Roles Of StNACb4 Gene Involved In Bacterial Wilt Resistance Of Potato

Potato(Solanum tuberosum)is one of the four main food crops in the world,but it is infected by Ralstonia solanacearum,and its quality and yield are seriously damaged.It is helpful to the related disease resistance practice by studying and mining the function of potato defense genes.NAC gene family is one of the main families of plant transcriptional regulatory factors,which has a variety of functions.Most of NAC TFs related to plant growth and development and responsed to biotic or abiotic stress.In the previous study on the defense mechanism of potato against bacterial wilt,it was found that the transcription factor NAC may be involved in the resistance to bacterial wilt.Based on this,a potato NAC gene(StNACb4)was cloned by RACE method,and the structure of the gene was analyzed by relevant softwares and online server.Potato seedlings were treated with R.solanacearum and plant hormones,and the expression characteristics of this gene and the disease resistance marker gene StPR10 were analyzed.Then the tolerance to R.solanacearum was verified by virus-induced homologous gene silencing(VIGS Nb NACb4)and overexpression of StNACb4 gene in tobacco.The function of the gene was verified by observing the disease symptoms,hypersensitive cell death and callose deposition after inoculation.The results are as follows:(1)According bioinformatics analysis,the length of the gene is 864 bp,open reading frame(ORF)is 705 bp,StNACb4 encodes a 235 aa NAC transcription factor,including a typical NAM superfamily conserved domain.StNACb4 protein is located in the nucleus and has no transmembrane region,so it is a stable hydrophilic protein.Phylogenetic analysis showed that the StNACb4 protein related closely to the homologous NAC of Solanaceae.A variety of cis-acting elements which responded to plant hormone signal pathway and defensed stress response were predicted in the promoter region of this gene.It was inferred that StNACb4 was involved in plant hormone signal pathway and defense stress response.(2)The transcriptional expression characteristics of StNACb4 gene and different exogenous hormones such as salicylic acid,methyl jasmonate and abscisic acid at different time points were identified by RT-qPCR.The results showed that StNACb4 gene was rapidly activated and expressed by R.solanacearum,which maintained the tolerance of potato to pathogens.At the same time,StNACb4 gene was also induced by a certain concentration of exogenous plant hormones,and maintained a continuous effect for a certain period of time.The results showed that the gene was involved in potato resistance to bacterial wilt and was regulated by plant hormone signals.(3)The tissue localization of StNACb4 expression was analyzed by fluorescence in situ hybridization,and it was found that it was mainly expressed in the vascular bundles of stems and leaves,indicating that the gene has vascular-specific tissue specificity.(4)VIGS-NAC-pTRV2 silencing vector and VOX-NAC-pTRV2 overexpression vector were constructed by VIGS and VOX respectively and transformed into Nicotiana benthamiana successfully.After treatment with R.solanacearum,it was found that the disease symptoms of plants silenced by NACb4 gene were the most serious at the same time.The silencing of NACb4 gene could enhance the sensitivity of tobacco to R.solanacearum.On the contrary,the overexpression of NACb4 gene enhanced the tolerance of tobacco to R.solanacearum.Gene functional deletion and overexpression experiments confirmed that the gene has the function of disease resistance.(5)Different tobacco leaves were stained with trypan blue stain.It was found that VOX-StNACb4 overexpression in tobacco obviously triggered cell death after R.solanacearum treatment.It was concluded that transient overexpression of StNACb4 gene could induce tobacco hypersensitivity(HR)cell death after stress by pathogens.Aniline blue staining was used to observe the deposition of callose in different tobacco leaves treated with flg22.It was concluded that the transient overexpression of StNACb4 gene could induce the immune(PTI)response of callose deposition in VOX-StNACb4 tobacco leaves.The results showed that NAC gene showed resistance to bacterial wilt by inducing HR and PTI immune response.(6)It was found that the expression of St RP10,a gene of pathogen-related protein PR10,increased or decreased rapidly with the upregulation or decline of StNACb4 gene.Combined with promoter analysis,it was inferred that StPR10 gene may be the target gene of StNACb4.Based on the above analysis,it is considered that StNACb4 gene as a positive factor is involved in the innate immunity of potato to bacterial wilt and may be related to systemic acquired resistance,which provides a theoretical basis for subsequent practical application.