Function Analysis Of Tomato E3 Ubiqutin Ligase SlSL1

The ubiquitination pathway plays an important role in the regulation of plant growth,development and responses to internal and external environmental signals.Ubiquitin molecules were ligated to target proteins under the cooperation of multiple enzymes to allow degradation or modification of ubiquitinated proteins.E3 ligases,which determine the specificity of target proteins,function in ubiquitin linkage in the form of complexes or single subunit,but relatively inadequate studies have been conducted on tomato single subunit form E3 ligases.Tomato SlSL1(Solanum lycopersicum SINA-like 1)gene has a RING type E3 ligase domain,but its ubiquitin ligase activity and biological functions have not been investigated.In the present work,tomato SlSL1 gene was cloned for prokaryotic expression plasmid construction and the recombinant protein of SlSL1 was purified by induced expression and affinity adsorption,then the ubiquitination activity of SlSL1 was detected in vitro.SDS-PAGE electrophoresis and Western blotting showed that the expected recombinant SlSL1 was obtained from the constructed prokaryotic expression plasmid of tomato SlSL1 and the polyubiquitination of SlSL1 was observed in vitro assay.The results indicated the E3 ubiquitin ligase activity of SlSL1 by its self ubiquitination.The E3 ligase gene of SlSL1 was overexpressed in tomato by pBI121-SlSL1 overexpression vector construction and Agrobacterium-mediated genetic transformation.SlSL1-overexpressed lines were determined by quantitative real-time PCR.Compared with wild type plalnts,there was no significant difference in the growth and development of transgenic seedlings.However,after inoculated with Pst(Pseudomonas syringae pv.tomato)strain DC3000,the resistance of SlSL1-overexpression plants to the bacteria was remarkably enhanced by reduced leaf specks and pathogen colony counting compared to wild type.It was suggested that SlSL1 was involved in the defense response of tomato through ubiquitination pathway.To explore the targets regulated by SlSL1,yeast two-hybrid library derived from tomato multi-tissues was used for screening of SlSL1 interaction proteins.Six candidates,polyubiquitin,ubiquitin-60 ribosomal protein L40,IQ-DOMAIN 14-like,glycine-rich protein 2-like,glycosyltransferase and hsp70-Hsp90 organizing protein 2 were preliminarily screened,which provided clues for revealing the mechanism of SlSL1.Additionly,overexpression of SlSL1 indicted its potential application in genetic improvement by enhancing the defense of tomato against pathogenic bacteria without obvious adverse effects on plant growth,flowering time,fruit weight and size,fruit and seed development.Taken together,this work laid an important foundation for further investigation of the regulation role and mechanism of E3 ubiquitin ligase SlSL1 in immune response and provided the new gene resource for genetic improvement of disease resistance in tomato.