In Silico Analyses of Rice Thionin Genes and the Antimicrobial Activity of OsTHION15 Against Phytopathogens.

Thionins are a family of antimicrobial peptides. We performed in silico expression analyses of the 44 rice (Oryza sativa) thionins (OsTHIONs). Modulated expression levels of OsTHIONs under different treatments suggest their involvement in many processes, including biotic, abiotic, and nutritional stress responses, and in hormone signaling. OsTHION15 (LOC_Os06g32600) was selected for further characterization based on several in silico analyses. OsTHION15 in O. sativa subsp. indica 'KDML 105' was expressed in all of the tissues and organs examined, including germinating seed, leaves, and roots of seedlings and mature plants, and inflorescences. To investigate the antimicrobial activity of OsTHION15, we produced a recombinant peptide in Escherichia coli Rosetta-gami (DE3). The recombinant OsTHION15 exhibited inhibitory activities toward rice-pathogenic bacteria such as Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum pv. atroseptica, with minimum inhibitory concentrations of 112.6 and 14.1 µg ml-1, respectively. A significant hyphal growth inhibition was also observed toward Fusarium oxysporum f. sp. cubense and Helminthosporium oryzae. In addition, we demonstrated the in planta antibacterial activity of this peptide in Nicotiana benthamiana against X. campestris pv. glycines. These activities suggest the possible application of OsTHION15 in plant disease control.

Heterologous expression and antimicrobial activity of OsGASR3 from rice (Oryza sativa L.).

According to an in silico analysis, OsGASR3 (LOC_Os03g55290) from rice (Oryza sativa L.) was predicted to be involved in plant defense mechanisms. A semi-quantitative reverse transcription polymerase chain reaction assay revealed that OsGASR3 is highly expressed in the inflorescences of Thai jasmine rice (O. sativa L. subsp. indica 'KDML 105'). To characterize the biological activity of OsGASR3, we produced an OsGASR3-glutathione S-transferase fusion protein in Escherichia coli Rosetta-gami (DE3) cells for a final purified recombinant OsGASR3 yield of 0.65 mg/L. The purified OsGASR3 inhibited the hyphal growth of Fusarium oxysporum f.sp. cubense and Helminthosporium oryzae at a relatively low concentration (7.5 µg/mL). Furthermore, OsGASR3 exhibited in planta inhibitory activity against Xanthomonas campestris, suggesting its involvement in defense mechanisms, in addition to its previously reported functions affecting growth and development. These observations indicate that recombinant OsGASR3 may be useful for protecting agriculturally important crops against pathogenic microbes.