Identification Of RALF-like Effector Of Endophytic Colletotrichum Tofieldiae And Preliminary Study On Its Function

Plants growing in the natural environment are affected by a variety of microorganisms.As a kind of beneficial bacteria,endophyte can obtain nutrients from plants to promote their own growth while providing nutrients for plants,and will not cause obvious disease symptoms of plants.Endophytes have a wide range of functions in agriculture and can promote the growth and development of crops.In the process of hypha colonization,the effector secreted by endophytes affect the immune response of plants to achieve the purpose of symbiosis establishment.The effectors can inhibit plant immunity and promote the establishment of hypha symbiosis from binding to plant receptor-like kinase(RLK)and regulating the transcription expression.The identification of new endophytic effectors is conducive to in-depth study of the mechanism of symbiosis establishment.The rapid alkalization factor(RALF)analogs identified in a variety of microorganisms have laid a theoretical foundation for us to study endophytic effectors.In this study,we identified and analyzed the endophytic Colletotrichum tofieldiae RALF-like protein through bioinformatics:(1)We used the typical motifs of plant RALF such as YISY motif and RGC(5N)C motif to search for a homologous protein sequence named Ct RALF.Ct RALF was cloned and expressed in vitro.Exogenous addition of Ct RALF can inhibit the root length of Arabidopsis wild-type Col-0 by the plant receptor FERONIA(FER)and promote extracellular alkalization.This indicated that the endophytic Colletotrichum tofieldiae secrete RALF-like effector which has the typical biological activity of plant RALF.(2)We constructed a prokaryotic expression vector of the extracellular domain of FER,and verified the interaction between Ct RALF and FER through Coimmunoprecipitation(CO-IP),GST pull-down,and competitive binding experiments.The results showed that Ct RALF can directly interact with FER like plant RALF.(3)Ct can express Ct RALF during the process of infecting plants through realtime quantitative PCR.We analyzed the effect of Ct RALF on plant immune signals combined with Western Blot.The results showed that exogenous addition of Ct RALF can inhibit the accumulation of reactive oxygen species(ROS)in plants and induce the burst of calcium signals within a short period of time,but its impact is less severe than that of plant RALF.Ct RALF can promote mitogen-activated protein kinase(MAPK)phosphorylation through FER and reduce the stability of the regulatory protein MYC2 in the jasmonic acid(JA)signaling pathway.(3)We used Arabidopsis wild-type Col-0/C24 and FERONIA function deficiency mutant fer-4/srn as plant materials to verify the effect of Ct RALF on hypha colonization through hypha infection experiments.Col-0 and C24 had more hyphae attached to the roots than fer-4 and srn roots without Ct RALF.After exogenous addition of Ct RALF,the hyphae attached to the roots of Col-0 increased,and the colonization rate of hyphae attached to the roots of fer-4 did not change significantly.This showed that Ct RALF can promote hyphae colonization through FER pathway.In summary,this study found that the endophytic Colletotrichum tofieldiae can secrete the RALF-like protein-Ct RALF.Ct RALF has the typical activity of plant RALF and affects plant immune signals through the RALF-FER signaling pathway to promote endophytic hypha colonization.