Functional Analysis Of Tomato Transcription Factor NAC29 In Plant Growth And Defense Against Pseudomonas Syringae

In recent years,the vegetable industry is flourishing,but it is threatened by various pathogens and pests,which will restrict the development of vegetable industry for a long time.Tomato leaf spot disease is highly prone to occuring in horticultural cultivation,which is caused by the Pseudomonas syringae pv.Tomato(Pst DC3000),a kind of biotroph bacteria disease.Chemical fungicide application is a common method to control the tomato leaf spot disease,which poses a serious threaten to the ecological environment and food safety in the long time.It is importent to explore intrinsic mechanisms in the defend against Pst DC3000 since it can provide new ideas and strategies for establishing disease resistance methods.The NAC(NAM/ATAF/CUC)transcription factors are one of the six major transcription factors in plant.Studies have shown that the NAC family is involved in the plant growth and defense response to biotic stresses such as pathogen infection.In this paper,tomato(Solanum lycopersicum)is used as the research material to study the role and mechanism of transcription factor NAC29 in tomato growth and defense against Pseudomonas syringae.The main results are as follows:1.We studied the response of tomato NAC transcription factor family members after Pst DC3000 infection.This chapter mainly uses the RNA sequencing(RNA-Seq)data available in the laboratory to analyze the response of tomato NAC family members under Pst DC3000 stress.The relative expression intensity of 101 tomato NAC genes was analyzed,and 15 NAC genes were found to be significantly up-regulated after inoculation with Pst DC3000(1.5-3750 times).The initial content of 15 genes was analyzed,and 4 genes with too low content were eliminated,and 11 up-regulated genes were finally identified.The Quantitative real-time PCR(qRT-PCR)was performed to verify that the experimental trend was similar to that of RNA-Seq data,and both were significantly up-regulated.Among them,the relative gene expression of NAC29 increased the most,up to 22.9 times.In order to clarify the location of NAC29,a subcellular localization test of NAC29 was performed,and confirmed that NAC29 is localized in the cell membrane and nucleus.2.In the last chapter,we analyzed the response of tomato NAC transcription factor family members after Pst DC3000 infection,and found that the expression level of NAC29 gene was the highest,22.9 times.Thus,we hypothesized that NAC29 is involved in the defense against Pst DC3000 and may play an important role.Therefore,in this chapter,we used NAC29 as a research object,we constructed and identified tomato NAC29 gene overexpression plants.We got two stable homozygous strains OE:NAC29-1,OE:NAC29-2with up-regulation multiples of 58.3 and 56.4,the overexpressed plants were inoculated with Pst DC3000,in order to study the effect of NAC29 gene on tomato plants against Pst DC3000.Colony-Forming Units(cfu)was performed,and the growth rate of Pst DC3000 was significantly decreased,with 74.3%and 93.1%,respectively.The results of ΦPSⅡ and trypan blue staining were consistent with the results of cfu assay.After overexpression of NAC29 gene,the Symptoms were reduced compared with wild type plants.It can be seen that the overexpression of NAC29 gene can significantly enhance the tomato plants defense capabilities to Pst DC3000.3.We proved that overexpression of NAC29 gene can enhance the defense ability of tomato plants against Pst DC3000 in the previous chapter,this chapter we studied the relationship between tomato transcription factor NAC29 and resistance-related genes in defense against Pst DC3000,and explored mechanism of NAC29 transcription factor in improving tomato defense ability.In order to further explore the mechanism,The promoter sequences of disease-resistant genes were screened,and all genes can be targeted by NAC29 were identified among 7 genes.Finally,MYB14(1 locus)with a large up-regulation range and βCA3,GS2,and Fd-GOGAT with more recognition sites were selected as follow-up objects.Further screening by qRT-PCR revealed that βCarbonic anhydrase 3(βCA3)was overexpressed in the overexpression plant and after infection of Pst DC3000(After the inoculation of Pst DC3000,the expression level ofβCA3 was up-regulated from 1383 times in the wild type to 2523 and 2272 times in the overexpression lines).We performed electrophoretic mobility shift assay(EMSA)subsequently,showed that NAC29 binds to the βCA3 promoter.Further studies in vivo binding by chromatin immunoprecipitation(ChIP)assay was conductded,proved that NAC29 specifically targets the target βCA3 promoter sequence.The results showed that among the 8 resistance genes,NAC29 can specifically recognize the βCA3 promoter sequence and regulate the transcription level of βCA3.Furthermore,the role of,βCA3 in defense against Pst DC3000 was verified,the result was consistent with previous studies.In conclusion,tomato NAC transcription factor NAC29 can enhance tomato resistance to Pst DC3000 by activating βPCA3’s expression.4.In the last chapter,The plant growth phenotype was found to be changed.So overexpression of NAC29 has an effect on plant growth.The NAC29 gene overexpression plants were short but strong in the early stage,and the growth in the late stage was similar to that in the wild type;Overexpression of NAC29 gene can reduce the quality of dry matter in leaves,increase the quality of dry matter in fruits,and increase the proportion of dry matter distribution to fruits.The results indicated that the transcription factor NAC29 increases yield by increasing the proportion of dry matter to the fruit.