The ethylene-responsive factor like protein 1 (CaERFLP1) of hot pepper (Capsicum annuum L.) interacts in vitro with both GCC and DRE/CRT sequences with different binding affinities: possible biological roles of CaERFLP1 in response to pathogen infection and high salinity conditions in transgenic tobacco plants

Abstract

From a pathogen-inoculated hot pepper (Capsicum annuum L. cv. Pukang) leaf EST, we identified a cDNA clone, pCaERFLP1, encoding a putative transcription factor that contains a single ERF/AP2 DNA binding domain. CaERFLP1 was most closely related to tomato LeERF2 (73%), both of which belong to the novel ERF class IV typified by the N-terminal MCGGAIL signature sequence, while it had a limited sequence identity (25-30%) with Arabidopsis AtERFs and tobacco NtERFs. Quantitative gel retardation assays revealed that bacterially expressed full-length CaERFLP1 was able to form a specific complex with both the GCC box and DRE/CRT motif, with its binding affinity for GCC being stronger than for DRE/CRT. When fused to the GAL4 DNA binding domain, the N-terminal CaERFLP1(1-37) and C-terminal CaERFLP1(198-264) mutant polypeptides could function individually as transactivators in yeast. This suggests that two separate domains of CaERFLP1 may play distinct roles in transcription activation. In particle co-bombardment experiments, CaERFLP1 activated the transcription of reporter genes containing the 4X[GCC] element in tobacco cells. In hot pepper plants, the steady-state level of CaERFLP1 mRNA was markedly induced by multiple environmental factors, such as pathogen infection, ethylene, mechanical wounding and high salinity. Furthermore, ectopic expression of CaERFLP1 in transgenic tobacco plants resulted in partially improved tolerance against the bacterial pathogen Pseudomonas syringae and salt stress (100 mM NaCl). Consistently, various defense-related genes, including GCC box-containing PR genes and the DRE/CRT-containing LTI45 (ERD10) gene, were constitutively expressed in 35S::CaERFLP1 tobacco plants. Thus, it appears that CaERFLP1 is functional in tobacco cells, where it induces the transactivation of some GCC- and DRE/CRT-genes to trigger a subset of stress response. Here, the possible biological role(s) of CaERFLP1 is discussed, especially with regard to the possibility that CaERFLP1 has multiple functions in the regulation of GCC- and DRE/CRT-mediated gene expression in hot pepper plants.