Gene Expression Analysis And Recombinant Defensins Production Of Rice Oryza Sativa L.

Abstract

Antimicrobial peptides (AMPs) are small cysteine-rich peptides which generally contain less than 100 amino acid residues. They have a non-specific antimicrobial activity against bacteria and fungi by invading their cell membranes. Defensin (DEF) represents a group of AMPs in plants including rice Oryza sativa L. Previously, rice DEF7 (OsDEF7) and rice DEF8 (OsDEF8) were heterologously produced in bacteria exhibiting a growth inhibitory effect against several phytopathogens. In this study, both DEFs were cloned and analysed for gene expression patterns using quantitative RT-PCR in Thai jasmine rice, Oryza sativa L. ssp. indica cv. KDML105 comparing to the results of those Japanese rice Oryza sativa L. ssp. japonica. Although the nucleotide sequences of both genes in the two cultivars show 100% identity and similar gene expression pattern at the examined developmental stages was observed, their expression levels were shown to be different between organs. Upon Xanthomonas oryzae pv. oryzae infection in rice leaves, both OsDEFs showed high upregulation at 8 days post-infection suggesting their involvement in pathogen defense. In addition, in silico analysis also demonstrated that the expression levels of these two OsDEFs were affected by abiotic stress including drought, cold, imbibition, anoxia, and dehydration. Using GFP fusion approach, OsDEFs were demonstrated to localize to the extracellular compartment. However, the recombinant OsDEFs cannot be detected in culture medium when expressed in the hairy roots of Nicotiana benthamiana and tobacco BY-2 suspension cells. Subsequently, transiently expression of these peptides in N. benthamiana leaves was performed by agroinfiltration and in planta inhibitory activity of these proteins was tested. Even though the low levels of these proteins were detected in the leaves, both recombinant proteins exhibited in planta inhibitory activity against phytopatogens, Xanthomonas campestris pv. glycines. These results suggested that these peptides might be exploited further for disease control and N. benthamiana could be used as an alternative host for screening other antimicrobial peptides.