The Role Of BrAtg6 In Chinese Cabbage Resisting To Plasmodiophora Brassicae Woroin Infection And The Screening Of Its Interaction Proteins

Chinese cabbage is an important vegetable crop in China,its yield is seriously affected by clubroot disease.In order to study the resistance mechanism of Chinese cabbage to Plasmodiophora brassicae woroin,our research group analyzed the differential proteome of Chinese cabbage roots between the before and after the inoculation of Plasmodiophora brassicae,and screened a Br Atg6 autophagy protein that was significantly up-regulated after inoculation in the early stage.The deletion of Arabidopsis thaliana Atg6 was significantly accelerated in the process of infection.In order to understand the mechanism of Br Atg6 protein in Chinese cabbage may have a role in preventing the infection of Plasmodiophora brassicae woronin.In this study,resistance/susceptibility near-isogenic lines of Chinese cabbage were used as experimental.The gene expression of Br Atg6 at different infection stages was quantitatively analyzed by q RT-PCR;The gene expression sites of Br Atg6 were analyzed by subcellular localization;MDC staining was used to observe the autophagic structure of Chinese cabbage roots;Yeast two-hybrid library was constructed and the proteins interacting with Br Atg6 were screened from the library by yeast two-hybrid.Online software(STRING)was used to predict the proteins interacting with Br Atg6.Finally,the interaction between Br Atg6 and these interacting proteins was verified by yeast two-hybrid one-to-one interaction verification.Get the following results:1.The c DNAs of root and stem from the resistant/sensitive materials at different infection stages were used to analyze the expression of Br Atg6.The results showed that after infection by P.brassicae,Br Atg6 were significantly increased in both root and stem parts of the susceptible material during infection or the disease periods.With the aggravation of the incidence degree,the degree of up-regulation was most significant in the root of the susceptible material.The expression of this gene in the root and stem of the resistant material was significantly higher in inoculated plants than that of the control group.However,the expression of Br Atg6 gene was not affected much by the P.brassicae inoculation during the disease period.It indicates that this gene may play a key role in resisting the infection P.brassicae in Chinese cabbage,and the expression difference of this gene is existed between resistant and sensitive materials.2.The GFP-containing over-expression vector p BWA(V)BS-Br Atg6-gfp was constructed.The subcellular localization results showed that the laser signal of GFP was distributed to several parts of cytoplasm and nucleus under confocal microscope,while the target gene only combine with DAIP nuclear dye diffused the fluorescence signal in the nucleus.Thus,the target gene is located in the nucleus.3.The roots of the resistant/susceptible materials in the infection period were stained by MDC and observed under laser confocal microscopy.At the same time,the uninoclulated roots at the same period were used as control.It was found that the autophagy signal was enhanced after the infection ofPlasmodiophora brassicae.In the sensitive materials,autophagy signal was strengthened with the extension of inoculation time.Autophagy signal was concentrated in the infected area of Plasmodiophora brassicae,and the signal was unusually strong.However,in the disease-resistant materials,the autophagy signal is not confined on the infected site,but extended outwardly,indicating that the autophagy process in the disease-resistant materials restricts the derivation of programmed cell death.4.Yeast two-hybrid library was constructed by Extract RNA from the inoculated roots of resistant/susceptible materials during infection period.The qualified library with a titer of 4.0x107cfu/m L was obtained.p GBKT7-Br Atg6 bait vector was constructed for yeast library screening.Finally,42 kinds of interacting proteins were obtained.Some proteins appeared more frequently,such as: transcription factor-related proteins(16 times);voltage-gated anion channel related proteins(18 times);important proteins associated with autophagy genes,synapsin A(SYNAPTOTAGMIN A)related proteins(14 times);calcium-dependent lipid-binding proteins(11 times)and the autophagy-related protein(7 times).5.The important proteins related to autophagy gene,synapse A protein Bra031157,calcium-dependent lipid-binding protein Bra028148,and autophagy protein VPS34,were selected as the main research objects,and the interactions between candidate proteins and Br Atg6 were confirmed by a pair of co-transformation.Finally,it was confirmed that all of them interact with Br Atg6.To sum up,Br Atg6 in Chinese cabbage may mediate the defense system of plants by means of cellular autophagy and calciumion transduction in the process of resistance to P.brassicae woronin infection.