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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.
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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.
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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.
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Proteína Exportina 1 , Hemípteros , Insectos Vectores , Carioferinas , Ovario , Interferencia de ARN , Receptores Citoplasmáticos y Nucleares , Animales , Femenino , Hemípteros/virología , Hemípteros/genética , Hemípteros/crecimiento & desarrollo , Carioferinas/metabolismo , Carioferinas/genética , Ovario/virología , Ovario/metabolismo , Ovario/crecimiento & desarrollo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Insectos Vectores/virología , Insectos Vectores/genética , Rhabdoviridae/fisiología , Proteínas de Insectos/metabolismo , Proteínas de Insectos/genética , Zea mays/virología , Zea mays/genética , Técnicas de Silenciamiento del GenRESUMEN
The first infectious agent to bear the name 'virus' was described in 1898: a plant pathogen called tobacco mosaic virus that infects a wide range of plants and results in a yellow mosaic of the leaves. Since then, the study of plant viruses has facilitated new discoveries in both virology and plant biology. Traditionally, research has focused on viruses that cause severe disease in plants used for human and animal food or recreation. However, closer inspection of the plant-associated virome is now revealing interactions that range from pathogenic to symbiotic. Although they are often studied in isolation, plant viruses are usually found as part of a broader community that includes other plant-associated microbes and pests. For example, biological vectors of plant viruses (arthropods, nematodes, fungi, and protists) can facilitate the transmission of viruses between plants in an intricate interaction. To enhance transmission, viruses can induce the plant to 'lure' the vector by modulating plant chemistry and defenses. Once delivered to a new host, viruses are dependent on specific proteins that modify the structural components of the cell to enable transport of viral proteins and genomic material. Links between antiviral plant defenses and key steps in virus movement and transmission are being revealed. Upon infection, a suite of antiviral responses is triggered, including the expression of resistance genes - a favored strategy to control plant viruses. In this primer, we discuss these features and more, highlighting the exciting world of plant-virus interactions.
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Enfermedades de las Plantas , Enfermedades de las Plantas/virología , Variación Genética , Fenómenos Fisiológicos de las PlantasRESUMEN
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.
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Compuestos Orgánicos Volátiles , Dispositivos Electrónicos Vestibles , Hojas de la Planta , Temperatura , Fenómenos Fisiológicos de las Plantas , PlantasRESUMEN
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.
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Thysanoptera , Tospovirus , Femenino , Animales , Tospovirus/genéticaRESUMEN
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.
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Hemípteros , Insecticidas , Femenino , Animales , Zea mays/genética , Transportadoras de Casetes de Unión a ATP/genética , Hemípteros/genética , Perfilación de la Expresión GénicaRESUMEN
Papaya sticky disease is caused by the association of a fusagra-like and an umbra-like virus, named papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), respectively. Both viral genomes are encapsidated in particles formed by the PMeV ORF1 product, which has the potential to encode a protein with 1563 amino acids (aa). However, the structural components of the viral capsid are unknown. To characterize the structural proteins of PMeV and PMeV2, virions were purified from Carica papaya latex. SDS-PAGE analysis of purified virus revealed two major proteins of ~40 kDa and ~55 kDa. Amino-terminal sequencing of the ~55 kDa protein and LC-MS/MS of purified virions indicated that this protein starts at aa 263 of the deduced ORF1 product as a result of either degradation or proteolytic processing. A yeast two-hybrid assay was used to identify Arabidopsis proteins interacting with two PMeV ORF1 product fragments (aa 321-670 and 961-1200). The 50S ribosomal protein L17 (AtRPL17) was identified as potentially associated with modulated translation-related proteins. In plant cells, AtRPL17 co-localized and interacted with the PMeV ORF1 fragments. These findings support the hypothesis that the interaction between PMeV/PMeV2 structural proteins and RPL17 is important for virus-host interactions.
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Proteínas de la Cápside , Carica , Aminoácidos , Cápside , Proteínas de la Cápside/genética , Cromatografía Liquida , Látex , Espectrometría de Masas en Tándem , Virus ARN/genéticaRESUMEN
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.
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The corn planthopper, Peregrinus maidis Ashmead (Hemiptera:Delphacidae), is a widely distributed insect pest which serves as a vector of two phytopathogenic viruses, Maize mosaic virus (MMV) and Maize stripe virus (MStV). It transmits the viruses in a persistent and propagative manner. MMV is an alphanucleorhabdovirus with a negative-sense, single-stranded RNA unsegmented genome. One identified insect vector protein that may serve as receptor to MMV is Syntaxin-18 (PmStx18) which belongs to the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins. SNAREs play major roles in the final stage of docking and subsequent fusion of diverse vesicle-mediated transport events. In this work, in silico analysis of the interaction of MMV glycoprotein (MMV G) and PmStx18 was performed. Various freely available protein-protein docking web servers were used to predict the 3 D complex of MMV G and PmStx18. Analysis and protein-protein interaction (PPI) count showed that the complex predicted by the ZDOCK server has the highest number of interaction and highest affinity, as suggested by the calculated solvation free energy gain upon formation of the interface (ΔiG = -31 kcal/mol). Molecular dynamics simulation of the complex revealed important interactions at the interface over the course of 25 ns. This is the first in silico analysis performed for the interaction on a putative receptor of P. maidis and MMV G. The results of the PPI prediction provide novel information for studying the role of Stx18 in the transport, docking and fusion events involved in virus particle transport in the insect vector cells and its release.Communicated by Ramaswamy H. Sarma.
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Hemípteros , Rhabdoviridae , Animales , Hemípteros/genética , Proteínas Qa-SNARE , GlicoproteínasRESUMEN
Recent reverse genetics technologies have enabled genetic manipulation of plant negative-strand RNA virus (NSR) genomes. Here, we report construction of an infectious clone for the maize-infecting Alphanucleorhabdovirus maydis, the first efficient NSR vector for maize. The full-length infectious clone was established using agrobacterium-mediated delivery of full-length maize mosaic virus (MMV) antigenomic RNA and the viral core proteins (nucleoprotein N, phosphoprotein P, and RNA-directed RNA polymerase L) required for viral transcription and replication into Nicotiana benthamiana. Insertion of intron 2 ST-LS1 into the viral L gene increased stability of the infectious clone in Escherichia coli and Agrobacterium tumefaciens. To monitor virus infection in vivo, a green fluorescent protein (GFP) gene was inserted in between the N and P gene junctions to generate recombinant MMV-GFP. Complementary DNA (cDNA) clones of MMV-wild type (WT) and MMV-GFP replicated in single cells of agroinfiltrated N. benthamiana. Uniform systemic infection and high GFP expression were observed in maize inoculated with extracts of the infiltrated N. benthamiana leaves. Insect vectors supported virus infection when inoculated via feeding on infected maize or microinjection. Both MMV-WT and MMV-GFP were efficiently transmitted to maize by planthopper vectors. The GFP reporter gene was stable in the virus genome and expression remained high over three cycles of transmission in plants and insects. The MMV infectious clone will be a versatile tool for expression of proteins of interest in maize and cross-kingdom studies of virus replication in plant and insect hosts.
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Hemípteros , Zea mays , Animales , ADN Complementario , Zea mays/genética , Insectos Vectores , Nicotiana/genética , Vectores GenéticosRESUMEN
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.
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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.
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Thysanoptera , Tospovirus , Animales , Femenino , Flores , Masculino , Enfermedades de las Plantas , Plantas , Proteoma/metabolismo , Proteómica , Glándulas Salivales , Thysanoptera/metabolismo , Tospovirus/fisiologíaRESUMEN
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.
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Rhabdoviridae , Animales , Aves , Peces , Genoma Viral , Mamíferos , Reptiles , Rhabdoviridae/genética , Virión , Replicación ViralRESUMEN
We demonstrate an integrated microneedle (MN)-smartphone nucleic acid amplification platform for "sample-to-answer" diagnosis of multiplexed plant pathogens within 30 min. This portable system consists of a polymeric MN patch for rapid nucleic acid extraction within a minute and a 3D-printed smartphone imaging device for loop-mediated isothermal amplification (LAMP) reaction and detection. We expanded the extraction of the MN technology for DNA targets as in the previous study (ACS Nano, 2019, 13, 6540-6549) to more fragile RNA biomarkers, evaluated the storability of the extracted nucleic acid samples on MN surfaces, and developed a smartphone-based LAMP amplification and fluorescent reader device that can quantify four LAMP reactions on the same chip. In addition, we have found that the MN patch containing as few as a single needle tip successfully extracted enough RNA for RT-PCR or RT-LAMP analysis. Moreover, MN-extracted RNA samples remained stable on MN surfaces for up to three days. The MN-smartphone platform has been used to detect both Phytophthora infestans DNA and tomato spotted wilt virus (TSWV) RNA down to 1 pg, comparable to the results from a benchtop thermal cycler. Finally, multiplexed detection of P. infestans and TSWV through a single extraction from infected tomato leaves and amplification on the smartphone without benchtop equipment was demonstrated.
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Técnicas Biosensibles , Teléfono Inteligente , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Enfermedades de las PlantasRESUMEN
The corn planthopper, Peregrinus maidis, is a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.
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Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Zea mays/química , Animales , Endonucleasas/genética , FemeninoRESUMEN
Plant rhabdoviruses are recognized by their large bacilliform particles and for being able to replicate in both their plant hosts and arthropod vectors. This review highlights selected, better studied examples of plant rhabdoviruses, their genetic diversity, epidemiology and interactions with plant hosts and arthropod vectors: Alfalfa dwarf virus is classified as a cytorhabdovirus, but its multifunctional phosphoprotein is localized to the plant cell nucleus. Lettuce necrotic yellows virus subtypes may differentially interact with their aphid vectors leading to changes in virus population diversity. Interactions of rhabdoviruses that infect rice, maize and other grains are tightly associated with their specific leafhopper and planthopper vectors. Future outbreaks of vector-borne nucleorhabdoviruses may be predicted based on a world distribution map of the insect vectors. The epidemiology of coffee ringspot virus and its Brevipalpus mite vector is illustrated highlighting the symptomatology and biology of a dichorhavirus and potential impacts of climate change on its epidemiology.
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Productos Agrícolas/virología , Insectos Vectores/virología , Enfermedades de las Plantas/virología , Virus de Plantas , Rhabdoviridae , Animales , Interacciones Microbiota-Huesped , Virus de Plantas/genética , Virus de Plantas/fisiología , Rhabdoviridae/genética , Rhabdoviridae/fisiologíaRESUMEN
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.
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Insectos/virología , Enfermedades de las Plantas/virología , Virus de Plantas , Plantas/virología , Virus ARN , Animales , Insectos Vectores/virologíaRESUMEN
The Great Plains of the United States is a region comprised of approximately 45 million hectares of grasslands where several economically important cereal crops are grown. Arthropod-transmitted, cereal-infecting viruses vary in incidence from year-to-year and are often difficult to detect in large acreages. To facilitate the detection of economically important viruses of cereals that often exist in co-infections, a multiplex reverse transcriptase PCR (RT-PCR) platform assay was developed. This method can be used in combination with high resolution melting (HRM) to detect and allow for discrimination between three arthropod-transmitted plant viruses; Wheat streak mosaic virus (WSMV), Maize mosaic virus (MMV) and Barley yellow dwarf virus (BYDV). Multiplex PCR in combination with HRM allowed for successful detection of WSMV, MMV, and BYDV, as well as discrimination between three BYDV species, BYDV-PAS, BYDV-PAV and BYDV-MAV. All primer pairs amplified products of the predicted size. The BYDV-RT-PCR primers amplified products of identical length for all three species of BYDV. HRM was then used to discriminate between these products by determining significant differences between the melting rates for each (p < 0.05). This study demonstrates the flexibility of combining multiplex PCR with HRM to increase the specificity of plant virus diagnostics based on the needs of the diagnostician performing the assay.