Abstract
BackgroundCotton leaf curl disease, caused by single-stranded DNA viruses of the genus Begomovirus (family Geminiviridae), is a major constraint to cotton cultivation across Pakistan and north-western India. At this time only cotton varieties with moderate tolerance are available to counter the disease. microRNAs (miRNAs) are a class of endogenous small RNA molecules that play an important role in plant development, signal transduction, and response to biotic and a biotic stress. Studies have shown that miRNAs can be engineered to alter their target specificity. Such artificial miRNAs (amiRNAs) have been shown to provide resistance against plant-infecting viruses.ResultsTwo amiRNA constructs, based on the sequence of cotton miRNA169a, were produced containing 21 nt of the V2 gene sequence of Cotton leaf curl Burewala virus (CLCuBuV) and transformed into Nicotiana benthamiana. The first amiRNA construct (P1C) maintained the miR169a sequence with the exception of the replaced 21 nt whereas in the second (P1D) the sequence of the miRNA169a backbone was altered to restore some of the hydrogen bonding of the mature miRNA duplex. P1C transgenic plants showed good resistance when challenge with CLCuBV; plants being asymptomatic with low viral DNA levels. The resistance to heterologous viruses was lower and correlated with the numbers of sequence mismatches between the amiRNA and the V2 gene sequence. P1D plants showed overall poorer resistance to challenge with all viruses tested.ConclusionsThe 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. Additionally the findings indicate that the levels of resistance depend upon the levels of complementarity between the amiRNA and the target sequence and the sequence of the miRNA backbone, consistent with earlier studies.
Highlights
Cotton leaf curl disease, caused by single-stranded DNA viruses of the genus Begomovirus, is a major constraint to cotton cultivation across Pakistan and north-western India
The key features of RNA interference (RNAi) include the production of 21–25 nt small RNAs by enzymes known as Dicers [1] and the formation of RNA-induced silencing complexes (RISCs) which contain Argonaute (Ago) proteins that directly carry out gene silencing at the transcriptional or posttranscriptional levels [2,3,4,5]
Production and analysis of transgenic N. benthamiana plants harbouring artificial miRNAs (amiRNAs) The amiRNA produced were based upon the sequence of the pre-miRNA169a (Figure 1A; [13])
Summary
Cotton leaf curl disease, caused by single-stranded DNA viruses of the genus Begomovirus (family Geminiviridae), is a major constraint to cotton cultivation across Pakistan and north-western India. At this time only cotton varieties with moderate tolerance are available to counter the disease. There are two major classes of sRNAs involved in RNAi, small interfering RNAs (siRNAs) and microRNAs (miRNAs). In the Old World a small number of begomoviruses have been identified, with genomes consisting of two ssDNA circles (known as DNA A and DNA B) the majority, including CLCuBuV [8], have genomes consisting of a single component which is a homolog of the DNA A of bipartite viruses. The virion-sense strand encodes the coat protein and the V2 gene whereas genes encoded on the complementary-sense strand are involved in viral DNA replication, regulating host and viral gene expression and overcoming host defences [7,8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
