Nitrogen-fixing bacteria establish symbiotic relationships with their host plants via two different entry systems: root hair-mediated (intracellular) entry and intercellular entry. However, the molecular mechanisms underlying the intercellular entry system have received relatively little research attention. In this study, we compared the transcriptomes of the nodules and roots of Myrica rubra, which forms an ancient type of symbiosis with Frankia via intercellular entry. We found that cysteine-rich receptor-like secreted protein 1 (CRRSP1) was highly upregulated in M. rubra nodules. We then investigated the function of MrCRRSP1 in Aeschynomene indica, which establishes symbiosis with Bradyrhizobium sp. ORS285 through an intercellular entry system. The overexpression of MrCRRSP1 and AiCRRSP1 in A. indica enhanced the nodule number and plant growth. Exogenous application of glutathione S-transferase (GST)-tagged MrCRRSP1 and AiCRRSP1 in A. indica promoted rhizobial attachment at cracks in the lateral root base, as well as rhizobial motility and biofilm formation. These results suggest that CRRSP1 promotes nodulation by enhancing rhizobial attachment to lateral root cracks. In addition to providing new insights into the molecular mechanisms underlying nodule formation through intercellular entry, this research enhances our understanding of actinorhizal plant–Frankia symbiosis.

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