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Pospiviroids infect a wide range of plant species, and many pospiviroids can be transmitted to potato and tomato. Pospiviroids continue to be a major production constraint as well as of quarantine concern for the movement of germplasm, and are regulated in several countries/regions. The USDA APHIS issued a federal order requiring all imported tomato and pepper seeds be certified free of six pospiviroids of quarantine significance. The six pospiviroids of quarantine interest include CLVd, PCFVd, PSTVd, TASVd, TCDVd, TPMVd. Currently, those six viroids are detected by real-time RT-PCR. CRISPR/Cas-based genome editing has been increasingly used for virus detection in the past five years. We used a rapid Cas13-based Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) platform for pospiviroid detection, determined the limits of detection and specificity of CRISPR-Cas13a assays. This platform combines recombinase polymerase amplification (RPA) with CRISPR and CRISPR-associated (CRISPR-Cas) RNA-guided endoribonuclease that is rapid and does not require expensive equipment, and can be adapted for on-site detection.
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Sistemas CRISPR-Cas , Enfermedades de las Plantas , Viroides , Enfermedades de las Plantas/virología , Viroides/genética , Viroides/aislamiento & purificación , Sensibilidad y Especificidad , Solanum lycopersicum/virología , Edición Génica/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Solanum tuberosum/virologíaRESUMEN
Potato mop-top virus (PMTV) is an emerging viral pathogen that causes tuber necrosis in potatoes. PMTV is composed of three single-stranded RNA segments: RNA1 encodes RNA-dependent RNA polymerase, RNA2 contains the coat protein (CP), and RNA3 harbors a triple gene block (TGB 1, TGB2, and TGB3). CP plays a role in viral transmission, while TGB is known to facilitate cell-to-cell and long-distance systemic movement. The role of CP in symptom development, specifically in the presence of TGB genes, was investigated using potato virus X (PVX) as a delivery vehicle to express PMTV genes in the model plant Nicotiana benthamiana. Plants expressing individual genes showed mild symptoms that included leaf curling and crumpling. Interestingly, symptom severity varied among plants infected with three different combinations: CP with TGB1, CP with TGB2, and CP with TGB3. Notably, the combination of CP and TGB3 induced a hypersensitive response, accompanied by stunted growth and downward curling and crumpling. These results suggest the potential role of TGB co-expressed with CP in symptom development during PMTV infection. Additionally, this study demonstrates the use of the PVX-based expression system as a valuable platform for assessing the role of unknown genes in viral pathogenicity.
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Proteínas de la Cápside , Nicotiana , Enfermedades de las Plantas , Potexvirus , Solanum tuberosum , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Nicotiana/genética , Nicotiana/virología , Nicotiana/metabolismo , Potexvirus/genética , Potexvirus/patogenicidad , Enfermedades de las Plantas/virología , Enfermedades de las Plantas/genética , Solanum tuberosum/virología , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Tomato mosaic virus (ToMV), an economically important virus that affects a wide range of crops, is highly contagious, and its transmission is mediated by mechanical means, and through contaminated seeds or planting materials, making its management challenging. To contain its wide distribution, early and accurate detection of infection is required. A survey was conducted between January and May, 2023 in major tomato growing counties in Kenya, namely, Baringo, Kajiado, Kirinyaga and Laikipia, to establish ToMV disease incidence and to collect samples for optimization of the reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) assay. A RT-LAMP assay, utilizing primers targeting the coat protein, was developed and evaluated for its performance. The method was able to detect ToMV in tomato samples within 4:45 minutes, had a 1,000-fold higher sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR) method and was specific to ToMV. Furthermore, the practical applicability of the assay was assessed using tomato samples and other solanaecous plants. The assay was able to detect the virus in 14 tomato leaf samples collected from the field, compared to 11 samples detected by RT-PCR, further supporting the greater sensitivity of the assay. To make the assay more amenable for on-site ToMV detection, a quick-extraction method based on alkaline polyethylene glycol buffer was evaluated, which permitted the direct detection of the target virus from crude leaf extracts. Due to its high sensitivity, specificity and rapidity, the RT-LAMP method could be valuable for field surveys and quarantine inspections towards a robust management of ToMV infections.
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Técnicas de Amplificación de Ácido Nucleico , Enfermedades de las Plantas , Solanum lycopersicum , Tobamovirus , Técnicas de Amplificación de Ácido Nucleico/métodos , Solanum lycopersicum/virología , Enfermedades de las Plantas/virología , Tobamovirus/genética , Tobamovirus/aislamiento & purificación , Transcripción Reversa , Sensibilidad y Especificidad , Kenia , ARN Viral/genética , ARN Viral/análisis , ARN Viral/aislamiento & purificación , Técnicas de Diagnóstico MolecularRESUMEN
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.
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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.
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Enfermedades de las Plantas , Plasmodiophorida , Almidón , Amilopectina , Metabolismo de los Hidratos de Carbono , Plasmodiophorida/genéticaRESUMEN
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.
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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.
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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.
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Proteínas , Saccharomyces cerevisiae , Mapeo de Interacción de Proteínas/métodos , Proteínas/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
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.
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Mapeo de Interacción de Proteínas , Proteínas , Unión Proteica , Proteínas/química , Espectrometría de Masas , Cromatografía de Afinidad , Inmunoprecipitación , Mapeo de Interacción de Proteínas/métodosRESUMEN
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.
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Glutatión Transferasa , Proteínas , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Proteínas/metabolismo , Cromatografía de Afinidad/métodos , Escherichia coli/metabolismo , Glutatión/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
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.
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Mapeo de Interacción de Proteínas , Fluorescencia , Microscopía Fluorescente/métodos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mapeo de Interacción de Proteínas/métodosRESUMEN
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.
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Transferencia Resonante de Energía de Fluorescencia , Células Vegetales , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Células Vegetales/metabolismo , Fenómenos BiofísicosRESUMEN
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.
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Virus de Plantas , Virosis , Sistemas CRISPR-Cas , Interferencia de ARN , Multiómica , Enfermedades de las Plantas , Fitomejoramiento , Plantas/genética , Virus de Plantas/genética , Virosis/genética , Manejo de la Enfermedad , Genoma de PlantaRESUMEN
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.
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Cebollas , Thysanoptera , Estados Unidos , Animales , Cebollas/genética , Thysanoptera/genética , Filogenia , Evolución Biológica , Variación GenéticaRESUMEN
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.
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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.
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Virus de Plantas , Solanum tuberosum , Enfermedades de las Plantas , Virus de Plantas/genética , SueloRESUMEN
Tospoviruses infect numerous crop species worldwide, causing significant losses throughout the supply chain. As a defence mechanism, plants use RNA interference (RNAi) to generate virus-derived small-interfering RNAs (vsiRNAs), which target viral transcripts for degradation. Small RNA sequencing and in silico analysis of capsicum and N. benthamiana infected by tomato spotted wilt virus (TSWV) or capsicum chlorosis virus (CaCV) demonstrated the presence of abundant vsiRNAs, with host-specific differences evident for each pathosystem. Despite the biogenesis of vsiRNAs in capsicum and N. benthamiana, TSWV and CaCV viral loads were readily detectable. In response to tospovirus infection, the solanaceous host species also generated highly abundant virus-activated small interfering RNAs (vasiRNAs) against many endogenous transcripts, except for an N. benthamiana accession lacking a functional RDR1 gene. Strong enrichment for ribosomal protein-encoding genes and for many genes involved in protein processing in the endoplasmic reticulum suggested co-localisation of viral and endogenous transcripts as a basis for initiating vasiRNA biogenesis. RNA-seq and RT-qPCR-based analyses of target transcript expression revealed an inconsistent role for vasiRNAs in modulating gene expression in N. benthamiana, which may be characteristic of this tospovirus-host pathosystem.
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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.
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This communication summarizes the presentations given at the 1st international conference of the World Society for Virology (WSV) held virtually during 16-18 June 2021, under the theme of tackling global viral epidemics. The purpose of this biennial meeting is to foster international collaborations and address important viral epidemics in different hosts. The first day included two sessions exclusively on SARS-CoV-2 and COVID-19. The other two days included one plenary and three parallel sessions each. Last not least, 16 sessions covered 140 on-demand submitted talks. In total, 270 scientists from 49 countries attended the meeting, including 40 invited keynote speakers.