Distinct signalling routes mediates intercellular and intracellular rhizobial infection in Lotus japonicus

Abstract

Rhizobial infection of legume roots during development of nitrogen fixing root nodules occurs either intracellularly though plant derived infection threads traversing the epidermal and cortical cell layers to deliver the bacteria or intercellularly via bacterial entry between epidermal plant cells. Although, around 25% of all legume genera are postulated to be intercellularly infected, the pathways and mechanisms supporting this process has remained virtually unexplored due to lack of genetically amenable legumes that have this infection mode. In this study, we report that the model legume Lotus japonicus is infected intercellularly by Rhizobium sp. IRBG74 and demonstrate that the resources available in Lotus enable insight into the genetic requirements and the fine-tuning of the pathway governing intercellular infection. Inoculation of Lotus mutants shows that Ern1 and RinRK1 are dispensable for intercellular infection in contrast to intracellular infection. Other symbiotic genes, including Nfr5, SymRK, CCaMK, Epr3, Cyclops, Nin, Nsp1, Nsp2, Cbs and Vpy1 are equally important for both entry modes. Comparative RNAseq analysis of roots inoculated with IRBG74 revealed a distinctive transcriptome response compared to intracellular colonization. In particular, a number of cytokinin-related genes were differentially regulated. Corroborating this observation cyp735A and ipt4 cytokinin biosynthesis mutants were significantly affected in their nodulation with IRBG74 while lhk1 cytokinin receptor mutants did not form any nodules. These results indicate that a differential requirement for cytokinin signalling conditions intercellular rhizobial entry and highlight the distinct modalities of the inter- and intra-cellular infection mechanisms.