Subtilisin-like gene Dt12 is specifically expressed in Discaria trinervis along intercellular root infection by Frankia and root nodule development

WALL LG; IMANISHI L; VAYSSAYRE V; PIRROLLES E; BROTTIER L; MUÑOZ N; LASCANO R; GHERBI H; HOCHER V; SVISTOONOFF S

Stockholm

Congreso; 13th European Congress on Nitrogen Fixation; 2018

ECNF

The actinorhizal host Discaria trinervis is initially colonized by Frankia via an exclusively intercellular pathway without infection thread formation. This mode of microsymbiont entry has received little attention until now. Cg12 gene encodes a putative apoplastic subtilisin-like serine protease strongly and specifically expressed in Casuarina glauca undergoing intracellular root hair infection by Frankia as well as during rhizobial infection of M. truncatula. Exploratory experiments introducing the ProCg12:GUS reporter into D. trinervis roots via A. rhizogenes-mediated transformation show strong GUS expression in root nodules following Frankia inoculation. The orthologue Dt12 was cloned from D. trinervis based on conserved regions of both Cg12 and the Alnus glutinosa orthologue Ag12. The corresponding Dt12 protein shares an overall similarity of 77% with both Cg12 and Ag12. Phylogeny analysis showed that Dt12 and other actinorhizal symbiosis-related subtilases cluster together in a well-supported group which also includes two Medicago truncatula proteins. Both ProCg12:GUS and ProDt12:GFP were found to be strongly activated in D. trinervis inoculated roots from early small scattered groups of cells in the epidermis and outer root cortex tissues, till mature nodule, especially during nodule development following the infection zone. GUS/GFP reporters were undetectable in developing lateral roots. Preliminary Dt12 RNAi experiments show a des-regulation of nodulation in the hairy roots of D. trinervis suggesting that subtilase activity is not only involved in reorganization and composition of the extracellular cell wall/matrix which surrounds the filamentous Frankia during intercellular colonization, but also would be involved in the autoregulation of nodulation. This result is supported by an old observation that autoregulation is triggered just after intercellular cortex invasion and before nodule primordia development.