Receptor Like-kinases PBL28 Involved In The Regulation Of Plant Root Growth By N-decanoyl Homoserine Lactone

N-acyl-homoserine lactones(AHLs)are a kind of signal molecules that can regulate bacterial colony behavior.These signal molecules affect the physiological activities of Gram-negative bacteria through Quorum sensing(QS),and thus make managing in the process of bacterial function.N-decanoyl-homoserine lactone(C10-HSL)is a kind of medium long chain AHLs.Recent studies have found that C10-HSL treatment can significantly change the root structure of plants,inhibit the growth of primary roots,and promote the formation of lateral roots and root hair.However,little is known about how plants have a sense of C10-HSL.There are many Receptor like-kinases(RLKs)in plants.The existence of these RLKs is not only beneficial for plants to obtain the internal changes of the body,but also can recept the information of living environment.Therefore,RLKs play an greater role in the whole process of plant life.However,it is not clear whether RLKs are involved in plant sensing bacterial QS signaling molecules and regulating plant physiological processes.In this study,C10-HSL and RLKs deletion mutants were selected to explore the role of PBL28 in C10-HSL managing of plant root growth.The main conclusions are:1.Firstly,the effects of C10-HSL on the root structure of Arabidopsis wild-type Col-0 were analyzed,and the optimal concentration of C10-HSL was determined to be 30?M.The content and distribution of NO and H₂O₂in Arabidopsis thaliana treated with C10-HSL were analyzed by DAF-2DA and DCFH-DA.It was found that C10-HSL could promote the production of NO and H₂O₂in plants.Further studies with the enzyme deletion mutants noa1 and rboh D/F,which are related to the synthesis of NO and H₂O₂,showed that the sensitivity of these two mutants to C10-HSL treatment was significantly reduced,and the inhibition degree of primary root was obviously reduced.It turns out to C10-HSL could affect plant root formation by regulating the synthesis of NO and H₂O₂.2.In order to study the role of RLKs in the regulation of plant root structure by C10-HSL,we observed and analyzed the response of root structure of Col-0 and RLKs mutants to C10-HSL treatment,The results showed that five RLKs deletion mutants pbl28,rgi5,rkf1,lecrk-ix.1 and crrk1-l showed different degrees of insensitive phenotypes.In this study,five RLKs deletion mutants pbl28,rgi5,rkf1,lecrk-ix.1 and crrlk1-l were significantly reduced after C10-HSL treatment,pbl28 is very insensitive to C10-HSL.In order to further study the role of receptor like protein kinase PBL28 in the regulation of C10-HSL on plant root growth,the changes of NO and H₂O₂levels in Col-0 and pbl28before and after C10-HSL treatment were detected.It was found that the absence of PBL28seriously hindered the production of NO and H₂O₂induced by C10-HSL.The results showed that PBL28 may be involved in the regulation of plant root growth by regulating the levels of NO and H₂O₂induced by C10-HSL.3.In order to further study the function and mechanism of PBL28 in C10-HSL regulating plant root structure by genetic method,we set up a plant binary expression vector p BI121-PBL28-GFP,Agrobacterium tumefaciens was used as the vehicle,The expression vector was transformed into Arabidopsis thaliana by flower dipping method,and eight single copy transgenic plants were obtained after resistance screening.In addition,We bulit prokaryotic expression vector p ET-28a-PBL28.IPTG was used to induce production,PBL28 can be existed in a soluble form.Western blot was used to check the specificity of the protein,it showed that the specificity was good.These results laid the experimental foundation for further verification of PBL28involved in the regulation of C10-HSL on roots,and also opened up a new path for the study of receptor like protein kinases sensing AHLs.