Inhibition of maize streak virus (MSV) replication by transient and transgenic expression of MSV replication-associated protein mutants

Abstract

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 pathogenderived 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.

The Use of African Indigenous Genes in the Development of Transgenic Maize Tolerant to Drought and Resistant to Maize Streak Virus

“…As discussed above, although several mutant MSV rep constructs were shown to inhibit virus replication in transient BMS assays and in transgenic D. sanguinalis, only one, prep 1-219Rb-, which retained amino acids 1-219 having had the C terminus deleted, and had a mutation in the pRBR binding domain, resulted in phenotypically normal, fertile, MSV-resistant plants (Shepherd et al 2007a). The construct cloned into the plant vector pAHC25 (Christensen and Quail 1996) was transformed into maize Hi-II.…”

A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay

“…Despite the fact that, theoretically, an antisense transcript could form dsRNA with the sense transcript from the virus, it has been shown previously that antisense MSV rep is ineffective at reducing viral loads, even when expressed from the maize ubiquitin promoter (Shepherd et al, 2007a).…”

“…This system has been used previously to screen for other MSV resistance genes (Shepherd et al, 2007a), as well as to determine the size of the MSV minimal replication origin (Willment et al, 2007).…”

“…One such model system that has proven to be an invaluable tool for evaluating and comparing different MSV resistance strategies utilizes transient expression of transgenes by black Mexican sweet (BMS) cells in suspension culture (Shepherd et al, 2005(Shepherd et al, , 2007a, combined with quantification by real-time PCR of MSV DNA accumulation during transient infection. In light of the growing need to diversify sources of maize streak disease resistance (reviewed by Shepherd et al, 2010), we evaluated the efficacy of two potentially silencing-inducing hairpin RNA constructs (i.e.…”

Reaction of Waxy and Opaque-2 Inbreds and their Derived Progenies to Multiple Foliar Diseases of Maize in Uganda

“…= Helminthosporium turcicum Pass.) and maize streak virus disease (MSVD) caused by Maize streak mastrevirus are major foliar diseases of maize that critically curtail production in sub-Saharan Africa (Adipala, Lipps, & Madden, 1993;Okori, Rubaihayo, Adipala, Fahleson, & Dixelius, 2004;Shepherd et al, 2007;Ward, Stromberg, Nowel, & Nutter, 1999). Losses from these diseases may exceed 30% depending on environment and host genotype resistance (Bosque-Perez, 2000;Raymundo & Hooker, 1981;Ward et al, 1999) and can occur simultaneously in a single crop resulting in severe yield losses.…”