An Endoglucanase Is Involved in Infection of Rice Roots by the Not-Cellulose-Metabolizing Endophyte Azoarcus Sp. Strain BH72

The nitrogen-fixing endophyte Azoarcus sp. strain BH72 infects roots of Kallar grass and rice inter- and intra-cellularly and can spread systemically into shoots without causing symptoms of plant disease. Although cellulose or its breakdown products do not support growth, this strain expresses an endoglucanase, which might be involved in infection. Sequence analysis of eglA places the secreted 34-kDa protein into the glycosyl hydrolases family 5, with highest relatedness (40% identity) to endoglucanases of the phytopathogenic bacteria Xanthomonas campestris and Ralstonia solanacearum. Transcriptional regulation studied by eglA:: gusA fusion was not significantly affected by cellulose or its breakdown products or by microaerobiosis. Strongest induction (threefold) was obtained in bacteria grown in close vicinity to rice roots. Visible sites of expression were the emergence points of lateral roots and root tips, which are the primary regions of ingress into the root. To study the role in endophytic colonization, eglA was inactivated by transposon mutagenesis. Systemic spreading of the eglA mutant and of a pilAB mutant into the rice shoot could no longer be detected by polymerase chain reaction. Microscopic inspection of infection revealed that the intracellular colonization of root epidermis cells was significantly reduced in the eglA- mutant BHE6 compared with the wild type and partially restored in the complementation mutant BHRE2 expressing eglA. This provides evidence that Azoarcus sp. endoglucanase is an important determinant for successful endophytic colonization of rice roots, suggesting an active bacterial colonization process.