DNA-Binding Activity of CAMTA3 Is Essential for Its Function: Identification of Critical Amino Acids for Its Transcriptional Activity

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 regulates diverse biotic and abiotic stress responses. A recent study has shown that CAMTA3 is a guardee of NLRs (Nucleotide-binding, Leucine-rich repeat Receptors) in modulating plant immunity, raising the possibility that CAMTA3 transcriptional activity is dispensable 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 six 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. In addition, transient assays showed that these residues are essential for the CAMTA3 function in activating the Rapid Stress Response Element (RSRE)-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.

A natural single‐nucleotide polymorphism in the CAMTA3 transcription factor regulates its function and transcription of its target genes

“…CAMTA3 regulates the expression of many stress-responsive genes (Chao et al, 2022;Du et al, 2009;Finkler et al, 2007;Kim et al, 2017;Reddy et al, 2000). Mutations in various domains and potential phosphorylation sites have been shown to have a significant impact on CAMTA3 activity in regulating the expression of target genes (Chao et al, 2022;Du et al, 2009;Jiang et al, 2020;Kim et al, 2017;Prasad et al, 2023). In this study, we identified a single amino acid substitution at position 386 where histidine (H) was replaced with aspartic acid (D) (File S1).…”

“…The chemiluminescent signal was detected using the gel imaging system (Bio-Rad). Luciferase reporter constructs driven by 4 × RSRE:NOS promoter were prepared essentially as described earlier (Prasad et al, 2023).…”

“…A. tumefaciens-mediated transient assays with reporter and effector constructs were performed in N. benthamiana leaves as described earlier (Prasad et al, 2023).…”

“…The frozen leaves were ground to a fine powder in a TissueLyser (Qiagen), and 100-200 mg of the powder was used for preparing protein extract. The powdered tissue was suspended in 500 μL of CCLR extraction buffer (Prasad et al, 2023) and kept on an orbital shaker for 15 min at 4°C for protein extraction. The extract was then clarified by centrifugation at 15,700 g for 15 min at 4°C and the luciferase activity was quantified with the luciferase assay kit (Prasad et al, 2023).…”

“…The powdered tissue was suspended in 500 μL of CCLR extraction buffer (Prasad et al, 2023) and kept on an orbital shaker for 15 min at 4°C for protein extraction. The extract was then clarified by centrifugation at 15,700 g for 15 min at 4°C and the luciferase activity was quantified with the luciferase assay kit (Prasad et al, 2023). Three infiltrated leaves per plant and three plants per biological replicate were used to determine luciferase activity.…”