The Effect Factor Hfq Of Soft Rot Pathogen Is Involved In The Defense Response Mechanism Of Soft Rot In Chinese Cabbage

Chinese cabbage(Brassica rapa L.ssp.pekinesis)is native to China and has been cultivated for more than 5000 years.It is an important Brassica vegetable of Cruciferae in China.Soft rot is one of the important diseases affecting the production of Chinese cabbage.With the increase of planting years and the annual production and supply of Chinese cabbage,soft rot diseases occur frequently in the field.At present,soft rot is mainly controlled by biological means.Due to the lack of resistant materials and the complexity of host plant resistance regulation to dead vegetative pathogens,there are few studies on the molecular regulatory network of soft rot resistance in Chinese cabbage.The research group of teacher Fan Jiaqin of Nanjing Agricultural University extracted Pectobacterium carotovorum ssp,the pathogen of Chinese cabbage soft rot.The extracellular protein of Pectobacterium.carotovorum ssp.carotovorum(Pcc)was obtained,and the effect factor Hfq with exocrine function was obtained.It was found that the strain Hfq almost lost its pathogenicity to Chinese cabbage by inoculating Chinese cabbage with Hfq deletion strain Hfq.In this study,the interaction protein with soft rot pathogen effect factor Hfq was obtained by screening Chinese cabbage cDNA yeast library,and the interaction protein was further verified by bimolecular fluorescence complementarity(BiFC)technique.Gene cloning,subcellular localization and transcriptome sequencing(RNA-Seq)were used to analyze the molecular mechanism of soft rot pathogen Hfq involved in the immune response of Chinese cabbage soft rot.To provide a new theoretical basis for the prevention and control of soft rot of Chinese cabbage.The main results are as follows:1.24 hours after inoculating Chinese cabbage with wild type strain Pcc,Hfq overexpression strain OE-Hfq and Hfq deletion strain Hfq with the same concentration(108cfu·mL-1),it was found that inoculation with Hfq deletion strain Hfq hardly caused the disease of Chinese cabbage.After inoculation with Hfq overexpression strain,the disease area of Chinese cabbage was 5%higher than that of wild type strain Pcc.The Hfq overexpression strain OE-Hfq with GFP tag was observed by laser confocal fluorescence microscope.It was found that the effect factor Hfq was secreted into the bacteria through the outer vesicles of soft rot bacteria Pcc during infection.2.Using effector protein Hfq as bait protein,seven proteins interacting with Hfq were screened from Chinese cabbage cDNA library by yeast two-hybrid technique.Further verification by yeast two-hybrid showed that aquaporin BrPip2;7,heat shock protein BrDnaj and bait protein Hfq interacted.3.The overexpression vectors containing Hfq,BrPip2;7 and BrDnaj fusion GFP tags were constructed and transferred into tobacco.Through laser confocal microscope observation,it was found that Hfq could be secreted and located on the plant cell membrane,while BrPip2;7 and BrDnaj were located on the cell membrane.BiFC results further proved that Hfq interacted with BrPip2;7 and BrDnaj in plant cell membrane.4.Chinese cabbage ’ A03’was inoculated with sterile water,wild type strain Pcc,Hfq overexpression strain OE-Hfq and Hfq deletion strain ΔHfq,respectively.RNA was extracted from inoculated leaves at 6h,12h and 24h after inoculation,and transcriptome analysis was carried out.The results showed that the number of differentially expressed genes was the most after inoculation with wild-type strain Pcc 12h,which was the critical time for plants to respond to soft rot pathogen,and the expression of Hfq interacting proteins BrPip2;7 and BrDnaj was down-regulated.The weighted co-expression network(Weighted correlation network analysis,WGCNA)of differentially expressed genes of three strains(ΔHfq,OEHfq,Pcc)12 hours after inoculation with sterile water(CK)was analyzed.The results showed that two differentially expressed gene modules(brown and yellow)were regulated by Hfq.Based on the gene pathway analysis of brown and yellow modules,it was found that the differentially expressed genes of these two modules were significantly enriched in five pathways,including MAPK signaling pathway and plant-pathogen interaction pathway,indicating that Hfq may further regulate the response of downstream disease resistance genes to Chinese cabbage to soft rot by interacting with BrPip2;7 and BrDnaj.