Characterization of cDNAs encoding two distinct miraculin-like proteins and stress-related modulation of the corresponding mRNAs in Citrus jambhiri Lush

Two distinct full-length cDNAs from rough lemon that encoded miraculin-like proteins were isolated by random amplification of cDNA ends (RACEs), based on sequence information from subtractive PCR previously described, and designated as RlemMLP1 and RlemMLP2. The transcripts of both RlemMLP1 and RlemMLP2 were not detected in leaves, or stems but accumulated in fruits. Transcripts accumulated to higher levels in leaves after wounding, inoculation with conidia of Alternaria alternata, or treatment with methyl jasmonate vapors. Treatment with methyl salicylate antagonized the signaling pathway of wounding. Treatment with methyl salicylate at 2 h after wounding significantly reduced wounding-induced gene expression of both RlemMLP1 and RlemMLP2. Protein products of these genes were obtained by using a prokaryotic expression system, and had protease inhibitor activity. RlemMLP2, but not RlemMLP1, contained a thaumatin motif, and only RlemMLP2 showed anti-fungal activity against Alternaria citri. Cellular localization analysis with RlemMLP1 or RlemMLP2 fused to a green fluorescence protein gene following transient translation using a particle bombardment in onion cells indicated that both RlemMLP1 and RlemMLP2 were localized to the cytosol. These evidences revealed that rough lemon RlemMLPs are likely to have defensive function against pathogens at least when host cells are broken by their infections.