Functional Analysis Of Two Key Effectors MIF And PDI During Melodigyne Incognita Parasitism

Root knot nematode(RKN)is widespread and devastating,which can infect more than 5,500 species of plants,causing huge economic losses every year and is one of the most important pathogens in agricultural production.In recent years,studies have found that plant parasitic nematodes secrete a variety of effector proteins to assist parasitism.Some of these effector proteins interact with host proteins in plant cells to affect immune responses to promote nematode parasitism.However,the function of most effector proteins and target proteins in plants remains unknown.In this thesis,the function of two parasitic key effector proteins of Meloidogyne incognita was investigated,and also analyzed the interaction betweent the effector proteins and their target proteins in the host to control the host defense response and assist parasitism.The four-macrophage migration inhibitory factors(MIFs)identified in Meloidogyne incognita(MiMIFs)are typical MIF-like proteins(i.e.resembling the MIFs secreted by human and animal parasites).We show here that MiMIFs are upregulated early in parasitism.MiMIF proteins are mainly expressed in the hypodermis and cuticle,and secreted into host plants.We show that MiBMIF has tautomerase activity and can protect nematodes against H2O2 damage.Using transient expression system in Nicotiana benthamiana,the expression of MiBMIF on tobacco leaves could inhibit the necrosis induced by Bax.In planta,RNA interference to block MiMIFs resulted in 60%fewer galls and nematodes,demonstrating the importance of these factors for nematode parasitism.MiBMIF overproduction led to 30%higher levels of plant infection,and impaired the[Ca2+]cyt influx induced by H2O2.MiBMIF ectopicly expressing in Arabidopsis thaliana suppressed the activation of the MAPK cascade,induction of defense marker genes and callose deposition triggered by treatment with the PAMP flg22.The immunoprecipitation(IP)of MiBMIF-interacting proteins,followed by Co-IP and BiFC assays,confired specific interactions between MiBMIF and two Arabidopsis annexins,AnnAtl and AnnAt4,involved in the transmembrane transport of calcium ions,stress responses and signal transduction.The first amino-terminal proline residue of MiBMIF and the Ca2+-binding sites of annexins are required for these interactions.Our results provide the first functional evidence that M.incognita uses MiMIFs to promote parasitism by down regulating annexin-mediated plant immune responses.Protein disulfide isomerase(PDI)is widely ranged in human,animal,plant and fungus,which functions as redox,isomerase and molecular chaperon.So far,the results for the function of human and animal parasites PDI show that it plays a role in evading host immune response during infection.In our research,we found there are two types of PDI like proteins in M.incognita(MiPDIl and MiPDI2),and they all contain a N-terminal signal peptide for secretion and share the same thioredoxin domains.The expression level of MiPDI1 is significantly up regulated in parasitic stages by RT-qPCR analysis.In situ hybridization showed both MiPDI1 and MiPDI2 specifically expressed in the subventral gland of M.incognita,and immunolocalization showed that MiPD1l was secreted into plant tissue during nematode early parastism.In planta RNA interference(RNAi)of MiPDI1 substantially reduced the nematodes parasitic ability,providing evidence that MiPDI1 plays a critical role in M.incognita parasitism.Using transient expression assays in Nicotiana benthamiana,we found that MiPDIl could induce obvious cell death,while MiPDI2 could not.Yeast two-hybrid and BiFC confirmed that a stress-associated zinc finger protein interacted with MiPDI1.The results lay an important foundation for further analysis of the pathogenic mechanism of root knot nematodes.