Expression Analysis of Hairpin RNA Carrying Sugarcane mosaic virus (SCMV) Derived Sequences and Transgenic Resistance Development in a Model Rice Plant.

Sehrish Akbar,Muhammad Tahir,Qing Liu,Ming-Bo Wang

doi:10.1155/2017/1646140

Sehrish Akbar, Muhammad Tahir + Show 2 more

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https://doi.org/10.1155/2017/1646140

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Abstract

Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research. Sugarcane mosaic virus (SCMV) is a serious pathogen of many monocotyledonous crops including sugarcane. The objective of present study was to analyze transgenic expression of hairpin RNA (hpRNA), targeting simultaneously CP (Coat Protein) and Hc-Pro (helper component-proteinase) genes of SCMV, in a model rice plant. Conserved nucleotide sequences, exclusive for DAG (Aspartic acid-Alanine-Glycine) and KITC (Lycine-Isoleucine-Threonine-Cysteine) motifs, derived from SCMV CP and Hc-Pro genes, respectively, were fused together and assembled into the hpRNA cassette under maize ubiquitin promoter to form Ubi-hpCP:Hc-Pro construct. The same CP:Hc-Pro sequence was fused with the β-glucuronidase gene (GUS) at the 3′ end under CaMV 35S promoter to develop 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. When delivered into rice callus tissues by particle bombardment, the Ubi-hpCP:Hc-Pro construct induced strong silencing of 35S-GUS:CP:Hc-Pro. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21–24 nt small interfering RNAs, which induced specific suppression against GUS:CP:Hc-Pro delivered by particle bombardment and conferred strong resistance to mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane.

Highlights

Sugarcane mosaic virus (SCMV), belonging to genus Potyvirus, family Potyviridae, is a serious pathogen of many monocotyledonous crops including sugarcane
This paper reports the expression analysis of hairpin RNA (hpRNA), targeting simultaneously coat protein (CP) and Hc-Pro genes of SCMV, in a model rice plant
The selected CP and Hc-Pro gene sequences were subsequently joined together to form a chimera sequence (CP:Hc-Pro) which was arranged in an inverted repeat configuration in the hpRNA construct and successfully cloned under the maize ubiquitin promoter (Ubi), to develop Ubi-hpCP:HcPro expression construct (Figure 1(a))

Summary

Introduction

Sugarcane mosaic virus (SCMV), belonging to genus Potyvirus, family Potyviridae, is a serious pathogen of many monocotyledonous crops including sugarcane. Infected plants showed mosaic pattern and are characterized by typical pinwheel shaped inclusion bodies in cell cytoplasm. The 10 kb single stranded RNA genome of SCMV encodes a single polypeptide which is cleaved either co- or post translationally into ten mature proteins (P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb, and CP) [2, 3]. Hc-Pro is a multifunctional protein and a proteinase, which is responsible for the multiplication of virus genome, systemic virus movement [5], and suppression of plant RNA silencing machinery [6] by interacting with numerous host factors

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