Molecular characterization of the gibberellin-stimulated transcript of GASA4 in Citrus

A Citrus clementina gene, CcGASA4, which is involved in the responses of citrus to stress, was characterized. The gene was induced by Citrus tristeza virus infection, wounding and gibberellic, salicylic and abscisic acid treatments. A qRT-PCR analysis showed that CcGASA4 had a very high basal expression in flowers yet was still able to be further induced independently in giberellic, salicylic and abscisic acid-treated flowers. Subcellular localization analysis revealed that the CcGASA4 protein localized to the cell membrane and nucleus. A yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC) assays showed that CcGASA4 interacted with two proteins, the large proline-rich protein bag6-A (PRPBAG6-A) and the general negative regulator of transcription subunit 3 (CNOT3). PRPBAG6 has been reported to be involved in disease resistance. Replacing some of CcGASA4's conserved cysteines with alanines (Cys → Ala) abolished the protein’s interaction with CNOT3 but did not show any effect on the protein’s interaction with PRPBAG6-A. Thus, CcGASA4 appears to play multiple roles in Citrus, probably by interacting with different proteins and/or by localizing to different subcellular compartments.