The Potential Mechanism Of SlATL31-and SlHSP90-1-regulated Bacterial Wilt Resistance In Tomato （Solanum Lycopersicum）

Tomato（Solanum lycopersicum）is the largest vegetable crop in the worldwide,and the Ralstonia solanacearum is one of the important soil-borne bacterial wilt in tomato production.R.solanacearum has a large host range,wide distribution and persistent existence in soil,so it is extremely difficult to control in actual production.In recent years,proteomics technology has been widely used in the mining and analysis of plant disease resistance genes.In order to analyze the potential molecular mechanism of tomato resistance against R.solanacearum,in this study,we use TMT（tandem mass tag）technology to analyze the quantitative proteomics of tomato infected/uninfected with R.solanacearum GMI1000,and obtained the differentially expressed proteins.GO annotation analysis,subcellular localization prediction,KEGG enrichment analysis etc.were performed on the differentially expressed proteins.Two differentially up-regulated proteins,ubiquitin ligase SlATL31 and heat shock protein SlHSP90-1 were screened,and they were used as research objects to carry out pathogenicity analysis,enzyme activity detection,disease resistance-related event analysis and gene expression detection.The result is as follows:1.Proteomic analysis of tomato response to R.solanacearumTomato seedlings at 6-leaf stage with consistent growth were selected and inoculated with GMI1000 by leaf injection.Control plants were inoculated with 10 mM MgCl₂.Samples were harvested 24 hours after inoculation.Based on TMT technology,a total of 6207 proteins were identified,and 438 differential proteins were screened,including 310 up-regulated proteins and 128 down-regulated proteins.GO annotations indicated that these proteins were involved in biological processes such as cellular biosynthesis metabolism and stress response.Subcellular localization predictions show that indicated that the differential proteins were localized in the chloroplast,cytoplasm,nucleus,cytoplasmic membrane and mitochondria,etc.KEGG analysis showed that some differential proteins were enriched in pathways such as plant-pathogen interaction,phenylpropane biosynthesis,and MAPK signaling.2.Mechanism analysis of SlATL31 regulating tomato bacterial wilt resistanceThe SlATL31 gene was successfully cloned,and the transient expression vector pEGAD-SlATL31 and virus-induced gene silencing vector pTRV2-SlATL31 were constructed.Subcellular localization analysis showed that the GFP-SlATL31 fusion protein was localized in the cytoplasm and nucleus of Nicotiana benthamiana leaves.Real-time quantitative PCR（RT-qPCR）detection showed that the expression of SlATL31 was significantly down-regulated after 14 days of TRV inoculation,and gene-silenced plants were obtained.Pathogenicity analysis showed that overexpression of the SlATL31 significantly inhibited the growth of GMI1000,while silenced plants were more susceptible to GMI1000.Immunohistochemistry showed that SlATL31 positively regulated cell death and reactive oxygen species（ROS）accumulation in tomato leaves after GMI1000 inoculation.Enzyme activity assay showed that SlATL31 had no effect on the activities of superoxide dismutase（SOD）,catalase（CAT）and peroxidase（POD）in tomato leaves after GMI1000inoculation.After overexpression of SlATL31,the content of salicylic acid（SA）did not change significantly,while the content of jasmonic acid（JA）was decreased.RT-qPCR detected SA and JA signaling pathway defense gene expressions respectively.The results showed that after GMI1000inoculation,SlATL31 had no significant effect on the transcription level of SA signaling pathway-related gene SlPR1a,but overexpression of SlATL31led to the JA signaling pathway-related gene SlPI-Ⅱ transcription level significantly decreased,consistent with the change in JA content,while the JA signaling pathway defense gene SlPI-Ⅱ in gene-silenced plants did not change significantly.The above results indicated that SlATL31,as a positive regulator of tomato bacterial wilt resistance,played a certain role in tomato PTI and ETI immune responses.3.Mechanism analysis of SlHSP90-1 regulating tomato bacterial wilt resistanceThe SlHSP90-1 gene was successfully cloned,and the transient expression pEGAD-SlHSP90-1 vector and virus-induced gene silencing vector pTRV2-SlHSP90-1 were constructed.Subcellular localization analysis showed that the GFP-SlHSP90-1 fusion protein was localized in the cytoplasm of N.benthamiana leaves.RT-qPCR detection showed that there was no significant change in the expression of SlHSP90-1 after 14days of TRV inoculation,we speculated that the tomato HSP90 family may contain multiple isoforms,resulting in functional redundancy.Pathogenicity analysis showed that overexpression of SlHSP90-1significantly inhibited the growth of GMI1000.Immunohistochemical analysis showed that overexpression of SlHSP90-1 positively regulated cell death and ROS accumulation in tomato leaves after GMI1000inoculation.Enzyme activity assay showed that overexpression of SlHSP90-1 had no effect on the activities of SOD,CAT and POD in tomato leaves after GMI1000 inoculation.After overexpression of SlHSP90-1,the SA content did not change significantly,but the JA content was decreased.The expression of SA and JA signaling pathway genes were detected by RT-qPCR,respectively.The results showed that overexpression of SlHSP90-1 led a significant up-regulation of SA signaling pathway gene SlPR1a and a down-regulation of JA signaling pathway defense gene SlPI-Ⅱ after GMI1000 inoculation.Overexpression of SlHSP90-1 positively regulated tomato bacterial wilt resistance.In summary,a total of 310 up-regulated and 128 down-regulated proteins were identified in this study.Among them,most of the differential proteins were enriched in the plant-pathogen interaction signaling pathway.Through transient expression and virus-induced gene silencing,pathogenicity,enzyme activity,disease resistance-related event and gene expression analysis showed that SlATL31 and SlHSP90-1 overexpression positively regulates tomato bacterial wilt resistance.The SlATL31 and SlHSP90-1 as positive regulators,involved in the innate immunity of tomato against bacterial wilt and related to systemic acquired resistance,which provide a theoretical basis for possibly subsequent practical applications in the future.