A laser dissection-RNAseq analysis highlights the activation of cytokinin pathways by Nod factors in the Medicago truncatula root epidermis

Abstract

Nod factors (NF) are lipochitooligosaccharidic signal molecules produced by rhizobia, which play a key role in the rhizobium-legume symbiotic interaction. In this study, we analyzed the gene expression reprogramming induced by purified NF (4h and 24h of treatment) in the root epidermis of the model legume Medicago truncatula. Tissue-specific transcriptome analysis was achieved by laser capture micro-dissection coupled to high depth RNA sequencing. Expression of 17,191 genes was detected in the epidermis, among which 1070 were found to be regulated by NF addition, including previously characterized NF-induced marker genes. Many genes exhibited strong levels of transcriptional activation, sometimes only transiently at 4 hours, indicating highly dynamic regulation. Expression reprogramming affected a variety of cellular processes, including perception, signaling, regulation of gene expression, as well as cell wall, cytoskeleton, transport, metabolism and defense, with numerous NF-induced genes never identified before. Strikingly, early epidermal activation of cytokinin (CK) pathways was evidenced, based on the induction of CK metabolic and signaling genes, including the CRE1 receptor essential to promote nodulation. These transcriptional activations were independently validated using promoter:GUS fusions with the MtCRE1 CK receptor gene and a CK response reporter (TCSn). A CK pre-treatment reduced the NF-induction of the ENOD11 symbiotic marker, while a CK degrading enzyme (AtCKX3, for cytokinin oxidase/deshydrogenase 3) ectopically expressed in the root epidermis led to increased NF-induction of ENOD11 and nodulation. CK may therefore play both positive and negative roles in M. truncatula nodulation.