Study Of Tomato LRR Receptor-like Kinase SlDALR1 In The Defence Against Bacterial Speck

With the improvements in structure of the agricultural industry and in the planting technologies,greenhouse vegetable cultivation has developed vigorously,and the planting area is still expanding.However,greenhouse cultivation is often threatened by a variety of pests and diseases,among them,bacterial speck caused by Pseudomonas syringae pv.tomato DC3000 has been reported all over China,and is responsible for up to 50% of the total loss occasionally.Current methods to control the disease is highly dependent on chemical fungicides,which seriously endanger food safety and ecological environment.Therefore,it's urgently required to study the molecular mechanism of plants' innate immunity in depth.Leucine rich-repeat receptor-like kinases are the largest family of transmembrane receptor-like kinases in plants,which serve as the pattern recognition receptors to sense bacterial infection and stimulates the plant's innate immune response.In this study,we investigated the role of LRR-RLK SlDALR1(Defence-associated LRR-RLK1)in defence against bacterial speck in tomato,and the mechanism involved.Our results are as follows:1.The response of tomatoes' LRR-RLK IV genes to Pst DC3000 infection is studied,and SlDALR1 and SlDALR2 amino acid sequences analyzed.This chapter mainly conducts qRT-PCR analysis over the expressions of LRR-RLK IV subfamily members after inoculating tomatoes against Pst DC3000.Meanwhile,the existed RNA-Seq data in the laboratory and PAMP experiment are analyzed.Consistent with the qRT-PCR result,these results suggest the relative transcript level of SlDALR1 gene sees the maximum rise among LRR-RLK IV genes after infection with Pst DC3000.Subcellular localization experiment help determine that the protein expressed by SlDALR1 is located on plasma membrane;the analysis over the LRR-RLK-ID amino acid sequences makes clear that the gene is highly conservative in different species and their perceived ligand may be some unknown component.2.On the basis of the analysis in the previous chapter about tomato SlDALR1 gene greatly increasing under the infection of Pst DC3000,we guess it may get involved in the defence process of tomatoes against Pst DC3000 and play a key role in it.This chapter uses VIGS to initially explore the effect of SlDALR1 on tomatoes' defence against Pst DC3000.According to the results,SlDALR1-silenced plants show much more serious diseases and increased bacterial colonies compared with control plants.The results of trypan blue staining and ?PS? are consistent with that of CFU.In addition,ROS burst,MAPK activation and immune-related hormones,three PTI signals are measured in this chapter.It is found that immune response of silenced plants is weaker than that of control plants.In summary,these findings show that SlDALR1 upregulates resistance against Pst DC3000 in tomato.3.Proteins interactive with SlDALR1 are screened out through yeast two-hybrid.The studies above have initially determined that SlDALR1 participate in the process of defence against Pst DC3000 in tomato,but the specific defence mechanism is still unknown.In this chapter,the yeast two-hybrid technology is used to screen cDNA libraries of tomato leaves by taking SlDALR1 as the bait.Positive clones are sequenced through blue-white screening,and preliminary analysis was performed using SGN and NCBI websites,then GO annotation was conducted on the obtained selected proteins.Meanwhile,SlSERK3 A and SlBSK1 were preliminarily selected as candidate genes through one to one yeast two-hybrid experiment.Through BiFC and VIGS experiments,the interaction between SlSERK3 A and SlDALR1 could be confirmed and SlSERK3 A shows a positive regulatory effect on plants' resistance against Pst DC3000.Therefore,it could be preliminarily determined that this gene is involved in SlDALR1-mediated defence process against Pst DC3000 in tomato.