: A Key Player in Regulating the - Symbiotic Interaction.

Cysteine-rich receptor-like kinases (CRKs) are a type of receptor-like kinases (RLKs) that are important for pathogen resistance, extracellular reactive oxygen species (ROS) signaling, and programmed cell death in plants. In a previous study, we identified 46 CRK family members in the genome and found that was highly upregulated under root nodule symbiotic conditions. To better understand the role of in the - symbiotic interaction, we functionally characterized this gene by overexpressing (-OE) and silencing (-RNAi) it in a hairy root system. We found that the constitutive expression of led to an increase in root hair length and the expression of root hair regulatory genes, while silencing the gene had the opposite effect. During symbiosis, -RNAi resulted in a significant reduction in nodule numbers, while -OE roots showed a dramatic increase in rhizobial infection threads and the number of nodules. Nodule cross sections revealed that silenced nodules had very few infected cells, while -OE nodules had enlarged infected cells, whose numbers had increased compared to controls. As expected, -RNAi negatively affected nitrogen fixation, while -OE nodules fixed 1.5 times more nitrogen than controls. Expression levels of genes involved in symbiosis and ROS signaling, as well as nitrogen export genes, supported the nodule phenotypes. Moreover, nodule senescence was prolonged in -overexpressing roots. Subcellular localization assays showed that the CRK12 protein localized to the plasma membrane, and the spatiotemporal expression patterns of the -promoter::GUS-GFP analysis revealed a symbiosis-specific expression of during the early stages of rhizobial infection and in the development of nodules. Our findings suggest that CRK12, a membrane RLK, is a novel regulator of symbiosis.