Mechanism Analysis Of Two Effector Genes And Identificate PAMP In Meloidogyne Graminicola

Root-knot nematodes(RKNs)are one of the most important plant pathogens,resulting serious losses in worldwide agriculture and economically crop.Rice serves as staple food of more than half of the global population,Meloidogyne graminicola is considered as a major prevalent plant-parasitic nematodes(PPNs)attacking rice,especially in the major paddy rice production areas of Asia.Numerous secretions are delivred by RKNs through their style in parasitism,which ultimately play key various functions during PPNs infecton.Extensive genome,transcriptome and proteome studies have shown that plant-parasitic nematodes secrete many additional effectors,while,the function of many of these is little known.Studing the detailed molecular roles of these secretions are better to understand this mechanisms of RNKs in the parasitism.In this papar,we identified and functional analysis two novel Meloidogyne graminicola effectors MgGPP and MgMO237,illustrating the two effects toxicity mechanism of parasitism.In addition,Mg-eEF1 A which as a Meloidogyne graminicola PAMP can induce rice immune resposing,providing a new strategy of the nematode disease prevention and control in the future.1.In this study,a novel effector MgGPP form the plant-parasitic nematode Meloidogyne graminicola was cloned by rapid amplification of cDNA ends(RACE).MgGPP is exclusively expressed within the nematode subventral esophageal gland cells and upregulated in the early parasitic stage of M.graminicola.Southern blot analysis showed that MgGPP is a single-copy gene in the genome of M.graminicola.Transgenic rice lines expressing MgGPP become significantly more susceptible to M.graminicola infection than wild-type control plants,and conversely,in planta,the silencing of MgGPP through RNAi technology substantially increases the resistance of rice to M.graminicola,rising the effector MgGPP plays a role in nematode parasitism.Immunofluorescence analysis shows that MgGPP is secreted into host plants and targeted to the ER,where the N-glycosylation and Cterminal proteolysis of MgGPP occur.C-terminal proteolysis promotes MgGPP to leave the ER,after which it is transported to the nucleus.In addition,N-glycosylation of MgGPP is required for suppressing the host response.The research data provide an intriguing example of in planta glycosylation in concert with proteolysis of a pathogen effector,which depict a novel mechanism by which parasitic nematodes could subjugate plant immunity and promote parasitism and may present a promising target for developing new strategies against nematode infections.2.In this study,a novel effector protein gene,MgMO237,which is exclusively expressed within the nematode dorsal esophageal gland cell and up-regulated in the parasitic stage of Meloidogyne graminicola.Transient expression of Mg MO237 in protoplasts from rice roots showed that MgMO237 was localized in the cytoplasm and nucleus of the host cell.Transgenic rice lines expressing MgMO237 affected plant morphology and increased susceptibility to M.graminicola infection.Instead,specific MgMO237 silence via RNA interference(RNAi)substantially reduced the expression of MgMO237 in feeding nematode and increased plant resistance to M.graminicola.Significantly,yeast two-hybrid assays and co-immunoprecipitation(Co-IP)have confirmed the specific interaction of MgMO237 with rice proteins 1,3-beta-glucan synthase component,OsCRRSP55 and OsBetv1.In addition,MgMO237 can disturb the host defence response by suppressing host defence-related marker genes expression,cell wall callose deposition and reactive oxygen species(ROS)burst.These results demonstrate that MgMO237 is a novel plant-parasitic nematode effector which promotes parasitism by interacting with host defence-related proteins to suppress immune system.3.We used a rice membrane receptor kinase OsCERK1 screened an interacting protein translation elongation factor(Mg-eEF1A)in M.graminicola yeast two-hybrid library.Co-IP was used to further comfirm that OsCERK1 can interacted with two regions of Mg-eEF1 A.In this study,Mg-eEF1 A have been determinded that can be secreted in vitro by M.graminicola and induced the plant resistence to pathogens.In GUS system,Os CERK1 significant up-regulated in the gaint cell of Meloidogyne graminicola.In the meanwhile,MgeEF1 A can induce rice the ROS,callose,defence genes up-regulated and MAPK signaling pathway.This research provide a new method to identificate plant-parasitic nematode PAMPs.