Transcriptional and functional predictors of potato virus Y-induced tuber necrosis in potato (Solanum tuberosum).

Introduction: Potato (Solanum tuberosum L.), the fourth most important food crop in the world, is affected by several viral pathogens with potato virus Y (PVY) having the greatest economic impact. At least nine biologically distinct variants of PVY are known to infect potato. These include the relatively new recombinant types named PVY-NTN and PVYN-Wi, which induce tuber necrosis in susceptible cultivars. To date, the molecular plant-virus interactions underlying this pathogenicity have not been fully characterized. We hypothesized that this necrotic behavior is supported by transcriptional and functional signatures that are unique to PVY-NTN and PVYN-Wi. Methods: To test this hypothesis, transcriptional responses of cv. Russet Burbank, a PVY susceptible cultivar, to three PVY strains PVY-O, PVY-NTN, and PVYN-Wi were studied using mRNA-Seq. A haploid-resolved genome assembly for tetraploid potato was used for bioinformatics analysis. Results: The study revealed 36 GO terms and nine KEGG 24 pathways that overlapped across the three PVY strains, making them generic features of PVY susceptibility in potato. Ten GO terms and three KEGG pathways enriched for PVY-NTN and PVYN-Wi only, which made them candidate functional signatures associated with PVY-induced tuber necrosis in potato. In addition, five other pathways were enriched for PVYNTN or PVYN-Wi. One carbon pool by folate was enriched exclusively in response to PVY-NTN infection; PVYN-Wi infection specifically impacted cutin, suberine and wax biosynthesis, phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and monoterpenoid biosynthesis. Discussion: Results suggest that PVYN-Wi-induced necrosis may be mechanistically distinguishable from that of PVY-NTN. Our study provides a basis for understanding the mechanism underlying the development of PVY-induced tuber necrosis in potato.

Starch Plays a Key Role in Sporosorus Formation by the Powdery Scab Pathogen Spongospora subterranea.

Powdery scab disease, caused by the soilborne protist Spongospora subterranea f. sp. subterranea, poses a major constraint to potato production worldwide. Disease symptoms include damage to the tuber skin and the formation of root galls. This study aimed to investigate the potential mechanism behind the formation of sporosori, which are aggregates of resting spores, within root galls. Scanning electron microscopy analysis revealed that the early stage of gall formation, characterized by a white color, involved the accumulation of starch grains, which later disappeared as the gall matured and turned brown. The mature brown galls were found to contain fully formed sporosori. Light microscopy examination of ultramicrotome sections of the root galls showed that the high-amylopectin starches were surrounded by a plasmodium, a precursor to sporosorus. These findings suggest that starch grains contribute to the formation of a sponge-like structure within the sporosori. A significant reduction in total starch levels in both the root galls and their associated roots was observed compared with healthy roots. These findings indicate starch consumption by sporosori during the maturation of root galls. Interestingly, analysis of the transcript levels of starch-related genes showed downregulation of genes encoding starch degrading enzymes and an amylopectin-debranching enzyme, whereas genes encoding a starch synthase and a protein facilitating starch synthesis were upregulated in the infected roots. Overall, our results demonstrate that starch is consumed during sporosorus formation, and the pathogen likely manipulates starch homeostasis to its advantage for sporosorus development within the root galls.

Current status, breeding strategies and future prospects for managing chilli leaf curl virus disease and associated begomoviruses in Chilli (Capsicum spp.).

Chilli leaf curl virus disease caused by begomoviruses, has emerged as a major threat to global chilli production, causing severe yield losses and economic harm. Begomoviruses are a highly successful and emerging group of plant viruses that are primarily transmitted by whiteflies belonging to the Bemisia tabaci complex. The most effective method for mitigating chilli leaf curl virus disease losses is breeding for host resistance to Begomovirus. This review highlights the current situation of chilli leaf curl virus disease and associated begomoviruses in chilli production, stressing the significant issues that breeders and growers confront. In addition, the various breeding methods used to generate begomovirus resistant chilli cultivars, and also the complicated connections between the host plant, vector and the virus are discussed. This review highlights the importance of resistance breeding, emphasising the importance of multidisciplinary approaches that combine the best of traditional breeding with cutting-edge genomic technologies. subsequently, the article highlights the challenges that must be overcome in order to effectively deploy begomovirus resistant chilli varieties across diverse agroecological zones and farming systems, as well as understanding the pathogen thus providing the opportunities for improving the sustainability and profitability of chilli production.

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.

CRISPR/Cas, Multiomics, and RNA Interference in Virus Disease Management.

Plant viruses infect a wide range of commercially important crop plants and cause significant crop production losses worldwide. Numerous alterations in plant physiology related to the reprogramming of gene expression may result from viral infections. Although conventional integrated pest management-based strategies have been effective in reducing the impact of several viral diseases, continued emergence of new viruses and strains, expanding host ranges, and emergence of resistance-breaking strains necessitate a sustained effort toward the development and application of new approaches for virus management that would complement existing tactics. RNA interference-based techniques, and more recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing technologies have paved the way for precise targeting of viral transcripts and manipulation of viral genomes and host factors. In-depth knowledge of the molecular mechanisms underlying the development of disease would further expand the applicability of these recent methods. Advances in next-generation/high-throughput sequencing have made possible more intensive studies into host-virus interactions. Utilizing the omics data and its application has the potential to expedite fast-tracking traditional plant breeding methods, as well as applying modern molecular tools for trait enhancement, including virus resistance. Here, we summarize the recent developments in the CRISPR/Cas system, transcriptomics, endogenous RNA interference, and exogenous application of dsRNA in virus disease management.

Yeast Two-Hybrid Technique to Identify Protein-Protein Interactions.

Protein-protein interactions are specific and direct physical contact between two or more proteins, and the interaction involves hydrogen bonding, electrostatic forces, and hydrophobic forces. Majority of biological processes in the living cell are executed by proteins, and any particular protein function is regulated by numerous other proteins. Thus, knowledge of protein-protein interaction is necessary to understand the biological processes. In this chapter, we explain the widely used yeast two-hybrid assay to identify the protein-interacting partners.

Affinity Purification-Mass Spectroscopy (AP-MS) and Co-Immunoprecipitation (Co-IP) Technique to Study Protein-Protein Interactions.

Affinity purification-Mass spectroscopy (AP-MS) is a biochemical technique to identify the novel protein-protein interaction that occurs in the most relevant physiological conditions, whereas co-immunoprecipitation (Co-IP) is used to study the interaction between two known protein partners that are expressed in the native physiological conditions. Both AP-MS and Co-IP techniques are based on the ability of the interacting partners to pull-down with protein of interest. In this chapter, we have explained the AP-MS and Co-IP methods to study protein-protein interactions in the plant cells.

Detection of Protein-Protein Interactions Using Glutathione-S-Transferase (GST) Pull-Down Assay Technique.

Pull-down assay is a technique to analyze direct protein-protein interaction under in vitro condition. Also, this technique is appropriate for investigating the direct interaction between two purified proteins. Glutathione-s-transferase (GST) protein is a widely used affinity tag for affinity purification. In this chapter, we explain the widely used GST pull-down assay to identify the protein-protein interaction between purified proteins.

Bimolecular Fluorescence Complementation (BiFC) Assay to Visualize Protein-Protein Interactions in Living Cells.

Bimolecular fluorescence complementation (BiFC) assay is a method to visualize the protein-protein interaction in living cells. This technique is based on ability of the non-fluorescent fragment of fluorescent protein to form fluorescent complex when they are fused to two interacting proteins. In this chapter, we describe the widely used split yellow fluorescent protein (YFP) system to visualize the protein-protein interaction in plant cells.

Forster Resonance Energy Transfer (FRET) to Visualize Protein-Protein Interactions in the Plant Cell.

Forster resonance energy transfer (FRET) is an efficient method to visualize the protein-protein interaction in living cells. This technique is based on transfer of energy between two different fluorophores that are fused to two interacting proteins. In this chapter, we described the FRET assay to visualize the protein-protein interaction in plant cells.

Mitochondrial genetic diversity of Thrips tabaci (Thysanoptera: Thripidae) in onion growing regions of the United States.

Onion thrips (Thrips tabaci Lindeman, Thysanoptera: Thripidae) causes severe damage to many horticultural and agronomic crops worldwide. It also acts as a vector of several plant viruses. T. tabaci is a key pest of Allium cepa in the United States. However, there is limited information available on the genetic variation within and between T. tabaci populations in the United States and its key evolutionary parameters. In the current study, 83 T. tabaci specimens were collected from A. cepa from 15 different locations comprising four states of the United States. A total of 92 mtCOI gene sequences of T. tabaci from A. cepa were analyzed to understand the genetic diversity and structure of T. tabaci collected from onion host. Seven distinct haplotypes of T. tabaci infesting A. cepa were identified from the current collection, while nine T. tabaci sequences retrieved from GenBank comprised 5 haplotypes. Overall, 15 haplotypes of T. tabaci infesting A. cepa were identified in the world that includes the ten haplotypes in the United States. In the phylogenetic analysis, all the populations collected during the study clustered with thelytokous lineage, while T. tabaci sequences retrieved from GenBank corresponded to leek-associated arrhenotokous lineage. The highest genetic variation was found in Elba and Malheur populations with 3 haplotypes identified in each. The results suggest that haplotypes 1 and 7 are more frequently prevailing haplotypes in the north-western United States, with haplotype 1 being the predominant all over the country. The eastern United States appears to have a more diverse group of haplotypes. The populations from Hungary constituted distinct haplotypes and a haplotype from Kingston linked it with the predominant haplotype.

Functional characterization of a new ORF ßV1 encoded by radish leaf curl betasatellite.

Whitefly-transmitted begomoviruses infect and damage a wide range of food, feed, and fiber crops worldwide. Some of these viruses are associated with betasatellite molecules that are known to enhance viral pathogenesis. In this study, we investigated the function of a novel ßV1 protein encoded by radish leaf curl betasatellite (RaLCB) by overexpressing the protein using potato virus X (PVX)-based virus vector in Nicotiana benthamiana. ßV1 protein induced lesions on leaves, suggestive of hypersensitive response (HR), indicating cell death. The HR reaction induced by ßV1 protein was accompanied by an increased accumulation of reactive oxygen species (ROS), free radicals, and HR-related transcripts. Subcellular localization through confocal microscopy revealed that ßV1 protein localizes to the cellular periphery. ßV1 was also found to interact with replication enhancer protein (AC3) of helper virus in the nucleus. The current findings suggest that ßV1 functions as a protein elicitor and a pathogenicity determinant.

An on-site adaptable test for rapid and sensitive detection of Potato mop-top virus, a soil-borne virus of potato (Solanum tuberosum).

Potato mop-top virus (PMTV) is considered an emerging threat to potato production in the United States. PMTV is transmitted by a soil-borne protist, Spongospora subterranean. Rapid, accurate, and sensitive detection of PMTV in leaves and tubers is an essential component in PMTV management program. A rapid test that can be adapted to in-field, on-site testing with minimal sample manipulation could help in ensuring the sanitary status of the produce in situations such as certification programs and shipping point inspections. Toward that goal, a rapid and highly sensitive recombinase polymerase amplification (RPA)-based test was developed for PMTV detection in potato tubers. The test combines the convenience of RPA assay with a simple sample extraction procedure, making it amenable to rapid on-site diagnosis of PMTV. Furthermore, the assay was duplexed with a plant internal control to monitor sample extraction and RPA reaction performance. The method described could detect as little as 10 fg of PMTV RNA transcript in various potato tissues, the diagnostic limit of detection (LOQ) similar to that of traditional molecular methods.

Genome Sequence of Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1 Found in the Fungus Penicillium olsonii Isolated from Washington State, USA.

We report the discovery of a Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) isolate, named SsHADV1_PO, from the fungus Penicillium olsonii isolated from Washington state, USA. The genome of SsHADV1_PO is 2,166 bp and contains two open reading frames, with more than 98% nucleotide identity with respect to reported SsHADV-1 isolates.

Phylogenetics of tobacco rattle virus isolates from potato (Solanum tuberosum L.) in the USA: a multi-gene approach to evolutionary lineage.

Tobacco rattle virus (TRV) is an important soil-borne virus of potato that is transmitted by stubby-root nematodes. TRV causes corky ringspot, a tuber disease of economic importance to potato production. Utilizing protein-coding regions of the whole genome and a range of computational tools, the genetic diversity, and population structure of TRV isolates from several potato-growing regions (Colorado, Idaho, Indiana, Minnesota, Nebraska, North Dakota, and Washington State) in the USA were determined. Phylogenetic analyses based on RNA2 nucleotide sequences, the coat protein (CP) and nematode transmission (2b) genes, showed geographical clustering of USA isolates with previously known American isolates, while European isolates grouped in a distinct cluster. This was corroborated by the observed genetic differentiation and infrequent gene flow between American and European isolates. Low genetic diversity was revealed among American isolates compared to European isolates. Phylogenetic clustering based on RNA1 genes (RdRp, RdRp-RT, and 1a) were all largely incongruent to that of 1b gene (virus suppressor of RNA silencing). This genetic incongruence suggested the influence of recombination. Furthermore, the RdRp, RdRp-RT, and 1a genes were predicted to be more conserved and under negative selection, while the 1b gene was less constrained. Different evolutionary lineages between TRV RNA1 and RNA2 genomic segments were revealed.

Pathogen-triggered metabolic adjustments to potato virus Y infection in potato.

Potato (Solanum tuberosum L) is affected by several viral pathogens with the most economically damaging being potato virus Y (PVY). At least nine biologically distinct variants of PVY are known to attack potato, with necrotic types named PVYNTN and PVYN-Wi being the most recent additions to the list. So far, the molecular plant-virus interactions underlying this pathogenicity are not fully understood. In this study, gas chromatography coupled with mass spectroscopy (GC-MS) was used for an untargeted investigation of the changes in leaf metabolomes of PVY-resistant cultivar Premier Russet, and a susceptible cultivar, Russet Burbank, following inoculation with three PVY strains, PVYNTN, PVYN-Wi, and PVYO. Analysis of the resulting GC-MS spectra with the online software Metaboanalyst (version 5.0) uncovered several common and strain-specific metabolites that are induced by PVY inoculation. In Premier Russet, the major overlap in differential accumulation was found between PVYN-Wi and PVYO. However, the 14 significant pathways occurred solely due to PVYN-Wi. In contrast, the main overlap in differential metabolite profiles and pathways in Russet Burbank was between PVYNTN and PVYO. Overall, limited overlap was observed between PVYNTN and PVYN-Wi. As a result, PVYN-Wi-induced necrosis may be mechanistically distinguishable from that of PVYNTN. Furthermore, 10 common and seven cultivar-specific metabolites as potential indicators of PVY infection and susceptibility/resistance were identified by using PLS-DA and ANOVA. In Russet Burbank, glucose-6-phosphate and fructose-6-phosphate were particularly affected by strain-time interaction. This highlights the relevance of the regulation of carbohydrate metabolism for defense against PVY. Some strain- and cultivar-dependent metabolite changes were also observed, reflecting the known genetic resistance-susceptibility dichotomy between the two cultivars. Consequently, engineering broad-spectrum resistance may be the most effective breeding strategy for managing these necrotic strains of PVY.

Viruses Without Borders: Global Analysis of the Population Structure, Haplotype Distribution, and Evolutionary Pattern of Iris Yellow Spot Orthotospovirus (Family Tospoviridae, Genus Orthotospovirus).

Iris yellow spot, caused by Iris yellow spot orthotospovirus (IYSV) (Genus: Orthotospovirus, Family: Tospoviridae), is an important disease of Allium spp. The complete N gene sequences of 142 IYSV isolates of curated sequence data from GenBank were used to determine the genetic diversity and evolutionary pattern. In silico restriction fragment length polymorphism (RFLP) analysis, codon-based maximum likelihood studies, genetic differentiation and gene flow within the populations of IYSV genotypes were investigated. Bayesian phylogenetic analysis was carried out to estimate the evolutionary rate. In silico RFLP analysis of N gene sequences categorized IYSV isolates into two major genotypes viz., IYSV Netherlands (IYSV NL ; 55.63%), IYSV Brazil (IYSV BR ; 38.73%) and the rest fell in neither group [IYSV other (IYSV other ; 5.63%)]. Phylogenetic tree largely corroborated the results of RFLP analysis and the IYSV genotypes clustered into IYSV NL and IYSV BR genotypes. Genetic diversity test revealed IYSV other to be more diverse than IYSV NL and IYSV BR . IYSV NL and IYSV BR genotypes are under purifying selection and population expansion, whereas IYSV other showed decreasing population size and hence appear to be under balancing selection. IYSV BR is least differentiated from IYSV other compared to IYSV NL genotype based on nucleotide diversity. Three putative recombinant events were found in the N gene of IYSV isolates based on RDP analysis, however, RAT substantiated two among them. The marginal likelihood mean substitution rate was 5.08 × 10-5 subs/site/year and 95% highest posterior density (HPD) substitution rate between 5.11 × 10-5 and 5.06 × 10-5. Findings suggest that IYSV continues to evolve using population expansion strategies. The substitution rates identified are similar to other plant RNA viruses.

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

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 (Nicotiana tabacum) 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.

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

Tomato spotted wilt virus (TSWV) is the type member of the genus Orthotospovirus in the family Tospoviridae 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.