Publications by authors named "Hanu R Pappu"

45 Publications

Induction of Plant Resistance in Tobacco against Tomato Spotted Wilt Orthotospovirus through Foliar Application of dsRNA.

Viruses 2021 04 12;13(4). Epub 2021 Apr 12.

Department of Plant Pathology, Washington State University, Pullman, WA 99163, USA.

Thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) continues to be a constraint to peanut, pepper, tobacco, and tomato production in Georgia and elsewhere. TSWV is being managed by an integrated disease management strategy that includes a combination of cultural practices, vector management, and growing virus-resistant varieties where available. We used a non-transgenic strategy to induce RNA interference (RNAi)-mediated resistance in tobacco () plants against TSWV. Double-stranded RNA (dsRNA) molecules for the NSs (silencing suppressor) and N (nucleoprotein) genes were produced by a two-step PCR approach followed by in vitro transcription. When topically applied to tobacco leaves, both molecules elicited a resistance response. Host response to the treatments was measured by determining the time to symptom expression, and the level of resistance by absolute quantification of the virus. We also show the systemic movement of dsRNA_N from the inoculated leaves to younger, non-inoculated leaves. Post-application, viral siRNAs were detected for up to nine days in inoculated leaves and up to six days in non-inoculated leaves. The topical application of dsRNAs to induce RNAi represents an environmentally safe and efficient way to manage TSWV in tobacco crops and could be applicable to other TSWV-susceptible crops.
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http://dx.doi.org/10.3390/v13040662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069313PMC
April 2021

Current Status and Potential of RNA Interference for the Management of Tomato Spotted Wilt Virus and Thrips Vectors.

Pathogens 2021 Mar 9;10(3). Epub 2021 Mar 9.

Centre for Horticultural Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4067, Australia.

Tomato spotted wilt virus (TSWV) is the type member of the genus in the family and order Bunyavirales. TSWV, transmitted by several species of thrips, causes significant disease losses to agronomic and horticultural crops worldwide, impacting both the yield and quality of the produce. Management strategies include growing virus-resistant cultivars, cultural practices, and managing thrips vectors through pesticide application. However, numerous studies have reported that TSWV isolates can overcome host-plant resistance, while thrips are developing resistance to pesticides that were once effective. RNA interference (RNAi) offers a means of host defence by using double-stranded (ds) RNA to initiate gene silencing against invading viruses. However, adoption of this approach requires production and use of transgenic plants and thus limits the practical application of RNAi against TSWV and other viruses. To fully utilize the potential of RNAi for virus management at the field level, new and novel approaches are needed. In this review, we summarize RNAi and highlight the potential of topical or exogenous application of RNAi triggers for managing TSWV and thrips vectors.
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http://dx.doi.org/10.3390/pathogens10030320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001667PMC
March 2021

Virus and Viroid-Derived Small RNAs as Modulators of Host Gene Expression: Molecular Insights Into Pathogenesis.

Front Microbiol 2020 14;11:614231. Epub 2021 Jan 14.

Department of Plant Pathology, Washington State University, Pullman, WA, United States.

Virus-derived siRNAs (vsiRNAs) generated by the host RNA silencing mechanism are effectors of plant's defense response and act by targeting the viral RNA and DNA in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) pathways, respectively. Contrarily, viral suppressors of RNA silencing (VSRs) compromise the host RNA silencing pathways and also cause disease-associated symptoms. In this backdrop, reports describing the modulation of plant gene(s) expression by vsiRNAs via sequence complementarity between viral small RNAs (sRNAs) and host mRNAs have emerged. In some cases, silencing of host mRNAs by vsiRNAs has been implicated to cause characteristic symptoms of the viral diseases. Similarly, viroid infection results in generation of sRNAs, originating from viroid genomic RNAs, that potentially target host mRNAs causing typical disease-associated symptoms. Pathogen-derived sRNAs have been demonstrated to have the propensity to target wide range of genes including host defense-related genes, genes involved in flowering and reproductive pathways. Recent evidence indicates that vsiRNAs inhibit host RNA silencing to promote viral infection by acting as decoy sRNAs. Nevertheless, it remains unclear if the silencing of host transcripts by viral genome-derived sRNAs are inadvertent effects due to fortuitous pairing between vsiRNA and host mRNA or the result of genuine counter-defense strategy employed by viruses to enhance its survival inside the plant cell. In this review, we analyze the instances of such cross reaction between pathogen-derived vsiRNAs and host mRNAs and discuss the molecular insights regarding the process of pathogenesis.
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http://dx.doi.org/10.3389/fmicb.2020.614231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874048PMC
January 2021

Identification and Characterization of Plant-Interacting Targets of Tomato Spotted Wilt Virus Silencing Suppressor.

Pathogens 2021 Jan 1;10(1). Epub 2021 Jan 1.

Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA.

Tomato spotted wilt virus (TSWV; species ) is an economically important plant virus that infects multiple horticultural crops on a global scale. TSWV encodes a non-structural protein NSs that acts as a suppressor of host RNA silencing machinery during infection. Despite extensive structural and functional analyses having been carried out on TSWV NSs, its protein-interacting targets in host plants are still largely unknown. Here, we systemically investigated NSs-interacting proteins in via affinity purification and mass spectrometry (AP-MS) analysis. Forty-three TSWV NSs-interacting candidates were identified in . Gene Ontology (GO) and protein-protein interaction (PPI) network analyses were carried out on their closest homologs in tobacco (), tomatoes () and (). The results showed that NSs preferentially interacts with plant defense-related proteins such as calmodulin (CaM), importin, carbonic anhydrase and two heat shock proteins (HSPs): HSP70 and HSP90. As two major nodes in the PPI network, CaM and importin subunit α were selected for the further verification of their interactions with NSs via yeast two-hybrid (Y2H) screening. Our work suggests that the downstream signaling, transportation and/or metabolic pathways of host-NSs-interacting proteins may play critical roles in NSs-facilitated TSWV infection.
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http://dx.doi.org/10.3390/pathogens10010027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823891PMC
January 2021

Emerging Molecular Links Between Plant Photomorphogenesis and Virus Resistance.

Front Plant Sci 2020 30;11:920. Epub 2020 Jun 30.

Department of Plant Pathology, Washington State University, Pullman, WA, United States.

Photomorphogenesis refers to photoreceptor-mediated morphological changes in plant development that are triggered by light. Multiple photoreceptors and transcription factors (TFs) are involved in the molecular regulation of photomorphogenesis. Likewise, light can also modulate the outcome of plant-virus interactions since both photosynthesis and many viral infection events occur in the chloroplast. Despite the apparent association between photosynthesis and virus infection, little is known about whether there are also interplays between photomorphogenesis and plant virus resistance. Recent research suggests that plant-virus interactions are potentially regulated by several photoreceptors and photomorphogenesis regulators, including phytochromes A and B (PHYA and PHYB), cryptochromes 2 (CRY2), phototropin 2 (PHOT2), the photomorphogenesis repressor constitutive photomorphogenesis 1 (COP1), the NAM, ATAF, and CUC (NAC)-family TF ATAF2, the Aux/IAA protein phytochrome-associated protein 1 (PAP1), the homeodomain-leucine zipper (HD-Zip) TF HAT1, and the core circadian clock component circadian clock associated 1 (CCA1). Particularly, the plant growth promoting brassinosteroid (BR) hormones play critical roles in integrating the regulatory pathways of plant photomorphogenesis and viral defense. Here, we summarize the current understanding of molecular mechanisms linking plant photomorphogenesis and defense against viruses, which represents an emerging interdisciplinary research topic in both molecular plant biology and virology.
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http://dx.doi.org/10.3389/fpls.2020.00920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338571PMC
June 2020

The (TSWV) Genome is Differentially Targeted in TSWV-Infected Tomato () with or without Gene.

Viruses 2020 03 26;12(4). Epub 2020 Mar 26.

Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA.

Tospoviruses cause significant losses to a wide range of agronomic and horticultural crops worldwide. The type member, (TSWV), causes systemic infection in susceptible tomato cultivars, whereas its infection is localized in cultivars carrying the 5 resistance gene. The response to TSWV infection in tomato cultivars with or without -5 was determined at the virus small RNA level in the locally infected leaf. Predicted reads were aligned to TSWV reference sequences. The TSWV genome was found to be differentially processed among each of the three-viral genomic RNAs-Large (L), Medium (M) and Small (S)-in the -5(+) compared to -5(-) genotypes. In the -5(+) cultivar, the L RNA had the highest number of viral small-interfering RNAs (vsiRNAs), whereas in the -5(-) cultivar the number was higher in the S RNA. Among the three-viral genomic RNAs, the distribution of hotspots showed a higher number of reads per million reads of vsiRNAs of 21 and 22 nt class at the 5' and 3' ends of the L and the S RNAs, with less coverage in the M RNA. In the -5(-) cultivar, the nature of the 5' nucleotide-end in the siRNAs varied significantly; reads with 5'-adenine-end were most abundant in the mock control, whereas cytosine and uracil were more abundant in the infected plants. No such differences were seen in case of the resistant genotype. Findings provided insights into the response of tomato cultivars to TSWV infection.
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http://dx.doi.org/10.3390/v12040363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232525PMC
March 2020

βC1, pathogenicity determinant encoded by Cotton leaf curl Multan betasatellite, interacts with calmodulin-like protein 11 (Gh-CML11) in Gossypium hirsutum.

PLoS One 2019 3;14(12):e0225876. Epub 2019 Dec 3.

National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan.

Begomoviruses interfere with host plant machinery to evade host defense mechanism by interacting with plant proteins. In the old world, this group of viruses are usually associated with betasatellite that induces severe disease symptoms by encoding a protein, βC1, which is a pathogenicity determinant. Here, we show that βC1 encoded by Cotton leaf curl Multan betasatellite (CLCuMB) requires Gossypium hirsutum calmodulin-like protein 11 (Gh-CML11) to infect cotton. First, we used the in silico approach to predict the interaction of CLCuMB-βC1 with Gh-CML11. A number of sequence- and structure-based in-silico interaction prediction techniques suggested a strong putative binding of CLCuMB-βC1 with Gh-CML11 in a Ca+2-dependent manner. In-silico interaction prediction was then confirmed by three different experimental approaches: The Gh-CML11 interaction was confirmed using CLCuMB-βC1 in a yeast two hybrid system and pull down assay. These results were further validated using bimolecular fluorescence complementation system showing the interaction in cytoplasmic veins of Nicotiana benthamiana. Bioinformatics and molecular studies suggested that CLCuMB-βC1 induces the overexpression of Gh-CML11 protein and ultimately provides calcium as a nutrient source for virus movement and transmission. This is the first comprehensive study on the interaction between CLCuMB-βC1 and Gh-CML11 proteins which provided insights into our understating of the role of βC1 in cotton leaf curl disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0225876PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890265PMC
March 2020

Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development.

PLoS One 2019 23;14(10):e0223765. Epub 2019 Oct 23.

Department of Plant Pathology, Washington State University, Pullman, WA, United States of America.

Whitefly-transmitted begomoviruses cause serious damage to many economically important food, feed, and fiber crops. Numerous vegetable crops are severely affected and chilli leaf curl virus (ChiLCV) is the most dominant and widely distributed begomovirus in chilli (Capsicum annuum) throughout the Indian subcontinent. Recently, CRISPR-Cas9 technology was used as a means to reduce geminivirus replication in infected plants. However, this approach was shown to have certain limitations such as the evolution of escape mutants. In this study, we used a novel, multiplexed guide RNA (gRNA) based CRISPR-Cas9 approach that targets the viral genome at two or more sites simultaneously. This tactic was effective in eliminating the ChiLCV genome without recurrence of functional escape mutants. Six individual gRNA spacer sequences were designed from the ChiLCV genome and in vitro assays confirmed the cleavage behaviour of these spacer sequences. Multiplexed gRNA expression clones, based on combinations of the above-mentioned spacer sequences, were developed. A total of nine-duplex and two-triplex CRISPR-Cas9 constructs were made. The efficacy of these constructs was tested for inhibition of ChiLCV infection in Nicotiana benthamiana. Results indicated that all the constructs caused a significant reduction in viral DNA accumulation. In particular, three constructs (gRNA5+4, gRNA5+2 and gRNA1+2) were most effective in reducing the viral titer and symptoms. T7E1 assay and sequencing of the targeted viral genome did not detect any escape mutants. The multiplexed genome-editing technique could be an effective way to trigger a high level of resistance against begemoviruses. To our knowledge, this is the first report of demonstrating the effectiveness of a multiplexed gRNA-based plant virus genome editing to minimize and eliminate escape mutant formation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0223765PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6808502PMC
March 2020

Putative Auxin and Light Responsive Promoter Elements From the Genome, When Expressed as cDNA, Are Functional in .

Front Plant Sci 2019 28;10:804. Epub 2019 Jun 28.

Department of Plant Pathology, Washington State University, Pullman, WA, United States.

Members of the virus order cause serious diseases in animals, humans and plants. Family in this order contains only one genus , and members in this genus exclusively infect plants. (TSWV) is considered one of the most economically important plants viruses. Little is known about the regulatory elements in the TSWV genome. Here we show that, when in the cDNA form, the 5'-upstream region of the TSWV-coded G/G gene (pG/G) possesses putative -regulatory elements, including an auxin responsive element (AuxRE) for binding of auxin response factors (ARFs), as well as a circadian clock-associated 1 (CCA1) protein binding site (CBS). Due to the lack of a reverse genetics system, we verified the functionality of these elements in Arabidopsis. pG/G showed light-suppressive promoter activity in transgenic Arabidopsis, and mutation in the CBS was sufficient to switch the activity to light inducible. Additionally, exogenous auxin treatments repressed the promoter activity of both wild type and CBS-mutated pG/G. Mutation in AuxRE in both promoters abolished their sensitivity to auxin. As transcriptional repressors, both CCA1 and ARF2 were able to bind to pG/G directly. To our knowledge, this is the first report that a 5'-terminal sequence of an RNA virus has light-and hormone-responsive promoter activities when expressed as cDNA in host plant's nuclear background. Our findings suggest new clues on the possible origin, evolution and function of the TSWV genomic sequence and its non-coding regions.
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http://dx.doi.org/10.3389/fpls.2019.00804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611158PMC
June 2019

Prediction and Validations of Domains Involved in SnRK1 Protein Interaction With Cotton Leaf Curl Multan Betasatellite Encoded βC1.

Front Plant Sci 2019 28;10:656. Epub 2019 May 28.

National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan.

Cotton leaf curl disease (CLCuD) caused by viruses of genus is a major constraint to cotton () 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 SnRK1 protein and its domains with CLCuMB-βC1. To verify and validate the 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 -encoded SnRK1 are involved in CLCuMB-βC1 interaction. This is the first comprehensive investigation that combined 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.
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http://dx.doi.org/10.3389/fpls.2019.00656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546731PMC
May 2019

Complete genome characterization and population dynamics of potato virus Y-NTN strain from India.

Virusdisease 2019 Jun 5;30(2):252-260. Epub 2019 Apr 5.

1Department of Plant Pathology, Washington State University, Pullman, WA USA.

Potato virus Y (PVY) is a major threat to potato cultivation worldwide. PVY exists as biologically and genetically distinct strains and causes varying degrees of pathogenicity and a wide range of symptoms in potato. Knowledge of the nature of PVY strains is essential for breeding PVY resistant cultivars that are durable against a wide range of strains. We report the complete genome of a PVY potato isolate (JK12) characterised from the potato production areas of Jammu and Kashmir, India. Nucleotide sequence comparisons and phylogenetic analysis with known PVY strains revealed that the isolate belongs to the NTN strain of PVY. At the whole genome sequence level, the JK12 isolate shared the highest identity (99.42%) with PVY-NTN strains reported from Germany, followed by those from United Kingdom (99.34%) and Japan (99.33%). Recombination detection analysis identified two recombination break points and JK12 appeared to have originated from a recombination event between a PVY-N strain from Belgium as a major parent and a PVY-O strain from China as the minor parent. Our results suggest possible mutation and recombination could be the basis for the evolution and the subsequent establishment of NTN in this region. Furthermore, a global evolutionary lineage analysis of all the known PVY strains showed relatively low nucleotide diversity among the PVY-NTN strains. Neutrality tests showed that all the genotypes of PVY are undergoing purifying selection suggesting population expansion of PVY. This is the first report of complete genomic characterization of an NTN strain of PVY isolated from commercial potato fields in India. The implications of the emergence of this strain in the Indian context are discussed.
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http://dx.doi.org/10.1007/s13337-019-00526-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531552PMC
June 2019

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

Virol J 2019 01 11;16(1). Epub 2019 Jan 11.

Department of Plant Pathology, Washington State University, Pullman, WA, 99164, USA.

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.
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http://dx.doi.org/10.1186/s12985-018-1106-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330412PMC
January 2019

A novel fungal effector from Puccinia graminis suppressing RNA silencing and plant defense responses.

New Phytol 2019 05 13;222(3):1561-1572. Epub 2019 Feb 13.

Department of Plant Pathology, Washington State University, Pullman, WA, 99164-6430, USA.

Fungal plant pathogens, like rust-causing biotrophic fungi, secrete hundreds of effectors into plant cells to subvert host immunity and promote pathogenicity on their host plants by manipulating specific physiological processes or signal pathways, but the actual function has been demonstrated for very few of these proteins. Here, we show that the PgtSR1 effector proteins, encoded by two allelic genes (PgtSR1-a and PgtSR1-b), from the wheat stem rust pathogen Puccinia graminis f. sp. tritici (Pgt), suppress RNA silencing in plants and impede plant defenses by altering the abundance of small RNAs that serve as defense regulators. Expression of the PgtSR1s in plants revealed that the PgtSR1s promote susceptibility to multiple pathogens and partially suppress cell death triggered by multiple R proteins. Overall, our study provides the first evidence that the filamentous fungus P. graminis has evolved to produce fungal suppressors of RNA silencing and indicates that PgtSR1s suppress both basal defenses and effector triggered immunity.
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http://dx.doi.org/10.1111/nph.15676DOI Listing
May 2019

Deriving Economic Models for Pea Aphid (Hemiptera: Aphididae) as a Direct-Pest and a Virus-Vector on Commercial Lentils.

J Econ Entomol 2018 09;111(5):2225-2232

Department of Entomology, Plant Pathology and Nematology, University of Idaho, Moscow, ID.

The pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), presents a dual threat to commercial pulse growers because it can inflict direct injury through feeding and indirect injury as a vector of two important viruses, Pea enation mosaic virus (PEMV) and Bean leafroll virus (BLRV). A decision support system is needed to help producers manage both of these threats in pulses. To address these gaps in lentil, Lens culinaris (Medikus) (Fabales: Fabaceae), we conducted field experiments near Moscow, Idaho in 2011 and 2012 with three objectives: 1) determine economic injury levels (EILs) for pea aphid in lentil based on the direct effects of their feeding on yield, 2) develop economic guidelines for treating aphids carrying PEMV or BLRV based on the impact on yield of virus inoculation at different times after crop emergence, and 3) provide a framework for using both of these decision tools as part of a comprehensive approach to pea aphid management in lentil. EILs were determined based on data from replicated field cage trials over 2 yr. Windows of economic vulnerability to viruses were determined based on artificial inoculation with viruses at different days after crop emergence over 2 yr. Both direct and indirect injury support tools can be parameterized with potential yields, market prices, and the costs of insecticide applications to guide treatment decisions. Together, the two tools comprise a decision support system for managing pea aphid acting as both a direct pest and as a vector of the viruses in lentils in the Palouse region of northern Idaho and southeastern Washington State.
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http://dx.doi.org/10.1093/jee/toy188DOI Listing
September 2018

Whole-Genome Characterization of Prunus necrotic ringspot virus Infecting Sweet Cherry in China.

Genome Announc 2018 Mar 1;6(9). Epub 2018 Mar 1.

Key Laboratory for Fruit Biotechnology Breeding of Shandong Province, Shandong Institute of Pomology, Taian, Shandong, China

(PNRSV) causes yield loss in most cultivated stone fruits, including sweet cherry. Using a small RNA deep-sequencing approach combined with end-genome sequence cloning, we identified the complete genomes of all three PNRSV strands from PNRSV-infected sweet cherry trees and compared them with those of two previously reported isolates.
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http://dx.doi.org/10.1128/genomeA.00060-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834323PMC
March 2018

Geminiviruses and Plant Hosts: A Closer Examination of the Molecular Arms Race.

Viruses 2017 09 15;9(9). Epub 2017 Sep 15.

Department of Plant Pathology, Washington State University, Pullman, WA 99163, USA.

Geminiviruses are plant-infecting viruses characterized by a single-stranded DNA (ssDNA) genome. Geminivirus-derived proteins are multifunctional and effective regulators in modulating the host cellular processes resulting in successful infection. Virus-host interactions result in changes in host gene expression patterns, reprogram plant signaling controls, disrupt central cellular metabolic pathways, impair plant's defense system, and effectively evade RNA silencing response leading to host susceptibility. This review summarizes what is known about the cellular processes in the continuing tug of war between geminiviruses and their plant hosts at the molecular level. In addition, implications for engineered resistance to geminivirus infection in the context of a greater understanding of the molecular processes are also discussed. Finally, the prospect of employing geminivirus-based vectors in plant genome engineering and the emergence of powerful genome editing tools to confer geminivirus resistance are highlighted to complete the perspective on geminivirus-plant molecular interactions.
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http://dx.doi.org/10.3390/v9090256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5618022PMC
September 2017

The effects of potato virus Y-derived virus small interfering RNAs of three biologically distinct strains on potato (Solanum tuberosum) transcriptome.

Virol J 2017 07 17;14(1):129. Epub 2017 Jul 17.

Department of Plant Pathology, Washington State University, Pullman, WA, 99164, USA.

Background: Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome.

Methods: The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR.

Results: The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3' UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic information processing, plant-pathogen interactions, plant defense and stress response processes in potato.

Conclusions: The findings suggested that the PVY-derived vsiRNAs could act as a pathogenicity determinant and as a counter-defense strategy to host RNA silencing in PVY-potato interactions. The broad range of host genes targeted by PVY vsiRNAs in infected potato suggests a diverse role for vsiRNAs that includes suppression of host stress responses and developmental processes. The interactome scenario is the first report on the interaction between one of the most important Potyvirus genome-derived siRNAs and the potato transcripts.
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http://dx.doi.org/10.1186/s12985-017-0803-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513076PMC
July 2017

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

Virus Res 2017 06 22;238:13-23. Epub 2017 May 22.

Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.virusres.2017.05.014DOI Listing
June 2017

The Tomato Spotted Wilt Virus Genome Is Processed Differentially in its Plant Host Arachis hypogaea and its Thrips Vector Frankliniella fusca.

Front Plant Sci 2016 7;7:1349. Epub 2016 Sep 7.

Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia QLD, Australia.

Thrips-transmitted tospoviruses are economically important viruses affecting a wide range of field and horticultural crops worldwide. Tomato spotted wilt virus (TSWV) is the type member of the Tospovirus genus with a broad host range of more than 900 plant species. Interactions between these viruses and their plant hosts and insect vectors via RNAi pathways are likely a key determinant of pathogenicity. The current investigation, for the first time, compares biogenesis of small RNAs between the plant host and insect vector in the presence or absence of TSWV. Unique viral small interfering RNA (vsiRNA) profiles are evident for Arachis hypogaea (peanut) and Frankliniella fusca (thrips vector) following infection with TSWV. Differences between vsiRNA profiles for these plant and insect species, such as the relative abundance of 21 and 22 nt vsiRNAs and locations of alignment hotspots, reflect the diverse siRNA biosynthesis pathways of their respective kingdoms. The presence of unique vsiRNAs in F. fusca samples indicates that vsiRNA generation takes place within the thrips, and not solely through uptake via feeding on vsiRNAs produced in infected A. hypogaea. The study also shows key vsiRNA profile differences for TSWV among plant families, which are evident in the case of A. hypogaea, a legume, and members of Solanaceae (S. lycopersicum and Nicotiana benthamiana). Distinctively, overall small RNA (sRNA) biogenesis in A. hypogaea is markedly affected with an absence of the 24 nt sRNAs in TSWV-infected plants, possibly leading to wide-spread molecular and phenotypic perturbations specific to this species. These findings add significant information on the host-virus-vector interaction in terms of RNAi pathways and may lead to better crop and vector specific control strategies.
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http://dx.doi.org/10.3389/fpls.2016.01349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5013717PMC
September 2016

Complete Genomic Characterization of Plum bark necrosis stem pitting-associated virus Infecting Sweet Cherry in China.

Genome Announc 2016 May 19;4(3). Epub 2016 May 19.

Key Laboratory for Fruit Biotechnology Breeding of Shandong Province, Shandong Institute of Pomology, Taian, Shandong, China

Plum bark necrosis stem pitting-associated virus (PBNSPaV) causes the plum bark necrosis stem pitting-associated disease. We obtained the complete genome of a PBNSPaV isolate (PBNSPaV-TA) using small RNA deep sequencing followed by overlapping RT-PCR. To our knowledge, this is the first report of a completed genome of PBNSPaV identified from cherry trees.
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http://dx.doi.org/10.1128/genomeA.00413-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888985PMC
May 2016

Structure and Genome Organization of Cherry Virus A (Capillovirus, Betaflexiviridae) from China Using Small RNA Sequencing.

Genome Announc 2016 May 12;4(3). Epub 2016 May 12.

Key Laboratory for Fruit Biotechnology Breeding of Shandong Province, Shandong Institute of Pomology, Taian, Shandong, China

Cherry virus A (CVA) (Capillovirus, Betaflexiviridae) is widely present in cherry-growing areas. We obtained the complete genome of a CVA isolate (CVA-TA) using small RNA deep sequencing, followed by overlapping reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The newly identified 5'-untranslated region (5'-UTR) from CVA-TA may form additional hairpin and loop structures to stabilize the CVA genome.
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http://dx.doi.org/10.1128/genomeA.00364-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4866854PMC
May 2016

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

BMC Res Notes 2016 Apr 1;9:200. Epub 2016 Apr 1.

Department of Plant Pathology, Washington State University, 123 Vogel Plant BiologicalSciences, Pullman, WA, 99164, USA.

Background: 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 identified.
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http://dx.doi.org/10.1186/s13104-016-1999-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4818514PMC
April 2016

Evaluation and identification of candidate genes for artificial microRNA-mediated resistance to tomato spotted wilt virus.

Virus Res 2016 Jan 11;211:151-8. Epub 2015 Nov 11.

Department of Plant Pathology, Washington State University, Pullman, WA, USA. Electronic address:

Tomato spotted wilt virus (TSWV) is an economically important viral pathogen of a wide range of field and horticultural crops. We developed an artificial microRNA (amiRNA) strategy against TSWV, targeting the nucleoprotein (N) and silencing suppressor (NSs) genes. The amiRNA constructs replaced the natural miRNA in a shortened Arabidopsis 173-nucleotide (nt) miR159a precursor backbone (athmiR159a) without the stem base extending beyond the miR/miR* duplex. Further, each amiRNA was modified to contain a mismatch (wobble) sequence at nucleotide position 12 and 13 on the complementary strand amiRNA*, mimicking the endogenous miR159a sequence structure. Transient expression in Nicotiana benthamiana demonstrated that the introduction of a wobble sequence did not alter amiRNA expression levels. Following challenge inoculation with TSWV, plants expressing N-specific amiRNAs with or without the wobble remained asymptomatic and were negative for TSWV by ELISA. In contrast, plants expressing the NSs-specific amiRNAs were symptomatic and accumulated high levels of TSWV. Similar findings were obtained in stably transformed Nicotiana tabacum plants. Our results show that a shortened 173-nt athmiR159a backbone is sufficient to express amiRNAs and that the presence of mismatch at position 12-13 does not influence amiRNA expression or conferring of resistance. We also show that selection of target gene and positional effect are critical in amiRNA-based approach for introducing resistance. These findings open the possibility of employing the amiRNA approach for broad-spectrum resistance to tospoviruses as well as other viruses.
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http://dx.doi.org/10.1016/j.virusres.2015.10.003DOI Listing
January 2016

Comparison of small RNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot tospovirus reveals host-specific responses to viral infection.

Virus Res 2016 Jan 30;211:38-45. Epub 2015 Sep 30.

Istituto per la Protezione Sostenibile delle Piante, CNR, Strada delle Cacce 73, 10135 Torino, Italy. Electronic address:

Viral small RNAs (vsRNAs) are one of the key elements involved in RNA silencing-based defense against viruses in plants. We analyzed the vsRNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot virus (PolRSV) (Tospovirus, Bunyaviridae). VsRNAs were abundant in both hosts, but a different size profile was observed, with an abundance peak at 21 in N. benthamiana and at 22 nt in tomato. VsRNAs mapping to the PolRSV L genomic segment were under-represented in both hosts, while S and M segments were differentially and highly targeted in N. benthamiana and tomato, respectively. Differences in preferential targeting of single ORFs were observed, with over-representation of NSs ORF-derived reads in N. benthamiana. Intergenic regions (IGRs)-mapping vsRNAs were under-represented, while enrichment of vsRNAs reads mapping to the NSs positive sense strand was observed in both hosts. Comparison with a previous study on tomato spotted wilt virus (TSWV) under the same experimental conditions, showed that the relative accumulation of PolRSV-specific and endogenous sRNAs was similar to the one observed for silencing suppressor-deficient TSWV strains, suggesting possible different properties of PolRSV NSs silencing suppressor compared to that of TSWV.
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http://dx.doi.org/10.1016/j.virusres.2015.09.019DOI Listing
January 2016

Use of Electrical Penetration Graph Technology to Examine Transmission of 'Candidatus Liberibacter solanacearum' to Potato by Three Haplotypes of Potato Psyllid (Bactericera cockerelli; Hemiptera: Triozidae).

PLoS One 2015 25;10(9):e0138946. Epub 2015 Sep 25.

USDA-ARS, Yakima Agricultural Research Laboratory, Wapato, Washington, United States of America.

The potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), is a vector of the phloem-limited bacterium 'Candidatus Liberibacter solanacearum' (Lso), the putative causal agent of zebra chip disease of potato. Little is known about how potato psyllid transmits Lso to potato. We used electrical penetration graph (EPG) technology to compare stylet probing behaviors and efficiency of Lso transmission of three haplotypes of potato psyllid (Central, Western, Northwestern). All haplotypes exhibited the full suite of stylet behaviors identified in previous studies with this psyllid, including intercellular penetration and secretion of the stylet pathway, xylem ingestion, and phloem activities, the latter comprising salivation and ingestion. The three haplotypes exhibited similar frequency and duration of probing behaviors, with the exception of salivation into phloem, which was of higher duration by psyllids of the Western haplotype. We manipulated how long psyllids were allowed access to potato ("inoculation access period", or IAP) to examine the relationship between phloem activities and Lso transmission. Between 25 and 30% of psyllids reached and salivated into phloem at an IAP of 1 hr, increasing to almost 80% of psyllids as IAP was increased to 24 h. Probability of Lso-transmission was lower across all IAP levels than probability of phloem salivation, indicating that a percentage of infected psyllids which salivated into the phloem failed to transmit Lso. Logistic regression showed that probability of transmission increased as a function of time spent salivating into the phloem; transmission occurred as quickly as 5 min following onset of salivation. A small percentage of infected psyllids showed extremely long salivation events but nonetheless failed to transmit Lso, for unknown reasons. Information from these studies increases our understanding of Lso transmission by potato psyllid, and demonstrates the value of EPG technology in exploring questions of vector efficiency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0138946PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4583427PMC
June 2016

Small RNA profiles of wild-type and silencing suppressor-deficient tomato spotted wilt virus infected Nicotiana benthamiana.

Virus Res 2015 Oct 3;208:30-8. Epub 2015 Jun 3.

Istituto per la Protezione Sostenibile delle Piante, CNR, Strada delle Cacce 73, 10135 Torino, Italy. Electronic address:

Tospoviruses are plant-infecting viruses belonging to the family Bunyaviridae. We used a collection of wild-type, phylogenetically distinct tomato spotted wilt virus isolates and related silencing-suppressor defective mutants to study the effects on the small RNA (sRNA) accumulation during infection of Nicotiana benthamiana. Our data showed that absence of a functional silencing suppressor determined a marked increase of the total amount of viral sRNAs (vsRNAs), and specifically of the 21 nt class. We observed a common under-representation of vsRNAs mapping to the intergenic region of S and M genomic segments, and preferential mapping of the reads against the viral sense open reading frames, with the exception of the NSs gene. The NSs-mutant strains showed enrichment of NSm-derived vsRNA compared to the expected amount based on gene size. Analysis of 5' terminal nucleotide preference evidenced a significant enrichment in U for the 21 nt- and in A for 24 nt-long endogenous sRNAs in all the samples. Hotspot analysis revealed a common abundant accumulation of reads at the 5' end of the L segment, mostly in the antiviral sense, for the NSs-defective isolates, suggesting that absence of the silencing suppressor can influence preferential targeting of the viral genome.
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http://dx.doi.org/10.1016/j.virusres.2015.05.021DOI Listing
October 2015

Maize Lethal Necrosis (MLN), an Emerging Threat to Maize-Based Food Security in Sub-Saharan Africa.

Phytopathology 2015 Jul 1;105(7):956-65. Epub 2015 Jul 1.

First, fifth, and seventeenth authors: International Maize and Wheat Improvement Center (CIMMYT), ICRAF Campus, UN Avenue, Gigiri, PO Box 1041-00621, Nairobi, Kenya; second author: Department of Plant Pathology, University of Minnesota, St. Paul; third and sixteenth authors: Kenya Agricultural and Livestock Research Organization (KALRO), Nairobi, Kenya; fourth, sixth, seventh, and eighteenth authors: United States Department of Agriculture-Agricultural Research Service Corn, Soybean and Wheat Quality Research and Department of Plant Pathology, Ohio State University, Wooster 44691; eighth, ninth, and tenth authors: Plant Health Division, International Centre of Insect Physiology and Ecology, P.O. Box 30772-00100, Nairobi, Kenya; eleventh author: International Institute of Tropical Agriculture (IITA), PMB 5320, Ibadan, Nigeria; twelfth author: Venganza, Inc., 9505 Ocean Shore Blvd., St. Augustine, FL 32080; thirteenth and fourteenth authors: National Agricultural Research Organization, Entebbe, Uganda; and fifteenth author: Department of Plant Pathology, Washington State University, Pullman 99164.

In sub-Saharan Africa, maize is a staple food and key determinant of food security for smallholder farming communities. Pest and disease outbreaks are key constraints to maize productivity. In September 2011, a serious disease outbreak, later diagnosed as maize lethal necrosis (MLN), was reported on maize in Kenya. The disease has since been confirmed in Rwanda and the Democratic Republic of Congo, and similar symptoms have been reported in Tanzania, Uganda, South Sudan, and Ethiopia. In 2012, yield losses of up to 90% resulted in an estimated grain loss of 126,000 metric tons valued at $52 million in Kenya alone. In eastern Africa, MLN was found to result from coinfection of maize with Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV), although MCMV alone appears to cause significant crop losses. We summarize here the results of collaborative research undertaken to understand the biology and epidemiology of MLN in East Africa and to develop disease management strategies, including identification of MLN-tolerant maize germplasm. We discuss recent progress, identify major issues requiring further research, and discuss the possible next steps for effective management of MLN.
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http://dx.doi.org/10.1094/PHYTO-12-14-0367-FIDOI Listing
July 2015

In vivo localization of iris yellow spot tospovirus (Bunyaviridae)-encoded proteins and identification of interacting regions of nucleocapsid and movement proteins.

PLoS One 2015 17;10(3):e0118973. Epub 2015 Mar 17.

Department of Plant Pathology, P.O. Box 646430, Washington State University, Pullman, Washington, United States of America.

Background: Localization and interaction studies of viral proteins provide important information about their replication in their host plants. Tospoviruses (Family Bunyaviridae) are economically important viruses affecting numerous field and horticultural crops. Iris yellow spot virus (IYSV), one of the tospoviruses, has recently emerged as an important viral pathogen of Allium spp. in many parts of the world. We studied the in vivo localization and interaction patterns of the IYSV proteins in uninfected and infected Nicotiana benthamiana and identified the interacting partners.

Principal Findings: Bimolecular fluorescence complementation (BiFC) analysis demonstrated homotypic and heterotypic interactions between IYSV nucleocapsid (N) and movement (NSm) proteins. These interactions were further confirmed by pull-down assays. Additionally, interacting regions of IYSV N and NSm were identified by the yeast-2-hybrid system and β-galactosidase assay. The N protein self-association was found to be mediated through the N- and C-terminal regions making head to tail interaction. Self-interaction of IYSV NSm was shown to occur through multiple interacting regions. In yeast-2-hybrid assay, the N- and C-terminal regions of IYSV N protein interacted with an N-terminal region of IYSV NSm protein.

Conclusion/significance: Our studies provide new insights into localization and interactions of IYSV N and NSm proteins. Molecular basis of these interactions was studied and is discussed in the context of tospovirus assembly, replication, and infection processes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0118973PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4363525PMC
February 2016