Downloaded from www.microbiologyresearch.org by IP: 54.146.78.28 On: Sat, 03 Dec 2016 07:56:05 Inhibition of maize streak virus (MSV) replication by transient and transgenic expression of MSV replication-associated protein mutants Dionne N. Shepherd, 1 Tichaona Mangwende, 1,2 Darren P. Martin, 3 Marion Bezuidenhout, 1 Jennifer A. Thomson 1 3 and Edward P. Rybicki 1,3 3 Correspondence Edward P. Rybicki ed@science.uct.ac.za 1 Department of Molecular and Cell Biology, University of Cape Town, Private Bag, Rondebosch, Cape Town 7701, South Africa 2 Division of Pharmacology, University of Cape Town, Cape Town, South Africa 3 Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, Anzio Road, Cape Town 7925, South Africa Received 28 June 2006 Accepted 7 September 2006 Maize streak disease is a severe agricultural problem in Africa and the development of maize genotypes resistant to the causal agent, Maize streak virus (MSV), is a priority. A transgenic approach to engineering MSV-resistant maize was developed and tested in this study. A pathogen- derived resistance strategy was adopted by using targeted deletions and nucleotide-substitution mutants of the multifunctional MSV replication-associated protein gene (rep). Various rep gene constructs were tested for their efficacy in limiting replication of wild-type MSV by co-bombardment of maize suspension cells together with an infectious genomic clone of MSV and assaying replicative forms of DNA by quantitative PCR. Digitaria sanguinalis, an MSV-sensitive grass species used as a model monocot, was then transformed with constructs that had inhibited virus replication in the transient-expression system. Challenge experiments using leafhopper-transmitted MSV indicated significant MSV resistance – from highly resistant to immune – in regenerated transgenic D. sanguinalis lines. Whereas regenerated lines containing a mutated full-length rep gene displayed developmental and growth defects, those containing a truncated rep gene both were fertile and displayed no growth defects, making the truncated gene a suitable candidate for the development of transgenic MSV-resistant maize. INTRODUCTION Maize (Zea mays L.) is Africa’s most important staple food crop and is the mainstay of most of the continent’s rural economies. Despite this, mean maize yields per hectare in Africa are extremely low compared with the developed world and, as a result, food shortages are a perpetual problem in most sub-Saharan countries (see www.fao.org and www.wfp.org). Whilst lack of access to modern farming techniques and provisions are the main reasons for low yields, maize pathogen epidemics can decrease yields further. Of the many viral pathogens infecting maize, the leafhopper-vectored Maize streak virus (MSV; family Geminiviridae, genus Mastrevirus) is considered the most important and widespread (Thottappilly et al., 1993; Bosque-Perez, 2000). In epidemic years, maize streak disease (MSD) can result in up to 100 % yield losses (Wambugu & Wafula, 1999; Bosque-Perez, 2000). The most effective control strategy for MSD prevention is frequent application of systemic insecticides for the Cicadulina spp. vector leafhoppers; this option is not generally open to poorer farmers. Although maize genotypes with varying degrees of MSV resistance have been developed (Kim et al., 1989; Van Rensburg et al., 1991; Barrow, 1992, 1993; Rodier et al., 1995; Welz et al., 1998; Buddenhagen & Bosque-Pe ´rez, 1999; Kyetere et al., 1999), their use has not been widespread among resource-poor farmers, due either to cost or their inability to yield as productively as MSV-sensitive genotypes in non-epidemic years. MSV replicates in the nuclei of infected cells by rolling-circle replication (Saunders et al., 1991; Stenger et al., 1991) and possibly via recombination-dependent mechanisms (Jeske et al., 2001). Rolling-circle replication is initiated by binding of the virus replication-associated protein (Rep) to the virion-strand origin of replication, where the protein initiates and terminates virion-strand DNA synthesis. MSV Rep is the translation product of two complemen- tary-sense open reading frames (ORFs), C1 and C2. The C1– C2 transcript, which contains an intron, is translated to yield either Rep (from the spliced transcript) or RepA (from the unspliced transcript). Rep and RepA share several distinct 3These authors contributed equally to this work. 0008-2338 G 2007 SGM Printed in Great Britain 325 Journal of General Virology (2007), 88, 325–336 DOI 10.1099/vir.0.82338-0