Molecular analysis of cellulolytic enzymes in plant-nematode interactions (Globodera tabacum, Meloidogyne incognita).

摘要

Feeding cells induced by cyst (Heterodera and Globodera spp.) and root-knot (Meloidogyne spp.) nematodes, termed syncytia and giant-cells, respectively, are initiated within host roots during parasitism to supply nutrition for nematode growth and reproduction. One of the most distinguishing characteristics of nematode-induced feeding cells is the extensive remodeling of their cell walls. Physiological processes including cell expansion, cell wall biosynthesis, and complete cell wall dissolution have been observed during feeding cell formation. It is unclear whether the extensive cell wall architectural modifications observed in nematode-induced feeding cells result from enzymes of nematode or plant origin. The objective of this research was to identify and characterize β-1,4-endoglucanases (EGases), enzymes that catalyze the hydrolyis of polysaccharides containing a β-1,4-glucan backbone, of nematode and plant origin and determine their roles during compatible plant-nematode interactions. Two β-1,4-endoglucanase genes, Gt-eng-1 and Gt-eng-2, were isolated from Globodera tabacum, the tobacco cyst nematode, and their expression was localized within the nematode's subventral esophageal gland cells (SvG). Expression of EGases within the SvG of G. tabacum occurs in second-stage juveniles (J2) within eggs prior to hatching and continues up until the late J2 parasitic life stage. EGase expression is not observed in subsequent female nematode life stages, but interestingly, expression resumes in males as they develop to adults. Immunolocalization studies confirmed the secretion of G. tabacum EGases into plant tissue during parasitism of tobacco roots. G. tabacum EGases were detected within the SvG cells, in plant tissue along the migratory path of J2 through the root cortex, and outside the tip of the head on the cell wall in contact with the stylet at 24 hours after infection. Secretion of EGases was not detected from nematodes at 48–96 hours after infection into developing syncytia, during which time cell wall breakdown is active. The developmental expression profile and the localization of secreted G. tabacum EGases into root tissue during migration, but not during the establishment of feeding cells, supports the hypothesis that plant enzymes are involved in the observed cell wall dissolution in developing syncytia. Five structurally-divergent tobacco endo-β-1,4-glucanase cDNA's corresponding to mRNA's expressed in G. tabacum-infected root tissue were isolated using degenerate primers to conserved regions of known plant EGases. All five EGase genes are upregulated in both cyst and root-knot nematode-infected root tissue as compared to healthy root tissue. In addition, an Arabidopsis thaliana elongation-specific EGase (Cel1) promoter is upregulated in root-knot nematode-induced giant cells in tobacco, but not within syncytia. In situ mRNA hybridization studies localized expression of tobacco EGases to nematode-induced syncytia, giant-cells, and root tips. This is the first direct evidence supporting a role for plant endo-β-1,4-glucanases in nematode-induced feeding cell development. The tobacco EGases are likely an integral component of a complex array of cell wall enzymes activated in response to nematode parasitism and involved in wall remodeling during cyst and root knot nematode-induced feeding cell development.