Construction Of Tomato SlMYC2 Knockout Lines By CRISPR/Cas9 Technology And Exploring Its Function In Fruit Disease Resistance

Tomato is not only as an economic crop with important nutritional value s and special flavor but also as a model crop for disease research.Disease is one of the prime reasons leading to postharvest loss of fruits and vegetables.So it is of great significance for disease control to study the mechanism of fruit-induced disease resistance using tomato as a material.Jasmonic acid(JA)and it?s methyl ester(methyl jasmonate,Me JA)are important hormones in plants,which can effectively regulate various biotic and abiotic stresses in postharvest fruits and vegetables.MYC2 is an important transcription factor of JA signaling pathway and participates in various metabolic processes regulated by Me JA.At present,there is still no in-depth study on the role of MYC2 in Me JA-mediated post-harvest disease resistance of fruits and vegetables.In this paper,we used Micro-Tom tomato as material and employed CRISPR/Cas9(clustered regularly interspaced short palindromic repeats and associated cas9 protein)system to generate loss-function mutants of SlMYC2 and the reporter gene SlPDS which encodes phytoene desaturase.Then,the wild type fruit and SlMYC2 mutants were treated with exogenous Me JA to further investigate the role of SlMYC2 in Me JA-induced tomato fruit resistance to Botrytis cinerea.The main results are as follows:(1)Using CRISPR/Cas9 gene editing technology,the SlPDS and SlMYC2 were successfully mutated.The slpds mutants showed photobleaching(white plants)phenotype.We obtained two strains of slmyc2 mutants,which were named Line 2(knockout 3 bp)and Line 7(knockout 10 bp),repectively.The leaves and fruits of slmyc2 mutants differed from those of wild plants.Compared with the wild type plants,the number of flowers were increased,but the fruit setting rate was reduced in slmyc2 mutants.In addition,the fruit shape of slmyc2 mutants were prolate while the wild fruits were nearly spherical.(2)The disease resistance was reduced in SlMYC2 mutation fruits,and the disease incidence and lesion diameter were significantly higher than wild type fruit.The disease incidence of controls and slmyc2 mutants was 33.3 % and 100 %,respectively,after 2 days of inoculation.Furt hermore,the lesion diameter of slmyc2 mutants was 6.4 mm,which was about three times higher than that of control fruits.(3)SlMYC2 mutation significantly inhibited the activities of fruit resistance-related enzymes(chitinase,β-1,3-glucanase,phenylalanine ammonia-lyase,polyphenol oxidase)and antioxidant enzymes(peroxidase,catalase,ascorbate peroxidase)and the expression of MYC2.Exogenous Me JA treatment effectively increased the activities of the above enzymes in wild-type and SlMYC2 mutation fruits,but the SlMYC2 mutation significantly inhibited the inductive effects of Me JA on the above enzyme activities,disease resistance of fruits and the expression of MYC2.Therefore,the results indicated that SlMYC2 played important positive regulatory roles in Me JA-induced disease resistance and antioxidant process in tomato fruit.From the above experimental results,it can be found that SlMYC2 played a vital important role in regulating tomato growth and development,and positively participated in the response of exogenous Me JA to tomato fruit resistance to gray mold.