First Report of Impatiens Necrotic Spot Virus (INSV) Infecting Tomatoes in North Carolina.

Impatiens necrotic spot virus (INSV) (Orthotospovirus impatiensnecromaculae) is a virus in the Order Bunyavirales and Family Tospoviridae. The virus is vectored by several species of thrips and is a serious pathogen of ornamentals and lettuce in the United States (Hasegawa & Del Pozo-Valdivia 2023; Daughtrey, M. L., et al. 1997; Webster, C. G., et al. 2015). In January 2023, tomato plants (Solanum lycopersicum,'Big Dena') with viral symptoms of reduced vigor, wilting, necrotic spots on leaves, and sunken lesions on the stem were observed in one greenhouse in Guilford County, North Carolina (NC) (Figure 1A-C). Disease incidence was low (2%), with only three symptomatic plants in the single greenhouse. Affected plants also had signs of thrips feeding (dead thrips, frass, and feeding scars) present across the whole plant (Figure 1D). Samples were submitted to the NC State Plant Disease and Insect Clinic and tested positive for INSV, but negative for TSWV, using Agdia ImmunoStrips®. RNA was extracted from symptomatic leaf tissue using the IBI Total RNA Mini kit (Plant), and complementary DNA (cDNA) was generated using the ThermoFisher Verso cDNA synthesis kit. A reverse transcriptase (RT)-PCR with INSV nucleocapsid (N) primers (F:5'-ATGAACAAAGCAAAGATTACC-3' and R:5'- TTAAATAGAATCATTTTTCCC-3') was used to confirm INSV presence (Hassani-Mehraban et al. 2016). Full length N cDNA amplicon sequencing [GenBank No. PP658213] revealed 99.62% nucleotide identity to NCBI GenBank accessions KF926828 (orchid in California), MH453554.1 (hosta from NY), and MH453552.1 (foxglove from NY), all of which are INSV N sequences. The infected leaf samples were used to mechanically inoculate Emilia sonchifolia and tomato (cv.'Moneymaker') using standard virological methods. We successfully infected E. sonchifolia with INSV (confirmed with visual mosaic symptoms and positive INSV ImmunoStrip). However, mechanical inoculation of the tomato plants proved unsuccessful. Using the INSV infected E. sonchifolia leaves as an inoculum source, we generated a viruliferous Frankliniella occidentalis (Western flower thrips) cohort and challenged three week old tomatoes using thrips mediated inoculation (adapted from Aramburu et al. 2009 and Rotenberg et al., 2009). Twenty days post-inoculation, tomatoes with thrips feeding scars were symptomatic for INSV infection with chlorotic and necrotic spots, stunting, and reduced vigor. INSV infection of these tomato plants was verified with a positive INSV ImmunoStrip® result, two-step RT-PCR amplification of N, and Sanger sequencing of N. Samples from thrips-inoculated tomato plants did not test positive for TSWV. Sequence alignment showed that the recovered virus sequence was 99.85% identical to the original INSV sequence from the diagnostic sample (a single nucleotide difference). To the best of our knowledge, this is the first instance of INSV infecting tomato in NC production systems. Although TSWV is more common in vegetable production in NC (253 cases of TSWV compared to 1 case of INSV in vegetable crops based on NC State Plant Disease and Insect Clinic records since 2008), INSV incursion into tomato producing areas is concerning and should be closely monitored, especially at the transplant stage. This report also underscores the importance of using thrips vectors to transmit virus in screening for susceptibility to orthotospoviruses.

Dehydration and tomato spotted wilt virus infection combine to alter feeding and survival parameters for the western flower thrips, Frankliniella occidentalis.

Dehydration and tomato spotted wilt virus (TSWV) infection substantially impact the feeding of western flower thrips, Frankliniella occidentalis. Until now, the dynamics between these biotic and abiotic stresses have not been examined for thrips. Here, we report water balance characteristics and changes in other biological parameters during infection with TSWV for the western flower thrips. There were no apparent differences in water balance parameters during TSWV infection of male or female thrips. Our results show that, although water balance characteristics of western flower thrips are minimally impacted by TSWV infection, the increase in feeding and activity when dehydration and TSWV are combined suggests that virus transmission could be increased under periods of drought. Importantly, survival and progeny generation were impaired during TSWV infection and dehydration bouts. The negative impact on survival and reproduction suggests that the interactions between TSWV infection and dehydration will likely reduce thrips populations. The opposite effects of dehydration on feeding/activity and survival/reproduction for virus infected thrips suggest the impact of vectorial capacity will likely be minor for TSWV transmission. As water stress significantly impacts insect-plant-virus dynamics, these studies highlight that all interactions and effects need to be measured to understand thrips-TSWV interactions in their role as viral vector to plants.

CRISPR/Cas9-mediated genome editing of Frankliniella occidentalis, the western flower thrips, via embryonic microinjection.

The western flower thrips, Frankliniella occidentalis, poses a significant challenge in global agriculture as a notorious pest and a vector of economically significant orthotospoviruses. However, the limited availability of genetic tools for F. occidentalis hampers the advancement of functional genomics and the development of innovative pest control strategies. In this study, we present a robust methodology for generating heritable mutations in F. occidentalis using the CRISPR/Cas9 genome editing system. Two eye-colour genes, white (Fo-w) and cinnabar (Fo-cn), frequently used to assess Cas9 function in insects were identified in the F. occidentalis genome and targeted for knockout through embryonic microinjection of Cas9 complexed with Fo-w or Fo-cn specific guide RNAs. Homozygous Fo-w and Fo-cn knockout lines were established by crossing mutant females and males. The Fo-w knockout line revealed an age-dependent modification of eye-colour phenotype. Specifically, while young larvae exhibit orange-coloured eyes, the colour transitions to bright red as they age. Unexpectedly, loss of Fo-w function also altered body colour, with Fo-w mutants having a lighter coloured body than wild type, suggesting a dual role for Fo-w in thrips. In contrast, individuals from the Fo-cn knockout line consistently displayed bright red eyes throughout all life stages. Molecular analyses validated precise editing of both target genes. This study offers a powerful tool to investigate thrips gene function and paves the way for the development of genetic technologies for population suppression and/or population replacement as a means of mitigating virus transmission by this vector.

Multigenic hairpin transgenes in tomato confer resistance to multiple orthotospoviruses including Sw-5 resistance-breaking tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) and related thrips-borne orthotospoviruses are a threat to food and ornamental crops. Orthotospoviruses have the capacity for rapid genetic change by genome segment reassortment and mutation. Genetic resistance is one of the most effective strategies for managing orthotospoviruses, but there are multiple examples of resistance gene breakdown. Our goal was to develop effective multigenic, broad-spectrum resistance to TSWV and other orthotospoviruses. The most conserved sequences for each open reading frame (ORF) of the TSWV genome were identified and comparison to other orthotospoviruses revealed sequence conservation within virus clades and some overlapped with domains with well-documented biological functions. We made six hairpin constructs, each of which incorporated sequences matching portions of all five ORFs. Tomato plants expressing the hairpin transgene were challenged with TSWV by thrips and leaf-rub inoculation and four constructs provided strong protection against TSWV in foliage and fruit. To determine if the hairpin constructs provided protection against other emerging orthotospoviruses, we challenged the plants with tomato chlorotic spot virus and resistance-breaking TSWV (RB-TSWV) and found that the same constructs also provided resistance to these related viruses. Antiviral hairpin constructs are an effective way to protect plants from multiple orthotospoviruses and are an important strategy in the fight against RB-TSWV and emerging viruses. Targeting of all five viral ORFs is expected to increase the durability of resistance and combining them with other resistance genes could further extend the utility of this disease control strategy.

Abaxial leaf surface-mounted multimodal wearable sensor for continuous plant physiology monitoring.

Wearable plant sensors hold tremendous potential for smart agriculture. We report a lower leaf surface-attached multimodal wearable sensor for continuous monitoring of plant physiology by tracking both biochemical and biophysical signals of the plant and its microenvironment. Sensors for detecting volatile organic compounds (VOCs), temperature, and humidity are integrated into a single platform. The abaxial leaf attachment position is selected on the basis of the stomata density to improve the sensor signal strength. This versatile platform enables various stress monitoring applications, ranging from tracking plant water loss to early detection of plant pathogens. A machine learning model was also developed to analyze multichannel sensor data for quantitative detection of tomato spotted wilt virus as early as 4 days after inoculation. The model also evaluates different sensor combinations for early disease detection and predicts that minimally three sensors are required including the VOC sensors.

A call to arms: novel strategies for thrips and tospovirus control.

Thrips and the tospoviruses they transmit are some of the most significant threats to food and ornamental crop production globally. Control of the insect and virus is challenging and new strategies are needed. Characterizing the thrips-virus interactome provides new targets for disrupting the transmission cycle. Viral and insect determinants of vector competence are being defined, including the viral attachment protein and its structure as well as thrips proteins that interact with and respond to tospovirus infection. Additional thrips control strategies such as RNA interference need further refinement and field-applicable delivery systems, but they show promise for the knockdown of essential genes for thrips survival and virus transmission. The identification of a toxin that acts to deter thrips oviposition on cotton also presents new opportunities for control of this important pest.

First Report of Resistance-Breaking Variants of Tomato Spotted Wilt Virus (TSWV) Infecting Tomatoes with the Sw-5 Tospovirus-Resistance Gene in North Carolina.

Widespread use of tomato cultivars with the Sw-5 resistance gene has led to the emergence of resistance-breaking (RB) strains of tomato spotted wilt virus across the globe. In June of 2022, tomato spotted wilt (TSW) symptoms were observed at two farms (A and B, within 15 miles of each other) in Rowan County, NC on several commercial TSW resistant tomato cultivars (all heterozygous for the Sw-5 gene). At farm A, ~10% of plants had symptomatic foliage with ~30% of fruit with symptoms, while at farm B, up to 50% of plants had symptomatic foliage with ~80% of fruit with symptoms. Visual symptoms included stunting, severe leaf curling and bronzing, necrotic lesions on leaves, petioles and stems, and concentric ring spots on fruit (Supplementary Fig. 1). TSWV ImmunoStrips (AgDia, Elkhart, IN) and reverse-transcription (RT)-PCR with NSm primers (di Rienzo et al 2018) confirmed the presence of TSWV in 12 symptomatic plants sampled across the two farms. Primers designed to detect Impatiens necrotic spot virus, groundnut ringspot virus, tomato chlorotic spot virus, tomato chlorosis virus, alfalfa mosaic virus, and tomato necrotic streak virus (ilarvirus, Badillo et al., 2016) failed to generate amplicons of the expected size from cDNA generated from these field samples. The amplicons from full-length NSm cDNA were sequenced from independent, single-leaflet isolates from the TSWV-positive plants (three from farm A, nine from farm B) with the expectation of finding an amino acid (aa) substitution associated with the Sw-5 RB phenotype identified previously in CA (C118Y, Batuman et al. 2017) or Spain (C118Y and T120N, Lopez et al. 2011). All three nucleotide sequences from farm A contained the NSm C118Y substitution reported in CA. All three sequences were 99% identical (including the C118Y mutation) to NCBI GenBank accession KU179600.1, a TSWV isolate collected from GA in 2014 with no cultivar information reported. The nine nucleotide sequences from farm B contained neither of the two previously reported aa substitutions associated with the RB phenotype. Instead, all contained a D122G substitution within a conserved region of the TSWV NSm protein reported to be involved in direct interaction with the Sw-5 protein (Zhu et al 2017). Likewise, Huang et al (2021) generated a D122A mutation in TSWV-NSm, resulting in failure to elicit a Sw-5 mediated hypersensitive response. Three NSm sequences retrieved from GenBank contained the D122G substitution (AY848921.1, HM015516.1, KU179582.1), however, this mutation was not implicated directly with RB phenotypes (Ciuffo et al., 2005; Lopez et al., 2011; Marshall, 2016). The RB phenotype was confirmed with the NC variants on 'Mountain Merit' (Sw-5) by two means of virus inoculation: mechanical, rub-inoculation with extracted sap from infected plants, and thrips transmission assays with lab colony-maintained, Frankliniella occidentalis, the western flower thrips. Symptomatic leaf tissue obtained from these inoculation assays tested positive for TSWV by DAS-ELISA (AgDia, Elkhart, IN) and RT-PCR with NSm primers, providing definitive evidence of the occurrence of RB-TSWV at both farms, and subsequent sequencing confirmed the C118Y and D122G substitutions. This report warrants further investigation of the putative origins, prevalence and epidemiological implications of RB-TSWV variants in NC tomato production, and the development of new sources of resistance to TSWV.

The physical interactome between Peregrinus maidis proteins and the maize mosaic virus glycoprotein provides insights into the cellular biology of a rhabdovirus in the insect vector.

Rhabdovirus glycoproteins (G) serve multifunctional roles in virus entry, assembly, and exit from animal cells. We hypothesize that maize mosaic virus (MMV) G is required for invasion, infection, and spread in Peregrinus maidis, the planthopper vector. Using a membrane-based yeast two-hybrid assay, we identified 107 P. maidis proteins that physically interacted with MMV G, of which approximately 53% matched proteins with known functions including endocytosis, vesicle-mediated transport, protein synthesis and turnover, nuclear export, metabolism and host defense. Physical interaction networks among conserved proteins indicated a possible cellular coordination of processes associated with MMV G translation, protein folding and trafficking. Non-annotated proteins contained predicted functional sites, including a diverse array of ligand binding sites. Cyclophilin A and apolipophorin III co-immunoprecipitated with MMV G, and each showed different patterns of localization with G in insect cells. This study describes the first protein interactome for a rhabdovirus spike protein and insect vector.

Sex-biased proteomic response to tomato spotted wilt virus infection of the salivary glands of Frankliniella occidentalis, the western flower thrips.

Successful transmission of tomato spotted wilt virus (TSWV) by Frankliniella occidentalis requires robust infection of the salivary glands (SGs) and virus delivery to plants during salivation. Feeding behavior and transmission efficiency are sexually-dimorphic traits of this thrips vector species. Proteins secreted from male and female SG tissues, and the effect of TSWV infection on the thrips SG proteome are unknown. To begin to discern thrips factors that facilitate virus infection of SGs and transmission by F. occidentalis, we used gel- and label-free quantitative and qualitative proteomics to address two hypotheses: (i) TSWV infection modifies the composition and/or abundance of SG-expressed proteins in adults; and (ii) TSWV has a differential effect on the male and female SG proteome and secreted saliva. Our study revealed a sex-biased SG proteome for F. occidentalis, and TSWV infection modulated the SG proteome in a sex-dependent manner as evident by the number, differential abundance, identities and generalized roles of the proteins. Male SGs exhibited a larger proteomic response to the virus than female SGs. Intracellular processes modulated by TSWV in males indicated perturbation of SG cytoskeletal networks and cell-cell interactions, i.e., basement membrane (BM) and extracellular matrix (ECM) proteins, and subcellular processes consistent with a metabolic slow-down under infection. Several differentially-abundant proteins in infected male SGs play critical roles in viral life cycles of other host-virus pathosystems. In females, TSWV modulated processes consistent with tissue integrity and active translational and transcriptional regulation. A core set of proteins known for their roles in plant cell-wall degradation and protein metabolism were identified in saliva of both sexes, regardless of virus infection status. Saliva proteins secreted by TSWV-infected adults indicated energy generation, consumption and protein turnover, with an enrichment of cytoskeletal/BM/ECM proteins and tricarboxylic acid cycle proteins in male and female saliva, respectively. The nonstructural TSWV protein NSs - a multifunctional viral effector protein reported to target plant defenses against TSWV and thrips - was identified in female saliva. This study represents the first description of the SG proteome and secretome of a thysanopteran and provides many candidate proteins to further unravel the complex interplay between the virus, insect vector, and plant host.