RNA Interference of Genes Encoding the Vacuolar-ATPase in Liriomyza trifolii

The leafminer fly, Liriomyza trifolii, is an invasive pest of vegetable and horticultural crops in China. In this study, a microinjection method based on dsRNA was developed for RNA interference (RNAi) in L. trifolii using genes encoding vacuolar-ATPase (V-ATPase). Expression analysis indicated that V-ATPase B and V-ATPase D were more highly expressed in L. trifolii adults than in larvae or pupae. Microinjection experiments with dsV-ATPase B and dsV-ATPase D were conducted to evaluate the efficacy of RNAi in L. trifolii adults. Expression analysis indicated that microinjection with 100 ng dsV-ATPase B or dsV-ATPase led to a significant reduction in V-ATPase transcripts as compared to that of the dsGFP control (dsRNA specific to green fluorescent protein). Furthermore, lower dsRNA concentrations were also effective in reducing the expression of target genes when delivered by microinjection. Mortality was significantly higher in dsV-ATPase B- and dsV-ATPase D-treated insects than in controls injected with dsGFP. The successful deployment of RNAi in L. trifolii will facilitate functional analyses of vital genes in this economically-important pest and may ultimately result in new control strategies.

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RNAi-Mediated Management of Whitefly Bemisia tabaci by Oral Delivery of Double-stranded RNAs

Proceedings ◽

10.3390/proceedings2019036011 ◽

2019 ◽

Vol 36 (1) ◽

pp. 11

Author(s):

Jain ◽

Robinson ◽

Mitter

Keyword(s):

Rna Interference ◽

Gene Silencing ◽

Bemisia Tabaci ◽

Oral Delivery ◽

Target Genes ◽

Insect Pests ◽

Control Strategies ◽

Plant Viruses ◽

Potential Game ◽

Viable Approach

The whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) is a significant global pest of economically important vegetable, fibre, and ornamental crops. Whiteflies directly damage the plants by piercing and sucking essential nutrients, indirectly through honeydew secretion and by transmitting more than 200 plant viruses that cause millions of dollars in produce losses per year. Whitefly management is mostly reliant on the heavy use of chemical insecticides. However, this ultimately leads to increasing resistance development, detrimental effects on beneficial insects and biomagnification of ecologically harmful chemicals in the environment. Responding to consumer demands for more selective, less toxic, non-GM insect control strategies, RNA interference (RNAi) has emerged as a potential game-changing solution. The RNA interference (RNAi) is a homology-dependent mechanism of gene silencing that represents a feasible and sustainable technology for the management of insect pests. In the present study, twenty-two whitefly genes were selected based on their essential function in the insect and tested in artificial diet bioassays for mortality and gene silencing efficacy. The nine most effective dsRNA constructs showed moderate-to-high whitefly mortality as compared to negative controls six days post-feeding. qPCR analysis further demonstrated significant knockdown of target gene mRNA expression. Additionally, uptake and spread of fluorescently labelled dsRNA was evident beyond the midgut of the whitefly supporting the systemic spreading of RNAi effectors. Taken together, the oral delivery of dsRNA shows effective RNAi mediated gene silencing of target genes and offers a viable approach for the development of dsRNA biopesticides against hemipteran pest.

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Application of RNA Interference Technology to Acroporid Juvenile Corals

Frontiers in Marine Science ◽

10.3389/fmars.2021.688876 ◽

2021 ◽

Vol 8 ◽

Author(s):

Ikuko Yuyama ◽

Tomihiko Higuchi ◽

Michio Hidaka

Keyword(s):

Gene Expression ◽

Rna Interference ◽

Stress Response ◽

Large Scale ◽

Elevated Temperatures ◽

Fluorescent Protein ◽

Fluorescence Emission ◽

Pcr Analysis ◽

Genes Encoding ◽

Green Fluorescent

Numerous genes involved in calcification, algal endosymbiosis, and the stress response have been identified in corals by large-scale gene expression analysis, but functional analysis of those genes is lacking. There are few experimental examples of gene expression manipulation in corals, such as gene knockdown by RNA interference (RNAi). The purpose of this study is to establish an RNAi method for coral juveniles. As a first trial, the genes encoding green fluorescent protein (GFP, an endogenous fluorophore expressed by corals) and thioredoxin (TRX, a stress response gene) were selected for knockdown. Synthesized double-stranded RNAs (dsRNAs) corresponding to GFP and TRX were transformed into planula larvae by lipofection method to attempt RNAi. Real-time PCR analysis to verify knockdown showed that GFP and TRX expression levels tended to decrease with each dsRNA treatment (not significant). In addition, stress exposure experiments following RNAi treatment revealed that planulae with TRX knockdown exhibited increased mortality at elevated temperatures. In GFP-knockdown corals, decreased GFP fluorescence was observed. However, the effect of GFP-knockdown was confirmed only in the coral at the initial stages of larval metamorphosis into polyps, but not in planulae and 1 month-old budding polyps. This study showed that lipofection RNAi can be applied to coral planulae and polyps after settlement, and that this method provides a useful tool to modify expression of genes involved in stress tolerance and fluorescence emission of the corals.

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Search for Candidate Genes Causing the Excessive Ca Accumulation in Roots of Tipburn-Damaged Lisianthus (Eustoma grandiflorum) Cultivars

Agriculture ◽

10.3390/agriculture11030254 ◽

2021 ◽

Vol 11 (3) ◽

pp. 254

Author(s):

Takanori Kuronuma ◽

Hitoshi Watanabe

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Target Genes ◽

Pectin Methylesterase ◽

Eustoma Grandiflorum ◽

Gene Encoding ◽

Severe Problem ◽

Horticultural Crops ◽

Genes Encoding ◽

Leaves And Roots

Occurrence of tipburn is a severe problem in the production of lisianthus cultivars. Previous studies have shown excessive Ca accumulation in the roots of tipburn-damaged cultivars, where the distribution of Ca to the tips of the top leaves is inhibited. However, few studies have investigated the association between Ca accumulation and gene expression in horticultural crops. To provide a list of candidate target genes that might be causing the excessive Ca accumulation in roots, we focused Ca2+ transporter and pectin methylesterase (PME) genes and RNA-seq of upper leaves and roots in tipburn-occurrence cultivar (“Voyage peach”: VP) and non-occurrence cultivar (“Umi honoka”: UH) was conducted. In both the upper leaves and roots of VP, genes encoding the glutamate receptors (GLRs), cation/Ca2+ exchangers 4 (CCX4), Na+/Ca2+ exchanger-like protein (NCL), and PMEs were upregulated, and a gene encoding the cyclic nucleotide-gated ion channel 9 (CNGC9) was downregulated. In contrast, genes encoding the vacuolar cation/proton exchanger 5 (CAX5), calcium-transporting ATPase 1 and 12 (ACA1 and ACA12) showed differential expression in each organ. Among them, only CAX5 was upregulated and ACA12 was downregulated in the roots of VP. Based on these results, we suggested that CAX5 and ACA12 are the candidate genes causing the excessive Ca accumulation in the roots of tipburn-occurrence lisianthus cultivars. Future studies should investigate the temporal changes in gene expression using quantitative PCR and conduct functional analysis of candidate genes in tipburn-damaged lisianthus cultivars.

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Cloning and Expression Analysis of a Vacuolar ATPase Subunit A Gene in Maize

ACTA AGRONOMICA SINICA ◽

10.3724/sp.j.1006.2008.00031 ◽

2008 ◽

Vol 34 (1) ◽

Author(s):

Yi-Hui MAO

Keyword(s):

Expression Analysis ◽

Vacuolar Atpase ◽

Cloning And Expression ◽

Atpase Subunit ◽

Subunit A

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Role of Satb1 and Satb2 Transcription Factors in the Glutamate Receptors Expression and Ca2+ Signaling in the Cortical Neurons In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms22115968 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5968

Author(s):

Egor A. Turovsky ◽

Maria V. Turovskaya ◽

Evgeniya I. Fedotova ◽

Alexey A. Babaev ◽

Viktor S. Tarabykin ◽

...

Keyword(s):

Transcription Factors ◽

Nmda Receptor ◽

Cortical Neurons ◽

Target Genes ◽

Cortical Cell ◽

Inhibitory System ◽

Selective Agonist ◽

Genes Encoding ◽

Deficient Mice

Transcription factors Satb1 and Satb2 are involved in the processes of cortex development and maturation of neurons. Alterations in the expression of their target genes can lead to neurodegenerative processes. Molecular and cellular mechanisms of regulation of neurotransmission by these transcription factors remain poorly understood. In this study, we have shown that transcription factors Satb1 and Satb2 participate in the regulation of genes encoding the NMDA-, AMPA-, and KA- receptor subunits and the inhibitory GABA(A) receptor. Deletion of gene for either Satb1 or Satb2 homologous factors induces the expression of genes encoding the NMDA receptor subunits, thereby leading to higher amplitudes of Ca2+-signals in neurons derived from the Satb1-deficient (Satb1fl/+ * NexCre/+) and Satb1-null mice (Satb1fl/fl * NexCre/+) in response to the selective agonist reducing the EC50 for the NMDA receptor. Simultaneously, there is an increase in the expression of the Gria2 gene, encoding the AMPA receptor subunit, thus decreasing the Ca2+-signals of neurons in response to the treatment with a selective agonist (5-Fluorowillardiine (FW)). The Satb1 deletion increases the sensitivity of the KA receptor to the agonist (domoic acid), in the cortical neurons of the Satb1-deficient mice but decreases it in the Satb1-null mice. At the same time, the Satb2 deletion decreases Ca2+-signals and the sensitivity of the KA receptor to the agonist in neurons from the Satb1-null and the Satb1-deficient mice. The Satb1 deletion affects the development of the inhibitory system of neurotransmission resulting in the suppression of the neuron maturation process and switching the GABAergic responses from excitatory to inhibitory, while the Satb2 deletion has a similar effect only in the Satb1-null mice. We show that the Satb1 and Satb2 transcription factors are involved in the regulation of the transmission of excitatory signals and inhibition of the neuronal network in the cortical cell culture.

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Current Status and Potential of RNA Interference for the Management of Tomato Spotted Wilt Virus and Thrips Vectors

Pathogens ◽

10.3390/pathogens10030320 ◽

2021 ◽

Vol 10 (3) ◽

pp. 320

Author(s):

Alexander Nilon ◽

Karl Robinson ◽

Hanu R. Pappu ◽

Neena Mitter

Keyword(s):

Rna Interference ◽

Tomato Spotted Wilt Virus ◽

Cultural Practices ◽

Management Strategies ◽

Current Status ◽

Horticultural Crops ◽

Yield And Quality ◽

Tomato Spotted Wilt ◽

Wilt Virus

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.

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Core RNA Interference Genes Involved in miRNA and Ta-siRNA Biogenesis in Hops and Their Expression Analysis after Challenging with Verticillium nonalfalfae

International Journal of Molecular Sciences ◽

10.3390/ijms22084224 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4224

Author(s):

Urban Kunej ◽

Jernej Jakše ◽

Sebastjan Radišek ◽

Nataša Štajner

Keyword(s):

Rna Interference ◽

Expression Analysis ◽

Cannabis Sativa ◽

Defense Responses ◽

Resistant Cultivar ◽

Post Inoculation ◽

Biotic Stresses ◽

Argonaute 2 ◽

Expression Of Genes ◽

Verticillium Nonalfalfae

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

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Chemosensory genes in the head of Spodoptera litura larvae

Bulletin of Entomological Research ◽

10.1017/s0007485321000109 ◽

2021 ◽

pp. 1-10

Author(s):

Lu-Lu Li ◽

Ji-Wei Xu ◽

Wei-Chen Yao ◽

Hui-Hui Yang ◽

Youssef Dewer ◽

...

Keyword(s):

Spodoptera Litura ◽

Phylogenetic Trees ◽

Target Genes ◽

Control Strategies ◽

Insect Pest ◽

Management Strategies ◽

Food Sources ◽

Larval Feeding ◽

Chemosensory System ◽

Chemosensory Genes

Abstract The tobacco cutworm Spodoptera litura (Lepidoptera: Noctuidae) is a polyphagous pest with a highly selective and sensitive chemosensory system involved in complex physiological behaviors such as searching for food sources, feeding, courtship, and oviposition. However, effective management strategies for controlling the insect pest populations under threshold levels are lacking. Therefore, there is an urgent need to formulate eco-friendly pest control strategies based on the disruption of the insect chemosensory system. In this study, we identified 158 putative chemosensory genes based on transcriptomic and genomic data for S. litura, including 45 odorant-binding proteins (OBPs, nine were new), 23 chemosensory proteins (CSPs), 60 odorant receptors (ORs, three were new), and 30 gustatory receptors (GRs, three were new), a number higher than those reported by previous transcriptome studies. Subsequently, we constructed phylogenetic trees based on these genes in moths and analyzed the dynamic expression of various genes in head capsules across larval instars using quantitative real-time polymerase chain reaction. Nine genes–SlitOBP8, SlitOBP9, SlitOBP25, SlitCSP1, SlitCSP7, SlitCSP18, SlitOR34, SlitGR240, and SlitGR242–were highly expressed in the heads of 3- to 5-day-old S. litura larvae. The genes differentially expressed in olfactory organs during larval development might play crucial roles in the chemosensory system of S. litura larvae. Our findings substantially expand the gene inventory for S. litura and present potential target genes for further studies on larval feeding in S. litura.

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The use of light spectrum blocking films to reduce populations of Drosophila suzukii Matsumura in fruit crops

Scientific Reports ◽

10.1038/s41598-020-72074-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Michelle T. Fountain ◽

Amir Badiee ◽

Sebastian Hemer ◽

Alvaro Delgado ◽

Michael Mangan ◽

...

Keyword(s):

Control Strategies ◽

Field Trials ◽

Biological Data ◽

Insect Vision ◽

Invasive Pest ◽

Effective Control ◽

Fruit Crops ◽

Light Spectrum ◽

Drosophila Suzukii ◽

The Impact

Abstract Spotted wing drosophila, Drosophila suzukii, is a serious invasive pest impacting the production of multiple fruit crops, including soft and stone fruits such as strawberries, raspberries and cherries. Effective control is challenging and reliant on integrated pest management which includes the use of an ever decreasing number of approved insecticides. New means to reduce the impact of this pest that can be integrated into control strategies are urgently required. In many production regions, including the UK, soft fruit are typically grown inside tunnels clad with polyethylene based materials. These can be modified to filter specific wavebands of light. We investigated whether targeted spectral modifications to cladding materials that disrupt insect vision could reduce the incidence of D. suzukii. We present a novel approach that starts from a neuroscientific investigation of insect sensory systems and ends with infield testing of new cladding materials inspired by the biological data. We show D. suzukii are predominantly sensitive to wavelengths below 405 nm (ultraviolet) and above 565 nm (orange & red) and that targeted blocking of lower wavebands (up to 430 nm) using light restricting materials reduces pest populations up to 73% in field trials.

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