Background: The Calcineurin B-like (CBL) and CBL-interacting protein kinase (CIPK) system constitute critical signaling modules mediating plant responses to abiotic stress. Although these families have been studied across various species, their evolutionary dynamics across grasses and the functional plasticity of specific isoforms remain elusive. Methods: A genome-wide analysis of CBL and CIPK families was conducted across five major Poaceae species (Oryza sativa, Triticum aestivum, Zea mays, Sorghum bicolor, and Saccharum spontaneum). Phylogenetic and synteny analyses were analyzed to family expansion and evolution. Cis-regulatory elements analysis in gene promoter regions were examined to predict potential stress-responsive features. Expression profiles of OsCBL and OsCIPK gene families were examined by qRT-PCR under conditions involving PEG-induced osmotic stress, pathogen strain P6 inoculation, and exogenous application of the phytohormones abscisic acid (ABA) and methyl jasmonate (MeJA). Protein–protein interactions between selected CBL (OsCBL4) and CIPK pairs were assessed via Yeast Two-Hybrid (Y2H) and Luciferase Complementation Imaging assays (LCI). Results: Phylogenetic and synteny analyses indicated that segmental duplications have contributed substantially to the expansion of these gene families. Promoter analysis revealed that the majority of CBL and CIPK family members, exemplified by OsCBL4, traditionally characterized as a salt sensor, possesses a cis-element architecture (rich in ABREs and MBS) heavily biased towards dehydration responsiveness. Expression profiling showed that OsCBL4 is significantly hyper-induced by direct osmotic stress (PEG) but exhibits almost no response to exogenous ABA. A subset of kinases genes (e.g., OsCIPK2, 9, 18) displayed PEG-induced expression patterns resembling those of OsCBL4, whereas OsCIPK30 remained transcriptionally unresponsive under the same conditions. Protein interaction assays demonstrated that OsCBL4 physically interacts exclusively with PEG-responsive transcriptionally activated kinases such as OsCIPK9, but failed to interact with the non-responsive OsCIPK30. Conclusions: Our study provides a genomic characterization of CBL and CIPK families across five major Poaceae species. The combined expression and interaction data reveal that OsCBL4-assembles with specific CIPKs into signaling modules during osmotic stress responses in rice, pointing to roles that go beyond salt stress responses. The findings establish a foundation for further functional dissection of CBL-CIPK pathway diversification in abiotic stress adaptation.

Bacterial blight (BB), caused by Xanthomonas oryzae pv. Oryzae (Xoo), is one of the most devastating diseases in rice worldwide. Common wild rice (Oryza rufipogon Griff.) inhabiting the high-temperature and high-humidity environments of Hainan Island has evolved strong disease resistance through natural selection, representing a valuable genetic reservoir for resistance breeding. However, large-scale characterization of resistance phenotypes, resistance genes, and their combinations remains limited. In this study, we evaluated BB resistance in 1511 Hainan common wild rice accessions against three Xoo strains (HNX004, PXO99^A, and Z173) and analyzed the distribution of ten major known resistance genes (Xa1, Xa3, Xa4, xa5, Xa7, Xa10, xa13, Xa21, Xa23, and Xa27). Phenotypic evaluation revealed distinct strain-specific resistance patterns. Broad-spectrum resistance analysis (defined as moderate resistance or higher) revealed that 35 accessions (2.32%) were resistant to all strains, and 378 accessions (25.02%) showed resistance to two strains. Genotyping of known resistance genes revealed that, except for one accession, which lacked all tested genes but showed resistance to strain PXO99^A, all other accessions carried every tested gene except Xa21 and xa13. Interestingly, different Xoo strains exhibited distinct requirements for resistance genes, revealing a clear strain-specific resistance pattern. Notably, the number of resistance genes did not correlate with resistance level. Instead, specific complementary combinations, particularly Xa1 + Xa10 + Xa23 + Xa4 + Xa7, conferred the strongest broad-spectrum resistance. Our results demonstrate that gene quality and specific complementary combinations are more important than the absolute number of resistance genes. The identified resistant accessions and favorable gene combinations provide valuable resources for rice breeding programs.