Identification of CBL and CIPK Gene Families and Functional Characterization of PvCIPK7 as an Essential Regulator of Root Nodule Development and Nitrogen Fixation in Phaseolus vulgaris

Abstract

Calcineurin B-like proteins (CBLs) act as Ca2+ sensors and activate CBL-interacting protein kinases (CIPKs) to form CBL-CIPK complexes, crucial for plant signal transduction and stress perception. Nevertheless, their functions in root nodule symbiosis (RNS) are largely unexplored. Herein, we classified previously identified SnRK3s as CIPKs and identified 10 CBLs in Phaseolus vulgaris. Additionally, we conducted a functional characterization of PvCIPK7 to acquire a comprehensive understanding of its involvement in RNS in P. vulgaris. Transcript downregulation of PvCIPK7 by RNA interference (RNAi) significantly affected primary and lateral root growth and density. During symbiosis, PvCIPK7-RNAi roots showed a reduction in infection events, nodule numbers, and, nodule size was significantly reduced. The cross section of PvCIPK7-RNAi nodule revealed a loss of nodule organization, a reduction in the number of symbiosomes in infected cells, and a failure of Rhizobium bacteria to differentiate into bacteroids. This severely affected the nitrogen-fixing ability of PvCIPK7-RNAi nodules, as evidenced by downregulated symbiosis-specific genes and genes related to nitrogen fixation. The spatiotemporal expression pattern of the CIPK7 promoter revealed symbiosis-associated expression in the early stages. In conclusion, PvCIPK7 is indispensable for root nodule development, bacterial differentiation, and, consequently, nitrogen fixation during the symbiotic interaction between rhizobia and P. vulgaris.