Characterisation and functional importance of β-1,4-endoglucanases from the potato rot nematode, Ditylenchus destructor

Abstract

Plant-parasitic nematodes have developed a series of enzymes to degrade the rigid plant cell wall; β-1,4-endoglucanase is a very important component. Ditylenchus destructor is a migratory endoparasite for which few molecular data have been published. Two novel β-1,4-endoglucanases (Dd-eng-1a and Dd-eng-2) were cloned and characterised from D. destructor. The DD-ENG-1A putative protein consists of a signal peptide, a catalytic domain and a carbohydrate-binding module (CBM). By contrast, the CBM domain is absent from DD-ENG-2. The exon/intron structure and phylogenetic tree indicate that both cellulase genes could have evolved from common ancestral genes. Southern blotting confirmed that the β-1,4-endoglucanases were of nematode origin and a member of a small multi-gene family. In situ hybridisation localised the expression of Dd-eng-1a and Dd-eng-2 to the subventral pharyngeal glands. RT-PCR showed that both genes were expressed in the adult female and second-stage juvenile. The stylet secretions of D. destructor showed clear cellulase activity in carboxymethylcellulose (CMC) plate assay, and similar results were observed in total homogenates and DD-ENG-1A and DD-ENG-2 recombinant proteins. These results demonstrated that D. destructor can produce and secrete functional cellulases. Silencing the putative β-1,4-endoglucanases by double-stranded RNA (dsRNA) resulted in an average decrease in infection of 50%. Successful RNAi in vitro was demonstrated in this study, which confirmed that Dd-eng-1a and Dd-eng-2 play important roles in nematode parasitism.