Researchers from the National Institute for Biotechnology and Genetic Engineering in Pakistan this month reported on the development of an artificial microRNA-based approach for creating pathogen resistance in cotton.
However, the approach was not able to create immunity in the plants, indicating that further work needs to be done.
In Pakistan and surrounding nations, cotton leaf curl disease, or CLCuD, has become a significant constraint on cotton production, according to the team. The disease is caused by monopartite begomoviruses, including cotton leaf curl Burewala virus.
In 2006, a team led by Rockefeller University investigators reported on the use of artificial miRNAs, or amiRNAs, to confer virus resistance in Arabidopsis thaliana. They modified the precursor to one of the plant’s miRNAs to express an artificial miRNA targeting viral mRNA sequences encoding two gene-silencing suppressors, and found that transgenic plants expressing these constructs were resistant to specific viruses.
And this year, a group from India described the creation of transgenic tomato plants expressing amiRNAs that silence key proteins of the bipartite begomovirus tomato leaf curl New Delhi virus, resulting in tolerance to infection.
Encouraged by these and other data, the Pakistani researchers aimed to use this approach to generate resistance against CLCuD — the first time amiRNAs have been used against a monopartite begomovirus, they wrote in Virology Journal.
They created two amiRNAs, both of which were based on the sequence of pre-miRNA-169a with the 21 nucleotides that form the mature miRNA being replaced with the sequence of the V2 gene of cotton leaf curl Burewala virus. The first, called P1C, was not changed further, while the other, called P1D, included alterations to the miR-169a backbone to restore some of the hydrogen bonding of the mature duplex.
The constructs were then transformed into Nicotiana benthamiana by agrobacterium-mediated transformation.
During experimentation, non-transgenic N. benthamiana plants that were inoculated with cotton leaf curl Burewala virus displayed early symptoms of infection — the downward curling of newly developing leaves — at 15 days. By 28 days, all these plants showed signs of infection.
Inoculated plants containing the P1C amiRNAs also showed “very mild” symptoms of infection at day 15, but these did not develop into severe symptoms, the team reported. P1D trangenic plants also showed the first mild symptoms of infection 15 days after inoculation, but these too failed to develop severe symptoms.
The scientists also tested the resistance of amiRNA-altered plants against cotton leaf curl Kokhran virus, and found that none of the P1C plants developed symptoms of the disease. However, the first symptoms of infection were visible on a small number of transgenic plants harboring P1D amiRNAs 21 days after inoculation, and the majority of plants showed severe symptoms at 28 days.
The team further examined challenges by Pedilanthus leaf curl virus, and found that both transgenic N. benthamiana plants experienced severe symptoms around three weeks after inoculation. Similarly, amiRNA plants infected with tomato leaf curl New Delhi virus developed symptoms of full infection on par with non-transgenic plants.
“The results show that the amiRNA approach can deliver efficient resistance in plants against a monopartite begomoviruses and that this has the potential to be broad-spectrum, providing protection from a number of viruses,” the researchers concluded in their paper.
However, even in cases where symptoms of infection were abrogated, viral DNA was still detected, indicating that amiRNA treatment was unable to provide immunity. As a result, “this approach is unlikely to yield a durable response,” they cautioned. “Although virus levels are low in transgenic plants, the presence of replicating virus would allow mutations in the target sequence to accumulate leading, eventually, to a virus able to overcome the resistance.”
To overcome this issue, the investigators suggested that the amiRNAs could be designed to contain sequences that are more essential to the virus than the ones they tested, such as the sequences of active sites of virus encoded proteins which are likely to prove less likely to change by mutation.
“Another possibility is to target both complementary and virion-sense sequences by multiple amiRNAs,” they added. The amiRNA resistance may be “pyramided with additional resistances that act by distinct mechanisms, making it less likely that mutation will lead to susceptibility.”