Gene transcriptional pattern, prokaryotic expression and functional analysis of an apoplastic, hydrophobic and small effector SCR82 from Phytophthora capsici

Abstract:

[Objective] The aim was to analyze the gene transcriptional profiles, express the protein in Escherichia coli and analyze functions of the PcF/SCR effector SCR82 from Phytophthora capsici. [Methods] Gene transcripts during the developmental stages (mycelia, sporangia, zoospores and germinated cysts) and infection period (1.5, 3, 6, 12, 24, 36, 72 h post-inoculation) of Nicotiana benthamiana by P. capsici were checked by semi-quantitative RT-PCR. The cDNA full length was cloned into a modified pET28a(+) expression vector in a way that a 6×His-SUMO tag was fused with the protein N-terminal. Protein was induced using the E. coli isolate Rossette 2 at two temperatures (22℃ and 37℃) combined with different IPTG concentrations (0.1, 0.2, 0.5, 1 mmol/L). Protein was purified by Ni²⁺ column affinity chromatography and infiltrated into the leaves of N. benthamiana and Arabidopsis thaliana to test its ability of inducing plant cell death. Meanwhile, the expression changes of three resistance-related marker genes (NbMC1, NbSOD and NbPOX) of N. benthamiana was examined at 12 and 24 h post-infiltration of the recombinant protein. [Results] The expression of scr82 was upregulated during the cyst germination and host infection. The gene was single-copy without any introns and its open reading frame was 249-bp long. The deduced protein was of 82 amino acids consisting of a 21-aa signal peptide and 7 cysteines. It was predicted to be hydrophobic and contain no known domains. The secondary structure consisted of random coil and α-helix. Its homologues existed only in Phytophthora spp. After inducing by 0.2 mmol/L IPTG at 22℃ for about 16 h, the recombinant protein of about 24 kDa was observed on SDS-PAGE gels. About 30 mg/mL of purified protein was obtained. The recombinant did not trigger plant cell death, but caused up-regulated expression of NbMC1, NbSOD and NbPOX genes. [Conclusion] The recombinant could not induce plant cell death, but triggered plant defense reaction.

Key words:Phytophthora capsici;PcF/SCR effector;hydrophobicity;RT-PCR;prokaryotic expression;plant defense reaction