Plant disease management is key to sustainable production of staple food crops. Calcium (Ca²⁺) signal and phytohormones play critical roles in regulating plant defense responses against pathogens. The Ca²⁺ signals are sensed, decoded and transduced by calmodulin and other Ca²⁺ -binding proteins, followed by interaction with and modulation of activities of target proteins such as calmodulin-binding proteins (CBPs). Members of the Arabidopsis CBP60 gene family, AtCBP60g and AtSARD1, have emerged as major regulators of immune responses. In this study, we identified a 15 member CBP60 gene family in rice (Oryza sativa) of which OsCBP60g-3, OsCBP60g-4, OsCBP60a and OsSARD-like1 genes were consistently upregulated in rice seedlings in response to infection with both fungal (Magnaporthe oryzae) and bacterial (Xanthomonas oryzae) pathogens as well as by salicylic acid (SA). OsCBP60g-4 and OsCBP60g-3 were induced maximally by SA and brassinosteroid (BR), respectively, and OsCBP60g-4 was expressed at 3-fold higher levels in the M. oryzae resistant rice genotype (IC-346004) as compared to the susceptible rice genotype (Rajendra Kasturi). The considerable expansion of the immunity clade and the up-regulation of several OsCBP60 genes in response to pathogens and defense hormones supports the importance of further investigating OsCBP60 genes as targets for increasing disease resistance in rice.

Brassinosteroids (BRs) are plant steroid hormones that not only play vital roles in plant growth and development, but also in mediating stress responses. A group of calmodulin-binding proteins, known as CBP60s are also involved in mediating the response of plants to stress. The aims of the present study were: (1) to investigate the effect of exogenous 24-epibrassinolide (EBR) on the phenotype of cotton (Gossypium hirsutum) seedlings under mild to moderate biotic and abiotic stresses, (2) to find and characterise cotton CBP60-encoding genes, orthologous to Arabidopsis CBP60s with known involvement in stress responses, and to investigate whether EBR may act by modulating the expression of GhCBP60 genes in cotton leaf tissue under salt stress. Experiments were designed to demonstrate the effects of EBR application from 0.1 to 2 µM on the phenotypic responses of cotton seedlings to mild/moderate salt, drought and pathogen (Verticillium dahliae) stresses. Results show that the exogenous application of EBR at low concentrations of 0.1 and 0.2 µM had no positive effect on seedling growth under all stresses. In addition, EBR at a higher concentration (0.5 µM) or with the surfactant Tween 20 caused toxic effects. Bioinformatics approaches revealed the presence of GhCBP60 orthologues of AtCBP60. Phylogenetic analysis indicated that CBP60a, CBP60g, andSARD1 from Arabidopsis each have four co-orthologues in cotton. AtCBP60f has two coorthologues, whereas CBP60b/c/d have nine co-orthologues. Multiple amino acid sequence alignments indicate that the DNA-binding and CaM-binding domains of AtCBP60 are highly conserved in GhCBP60, suggesting similar protein structures to AtCBP60. Prediction of subcellular localisation suggested that all GhCBP60 proteins contain a nuclear localisation signal. This, together with the highly conserved putative DNA binding region, suggests that all GhCBP60 are transcription factors. The results of qRT-PCR demonstrated that EBR treatment of cotton up-regulated the expression of GhCBP60a/f/g. On the other hand, salt down-regulated the expression of GhCBP60a but up-regulated the expression of GhCBP60f/g. Interestingly, treatment with EBR in the absence of salt restored the expression of GhCBP60a to levels similar to the control tissue. Analysis of promoters of GhCBP60 genes for putative BR-related transcription factor binding motifs indicated the presence of CANNTG and GGTCC elements. However, these were not significantly enriched in stress-regulated genes. Furthermore, higher stringency BR-signalling-related elements: BRRE (CGTGTG/CGTGCG), G-box (CACGTG) and transcription factors TGA 1/TGA4 (TGACG) sense strands were absent in stress responsive genes GhCBP60a/f/g and GhSARD1 as compared to other groups. In the light of these results, I concluded that brassinosteroids (BRs) positively regulates the expression of novel GhCBP60 genes suggesting a possible connection between BR signalling and GhCBP60 transcription factors in mediating abiotic stress responses in cotton. However, the results from the cis-element search suggest that this connection is likely to be indirect rather than via a direct interaction with the BR signal transduction pathway.