Omics Identification And Function Analysis Of Bacterial Wilt Resistance Genes In Tomato

Bacterial wilt is one of the most destructive bacterial diseases in the production of tomato(Solanum lycopersicum).However,the exploitation of resistant germplasm,especially broad-spectrum resistance genes,is very limited at present.To characterize the transcriptomes and further explore the resistance genes,several defense enzyme activity detection and RNA sequencing were performed in resistant and susceptible tomato inbred lines inoculated with Ralstonia solanacearum(Rs).Especially,to understand the genetic characteristics of NBS-LRR and Mlo genes and accelerate the utilization on genetic improvement of tomato bacterial wilt resistance,they were systematically identified and analyzed by bioinformatics,comparative genomics and molecular biology methods.1.On the whole,the activities of POD and PPO showed a downward trend in different tomato lines,but they were slower in the resistant line than that in the susceptible line.The PAL activity in the resistant line showed an upward trend,while the susceptible line showed a downward trend.The GST activity in the resistant line was lower than that in the susceptible line.The activities of β-1,3-GA and chitinase showed an upward trend in the resistant line.However,in the susceptible line,β-1,3-GA activity did not change significantly in the early stage,but showed an upward trend in the later stage,while chitinase activity showed a downward trend.2.A total of 1312 differentially expressed genes(DEGs,including32 new genes)were identified,including 693 and 621 up-and down-regulated genes,respectively.And further,836 genotype-specific DEGs(containing 18 new genes and Sl NLR5)were ascertained.Combined with gene co-expression,27 key DEGs were further ascertained.In total,1290 DEGs were annotated in eight databases,including 9 NBS-LRR genes,11 plant-pathogen interaction genes,16 plant hormone signal transduction genes,37 defense response genes,70 protein kinases,and 95 transcription factors.Sl NLR5,Sl Mlo1 and Sl Mlo6 were identified as core defense genes by gene set enrichment analysis.The output was confirmed using RT-q PCR for 62 representative genes.Solyc01g080220.3,Solyc02g086980.3,Solyc04g011670.3,Solyc04g058170.1,Solyc08g068480.1,and Solyc10g006710.4 may be involved in the Rs resistance response,whereas Solyc01g073985.1,Solyc03g093560.1,Solyc07g040960.1,Solyc08g079430.3,Solyc09g092580.4,Solyc09g098100.4,and Solyc10g081300.1 being the opposite.3.A total of 336 miRNA including 193 novel miRNA were identified.Among them,31 differentially expressed miRNA(DE miRNA)could target 575 genes(containing 31 DEGs),especially in 17genotype-specific miRNA-Targets.In total,556 DE miRNA targets were annotated,including 39 NBS-LRR genes,4 plant-pathogen interaction genes,5 plant hormone signal transduction genes,42 defense response genes,35 protein kinases,73 TFs,and some other important functional genes.In addition to typical TATA-box and CAAT-box with the transcriptional initiation role,as well as W box and TC-rich repeats related to biotic stresses,there were also hormone,light,abiotic stress,and other response elements in the downstream regulatory gene promoters.Six pairs of miRNA-Targets had three bacterial wilt response patterns in tomato: positive-negative,negative-positive and negative-negative,by RT-q PCR confirmation.4.Combined with RNA-seq results,two important genes were selected for further genome-wide identification.A total of 238,202 and217 NBS-LRR genes were identified in cultivated tomato and two wild tomato species(S.pennellii and S.pimpinellifolium),with 58,87 and 13 TNL,CNL and RNL genes,respectively.The NBS-LRR genes were mainly located in the nucleus,cytoplasm and chloroplast.They were unevenly distributed on different chromosomes in tomato,and mainly located at both ends,forming multiple gene clusters and tandem repeats,especially chromosomes 4 and 6.Phylogenetic relationship revealed that more than 50% of NBS-LRR had a homologous gene,including 115 pairs of orthologous genes and 8 pairs of paralogous genes,with most Ka/Ks values being less than 1,indicating that purification selection had occurred in evolution.It revealed the NBS-LRR genes were mainly expressed in root and leaf in tomato,and most could respond to different biotic stresses,by EST and RNA-seq date.RT-q PCR revealed that Solyc01g008800.2 probably involved in the Rs resistance response,whereas the others being the opposite.The transcription factors and interaction proteins that regulate target genes were mainly Dof,NAC and MYB families and kinases.5.A total of 197 Mlo genes were identified.Some Mlo proteins contained a signal peptide and a calmodulin binding domain.The vast majority of Mlo proteins were located in the cell membrane,and the transmembrane domains ranged from 5 to 7.A total of 359 polymorphic sites were detected from 143 sequences,from which 142 haplotypes were sorted,suggesting a rich nucleotide diversity of Mlo genes.Chromosomal localization revealed that 174 Mlo genes were unevenly distributed on different chromosomes or scaffolds,and mainly located at both ends,forming 19(24%)gene clusters,5 pairs(6%)tandem duplicates,and 61pairs(36%)segment duplicates.The Ka/Ks values of 25 pairs of orthologous genes and 28 pairs of paralogous genes ranged from 0.01 to0.78,indicating that purification selection had occurred in evolution.Sl Mlo genes contained multiple biotic and abiotic stress response elements,especially Sl Mlo1,Sl Mlo4,Sl Mlo5 and Sl Mlo12.RT-q PCR revealed that Sl Mlo1,Sl Mlo2,Sl Mlo4 and Sl Mlo6 probably involved in the Rs susceptibility response,whereas Sl Mlo14 and Sl Mlo16 being the opposite.Moreover,26 miRNAs were identified to target 12 Sl Mlo genes.6.Based on the omics characteristics of DEGs,further gene function studies were carried out.The target gene Sl NLR5 was integrated into the plant expression vector p BGV008 driven by the 35 S promoter,the interference fragment of Sl Mlo1/6 was integrated into the VIGS vector p TRV2,and the DNA fragment containing the high efficiency target was integrated into the CRISPR vector p BGK01.Three vectors were constructed,and the T0 generation tomato seedlings were further obtained by transformation.Thirteen(68%)overexpressed transformed plants showed a 284 bp specific band by PCR amplification,indicating the entry of target gene.Sequencing of target sites in 9 gene-edited plants revealed that M1 was a heterozygous Sl Mlo6 mutant.M2 and M8 lost a177 bp and 7bp Sl Mlo1 fragment respectively,with a heterozygous Sl Mlo6 mutation.M3 and M10 were heterozygous mutants of Sl Mlo1.Both M4 and M6 were heterozygous mutants with double genes.M7 lost a 12 bp Sl Mlo6 fragment,with a heterozygous Sl Mlo1 mutation.A Sl Mlo6 single base T insertion and Sl Mlo1 heterozygous mutation occurred in M9.RT-q PCR showed that compared with the wild type,Sl NLR5 gene expression level was significantly increased,while that of Sl Mlo1 and Sl Mlo6 were significantly decreased,in transformed plants(P<0.01).OE4 and OE5,and M2,M4 and M8 were more obvious.The phenotype of resistance to bacterial wilt of gene-silenced plants was preliminarily identified,suggesting that Sl Mlo1/6 may negatively regulate tomato bacterial wilt resistance.In conclusion,PAL,β-1,3-GA and chitinase may be used as a reference for early resistance identification in tomato.In addition,multiple resistance genes(negative regulation)and their miRNA play important roles in response to Rs infection.The specific regulation of new genes on the cell wall of the resistant line and the up-regulated expression of some DEGs in the susceptible line may be the main sources of different resistance.Some genes may co-regulate different stresses such as disease and drought.NBS-LRR and Mlo genes have a rich genetic diversity and are existed in cluster.During the conservative evolution,a large number of homologous genes have appeared through gene expansion and purification selection.Sl NLR5 and Sl Mlo1/6 may be involved in tomato bacterial wilt resistance.Functional genes are regulated by a variety of factors such as variation,alternative splicing,non-coding RNA,gene interaction,and epigenetic modification.By genetic transformation,13 tomato Sl NLR5 overexpressed seedlings and 9tomato Sl Mlo1/6 gene-edited seedlings were obtained,respectively.Phenotypic identification indicates that Sl Mlo1/6 may negatively regulate tomato bacterial wilt resistance.The functional transformation is mainly due to amino acid loss,premature termination of translation and frameshift mutation.In this study,resistance genes were systematically explored,and the possible application of broad-spectrum resistance was analyzed for the first time.The gene expression profile lays a foundation for the potential molecular regulation research,isolation and application of related genes(especially S gene)in tomato disease resistance breeding.