ISB News Report OCTOBER 2007 Maize Streak Virus Resistant Transgenic Maize: A First for Africa Dionne N Shepherd, Edward P Rybicki, & Jennifer A Thomson Maize is Africa’s staple food crop, comprising more than 50% of the total caloric intake in local diets. 1 However, average maize yields of 1.2 tons per hectare are just a quarter of global averages (http://faostat.fao.org), a disparity exacerbated by the susceptibility of the crop to pathogens. Maize streak disease (MSD), caused by the geminivirus maize streak virus (MSV), is the major viral pathogenic constraint on maize production in Africa, 2,3 making resistance to MSV a key target for crop improvement. Conventional breeding for the trait, however, is complicated because there is 1) more than one gene involved and 2) an association of undesirable traits with resistance. There are numerous reports of genetically engineered virus resistance in crops, mostly derived from the introduction of coat protein genes for RNA viruses. Geminiviruses have DNA genomes, so coat protein resistance is less likely to be successful due to fundamental differences in the ways viruses replicate. Accordingly, we have used dominant negative mutants of the MSV replication-associated protein gene (rep) to develop resistance in maize. The multifunctional Rep protein is essential to viral replication. Rep is required early in the viral lifecycle and is a limiting factor because it is expressed at low levels. Rep functions as an oligomer, making it an ideal pathogen-derived resistance target. If target plants constitutively express mutant forms of the rep gene, incoming viruses will have their Rep proteins “swamped” by the mutants, which will render the invading viruses incapable of replicating MSV DNA. We irst tested a variety of rep mutants in the MSV-susceptible grass, Digitaria sanguinalis, which is easier to transform than maize. 4 Although a number of mutant reps inhibited MSV replication, only one resulted in phenotypically normal, fertile MSV-resistant plants. This was a C-terminal truncated Rep with a mutation in the retinoblastoma-related protein (pRBR) interaction motif. An intact pRBR interaction motif creates a cellular environment permissive for virus replication and interferes with plant cell development; therefore, it was essential to render pRBR non-functional. The construct, whose expression was driven by the maize ubiquitin promoter, 5 was bombarded into Hi-II maize (a non- commercial variety developed for relative ease of transformation), and T 2 plants were seed tested for MSV resistance. In the initial screen for resistance, 110 T 2 plants and 50 non-transgenic controls were tested for MSV resistance by infection of 3-day old seedlings: in general, the younger the maize at the time of virus inoculation, the more susceptible the plant is. All trials were blind, with transgene presence/absence only determined by PCR following symptom analysis. Percentages of chlorotic leaf areas in infected plants were estimated using both a visual key and a microcomputer-assisted image analysis technique. 6,7 Based on three criteria—no obvious phenotypic side effects, fertility, and likely MSV-resistance—T 3 seed resulting from self-pollination of two T 2 parents were selected for further trials. In this second screen, 50 T 3 and 20 non-transgenic 3-day old seedlings were infected with MSV. Resistance phenotypes amongst the transgenics included signiicantly lower infection rates, higher survival rates, and attenuated symptoms (Fig. 1). Signiicantly delayed symptom development was evidenced by a reduction in chlorotic leaf areas by a factor of 61 in transgenics compared to non-transgenic plants. Transgenic plants were also signiicantly taller than non-transgenics (17 cm ± 2.4) at 28 days post-inoculation. 0 20 40 60 80 100 20 25 30 35 40 45 50 Day pi Percentage alive 0 20 40 60 80 100 0 5 10 15 20 25 30 Day pi Percantage infected Transgenic Non-Transgenic a b c d Transgenic Non-Transgenic Figure 1. MSV resistance in challenged T 3 transgenic Hi-II maize. (a) Transgenics (n = 47) become symptomatic at a signiicantly lower rate than non-transgenics (n = 18; P = 0.0047; Mann Whitney u-test). (b) Transgenics have a signiicantly higher survival rate than non-transgenics between 1 and 50 days post inoculation (dpi) (P<0.0001; Kaplan-Meier log rank test). (c-d) Comparison of non-symptomatic (c) and symptomatic (d) transgenic plants with non-transgenics at 20 dpi.