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

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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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

Movement and nucleocapsid proteins coded by two tospovirus species interact through multiple binding regions in mixed infections

Negative-stranded tospoviruses (family: Bunyaviridae) are among the most agronomically important viruses. Some of the tospoviruses are known to exist as mixed infections in the same host plant. Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV) were used to study virus-virus interaction in dually infected host plants. Viral genes of both viruses were separately cloned into binary pSITE-BiFC vectors. BiFC results showed that the N and NSm proteins of IYSV interact with their counterparts coded by TSWV in dually infected Nicotiana benthamiana plants. BiFC results were further confirmed by pull down and yeast-2-hybrid (Y2H) assays. Interacting regions of the N and NSm proteins were also identified by Y2H system and β-galactosidase activity. Several regions of the N and NSm were found interacting with each other. The regions involved in these interactions are presumed to be critical for the functioning of the tospovirus N and NSm proteins. This is the first report of in vivo protein interactions of distinct tospoviruses in mixed infection

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Not AvailablePotato mop-top virus (PMTV) is the type species of genus Pomovirus, family Virgaviridae. The occurrence of PMTV was reported recently in the Pacific Northwestern USA. PMTV genome is characterized by three linear positive sense ssRNA molecules. As part of a study to better understand the virus biology and molecular biology, the genome of a WA isolate was characterized and we report the complete nucleotide (nt) sequence of the genomic components RNA-2, and RNA-3. RNA2 was 3134nt in length with an ORF between nt positions 314-844 that codes for the coat protein and the same ORF as a read-through extends further to nt position 2791 coding for the CPread through protein. RNA 3 was 2964nt in length and potentially encodes four ORFs – three of them forming the triple gene bock (TGB) that was shown to be involved in cell to cell movement. RNA2 also contains a 206 nt ORF present toward the 3’ end that encodes a cysteine-rich 8K protein. RFLP analysis of RNA2 and RNA 3 of the WA isolate revealed the combination of RNA2-II and RNA3-B types, the combination that is prevalent in Europe and elsewhere. The sequenced regions had 99% sequence identity with the corresponding RNA components reported from Europe. The high degree of sequence identity could be useful in developing virus detection tools and for designing RNAi-based virus management strategies.Not Availabl

Complementation between two tospoviruses facilitates the systemic movement of a plant virus silencing suppressor in an otherwise restrictive host

New viruses pathogenic to plants continue to emerge due to mutation, recombination, or reassortment among genomic segments among individual viruses. Tospoviruses cause significant economic damage to a wide range of crops in many parts of the world. The genetic or molecular basis of the continued emergence of new tospoviruses and new hosts is not well understood though it is generally accepted that reassortment and/or genetic complementation among the three genomic segments of individual viruses could be contributing to this variability since plants infected with more than one tospovirus are not uncommon in nature. Two distinct and economically important tospoviruses, Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV), were investigated for inter-virus interactions at the molecular level in dually-infected plants. Datura (Datura stramonium) is a permissive host for TSWV, while it restricts the movement of IYSV to inoculated leaves. In plants infected with both viruses, however, TSWV facilitated the selective movement of the viral gene silencing suppressor (NSs) gene of IYSV to the younger, uninoculated leaves. The small RNA expression profiles of IYSV and TSWV in single- and dually-infected datura plants showed that systemic leaves of dually-infected plants had reduced levels of TSWV N gene-specific small interfering RNAs (siRNAs). No TSWV NSs-specific siRNAs were detected either in the inoculated or systemic leaves of dually-infected datura plants indicating a more efficient suppression of host silencing machinery in the presence of NSs from both viruses as compared to the presence of only TSWV NSs. Our study identifies a new role for the viral gene silencing suppressor in potentially modulating the biology and host range of viruses and underscores the importance of virally-coded suppressors of gene silencing in virus infection of plants. This is the first experimental evidence of functional complementation between two distinct tospoviruses in the Bunyaviridae family