Functional Analysis Of Key Sugar Transporters SWEETs Ininteraction Between Chinese Cabbage And Plasmodiophora.brassicae

Clubroot caused by Plasmodiophora.brassicae infection is a highly infectious world-class soil-borne disease,which has caused a large reduction in the yield of Brassica crops.After the P.brassicae infects the host’s roots,it will ingest a large amount of energy substances from the host for its own growth and reproduction and eventually form root tumors.Sugars will eventually be exported transporters(SWEET)are a class of sugar transporters newly discovered in recent years.In addition to participating in the regulation of phloem loading,stress,growth and development,they also play an important role in host-pathogen interactions.Many studies have shown that the invasion of host plants by plant pathogens will induce the up-regulation of the SWEEET gene expression in the host to participate in the pathogen-host interaction.According to the previous research results of our research group,we found some SWEET genes whose expression levels were significantly up-regulated after P.brassicae infection,and initially identified these genes as the key Bra SWEETs genes for the interaction between Chinese cabbage and P.brassicae.Based on this research,this paper uses a yeast hexose-deficient mutant to verify the sugar transport properties of the key Bra SWEET protein in the interaction between Chinese cabbage and P.brassicae.The mb SUS ubiquitin-cleaved yeast membrane protein two-hybrid system and Bi FC bimolecular fluorescence complementary system were used to analyze the selected Bra SWEETs proteins.The main findings are as follows:1.Five yeast-deficient expression vectors p DRf1_GW-Bra SWEET1a/2a/11a/12a/14 a were successfully constructed.The constructed recombinant vector Bra SWEETs-p DRf1_GW was transformed into yeast mutant strain EBY.VW4000,and cultured on a uracil-deficient medium SD-ura with maltose,glucose and fructose as the sole carbon sources,and its sugar transport capacity was determined.Among them,Bra SWEET1a/11a/12a/14 a were confirmed to have the characteristics of transporting glucose and fructose,Bra SWEET2 a does not have the ability to transport glucose and fructose.2.Using the mb SUS ubiquitin cleavage membrane protein yeast two-hybrid system,the four key genes Bra SWEET1a/2a/11a/12 a were respectively ligated into the bait vector p Met YC_GW and the capture vector p XN22_GW,and transformed into THY.AP4 and In THY.AP5 yeasts.Two types of yeasts were fused in pairs and tested for self-activation and toxicity on SD-Leu-Trp medium.Finally,SD-Leu-Trp-His selection medium was used for screening.The results show that Bra SWEET1 a,Bra SWEET11 a and Bra SWEET12 a form homodimers through self-binding,and Bra SWEET2a/1a,Bra SWEET11a/1a,Bra SWEET12a/1a,Bra SWEET12a/11 a,Bra SWEET2a/12 a and Bra SWEET11a/12 a can form heterodimers.3.The Bra SWEET1 a and Bra SWEET11 a genes were ligated into the p XCGW and p XNGW vectors using the Bi FC dual-molecule fluorescence complementary system,respectively.The recombinant plasmid was transformed into Agrobacterium and injected into tobacco leaves.Microscopic observations revealed that Bra SWEET1 a and Bra SWEET11 a can form homodimers by self-binding to each other and that both Bra SWEET1 a and Bra SWEET11 a are still active can interact to form heterodimers.This result supports the argument that higher plant SWEETs proteins generally function through multimerization.