Functional Research And Resistance Mechanism Analysis Of LEACT1 From Tomato

Tomato yellow leaf curl disease is a serious disease to tomato production,which is caused by geminivius Tomato yellow leaf curl virus.The leaves of infected plant appeared curl,shrinking and chlorotic,the fruit set percentage fell drastically,which caused the production and quality dropped seriously.The resistant genes have only been identified in the wild tomato varieties but not in cultivated tomatoes,which were nominated as Ty-1,Ty-2,Ty-3,Ty-4 and Ty-5.Among them,Ty-2has the best resistant effect.Thecommonly method ofresistant varieties breeding is transferring the resistant genes to commercial cultivars by crossing them with wild materialsat present.By transcriptomic profiling of the resistant material CLN2777 A containing Ty-2 and susceptible 4840,we have identified a gene named Le ACT1 induced by TYLCV inoculation in CLN2777 A but suppressed in 4840.Le ACT1 was speculated been involved in TYLCV resistance.It was cloned by RT-PCR and bioinformatics analyzed firstly,then Real time RT-PCR was used to analyze its tissue and induction expression pattern.At the same time,the function of Le ACT1 was verified by virus induced gene silencing(VIGS)and transgenic analysis.In addition,the characteristics of the promoter of Le ACT1 were analyzed by transientinfection on tobacco.Finally,the PAL enzyme activity assay of transgenic plants was carried out to elucidate the resistant mechanism preliminarily.The main results were as follows:By transcriptomic profiling,a tomato gene Solyc11g071480.1 was found induced in the resistant material CLN2777 A,its RPKM(Reads Per Kilobase of exon model per Million mapped reads)value was 3.97 and 25.62 respectively before and after induction,but which was 32.59 to 4.01 in 4840 after infection.The significant different expression pattern in resistant and susceptible materials indicated the gene may play important role in the resistance to TYLCV.BLAST analysis showed that the gene encoding agmatine coumaroyltransferase,which was nominated as Le ACT1.It was cloned by RT-PCR,the sequencing results showed that the length of the open reading frame was 1332 bp,encoding a protein of 443 amino acids,and the molecular weight was 50.5 KD and isoelectric point was 6.07.Although there was no difference along the encoding area of the gene between the resistant and susceptible materials,but the similarity of the promoter region is only 89%.The expression pattern of Le ACT1 was analyzed by Realtime RT-PCR,the results showed the expression in leaves was lower than other organizations andthe highest expression was inbud,cortex and root.In addition,Le ACT1 was induced by TYLCV inoculation,the expression was increased at 3 days after inoculationand the apex appearedat the 5th day.The expression vector of Le ACT1 fused with GFP4 was constructed to analyze its subcellular localization and Le ACT1 was showed located in the nucleus and on the cell membrane.The promoter of Le ACT1 derived from the resistant plants was ligated to PBI101,and the construct was injected to tobacco combined with individual protein of TYLCV to analyze its characteristics by GUS staining.The results showed that each protein of TYLCV can induce the activity of the promoter of Le ACT1,and the AC2 and C1 has the most significant effect.The VIGS vector of Le ACT1 was constructed and was inoculated on the TYLCV resistant and susceptible tomato cultivars.The gene silence efficiency was analyzed by Real-time RT-PCR,and the results indicated the expression of subject gene decreased by 60%,but the phenotype did not change significantly.The silenced plants were inoculated with TYLCV,and the amount of virus in the silenced plants was about 30 times compared with control at 15 d after inoculation.At the same time,the leaves appeared some abnormalphenotype,such as warping and shrinking.The over expression vector of Le ACT1 was constructed and transformed to Nicotiana benthamiana,9 positive plants were identified and among which 4 plants with the highestexpression of subject gene were selected for disease resistance analysis.The results of resistance identification showed that the resistance to TYLCV of the transgenic plants increased significantly,the relative amount of virus dropped about 40%-90% compared to the wild type plants.At the same time,the resistance of Le ACT1 toverticillium wilt was also analyzed to verify whether its resistance is broad spectrum or not.The disease index of the transgenic plants when inoculated by defoliating type verticillium wilt V991 was only 24.35%,38.19% and 58.33%,while the wild type reached to 85.36%.In addition,the amount of TYLCV decreased significantly in transgenic plants than the control.The resistant mechanism toverticillium wilt was also determined preliminary,when the in vitro leaves inoculated with verticillium wilt,the PAL activity of the transgenic plants increased significantly than the control.All the results showed that the promoter sequence of the Le ACT1 differed significantly in the resistant and susceptible materials and could be induced by TYLCV genes,which illustrated the disease-resistant promoter recognition played a key role in resistance function.At the same time,Le ACT1 of tomato confers resistance to TYLCV and can be applied to new germplasm creation for disease resistance.