Study On A Strategy To Facilitate Nematode Parasitism Via Plant RALF-FERONIA Signaling

Plant-parasitic nematodes(PPNs)are devastating pathogens that cause crop diseases and subsequently large area of infections leading to severe national economic losses(up to tens of billions of RMB).Plants and PPNs are always competing in an arms race.PPNs can secret a number of effectors via stylet to benefit its parasitism and simultaneously aid evade the attack of their host immune system.However,in this field,our understanding of how plant-parasitic nematodes interact with its host is limited and warrants further studies.As a receptor kinase on plasma membrane,FERONIA(FER)is one of well-studied signaling networks in plants so far.FER modulates a multitude of essential biological processes in plants(e.g.,growth,reproduction,and stress responses)and influences the yield and quality of crops.RALFs,one of the peptide ligands perceived by FER,trigger the casecade downstream,modulate the activity of AHA2 and Ca²⁺transport,and influence the stability of immune-related proteins and immune complex,further regulating plant growth and development and immune responses.Root-knot nematode(e.g.,Meloidogyne incognita)is most well-studied in PPNs so far.It has a similarity for the lifestyle of root-knot nematode,but a significant difference in the feeding site,with other species.Compared with cyst nematode,root-knot nematode modulates the cell size(e.g.,modulating the vacuole size),further forming the feeding site.As previously reported,RALF-FER signaling controls plant cell size and regulates its development of vacuole,both of which benefit the parasitism of nematode.In plants,RALF23 peptide regulates the immune response via modulating the formation of immune complex(FLS2-BAK1)through FER and influencing the flg22-induced ROS burst.Meanwhile,RALF23 peptide inhibits FER-dependent phosphorylation of MYC2,a key transcription factor in Jasmonic Acid(JA)signaling,further affecting the JA signaling and immune responses.It has been shown that Fusarium oxysporum encodes RALF-likes that can modulate plant immune response via FER,consequentially inhibits the activity of AHA2,alkalinizes the apoplast and phosphorylates MAPK in Fusarium oxysporum.This indicates that RALF peptides have the potency of cross-kingdom signal transduction.Based on this,we aim to(1)confirm the interaction of nematode parasitism with receptor kinase FER;(2)explore whether there are ligands such as RAFL peptides that can bind to plant receptor protein in the secreted effectors during nematode parasitism;(3)determine how PPN invovle in and regulate the RALF signaling pathway mediated by plant receptor protein FER.A series of molecular,biochemical and genetic experiments were conducted,and the results of this study are as follows:(1)Loss or mutation of FERONIA in Arabidopsis or rice lost susceptibility to root-knot nematodes parasitism.These mutants reduced the capacity to attract Meloidogyne incognita,inhibited the efficiency of infection,delayed the development of Meloidogyne incognita and suppressed the reproduction of Meloidogyne incognita in plants.These results indicated that the key node of FER in the plant signaling pathway also plays an important role in the parasitism of Meloidogyne incognita.(2)To confirm the underlying mechanisms associated with above-mentioned findings,we have identified 18 novel RALF-likes from existing databases.These RALF-likes had high similarity with plant RALFs and conserved domain with plant RALFs,and nematode RALF-likes all originated from root-knot nematodes,not cyst nematodes.From the evolutionary relationship,18 RALF-like scattered,rather than clustered,in different branches of the evolutionary tree,which indicates nematode RALF-likes may originate from horizontal transfer or coevolution,instead of vertical genetics.Taking Mi RALF1 and Mi RALF3 as representatives,the temporal and spatial specific expression of RALF-likes in Meloidogyne incognita was studied.It showed that Mi RALF1 and Mi RALF3 were expressed in esophageal gland cells and especially higher during parasitism.(3)RALF-likes had the typical biological activity of plant RALF peptides:root elongation inhibition,extracellular alkalization.They also had a simialr transcriptional expression profile with plant RALF.RALF-likes can bind to the extracellular domain of receptor protein FER in GST pull-down,competitive binding and Co-IP assays.Meanwhile,docking assay and MST assay also confirm this from the aspects of structure simulation and molecular thermodynamics.The affinities between RALF/RALF-likes and FER extracellular domain were measured as well.(4)Our results showed that RALF-likes could induce MAPK phosphorylation in plants and inhibit the stability of MYC2 protein,a key transcription factor in JA signaling pathway.It was found that RALF-like of nematodes could inhibit the flg22induced ROS signal pathway similar to plant RALF.Knock-down of RALF-like expression by host induced gene silencing(HIGS)delayed the development and reproduction of Meloidogyne incognita in plants.Meanwhile,overexpression of RALF-like in plants increased the sensitivity of plants to Meloidogyne incognita.In summary,this study indicated that nematode-encoded RALFs facilitate parasitism via plant-encoded FERONIA and provides a novel paradigm for studying host-pathogen interactions.