| Tomato bacterial speck is a common tomato disease.As its high infectivity,rapid onset and difficult to control,this disease affects the quality of fruit and reduces the yield of tomato.The pathogenic microorganism causing the disease is Pseudomonas syringae pv.tomato(Pst).In resistant tomato,the resistance gene Pto,which encodes a kind of serine/threonine protein kinase,specifically recognizes the effectors released by Pseudomonas syringae pv.tomato,thus activating the downstream defense response.As a vital protein in the tomato disease resistance and immunity mechanism,Pto interacts with three transcription factors,named Pti4,Pti5 and Pti6 respectively,to function downstream of Pto-mediated pathway.The study on these Pto interaction proteins used the model plant Arabidopsis thaliana,but their phenotypes in tomato still need further analysis and comparison.In this work,the functions of Pti4,Pti5 and Pti6 were performed by obtaining stable and homozygous overexpression genetic transformation tomato lines.The results of q RTPCR showed that the expression levels of pathogenesis-related PR1,PR2 and PR5 were significantly increased in Pti4/5/6-overexpressed tomato plants.After inoculated with pathogen Pst DC3000,the number of disease specks and bacterial colonies in tomato leaves were significantly lower than that of wild type to exhibit improved disease resistance.As the sequences of these transcription factors are homologous with plant specific ethylene response element binding protein(EREBP),the possible effects of Pti4,Pti5 and Pti6 on fruit ripening were also explored in this study.The results of field observation and ripening experiment indicated that overexpression of Pti4,Pti5 and Pti6 resulted in fast color change and early ripening of tomato fruit,and enhanced response to ethylene.But there were no remarkabke changes on flowering time and seed setting rate,and also no obvious adverse effects on fruit characteristics of weight,size,shape,hardness,soluble solids content,and acid-sugar ratio.In addition,Pti4/5/6-overexpressed tomato showed delayed decay of fruits.The activities of related enzymes were further determined.The results indicated that the activities of phenylalanine ammonia lyase,catalase and peroxidase,which play important roles in synthesis of plant disease resistant metabolites and removal of intracellular active oxygen,significantly increased in overexpression lines.Accordingly,the content of malondialdehyde,reflecting the degree of cell membrane damage,markedly decreased.Additionally,the cell-free protein instablilty and in vivo ubiquitination assay showed that Pti4,Pti5 and Pti6 were degraded by ubiquitin-proteasome system,which suggested that these transcription factors themselves are also regulated when they involve in regulation of disease resistance and ethylene response.Therefore,the functional analysis of Pti4/5/6 indicated that they play a regulatory role in disease resistance and fruit ripening.The up-regulation of Pti4,Pti5 and Pti6 can improve resistance of tomato plants to pathogen,promote the fruits ripening but without obvious adverse effects on the yield and quality characteristics,and even have a certain effect on prolonging the shelf-life of tomatoes,which suggested their potential application in genetic improvement.In addition,the performances of Pti4,Pti5 and Pti6 imply the crossing of basic defense,hormones response and post-translatinal regulation.In this study,we futherly constructed the c DNA library of tomato leaves used yeast two-hybrid and screened the Pti interaction proteins to provide a foundation for investigation of its molecular mechanism. |
PDF Full Text Request