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Regional changes in the sequence of cotton leaf curl multan betasatellite.
Viruses
PUBLISHED: 03-24-2014
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Cotton leaf curl disease (CLCuD) in Pakistan and northwestern India is caused by monopartite begomoviruses in association with an essential, disease-specific satellite, Cotton leaf curl Multan betasatellite (CLCuMB). Following a recent upsurge in CLCuD problems in Sindh province (southern Pakistan), sequences of clones of CLCuMB were obtained from Sindh and Punjab province (central Pakistan), where CLCuD has been a problem since the mid-1980s. The sequences were compared to all sequences of CLCuMB available in the databases. Analysis of the sequences shows extensive sequence variation in CLCuMB, most likely resulting from recombination. The range of sequence variants differ between Sindh, the Punjab and northwestern India. The possible significance of the findings with respect to movement of the CLCuD between the three regions is discussed. Additionally, the lack of sequence variation within the only coding sequence of CLCuMB suggests that the betasatellite is not involved in resistance breaking which became a problem after 2001 in the Punjab and subsequently also in northwestern India.
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Nutrients Can Enhance the Abundance and Expression of Alkane Hydroxylase CYP153 Gene in the Rhizosphere of Ryegrass Planted in Hydrocarbon-Polluted Soil.
PLoS ONE
PUBLISHED: 01-01-2014
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Plant-bacteria partnership is a promising strategy for the remediation of soil and water polluted with hydrocarbons. However, the limitation of major nutrients (N, P and K) in soil affects the survival and metabolic activity of plant associated bacteria. The objective of this study was to explore the effects of nutrients on survival and metabolic activity of an alkane degrading rhizo-bacterium. Annual ryegrass (Lolium multiflorum) was grown in diesel-contaminated soil and inoculated with an alkane degrading bacterium, Pantoea sp. strain BTRH79, in greenhouse experiments. Two levels of nutrients were applied and plant growth, hydrocarbon removal, and gene abundance and expression were determined after 100 days of sowing of ryegrass. Results obtained from these experiments showed that the bacterial inoculation improved plant growth and hydrocarbon degradation and these were further enhanced by nutrients application. Maximum plant biomass production and hydrocarbon mineralization was observed by the combined use of inoculum and higher level of nutrients. The presence of nutrients in soil enhanced the colonization and metabolic activity of the inoculated bacterium in the rhizosphere. The abundance and expression of CYP153 gene in the rhizosphere of ryegrass was found to be directly associated with the level of applied nutrients. Enhanced hydrocarbon degradation was associated with the population of the inoculum bacterium, the abundance and expression of CYP153 gene in the rhizosphere of ryegrass. It is thus concluded that the combination between vegetation, inoculation with pollutant-degrading bacteria and nutrients amendment was an efficient approach to reduce hydrocarbon contamination.
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Effects of genetic changes to the begomovirus/betasatellite complex causing cotton leaf curl disease in South Asia post-resistance breaking.
Virus Res.
PUBLISHED: 07-20-2013
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Cotton leaf curl disease (CLCuD) has been a problem for cotton production across Pakistan and north-eastern India since the early 1990s. The appearance of the disease has been attributed to the introduction, and near monoculture of highly susceptible cotton varieties. During the intervening period the genetic make-up of the virus(es) causing the disease has changed dramatically. The most prominent of these changes has been in response to the introduction of CLCuD-resistant cotton varieties in the late 1990s, which provided a brief respite from the losses due to the disease. During the 1990s the disease was shown to be caused by multiple begomoviruses and a single, disease-specific betasatellite. Post-resistance breaking the complex encompassed only a single begomovirus, Cotton leaf curl Burewala virus (CLCuBuV), and a recombinant version of the betasatellite. Surprisingly CLCuBuV lacks an intact transcriptional-activator protein (TrAP) gene. The TrAP gene is found in all begomoviruses and encodes a product of ?134 amino acids that is important in virus-host interactions; being a suppressor of post-transcriptional gene silencing (host defence) and a transcription factor that modulates host gene expression, including microRNA genes. Recent studies have highlighted the differences between CLCuBuV and the earlier viruses that are part of on-going efforts to define the molecular basis for resistance breaking in cotton.
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Artificial microRNA-mediated resistance against the monopartite begomovirus Cotton leaf curl Burewala virus.
Virol. J.
PUBLISHED: 04-06-2013
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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.
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Molecular characterization of a new synthetic cry2ab gene in Nicotiana tabacum.
Biotechnol. Lett.
PUBLISHED: 01-23-2013
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A newly-synthesized cry2Ab gene was characterized in Nicotiana tabacum, before its further transformation in cotton. Synthetic cry2Ab gene was cloned in pGreen0029 and its expression was transiently analyzed at mRNA level through agroinfiltration in tobacco. The mRNA of cry2Ab was detected after 72 h agroinfiltration through PCR using total plant RNA. This construct was then transformed into N. tabacum through Agrobacterium. Insect bioassays were conducted on detached leaves using first instar Spodoptera exigua larvae; after 96 h significant insect mortality was recorded. This newly synthesized gene was effective in controlling S. exigua first instar larvae. It can be used in combinations with other Bt genes like cry1Ac for developing resistance against major insect pests of cotton and further widening the insect control spectrum.
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Suppressors of RNA silencing encoded by the components of the cotton leaf curl begomovirus-betasatellite complex.
Mol. Plant Microbe Interact.
PUBLISHED: 07-15-2011
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Begomoviruses (family Geminiviridae) are single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. Many economically important diseases in crops are caused by begomoviruses, particularly in tropical and subtropical environments. These include the betasatellite-associated begomoviruses causing cotton leaf curl disease (CLCuD) that causes significant losses to a mainstay of the economy of Pakistan, cotton. RNA interference (RNAi) or gene silencing is a natural defense response of plants against invading viruses. In counter-defense, viruses encode suppressors of gene silencing that allow them to effectively invade plants. Here, we have analyzed the ability of the begomovirus Cotton leaf curl Multan virus (CLCuMV) and its associated betasatellite, Cotton leaf curl Multan ?-satellite (CLCuMB) which, together, cause CLCuD, and the nonessential alphasatellite (Cotton leaf curl Multan alphasatellite [CLCuMA]) for their ability to suppress gene silencing in Nicotiana benthamiana. The results showed that CLCuMV by itself was unable to efficiently block silencing. However, in the presence of the betasatellite, gene silencing was entirely suppressed. Silencing was not affected in any way when infections included CLCuMA, although the alphasatellite was, for the first time, shown to be a target of RNA silencing, inducing the production in planta of specific small interfering RNAs, the effectors of silencing. Subsequently, using a quantitative real-time polymerase chain reaction assay and Northern blot analysis, the ability of all proteins encoded by CLCuMV and CLCuMB were assessed for their ability to suppress RNAi and the relative strengths of their suppression activity were compared. The analysis showed that the V2, C2, C4, and ?C1 proteins exhibited suppressor activity, with the V2 showing the strongest activity. In addition, V2, C4, and ?C1 were examined for their ability to bind RNA and shown to have distinct specificities. Although each of these proteins has, for other begomoviruses or betasatellites, been previously shown to have suppressor activity, this is the first time all proteins encoded by a geminiviruses (or begomovirus-betasatellite complex) have been examined and also the first for which four separate suppressors have been identified.
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Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs.
Virol. J.
PUBLISHED: 05-19-2011
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Whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs) are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses.
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RNA interference-based resistance against a legume mastrevirus.
Virol. J.
PUBLISHED: 03-15-2011
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RNA interference (RNAi) is a homology-dependant gene silencing mechanism and has been widely used to engineer resistance in plants against RNA viruses. However, its usefulness in delivering resistance against plant DNA viruses belonging to family Geminiviridae is still being debated. Although the RNAi approach has been shown, using a transient assay, to be useful in countering monocotyledonous plant-infecting geminiviruses of the genus Mastrevirus, it has yet to be investigated as a means of delivering resistance to dicot-infecting mastreviruses. Chickpea chlorotic dwarf Pakistan virus (CpCDPKV) is a legume-infecting mastrevirus that affects chickpea and other leguminous crops in Pakistan.
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The merging of two dynasties--identification of an African cotton leaf curl disease-associated begomovirus with cotton in Pakistan.
PLoS ONE
PUBLISHED: 02-20-2011
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Cotton leaf curl disease (CLCuD) is a severe disease of cotton that occurs in Africa and Pakistan/northwestern India. The disease is caused by begomoviruses in association with specific betasatellites that differ between Africa and Asia. During survey of symptomatic cotton in Sindh (southern Pakistan) Cotton leaf curl Gezira virus (CLCuGV), the begomovirus associated with CLCuD in Africa, was identified. However, the cognate African betasatellite (Cotton leaf curl Gezira betasatellite) was not found. Instead, two Asian betasatellites, the CLCuD-associated Cotton leaf curl Multan betasatellite (CLCuMB) and Chilli leaf curl betasatellite (ChLCB) were identified. Inoculation of the experimental plant species Nicotiana benthamiana showed that CLCuGV was competent to maintain both CLCuMB and ChLCB. Interestingly, the enations typical of CLCuD were only induced by CLCuGV in the presence of CLCuMB. Also in infections involving both CLCuMB and ChLCB the enations typical of CLCuMB were less evident. This is the first time an African begomovirus has been identified on the Indian sub-continent, highlight the growing threat of begomoviruses and particularly the threat of CLCuD causing viruses to cotton cultivation in the rest of the world.
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A common set of developmental miRNAs are upregulated in Nicotiana benthamiana by diverse begomoviruses.
Virol. J.
PUBLISHED: 01-04-2011
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Begomoviruses are single-stranded DNA viruses that cause economically important diseases of many crops throughout the world and induce symptoms in plants, including enations, leaf curling and stunting, that resemble developmental abnormalities. MicroRNAs (miRNAs) are small endogenous RNAs that are involved in a variety of activities, including plant development, signal transduction and protein degradation, as well as response to environmental stress, and pathogen invasion.
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Transient expression of ?C1 protein differentially regulates host genes related to stress response, chloroplast and mitochondrial functions.
Virol. J.
PUBLISHED: 10-10-2010
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Geminiviruses are emerging plant pathogens that infect a wide variety of crops including cotton, cassava, vegetables, ornamental plants and cereals. The geminivirus disease complex consists of monopartite begomoviruses that require betasatellites for the expression of disease symptoms. These complexes are widespread throughout the Old World and cause economically important diseases on several crops. A single protein encoded by betasatellites, termed ?C1, is a suppressor of gene silencing, inducer of disease symptoms and is possibly involved in virus movement. Studies of the interaction of ?C1 with hosts can provide useful insight into virus-host interactions and aid in the development of novel control strategies. We have used the differential display technique to isolate host genes which are differentially regulated upon transient expression of the ?C1 protein of chili leaf curl betasatellite (ChLCB) in Nicotiana tabacum.
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Identification of a major pathogenicity determinant and suppressors of RNA silencing encoded by a South Pacific isolate of Banana bunchy top virus originating from Pakistan.
Virus Genes
PUBLISHED: 09-02-2010
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Five genes encoded by Banana bunchy top virus (BBTV) originating from Pakistan were expressed in Nicotiana benthamiana using a Potato virus X (PVX) vector. Expression of the master replication-associated protein (mRep) and movement protein (MP) resulted in necrotic cell death of inoculated tissues, as well as leaf curling and necrosis along the veins in newly emerging leaves. The systemic necrosis induced by the expression of MP was discolored (dark) in comparison to that induced by mRep. Expression of the cell-cycle link protein (Clink), the coat protein (CP), and the nuclear shuttle protein from the PVX vector induced somewhat milder symptoms, consisting of mild leaf curling and mosaic, although expression of the CP caused a necrotic response in inoculated leaf. The accumulation of viral RNA was enhanced by MP, Clink, and CP. Of the five BBTV-encoded gene products two, the MP and Clink, stabilized GFP-specific mRNA and reduced GFP-specific small interfering RNA in N. benthamiana line 16c when expressed under the control of the 35S promoter and co-inoculated with a construct for the expression of GFP hairpin RNA construct. These results identified MP and Clink as suppressors of RNA silencing. Taken together the ability of MP to induce severe symptoms in plants and suppress RNA silencing implicates this product as a major pathogenicity determinant of BBTV.
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Cotton leaf curl disease in Sindh province of Pakistan is associated with recombinant begomovirus components.
Virus Res.
PUBLISHED: 06-14-2010
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Cotton leaf curl disease (CLCuD) is a devastating disease of cotton causing severe losses to cotton across the Punjab province of Pakistan and northeastern India. Although the disease has been reported as occurring sporadically in Sindh province, Pakistan, this has not caused significant losses. However, in the last few years the disease has become more significant in Sindh province. CLCuD is caused by begomoviruses in association with a disease-specific symptom determining satellite (Cotton leaf curl Multan betasaellite [CLCuMB]) and, in some cases, a non-essential alphasatellite (the function of which remains unclear). These components were cloned from six samples collected in Sindh. Analysis of the full-length sequences of six begomovirus clones showed one to be an isolate of Cotton leaf curl Kokhran virus (CLCuKV), a virus previously shown to be associated with CLCuD in the Punjab, whereas the other five clones showed less than approximately 90% nucleotide sequence identity to several known begomoviruses associated with CLCuD. We take this to indicate that these are isolates of a newly identified begomovirus, for which we propose the name Cotton leaf curl Shahdadpur virus (CLCuShV). Closer inspection of the sequence of CLCuShV showed it to have a recombinant origin. For only two of the cotton samples was the presence of an alphasatellite detected. The sequences of clones of these alphasatellites indicate them to be newly identified species. A betasatellite was shown to be present in all six plants examined and sequence analysis of seven full-length clones indicated that two types of CLCuMB are present in Sindh and both are recombinant. These results indicate that the virus complex causing CLCuD in Sindh is distinct from that in the adjacent Punjab province. Possible reasons for these differences are discussed.
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Both malvaceous and non-malvaceous betasatellites are associated with two wild cotton species grown under field conditions in Pakistan.
Virus Genes
PUBLISHED: 05-23-2010
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Betasatellites are symptom-modulating DNA satellites that are associated with the majority of monopartite begomoviruses in the Old World. Betasatellites show both geographical and host-specific relatedness; those isolated from malvaceous hosts form one group and those isolated from non-malvaceous hosts form the second group. Real-time PCR based estimation of begomovirus DNA in cotton species grown in a living herbarium under field conditions at the Central Cotton Research Institute (CCRI), Multan was carried out to understand their role in the ongoing cotton leaf curl disease (CLCuD) epidemic in Pakistan. The levels of begomovirus DNA in cotton species from the herbarium was found to be several folds lower than symptomatic plants of commercially grown cotton. Among herbarium species Gossypium anomalum, G. robinsoni and G. somalense contained higher levels of begomovirus DNA in comparison to other wild species. Characterization of betasatellites from two cotton species (G. anomalum and G. somalense) was carried out to understand their role in accumulation of begomovirus DNA. Here, we show that the two wild species of cotton are associated with Cotton leaf Multan betasatellite (CLCuMB), a malvaceous betasatellite associated with CLCuD, as well as Chili leaf curl betasatellite, a non-malvaceous betasatellite not previously identified in a malvaceous host. This is the first evidence of the presence of a non-malvaceous betasatellite in malvaceous hosts.
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The hypersensitive response induced by the V2 protein of a monopartite begomovirus is countered by the C2 protein.
Mol. Plant Pathol.
PUBLISHED: 05-08-2010
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A functional analysis of the V2 protein of two monopartite begomoviruses, Papaya leaf curl virus (PaLCuV) and Cotton leaf curl Kokhran virus (CLCuKoV), has been performed. Expression of the V2 gene from a Potato virus X (PVX) vector resulted in severe leaf curling followed by a hypersensitive response (HR) in Nicotiana benthamiana and N. tabacum, demonstrating that the V2 protein is a pathogenicity determinant and a target of host defence responses. Agroinfiltration of a PVX vector expressing the V2 protein resulted in cell death in the infiltrated area. Subsequently, a systemic HR developed that was associated with the long-distance spread of the virus and led to the death of the plant. V2 amino acid sequences encompassing a conserved putative protein kinase C (PKC) phosphorylation motif were shown to be essential for the elicitation of cell death. In co-inoculation experiments, the transient expression of the C2 protein of PaLCuV or Cotton leaf curl Multan virus under the control of the Cauliflower mosaic virus 35S promoter inhibited the HR induced by V2 in the agroinfiltrated area. These findings demonstrate that the V2 protein of monopartite begomoviruses is a pathogenicity determinant and induces an HR that can be suppressed by the C2 protein. The induction and suppression of HR have been demonstrated previously in bipartite begomoviruses and our results extend this to monopartite begomoviruses.
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Cotton leaf curl disease in resistant cotton is associated with a single begomovirus that lacks an intact transcriptional activator protein.
Virus Res.
PUBLISHED: 05-02-2010
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Cotton leaf curl disease (CLCuD) is the major limitation to cotton production across Pakistan and northwestern India. The disease first appeared in epidemic form in the 1980s and was shown to be caused by monopartite begomoviruses (seven distinct species have thus far been shown to be involved), frequently as multiple infections. Additionally, the viruses are associated with a specific satellite, the CLCuD betasatellite Cotton leaf curl Multan betasatellite (CLCuMB), which is responsible for the distinctive disease symptoms, and a satellite-like molecule (termed an alphasatellite), the function of which is unclear. During the late 1990s, cotton varieties with conventional resistance were introduced, alleviating losses to cotton production. However, during 2001 a resistance breaking strain of CLCuD (known as the "Burewala" strain) appeared which spread across most cotton producing areas of Pakistan. We have conducted an analysis of the Burewala strain and show that, contrary to the earlier (Multan) strain, it consists of a single begomovirus. The virus is associated with a recombinant betasatellite, derived from the Multan strain, but we were unable to detect the presence of an alphasatellite. Sequence comparisons show the virus to be a new recombinant species, consisting of sequences derived from two of the viruses associated with the first epidemic, for which we propose the name Cotton leaf curl Burewala virus (CLCuBuV). Surprisingly the virus lacks an intact C2 gene, encoding the transcriptional activator protein, which is invariably present in begomoviruses. The possible mechanisms for the selection of a "defective" begomovirus are discussed.
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