Molecular characterization and functional analysis of two new β-1,4-encloglucanase genes (Ha-eng-2, Ha-eng-3) from the cereal cyst nematode Heterodera avenae

摘要

Parasitism genes encoding secretory proteins expressed in the pharyngeal glands of plant-parasitic nematodes play a crucial role in nematode parasitism of plants. Two new β-1,4-endoglucanase genes (Ha-eng-2 and Ha-eng-3) expressed in the pharyngeal glands of the sedentary cyst nematode, Heterodera avenae, were cloned. Both of the predicted proteins have a putative signal peptide for secretion and a catalytic domain. Neither peptide linkers nor cellulose binding domains were present. In situ hybridization showed that the transcripts of Ha-eng-2 and Ha-eng-3 accumulated specifically in the two subventral gland cells of H. avenae. RT-PCR analysis confirmed that their transcriptions were strong in the preparasitic and early parasitic second-stage juveniles, and were undetectable at the late parasitic stages of the nematode. Cellulase activities of the recombinant proteins HA-ENG-2 and HA-ENG-3 were confirmed in vitro. Knocking down Ha-eng-2 using RNA interference reduced nematode infectivity by 40%. The results indicate that these β-1,4-endoglucanases can be secreted into plant tissues and play an important role in the wall degradation of plant cells during penetration and the migration of second-stage juveniles in host roots.