Functional Characterization Of Homologous Genes Of Calmodulin-binding Protein 60(CBP60)

In order to survive and reproduce,plants have evolved a complex immune system to resist the invasion of pathogens.Plants use a dual innate immune system to fight off invading pathogens.The first layer plant immunity is the sensing of pathogen-associated molecular patterns（PAMPs/DAMPs）through plasma membrane-directed pattern recognition receptors（PRRs）,which is called PAMPs-triggered immunity（PTI）response.The second layer plant immunity involves the recognition of effectors released by pathogens.The intracellular immune receptor R proteins directly or indirectly recognize effectors,which triggers effectors-triggered immunity（ETI）response.Both PTI and ETI responses will result in cascade of immune signals and eventually lead to expression of a large number of immune genes called transcriptional reprogramming,to resist the attack of pathogens.Transcriptional reprogramming is an important feature of plant immunity,which is controlled by immune-related transcription factors and co-regulatory proteins.CBP60（Calmodulin binding protein 60）is a transcription factor family unique to plants.There are eight members in Arabidopsis thaliana,CBP60a-g and SARD1.The function of CBP60a,CBP60g and SARD1 in the immunity has been reported.Previous work in our laboratory has shown that CBP60b-f can bind to promoter of key immune genes to activate their expression and CBP60b-f are positive regulators of plant immune pathways.Based on searching databases and phylogenetic analysis revealed that the CBP60ortholog was the first to appear in bryophytes,belonging to the clade CBP60b-f.Whether CBP60b-f is functionally conservative during evolution has not been reported.In this study,we performed functional analysis of CBP60 in Physcomitrella patens and Selaginella moellendorffii as well as two cash crops,cucumber and tomato.We found that the CBP60b-f clade can participate in plant immune response functionally conserved in different species.The main results and conclusions are as follows:（1）CBP60 gene is functionally conserved in evolutionFirstly,we searched the database of CBP60 homologous genes of different species,and found that it first appeared in Bryophyta.Then,through phylogenetic analysis,we found that CBP60 in the Physcomitrella patens and Selaginella moellendorffii belongs to the clade CBP60b-f.We selected XP＿024367012.1（PpCBP60b）in Physcomitrella patens and EFJ05811.1（Sm CBP60b）in Selaginella moellendorffii as research objects.We observed the localization of PpCBP60b and Sm CBP60b in the mesophyll protoplasts of Arabidopsis and found that both PpCBP60b and Sm CBP60b were located in the nucleus.Subsequently,we found that PpCBP60b and Sm CBP60b can interact with Ca M.We then conducted LUC transcriptional activation experiments to verify that PpCBP60b and Sm CBP60b could activate the expressions of immune marker genes SID2 and EDS1.Finally,PpCBP60b and Sm CBP60b were transformed into Arabidopsis cbp60b and it was found that PpCBP60b and Sm CBP60b could complement the autoimmune phenotype of cbp60b.These results indicate that the prototypical CBP60 gene is functionally conserved in evolution.（2）The homologous genes of CBP60b-f clade in tomato and cucumber are involved in immune regulationBy searching the database,we found that there are multiple CBP60b homologous genes in tomato and cucumber.In order to explore whether CBP60 gene in different higher plants can participate in immune response,we selected Solyc07g006830（SlCBP60b）in tomato and Csa V3＿1G031390（CsCBP60b）in cucumber for study.Similarly,we first observed the localization of SlCBP60b and CsCBP60b in Arabidopsis mesophyll protoplasts,and found that both SlCBP60b and CsCBP60b were located in the nucleus.And then we found that SlCBP60b and CsCBP60b can bind Ca M.Moreover,LUC transcriptional activation experiment verified that SlCBP60b and CsCBP60b could activate the expressions of immune marker genes SID2and EDS1.The key site of DNA binding domain or Ca M binding domain of SlCBP60b was mutated.It was found that SlCBP60b△^204-208 could not activate the expression of immune genes,and SlCBP60b^W616P showed a decreased ability to activate immune genes.Finally,when SlCBP60b and CsCBP60b were transformed to Arabidopsis cbp60b,it was found that they could complement the autoimmune phenotype of cbp60b.Meanwhile,we found that overexpression of SlCBP60b and CsCBP60b could enhance plant resistance to pathogen Pto DC3000.These results demonstrated that CBP60b as a transcription factor functionally activates immune gene expression and CBP60b may participate in the immune response process by responding to Ca²⁺signal at the protein level.In conclusion,the function of CBP60 homologous genes are analyzed by bioinformatics,molecular biology and genetics in this paper.Our study shows that the protein structure and function of CBP60 are relatively conserved during evolution and can participate in plant immune regulation in higher plants.