| Plant parasitic nematodes(PPNs)was an important parasitic nematodes in agricultural production.Pratylenchus spp.,also known as root rot nematode,is one of the three most harmful plant parasitic nematodes on crops.Pratylenchus coffeae,as one of the main pathogens causing root rot of Chinese crops,seriously harms corn,wheat,soybean,yam,tomato,sesame and other food crops and cash crops.Due to its strong colonization,adaptability and wide host range,there is still a lack of effective targets and methods for the control of this disease,and there is an urgent need for effective prevention and control methods in production.At present,the pathogenicity of effector in plant-parasitic nematodes and their interaction with hosts have become a hot topic in plant nematology.As one of the most harmful root rot nematodes,the studies on the pathogenic proteins of P.coffeae have just begun.In this study,Pc-NEX-1 and Pc-NEX-2 gene sequences were obtained by targeting PCNEXs,a effector proteins in P.coffeae.The purpose of this study was to investigate the function of Pc-NEX-1 and Pc-NEX-2 in the pathogenic process of P.coffeae infection and the molecular mechanism of inhibition of host defense response.The main results are as follows:1.The sequences of the Pc-NEX-1 and Pc-NEX-2 genes were obtained by transcriptome sequencing.The length of Pc-NEX-1 DNA coding region is 1596 bp,ORF is 987 bp,6 introns,encodes 328 aa,no signal peptide with transmembrane structural domain,the theoretical molecular weight of this protein is 37.81 k Da,isoelectric point PI 7.04,hydrophilicity-0.425;Pc-NEX-2 has a coding region sequence length of 1276 bp,an ORF of 975 bp,and 6 introns encoding 324 aa,no signal peptide with transmembrane structural domain,molecular weight size is 36.8 k Da,isobaric site PI 7.12,hydrophilicity-0.437.2.The transcript levels of Pc-NEX-1 and Pc-NEX-2 were examined by q RT-PCR in four worm states(females,males,larvae and eggs)of P.coffeae.The expression of both Pc-NEX-1 and Pc-NEX-2 genes was lowest in females,highest in eggs and highest in larvae.In situ hybridization showed that Pc-NEX-1 was expressed in both esophageal glands and eggs of P.coffeae,whereas Pc-NEX-2 was specifically expressed only in the esophageal glands of nematodes.3.The recombinant proteins of Pc-NEX-1 and Pc-NEX-2 with e GFP were transiently expressed on tobacco leaves using an Agrobacterium-mediated method,and the subcellular localization of the proteins was analysed.The results showed that Pc-NEX-1 was localized in the cytoplasm and nucleus of tobacco leaves,while Pc-NEX-2 was localized in the cytoplasm of tobacco leaves.4.Using the in vitro RNAi method,the silencing efficiency of the membrane linked protein genes,Pc-NEX-1 and Pc-NEX-2,was confirmed to decrease with increasing treatment time in P.coffeae.The highest silencing efficiency was observed in P.coffeae after 12 h of Pc-NEX-1 ds RNA treatment compared to the control,and the nematode reproduction rate in carrot healing tissues was reduced by 84.9% in the Pc-NEX-1 RNAi 12 h treatment group compared to the e GFP control,a significant difference(P<0.001),and the nematode infestationand(P<0.05)and pathogenicity(P<0.05)of the host maize were significantly reduced in this treatment group.Pc-NEX-2 ds RNA treatment for 12 h had the highest silencing efficiency of Pratyenchus spp in the Pc-NEX-2 RNAi 12 h treatment group,and nematode fecundity in carrot healing tissues was reduced by 48% compared to the e GFP control,a significant difference(P<0.05),and nematode infestation(P<0.01)and pathogenicity of maize in this treatment group were significantly reduced(P<0.05).were significantly reduced.These results suggest that Pc-NEX-1 and Pc-NEX-2 effector proteins play an important role in the infestation pathogenicity and reproduction of P.coffeae.5.Transient expression of both P.coffeae PC-NEX-1 and PC-NEX-2 proteins in leaf fragments of N.benthamiana using an Agrobacterium-mediated method significantly inhibited the expression of immune-related genes in the host plant.Transient expression of PC-NEX-1 in N benthamiana significantly reduced the transcript levels of PTI-related genes Nb-Pti5,Nb-Grants2,Nb-Acre3 and Nb-PAL and Nb-PR1 of the salicylic acid pathway and Nb-LOX of the jasmonic acid pathway compared to e GFP(P<0.05).Pc-NEX-1 and Pc-NEX-2 significantly inhibited the burst of reactive oxygen species ROS in tobacco induced by the bacterial flagellin Flg22 and also inhibited the RBP1/GPA2 protein-induced tobacco leaf cell necrosis response.6.A Pc-NEX-1 decoy vector was constructed and Pc-NEX-1 was assayed for its toxicity and self-activating activity against yeast.A reciprocal protein Zmpifi(Zea mays chloroplast post-illumination chlorophyll fluorescence increase protein)was screened from a maize library by yeast two-hybrid library screening.The ability of PC-NEX-1 to interact with this protein was tentatively demonstrated. |
PDF Full Text Request