microinjection-corn-planthopper-peregrinus-maidis-embryos-for

Microinjection of Corn Planthopper, <em>Peregrinus maidis</em>, Embryos for CRISPR/Cas9 Genome Editing

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the ...

North Carolina State University

Maize mosaic virus (MMV) is a plant-pathogenic rhabdovirus that is transmitted by the corn planthopper, Peregrinus maidis, in a propagative manner. P. maidis supports long-term MMV infections with no negative effects on insect performance. To elucidate whole-body transcriptome responses to virus infection, RNA-Seq was used to examine differential gene expression of virus-infected adult insects, and libraries were prepared from replicated groups of virus-exposed insects and non-exposed insects. From the 68,003 de novo-assembled transcripts, 144 were differentially-expressed (DE) during viral infection with comparable numbers up- and down-regulated. DE transcripts with similarity to genes associated with transposable elements (i.e., RNA-directed DNA polymerases) were enriched and may represent a mechanisim for modulating virus infection. Comparison of the P. maidis DE transcripts to published propagative virus-responsive transcript databases for two other hopper vectors revealed that 16% of the DE transcripts were shared across the three systems and may represent conserved responses to propagative viruses.

Transcriptomic response of the insect vector, Peregrinus maidis, to Maize mosaic rhabdovirus and identification of conserved responses to propagative viruses in hopper vectors.

Thrips-transmitted tospoviruses are an emerging and re-emerging threat to crop production worldwide. Tospoviruses are transstadially transmitted from larval to pupal stages of development, with adults serving as the primary inoculators of plants. A unique feature of the transmission cycle is that adults-while they can acquire virus from plants directly-are competent as vectors only if they acquire virus as larvae. Thrips vectors also serve as hosts for the virus, supporting its replication in midgut tissues and salivary glands. There is a tight link between thrips development and virus dissemination in the insect, and recent transcriptome studies point to stage-specific responses that coincide with localization of the virus in the insect body. Transcriptome sequencing of thrips vectors is leading to identification of virus-responsive thrips genes and possibly new targets to disrupt the virus transmission cycle. Accumulation of thrips-omics resources and advancements in functional biology tools will propel new and exciting molecular studies of thrips-tospoviruses interactions.

Molecular interactions between tospoviruses and thrips vectors.

Thrips palmi is a widely distributed major agricultural pest in the tropics and subtropics, causing significant losses in cucurbit and solanaceous crops through feeding damage and transmission of tospoviruses. Thrips palmi is a vector of capsicum chlorosis virus (CaCV) in Australia. The present understanding of transmission biology and potential effects of CaCV on T. palmi is limited. To gain insights into molecular responses to CaCV infection, we performed RNA-Seq to identify thrips transcripts that are differentially-abundant during virus infection of adults. De-novo assembly of the transcriptome generated from whole bodies of T. palmi adults generated 166,445 contigs, of which ~24% contained a predicted open reading frame. We identified 1,389 differentially-expressed (DE) transcripts, with comparable numbers up- (708) and down-regulated (681) in virus-exposed thrips compared to non-exposed thrips. Approximately 59% of these DE transcripts had significant matches to NCBI non-redundant proteins (Blastx) and Blast2GO identified provisional functional categories among the up-regulated transcripts in virus-exposed thrips including innate immune response-related genes, salivary gland and/or gut-associated genes and vitellogenin genes. The majority of the immune-related proteins are known to serve functions in lysosome activity and melanisation in insects. Most of the up-regulated oral and extra-oral digestion-associated genes appear to be involved in digestion of proteins, lipids and plant cell wall components which may indirectly enhance the likelihood or frequency of virus transmission or may be involved in the regulation of host defence responses. Most of the down-regulated transcripts fell into the gene ontology functional category of 'structural constituent of cuticle'. Comparison to DE genes responsive to tomato spotted wilt virus in Frankliniella occidentalis indicates conservation of some thrips molecular responses to infection by different tospoviruses. This study assembled the first transcriptome in the genus Thrips and provides important data to broaden our understanding of networks of molecular interactions between thrips and tospoviruses.

Transcriptome-wide responses of adult melon thrips (Thrips palmi) associated with capsicum chlorosis virus infection.

The corn planthopper, Peregrinus maidis, not only causes direct damage to plants by feeding, but also transmits maize mosaic virus (MMV) to the plant hosts. The virus is transmitted in a propagative manner but the acquisition of MMV by the vector feeding on infected plants can result in low acquisition and inoculation efficiency. Here, we increased the acquisition efficiency by delivering the virus directly into the hemocoel through microinjection, which resulted in efficient virus infection of the insect and transmission to maize. We found that delivery of virus by injection of 10 ng MMV (50 nl, 200 μg/ml virions) into P. maidis resulted in 93% transmission efficiency. In dose-response experiments, MMV abundance in insects and transmission efficiency decreased as the amount of virus inoculum delivered into the hemocoel was reduced. Examination of virus distribution in the vector using immunolabeling and confocal microscopy revealed similar tissue distributions in the injected insects when compared to those of previous studies using feeding on plants for virus acquisition. The utility of virus inoculation by microinjection for functional analysis in virus-vector interaction was explored. Co-microinjection of MMV virions and the dsRNA of PI3Kδ (a transcript that is less abundant in MMV-infected insects), resulted in a reduction in PI3Kδ expression and higher virus titers in P. maidis. These findings demonstrated that virus microinjection is a robust method for obtaining large numbers of infected planthoppers that are competent in transmitting MMV and, in combination with RNAi, could significantly facilitate the functional analysis of P. maidis-MMV interactions.

Delivery of maize mosaic virus to planthopper vectors by microinjection increases infection efficiency and facilitates functional genomics experiments in the vector.

The plant-pathogenic virus tomato spotted wilt virus (TSWV) encodes a structural glycoprotein (G) 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  is one of nine competent thrips vectors of TSWV transmission to plant hosts. However, the insect molecules that interact with viral proteins, such as G, 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 G 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 , the tissues most important during virus infection, replication, and plant inoculation. The TIPs and G 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 G These newly discovered thrips protein-G 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. 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  first-instar larvae. Two proteins, an endocuticle structural glycoprotein and cyclophilin, were able to interact directly with the TSWV attachment protein, G, in insect cells. The TSWV G-interacting proteins provide new targets for disrupting the viral disease cycle in the arthropod vector and could be putative determinants of vector competence.

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

The majority of plant-infecting viruses are transmitted by arthropod vectors that deliver them directly into a living plant cell. There are diverse mechanisms of transmission ranging from direct binding to the insect stylet (non-persistent transmission) to persistent-propagative transmission in which the virus replicates in the insect vector. Despite this diversity in interactions, most arthropods that serve as efficient vectors have feeding strategies that enable them to deliver the virus into the plant cell without extensive damage to the plant and thus effectively inoculate the plant. As such, the primary virus entry mechanism for plant viruses is mediated by the biological vector. Remarkably, viruses that are transmitted in a propagative manner (bunyaviruses, rhabdoviruses, and reoviruses) have developed an ability to replicate in hosts from two kingdoms. Viruses in the order Bunyavirales are of emerging importance and with the advent of new sequencing technologies, we are getting unprecedented glimpses into the diversity of these viruses. Plant-infecting bunyaviruses are transmitted in a persistent, propagative manner must enter two unique types of host cells, plant and insect. In the insect phase of the virus life cycle, the propagative viruses likely use typical cellular entry strategies to traverse cell membranes. In this review, we highlight the transmission and entry strategies of three genera of plant-infecting bunyaviruses: orthotospoviruses, tenuiviruses, and emaraviruses.

Entry of bunyaviruses into plants and vectors.

Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods.

Gene content evolution in the arthropods.

The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set.

Genome-enabled insights into the biology of thrips as crop pests.

An amendment to this paper has been published and can be accessed via the original article.

Correction to: Genome-enabled insights into the biology of thrips as crop pests.

Several plant viruses modulate vector fitness and behavior in ways that may enhance virus transmission. Previous studies have documented indirect, plant-mediated effects of tomato spotted wilt virus (TSWV) infection on the fecundity, growth and survival of its principal thrips vector, , the western flower thrips. We conducted thrips performance and preference experiments combined with plant gene expression, phytohormone and total free amino acid analyses to determine if systemically-infected tomato plants modulate primary metabolic and defense-related pathways to culminate into a more favorable environment for the vector. In a greenhouse setting, we documented a significant increase in the number of offspring produced by  on TSWV-infected tomato plants compared to mock-inoculated plants, and in choice test assays, females exhibited enhanced settling on TSWV-infected leaves. Microarray analysis combined with phytohormone signaling pathway analysis revealed reciprocal modulation of key phytohormone pathways under dual attack, possibly indicating a coordinated and dampening defense against the vector on infected plants. TSWV infection, alone or in combination with thrips, suppressed genes associated with photosynthesis and chloroplast function thereby significantly impacting primary metabolism of the host plant, and hierarchical cluster and network analyses revealed that many of these genes were co-regulated with phytohormone defense signaling genes. TSWV infection increased expression of genes related to protein synthesis and degradation which was reflected in the increased total free amino acid content in virus-infected plants that harbored higher thrips populations. These results suggest coordinated gene networks that regulate plant primary metabolism and defense responses rendering virus-infected plants more conducive for vector colonization, an outcome that is potentially beneficial to the vector and the virus when considered within the context of the complex transmission biology of TSWV. To our knowledge this is the first study to identify global transcriptional networks that underlie the TSWV-thrips interaction as compared to a single mechanistic approach. Findings of this study increase our fundamental knowledge of host plant-virus-vector interactions and identifies underlying mechanisms of induced host susceptibility to the insect vector.

Dorith Rotenberg

Department of Entomology and Plant Pathology

North Carolina State University

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Dorith Rotenberg

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Entomology and Plant Pathology

North Carolina State University

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Department of Entomology and Plant Pathology