The Genetic And Biochemical Analysis Of Arabidopsis Receptor-like Kinase RIPK And FERONIA Co-responding To RALF Signal

Plant cell growth is regulated by multiple signaling factors,the clarification of molecular signaling mechanisms will help deepen the understanding of signal transduction and overcome the constraints of agriculture,forestry and other applications development.Rapid alkalinization factor(RALF)which is endogenous plant growth-inhibiting peptides is found throughout the plant kingdom,it can quickly suppress plasma membrane hydrogen pump(PM-H+ATPase)activity to inhibit cell elongation.Recent studies showed that Arabidopsis FERONIA(FER)protein kinase as a RALF1 receptor through the extracellular domain direct binding RALF1 polypeptide to response to and transduce the RALF1 signal into the cytoplasm,resulting in the regulation of the PM-H+ATPase activity(e.g.AHA2)to control cell elongation.However,the detailed molecular mechanism of RALF1 signal reception and transduction is not unclear.For example,how FER receptor protein kinases transduces RALF1 signal into the cytoplasm?Are there some downstream parterners such as receptor-like cytoplasmic protein kinases(RLCKs),involved in the intracellular signaling pathways?If present,how these downstream members are involved in it?Therefore,in-depth research on downstream direct interaction molecules of FER protein kinase,such as RLCKs,will help us to reveal the molecular mechanisms that how FER receptor protein kinase perceives and transduces early RALF1 signal and ultimately fine regulation of cell growth.Based on this hypothesis,a series of biochemical and genetic experiments was carried out,results were as follows:(1)Mass spectrometry(MS),yeast two-hybrid(Y2H),bimolecular fluorescence complementation(BiFC),GST-pull down and Co-IP analysis demonstrated that an Arabidopsis thaliana receptor-like cytosolic receptor kinase(RLCK-RIPK)can specificly interacts with FER,and the interaction is dependent on each of the kinase activity.(2)Genetic analysis showed that RIPK mutation exhibits similar phenotypes as FER mutant,such as shorter root hairs,roots acidification rate enhancement,smaller plant morphology and leaves,insensitive to NAA while sensitive to ABA.Besides,phenotypic recovery results confirmed that RIPK overexpression in fer-4 mutant can partially restore the function of FER,proving the genetic relationship between FER and RIPK.(3)Using prokaryotic efficient expression system,we produced GST-tagged fusion recombinant protein GST-RIPK,and immunized mice using purified recombinant protein GST-RIPK as an antigen for RIPK polyclonal antibody preparation.Western blot combined with the alkaline phosphatase(Calf intestinal alkaline phosphatase;CIP)analysis proved that RIPK antibody can specifically react with RIPK protein,interestingly,two bands between 70 kDa and 55 kDa were detected in the Col.O,but not in the ripk mutant.We named the phosphorylated form(the upper band)P-RIPK,and the other non-phosphorylated form(the lower band)RIPK.(4)We measured RIPK phosphorylation status in the Co1.0 and fer-4 mutant and found that RIPK phosphorylation level was reduced in the fer-4 mutant as compared to the wild type Co1.0 plants.Meanwhile,we also examined FER phosphorylation status in the Co1.0 and ripk-Co1.0 mutant.The phosphorylated band was reduced in the ripk mutant as compared to that in the Co1.0.We further confirmed the FER-RIPK transphosphorylation using FER or RIPK dead kinase as a substrate in vitro.Results suggested that RIPK phosphorylated FER kinase,and the FER kinase domain also phosphorylated RIPK kinase.Taken together,RIPK and FER may form a protein kinase complex and phosphorylate each other in a mutually dependent manner.(5)We analyzed the time course and do sage-dependence of RIPK phosphorylation under RALF1 treatments and found that RIPK phosphorylation was elevated after RALF1 treatment,and the RIPK phosphorylation level was up-regulated in a RALF1 do sage-dependent fashion,proving that RIPK can direct response to RALF1 peptide.Compared with wild-type,RIPK mutants reduce the sensitivity of RALF1 peptide.(6)Using bimolecular fluorescence complementation(BiFC)and Co-IP methods in the present or absent of exogenous RALF1 peptide,the interaction between FER and RIPK is enhanced by RALF1 peptide.In addition,we found that in monocot model plant rice FER homolog OsFLR2 and RIPK homologous protein OsRIPK-A also can directly interact with each other in yeast AH109.Bioinformatics and Q-PCR analysis further demonstrated that RALF1,FER and RIPK were highly conserved and overlapped expression patterns on the plant kingdom,so RALF1-FER-RIPK may represent a common RALF1 small peptide signaling mode in plant kingdom.In summary,our study will greatly enrich the RALF1-FER signaling network mechanism regulating cell elongation.