The ex planta signal activity of a Medicago ribosomal uL2 protein suggests a moonlighting role in controlling secondary rhizobial infection

We recently described a regulatory loop, which we termed autoregulation of infection (AOI), by which Sinorhizobium meliloti, a Medicago endosymbiont, downregulates the root susceptibility to secondary infection events via ethylene. AOI is initially triggered by so-far unidentified Medicago nodule signals named signal 1 and signal 1’ whose transduction in bacteroids requires the S. meliloti outer-membrane-associated NsrA receptor protein and the cognate inner-membrane-associated adenylate cyclases, CyaK and CyaD1/D2, respectively. Here, we report on advances in signal 1 identification. Signal 1 activity is widespread as we robustly detected it in Medicago nodule extracts as well as in yeast and bacteria cell extracts. Biochemical analyses indicated a peptidic nature for signal 1 and, together with proteomic analyses, a universally conserved Medicago ribosomal protein of the uL2 family was identified as a candidate signal 1. Specifically, MtRPuL2A (MtrunA17Chr7g0247311) displays a strong signal activity that requires S. meliloti NsrA and CyaK, as endogenous signal 1. We have shown that MtRPuL2A is active in signaling only in a non-ribosomal form. A Medicago truncatula mutant in the major symbiotic transcriptional regulator MtNF-YA1 lacked most signal 1 activity, suggesting that signal 1 is under developmental control. Altogether, our results point to the MtRPuL2A ribosomal protein as the candidate for signal 1. Based on the Mtnf-ya1 mutant, we suggest a link between root infectiveness and nodule development. We discuss our findings in the context of ribosomal protein moonlighting.