Dual gene expression cassette is superior than single gene cassette for enhancing sheath blight tolerance in transgenic rice

Subhasis Karmakar,Swapan K Datta,Kutubuddin A Molla,Karabi Datta,Kaushik Das,Sailendra Nath Sarkar

doi:10.1038/s41598-017-08180-x

Subhasis Karmakar, Swapan K Datta + Show 4 more

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https://doi.org/10.1038/s41598-017-08180-x

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Abstract

Sheath blight, caused by the necrotrophic fungal pathogen Rhizoctonia solani, is a serious and destructive disease of the rice. In order to improve sheath blight resistance, we developed three different kinds of transgenic rice lines. The first transgenic line overexpresses the rice chitinase gene (OsCHI11); the second contains the Arabidopsis NPR1 (AtNPR1) gene and, the third has pyramided constructs with both the genes (OsCHI11 and AtNPR1). This is a comparative study between the single-gene transgenic lines and the double gene transgenic in terms of their ability to activate the plant defense system. Rice plants of each individual construct were screened via PCR, Southern hybridization, activity assays, and expression analysis. The best transgenic lines of each construct were chosen for comparative study. The fold change in qRT-PCR and activity assays revealed that the pyramided transgenic rice plants show a significant upregulation of defense-related genes, PR genes, and antioxidant marker genes as compared to the single transgene. Simultaneous co-expression of both the genes was found to be more efficient in tolerating oxidative stress. In R. solani (RS) toxin assay, mycelial agar disc bioassay, and in vivo plant bioassay, pyramided transgenic plant lines were more competent at restricting the pathogen development and enhancing sheath blight tolerance as compared to single gene transformants.

Highlights

Rice (Oryza sativa L.), being the most important staple food crop, is a major nutritional food supplement for more than half of the global population
PCR analysis of the T2 transgenic lines, using partial gene specific primers of OsCHI11-AtNPR1 rice lines using gene-specific (OsCHI11) and AtNPR1 rice lines using gene-specific (AtNPR1) genes, revealed amplification of 490 bp and 1.7 kbp fragments, respectively, while no amplification was observed in non-transformed plants [Fig. 1b(I),(II),(III),(IV)]
The integration and transgene copy numbers were further examined by Southern hybridization and the transgenic lines containing a single copy of the transgene for each of the constructs were selected for further evaluation and comparison [Fig. 1c(I),(II),(III)]

Summary

Introduction

Rice (Oryza sativa L.), being the most important staple food crop, is a major nutritional food supplement for more than half of the global population. In several crop plants such as tomato, tobacco, and rice[14,15,16,17,18], AtNPR1 or its orthologs have been found to be associated with the phenomenon of conferring enhanced resistance against a wide variety of pathogens These observations strongly suggest that AtNPR1 and its homologs could serve as important candidates for providing resistance against rice sheath blight. On the basis of aforementioned studies, the inhibition of chitin metabolism in fungi such as R. solani, via the expression of rice chitinase, is a promising strategy for controlling the disease like rice sheath blight

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