene expression patterns of induced genes by necrotrophic and biotrophic pathogens.We infected the tomato plants with B.cinerea and C.fulvum and detect the spatio-temporal expression of resistance gene expression by fluorescence quantitative PCR(qPCR).The results showed that the expression of resistance genes induced by B.cinerea and C.fulvum had distinct spatio-temporal patterns.The protease inhibitor(PI-II)gene and Threonine Deaminase(TD)were induced within 24 hours after infection,termed as early induced genes.PR-STH2 and PR1 genes were induced 3 days after infection and termed as late induction genes.At the same time,it was found that B.cinerea and C.fulvum infect could only induce the expression of resistance genes at the infected sites,but not in uninfected sites.To reveal the molecular basis of early responses of plant to necrotrophic pathogens,we constructed transgenic lines using PI-II as a marker gene.The major results from this study are described as followings:1.Two types of pathogens were used to infect tomato cultivar CM.Samples were collected at different time points with three biological replicates.Based on fluorescence quantitative PCR(qPCR)technology data,different inducible genes expressed at different time points.We found that PI-II and TD were early responsive genes,while ERF.C3,JA2 L,PR-STH2 and PR1 were late responsive genes during infection of B.cinerea.2.Only one leaf of two fully expanded leaves of tomato were infected with B.cinerea and C.fulvum.Both infected and uninfected leaves were harvested at different time points after pathogen infection.Each experiment has three biological replicates.qPCR analysis showed that PI-II,TD,ERF.C3,JA2 L,PR-STH2 and PR1 were induced in infected leaves but not in uninfected leaves.3.To further investigate the mechanism of early response of tomato to pathogens,we used early responsive genes PI-II as marker to construct YFP reporter lines.Gateway system was used to construct the PI-II overexpression vector with YFP fluorescent tag.Transgenic tomato lines were obtained by agrobacterium transformation.The PI-II-YFP expression of T0 plants was verified by PCR.We found that two transgenic lines have been transformed by the vector,which were used for future study on plant immune.