Effects Of GCR2 Myristoylation On Subcellular Localization And Transduction Of Bacterial Signaling Molecules In Plants

N-acyl-homoserine lactones(AHLs),one of the quorum sensing(QS)signal molecules,which mediated communication between Gram-negative bacteria and host.Recently,it has become evident that plants can not only sense to bacterial AHLs but also respond appropriately such as regulating plant cell growth.Our pervious data showed that GCR2was involved in 3OC6-HSL signal transduction and GCR2 binding to 3OC6-HSL specificly.However,the molecular mechanism of GCR2 signal transduction after perception to3OC6-HSL remains unclearly.Recently,Lan CL2 in mammals,which is highly homologous to GCR2 protein in plant on the amino acid sequence,was proved to be a receptor of a important plant hormone ABA.The myristoylation enables Lan CL2 protein localized in plasma membrane and transfered from cytoplasm to nucleus which mediated the signal transduction of ABA.We found that the amino acid sequence of plant GCR2 protein contains potential myristoylation sites,so we speculate that myristoylation of GCR2 protein may play an important role in the process of 3OC6-HSL signal transduction.Therefore,our study focus on the myristoylation of GCR2 protein and molecular mechanism of bacterial signal 3OC6-HSL perception and transduction.Fristly,Arabidopsis seedlings pretreated by 3OC6-HSL and myristoylation inhibitor2-Hydroxy Myristic Acid(HMA)inhibited the plant root growth.The results showed that the main root growth was inhibited by the interaction between 3OC6-HSL and GCR2.And myristoylation also participated in signal sensing and transduction.Secondly,I constructed two recombinant expression vectors Flexi-GCR2 and Flexi-G2A which the GCR2myristylation site was mutateded.GCR2 and G2A proteins were synthesized by TNT wheat germ acellular transcription and translation system which doping with ³⁵S-methionine and[9,10-³H]-myristic acid,but none of myristicated proteins were detected by autoradiography.Thirdly,The protein was myristicated by click chemistry in GCR2overexpressing plants treated with Alk14 derivatives and 3OC6-HSL.The results showed that there were none of myristicated protein detected in plant too.Last but not least,two recombinant expression vectors p BI121-GCR2-GFP and p BI121-G2A-GFP were constructed and transformed into Agrobacterium tumefaciens GV3101.Agrobacterium tumefaciens GV3101 was used to infect Columbia wild type Arabidopsis Col-0 and gcr2-2mutant plants.Seventeen T₀ generation transgenic plants were obtained by resistance screening.In order to supplement the previous study on the effect of intracellular calcium changes in Arabidopsis thaliana by N-decanoyl-HSL(C10-HSL).We detected the changes of intracellular calcium ions in Arabidopsis thaliana by fluorescent probe method(Fluo-4,AM)with different calcium ions inhibitors pretreated,such as Verapamil(VP),EGTA,LiCl and La Cl₃.The results showed that C10-HSL pretreatment induced intracellular calcium changes and inhibited the growth of Arabidopsis main roots.The inhibition of main roots was weakened after adding inhibitors,and the LiCl recovery was obvious,suggesting that the intracellular calcium release was involved in the signals transduction of C10-HSL.