A sugarcane mosaic virusvector for gene expression in maize.

Yu Mei,John H Hill,Guanjun Liu,Steven A Whitham,Chunquan Zhang

doi:10.1002/pld3.158

Abstract

Zea mays L. ssp. mays (maize) is an important crop plant as well as model system for genetics and plant biology. The ability to select among different virus‐based platforms for transient gene silencing or protein expression experiments is expected to facilitate studies of gene function in maize and complement experiments with stable transgenes. Here, we describe the development of a sugarcane mosaic virus (SCMV) vector for the purpose of protein expression in maize. An infectious SCMV cDNA clone was constructed, and heterologous genetic elements were placed between the protein 1 (P1) and helper component‐proteinase (HC‐Pro) cistrons in the SCMV genome. Recombinant SCMV clones engineered to express green fluorescent protein (GFP), β‐glucuronidase (GUS), or bialaphos resistance (BAR) protein were introduced into sweet corn (Golden × Bantam) plants. Documentation of developmental time courses spanning maize growth from seedling to tasseling showed that the SCMV genome tolerates insertion of foreign sequences of at least 1,809 nucleotides at the P1/HC‐Pro junction. Analysis of insert stability showed that the integrity of GFP and BAR coding sequences was maintained longer than that of the much larger GUS coding sequence. The SCMV isolate from which the expression vector is derived is able to infect several important maize inbred lines, suggesting that this SCMV vector has potential to be a valuable tool for gene functional analysis in a broad range of experimentally important maize genotypes.

Highlights

Virus‐based expression vectors are used to transiently and rap‐ idly express a wide range of recombinant proteins in plants (Gleba, Klimyuk, & Marillonnet, 2007)
The mature viral proteins occur in the following order in the viral polypro‐ tein: protein 1 (P1), helper component‐proteinase (HC‐Pro), protein 3 (P3), 6 kilo dalton 1 (6K1), cylindrical inclusion (CI), 6 kilo dalton 2 (6K2), viral protein genome‐linked (VPg), nuclear inclusion proteinase a (NIa‐Pro), nuclear inclusion b (NIb), and capsid protein (CP)
We report the development of a full‐length sugarcane mosaic virus (SCMV) infectious clone and its modifications for gene expression in maize

Summary

| INTRODUCTION

Many viruses that infect dicot plants and belong to the Potyvirus genus have been engineered to express foreign proteins. Cloning sites using the P1/HC‐Pro junction are engineered im‐ mediately after the cleavage site, which results in cleavage of the P1 C‐terminus from the N‐terminus of the foreign protein. A seven amino acid NIa‐Pro cleavage site is added after the cloning site to process the C‐terminus of the foreign protein away from the N‐terminus of HC‐Pro (Carrington, Haldeman, Dolja, & Restrepo‐Hartwig, 1993). The viral genome was placed under control of the cauliflower mosaic virus 35S promoter (P35S) and the nopaline synthase terminator (Tnos), and the SCMV cDNA clone was modified to systemically express proteins from the P1/HC‐Pro junction in maize plants. The ability of SCMV to express foreign proteins was tested using green fluorescent protein (GFP), β‐glucuronidase (GUS), and bialaphos re‐ sistance (BAR) protein

| MATERIALS AND METHODS

| DISCUSSION

CONFLICT OF INTEREST