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An in-house tomato inbred line, YNAU335, was planted in a greenhouse in spring from 2014 to 2017, and showed immunity to tomato spotted wilt virus (TSWV). YNAU335 was infected with TSWV in the spring from 2018 to 2020, and disease was observed on the leaves, sepals, and fruits. In 2021 and 2022, YNAU335 was planted in spring in the same greenhouse, which was suspected of being infected with TSWV, and visible disease symptoms were observed on the fruits. Transmission electron microscopy, deep sequencing of small RNAs, and molecular mutation diagnosis were used to analyze the pathological features and genetic polymorphism of TSWV infecting tomato fruit. Typical TSWV virions were observed in the infected fruits, but not leaves from YNAU335 grown between 2021 and 2022, and cross-infection was very rarely observed. The number of mitochondria and chloroplasts increased, but the damage to the mitochondria was greater than that seen in the chloroplasts. Small RNA deep sequencing revealed the presence of multiple viral species in TSWV-infected and non-infected tomato samples grown between 2014–2022. Many virus species, including TSWV, which accounted for the largest proportion, were detected in the TSWV-infected tomato leaves and fruit. However, a variety of viruses other than TSWV were also detected in the non-infected tissues. The amino acids of TSWV nucleocapsid proteins (NPs) and movement proteins (MPs) from diseased fruits of YNAU335 picked in 2021–2022 were found to be very diverse. Compared with previously identified NPs and MPs from TSWV isolates, those found in this study could be divided into three types: non-resistance-breaking, resistance-breaking, and other isolates. The number of positive clones and a comparison with previously identified amino acid mutations suggested that mutation F at AA118 of the MP (GenBank OL310707) is likely the key to breaking the resistance to TSWV, and this mutation developed only in the infected fruit of YNAU335 grown in 2021 and 2022. [ABSTRACT FROM AUTHOR]

We deployed field-based high-throughput phenotyping (HTP) techniques to acquire trait data for a subset of a peanut chromosome segment substitution line (CSSL) population. Sensors mounted on an unmanned aerial vehicle (UAV) were used to derive various vegetative indices as well as canopy temperatures. A combination of aerial imaging and manual scoring showed that CSSL 100, CSSL 84, CSSL 111, and CSSL 15 had remarkably low tomato spotted wilt virus (TSWV) incidence, a devastating disease in South Georgia, USA. The four lines also performed well under leaf spot pressure. The vegetative indices showed strong correlations of up to 0.94 with visual disease scores, indicating that aerial phenotyping is a reliable way of selecting under disease pressure. Since the yield components of peanut are below the soil surface, we deployed ground penetrating radar (GPR) technology to detect pods non-destructively. Moderate correlations of up to 0.5 between pod weight and data acquired from GPR signals were observed. Both the manually acquired pod data and GPR variables highlighted the three lines, CSSL 84, CSSL 100, and CSSL 111, as the best-performing lines, with pod weights comparable to the cultivated check Tifguard. Through the combined application of manual and HTP techniques, this study reinforces the premise that chromosome segments from peanut wild relatives may be a potential source of valuable agronomic traits. [ABSTRACT FROM AUTHOR]

The Polish germplasm collection of Nicotiana tabacum was started in the 1920s. Up to now, more than eight hundred accessions originating from different regions of the world have been gathered in the collection. It includes valuable breeding lines and obsolete cultivars, among them cytoplasmic male-sterile lines. Numerous cultivars are rich sources of features desired in tobacco breeding. Therefore, the accessions are continually characterised in terms of their various features, one of the most important of which is disease resistance. Much research is being done to explain the nature of resistance and its genetic basis. Moreover, cultivars with good agronomic characteristics are used in wide hybridisation, being recipients of resistance genes from wild species or are genetically modified with transgenes conditioning resistance. The biological diversity of cultivars also allows a proper selection of plant material for pathogen studies, while the large number of the accessions facilitates research into the conditions for long seed storage. Numerous examples of the use of Polish tobacco germplasm in research and breeding, specifically in disease resistance, have been presented in this paper. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plant viruses are inducers and targets of antiviral RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressor proteins that interfere with antiviral RNA silencing. The NSs protein is an RNA silencing suppressor in orthotospoviruses, such as the tomato spotted wilt virus (TSWV). The mechanism of RNA silencing suppression by NSs and its role in virus infection and movement are poorly understood. Here, we cloned and tagged TSWV NSs and expressed it from a GFP-tagged turnip mosaic virus (TuMV-GFP) carrying either a wild-type or suppressor-deficient (AS9) helper component proteinase (HC-Pro). When expressed in cis, NSs restored pathogenicity and promoted systemic infection of suppressor-deficient TuMV-AS9-GFP in Nicotiana benthamiana and Arabidopsis thaliana. Inactivating mutations were introduced in NSs RNA-binding domain one. A genetic analysis with active and suppressor-deficient NSs, in combination with wild-type and mutant plants lacking essential components of the RNA silencing machinery, showed that the NSs insert is stable when expressed from a potyvirus. NSs can functionally replace potyviral HC-Pro, condition virus susceptibility, and promote systemic infection and symptom development by suppressing antiviral RNA silencing through a mechanism that partially overlaps that of potyviral HC-Pro. The results presented provide new insight into the mechanism of silencing suppression by NSs and its effect on virus infection. [ABSTRACT FROM AUTHOR]

Tomato spotted wilt virus (TSWV) is one of the most destructive diseases affecting tomato (Solanum lycopersicum) cultivation and production worldwide. As defenses against TSWV, natural resistance genes have been identified in tomato, including Sw-1a, Sw-1b, sw-2, sw-3, sw-4, Sw-5, Sw-6, and Sw-7. However, only Sw-5 exhibits a high level of resistance to the TSWV. Thus, it has been cloned and widely used in the breeding of tomato with resistance to the disease. Due to the global spread of TSWV, resistance induced by Sw-5 decreases over time and can be overcome or broken by a high concentration of TSWV. How to utilize other resistance genes and identify novel resistance resources are key approaches for breeding tomato with resistance to TSWV. In this review, the characteristics of natural resistance genes, natural resistance resources, molecular markers for assisted selection, and methods for evaluating resistance to TSWV are summarized. The aim is to provide a theoretical basis for identifying, utilizing resistance genes, and developing tomato varieties that are resistant to TSWV. [ABSTRACT FROM AUTHOR]

The tomato Sw-5b gene confers resistance to tomato spotted wilt virus (TSWV) and encodes a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal Solanaceae-specific domain (SD). Although our understanding of how Sw-5b recognizes the viral NSm elicitor has increased significantly, the process by which Sw-5b activates downstream defense signaling remains to be elucidated. In this study, we used a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system to investigate the roles of the SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 genes in the Sw-5b-mediated signaling pathway. We found that chaperone SGT1 was required for Sw-5b function, but co-chaperone RAR1 was not. Sw-5b-mediated immune signaling was independent of both EDS1 and NDR1. Silencing NPR1, which is a central component in SA signaling, did not result in TSWV systemic infection in Sw-5b-transgenic N. benthamiana plants. Helper NLR NRCs (NLRs required for cell death) were required for Sw-5b-mediated systemic resistance to TSWV infection. Suppression of NRC2/3/4 compromised the Sw-5b resistance. However, the helper NLRs ADR1 and NRG1 may not participate in the Sw-5b signaling pathway. Silencing ADR1, NRG1, or both genes did not affect Sw-5b-mediated resistance to TSWV. Our findings provide new insight into the requirement for conserved key components in Sw-5b-mediated signaling pathways. [ABSTRACT FROM AUTHOR]

Plant-derived nanovesicles (NVs) have attracted interest due to their anti-inflammatory, anticancer and antioxidative properties and their efficient uptake by human intestinal epithelial cells. Previously we showed that tomato (Solanum lycopersicum L.) fruit is one of the interesting plant resources from which NVs can be obtained at a high yield. In the course of the isolation of NVs from different batches of tomatoes, using the established differential ultracentrifugation or size-exclusion chromatography methods, we occasionally observed the co-isolation of viral particles. Density gradient ultracentrifugation (gUC), using sucrose or iodixanol gradient materials, turned out to be efficient in the separation of NVs from the viral particles. We applied cryogenic transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM) for the morphological assessment and LC–MS/MS-based proteomics for the protein identification of the gradient fractions. Cryo-TEM showed that a low-density gUC fraction was enriched in membrane-enclosed NVs, while the high-density fractions were rich in rod-shaped objects. Mass spectrometry–based proteomic analysis identified capsid proteins of tomato brown rugose fruit virus, tomato mosaic virus and tomato mottle mosaic virus. In another batch of tomatoes, we isolated tomato spotted wilt virus, potato virus Y and southern tomato virus in the vesicle sample. Our results show the frequent co-isolation of plant viruses with NVs and the utility of the combination of cryo-TEM, SEM and proteomics in the detection of possible viral contamination. [ABSTRACT FROM AUTHOR]

Negative-strand (-) RNA viruses (NSVs) comprise a large and diverse group of viruses that are generally divided in those with non-segmented and those with segmented genomes. Whereas most NSVs infect animals and humans, the smaller group of the plant-infecting counterparts is expanding, with many causing devastating diseases worldwide, affecting a large number of major bulk and high-value food crops. In 2018, the taxonomy of segmented NSVs faced a major reorganization with the establishment of the order Bunyavirales. This article overviews the major plant viruses that are part of the order, i.e., orthospoviruses (Tospoviridae), tenuiviruses (Phenuiviridae), and emaraviruses (Fimoviridae), and provides updates on the more recent ongoing research. Features shared with the animal-infecting counterparts are mentioned, however, special attention is given to their adaptation to plant hosts and vector transmission, including intra/intercellular trafficking and viral counter defense to antiviral RNAi. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Tomato spotted wilt virus (TSWV) is one of the most destructive plant viruses, causing severe losses in many important crops worldwide. The non-structural protein NSm of TSWV is a viral movement protein that induces viral symptoms. However, the molecular mechanisms by which NSm contributes to symptom development are unclear. Here, we present evidence that NSm directly interacts with Nicotiana benthamiana chloroplast thylakoid membrane protein TMP14 (NbTMP14) by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. The interaction between NSm and NbTMP14 led to the translocation of the NbTMP14 protein from the chloroplast to the cytoplasm in TSWV-infected plants, and overexpressing NSm decreased NbTMP14 mRNA accumulation. In addition, abnormal chloroplasts and starch accumulation were observed in TSWV-infected plants. Silencing of NbTMP14 by TRV VIGS also showed similar results to those of TSWV-infected plants. Overexpressing NbTMP14 in transgenic N. benthamiana plants impeded TSWV infection, and silencing NbTMP14 in N. benthamiana plants increased disease symptom severity and virus accumulation. To our knowledge, this is the first report showing that the plant chloroplast TMP14 protein is involved in viral infection. Knowledge of the interaction between NSm and NbTMP14 advances our understanding of the molecular mechanisms underlying TSWV symptom development and infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Tomato spotted wilt virus (TSWV; species Tomato spotted wilt orthotospovirus) 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 Nicotiana benthamiana via affinity purification and mass spectrometry (AP-MS) analysis. Forty-three TSWV NSs-interacting candidates were identified in N. benthamiana. Gene Ontology (GO) and protein–protein interaction (PPI) network analyses were carried out on their closest homologs in tobacco (Nicotiana tabacum), tomatoes (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). 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. [ABSTRACT FROM AUTHOR]

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco. [ABSTRACT FROM AUTHOR]

Tospovirus is a tripartite negative stranded RNA virus and is considered as one of the most devastating plant viruses. Successful virus infection in plant requires many host factors. To date, very few host factors have been identified as important in Tospovirus infection in plants. We reported earlier that NSm protein encoded by Tomato spotted wilt virus (TSWV), a type species of the genus Orthotospovirus, plays critical roles in viral cell-to-cell and long-distance movement. In this study, we determined that molecular co-chaperone NbSGT1 interacted with TSWV NSm in Nicotiana benthamiana. TSWV infection significantly upregulated the expression of NbSGT1 gene and transient overexpression of NbSGT1 in N. benthamiana leaves accelerated TSWV infection. In contrast, silencing the NbSGT1 gene expression using a virus-induced gene silencing (VIGS) approach strongly inhibited TSWV NSm cell-to-cell movement, as well as TSWV local and systemic infection in N. benthamiana plants. Furthermore, NbSGT1 was found to regulate the infection of both American and Euro/Asia type tospoviruses in N. benthamiana plant. Collectively, our findings presented in this paper and the results published previously indicated that molecular co-chaperone NbSGT1 plays important roles in modulating both positive stranded and tripartite negative stranded RNA virus infection in plants. [ABSTRACT FROM AUTHOR]