Nitrogen-fixing symbiosis between legumes and rhizobia represents a crucial biological process with significant agricultural and economic importance, enabling the conversion of atmospheric nitrogen into ammonia, a usable form for plants. This highly regulated interaction relies on calcium-mediated signaling pathways. Although calcium oscillations are essential for nodule formation, the downstream proteins responsible for translating these signals into a developmental response remain poorly understood. This project investigates the role of the Calmodulin Binding Protein (CaMBP) in Medicago truncatula during legume-rhizobia symbiosis using CRISPR/Cas9-mediated gene disruption. CaMBP is hypothesized to function downstream of calcium signaling to regulate rhizobial infection and nodule development. 

To test this hypothesis, CRISPR/Cas9 constructs targeting CaMBP will be introduced into legume roots via Agrobacterium rhizogenes-mediated hairy root transformation, and successful edits will be confirmed through PCR-based genotyping. Following rhizobial inoculation, symbiotic phenotypes such as nodule formation and infection thread formation will be assessed. If CaMBP functions as a downstream effector of calcium signaling, loss-of-function mutants are expected to exhibit impaired infection thread formation and reduced or abnormal nodule development compared to control roots. By linking targeted gene disruption to measurable developmental phenotypes, this study aims to determine whether CaMBP is necessary to translate nuclear calcium oscillations into localized cellular responses required for successful nitrogen fixation. Clarifying the role of CaMBP in legume symbiosis would advance understanding of how calcium signaling regulates symbiotic development.