Identification and localization of Tospovirus genus-wide conserved residues in 3D models of the nucleocapsid and the silencing suppressor proteins

Background: Tospoviruses (genus Tospovirus, family Peribunyaviridae, order Bunyavirales) cause significant losses to a wide range of agronomic and horticultural crops worldwide. Identification and characterization of specific sequences and motifs that are critical for virus infection and pathogenicity could provide useful insights and targets for engineering virus resistance that is potentially both broad spectrum and durable. Tomato spotted wilt virus (TSWV), the most prolific member of the group, was used to better understand the structure-function relationships of the nucleocapsid gene (N), and the silencing suppressor gene (NSs), coded by the TSWV small RNA. Methods: Using a global collection of orthotospoviral sequences, several amino acids that were conserved across the genus and the potential location of these conserved amino acid motifs in these proteins was determined. We used state of the art 3D modeling algorithms, MULTICOM-CLUSTER, MULTICOM-CONSTRUCT, MULTICOM-NOVEL, I-TASSER, ROSETTA and CONFOLD to predict the secondary and tertiary structures of the N and the NSs proteins. Results: We identified nine amino acid residues in the N protein among 31 known tospoviral species, and ten amino acid residues in NSs protein among 27 tospoviral species that were conserved across the genus. For the N protein, all three algorithms gave nearly identical tertiary models. While the conserved residues were distributed throughout the protein on a linear scale, at the tertiary level, three residues were consistently located in the coil in all the models. For NSs protein models, there was no agreement among the three algorithms. However, with respect to the localization of the conserved motifs, G was consistently located in coil, while H was localized in the coil in three models. Conclusions: This is the first report of predicting the 3D structure of any tospoviral NSs protein and revealed a consistent location for two of the ten conserved residues. The modelers used gave accurate prediction for N protein allowing the localization of the conserved residues. Results form the basis for further work on the structure-function relationships of tospoviral proteins and could be useful in developing novel virus control strategies targeting the conserved residues. 18 11

A plant virus protein, NIa-pro, interacts with Indole-3-acetic acid-amido synthetase, whose levels positively correlate with disease severity

Potato virus Y (PVY) is an economically important plant pathogen that reduces the productivity of several host plants. To develop PVY-resistant cultivars, it is essential to identify the plant-PVY interactome and decipher the biological significance of those molecular interactions. We performed a yeast two-hybrid (Y2H) screen of Nicotiana benthamiana cDNA library using PVY-encoded NIa-pro as the bait. The N. benthamiana Indole-3-acetic acid-amido synthetase (IAAS) was identified as an interactor of NIa-pro protein. The interaction was confirmed via targeted Y2H and bimolecular fluorescence complementation (BiFC) assays. NIa-pro interacts with IAAS protein and consequently increasing the stability of IAAS protein. Also, the subcellular localization of both NIa-pro and IAAS protein in the nucleus and cytosol was demonstrated. By converting free IAA (active form) to conjugated IAA (inactive form), IAAS plays a crucial regulatory role in auxin signaling. Transient silencing of IAAS in N. benthamiana plants reduced the PVY-mediated symptom induction and virus accumulation. Conversely, overexpression of IAAS enhanced symptom induction and virus accumulation in infected plants. In addition, the expression of auxin-responsive genes was found to be downregulated during PVY infection. Our findings demonstrate that PVY NIa-pro protein potentially promotes disease development via modulating auxin homeostasis

Transcriptome-wide identification of host genes targeted by tomato spotted wilt virus-derived small interfering RNAs

RNA silencing mechanism functions as a major defense against invading viruses. The caveat in the RNA silencing mechanism is that the effector small interfering RNAs (siRNAs) act on any RNA transcripts with sequence complementarity irrespective of target's origin. A subset of highly expressed viral small interfering RNAs (vsiRNAs) derived from the tomato spotted wilt virus (TSWV; Tospovirus: Bunyaviridae) genome was analyzed for their propensity to downregulate the tomato transcriptome. A total of 11898 putative target sites on tomato transcripts were found to exhibit a propensity for down regulation by TSWV-derived vsiRNAs. In total, 2450 unique vsiRNAs were found to have potential cross-reacting capability with the tomato transcriptome. VsiRNAs were found to potentially target a gamut of host genes involved in basal cellular activities including enzymes, transcription factors, membrane transporters, and cytoskeletal proteins. KEGG pathway annotation of targets revealed that the vsiRNAs were mapped to secondary metabolite biosynthesis, amino acids, starch and sucrose metabolism, and carbon and purine metabolism. Transcripts for protein processing, hormone signalling, and plant-pathogen interactions were the most likely targets from the genetic, environmental information processing, and organismal systems, respectively. qRT-PCR validation of target gene expression showed that none of the selected transcripts from tomato cv. Marglobe showed up regulation, and all were down regulated even upto 20 folds (high affinity glucose transporter). However, the expression levels of transcripts from cv. Red Defender revealed differential regulation as three among the target transcripts showed up regulation (Cc-nbs-lrr, resistance protein, AP2-like ethylene-responsive transcription factor, and heat stress transcription factor A3). Accumulation of tomato target mRNAs of corresponding length was proved in both tomato cultivars using 5′ RACE analysis. The TSWV-tomato interaction at the sRNA interface points to the ability of tomato cultivars to overcome vsiRNA-mediated targeting of NBS-LRR class R genes. These results suggest the prevalence of vsiRNA-induced RNA silencing of host transcriptome, and the interactome scenario is the first report on the interaction between tospovirus genome-derived siRNAs and tomato transcripts, and provide a deeper understanding of the role of vsiRNAs in pathogenicity and in perturbing host machinery

In silico Prediction and Validations of Domains Involved in Gossypium hirsutum SnRK1 Protein Interaction With Cotton Leaf Curl Multan Betasatellite Encoded βC1

Cotton leaf curl disease (CLCuD) caused by viruses of genus Begomovirus is a major constraint to cotton (Gossypium hirsutum) production in many cotton-growing regions of the world. Symptoms of the disease are caused by Cotton leaf curl Multan betasatellite (CLCuMB) that encodes a pathogenicity determinant protein, βC1. Here, we report the identification of interacting regions in βC1 protein by using computational approaches including sequence recognition, and binding site and interface prediction methods. We show the domain-level interactions based on the structural analysis of G. hirsutum SnRK1 protein and its domains with CLCuMB-βC1. To verify and validate the in silico predictions, three different experimental approaches, yeast two hybrid, bimolecular fluorescence complementation and pull down assay were used. Our results showed that ubiquitin-associated domain (UBA) and autoinhibitory sequence (AIS) domains of G. hirsutum-encoded SnRK1 are involved in CLCuMB-βC1 interaction. This is the first comprehensive investigation that combined in silico interaction prediction followed by experimental validation of interaction between CLCuMB-βC1 and a host protein. We demonstrated that data from computational biology could provide binding site information between CLCuD-associated viruses/satellites and new hosts that lack known binding site information for protein–protein interaction studies. Implications of these findings are discussed

Ortervirales: A new viral order unifying five families of reverse-transcribing viruses

Reverse-transcribing viruses, which synthesize a copy of genomic DNA from an RNA template, are widespread in animals, plants, algae and fungi (1, 2).

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Not AvailableBackground Tomato spotted wilt virus (TSWV; Tospovirus: Bunyaviridae) has been an economically important virus in the USA for over 30 years. However the complete sequence of only one TSWV isolate PA01 characterized from pepper in Pennsylvania is available. Results The large (L) RNA of a TSWV WA-USA isolate was cloned and sequenced. It consisted of 8914 nucleotides (nt) encoding a single open reading frame of 8640 nts in the viral-complementary sense. The ORF potentially codes for RNA-dependent RNA polymerase (RdRp) of 330.9 kDa. Two untranslated regions of 241 and 33 nucleotides were present at the 5′ and 3′ termini, respectively that shared conserved tospoviral sequences. Phylogenetic analysis using nucleotide sequences of the complete L RNA showed that TSWV WA-USA isolate clustered with the American and Asian TSWV isolates which formed a distinct clade from Euro-Asiatic Tospoviruses. Phylogeny of the amino acid sequence of all tospoviral RdRps used in this study showed that all the known TSWV isolates including the USA isolate described in this study formed a distinct and a close cluster with that of Impateins necrotic spot virus. Multiple sequence alignment revealed conserved motifs in the RdRp of TSWV. Recombination analysis identified two recombinants including the TSWV WA-USA isolate. Among them, three recombination events were detected in the conserved motifs of the RdRp. Conclusions Sequence analysis and phylogenetic analysis of the L RNA showed distinct clustering with selected TSWV isolates reported from elsewhere. Conserved motifs in the core polymerase region of the RdRp and recombination events were identifiedDBT CREST AWAR

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Annual meeting of the American Phytopathological Society, Minneapolis Convention Center, Minneapolis, MN, August 9-13, 2014Small non-coding RNAs are important effector molecules in response to pathogen invasion in plants and animals. We conducted in silico analysis of the DNA genomes of two distinct species of genus Begomovirus (family Geminiviridae)-Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV)-that infect soybean using a micro-RNA (miRNA) target prediction algorithm, plant small RNA target analysis server. MYMV displays greater vulnerability to plant miRNAs with 99 miRNAs targeting its genome, whereas 70 miRNAs appear to be targeting the MYMIV genome. miRNAs derived from Glycine max, Glycine soja and Cajanus cajan display 63, 18, and 8 potential target sites on the begomovirus genomes. Among the non-host plants, begomoviruses exhibit seven and six potential target sites for O. sativa, and P. trichocarpa-derived miRNAs respectivelyNot Availabl

Sequence characterization, molecular phylogeny reconstruction and recombination analysis of the large RNA of Tomato spotted wilt virus (Tospovirus: Bunyaviridae) from the United States

Tomato spotted wilt virus (TSWV; Tospovirus: Bunyaviridae) has been an economically important virus in the USA for over 30 years. However the complete sequence of only one TSWV isolate PA01 characterized from pepper in Pennsylvania is available. The large (L) RNA of a TSWV WA-USA isolate was cloned and sequenced. It consisted of 8914 nucleotides (nt) encoding a single open reading frame of 8640 nts in the viral-complementary sense. The ORF potentially codes for RNA-dependent RNA polymerase (RdRp) of 330.9 kDa. Two untranslated regions of 241 and 33 nucleotides were present at the 5' and 3' termini, respectively that shared conserved tospoviral sequences. Phylogenetic analysis using nucleotide sequences of the complete L RNA showed that TSWV WA-USA isolate clustered with the American and Asian TSWV isolates which formed a distinct clade from Euro-Asiatic Tospoviruses. Phylogeny of the amino acid sequence of all tospoviral RdRps used in this study showed that all the known TSWV isolates including the USA isolate described in this study formed a distinct and a close cluster with that of Impateins necrotic spot virus. Multiple sequence alignment revealed conserved motifs in the RdRp of TSWV. Recombination analysis identified two recombinants including the TSWV WA-USA isolate. Among them, three recombination events were detected in the conserved motifs of the RdRp. Sequence analysis and phylogenetic analysis of the L RNA showed distinct clustering with selected TSWV isolates reported from elsewhere. Conserved motifs in the core polymerase region of the RdRp and recombination events were identified

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Not AvailableTomato spotted wilt virus (TSWV; Tospovirus:Bunyaviridae) is one of the most prolific and economically important viruses of field and horticultural crops. In the plant-virus interactions, virus-derived siRNAs (vsiRNAs) are the result of activity of host-mediated silencing mechanism. We recently obtained the TSWV-specific small RNA profiles from virus-infected tomato. A subset of siRNAs derived from the TSWV genome hotspots was analyzed in silico for their propensity to down regulate tomato transcriptome. vsiRNAs were found to interact with a gamut of host genes involved in basal cellular activities such as nucleic acid metabolism, ribosomal turnover, translational factors, cytoskeletal proteins, phenylpropanoid biosynthesis, glycosyl transferases, peptidases, hormonal signalling, protein kinases, intercellular transporter genes, and stress-related proteins. Notably, vsiRNAs derived from the TSWV NSs gene binds with transcripts generally associated with stress signalling, whereas siRNAs from NSm were predominantly found to bind the transcripts involved in abiotic stress responses such as dehydration responsive protein, ion exchange transporters, and low temperature and salt responsive proteins. The predicted interactome scenario when validated through gene expression studies using RNAseq could provide a detailed picture on the molecular mechanism underlying the tospovirus-plant interactions.Not Availabl