Functional Characterization of Replication-Associated Proteins Encoded by Alphasatellites Identified in Yunnan Province, China.

Alphasatellites, which encode only a replication-associated protein (alpha-Rep), are frequently found to be non-essential satellite components associated with begomovirus/betasatellite complexes, and their presence can modulate disease symptoms and/or viral DNA accumulation during infection. Our previous study has shown that there are three types of alphasatellites associated with begomovirus/betasatellite complexes in Yunnan province in China and they encode three corresponding types of alpha-Rep proteins. However, the biological functions of alpha-Reps remain poorly understood. In this study, we investigated the biological functions of alpha-Reps in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) using 16c and 16-TGS transgenic Nicotiana benthamiana plants. Results showed that all the three types of alpha-Rep proteins were capable of suppressing the PTGS and reversing the TGS. Among them, the alpha-Rep of Y10DNA1 has the strongest PTGS and TGS suppressor activities. We also found that the alpha-Rep proteins were able to increase the accumulation of their helper virus during coinfection. These results suggest that the alpha-Reps may have a role in overcoming host defense, which provides a possible explanation for the selective advantage provided by the association of alphasatellites with begomovirus/betasatellite complexes.

The Peptide AmPep1 Derived from Amaranth Recognizes the Replication Hairpin of TYLCV Disturbing Its Replication Process in Host Plants.

Antiviral compounds targeting viral replicative processes have been studied as an alternative for the control of begomoviruses. Previously, we have reported that the peptide AmPep1 has strong affinity binding to the replication origin sequence of tomato yellow leaf curl virus (TYLCV). In this study, we describe the mechanism of action of this peptide as a novel alternative for control of plant-infecting DNA viruses. When AmPep1 was applied exogenously to tomato and Nicotiana benthamiana plants infected with TYLCV, a decrease in the synthesis of the two viral DNA strands (CS and VS) was observed, with a consequent delay in the development of disease progress in treated plants. The chemical mechanism of action of AmPep1 was deduced using Raman spectroscopy and molecular modeling showing the formation of chemical interactions such as H bonds and electrostatic interactions and the formation of π-π interactions between both biomolecules contributing to tampering with the viral replication.

A virus-targeted plant receptor-like kinase promotes cell-to-cell spread of RNAi.

RNA interference (RNAi) in plants can move from cell to cell, allowing for systemic spread of an antiviral immune response. How this cell-to-cell spread of silencing is regulated is currently unknown. Here, we describe that the C4 protein from Tomato yellow leaf curl virus can inhibit the intercellular spread of RNAi. Using this viral protein as a probe, we have identified the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) as a positive regulator of the cell-to-cell movement of RNAi, and determined that BAM1 and its closest homolog, BAM2, play a redundant role in this process. C4 interacts with the intracellular domain of BAM1 and BAM2 at the plasma membrane and plasmodesmata, the cytoplasmic connections between plant cells, interfering with the function of these RLKs in the cell-to-cell spread of RNAi. Our results identify BAM1 as an element required for the cell-to-cell spread of RNAi and highlight that signaling components have been coopted to play multiple functions in plants.

Begomoviral Movement Protein Effects in Human and Plant Cells: Towards New Potential Interaction Partners.

Geminiviral single-stranded circular DNA genomes replicate in nuclei so that the progeny DNA has to cross both the nuclear envelope and the plasmodesmata for systemic spread within plant tissues. For intra- and intercellular transport, two proteins are required: a nuclear shuttle protein (NSP) and a movement protein (MP). New characteristics of ectopically produced Abutilon mosaic virus (AbMV) MP (MPAbMV), either authentically expressed or fused to a yellow fluorescent protein or epitope tags, respectively, were determined by localization studies in mammalian cell lines in comparison to plant cells. Wild-type MPAbMV and the distinct MPAbMV: reporter protein fusions appeared as curled threads throughout mammalian cells. Co-staining with cytoskeleton markers for actin, intermediate filaments, or microtubules identified these threads as re-organized microtubules. These were, however, not stabilized by the viral MP, as demonstrated by nocodazole treatment. The MP of a related bipartite New World begomovirus, Cleome leaf crumple virus (ClLCrV), resulted in the same intensified microtubule bundling, whereas that of a nanovirus did not. The C-terminal section of MPAbMV, i.e., the protein's oligomerization domain, was dispensable for the effect. However, MP expression in plant cells did not affect the microtubules network. Since plant epidermal cells are quiescent whilst mammalian cells are proliferating, the replication-associated protein RepAbMV protein was then co-expressed with MPAbMV to induce cell progression into S-phase, thereby inducing distinct microtubule bundling without MP recruitment to the newly formed threads. Co-immunoprecipitation of MPAbMV in the presence of RepAbMV, followed by mass spectrometry identified potential novel MPAbMV-host interaction partners: the peptidyl-prolyl cis-trans isomerase NIMA-interacting 4 (Pin4) and stomatal cytokinesis defective 2 (SCD2) proteins. Possible roles of these putative interaction partners in the begomoviral life cycle and cytoskeletal association modes are discussed.

Near-Atomic Resolution Structure of a Plant Geminivirus Determined by Electron Cryomicroscopy.

African cassava mosaic virus is a whitefly-transmitted geminivirus which forms unique twin particles of incomplete icosahedra that are joined at five-fold vertices, building an unusual waist. How its 22 capsomers interact within a half-capsid or across the waist is unknown thus far. Using electron cryo-microscopy and image processing, we determined the virion structure with a resolution of 4.2 Å and built an atomic model for its capsid protein. The inter-capsomer contacts mediated by the flexible N termini and loop regions differed within the half-capsids and at the waist, explaining partly the unusual twin structure. The tip of the pentameric capsomer is sealed by a plug formed by a turn region harboring the evolutionary conserved residue Y193. Basic amino acid residues inside the capsid form a positively charged pocket next to the five-fold axis of the capsomer suitable for binding DNA. Within this pocket, density most likely corresponding to DNA was resolved.

Differential methylation of the circular DNA in geminiviral minichromosomes.

Geminiviral minichromosomes were purified to explore epigenetic modifications. The levels of methylation in their covalently closed circular DNA were examined with the help of methylation-dependent restriction (MdR). DNA with 12 superhelical turns was preferentially modified, indicating minichromosomes with 12 nucleosomes leaving an open gap. MdR digestion yielded a specific product of genomic length, which was cloned and Sanger-sequenced, or amplified following ligation-mediated rolling circle amplification and deep-sequenced (circomics). The conventional approach revealed a single cleavage product indicating specific methylations at the borders of the common region. The circomics approach identified considerably more MdR sites in a preferential distance to each other of ~200 nts, which is the DNA length in a nucleosome. They accumulated in regions of nucleosome-free gaps, but scattered also along the genomic components. These results may hint at a function in specific gene regulation, as well as in virus resistance.

Identification of amino acid residues of the coat protein of Sri Lankan cassava mosaic virus affecting symptom production and viral titer in Nicotiana benthamiana.

Sri Lankan cassava mosaic virus (SLCMV) is bipartite begomovirus infecting cassava in India and Sri Lanka. Interestingly, the DNA-A component of the SLCMV alone is able to infect Nicotiana benthamiana causing symptoms of upward leaf rolling and stunting. One of the differences between monopartite and bipartite begomoviruses is the requirement of Coat Protein (CP) for infectivity; CP being essential for the former, but dispensable in the latter. This investigation was aimed to determine the importance of CP in the infectivity of the bipartite SLCMV, behaving as a monopartite virus in N. benthamiana. We tested CP-null mutants, single amino acid replacement mutants and double, triple and quadruple combinations of the above in SLCMV DNA-A, for infectivity, symptom development and viral DNA accumulation in N. benthamiana. While CP-null mutants were non-infectious, a majority of the single amino acid replacement mutants and their combinations retained infectivity, some with attenuated symptoms and reduced viral titers. Some of the combined mutations restored the attenuated symptoms to wild type levels. Some of the mutations were predicted to cause changes in the secondary structure of the CP, which roughly correlated with the attenuation of symptoms and the reduction in viral titers.

Association of tomato leaf curl New Delhi virus DNA-B with bhendi yellow vein mosaic virus in okra showing yellow vein mosaic disease symptoms.

Okra samples showing yellow vein mosaic, vein twisting and bushy appearance were collected from different locations of India during the surveys conducted between years 2005-2009. The dot blot and PCR detection revealed that 75.14% of the samples were associated with monopartite begomovirus and remaining samples with bipartite virus. Whitefly transmission was established for three samples representing widely separated geographical locations which are negative to betasatellites and associated with DNA-B. Genome components of these three representative isolates were cloned and sequenced. The analysis of DNA-A-like sequence revealed that three begomovirus isolates shared more than 93% nucleotide sequence identity with bhendi yellow vein mosaic virus from India (BYVMV), a monopartite begomovirus species that was reported previously as causative agent of bhendi yellow mosaic disease in association of bhendi yellow vein mosaic betasatellite. Further, the DNA-B-like sequences associated with the three virus isolates shared no more than 90% sequence identity with tomato leaf curl New Delhi virus (ToLCNDV). Analyses of putative iteron-binding sequence required for trans-replication suggests that begomovirus sequences shared compatible rep-binding iterons with DNA-B of ToLCNDV. Our data suggest that the monopartite begomovirus associated with okra yellow vein disease has captured DNA-B of ToLCNDV to infect okra. Widespread distribution of the complex shows the increasing trend of the capturing of DNA-B of ToLCNDV by monopartite begomoviruses in the Indian subcontinent. The recombination analysis showed that the DNA-A might have been derived from the inter-specific recombination of begomoviruses, while DNA-B was derived from the ToLCNDV infecting different hosts.

SnRK1 phosphorylation of AL2 delays Cabbage leaf curl virus infection in Arabidopsis.

UNLABELLED: Geminivirus AL2/C2 proteins play key roles in establishing infection and causing disease in their plant hosts. They are involved in viral gene expression, counter host defenses by suppressing transcriptional gene silencing, and interfere with the host signaling involved in pathogen resistance. We report here that begomovirus and curtovirus AL2/C2 proteins interact strongly with host geminivirus Rep-interacting kinases (GRIKs), which are upstream activating kinases of the protein kinase SnRK1, a global regulator of energy and nutrient levels in plants. We used an in vitro kinase system to show that GRIK-activated SnRK1 phosphorylates recombinant AL2/C2 proteins from several begomoviruses and to map the SnRK1 phosphorylation site to serine-109 in the AL2 proteins of two New World begomoviruses: Cabbage Leaf Curl Virus (CaLCuV) and Tomato mottle virus. A CaLCuV AL2 S109D phosphomimic mutation did not alter viral DNA levels in protoplast replication assays. In contrast, the phosphomimic mutant was delayed for symptom development and viral DNA accumulation during infection of Arabidopsis thaliana, demonstrating that SnRK1 contributes to host defenses against CaLCuV. Our observation that serine-109 is not conserved in all AL2/C2 proteins that are SnRK1 substrates in vitro suggested that phosphorylation of viral proteins by plant kinases contributes to the evolution of geminivirus-host interactions. IMPORTANCE: Geminiviruses are single-stranded DNA viruses that cause serious diseases in many crops. Dicot-infecting geminiviruses carry genes that encode multifunctional AL2/C2 proteins that are essential for infection. However, it is not clear how AL2/C2 proteins are regulated. Here, we show that the host protein kinase SnRK1, a central regulator of energy balance and nutrient metabolism in plants, phosphorylates serine-109 in AL2 proteins of three subgroups of New World begomoviruses, resulting in a delay in viral DNA accumulation and symptom appearance. Our results support SnRK1's antiviral role and reveal a novel mechanism underlying this function. Phylogenetic analysis suggested that AL2 S109 evolved as begomoviruses migrated from the Old World to the New World and may have provided a selective advantage as begomoviruses adapted to a different environment and different plant hosts. This study provides new insights into the interaction of viral pathogens with their plant hosts at the level of viral protein modification by the host.

Viral metagenomics: analysis of begomoviruses by illumina high-throughput sequencing.

Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions.

Inhibiting replication of begomoviruses using artificial zinc finger nucleases that target viral-conserved nucleotide motif.

Geminiviridae consists of a large group of single-stranded DNA viruses that cause tremendous losses worldwide. Frequent mixed infection and high rates of recombination and mutation allow them to adapt rapidly to new hosts and overcome hosts' resistances. Therefore, an effective strategy able to confer plants with resistance against multiple begomoviruses is needed. In the present study, artificial zinc finger proteins were designed based on a conserved sequence motif of begomoviruses. DNA-binding affinities and specificities of these artificial zinc fingers were evaluated using electrophoretic mobility shift assay. Artificial zinc finger nuclease (AZFNs) were then constructed based on the ones with the highest DNA-binding affinities. In vitro digest and transient expression assay showed that these AZFNs can efficiently cleave the target sequence and inhibit the replication of different begomoviruses. These results suggest that artificial zinc finger protein technology may be used to achieve resistance against multiple begomoviruses.

A novel prokaryotic promoter identified in the genome of some monopartite begomoviruses.

Geminiviruses are known to exhibit both prokaryotic and eukaryotic features in their genomes, with the ability to express their genes and even replicate in bacterial cells. We have demonstrated previously the existence of unit-length single-stranded circular DNAs of Ageratum yellow vein virus (AYVV, a species in the genus Begomovirus, family Geminiviridae) in Escherichia coli cells, which prompted our search for unknown prokaryotic functions in the begomovirus genomes. By using a promoter trapping strategy, we identified a novel prokaryotic promoter, designated AV3 promoter, in nts 762-831 of the AYVV genome. Activity assays revealed that the AV3 promoter is strong, unidirectional, and constitutive, with an endogenous downstream ribosome binding site and a translatable short open reading frame of eight amino acids. Sequence analyses suggested that the AV3 promoter might be a remnant of prokaryotic ancestors that could be related to certain promoters of bacteria from marine or freshwater environments. The discovery of the prokaryotic AV3 promoter provided further evidence for the prokaryotic origin in the evolutionary history of geminiviruses.

Identification of sequences required for AL2-mediated activation of the tomato golden mosaic virus-yellow vein BR1 promoter.

A 108 bp sequence has been identified in the tomato golden mosaic virus-yellow vein (yvTGMV) B component that is necessary and sufficient for AL2-mediated activation of the BR1 promoter. The sequence appears to have a bipartite arrangement, with elements located between -144 to -77 and -59 to -36 from the transcription start site, with both being required for activation by AL2. These sequences are located upstream of a TATA box and bind nuclear proteins from spinach, tomato and Arabidopsis. These sequences are also capable of binding Arabidopsis PPD2, which has been shown previously to interact with the yvTGMV coat protein (CP) promoter. We have identified two putative transcription factor-binding sites (CCAAT and GTGANTG10) that are conserved in sequences necessary for activation of the yvTGMV BR1, as well as the yvTGMV and cabbage leaf curl virus (CabLCV) CP promoters, which are all activated by AL2. The yvTGMV BR1 promoter exhibits AL2-independent expression in vascular tissue, similar to the yvTGMV, CabLCV and spinach curly top virus CP promoters. Together, this further confirms a common regulatory mechanism for AL2-mediated activation of bipartite begomovirus promoters.

Mapping of functional region conferring nuclear localization and karyopherin α-binding activity of the C2 protein of bhendi yellow vein mosaic virus.

Bhendi yellow vein mosaic disease is caused by a complex consisting of a monopartite begomovirus associated with a ß-satellite. The C2 protein of bhendi yellow vein mosaic virus (BYVMV) is a suppressor of post-transcriptional gene silencing and also functions as a transcriptional activator. To explore the molecular mechanisms of its nuclear trafficking and self-interaction, fusion proteins of fluorescent proteins with wild-type or mutated constructs of BYVMV C2 were expressed in tobacco protoplasts. Analyses revealed that the BYVMV C2 nuclear localization signal (NLS) was located in the N terminus of the protein, comprising aa 17-31 of C2. NLSs are recognized by a class of soluble transport receptors termed karyopherins α and ß. The BYVMV C2 NLS was found to be necessary for this protein's interaction with its nuclear import mediator, karyopherin α, ensuring its nuclear localization. Nevertheless, when deleted, C2 was found in both the cytoplasm and the nucleus, suggesting NLS-independent nuclear import of this protein. Homotypic interaction of BYVMV C2 was also found, which correlates with the nuclear localization needed for efficient activation of transcription.

Self-interaction of Abutilon mosaic virus replication initiator protein (Rep) in plant cell nuclei.

Geminiviruses replicate their circular single-stranded DNA genome in nuclei of infected plant cells. Their replication initiator proteins (Reps) possess interaction domains for homo- and hetero-oligomerization as shown previously by in vitro studies and yeast two hybrid assays. Here, homo-oligomerization and cellular localization of the Abutilon mosaic virus (AbMV) Rep was analysed with bimolecular fluorescence complementation (BiFC) in epidermal tissues of Nicotiana benthamiana. BiFC revealed that Rep oligomers accumulated within the nucleoplasm, but were excluded from nucleoli as indicated by a nucleoli/cajal body marker. A similar subcellular distribution was observed for Rep fused to full-length cyan fluorescent protein. To examine whether tagged Reps were functionally active, N. benthamiana plants transgenic for a dimeric AbMV DNA B were inoculated with the BiFC expression constructs and nucleic acids were analysed by rolling circle amplification/restriction fragment length polymorphism as well as Southern blot hybridization. The results confirmed that the modified AbMV Rep was able to transreplicate DNA B.

Detección, identificación y localización geográfica de Begomovirus que afectan al tomate en Colombia / Detection, identification and geographical localization of tomato-infecting Begomovirus in Colombia

En la actualidad, los Begomovirus (Familia Geminiviridae) se han convertido en la mayor amenaza para los cultivos de interés agrícola ubicados en las zonas tropicales y templadas del planeta. Estos virus son transmitidos por la mosca blanca Bemisia tabaci, la cual en los últimos años en Colombia ha tenido un aumento significativo en sus poblaciones y se ha asociado con la aparición de síntomas virales en cultivos de tomate. Muestras de tomate con síntomas virales típicos fueron recolectadas en las cinco principales zonas productoras de esta solanácea en el país. Los Begomovirus fueron detectados por medio de la técnica de hibridación de ácidos nucleicos tipo Dot blot así como por medio de la reacción en cadena de la polimerasa (PCR) en todas las muestras colectadas. Con la excepción de un reporte previo en el Valle del Cauca, este es el primer reporte de Begomovirus afectando cultivos de tomate en los departamentos de Antioquia, Santander, Boyacá y Cundinamarca. Asimismo, es la primera vez que se informa sobre Begomovirus que afectan cultivos de tomate localizados por encima de 1500 msnm en Colombia.

The begomoviruses (Family Geminiviridae) have recently emerged as samples with typical Begomovirus symptoms were collected in five different departments, comprising the mayor tomato growing areas of the country. Begomovirus were detected by Polymerase Chain Reaction (PCR) or Dot Blot Hybridization in all tomato samples collected in whole tomato growing areas of the country. With exception for Valle del Cauca, this is the first report of tomato-infecting Begomovirus in Antioquia, Santander, Boyacá and Cundinamarca departments. Also this is the first report of tomato-infecting Begomovirus crops located above 1500 masl in Colombia.

Tomato leaf curl Kerala virus (ToLCKeV) AC3 protein forms a higher order oligomer and enhances ATPase activity of replication initiator protein (Rep/AC1).

BACKGROUND: Geminiviruses are emerging plant viruses that infect a wide variety of vegetable crops, ornamental plants and cereal crops. They undergo recombination during co-infections by different species of geminiviruses and give rise to more virulent species. Antiviral strategies targeting a broad range of viruses necessitate a detailed understanding of the basic biology of the viruses. ToLCKeV, a virus prevalent in the tomato crop of Kerala state of India and a member of genus Begomovirus has been used as a model system in this study. RESULTS: AC3 is a geminiviral protein conserved across all the begomoviral species and is postulated to enhance viral DNA replication. In this work we have successfully expressed and purified the AC3 fusion proteins from E. coli. We demonstrated the higher order oligomerization of AC3 using sucrose gradient ultra-centrifugation and gel-filtration experiments. In addition we also established that ToLCKeV AC3 protein interacted with cognate AC1 protein and enhanced the AC1-mediated ATPase activity in vitro. CONCLUSIONS: Highly hydrophobic viral protein AC3 can be purified as a fusion protein with either MBP or GST. The purification method of AC3 protein improves scope for the biochemical characterization of the viral protein. The enhancement of AC1-mediated ATPase activity might lead to increased viral DNA replication.

A plastid-targeted heat shock cognate 70kDa protein interacts with the Abutilon mosaic virus movement protein.

The movement protein (MP) of bipartite geminiviruses facilitates cell-to-cell as well as long-distance transport within plants and influences viral pathogenicity. Yeast two-hybrid assays identified a chaperone, the nuclear-encoded and plastid-targeted heat shock cognate 70kDa protein (cpHSC70-1) of Arabidopsis thaliana, as a potential binding partner for the Abutilon mosaic virus (AbMV) MP. In planta, bimolecular fluorescence complementation (BiFC) analysis showed cpHSC70-1/MP complexes and MP homooligomers at the cell periphery and co-localized with chloroplasts. BiFC revealed cpHSC70-1 oligomers associated with chloroplasts, but also distributed at the cellular margin and in filaments arising from plastids reminiscent of stromules. Silencing the cpHSC70 gene of Nicotiana benthamiana using an AbMV DNA A-derived gene silencing vector induced minute white leaf areas, which indicate an effect on chloroplast stability. Although AbMV DNA accumulated within chlorotic spots, a spatial restriction of these occurred, suggesting a functional relevance of the MP-chaperone interaction for viral transport and symptom induction.

An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants.

A relatively quick and inexpensive modified cetyl trimethylammonium bromide method for extraction of DNA from leaf materials containing large quantities of mucilage is described. The modification including use of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 M NaCl, reduced markedly the viscosity of the mucilage and thus in the final purification step yielded a larger quantity of mucilage-free DNA suitable for subsequent PCR-based detection of begomoviruses. The method was standardized with jute samples with yellow mosaic disease and validated with different other mucilaginous-hosts with low titre of begomoviruses. DNA isolated using this method showed consistency in yield and compatibility with PCR for detection of begomoviruses from different mucilaginous plant species. The method was compared for efficacy with other reported methods and it was found to be superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus the method described could be used on a wider scale for reliable and consistent detection of begomoviruses from mucilaginous hosts for characterization and variability study.

Morphogenesis of growing soft tissues.

Recently, much attention has been given to a noteworthy property of some soft tissues: their ability to grow. Many attempts have been made to model this behavior in biology, chemistry, and physics. Using the theory of finite elasticity, Rodriguez has postulated a multiplicative decomposition of the geometric deformation gradient into a growth-induced part and an elastic one needed to ensure compatibility of the body. In order to fully explore the consequences of this hypothesis, the equations describing thin elastic objects under finite growth are derived. Under appropriate scaling assumptions for the growth rates, the proposed model is of the Föppl-von Kármán type. As an illustration, the circumferential growth of a free hyperelastic disk is studied.