Single amino acid change in tomato brown rugose fruit virus breaks virus-specific resistance in new resistant tomato cultivar.

Introduction: Tomato cultivation across the world is severely affected by emerging plant viruses. An effective method for protection of commercial crops against viral threats is the use of cultivars harboring resistance genes. Tomato brown rugose fruit virus (ToBRFV), a recently emerged tobamovirus, is able to overcome the dominant Tm-22 resistance that is present in the majority of commercial tomato cultivars. In an effort to alleviate the severe consequences of ToBRFV on tomato production, tomato breeding companies are developing new cultivars with varying levels of resistance against ToBRFV. Methods: In the present study, cultivars with a new resistant phenotype against ToBRFV were screened against a wild-type isolate of ToBRFV, and subsequently, their performance under commercial greenhouse conditions was monitored. Following the identification of ToBRFV symptoms in a commercial greenhouse-where both new resistant and susceptible cultivars were interplanted-these cultivars were more closely examined. Results: The presence of ToBRFV was molecularly confirmed on both cultivar types suggesting that the new resistance had been broken. High-throughput sequencing (HTS) was used to study the complete genomes of viral isolates present in the two cultivar types. The analysis revealed a single amino acid change at position 82 of the movement protein of ToBRFV in the isolate present in the new resistant cultivar compared with the isolate identified in the susceptible cultivar. Discussion: A screening bioassay, that was performed to compare the infectivity of the two ToBRFV isolates, confirmed that only the isolate with this specific amino acid change could successfully infect the resistant cultivar, overcoming the new resistance against ToBRFV.

First report of infections with isolates of the Asian genotype of Cucumber green mottle mosaic virus in cucumber crops in Belgium.

In July 2019, four rows of cucumber plants (Cucumis sativus) in a commercial glasshouse in the north of Belgium showed severe mosaic, blistering and distortion of the leaves, with symptoms resembling those caused by Cucumber green mottle mosaic virus (CGMMV). CGMMV is a Tobamovirus that mainly affects cucurbit crops worldwide (Dombrovsky et al., 2017). Phylogenetic analyses in previous studies have shown two major clades, one including isolates that were initially identified in Europe and Russia (European genotype) and the second one with isolates initially identified in Asia and Israel (Asian genotype) (Dombrovsky et al., 2017; Pitman et al., 2022; Mackie et al., 2023). A symptomatic leaf sample was collected and total RNA was isolated from 100 mg of leaf tissue (Spectrum™ Plant Total RNA kit, Sigma-Aldrich). CGMMV was detected using a one-step TaqMan RT-qPCR (Hongyun et al., 2008). High-throughput sequencing (HTS) confirmed the presence of CGMMV. The sample was prepared using the Novel enrichment technique of viromes protocol (NETOVIR protocol, Conceição-Neto et al., 2015). The leaf material was homogenized, enriched for virus-like particles and the RNA was extracted (QIAamp Viral RNA mini kit, QIAGEN). The extract was randomly amplified (Whole Transcriptome Amplification kit, Sigma Aldrich), used for library preparation (Nextera XT DNA library preparation kit, Illumina) and sequenced on a NovaSeq platform. HTS data analysis was performed using Geneious Prime software (Biomatters, Auckland, New Zealand, version 2023.2). After quality filtering and trimming, 26.7M reads were obtained (132 nt mean length). In total, 20.6M reads were mapped to two genomes KP772568 and GQ411361 (considered as reference for the Asian and European genotypes respectively) with Geneious. This revealed 100% coverage of the full sequences (6422 nt) with 99.4% and 90% nucleotide identities to the reference genomes of Asian and European genotypes, respectively. Phylogenetic analyses confirmed that isolate 2019-26A-BE, with GenBank ID OR724740, relates to the Asian genotype. The HTS data were additionally processed using the ViPER pipeline (De Coninck, 2021). The raw reads were quality filtered and trimmed, (Trimmomatic) and then used to perform de novo assembly (metaSPAdes). The produced contigs were classified using DIAMOND and visualized with KronaTools. The results showed that no other virus was detected in the sample. Finally, cucumber seedlings were inoculated using the original symptomatic sample and were grown in a research glasshouse. After 3 weeks, severe CGMMV symptoms, similar to the original symptoms observed in the commercial glasshouse, were observed in the inoculated plants. Infection with CGMMV was verified via RT-qPCR, and the isolate present in the inoculated plants was confirmed to belong to the Asian genotype via RT-PCR-RFLP (Crespo et al., 2017). Later samplings of symptomatic leaves confirmed the presence of isolates belonging to the Asian genotype of CGMMV at four other commercial glasshouse locations, specializing in cucumber crop, in Belgium in 2020, 2021 and 2023 by RT-PCR-RFLP. While the Asian genotype was previously found elsewhere in Europe (Pitman et al., 2022), to our knowledge, this is the first report of infections with isolates of this genotype in glasshouse cucumber crops in Belgium. Further investigation is required to determine the spread and impact of infections with isolates of the Asian genotype in cucumber crops in Belgium.

The Study of Cell-Penetrating Peptides to Deliver dsRNA and siRNA by Feeding in the Desert Locust, Schistocerca gregaria.

RNA(i) interference is a gene silencing mechanism triggered by double-stranded (ds)RNA, which promises to contribute to species-specific insect pest control strategies. The first step toward the application of RNAi as an insecticide is to enable efficient gene silencing upon dsRNA oral delivery. The desert locust, Schistocerca gregaria is a devastating agricultural pest. While this species is responsive to dsRNA delivered by intra-hemocoelic injection, it is refractory to orally delivered dsRNA. In this study, we evaluated the capacity of five cell-penetrating peptides (CPPs) to bind long dsRNA and protect it from the locust midgut environment. We then selected the CPP EB1 for further in vivo studies. EB1:dsRNA complexes failed to induce RNAi by feeding. Interestingly, we observed that intra-hemocoelic injection of small-interfering (si)RNAs does not result in a silencing response, but that this response can be obtained by injecting EB1:siRNA complexes. EB1 also protected siRNAs from midgut degradation activity. However, EB1:siRNA complexes failed as well in triggering RNAi when fed. Our findings highlight the complexity of the dsRNA/siRNA-triggered RNAi in this species and emphasize the multifactorial nature of the RNAi response in insects. Our study also stresses the importance of in vivo studies when it comes to dsRNA/siRNA delivery systems.

Tomato brown rugose fruit virus in aqueous environments - survival and significance of water-mediated transmission.

Tomato brown rugose fruit virus (ToBRFV) has recently emerged as a major disease of tomatoes and peppers. ToBRFV is a seed- and contact-transmitted virus. In Slovenia, ToBRFV RNA was detected in samples of wastewater, river, and water used to irrigate plants. Even though the source of detected RNA could not be clearly established, this raised the question of the significance of the detection of ToBRFV in water samples and experimental studies were performed to address this question. The data presented here confirm that the release of virus particles from the roots of infected plants is a source of infectious ToBRFV particles in water and that the virus can remain infective up to four weeks in water stored at room temperature, while its RNA can be detected for much longer. These data also indicate that irrigation with ToBRFV-contaminated water can lead to plant infection. In addition, it has been shown that ToBRFV circulated in drain water in commercial tomato greenhouses from other European countries and that an outbreak of ToBRFV can be detected by regular monitoring of drain water. A simple method for concentrating ToBRFV from water samples and a comparison of the sensitivity of different methods, including the determination of the highest ToBRFV dilution still capable of infecting test plants, were also investigated. The results of our studies fill the knowledge gaps in the epidemiology and diagnosis of ToBRFV, by studying the role of water-mediated transmission, and provide a reliable risk assessment to identify critical points for monitoring and control.

Accelerated delivery of dsRNA in lepidopteran midgut cells by a Galanthus nivalis lectin (GNA)-dsRNA-binding domain fusion protein.

Lepidopteran insects are highly refractory to oral RNA interference (RNAi). Degradation, impaired cellular uptake and intracellular transport of double-stranded RNA (dsRNA) are considered the major factors responsible for the reduced RNAi efficiency in these insects. In this study, the potential of lectins to improve dsRNA delivery and RNAi efficacy was evaluated. First, a fusion protein consisting of the Galanthus nivalis agglutinin (GNA) and a dsRNA binding domain was developed, further referred to as GNA:dsRBD (GNAF). Then, its ability to increase dsRNA uptake and transfection efficiency in lepidopteran midgut cells was evaluated, as well as its ability to protect and promote the RNAi response in the beet armyworm Spodoptera exigua. Confocal microscopy analysis showed that GNAF-complexed dsRNA was internalized faster in Choristoneura fumiferana midgut CF1 cells (1 min) compared to naked dsRNA (>1 h). The faster uptake was also correlated with an increased RNAi efficiency in these CF1 cells. In vivo feeding bioassays with GNAF-complexed dsRNA led to an increased mortality in S. exigua compared to the controls. By targeting the essential gene V-ATPase A, we observed that the mortality increased to 48% in the GNAF-dsRNA treatment compared to only 8.3% and 6.6% in the control treatments with the naked dsRNA and the GNAF, respectively.

Can BRET-based biosensors be used to characterize G-protein mediated signaling pathways of an insect GPCR, the Schistocerca gregaria CRF-related diuretic hormone receptor?

G protein-coupled receptors (GPCRs) are membrane-bound receptors that are considered prime candidates for the development of novel insect pest management strategies. However, the molecular signaling properties of insect GPCRs remain poorly understood. In fact, most studies on insect GPCR signaling are limited to analysis of fluctuations in the secondary messenger molecules calcium (Ca2+) and/or cyclic adenosine monophosphate (cAMP). In the current study, we characterized a corticotropin-releasing factor-related diuretic hormone (CRF-DH) receptor of the desert locust, Schistocerca gregaria. This Schgr-CRF-DHR is mainly expressed in the nervous system and in brain-associated endocrine organs. The neuropeptide Schgr-CRF-DH induced Ca2+-dependent aequorin-based bioluminescent responses in CHO cells co-expressing this receptor with the promiscuous Gα16 protein. Furthermore, when co-expressed with the cAMP-dependent bioluminescence resonance energy transfer (BRET)-based CAMYEL biosensor in HEK293T cells, this receptor elicited dose-dependent agonist-induced responses with an EC50 in the nanomolar range (4.02 nM). In addition, we tested if vertebrate BRET-based G protein biosensors, can also be used to detect direct Gα protein subunit activation by an insect GPCR. Therefore, we analyzed ten different human BRET-based G protein biosensors, representing members of all four Gα protein subfamilies; Gαs, Gαi/o, Gαq/11 and Gα12/13. Our data demonstrate that stimulation of Schgr-CRF-DHR by Schgr-CRF-DH can dose-dependently activate Gαi/o and Gαs biosensors, while no significant effects were observed with the Gαq/11 and Gα12/13 biosensors. Our study paves the way for future biosensor-based studies to analyze the signaling properties of insect GPCRs in both fundamental science and applied research contexts.

Generation of Virus- and dsRNA-Derived siRNAs with Species-Dependent Length in Insects.

Double-stranded RNA (dsRNA) molecules of viral origin trigger a post-transcriptional gene-silencing mechanism called RNA interference (RNAi). Specifically, virally derived dsRNA is recognized and cleaved by the enzyme Dicer2 into short interfering RNAs (siRNAs), which further direct sequence-specific RNA silencing, ultimately silencing replication of the virus. Notably, RNAi can also be artificially triggered by the delivery of gene-specific dsRNA, thereby leading to endogenous gene silencing. This is a widely used technology that holds great potential to contribute to novel pest control strategies. In this regard, research efforts have been set to find methods to efficiently trigger RNAi in the field. In this article, we demonstrate the generation of dsRNA- and/or virus-derived siRNAs-the main RNAi effectors-in six insect species belonging to five economically important orders (Lepidoptera, Orthoptera, Hymenoptera, Coleoptera, and Diptera). In addition, we describe that the siRNA length distribution is species-dependent. Taken together, our results reveal interspecies variability in the (antiviral) RNAi mechanism in insects and show promise to contribute to future research on (viral-based) RNAi-triggering mechanisms in this class of animals.

RNA Interference in Insects: Protecting Beneficials and Controlling Pests.

Insects constitute the largest and most diverse group of animals on Earth with an equally diverse virome. The main antiviral immune system of these animals is the post-transcriptional gene-silencing mechanism known as RNA(i) interference. Furthermore, this process can be artificially triggered via delivery of gene-specific double-stranded RNA molecules, leading to specific endogenous gene silencing. This is called RNAi technology and has important applications in several fields. In this paper, we review RNAi mechanisms in insects as well as the potential of RNAi technology to contribute to species-specific insecticidal strategies. Regarding this aspect, we cover the range of strategies considered and investigated so far, as well as their limitations and the most promising approaches to overcome them. Additionally, we discuss patterns of viral infection, specifically persistent and acute insect viral infections. In the latter case, we focus on infections affecting economically relevant species. Within this scope, we review the use of insect-specific viruses as bio-insecticides. Last, we discuss RNAi-based strategies to protect beneficial insects from harmful viral infections and their potential practical application. As a whole, this manuscript stresses the impact of insect viruses and RNAi technology in human life, highlighting clear lines of investigation within an exciting and promising field of research.

Increasing adult hippocampal neurogenesis in mice after exposure to unpredictable chronic mild stress may counteract some of the effects of stress.

Major depression is hypothesized to be associated with dysregulations of the hypothalamic-pituitary-adrenal (HPA) axis and impairments in adult hippocampal neurogenesis. Adult-born hippocampal neurons are required for several effects of antidepressants and increasing the rate of adult hippocampal neurogenesis (AHN) before exposure to chronic corticosterone is sufficient to protect against its harmful effects on behavior. However, it is an open question if increasing AHN after the onset of chronic stress exposure would be able to rescue behavioral deficits and which mechanisms might be involved in recovery. We investigated this question by using a 10-week unpredictable chronic mild stress (UCMS) model on a transgenic mouse line (iBax mice), in which the pro-apoptotic gene Bax can be inducibly ablated in neural stem cells following Tamoxifen injection, therefore enhancing the survival of newborn neurons in the adult brain. We did not observe any effect of our treatment in non-stress conditions, but we did find that increasing AHN after 2 weeks of UCMS is sufficient to counteract the effects of UCMS on certain behaviors (splash test and changes in coat state) and endocrine levels and thus to display some antidepressant-like effects. We observed that increasing AHN lowered the elevated basal corticosterone levels in mice exposed to UCMS. This was accompanied by a tamoxifen-induced reversal of the lack of stress-induced decrease in neuronal activation in the anteromedial division of the bed nucleus of the stria terminalis (BSTMA) after intrahippocampal dexamethasone infusion, pointing to a possible mechanism through which adult-born neurons might have exerted their effects. Our results contribute to the neurogenesis hypothesis of depression by suggesting that increasing AHN may be beneficial not just before, but also after exposure to stress by counteracting several of its effects, in part through regulating the HPA axis.

Transcriptional Analysis of The Adaptive Digestive System of The Migratory Locust in Response to Plant Defensive Protease Inhibitors.

Herbivorous insects evolved adaptive mechanisms to compensate for the presence of plant defensive protease inhibitors (PI) in their food. The underlying regulatory mechanisms of these compensatory responses remain largely elusive. In the current study, we investigated the initiation of this adaptive response in the migratory locust, Locusta migratoria, via microarray analysis of gut tissues. Four hours after dietary uptake of PIs, 114 and 150 transcripts were respectively found up- or downregulated. The results suggest a quick trade-off between compensating for potential loss of digestive activity on the one hand, and stress tolerance, defense, and structural integrity of the gut on the other hand. We additionally addressed the role of a group of related upregulated hexamerin-like proteins in the PI-induced response. Simultaneous knockdown of corresponding transcripts by means of RNA interference resulted in a reduced capacity of the locust nymphs to cope with the effects of PI. Moreover, since insect hexamerins have been shown to bind Juvenile Hormone (JH), we also investigated the effect of JH on the proteolytic digestion in L. migratoria. Our results indicate that JH has a stimulatory effect on the expression of three homologous chymotrypsin genes, while knocking down the JH receptor (methoprene tolerant) led to opposite effects.

Attenuated behavioural responses to acute and chronic cocaine in GASP-1-deficient mice.

G protein-coupled receptor (GPCR) associated sorting protein 1 (GASP-1) interacts with GPCRs and is implicated in their postendocytic sorting. Recently, GASP-1 has been shown to regulate dopamine (D(2)) and cannabinoid (CB1) receptor signalling, suggesting that preventing GASP-1 interaction with GPCRs might provide a means to limit the decrease in receptor signalling upon sustained agonist treatment. In order to test this hypothesis, we have generated and behaviourally characterized GASP-1 knockout (KO) mice and have examined the consequences of the absence of GASP-1 on chronic cocaine treatments. GASP-1 KO and wild-type (WT) mice were tested for sensitization to the locomotor effects of cocaine. Additional mice were trained to acquire intravenous self-administration of cocaine on a fixed ratio 1 schedule of reinforcement, and the motivational value of cocaine was then assessed using a progressive ratio schedule of reinforcement. The dopamine and muscarinic receptor densities were quantitatively evaluated in the striatum of WT and KO mice tested for sensitization and self-administration. Acute and sensitized cocaine-locomotor effects were attenuated in KO mice. A decrease in the percentage of animals that acquired cocaine self-administration was also observed in GASP-1-deficient mice, which was associated with pronounced down-regulation of dopamine and muscarinic receptors in the striatum. These data indicate that GASP-1 participates in acute and chronic behavioural responses induced by cocaine and are in agreement with a role of GASP-1 in postendocytic sorting of GPCRs. However, in contrast to previous studies, our data suggest that upon sustained receptor stimulation GASP-1 stimulates recycling rather than receptor degradation.

Middle-aged human apoE4 targeted-replacement mice show retention deficits on a wide range of spatial memory tasks.

Apolipoprotein (apo) E4, one of three human apoE (h-apoE) isoforms, has been identified as a major genetic risk factor for Alzheimer's disease and for cognitive deficits associated with aging. However, the biological mechanisms involving apoE in learning and memory processes are unclear. A potential isoform-dependent role of apoE in cognitive processes was studied in human apoE targeted-replacement (TR) mice. These mice express either the human apoE3 or apoE4 gene under the control of endogenous murine apoE regulatory sequences, resulting in physiological expression of h-apoE in both a temporal and spatial pattern similar to humans. Male and female apoE3-TR, apoE4-TR, apoE-knockout and C57BL/6J mice (15-18 months) were tested with spatial memory and avoidance conditioning tasks. Compared to apoE3-TR mice, spatial memory in female apoE4-TR mice was impaired based on their poor performances in; (i) the probe test of the water-maze reference memory task, (ii) the water-maze working memory task and (iii) an active avoidance Y-maze task. Retention performance on a passive avoidance task was also impaired in apoE4-TR mice, but not in other genotypes. These deficits in both spatial and avoidance memory tasks may be related to the anatomical and functional abnormalities previously reported in the hippocampus and the amygdala of apoE4-TR mice. We conclude that the apoE4-TR mice provide an excellent model for understanding the mechanisms underlying apoE4-dependent susceptibility to cognitive decline.

Early life genetic, epigenetic and environmental factors shaping emotionality in rodents.

Childhood trauma is known to increase risk for emotional disorders and addiction. However, little is currently understood about the neurodevelopmental basis of these effects, or how genetic and epigenetic factors interact with the environment to shape the systems subserving emotionality. In this review, we discuss the use of rodent models of early life emotional experience to study these issues in the laboratory and present some of our pertinent findings. In rats, postnatal maternal separation can produce lasting increases in emotional behavior and stressor-reactivity, together with alterations in various brain neurotransmitter systems implicated in emotionality, including corticotropin-releasing factor, serotonin, norepinephrine, and glutamate. Genetic differences between inbred mouse strains have been exploited to further study how maternal behavior affects emotional development using techniques such as cross-fostering and generation of inter-strain hybrids. Together with our own recent data, the findings of these studies demonstrate the pervasive influence of maternal and social environments during sensitive developmental periods and reveal how genetic factors determine how these early life experiences can shape brain and behavior throughout life.

Human apoE targeted replacement mouse lines: h-apoE4 and h-apoE3 mice differ on spatial memory performance and avoidance behavior.

Apolipoprotein E4 (apoE4), one of the three most common human apoE (h-apoE) isoforms, is a major genetic risk factor for Alzheimer's disease and for cognitive deficits associated with aging. The biological mechanisms involving apoE in learning and memory processes are unclear. A potential isoform-dependent effect of h-apoE on cognitive performance was studied in gene-targeted mice, which show physiological expression levels and distribution of h-apoE3 or h-apoE4. Male and female h-apoE3 and h-apoE4, apoE-deficient and C57BL/6J mice (4-5 months) were subjected to tasks evaluating spatial memory and avoidance conditioning. Female h-apoE4 mice did not detect changes in the spatial configuration of objects as opposed to female h-apoE3 mice. Female h-apoE3 mice failed to improve their performance during training in a reference memory version of the spatial water-maze task, but performed well during the probe trial 24 h after the last training trial. Memory retention performances of h-apoE4 mice were impaired during this probe trial. Both h-apoE3 and h-apoE4 mice did not improve their performance in a water-maze delayed matching to place task. Finally, h-apoE3 mice showed mild perturbations in a Y-maze active avoidance task, whereas both h-apoE mouse lines performed well in a passive avoidance task. Thus, spatial memory performances appeared particularly sensitive to h-apoE-isoform-dependent effects. Deficits occurred predominantly in female h-apoE4 mice, which support the hypothesis that humans carrying h-apoE4, especially women, have impaired spatial memory compared to those carrying h-apoE3.