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Arthropods from class Arachnida constitute a large and diverse group with over 100,000 described species, and they are sources of many proteins that have a direct impact on human health. Despite the importance of Arachnida, few proteins originating from these organisms have been characterized in terms of their structure. Here we present a detailed analysis of Arachnida proteins that have their experimental structures determined and deposited to the Protein Data Bank (PDB). Our results indicate that proteins represented in the PDB are derived from a small number of Arachnida families, and two-thirds of Arachnida proteins with experimental structures determined are derived from organisms belonging to Buthidae, Ixodidae, and Theraphosidae families. Moreover, 90% of the deposits come from just a dozen of Arachnida families, and almost half of the deposits represent proteins originating from only fifteen different species. In summary, our analysis shows that the structural analysis of proteins originating from Arachnida is not only limited to a small number of the source species, but also proteins from this group of animals are not extensively studied. However, the interest in Arachnida proteins seems to be increasing, which is reflected by a significant increase in the related PDB deposits during the last ten years.

Plant-pest interactions involve multifaceted processes encompassing a complex crosstalk of pathways, molecules, and regulators aimed at overcoming defenses developed by each interacting organism. Among plant defensive compounds against phytophagous arthropods, cyanide-derived products are toxic molecules that directly target pest physiology. Here, we identified the Arabidopsis (Arabidopsis thaliana) gene encoding hydroxynitrile lyase (AtHNL, At5g10300) as one gene induced in response to spider mite (Tetranychus urticae) infestation. AtHNL catalyzes the reversible interconversion between cyanohydrins and derived carbonyl compounds with free cyanide. AtHNL loss- and gain-of-function Arabidopsis plants showed that specific activity of AtHNL using mandelonitrile as substrate was higher in the overexpressing lines than in wild-type (WT) and mutant lines. Concomitantly, mandelonitrile accumulated at higher levels in mutant lines than in WT plants and was significantly reduced in the AtHNL overexpressing lines. After mite infestation, mandelonitrile content increased in WT and overexpressing plants but not in mutant lines, while hydrogen cyanide (HCN) accumulated in the three infested Arabidopsis genotypes. Feeding bioassays demonstrated that the AtHNL gene participated in Arabidopsis defense against T. urticae. The reduced leaf damage detected in the AtHNL overexpressing lines reflected the mite's reduced ability to feed on leaves, which consequently restricted mite fecundity. In turn, mites upregulated TuCAS1 encoding β-cyanoalanine synthase to avoid the respiratory damage produced by HCN. This detoxification effect was functionally demonstrated by reduced mite fecundity observed when dsRNA-TuCAS-treated mites fed on WT plants and hnl1 mutant lines. These findings add more players in the Arabidopsis-T. urticae interplay to overcome mutual defenses., Grants BIO2017-83472-R, PDC2021-121055-I00, RED2018-102407-T, RED2018-102397-T and RyC17MESFB funded by MCIN/AEI/10.13039/501100011033, as appropriate, by “ERDF A way of making Europe” and by the “European Union” supported this work. The Convenio Plurianual between Comunidad de Madrid and UPM through the Apoyo-Jóvenes investigadores Program (grant SUR6Q9-22-YTFC3Z) supported this work. Grants LCF/PR18/51130007 from “La Caixa” Foundation, RyC2017-21814 and PRE2018-083375 from MCIN/AEI supported A.A., M.E.S., and I.R-D, respectively. The Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2018.04538) supported S.D.

Arabidopsis (Arabidopsis thaliana) defenses against herbivores are regulated by the jasmonate (JA) hormonal signaling pathway, which leads to the production of a plethora of defense compounds. Arabidopsis defense compounds include tryptophan-derived metabolites, which limit Arabidopsis infestation by the generalist herbivore two-spotted spider mite, Tetranychus urticae. However, the phytochemicals responsible for Arabidopsis protection against T. urticae are unknown. Here, we used Arabidopsis mutants disrupted in the synthesis of tryptophan-derived secondary metabolites to identify phytochemicals involved in the defense against T. urticae. We show that of the three tryptophan-dependent pathways found in Arabidopsis, the indole glucosinolate (IG) pathway is necessary and sufficient to assure tryptophan-mediated defense against T. urticae. We demonstrate that all three IGs can limit T. urticae herbivory, but that they must be processed by myrosinases to hinder T. urticae oviposition. Putative IG breakdown products were detected in mite-infested leaves, suggesting in planta processing by myrosinases. Finally, we demonstrate that besides IGs, there are additional JA-regulated defenses that control T. urticae herbivory. Together, our results reveal the complexity of Arabidopsis defenses against T. urticae that rely on multiple IGs, specific myrosinases, and additional JA-dependent defenses.

Plant immunity depends on fast and specific transcriptional reprogramming triggered by the perception of biotic stresses. Numerous studies have been conducted to better understand the response of plants to the generalist herbivore two-spotted spider mite (Tetranychus urticae). However, how plants perceive mites and how this perception is translated into changes in gene expression are largely unknown. In this work, we identified a gene induced in Arabidopsis (Arabidopsis thaliana) upon spider mite attack that encodes a two-domain protein containing predicted lectin and Toll/Interleukin-1 receptor domains. The gene, previously named PP2-A5, belongs to the Phloem Protein2 family. Biotic assays showed that PP2-A5 confers tolerance to T. urticae. Overexpression or knockout of PP2-A5 leads to transcriptional reprogramming that alters the balance of hormone accumulation and corresponding signaling pathways. The nucleocytoplasmic location of this protein supports a direct interaction with regulators of gene transcription, suggesting that the combination of two putative signaling domains in a single protein may provide a novel mechanism for regulating gene expression. Together, our results suggest that PP2-A5 improves the ability to defend against T. urticae by participating in the tight regulation of hormonal cross talk upon mite feeding. Further research is needed to determine the mechanism by which this two-domain protein functions and to clarify its molecular role in signaling following a spider mite attack.

Arabidopsis defenses against herbivores are regulated by the jasmonate hormonal signaling pathway, which leads to the production of a plethora of defense compounds, including tryptophan-derived metabolites produced through CYP79B2/CYP79B3. Jasmonate signaling and CYP79B2/CYP79B3 limit Arabidopsis infestation by the generalist herbivore two-spotted spider mite, Tetranychus urticae. However, the phytochemicals responsible for Arabidopsis protection against T. urticae are unknown. Here, using Arabidopsis mutants that disrupt metabolic pathways downstream of CYP79B2/CYP79B3, and synthetic indole glucosinolates, we identified phytochemicals involved in the defense against T. urticae. We show that Trp-derived metabolites depending on CYP71A12 and CYP71A13 are not affecting mite herbivory. Instead, the supplementation of cyp79b2 cyp79b3 mutant leaves with the 3-indolylmethyl glucosinolate and its derived metabolites demonstrated that the indole glucosinolate pathway is sufficient to assure CYP79B2/CYP79B3-mediated defenses against T. urticae. We demonstrate that three indole glucosinolates can limit T. urticae herbivory, but that they have to be processed by the myrosinases to hinder T. urticae oviposition. Finally, the supplementation of the mutant myc2 myc3 myc4 with indole glucosinolates indicated that the transcription factors MYC2/MYC3/MYC4 induce additional indole glucosinolate-independent defenses that control T. urticae herbivory. Together, these results reveal the complexity of Arabidopsis defenses against T. urticae that rely on multiple indole glucosinolates, specific myrosinases, and additional MYC2/MYC3/MYC4-dependent defenses.One sentence summaryThree indole glucosinolates and the myrosinases TGG1/TGG2 help protect Arabidopsis thaliana against the herbivory of the two-spotted spider mite Tetranychus urticae.

Genetic adaptation, occurring over a long evolutionary time, enables host-specialized herbivores to develop novel resistance traits and to efficiently counteract the defenses of a narrow range of host plants. In contrast, physiological acclimation, leading to the suppression and/or detoxification of host defenses, is hypothesized to enable broad generalists to shift between plant hosts. However, the host adaptation mechanisms used by generalists composed of host-adapted populations are not known. Two-spotted spider mite (TSSM; Tetranychus urticae) is an extreme generalist herbivore whose individual populations perform well only on a subset of potential hosts. We combined experimental evolution, Arabidopsis thaliana genetics, mite reverse genetics, and pharmacological approaches to examine mite host adaptation upon the shift of a bean (Phaseolus vulgaris)-adapted population to Arabidopsis. We showed that cytochrome P450 monooxygenases are required for mite adaptation to Arabidopsis. We identified activities of two tiers of P450s: general xenobiotic-responsive P450s that have a limited contribution to mite adaptation to Arabidopsis and adaptation-associated P450s that efficiently counteract Arabidopsis defenses. In approximately 25 generations of mite selection on Arabidopsis plants, mites evolved highly efficient detoxification-based adaptation, characteristic of specialist herbivores. This demonstrates that specialization to plant resistance traits can occur within the ecological timescale, enabling the TSSM to shift to novel plant hosts., Mites can evolve highly efficient detoxification-based adaptation in approximately 25 generations on an initially unfavorable plant host, revealing that specialization can occur within the ecological timescale.

Spider mites constitute an assemblage of well-known pests in agriculture, but are less known for their ability to spin silk of nanoscale diameters and high Young’s moduli. Here, we characterize silk of the gorse spider mite Tetranychus lintearius, which produces copious amounts of silk with nano-dimensions. We determined biophysical characteristics of the silk fibres and manufactured nanoparticles and biofilm derived from native silk. We determined silk structure using attenuated total reflectance Fourier transform infrared spectroscopy, and characterized silk nanoparticles using field emission scanning electron microscopy. Comparative studies using T. lintearius and silkworm silk nanoparticles and biofilm demonstrated that spider mite silk supports mammalian cell growth in vitro and that fluorescently labelled nanoparticles can enter cell cytoplasm. The potential for cytocompatibility demonstrated by this study, together with the prospect of recombinant silk production, opens a new avenue for biomedical application of this little-known silk.

Comprehensive understanding of pleiotropic roles of RNAi machinery highlighted the conserved chromosomal functions of RNA interference. The consequences of the evolutionary variation in the core RNAi pathway genes are mostly unknown, but may lead to the species-specific functions associated with gene silencing. The two-spotted spider mite, Tetranychus urticae, is a major polyphagous chelicerate pest capable of feeding on over 1100 plant species and developing resistance to pesticides used for its control. A well annotated genome, susceptibility to RNAi and economic importance, make T. urticae an excellent candidate for development of an RNAi protocol that enables high-throughput genetic screens and RNAi-based pest control. Here, we show that the length of the exogenous dsRNA critically determines its processivity and ability to induce RNAi in vivo. A combination of the long dsRNAs and the use of dye to trace the ingestion of dsRNA enabled the identification of genes involved in membrane transport and 26S proteasome degradation as sensitive RNAi targets. Our data demonstrate that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the components of the RNAi machinery make T. urticae a potent experimental system to study the evolution of RNAi pathways., Tis work was supported by the Government of Canada through the Ontario Research Fund (RE08-067), the Natural Sciences and Engineering Research Council of Canada (NSERC) and by the European Union’s Horizon 2020 research and innovation program (773902-SuperPests) awarded to MG and VG; and by the Japan Society for the Promotion of Science KAKENHI (Grant Nos. 18H02203 to TS, 19K22304 to TF and 19K23674 to MT) and the Institute of Global Innovation Research in TUAT to MT, TS, TF and VG. MA was funded through the Global Tesis program, the University of Bari Aldo Moro, Italy.

GSTs (Glutathione S-transferases) are known to catalyze the nucleophilic attack of the sulfhydryl group of reduced glutathione (GSH) on electrophilic centers of xenobiotic compounds, including insecticides and acaricides. Genome analyses of the polyphagous spider mite herbivore Tetranychus urticae (two-spotted spider mite) revealed the presence of a set of 32 genes that code for secreted proteins belonging to the GST family of enzymes. To better understand the role of these proteins in T. urticae, we have functionally characterized TuGSTd01. Moreover, we have modeled the structure of the enzyme in apo form, as well as in the form with bound inhibitor. We demonstrated that this protein is a glutathione S-transferase that can conjugate glutathione to 1-chloro-2,4-dinitrobenzene (CDNB). We have tested TuGSTd01 activity with a range of potential substrates such as cinnamic acid, cumene hydroperoxide, and allyl isothiocyanate; however, the enzyme was unable to process these compounds. Using mutagenesis, we showed that putative active site variants S11A, E66A, S67A, and R68A mutants, which were residues predicted to interact directly with GSH, have no measurable activity, and these residues are required for the enzymatic activity of TuGSTd01. There are several reports that associate some T. urticae acaricide resistance with increased activity of GSTs . However, we found that TuGSTd01 is not able to detoxify abamectin; in fact, the acaricide inhibits the enzyme with Ki = 101 μM. Therefore, we suggest that the increased GST activity observed in abamectin resistant T. urticae field populations is a part of the compensatory feedback loop. In this case, the increased production of GSTs and relatively high concentration of GSH in cells allow GSTs to maintain physiological functions despite the presence of the acaricide.

Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels., Hypomorphic mutations are a valuable tool for analysing gene function, but current methods have difficult to predict effects on gene expression and are organism specific. Here the authors present a pan-species approach using variable polyA tracks to modulate expression of a target protein.

Phytophagous mites and thrips are global pests affecting a wide range of agricultural crops (Mouden et al., 2017; Agut et al., 2018). Among the arthropods, they are phylogenetically distant, but both classes harbor species ranging from highly specialized to extremely polyphagous (Rioja et al., 2017; Wu et al., 2018). Through convergent evolution, mites and thrips evolved stylets to facilitate feeding from mesophyll or epidermal cells (Bensoussan et al., 2016; Rioja et al., 2017; Wu et al., 2018). Despite large crops losses (Agut et al., 2018; Steenbergen et al., 2018) that are expected to become more severe with global warming (Ximenez-Embun et al., 2017; Urbaneja-Bernat et al., 2019), the interactions between mites/thrips and their host plants have been understudied. Hence, understanding how plants defend themselves against these pests is essential for developing crop protection strategies. This Research Topic provides an update on recent advances in the plant molecular and physiological mechanisms associated with phytophagous mite/thrips-plant interactions, and provides an overview of different approaches for improving crop resistance sustainably, either through repellence, feeding disruption or prevention of feeding damage. Here, we highlight some of the major points arising from these reports.

Two-spotted spider mite (TSSM) Tetranychus urticae (Koch) is an important agricultural pest that causes considerable yield losses to over 150 field and greenhouse crops. Mitochondrial electron transport inhibitors (METI) acaricides are commonly used to control mite species in commercial Canadian greenhouses. Development of resistance to METIs in TSSM populations have been reported worldwide, but not until recently in Canada. The objectives of this study were to: 1) monitor the acaricide-susceptibility in greenhouse TSSM populations, and 2) investigate the resistance to pyridaben, a METI acaricide, in greenhouse resistant and pyridaben-selected (SRS) mite strains. The increased mortality to the pyridaben sub-lethal concentration (LC30) when SRS mites were exposed to piperonyl butoxide (PBO), a general cytochrome P450 monooxygenase inhibitor, and higher P450 activity compared to the greenhouse strain (RS) mites, indicated that P450s may be at least partially responsible for the resistance. The molecular mechanisms of target site insensitivity-mediated resistance in the pyridaben resistant strain of TSSM were investigated by comparing the DNA sequence of NADH dehydrogenase subunits TYKY and PSST, NADH-ubiquinone oxidoreductase chain 1 and 5 (ND1, ND5) and the NADH-ubiquinone oxidoreductase subunit 49 kDa from SRS to the reference strain (SS) and RS. Despite a number of nucleotide substitutions, none correlated with the pyridaben resistance. Understanding the underlying mechanisms of TSSM adaptation to acaricides is an essential part of resistance management strategy in any IPM program. The findings of this study will encourage growers to apply acaricides with different modes of action to reduce the rate at which acaricide resistance will occur in greenhouse TSSM populations.

The two-spotted spider mite (TSSM), Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most polyphagous herbivores, feeding on more than 1,100 plant species. Its wide host range suggests that TSSM has an extraordinary ability to modulate its digestive and xenobiotic physiology. The analysis of the TSSM genome revealed the expansion of gene families that encode proteins involved in digestion and detoxification, many of which were associated with mite responses to host shifts. The majority of plant defense compounds that directly impact mite fitness are ingested. They interface mite compounds aimed at counteracting their effect in the gut. Despite several detailed ultrastructural studies, our knowledge of the TSSM digestive tract that is needed to support the functional analysis of digestive and detoxification physiology is lacking. Here, using a variety of histological and microscopy techniques, and a diversity of tracer dyes, we describe the organization and properties of the TSSM alimentary system. We define the cellular nature of floating vesicles in the midgut lumen that are proposed to be the site of intracellular digestion of plant macromolecules. In addition, by following the TSSM's ability to intake compounds of defined sizes, we determine a cut off size for the ingestible particles. Moreover, we demonstrate the existence of a distinct filtering function between midgut compartments which enables separation of molecules by size. Furthermore, we broadly define the spatial distribution of the expression domains of genes involved in digestion and detoxification. Finally, we discuss the relative simplicity of the spider mite digestive system in the context of mite's digestive and xenobiotic physiology, and consequences it has on the effectiveness of plant defenses.

Reactive oxygen species (ROS) are molecules that play a prominent role in plant response to numerous stresses, including plant interactions with herbivores. Previous findings indicate that Arabidopsis plants showed an increase in H2O2 accumulation after Tetranychus urticae infestation. Despite its importance, no information has been reported on the relationships between ROS-metabolizing systems and the spider mite-triggered plant-induced responses. In this work, four ROS-related genes that were differentially expressed between the resistant Bla-2 and the susceptible Kon Arabidopsis accessions were selected for the analysis. These genes encode proteins putatively involved in the generation (BBE22) and degradation (GPX7 and GSTU4) of H2O2, and in the degradation of ascorbate (AO). Overexpressing BBE22 and silencing GPX7, GSTU4 and AO resulted in higher leaf damage and better mite performance relative to the wild-type plants. Minor effects on H2O2 accumulation obscure major effects on the expression of genes related to ROS-metabolism and JA and SA signaling pathways, and on ROS-related enzymatic activities. In conclusion, the integration of ROS and ROS-related compounds and enzymes in the response of Arabidopsis to the spider mite T. urticae was confirmed. However, the complex network involved in ROS signaling makes difficult to predict the impact of a specific genetic manipulation.

Generalist arthropod herbivores rapidly adapt to a broad range of host plants. However, the extent of transcriptional reprogramming in the herbivore and its hosts associated with adaptation remains poorly understood. Using the spider mite Tetranychus urticae and tomato as models with available genomic resources, we investigated the reciprocal genomewide transcriptional changes in both spider mite and tomato as a consequence of mite's adaptation to tomato. We transferred a genetically diverse mite population from bean to tomato where triplicated populations were allowed to propagate for 30 generations. Evolving populations greatly increased their reproductive performance on tomato relative to their progenitors when reared under identical conditions, indicative of genetic adaptation. Analysis of transcriptional changes associated with mite adaptation to tomato revealed two main components. First, adaptation resulted in a set of mite genes that were constitutively downregulated, independently of the host. These genes were mostly of an unknown function. Second, adapted mites mounted an altered transcriptional response that had greater amplitude of changes when re-exposed to tomato, relative to nonadapted mites. This gene set was enriched in genes encoding detoxifying enzymes and xenobiotic transporters. Besides the direct effects on mite gene expression, adaptation also indirectly affected the tomato transcriptional responses, which were attenuated upon feeding of adapted mites, relative to the induced responses by nonadapted mite feeding. Thus, constitutive downregulation and increased transcriptional plasticity of genes in a herbivore may play a central role in adaptation to host plants, leading to both a higher detoxification potential and reduced production of plant defence compounds.

Nature Communications 8: Article number: 14112 (2017); Published: 20 January 2017; Updated: 16 February 2017 The original version of this Article contained a typographical error in the spelling of the Author Preetam Janakirama, which was incorrectly given as Preetam Jankirama. This has now been corrected in both the PDF and HTML versions of the Article.

Plant-herbivore interactions evolved over long periods of time, resulting in an elaborate arms race between interacting species. While specialist herbivores evolved specific strategies to cope with the defenses of a limited number of hosts, our understanding of how generalist herbivores deal with the defenses of a plethora of diverse host plants is largely unknown. Understanding the interaction between a plant host and a generalist herbivore requires an understanding of the plant's mechanisms aimed at defending itself and the herbivore's mechanisms intended to counteract diverse defenses. In this review, we use the two-spotted spider mite (TSSM), Tetranychus urticae (Koch) as an example of a generalist herbivore, as this chelicerate pest has a staggering number of plant hosts.We first establish that the ability of TSSM to adapt to marginal hosts underlies its polyphagy and agricultural pest status. We then highlight our understanding of direct plant defenses against spider mite herbivory and review recent advances in uncovering mechanisms of spider mite adaptations to them. Finally, we discuss the adaptation process itself, as it allows TSSM to overcome initially effective plant defenses. A highquality genome sequence and developing genetic tools, coupled with an ease of mite experimental selection to new hosts, make TSSM an outstanding systemto study the evolution of host range, mechanisms of pest xenobiotic resistance and plant-herbivore interactions. In addition, knowledge of plant defense mechanisms that affect mite fitness are of practical importance, as it can lead to development of new control strategies against this important agricultural pest. In parallel, understanding mechanisms of mite counter adaptations to these defenses is required to maintain the efficacy of these control strategies in agricultural practices. © 2017 The American Phytopathological Society.

RNA interference (RNAi) can be used for the protection against agricultural pests through the silencing of genes required for pest fitness. To assess the potential of RNAi approaches in the two-spotted spider mite, Tetranychus urticae, we compared 5 methods for the delivery of double-stranded RNA (dsRNA). These methods include mite feeding on either (i) leaves floating on a dsRNA solution, (ii) dsRNA-expressing plants, (iii) artificial diet supplemented with dsRNA, or (iv) dsRNA-coated leaves, and (v) mite soaking in a dsRNA solution. In all cases, the gene targeted for method validation was the Vacuolar-type H+-ATPase (TuVATPase), encoding a constitutively expressed ATP-driven proton pump located in the membrane. Down-regulation of TuVATPase increased mortality and/or reduced fecundity in all methods, but with variable efficiency. The most efficient methods for dsRNA delivery were direct soaking of mites in the dsRNA solution and mite feeding on dsRNA-coated leaves that mimics dsRNA application as a sprayable pesticide. Both resulted in a dark-body phenotype not observed in mites treated with a control dsRNA. Although with lower efficiency, dsRNA designed for TuVATPase silencing and expressed in transgenic Arabidopsis plants impacted the fitness of mites feeding on these plants. RNAi may thus be a valuable strategy to control spider mite populations, either as a sprayable pesticide or through transgenic crops. This comparative methodological study focusing on the induction of RNAi-based gene silencing in T. urticae paves the way for reverse genetics approaches in this model chelicerate system and prepares large-scale systematic RNAi screens as a first step towards the development of specific RNA-based pesticides. Such alternative molecules may help control spider mites that cause significant damages to crops and ornamental plant species, as well as other chelicerates detrimental to agriculture and health. © 2017 Suzuki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The two-spotted spider mite, Tetranychus urticae, is a chelicerate herbivore with an extremely wide host range and an extraordinary ability to develop pesticide resistance. Due to its responsiveness to natural and synthetic xenobiotics, the spider mite is becoming a prime pest herbivore model for studies of the evolution of host range, plant-herbivore interactions and mechanisms of xenobiotic resistance. The spider mite genome has been sequenced and its transcriptional responses to developmental and various biotic and abiotic cues have been documented. However, to identify biological and evolutionary roles of T. urticae genes and proteins, it is necessary to develop methods for the efficient manipulation of mite gene function or protein activity. Here, we describe protocols developed for the delivery of small molecules into spider mites. Starting with mite maintenance and the preparation of the experimental mite populations of developmentally synchronized larvae and adults, we describe 3 methods for delivery of small molecules including artificial diet, leaf coating, and soaking. The presented results define critical steps in these methods and demonstrate that they can successfully deliver tracer dyes into mites. Described protocols provide guidelines for high-throughput setups for delivery of experimental compounds that could be used in reverse genetics platforms to modulate gene expression or protein activity, or for screens focused on discovery of new molecules for mite control. In addition, described protocols could be adapted for other Tetranychidae and related species of economic importance such as Varroa, dust and poultry mites., Correction -- 15 Dec 2017: Suzuki T, España MU, Nunes MA, Zhurov V, Dermauw W, et al. (2017) Correction: Protocols for the delivery of small molecules to the two-spotted spider mite, Tetranychus urticae. PLOS ONE 12(12): e0190025.

In Arabidopsis thaliana, the HUA2 gene is required for proper expression of FLOWERING LOCUS C (FLC) and AGAMOUS, key regulators of flowering time and reproductive development, respectively. Although HUA2 is broadly expressed, plants lacking HUA2 function have only moderately reduced plant stature, leaf initiation rate and flowering time. To better understand HUA2 activity, and to test whether redundancy with similar genes underlies the absence of strong phenotypes in HUA2 mutant plants, we identified and subsequently characterized three additional HUA2-LIKE (HULK) genes in Arabidopsis. These genes form two clades (HUA2/HULK1 and HULK2/HULK3), with members broadly conserved in both vascular and non-vascular plants, but not present outside the plant kingdom. Plants with progressively reduced HULK activity had increasingly severe developmental defects, and plants homozygous for loss-of-function mutations in all four HULK genes were not recovered. Multiple mutants displayed reproductive, embryonic and post-embryonic abnormalities, and provide detailed insights into the overlapping and unique functions of individual HULK genes. With regard to flowering time, opposing influences were apparent: hua2 hulk1 plants were early-flowering, while hulk2 hulk3 mutants were late-flowering, and hua2 acted epistatically to cause early flowering in all combinations. Genome-wide expression profiling of mutant combinations using RNA-Seq revealed complex transcriptional changes in seedlings, with FLC, a known target of HUA2, among the most affected. Our studies, which include characterization of HULK expression patterns and subcellular localization, suggest that the HULK genes encode conserved nuclear factors with partially redundant but essential functions associated with diverse genetic pathways in plants.

Most molecular-genetic studies of plant defense responses to arthropod herbivores have focused on insects. However, plant-feeding mites are also pests of diverse plants, and mites induce different patterns of damage to plant tissues than do well-studied insects (e.g. lepidopteran larvae or aphids). The two-spotted spider mite (Tetranychus urticae) is among the most significant mite pests in agriculture, feeding on a staggering number of plant hosts. To understand the interactions between spider mite and a plant at the molecular level, we examined reciprocal genome-wide responses of mites and its host Arabidopsis (Arabidopsis thaliana). Despite differences in feeding guilds, we found that transcriptional responses of Arabidopsis to mite herbivory resembled those observed for lepidopteran herbivores. Mutant analysis of induced plant defense pathways showed functionally that only a subset of induced programs, including jasmonic acid signaling and biosynthesis of indole glucosinolates, are central to Arabidopsis’s defense to mite herbivory. On the herbivore side, indole glucosinolates dramatically increased mite mortality and development times. We identified an indole glucosinolate dose-dependent increase in the number of differentially expressed mite genes belonging to pathways associated with detoxification of xenobiotics. This demonstrates that spider mite is sensitive to Arabidopsis defenses that have also been associated with the deterrence of insect herbivores that are very distantly related to chelicerates. Our findings provide molecular insights into the nature of, and response to, herbivory for a representative of a major class of arthropod herbivores.

International audience; In indeterminate inflorescences, floral meristems develop on the flanks of the shoot apical meristem, at positions determined by auxin maxima. The floral identity of these meristems is conferred by a handful of genes called floral meristem identity genes, among which the LEAFY (LFY) transcription factor plays a prominent role. However, the molecular mechanism controlling the early emergence of floral meristems remains unknown. A body of evidence indicates that LFY may contribute to this developmental shift, but a direct effect of LFY on meristem emergence has not been demonstrated. We have generated a LFY allele with reduced floral function and revealed its ability to stimulate axillary meristem growth. This role is barely detectable in the lfy single mutant but becomes obvious in several double mutant backgrounds and plants ectopically expressing LFY. We show that this role requires the ability of LFY to bind DNA, and is mediated by direct induction of REGULATOR OF AXILLARY MERISTEMS1 (RAX1) by LFY. We propose that this function unifies the diverse roles described for LFY in multiple angiosperm species, ranging from monocot inflorescence identity to legume leaf development, and that it probably pre-dates the origin of angiosperms.

As a resource allocation strategy, plant growth and defense responses are generally mutually antagonistic. Brassinosteroid (BR) regulates many aspects of plant development and stress responses, however, genetic evidence of its integrated effects on plant growth and stress tolerance is lacking. We overexpressed the Arabidopsis BR biosynthetic gene AtDWF4 in the oilseed plant Brassica napus and scored growth and stress response phenotypes. The transgenic B. napus plants, in comparison to wild type, displayed increased seed yield leading to increased overall oil content per plant, higher root biomass and root length, significantly better tolerance to dehydration and heat stress and enhanced resistance to necrotrophic fungal pathogens Leptosphaeria maculans and Sclerotinia sclerotiorum. Transcriptome analysis supported the integrated effects of BR on growth and stress responses; in addition to BR responses associated with growth, a predominant plant defense signature, likely mediated by BES1/BZR1, was evident in the transgenic plants. These results establish that BR can interactively and simultaneously enhance abiotic and biotic stress tolerance and plant productivity. The ability to confer pleiotropic beneficial effects that are associated with different agronomic traits suggests that BR–related genes may be important targets for simultaneously increasing plant productivity and performance under stress conditions.

Method-Preparation of grapevine plantlets. (DOCX 811 kb)

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most polyphagous herbivores feeding on cell contents of over 1100 plant species including more than 150 crops. It is being established as a model for chelicerate herbivores with tools that enable tracking of reciprocal responses in plant-spider mite interactions. However, despite their important pest status and a growing understanding of the molecular basis of interactions with plant hosts, knowledge of the way mites interface with the plant while feeding and the plant damage directly inflicted by mites is lacking. Here, utilizing histology and microscopy methods, we uncovered several key features of T. urticae feeding. By following the stylet path within the plant tissue, we determined that the stylet penetrates the leaf either in between epidermal pavement cells or through a stomatal opening, without damaging the epidermal cellular layer. Our recordings of mite feeding established that duration of the feeding event ranges from several minutes to more than half an hour, during which time mites consume a single mesophyll cell in a pattern that is common to both bean and Arabidopsis plant hosts. In addition, this study determined that leaf chlorotic spots, a common symptom of mite herbivory, do not form as an immediate consequence of mite feeding. Our results establish a cellular context for the plant-spider mite interaction that will support our understanding of the molecular mechanisms and cell signaling associated with spider mite feeding. © 2016 Bensoussan, Santamaria, Zhurov, Diaz, Grbi and Grbi.

The MADS-AFFECTING FLOWERING 2 (MAF2) gene of Arabidopsis thaliana has been characterized as a repressor of flowering. The molecular basis of MAF2 gene function and role of alternative MAF2 transcripts in flowering time modulation is not understood. MAF2 splice variant expression was quantified in cold-acclimated plants by quantitative RT-PCR. Cold influenced the abundance of splice variants and prompted a functional study of splice forms. Individual variants were overexpressed in the Col background and were assayed for their ability to delay flowering. Overexpression of MAF2 variants 2 and 4 had limited effect on flowering time. Overexpression of MAF2 splice variant 1 resulted in early flowering and affected the expression of the endogenous MAF2 gene and its paralogues, confounding functional assessment. In the Ll-2 Arabidopsis accession, a MAF2, MAF3, MAF4 and FLC null line, MAF2 var1 was consistent in its effect on reproductive delay under ambient and reduced temperatures, indicating that it acts as a repressor of flowering. Electronic supplementary material The online version of this article (doi:10.1007/s11103-012-9982-2) contains supplementary material, which is available to authorized users.

Tandemly arrayed genes that belong to gene families characterize genomes of many organisms. Gene duplication and subsequent relaxation of selection can lead to the establishment of paralogous cluster members that may evolve along different trajectories. Here, we report on the structural variation in MADS AFFECTING FLOWERING 2 (MAF2) gene, one member of the tandemly duplicated cluster of MADS-box-containing transcription factors in Arabidopsis thaliana. The altered gene structure at the MAF2 locus is present as a moderate-frequency polymorphism in Arabidopsis and leads to the extensive diversity in transcript patterns due to alternative splicing. Rearrangements at the MAF2 locus are associated with an early flowering phenotype in BC5 lines. The lack of suppression of flowering time in a MAF2-insertion line expressing the MAF2-specific artificial miRNA suggests that these MAF2 variants are behaving as loss-of-function alleles. The variation in gene architecture is also associated with segregation distortion, which may have facilitated the spread and the establishment of the corresponding alleles throughout the Eurasian range of the A. thaliana population.

MADS-domain transcription factors play pivotal roles in various developmental processes. The lack of simple loss-of-function phenotypes provides impediments to understand the biological function of some of the MADS-box transcription factors. Here we have characterized the potential role of the Arabidopsis thaliana AGAMOUS-LIKE6 (AGL6) gene by fusing full-length coding sequence with transcriptional activator and repressor domains and suggest a role for AGL6 in lateral organ development and flowering. Upon photoperiodic induction of flowering, AGL6 becomes expressed in abaxial and proximal regions of cauline leaf primordia, as well as the cryptic bracts subtending flowers. In developing flowers, AGL6 is detected in the proximal regions of all floral organs and in developing ovules. Converting AGL6 into a strong activator through fusion to the VP16 domain triggers bract outgrowth, implicating AGL6 in the development of bractless flowers in Arabidopsis. In addition, ectopic reproductive structures form on both bracts and flowers in gAGL6::VP16 transgenic plants, which is dependent on B and C class homeotic genes, but independent of LEAFY. Overexpression of both AGL6 and its transcriptional repressor form, AGL6::EAR, causes precocious flowering and terminal flower formation, suggesting that AGL6 suppresses the function of a floral repressor.

The two-spotted spider mite, Tetranychus urticae, is a chelicerate herbivore with an extremely wide host range and an extraordinary ability to develop pesticide resistance. Due to its responsiveness to natural and synthetic xenobiotics, the spider mite is becoming a prime pest herbivore model for studies of the evolution of host range, plant-herbivore interactions and mechanisms of xenobiotic resistance. The spider mite genome has been sequenced and its transcriptional responses to developmental and various biotic and abiotic cues have been documented. However, to identify biological and evolutionary roles of T. urticae genes and proteins, it is necessary to develop methods for the efficient manipulation of mite gene function or protein activity. Here, we describe protocols developed for the delivery of small molecules into spider mites. Starting with mite maintenance and the preparation of the experimental mite populations of developmentally synchronized larvae and adults, we describe 3 methods for delivery of small molecules including artificial diet, leaf coating, and soaking. The presented results define critical steps in these methods and demonstrate that they can successfully deliver tracer dyes into mites. Described protocols provide guidelines for high-throughput setups for delivery of experimental compounds that could be used in reverse genetics platforms to modulate gene expression or protein activity, or for screens focused on discovery of new molecules for mite control. In addition, described protocols could be adapted for other Tetranychidae and related species of economic importance such as Varroa, dust and poultry mites.

31 p.-3 fig.-3 tab.-2 fig. supl.-1 tab. supl., Digestive proteases of the phytophagous mite Tetranychus urticae have been characterised by comparing their activity in body and faecal extracts. Aspartyl, cathepsin B- and L-like and legumain activities were detected in both mite bodies and faeces, with a specific activity of aspartyl and cathepsin L-like proteases about 5- and 2-fold higher, respectively, in mite faeces than in bodies. In general, all these activities were maintained independently of the host plant where the mites were reared (bean, tomato or maize). Remarkably, this is the first report in a phytophagous mite of legumain-like activity, which was characterised for its ability to hydrolyse the specific substrate Z-VAN-AMC, its activation by DTT and inhibition by IAA but not by E-64. Gel free nanoLC–nanoESI-QTOF MS/MS proteomic analysis of mite faeces resulted in the identification of four cathepsins L and one aspartyl protease (from a total of the 29 cathepsins L, 27 cathepsins B, 19 legumains and two aspartyl protease genes identified the genome of this species). Gene expression analysis reveals that four cathepsins L and the aspartyl protease identified in the mite faeces, but also two cathepsins B and two legumains that were not detected in the faeces, were expressed at high levels in the spider mite feeding stages (larvae, nymphs and adults) relative to embryos. Taken together, these results indicate a digestive role for cysteine and aspartyl proteases in T. urticae. The expression of the cathepsins B and L, legumains and aspartyl protease genes analysed in our study increased in female adults after feeding on Arabidopsis plants over-expressing the HvCPI-6 cystatin, that specifically targets cathepsins B and L, or the CMe trypsin inhibitor that targets serine proteases. This unspecific response suggests that in addition to compensation for inhibitor-targeted enzymes, the increase in the expression of digestive proteases in T. urticae may act as a first barrier against ingested plant defensive proteins., This work was supported by a Grant from CSIC (Grant CSIC-201040E049 to F.O), Ministerio de Ciencia e Innovación (Grant AGL11-23650 to I.D.) and the Government of Canada through Genome Canada and the Ontario Genomics Institute (Grant OGI-046 to V.G.), and the Ontario Research Fund-Global Leadership in Genomics and Life Sciences (Grant GL2-01-035 to V.G.).

International audience; The two-spotted spider mite Tetranychus urticae is one of the most significant mite pests in agriculture, feeding on more than 1,100 plant hosts, including model plants Arabidopsis thaliana and tomato, Solanum lycopersicum. Here, we describe timecourse tomato transcriptional responses to spider mite feeding and compare them with Arabidopsis in order to determine conserved and divergent defense responses to this pest. To refine the involvement of jasmonic acid (JA) in mite-induced responses and to improve tomato Gene Ontology annotations, we analyzed transcriptional changes in the tomato JA-signaling mutant defenseless1 (def-1) upon JA treatment and spider mite herbivory. Overlay of differentially expressed genes (DEG) identified in def-1 onto those from the timecourse experiment established that JA controls expression of the majority of genes differentially regulated by herbivory. Comparison of defense responses between tomato and Arabidopsis highlighted 96 orthologous genes (of 2,133 DEG) that were recruited for defense against spider mites in both species. These genes, involved in biosynthesis of JA, phenylpropanoids, flavonoids, and terpenoids, represent the conserved core of induced defenses. The remaining tomato DEG support the establishment of tomato-specific defenses, indicating profound divergence of spider mite–induced responses between tomato and Arabidopsis.

During leaf senescence, nutrients are remobilized from the senescing tissues to the growing parts of the plant. Many senescence-associated genes (SAGs) were identified based on the induction of their transcripts. However, little is known about the protein expression of the corresponding genes. We have raised antibodies against two Arabidopsis SAGs, SAG2 and SAG12, which encode putative cysteine proteases. The SAG2 antibodies recognized a 29-kDa protein that was abundant in senescing leaves, but was also present at low levels in green tissues. SAG12 antibodies labelled a 38-kDa protein present only in senescent leaves. The protein expression of these SAGs parallels their mRNA expression patterns, indicating that control of SAG2 and SAG12 is at the level of transcription or transcript stability. In addition, we found that SAGs are induced during stem senescence with delayed kinetics of their expression relative to leaf expression, suggesting that age-dependent factor(s) regulating the onset of senescence in Arabidopsis may act in tissue-dependent manner.