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Artificial microRNA-mediated resistance against the monopartite begomovirus Cotton leaf curl Burewala virus.

ABSTRACT: BACKGROUND: 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. 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. RESULTS: Two 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. CONCLUSIONS: 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. 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.

SUBMITTER: Ali I

PROVIDER: S-EPMC3765727 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Artificial microRNA-mediated resistance against the monopartite begomovirus Cotton leaf curl Burewala virus.

Ali Irfan I Amin Imran I Briddon Rob W RW Mansoor Shahid S

Virology journal 20130712

<h4>Background</h4>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. 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. Studie ...[more]

PMID: 23844988

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Project description:BACKGROUND: Whitefly-transmitted geminiviruses (family Geminiviridae, genus Begomovirus) are major limiting factors for the production of numerous dicotyledonous crops throughout the warmer regions of the world. In the Old World a small number of begomoviruses have genomes consisting of two components whereas the majority have single-component genomes. Most of the monopartite begomoviruses associate with satellite DNA molecules, the most important of which are the betasatellites. Cotton leaf curl disease (CLCuD) is one of the major problems for cotton production on the Indian sub-continent. Across Pakistan, CLCuD is currently associated with a single begomovirus (Cotton leaf curl Burewala virus [CLCuBuV]) and the cotton-specific betasatellite Cotton leaf curl Multan betasatellite (CLCuMuB), both of which have recombinant origins. Surprisingly, CLCuBuV lacks C2, one of the genes present in all previously characterized begomoviruses. Virus-specific transcripts have only been mapped for few begomoviruses, including one monopartite begomovirus that does not associate with betasatellites. Similarly, the transcripts of only two betasatellites have been mapped so far. The study described has investigated whether the recombination/mutation events involved in the evolution of CLCuBuV and its associated CLCuMuB have affected their transcription strategies. RESULTS: The major transcripts of CLCuBuV and its associated betasatellite (CLCuMuB) from infected Nicotiana benthamiana plants have been determined. Two complementary-sense transcripts of ~1.7 and ~0.7 kb were identified for CLCuBuV. The ~1.7 kb transcript appears similar in position and size to that of several begomoviruses and likely directs the translation of C1 and C4 proteins. Both complementary-sense transcripts can potentially direct the translation of C2 and C3 proteins. A single virion-sense transcript of ~1 kb, suitable for translation of the V1 and V2 genes was identified. A predominant complementary-sense transcript was also confirmed for the betasatellite. CONCLUSIONS: Overall, the transcription of CLCuBuV and the recombinant CLCuMuB is equivalent to earlier mapped begomoviruses/betasatellites. The recombination events that featured in the origins of these components had no detectable effects on transcription. The transcripts spanning the mutated C2 gene showed no evidence for involvement of splicing in restoring the ability to express intact C2 protein.

Project description:Background Cotton leaf curl disease (CLCuD) is a disease of cotton caused by begomoviruses, leading to a drastic loss in the annual yield of the crop. Pakistan has suffered two epidemics of this disease leading to the loss of billions in annual exports. The speculation that a third epidemic of CLCuD may result as consequence of the frequent occurrence of Tomato leaf curl New Delhi virus (ToLCNDV) and Cotton leaf curl Kokhran Virus-Burewala Strain (CLCuKoV-Bu) in CLCuD infected samples, demand that the interactions taking between the two viruses be properly evaluated. This study is designed to assess virus-virus interactions at the molecular level and determine the type of co-infection taking place. Methods Based on the amino acid sequences of the gene products of both CLCuKoV-Bu and ToLCNDV, protein structures were generated using different software, i.e., MODELLER, I-TASSER, QUARKS, LOMETS and RAPTORX. A consensus model for each protein was selected after model quality assessment using ERRAT, QMEANDisCo, PROCHECK Z-Score and Ramachandran plot analysis. The active and passive residues in the protein structures were identified using the CPORT server. Protein–Protein Docking was done using the HADDOCK webserver, and 169 Protein–Protein Interaction (PPIs) were performed between the proteins of the two viruses. The docked complexes were submitted to the PRODIGY server to identify the interacting residues between the complexes. The strongest interactions were determined based on the HADDOCK Score, Desolvation energy, Van der Waals Energy, Restraint Violation Energy, Electrostatic Energy, Buried Surface Area and Restraint Violation Energy, Binding Affinity and Dissociation constant (Kd). A total of 50 ns Molecular Dynamic simulations were performed on complexes that exhibited the strongest affinity in order to validate the stability of the complexes, and to remove any steric hindrances that may exist within the structures. Results Our results indicate significant interactions taking place between the proteins of the two viruses. Out of all the interactions, the strongest were observed between the Replication Initiation protein (Rep) of CLCuKoV-Bu with the Movement protein (MP), Nuclear Shuttle Protein (NSP) of ToLCNDV (DNA-B), while the weakest were seen between the Replication Enhancer protein (REn) of CLCuKoV-Bu with the REn protein of ToLCNDV. The residues identified to be taking a part in interaction belonged to domains having a pivotal role in the viral life cycle and pathogenicity. It maybe deduced that the two viruses exhibit antagonistic behavior towards each other, and the type of infection may be categorised as a type of Super Infection Exclusion (SIE) or homologous interference. However, further experimentation, in the form of transient expression analysis, is needed to confirm the nature of these interactions and increase our understanding of the direct interactions taking place between two viruses.

Dataset Information

Artificial microRNA-mediated resistance against the monopartite begomovirus Cotton leaf curl Burewala virus.

Publications

Artificial microRNA-mediated resistance against the monopartite begomovirus Cotton leaf curl Burewala virus.

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