The Interaction Between Bip130 And OSA7 Regulates The Activity Of Plasma Membrane H~+-ATPase In Rice

bip130(BR11-interacting protein 130)is a putative protein interacting with the receptor protein BRI1 of brassinolides(BRs),identified first from a direct phosphorylation screening of a rice cDNA library.Previous studies have shown that bip130 is involved in the antioxidant defense induced by the plant hormone abscisic acid(ABA),and enhances crop tolerance to drought,salt stress,and oxidative stress.However,the molecular mechanism by which bip130 regulates antioxidant defense in ABA signaling remains to be determined.In order to clarify the mechanism of action of bip130,bip130 was used as a bait to screen a rice cDNA library using yeast twohybrid(Y2H)in our laboratory,and a putative bip130-interacting protein,OSA7,was identified.OSA7 is a member of the plasma membrane H+-ATPase family in rice.The plasma membrane H+-ATPase has been shown to play an important role in plant growth and development and in plant response to environmental stresses.In this study,we validated the interaction between bip130 and OSA7 in vivo and in vitro,and then analyzed which domain of OSA7 interacts with bip130.Finally,we investigated the effect of their interaction on the activity of plasma membrane H+-ATPase induced by ABA,drought,and salt stress.The main results of this paper are as follows:Using glutathione S-transferase(GST)pull-down assay,Y2H assay,firefly luciferase complementary imaging(LCI)assay,and bimolecular fluorescence complementary analysis(BiFC)assay,we confirmed the interaction between bip130 and OSA7 in vivo and in vitro.Analysis of transient expression of rice protoplasts revealed that OSA7 was localized in the plasma membrane,while bip130 has been shown to be localized in the plasma membrane,the cytoplasm and the nucleus,which spatially indicates the possibility of interaction between them.In order to further reveal which domain of OSA7 interacts with bip130,the protein sequence and structure of OSA7 were analyzed by bioinformatics,and OSA7 was truncated into 4 different functional domains,namely transmembrane domain,A domain superfamily,and haloacid dehydrogenase(HAD)superfamily,cytoplasmic domain N domain.Y2H and LCI assays showed that the cytoplasmic domain N domain of OSA7 interacted with bip130.Further,compared with the homologous family protein of OSA7,5 homologous protein OSA1～5 with high homology to OSA7 were selected,and their cytoplasmic domain N domain was cloned.Y2H and LCI analyses showed that the cytoplasmic domain N domain of these five homologous proteins did not interact with bip 130.In order to explore whether the interaction of bip130 and OSA7 is involved in the response of rice plants to salt stress,we first analyzed the effects of ABA and NaCl treatments on the expression of OSA7 and the activity of plasma membrane H+ATPase,qRT-PCR was used to analyze the upstream and downstream relationship between bip130 and OSA7 during ABA treatment,and then used bip130 overexpression and interference(RNAi)mutants to analyze its effect on the activity of plasma membrane H+-ATPase under the stress conditions.Finally,the protoplast transient expression system was uesed to detect the activity of plasma membrane H+-ATPase after transient expression and silencing of OSA7 in the bip 130 mutant Our results showed that the treatments with ABA and NaCl induce the increases in the expression of OSA 7 and the activity of plasma membrane H+-ATPase in rice roots.In the ABA signaling pathway,bip130 is located upstream of OSA 7;Mutant analysis showed that bip130 overexpression further enhanced salt stress-induced plasma membrane H+ATPase activity,while bip130-RNAi prevented salt stress-induced increase in the activity of plasma membrane H+-ATPase.The analysis of the protoplast transient expression system showed that bip 130 can regulate OSA7 activity in the ABA signaling pathway.These results imply that bip130 is involved in the regulation of plasma membrane H+-ATPase activity under salt stress.This study provides a basis for further research on the mechanism of action of bip130 under stress conditions.