By 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. 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, 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 first tested a variety of rep mutants in the MSV-susceptible grass,
Digitaria sanguinalis, which is easier to transform than maize. 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, was
bombarded into Hi-II maize (a non-commercial variety developed for relative
ease of transformation), and T2 plants were seed tested for MSV resistance.

In the initial screen for resistance, 110 T2 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. Based on three criteria-no obvious phenotypic side
effects, fertility, and likely MSV-resistance-T3 seed resulting from
self-pollination of two T2 parents were selected for further trials.

In this second screen, 50 T3 and 20 non-transgenic 3-day old seedlings
were infected with MSV. Resistance phenotypes amongst the transgenics
included significantly lower infection rates, higher survival rates, and
attenuated symptoms. Significantly 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 significantly
taller than non-transgenics (17 cm ? 2.4) at 28 days post-inoculation.

To test the efficacy of our transgenic resistance mechanism in a more
agriculturally relevant genetic background, a highly MSV-susceptible elite
white maize genotype, WM3, was crossed with one of the transgenic Hi-II
lines. Hybrid offspring were challenged at 14 days old using
leafhopper-transmission of an extremely severe MSV field isolate. The
cicadellid leafhopper Cicadulina mbila is the natural insect vector of MSV.
In two initial challenges, only plants from hybrid populations were
completely resistant, whereas all non-transgenic controls were sensitive.

To further investigate the association between resistance and our
transgene in these hybrids, we challenged 58 transgenic and 24
non-transgenic seedlings a third time and extracted genomic DNA and RNA pre-
and post-infection. We also recorded symptom severities for each plant 28
days post infection. The transgenics had significantly lower infection rates
than the controls. In addition, symptoms in infected transgenics were
significantly milder than in the non-transgenic plants. Chlorotic leaf areas
were reduced by a factor of between 6 and 12 relative to the
non-transgenics. Resistance phenotypes included no symptom development at
all, or a delayed symptom development often associated with milder symptoms.
A third resistance phenotype had mild symptoms. Combining data for the three
resistance phenotypes, 90% of transgenic plants showed some resistance
compared with 29% of non-transgenics. Importantly, the transgene transcript
was detected in all transgenics both pre- and post-infection.

To gain insight into possible yield differences between the
MSV-infected transgenic and non-transgenic groups, challenged greenhouse
plants that reached maturity were self pollinated. While 62% of challenged
transgenics reached the flowering stage, only 15% of control plants
flowered. The rest of the plants either died before reaching maturity or
never developed tassels. Interestingly, 48% of symptomatic transgenics grew
to maturity, and 21% yielded seed compared with less than 6% of the control
plants. While yields were low due to non-ideal greenhouse conditions, these
data show that when symptomatically infected with MSV almost 4-fold more
transgenic plants than control plants yielded seed.

In conclusion, we have successfully developed both the world?s first
maize with transgenic MSV resistance, and, to our knowledge, the first
all-African produced GE crop plant. According to Sinha, our MSV-resistant
maize is also the first GE crop developed wholly by a developing country. We
have shown that the resistance is inherited until the T3 generation and have
subsequently confirmed that the transgene is stably inherited and expressed
up to the T4 generation (data not shown). We have also shown that
single-gene resistance can be transferred to an elite maize breeding line,
an achievement that should greatly simplify strategies for dissemination of
the trait.


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