Construction Of Virus-mediated Tomato Gene Knockout System And Study On The Function Of Small G Protein Of ROP Family Regulating Bacterial Wilt Resistance

Tomato is an important vegetable crop worldwide.Since tomato production is seriously threatened by bacterial wilt disease,breeding of disease-resistant tomato varieties is a vital research objective.Improving tomato breeding techniques and exploring new disease resistance related genes are the key to solving this problem.Previous researchers have developed virus-induced genome editing(VIGE)to achieve mutagenesis of target genes in tobacco,cotton,wheat and maize.However,such gene editing tool has not been reported in tomato so far.In this study,we transferred tobacco rattle virus(TRV)vectors carrying the sgRNA of the target gene into tomato seedlings stably overexpressing Cas9 through agroinfiltration to establish a TRV-based gRNA delivery system for CRISPR/Cas9-mediated gene mutagenesis in tomato.We chose SlPDS1 as target and found the editing efficiency of this gene by VIGE was up to 86%.In addition,TRV vectors were detected both in the injection area of agroinfiltration and the apical meristem far away from the injected sites,and the young leaves also showed obvious knockout phenotype,indicating that sgRNA can move with the virus.In this VIGE system,more than half of the cells had DNA mutations at the genome level in the target gene,so the normal translated target protein reduced significantly.We believe gene silencing caused by this method would be more stable than normal VIGS technology and can be utilized for quickly functional analysis of genes in tomato.ROP small G proteins belong to the plant-specific Rho subfamily and serve as molecular switches for regulating diverse cellular events,including morphogenesis and stress responses.However,the immune functions of ROPs in Solanum lycopersicum L.(tomato)is still largely unclear.The tomato genome contains 9 genes encoding ROP-type small GTPase family proteins(namely SlRop1-9)that fall into five distinct groups as revealed by phylogenetic tree.We studied the subcellular localization and immune response induction of nine SlRops by using a transient overexpression system in Nicotiana benthamiana.Except for SlRopl and SlRop3,which are predominantly localized at the plasma membrane,most of the remaining ROPs have additional nuclear and/or cytoplasmic distributions.Interestingly,even though nine ROPs in tomato showed highly conserved and canonical RHO domains,only seven constitutively active forms of SlRops were able to trigger hypersensitive responses.Furthermore,we analyzed the tissue-specific expression patterns of nine ROPs and found that the expression levels of SlRop3,4 and 6 were generally high in different tissues.The expression levels of SlRop1,2 and 7 significantly decreased in tomato seedlings after infection with Ralstonia solanacearum,while the others did not respond.Transient overexpression of SlRop3 and SlRop4 suppressed the growth of R.solanacearum(Y45)in tobacco,whereas the rest of tomato ROPs may not contribute to defense.Compared to the WT plants,SlRop3 knockout plants exhibited more susceptible to R.solanacearum(GMI1000),confirming that SlRop3 positively regulates tomato bacterial wilt disease resistance.By yeast-two hybrid screening,we found SlRop3 interacts with several guanine nucleotide exchange factors(GEFs)and the interaction between SlRop3 and PRONE-type SlRopGEF7 is the strongest.Our study provides systematic evidence of tomato Rho-related small GTPases for localization,immune response,and disease resistance.In addition,we raised the possibility that SlRop3 may control tomato defense against bacterial wilt through associating with the GEF protein SlRopGEF7 and also provided new genetic resources and technique for breeding tomato disease resistance varieties.