Transgenic viral resistance is short-lived while marker assisted selective breeding can offer long-term protection
Prof. Joe Cummins
Maize makes up more than 50 percent of the caloric intake of Africans; but it suffers substantial losses from pathogens. The main viral pathogen is the maize streak virus (MSV). The first transgenic maize crop to be created in Africa is one that resists MSV [1, 2].
The maize streak virus belongs to the family of Gemini viruses with small circular single stranded DNA genomes wrapped into twinned (geminate) particles. There are four Gemini virus genera, MSV is a member of the Mastrevirus genus, transmitted by specific leafhopper insects. Most Mastreviruses infect cereals and grasses but two species infect dicotyledons. The circular genomes are only two to three kilobases in size. Within host cells, the single stranded DNA genomes are converted to double stranded intermediates in the plant cell nucleus, which replicate into many single strand circular copies, by a ‘rolling circle’ mechanism. The double stranded DNA form of the genome also acts as a template for bidirectional transcription to make four RNA messages that are translated in the plant cell cytoplasm into viral proteins Rep A and Rep B for viral replication, a coat protein (Cp) and movement protein (MP).
The replication of viral DNA depends on the MSV’s ability to recruit maize enzymes and factors for DNA replication and transcription. When the virus is injected into mature non-dividing plant cells the viral protein Rep A interacts with a plant retinoblastoma-related protein which causes the mature plant cell to initiate passage through the cell cycle to DNA replication providing the enzymes and precursors for viral as well as cell DNA replication . MSV is small but clever; it recruits complex cell replication systems from the host allowing the virus to overcome its host.
The construction of MSV resistant maize involved extensive experimentation on genetic modification undertaken on a grass that could be infected by the virus and was amenable to genetic modification. The MSV rep A was a prime target for genetic modification, the aim of which was to produce genes that prevented virus replication. A number of site-specific mutations and truncations of the rep A gene were tested using the gene gun to inject multiple copies of the mutant viral genomes into the grass plants, which transiently expressed the injected viral plasmids. The mutant rep A genes that interfered with the replication of virus inoculations were then used to modify the grass. Most of the mutant rep A genes that interfered with virus replication also caused aberrations in the growth of the grass. However, one truncated rep A gene was found that interfered with the replication of MSV without causing anomalies in the growth and reproduction of the grass .
The final MSV resistant maize line was produced using a gene gun transformation of the callus from hybrid maize. The plasmids used in transformation included one with the truncated-Rep A mutant placed between the maize ubiquitin promoter and the nos terminator, and another carrying a bar selectable marker that provided resistance to glufosinate herbicides . The transformed maize showed resistance to MSV for four generations. The MSV resistant maize showed delayed symptoms and greater survival than did the unmodified maize .
MSV resistant maize has been produced, but there remain concerns about its long term stability in the face of the high mutability of MSV. MSV is typical of the single stranded RNA and DNA viruses in its high mutation rate, higher in the viral strand than in its complementary strand .
Along with its high mutation rate, MSV shows rapid host adaptation by extensive recombination . Analysis of MSV isolates in Uganda show widespread distribution of a recombinant variant .
Maize cell lines have been developed containing autonomously replicating MSV based gene vectors . Vectors based on MSV can replicate to high copy number in maize plants . The possibility that MSV could be engineered to make a gene replacement vector (gene therapy) has been discussed.
Mixtures of MSV vector and wild type virus were studied but failed to move through the plant. Complementing MSV vectors recombined to form wild type infectious virus . The replication A gene inserted into MSV resistant maize may also be affected by recombination with MSV
The high rate of mutation and recombination in MSV suggests that the genetically modified maize strains may retain their ability to resist MSV for only a few years at best and then have to be replaced with newly designed resistant strains.
There are a number of natural gene loci called quantitative trait loci (QTL) that have been identified and mapped to the maize chromosomes , and may be combined by conventional selective breeding to produce substantial resistance to the virus. Such combined resistance offers long term protection against the erosion of resistance by mutation and recombination.
In conclusion, genetic modification may offer resistance to MSV crop destruction in Africa. However that resistance may be relatively short-lived on account of rapid mutation and recombination of the virus. QTL marker assisted breeding offers long term protection of maize varieties, provided that area of research is not ignored.
Article first published 15/05/09
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