RNAi-mediated knockdown of exportin 1 negatively affected ovary development, survival and maize mosaic virus accumulation in its insect vector Peregrinus maidis.

Exportin 1 (XPO1) is the major karyopherin-ß nuclear receptor mediating the nuclear export of hundreds of proteins and some classes of RNA and regulates several critical processes in the cell, including cell-cycle progression, transcription and translation. Viruses have co-opted XPO1 to promote nucleocytoplasmic transport of viral proteins and RNA. Maize mosaic virus (MMV) is a plant-infecting rhabdovirus transmitted in a circulative propagative manner by the corn planthopper, Peregrinus maidis. MMV replicates in the nucleus of plant and insect hosts, and it remains unknown whether MMV co-opts P. maidis XPO1 (PmXPO1) to complete its life cycle. Because XPO1 plays multiple regulatory roles in cell functions and virus infection, we hypothesized that RNAi-mediated silencing of XPO1 would negatively affect MMV accumulation and insect physiology. Although PmXPO1 expression was not modulated during MMV infection, PmXPO1 knockdown negatively affected MMV accumulation in P. maidis at 12 and 15 days after microinjection. Likewise, PmXPO1 knockdown negatively affected P. maidis survival and reproduction. PmXPO1 exhibited tissue-specific expression patterns with higher expression in the ovaries compared with the guts of adult females. Survival rate was significantly lower for PmXPO1 knockdown females, compared with controls, but no effect was observed for males. PmXPO1 knockdown experiments revealed a role for PmXPO1 in ovary function and egg production. Oviposition and egg hatch on plants were dramatically reduced in females treated with dsRNA PmXPO1. These results suggest that PmXPO1 is a positive regulator of P. maidis reproduction and that it plays a proviral role in the insect vector supporting MMV infection.

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.

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.

Structural and functional insights into the ATP-binding cassette transporter family in the corn planthopper, Peregrinus maidis.

The corn planthopper, Peregrinus maidis, is an economically important pest of maize and sorghum. Its feeding behaviour and the viruses it transmits can significantly reduce crop yield. The control of P. maidis and its associated viruses relies heavily on insecticides. However, control has proven difficult due to limited direct exposure of P. maidis to insecticides and rapid development of resistance. As such, alternative control methods are needed. In the absence of a genome assembly for this species, we first developed transcriptomic resources. Then, with the goal of finding targets for RNAi-based control, we identified members of the ATP-binding cassette transporter family and targeted specific members via RNAi. PmABCB_160306_3, PmABCE_118332_5 and PmABCF_24241_1, whose orthologs in other insects have proven important in development, were selected for knockdown. We found that RNAi-mediated silencing of PmABCB_160306_3 impeded ovary development; disruption of PmABCE_118332_5 resulted in localized melanization; and knockdown of PmABCE_118332_5 or PmABCF_24241_1 each led to high mortality within five days. Each phenotype is similar to that found when targeting the orthologous gene in other species and it demonstrates their potential for use in RNAi-based P. maidis control. The transcriptomic data and RNAi results presented here will no doubt assist with the development of new control methods for this 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.

ICTV Virus Taxonomy Profile: Rhabdoviridae 2022.

The family Rhabdoviridae comprises viruses with negative-sense (-) RNA genomes of 10-16 kb. Virions are typically enveloped with bullet-shaped or bacilliform morphology but can also be non-enveloped filaments. Rhabdoviruses infect plants or animals, including mammals, birds, reptiles, amphibians or fish, as well as arthropods, which serve as single hosts or act as biological vectors for transmission to animals or plants. Rhabdoviruses include important pathogens of humans, livestock, fish or agricultural crops. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Rhabdoviridae, which is available at ictv.global/report/rhabdoviridae.

New Technologies for Studying Negative-Strand RNA Viruses in Plant and Arthropod Hosts.

The plant viruses in the phylum Negarnaviricota, orders Bunyavirales and Mononegavirales, have common features of single-stranded, negative-sense RNA genomes and replication in the biological vector. Due to the similarities in biology, comparative functional analysis in plant and vector hosts is helpful for understanding host-virus interactions for negative-strand RNA viruses. In this review, we will highlight recent technological advances that are breaking new ground in the study of these recalcitrant virus systems. The development of infectious clones for plant rhabdoviruses and bunyaviruses is enabling unprecedented examination of gene function in plants and these advances are also being transferred to study virus biology in the vector. In addition, genome and transcriptome projects for critical nonmodel arthropods has enabled characterization of insect response to viruses and identification of interacting proteins. Functional analysis of genes using genome editing will provide future pathways for further study of the transmission cycle and new control strategies for these viruses and their vectors.

Discovery of Novel Thrips Vector Proteins That Bind to the Viral Attachment Protein of the Plant Bunyavirus Tomato Spotted Wilt Virus.

The plant-pathogenic virus tomato spotted wilt virus (TSWV) encodes a structural glycoprotein (GN) that, like with other bunyavirus/vector interactions, serves a role in viral attachment and possibly in entry into arthropod vector host cells. It is well documented that Frankliniella occidentalis is one of nine competent thrips vectors of TSWV transmission to plant hosts. However, the insect molecules that interact with viral proteins, such as GN, during infection and dissemination in thrips vector tissues are unknown. The goals of this project were to identify TSWV-interacting proteins (TIPs) that interact directly with TSWV GN and to localize the expression of these proteins in relation to virus in thrips tissues of principal importance along the route of dissemination. We report here the identification of six TIPs from first-instar larvae (L1), the most acquisition-efficient developmental stage of the thrips vector. Sequence analyses of these TIPs revealed homology to proteins associated with the infection cycle of other vector-borne viruses. Immunolocalization of the TIPs in L1 revealed robust expression in the midgut and salivary glands of F. occidentalis, the tissues most important during virus infection, replication, and plant inoculation. The TIPs and GN interactions were validated using protein-protein interaction assays. Two of the thrips proteins, endocuticle structural glycoprotein and cyclophilin, were found to be consistent interactors with GN These newly discovered thrips protein-GN interactions are important for a better understanding of the transmission mechanism of persistent propagative plant viruses by their vectors, as well as for developing new strategies of insect pest management and virus resistance in plants.IMPORTANCE Thrips-transmitted viruses cause devastating losses to numerous food crops worldwide. For negative-sense RNA viruses that infect plants, the arthropod serves as a host as well by supporting virus replication in specific tissues and organs of the vector. The goal of this work was to identify thrips proteins that bind directly to the viral attachment protein and thus may play a role in the infection cycle in the insect. Using the model plant bunyavirus tomato spotted wilt virus (TSWV), and the most efficient thrips vector, we identified and validated six TSWV-interacting proteins from Frankliniella occidentalis first-instar larvae. Two proteins, an endocuticle structural glycoprotein and cyclophilin, were able to interact directly with the TSWV attachment protein, GN, in insect cells. The TSWV GN-interacting proteins provide new targets for disrupting the viral disease cycle in the arthropod vector and could be putative determinants of vector competence.

ICTV Virus Taxonomy Profile: Rhabdoviridae.

The family Rhabdoviridae comprises viruses with negative-sense (-) single-stranded RNA genomes of 10.8-16.1 kb. Virions are typically enveloped with bullet-shaped or bacilliform morphology but can also be non-enveloped filaments. Rhabdoviruses infect plants and animals including mammals, birds, reptiles and fish, as well as arthropods which serve as single hosts or act as biological vectors for transmission to animals or plants. Rhabdoviruses include important pathogens of humans, livestock, fish and agricultural crops. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Rhabdoviridae, which is available at www.ictv.global/report/rhabdoviridae.

Transcriptome changes associated with Tomato spotted wilt virus infection in various life stages of its thrips vector, Frankliniella fusca (Hinds).

Persistent propagative viruses maintain intricate interactions with their arthropod vectors. In this study, we investigated the transcriptome-level responses associated with a persistent propagative phytovirus infection in various life stages of its vector using an Illumina HiSeq sequencing platform. The pathosystem components included a Tospovirus, Tomato spotted wilt virus (TSWV), its insect vector, Frankliniella fusca (Hinds), and a plant host, Arachis hypogaea (L.). We assembled (de novo) reads from three developmental stage groups of virus-exposed and non-virus-exposed F. fusca into one transcriptome consisting of 72 366 contigs and identified 1161 differentially expressed (DE) contigs. The number of DE contigs was greatest in adults (female) (562) when compared with larvae (first and second instars) (395) and pupae (pre- and pupae) (204). Upregulated contigs in virus-exposed thrips had blastx annotations associated with intracellular transport and virus replication. Upregulated contigs were also assigned blastx annotations associated with immune responses, including apoptosis and phagocytosis. In virus-exposed larvae, Blast2GO analysis identified functional groups, such as multicellular development with downregulated contigs, while reproduction, embryo development and growth were identified with upregulated contigs in virus-exposed adults. This study provides insights into differences in transcriptome-level responses modulated by TSWV in various life stages of an important vector, F. fusca.