DNA binding activity of CAMTA3 is essential for its function

Abstract

Calmodulin-binding transcription activators (CAMTAs), a small family of highly conserved transcription factors, function in calcium-mediated signaling pathways. Of the six CAMTAs in Arabidopsis, CAMTA3 (also referred to as SR1) regulates diverse biotic and abiotic stress responses. A recent study has shown that CAMTA3 is a guardee of NLR ((nucleotide-binding, leucine-rich repeat domain) immune receptors in modulating plant immunity, thereby suggesting that CAMTA3 transcriptional activity is not necessary for its function. Here, we show that the DNA-binding activity of CAMTA3 is essential for its role in mediating plant immune responses. Analysis of the DNA binding (CG-1) domain of CAMTAs in plants and animals showed strong conservation of several amino acids. We mutated several conserved amino acids in the CG-1 domain to investigate their role in CAMTA3 function. Electrophoretic mobility shift assays using these mutants with a promoter of its target gene identified critical amino acid residues necessary for DNA binding activity. Furthermore, transient assays showed that these residues are essential for the CAMTA3 function in activating the RSRE (Rapid Stress Response Element)-driven reporter gene expression. In line with this, transgenic lines expressing the CG-1 mutants of CAMTA3 in the camta3 mutant failed to rescue the mutant phenotype and restore the expression of CAMTA3 downstream target genes. Collectively, our results provide biochemical and genetic evidence that the transcriptional activity of CAMTA3 is indispensable for its function.