Reactions of Nicotiana species to inoculation with monopartite and bipartite begomoviruses

<p>Abstract</p> <p>Background</p> <p>Some <it>Nicotiana </it>species are widely used as experimental hosts for plant viruses. <it>Nicotiana </it>species differ in ploidy levels, chromosome numbers and have diverse geographical origins. Thus, these species are useful model systems to investigate virus-host interactions, co-evolution of pathogens and hosts and the effects of ploidy level on virus resistance/susceptibility.</p> <p>Results</p> <p>Here we have studied the responses of seven <it>Nicotiana </it>species to inoculation with <it>Cotton leaf curl Multan virus </it>(CLCuMV), a monopartite begomovirus, and <it>Tomato leaf curl New Delhi virus </it>(ToLCNDV), a bipartite begomovirus, both from the Indian subcontinent. All <it>Nicotiana </it>species supported the replication of both begomoviruses in inoculated leaves. However, only three <it>Nicotiana </it>species, namely <it>N. benthamiana</it>, <it>N. tabacum </it>and <it>N. sylvestris </it>showed symptoms when inoculated with ToLCNDV, while <it>N. benthamiana </it>was the only species that developed leaf curl symptoms when inoculated with CLCuMV. CLCuMV accumulated to detectable levels in <it>N. tabacum</it>, but plants remained asymptomatic. A previously identified mutation of RNA dependent RNA polymerase 1 was shown to be present only in <it>N. benthamiana</it>. The finding is in line with earlier results showing that the susceptibility of this species to a diverse range of plant viruses correlates with a defective RNA silencing-mediated host defense.</p> <p>Conclusions</p> <p>The results presented show that individual <it>Nicotiana </it>species respond differently to inoculation with begomoviruses. The inability of begomoviruses to systemically infect several <it>Nicotiana </it>species is likely due to inhibition of virus movement, rather than replication, and thus provides a novel model to study virus-host interactions in resistant/susceptible hosts.</p

Infectious clones of Tomato leaf curl Palampur virus with a defective DNA B and their pseudo-recombination with Tomato leaf curl New Delhi virus

<p>Abstract</p> <p>Background</p> <p><it>Tomato leaf curl Palampur virus </it>(ToLCPMV) is a bipartite begomovirus which has been reported from India and Iran but infectious clones have not been obtained. We have previously shown the association of <it>Zucchini yellow mosaic virus </it>(ZYMV), a potyvirus, with severe leaf curl disease of muskmelon in Pakistan. However, the severity of symptoms in the field and yield losses led us to believe that some other agent, such as a begomovirus, could be associated with the disease.</p> <p>Results</p> <p>A bipartite begomovirus associated with a severe yellow leaf curl disease on muskmelon in Pakistan has been characterized. Analysis of the complete nucleotide sequence of the DNA A and DNA B components of the begomovirus showed that it has the highest DNA sequence identity with ToLCPMV. However, the gene encoding the nuclear shuttle protein (NSP) was truncated in comparison to previously characterised isolates. <it>Agrobacterium</it>-mediated inoculation of <it>Nicotiana benthamiana </it>with the ToLCPMV clones obtained here did not result in symptoms. However, inoculation of plants with the DNA A component of ToLCPMV and the DNA B component of <it>Tomato leaf curl New Delhi virus </it>(ToLCNDV) lead to systemic infection with leaf curl symptoms. This suggested that the lack of infectivity of the ToLCPMV clones was due to the defect in DNA B. The DNA B of ToLCPMV was able to move systemically when inoculated with DNA A of the either virus. Agro-infiltration of muskmelon with the DNA A and DNA B components of ToLCPMV did not lead to symptomatic infection whereas inoculation with the DNA A with the DNA B of ToLCNDV resulted in a hypersensitive response (HR) along the veins. Additionally, agro-infiltration of muskmelon with a construct for the expression of the NSP gene of ToLCNDV under the control of the cauliflower mosaic virus 35S promoter induced a HR, suggesting that this is the gene causing the HR.</p> <p>Conclusions</p> <p>Both ToLCPMV and ZYMV are associated with muskmelon leaf curl disease in Pakistan. However, the ToLCPMV variant identified in association with ZYMV has a defective NSP. The results suggest that a variant with a defective NSP may have been selected for in muskmelon, as this protein is an avirulence determinant in this species, and possibly that infection requires the synergistic interaction with ZYMV.</p

RNA interference-based resistance against a legume mastrevirus

<p>Abstract</p> <p>Background</p> <p>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 <it>Geminiviridae </it>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 <it>Mastrevirus</it>, it has yet to be investigated as a means of delivering resistance to dicot-infecting mastreviruses. <it>Chickpea chlorotic dwarf Pakistan virus </it>(CpCDPKV) is a legume-infecting mastrevirus that affects chickpea and other leguminous crops in Pakistan.</p> <p>Results</p> <p>Here a hairpin (hp)RNAi construct containing sequences encompassing part of replication-associated protein gene, intergenic region and part of the movement protein gene of CpCDPKV under the control of the <it>Cauliflower mosaic virus </it>35S promoter has been produced and stably transformed into <it>Nicotiana benthamiana</it>. Plants harboring the hairpin construct were challenged with CpCDPKV. All non-transgenic <it>N. benthamiana </it>plants developed symptoms of CpCDPKV infection within two weeks post-inoculation. In contrast, none of the inoculated transgenic plants showed symptoms of infection and no viral DNA could be detected by Southern hybridization. A real-time quantitative PCR analysis identified very low-level accumulation of viral DNA in the inoculated transgenic plants.</p> <p>Conclusions</p> <p>The results presented show that the RNAi-based resistance strategy is useful in protecting plants from a dicot-infecting mastrevirus. The very low levels of virus detected in plant tissue of transgenic plants distal to the inoculation site suggest that virus movement and/or viral replication was impaired leading to plants that showed no discernible signs of virus infection.</p

Selection of target sequences as well as sequence identity determine the outcome of RNAi approach for resistance against cotton leaf curl geminivirus complex

Cotton leaf curl disease is caused by a geminivirus complex that involves multiple distinct begomoviruses and a disease-specific DNA satellite, cotton leaf curl Multan betasatellite (CLCuMB), which is essential to induce disease symptoms. Here we have investigated the use of RNA interference (RNAi) for obtaining resistance against one of the viruses, Cotton leaf curl Multan virus (CLCuMV), associated with the disease. Three hairpin RNAi constructs were produced containing either complementary-sense genes essential for replication/pathogenicity or non-coding regulatory sequences of CLCuMV. In transient assays all three RNAi constructs significantly reduced the replication of the virus in inoculated tissues. However, only one of the constructs, that targeting the overlapping genes involved in virus replication and pathogenicity (the replication-associated protein (Rep), the transcriptional activator protein and the replication enhancer protein) was able to prevent systemic movement of the virus, although the other constructs significantly reduced the levels of virus in systemic tissues. In the presence of CLCuMB, however, a small number of plants co-inoculated with even the most efficient RNAi construct developed symptoms of virus infection, suggesting that the betasatellite may compromise resistance. Further analyses, using Rep gene sequences of distinct begomoviruses expressed from a PVX vector as the target, are consistent with the idea that the success of the RNAi approach depends on sequence identity to the target virus. The results show that selection of both the target sequence, as well as the levels of identity between the construct and target sequence, determine the outcome of RNAi-based resistance against geminivirus complexes

Transient expression of βC1 protein differentially regulates host genes related to stress response, chloroplast and mitochondrial functions

<p>Abstract</p> <p>Background</p> <p>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 βC1, is a suppressor of gene silencing, inducer of disease symptoms and is possibly involved in virus movement. Studies of the interaction of βC1 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 βC1 protein of chili leaf curl betasatellite (ChLCB) in <it>Nicotiana tabacum</it>.</p> <p>Results</p> <p>Through differential display analysis, eight genes were isolated from <it>Nicotiana tabacum</it>, at two and four days after infitration with βC1 of ChLCB, expressed under the control of the <it>Cauliflower mosaic virus </it>35S promoter. Cloning and sequence analysis of differentially amplified products suggested that these genes were involved in ATP synthesis, and acted as electron carriers for respiration and photosynthesis processes. These differentially expressed genes (DEGs) play an important role in plant growth and development, cell protection, defence processes, replication mechanisms and detoxification responses. Kegg orthology based annotation system analysis of these DEGs demonstrated that one of the genes, coding for polynucleotide nucleotidyl transferase, is involved in purine and pyrimidine metabolic pathways and is an RNA binding protein which is involved in RNA degradation.</p> <p>Conclusion</p> <p>βC1 differentially regulated genes are mostly involved in chloroplast and mitochondrial functions. βC1 also increases the expression of those genes which are involved in purine and pyrimidine metabolism. This information gives a new insight into the interaction of βC1 with the host and can be used to understand host-virus interactions in follow-up studies.</p

Pepper leaf curl Lahore virus requires the DNA B component of Tomato leaf curl New Delhi virus to cause leaf curl symptoms

<p>Abstract</p> <p>Background</p> <p>Begomoviruses are whitefly-transmitted geminiviruses with genomes that consist of either two components (known as DNA A and DNA B) or a single component (homologous to the DNA A component of bipartite begomoviruses). Monopartite begomoviruses are often associated with a symptom-modulating DNA satellite (collectively known as betasatellites). Both bipartite and monopartite begomoviruses with associated satellites have previously been identified in chillies showing leaf curl symptoms in Pakistan.</p> <p>Results</p> <p><b>A </b>chilli plant (<it>Capsicum annum</it>) with chilli leaf curl disease symptoms was found to contain a begomovirus, a betasatellite and the DNA B component of <it>Tomato leaf curl New Delhi virus </it>(ToLCNDV). The begomovirus consisted of 2747 nucleotides and had the highest sequence identity (99%) <it>with Pepper leaf curl Lahore virus </it>(PepLCLV-[PK: Lah:04], acc. no. AM404179). <it>Agrobacterium</it>-mediated inoculation of the clone to <it>Nicotiana benthamiana</it>, induced very mild symptoms and low levels of viral DNA, detected in systemically infected leaves by PCR. No symptoms were induced in <it>Nicotiana tabacum </it>or chillies either in the presence or absence of a betasatellite. However, inoculation of PepLCLV with the DNA B component of ToLCNDV induced leaf curl symptoms in <it>N. benthamiana</it>, <it>N. tabacum </it>and chillies and viral DNA accumulated to higher levels in comparison to plants infected with just PepLCLV.</p> <p>Conclusions</p> <p>Based on our previous efforts aimed at understanding of diversity of begomoviruses associated with chillies, we propose that PepLCLV was recently mobilized into chillies upon its interaction with DNA B of ToLCNDV. Interestingly, the putative rep-binding iterons found on PepLCLV (GGGGAC) differ at two base positions from those of ToLCNDV (GGTGTC). This is the first experimental demonstration of the infectivity for a bipartite begomovirus causing chilli leaf curl disease in chillies from Pakistan and suggests that component capture is contributing to the emerging complexity of begomovirus diseases in the region.</p

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>All genes encoded by four distinct begomoviruses (<it>African cassava mosaic virus </it>[ACMV], <it>Cabbage leaf curl virus </it>[CbLCuV], <it>Tomato yellow leaf curl virus </it>[TYLCV] and <it>Cotton leaf curl virus</it>/Cotton leaf curl betasatellite [CLCuV/CLCuMB]) were expressed from a <it>Potato virus X </it>(PVX) vector in <it>Nicotiana benthamiana</it>. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV) the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV) more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct.</p> <p>Conclusions</p> <p>Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to be the case.</p

Analysis of the Nucleotide Sequence of the Treehopper-Transmitted Geminivirus, Tomato Pseudo-Curly Top Virus, Suggests a Recombinant Origin

AbstractThe genome of tomato pseudo-curly top virus (TPCTV), originating from Florida, has been cloned and sequenced. TPCTV is the only geminivirus identified with a vector specificity which falls outside the Cicadellidae (leafhoppers) and Aleyrodidae (whiteflies). Infectivity of the cloned viral genome was demonstrated byAgrobacterium-mediated inoculation of several host species. Progeny virus was transmissible by the treehopper vector of TPCTV,Micrutalis malleifera(Fowler). The genome of TPCTV shows features typical of both subgroups I and III genera of the family Geminiviridae. The coat protein of TPCTV, although distinct from all previously characterized geminiviruses, exhibits features more akin to the leafhopper-transmitted geminiviruses than those transmissible by the whiteflyBemisia tabaciGenn. The relationship of TPCTV to other geminiviruses, particularly beet curly top virus, is discussed in relation to the possible evolutionary origins of this virus

Temporal changes in the levels of virus and betasatellite DNA in B. tabaci feeding on CLCuD affected cotton during the growing season

Cotton, a key source of income for Pakistan, has suffered significantly by cotton leaf curl disease (CLCuD) since 1990. This disease is caused by a complex of phylogenetically-related begomovirus (genus Begomovirus, family Geminiviridae) species and a specific betasatellite (genus Betasatellite, family Tolecusatellitidae), cotton leaf curl Multan betasatellite. Additionally, another DNA satellite called alphasatellite (family Alphasatellitidae), is also frequently associated. All these virus components are vectored by a single species of whitefly (Bemisia tabaci). While many factors affect cotton productivity, including cotton variety, sowing time, and environmental cues such as temperature, humidity, and rainfall, CLCuD is a major biotic constraint. Although the understanding of begomoviruses transmission by whiteflies has advanced significantly over the past three decades, however, the in-field seasonal dynamics of the viruses in the insect vector remained an enigma. This study aimed to assess the levels of virus and betasatellite in whiteflies collected from cotton plants throughout the cotton growing season from 2014 to 2016. Notably, begomovirus levels showed no consistent pattern, with minimal variations, ranging from 0.0017 to 0.0074 ng.μg–1 of the genomic DNA in 2014, 0.0356 to 0.113 ng.μg–1 of the genomic DNA in 2015, and 0.0517 to 0.0791 ng.μg–1 of the genomic DNA in 2016. However, betasatellite levels exhibited a distinct pattern. During 2014 and 2015, it steadily increased throughout the sampling period (May to September). While 2016 showed a similar trend from the start of sampling (July) to September but a decline in October (end of sampling). Such a study has not been conducted previously, and could potentially provide valuable insights about the epidemiology of the virus complex causing CLCuD and possible means of controlling losses due to it

Diversity and phylogeography of begomovirus-associated beta satellites of okra in India

<p>Abstract</p> <p>Background</p> <p>Okra (<it>Abelmoschus esculentus</it>; family <it>Malvaceae</it>) is grown in temperate as well as subtropical regions of the world, both for human consumption as a vegetable and for industrial uses. Okra yields are affected by the diseases caused by phyopathogenic viruses. India is the largest producer of okra and in this region a major biotic constraint to production are viruses of the genus <it>Begomovirus</it>. Begomoviruses affecting okra across the Old World are associated with specific, symptom modulating satellites (beta satellites). We describe a comprehensive analysis of the diversity of beta satellites associated with okra in India.</p> <p>Results</p> <p>The full-length sequences of 36 beta satellites, isolated from okra exhibiting typical begomovirus symptoms (leaf curl and yellow vein), were determined. The sequences segregated in to four groups. Two groups correspond to the beta satellites Okra leaf curl beta satellite (OLCuB) and Bhendi yellow vein beta satellite (BYVB) that have previously been identified in okra from the sub-continent. One sequence was distinct from all other, previously isolated beta satellites and represents a new species for which we propose the name Bhendi yellow vein India beta satellite (BYVIB). This new beta satellite was nevertheless closely related to BYVB and OLCuB. Most surprising was the identification of Croton yellow vein mosaic beta satellite (CroYVMB) in okra; a beta satellite not previously identified in a malvaceous plant species. The okra beta satellites were shown to have distinct geographic host ranges with BYVB occurring across India whereas OLCuB was only identified in northwestern India. Okra infections with CroYVMB were only identified across the northern and eastern central regions of India. A more detailed analysis of the sequences showed that OLCuB, BYVB and BYVIB share highest identity with respect βC1 gene. βC1 is the only gene encoded by beta satellites, the product of which is the major pathogenicity determinant of begomovirus-beta satellite complexes and is involved in overcoming host defenses based on RNAi.</p> <p>Conclusion</p> <p>The diversity of beta satellites in okra across the sub-continent is higher than previously realized and is higher than for any other malvaceous plant species so far analyzed. The beta satellites identified in okra show geographic segregation, which has implications for the development and introduction of resistant okra varieties. However, the finding that the βC1 gene of the major okra beta satellites (OLCuB, BYVB and BYVIB) share high sequence identity and provides a possible avenue to achieve a broad spectrum resistance.</p