Molecular Characterization And Function Analysis Of Expansion(HaEXPB2) And Calreticulin(HaCRT1) Protein From The Cereal Cyst Nematode Heterodera Avenae

Cereal cyst nematode,Heterodera avenae,is one of the most important plant parasitic nematode disease in China,and seriously threatens our food security.H.avenae are sedentary biotrophic endoparasites,and establish a complex interaction when they parasitic the host plants.Nematode synthesize effector proteins in the oesophageal glands,then secrete them into plant tissues through the stylets.To study the function of nematode effectors in parasitic process,46 predicted effectors genes from Heterodera avenae were cloned and transiently expressed in Nicotiana benthamiana.We cloned putative secreted protein genes from the transcript libraries,and got 46 genes with signal peptide and without transmembrane domain.Those candidate genes were cloned to the plant expression vector and transexpression in Nicotiana benthamiana.Our result showed that the expansin-like protein HaEXPB2 caused visible cell death in N.benthamiana leaves.The full length cDNA of HaEXPB2 was cloned from Heterodera avenae,with 870 bp ORF encoding a 289 amino acid protein.The HaEXPB2 protein had 89%homology with Ha-EXPB1,containing a signal peptide at N-terminal,a CBM II and an expansin domain.The in situ hybridization results showed that the transcripts of HaEXPB2 were localised within the subventral gland cells of the J2s.Real-time PCR assays indicated the expression of HaEXPB2 was highest in parJ2.HaEXPB2 was fused with RFP and expressed in N.benthamiana revealed that it could be localized in the cell wall.HaEXPB2 could bound to cellulose substrate by the carbohydrate-binding domain.When HaEXPB2 was knocked down by RNA interference in vitro,the infectivity of H.avenae was reduced by 53%.Thses results indicated that HaEXPB2 may play a role through targeting the host cell wall when H.avenae parasitic host plant.In this study,we cloned another effector gene HaCRT1,encoded a calreticulin protein,which could suppress the cell death induced by Bax when expressed in N.benthamiana.The result showed that HaCRT1 was localized in the Endoplasmic Reticulum(ER)in plant cell.HaCRT1 gene expression in Arabidopsis.thaliana increases susceptibility to Pseudomonas syringae.We trid to identify which signaling HaCRT1 inhabit Bax induce cell death depend on in yeast,and study the function of HaCRT1 in host innate immunity.The results will supply evidences to understand the interaction mechanism between cereal cyst nematode and their hosts,and the plant nematode pathogenic molecular mechanism,and is very important to make control strategies and protect our food security.