| Bacterial spot caused by several Xanthomonas sp. is one of the most devastating diseases in tomato (Solanum lycopersicum L.). Due to the existence of multiple pathogen species and races, marginal efficacy of commonly applied chemicals, development of resistance to these chemicals in bacterial populations, and a lack of available disease resistance traits in commercial cultivars, control of the disease has not been effective once epidemics start. Exploiting host resistance gene(s) combined with important defense response genes for developing durable resistant cultivars is considered as an effective approach to manage the disease. The resistance response to bacterial spot can be divided into hypersensitive response and field resistance response. The genetics of hypersensitive resistance to X. perforans race T3has been intensively investigated, and regulatory genes during the infection of race T3have been identified through transcriptional profiling. However, no work on isolating regulatory genes for field resistance has been reported.In this study, cDNA-amplified fragment length polymorphism technique was used to identify differentially expressed transcripts between resistant tomato accession PI114490and susceptible variety OH88119at3,4and5days post-inoculation of the pathogen. Using256selective primer combinations, a total of79differentially expressed transcript-derived fragments (TDFs) representing71genes were obtained. Of which,60were up-regulated at different levels in two tomato lines,4were down-regulated in both tomato lines,4were uniquely up-regulated and2were uniquely down-regulated in PI114490, and1was specific up-regulated in OH88119. The expression patterns of19representative TDFs were further confirmed by semi-quantitative and/or quantitative real time RT-PCR. These results suggested that the two tomato lines involve activation of partly similar defensive mechanism in response to race T3.RNA-seq was used to investigate transcript dynamics response to X. perforans race T3in the resistant line PI114490and susceptible line OH88119. A total of six samples were examined, which were mock-treatment and race T3post-inoculation at6h (6HPI) and6d (6DPI) for both two tomato lines. An average of7million reads was generated with approximately21,526mapped genes in each samples, accounting for approximately62%of reference tomato genes. The differentially expressed genes (DEGs) between two tomato lines and two time-points were compared through venn diagram and cluster analysis. Overall, the number of DEGs response to race T3was more in PI114490than that in OH88119at both two inoculation stages, and the number of DEGs at6DPI was significantly more than6HPI in both tomato lines. Only78and15genes were up-regulated at6HPI in PI114490and OH88119, respectively, while the down-regulated genes were numerically greater, demonstrating that the inducible defense response against race T3might have not been activated at6HPI. Accumulation expression levels of326co-up regulated genes in two tomato lines at6DPI were likely to involve in basal inducible defense response, while the specific and strongly induced genes at6DPI might be correlated with the absence of symptom in PI114490. KEGG enrichment analysis indicated the number of up-regulated genes which related to the process of defense response including plant hormone signal transduction and plant-pathogen interaction pathway were more in PI114490than that in OH88119at6DPI. The differentially expressed genes containing NBS-LRR domain, defense-related WRKY transcription factor were presented, which might take prominent roles in defense process. Comparing the cDNA-AFLP analysis and RNA-seq data discovered14common up-regulated genes. These data will provide a valuable resource for mechanism studies underlying X. perforans interaction with tomato plants.Virus-induced gene silencing (VIGS) was used to validate the function of up-regulated genes. A gene encoding U-box protein, tentatively named SIPUB24, that may be related to the process of resistance was identified. SIPUB24VIGS-silenced plants showed numerically greater small black foliar lesions after inoculation with race T3than control plants. Using Agrobacterium-mediated transient expression assay to over-expressed SIPUB24in tomato leaves results in less race T3bacterial growth than control. So we speculated that SIPUB24gene might play a positive regulation role in tomato field-resistance to bacterial spot race T3. Subcellular localization showed the S1PUB24-eGFP fusion protein accumulated in the cytoplasm of transiently transformed onion epidermal. To further validate the function of SIPUB24, silencing vector of S1PUB24-RNAi and over-expression vector of35S-S1PUB24was transformated into tomato line PI114490and OH88119, respectively. Integration of S1PUB24-RNAi and35S-S1PUB24into tomato genome was confirmed by PCR, and at least twenty positive generation plants were obtained for disease evaluation.The results of differentially expressed profiling and SIPUB24, obtained in this study, will help to uncover the mechanisms of tomato-X.perforans interaction, and have potential application value on tomato resistance breeding for bacterial spot. |
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