Fine Mapping And Functional Analysis Of The Sl5R-1 Gene Conferring Resistance To Tomato Spotted Wilt Virus (TSWV) In Tomato

Tomato（Solanum lycopersicum）is the world’s most economically important vegetable crop.The global annual planting area has exceeded 5 million hectares,and the output is as high as 180 million tons,accounting for 72%of the worldwide economic value of the global fresh vegetables.Diseases have always been the main factors affecting tomato production,Tomato spotted wilt is a worldwide disease.In recent years,it has generally occurred in China,resulting in a large-scale reduction of tomato production by more than 80%or even a failure of the harvest.The pathogenic Tomato spotted wilt virus（TSWV）is one of the top ten plant viruses recognized globally.Sw-5 is the only cloned TSWV resistance gene in tomatoes,which has broad-spectrum resistance to a variety of Tospovirus isolates and has been widely used in disease resistance breeding in tomatoes.However,the resistance of Sw-5 has been overcome or broken at high concentrations and specific TSWV isolates after years of long-term application and evolutionary variation of TSWV.Therefore,screening new TSWV-resistant tomato breeding materials,mining new TSWV-resistant genes,and cultivating new disease-resistant tomato varieties are effective ways to alleviate the current damage of TSWV,which are of great significance for tomato resistance breeding and sustainable development of the industry.In this study,based on the study group’s previous works,homozygous cherry tomato plants H149（S.lycopersicum var.cerasiforme）with high resistance and homozygous common tomato M82（S.lycopersicum cv.M82）with high susceptible to TSWV were utilized as parent materials for resistance evaluation,population construction,major QTL fine mapping,candidate gene identification,and the resistance gene Sl5R-1 function verification,and research on the regulatory mechanism of TSWV resistance was investigated.The main results are as follows:1.The cherry tomato H149 was new germplasm with high TSWV resistance.The artificial identification of TSWV results showed that the disease index（DI）value of H149 was3.26±1.04,which belonged to the high-resistant（0<DI≤5）;Trypan blue and diaminobenzidine（DAB）staining showed that H149 showed strong hypersensitive responses（HR）to TSWV infection;Ion deposition experiments showed that inoculation with TSWV caused H₂O₂accumulation in H149 plants.Molecular markers identified that H149 did not carry the known disease resistance gene,which was new tomato germplasm with high TSWV resistance and stable resistance without existing genes,and could be used as a resistance parent for the construction of resistance gene mapping population.At the same time,the tomato spotted wilt resistance evaluation system was further improved,including TSWV isolation detection,artificial inoculation of TSWV,plant resistance identification,molecular marker screening,and HR experiment,etc.,which laid the material and method foundation for the mining of new resistance genes.2.The major QTL Sl SW5-1 for TSWV resistance was located in the 145 Kb region of chromosome 05 on tomato.The six-generations of F₁,BC₁F₁-P₁,BC₁F₁-P₂,and F₂ populations were constructed with M82 and H149 as parents.Through TSWV inoculation and disease resistance rating analysis,the disease resistance phenotype of the F₂ population of each plant in three years（2017,2018,and 2020）conformed to the standard normal distribution curve,and the resistance traits were segregated and stable,which belonged to quantitative traits controlled by multiple genes.Within the preliminary mapping interval（1.37 Mb）of the previous study group,six new SSR polymorphism markers were designed and developed to carry out and polymorphism analysis of 1971 individual plants of the F₂ population in 2020was carried out,and combined with the resistance phenotype of each plant artificially inoculated with TSWV,the main QTL locus Sl SW5-1 was fine-mapped in the 145 Kb region on chromosome 05 of tomato,and there were 14 candidate annotation genes in this interval.3.Sl5R-1 is a candidate gene for tomato TSWV resistance.q RT-PCR analysis of the transcription levels of 14 candidate genes in the resistant and susceptible plants after inoculation with TSWV showed that the expression level of Sl5R-1（Solyc05g0097401.1）at7-28 days post-infection（dpi）in the resistant plants H149 was significantly higher than that in the susceptible plants M82.VIGS was used to silence Sl5R-1 in resistant plants H149,and the silencing plants showed obvious disease phenotypes at 35 dpi after inoculation with TSWV,and DI and incidence rate（IR）were increased by 1.8 times compared with the control plants,and the expression of TSWV-cp was also significantly higher than that of the control plants.The above results suggest that Sl5R-1 was a key gene involved in the regulation of TSWV resistance.4.Cloning and functional domain analysis of Sl5R-1 gene.The CDS of Sl5R-1 is 2,244bp,encoding 747 amino acids.Amino acid sequence alignment,cluster analysis,and domain analysis showed that it contained NB-ARC-LRR disease-resistance domain,clustered into a subfamily with the Sw-5 gene,and was highly conserved with Sw-5b protein,which was a typical NLR disease-resistance gene.The analysis of cis-acting regulatory elements（CARE）and transcription factor binding sites（TFBS）showed that the Sl5R-1 promoter contained the active elements involved in the regulation of SA and JA;When compared to the susceptible plant M82,the Sl5R-1 promoter sequence in the resistant plant H149 was deleted by 1 bp’A’at the-1,039 position,forming a binding site（CCTATGTCAG）that interacts with the transcription factor Sl TGA9（Solyc06g074320.2.1）.Sl TGA9 belongs to the b ZIP family and plays a key role in plant primary immune response PAMPs or SA signaling pathway.5.Sl TGA9 may be involved in the transcriptional regulation of the Sl5R-1 gene and confer H149 plant resistance to TSWV by affecting the SA-and JA-signaling pathways.q RT-PCR analysis showed that the expression of Sl TGA9 in resistant plants H149 was significantly higher than that in susceptible plants M82 at 7-14 dpi after inoculation with TSWV.The difference in phytohormones content between resistant and susceptible parents after TSWV inoculation was determined by liquid chromatography-mass spectrometry（LC-MS）.The results showed that SA and JA were significantly higher in H149 than in M82,and the transcription levels of key genes in SA-and JA-signaling pathways and defense-related genes Sl PR1,Sl NPR1,Sl POD,Sl SOD,and Sl CAT in resistant plants H149 were also significantly higher than in M82.After transient overexpression of Sl5R-1 in tobacco by injection,it was found that the injected area of tobacco leaves produced a strong HR to TSWV.The above results indicate that Sl TGA9 may be involved in regulating the transcription of the TSWV resistance gene Sl5R-1,and Sl5R-1 plays an important role in the regulation of SA-and JA-signaling pathways.In summary,tomato H149 is a new TSWV resistance source material,Sl5R-1 is a new gene with TSWV resistance,and Sl TGA9 has transcriptional regulatory effect on Sl5R-1resistance.Sl TGA9 could activate the up-regulated expression of Sl5R-1 by binding to the binding site in the promoter region of Sl5R-1,resulting in a strong HR at the TSWV infection site.Simultaneously,Sl TGA9 was also involved in the expression of genes related to SA-and JA-signaling pathways,which led to the accumulation of SA,strongly induced the up-regulation of resistance defense genes and enhanced SAR response;The pathogen-associated molecular pattern（PAMP）-triggered immune response（PTI）and effector-triggered immune response（ETI）of the two-layered immune system in tomato plants were triggered,and the induction of systemic resistance ISR mediated by JA was greatly enhanced,finally conferring Sl5R-1 with the resistance regulatory function for TSWV.The results of this study are of great significance for the study of tomato TSWV disease resistance genes and disease resistance breeding.