The Mechanism Of BrRGF6,a Root Meristem Growth Factor Of Chinese Cabbage,in Response To Infection By Plasmodiophora Brassicae Woroin

As the main vegetable crop in China,Chinese cabbage has a high annual demand,but its output has dropped sharply due to the infection of plasmodiophora brassicae.In previous research,we used transcriptome analysis obtaining a gene BrRGF6 that was significantly up-regulated in infected roots by p.brassicae.In this study,we used resistant and susceptible varieties of Chinese cabbage as experimental materials,and the expression pattern of BrRGF6 gene was analyzed by reverse transcription-q PCR(RT-q PCR)technology and verified by tissue in situ hybridization.The Arabidopsis mutant(rgf6)was identified for its disease resistance function to plasmodiophora brassicae.The specific expression position of BrRGF6 protein in tobacco plant cells was determined by protein fusion GFP subcellular localization technology,and the yeast two-hybrid library was used to screen out the proteins that interact with BrRGF6.The following results were obtained:1.Firstly,the full-length c DNA of candidate gene was cloned and identified as Root Meristem Growth Factor 6(RGF6).Protein structure prediction showed that BrRGF6 protein contained a signal peptide at amino acid positions 1–19,with no transmembrane structure and no conservative domain.The Arabidopsis homologous mutant(rgf6)was purchased,and the resistance to clubroot was identified between the mutant rgf6 and the wild type(WT).Microscopic observation revealed that the infection process in the mutant with RGF6 gene deletion was delayed,that is,the wild-type(WT)was infected at about 36 h after infected with p.brassicae,while the mutant rgf6 was infected at about 60 h after infected with p.brassicae.2.The root of the susceptible material "742" at different infected periods was observed under a scanning electron microscope.It was found that resting spore of p.brassicae firstly infected the root cortex.With the aggravation of the disease,resting spore would attach to the hypocotyl,the root cortex and near the stele,and then the cortical tissue structure of host would be changed.RT-q PCR analysis showed that the expression level of BrRGF6 gene in Chinese cabbage roots was relatively higher than that in stems and leaves.The expression patterns of BrRGF6 gene in Chinese cabbage resistant/ susceptible materials after infection with p.brassicae at different periods were analyzed,and the results showed that the expression of BrRGF6 in the susceptible material increased significantly with the aggravation of the diseased period,and the rate of in crease was significant in the diseased root,while the expression of BrRGF6 in the resistant material was less affected by infection with p.brassicae.It was speculated that BrRGF6 might play a role in the pathogenesis of clubroot in Chinese cabbage.3.The results of in situ hybridization showed that no hybridization signal was observed in the roots,stems and leaves of Chinese cabbage before infecting by plasmodiophora brassicae,indicating that the basal expression of BrRGF6 gene in each part of Chinese cabbage was low.The blue signal appeared in the susceptible material after infected by p.brassicae,and the signal was significantly higher in the diseased period than that in the early infected period.It was further proved that the expression level of BrRGF6 increased with the aggravation of the disease severity,indicating that it might play a role in responding to the infection process of p.brassicae.4.A gene expression vector,BrRGF6-p BWA(V)BS-GFP(in which BrRGF6 gene was fused with GFP)was constructed and transiently expressed in tobacco.After staining with DAIP dye,the fluorescence localization of the translation product of BrRGF6 was detected by laser confocal microscope.The results showed that the recombinant plasmid containing the target gene showed fluorescence signal expression in the nucleus.In view of this it is deduced that BrRGF6 is located on the nucleus.5.In this experiment,the pGBKT7-BrRGF6 bait vector was constructed,and the interacting protein of BrRGF6 was further screened from the library.Five interacting proteins were obtained,which were nuclear transcription factor Y subunit,N-(5’-phosphoribosyl)anthranilate isomerases,isocitrate dehydrogenase [NAD]catalyzed subunit,threonine synthase,and 60 S ribosomal protein L22-2.Among them,the nuclear transcription factor Y subunit achieved the repeated detection rate of 19,indicating that the nuclear transcription factor Y subunit played an important role in the regulatory mechanism of BrRGF6.The yeast rotation test verified that BrRGF6 interacted with the nuclear transcription factor Y subunit.6.Sequence alignment of the BrRGF6 promoter in the resistant/susceptible material revealed an intermittent difference within the 100 bp at upstream region of ATG.There is a MYB transcription factor binding site in this region.The full-length promoter region contains lots of transcription factor elements such as MYB and CAATT-box.In conclusion,BrRGF6 gene plays a role in the response of infected Chinese cabbage by p.brassicae.Nuclear transcription factor Y subunit may be related to the regulation of BrRGF6,or it may be upstream regulated by other transcription factors,which needs to be further studied.