We report a simple protocol for the photochemical Giese addition of C(sp3)‐centered radicals to a variety of electron‐poor olefins. The chemistry does not require external photoredox catalysts. ...Instead, it harnesses the excited‐state reactivity of 4‐alkyl‐1,4‐dihydropyridines (4‐alkyl‐DHPs) to generate alkyl radicals. Crucial for reactivity is the use of a catalytic amount of Ni(bpy)32+ (bpy=2,2′‐bipyridyl), which acts as an electron mediator to facilitate the redox processes involving fleeting and highly reactive intermediates.
A time to get, and a time to lose electrons: Electron mediators can alleviate limitations associated with the photochemistry of excited‐state intermediates, including short lifetimes and low electron transfer rates. The ability of a nickel complex to act as an electron shuttle assists the excited‐state reactivity of dihydropyridines 1 to enable a photochemical Giese addition of C(sp3)‐centered radicals to electron‐poor olefins.
Viroids are self replicating non-coding RNAs capable of infecting a wide range of plant hosts. They do not encode any proteins, thus the mechanism by which they escape plant defenses remains unclear. ...RNAi silencing is a major defense mechanism against virus infections, with the four DCL proteins being principal components of the pathway. We have used Nicotiana benthamiana as a model to study Potato spindle tuber viroid infection. This viroid is a member of the Pospiviroidae family and replicates in the nucleus via an asymmetric rolling circle mechanism. We have created knock-down plants for all four DCL genes and their combinations. Previously, we showed that DCL4 has a positive effect on PSTVd infectivity since viroid levels drop when DCL4 is suppressed. Here, we show that PSTVd levels remain decreased throughout infection in DCL4 knockdown plants, and that simultaneous knockdown of DCL1, DCL2 or DCL3 together with DCL4 cannot reverse this effect. Through infection of plants suppressed for multiple DCLs we further show that a combined suppression of DCL2 and DCL3 has a major effect in succumbing plant antiviral defense. Based on our results, we further suggest that Pospoviroids may have evolved to be primarily processed by DCL4 as it seems to be a DCL protein with less detrimental effects on viroid infectivity. These findings pave the way to delineate the complexity of the relationship between viroids and plant RNA silencing response.
Chelicerate mites diverged from other arthropod lineages more than 400 million years ago and subsequently developed specific and remarkable xenobiotic adaptations. The study of the two-spotted spider ...mite,
Tetranychus urticae
, for which a high-quality Sanger-sequenced genome was first available, revealed expansions and radiations in all major detoxification gene families, including P450 monooxygenases, carboxyl cholinesterases, glutathione-
S
-transferases, and ATP-binding cassette transporters. Novel gene families that are not well studied in other arthropods, such as major facilitator family transporters and lipocalins, also reflect the evolution of xenobiotic adaptation. The acquisition of genes by horizontal gene transfer provided new routes to handle toxins, for example, the β-cyanoalanine synthase enzyme that metabolizes cyanide. The availability of genomic resources for other mite species has allowed researchers to study the lineage specificity of these gene family expansions and the distinct evolution of genes involved in xenobiotic metabolism in mites. Genome-based tools have been crucial in supporting the idiosyncrasies of mite detoxification and will further support the expanding field of mite-plant interactions.
About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene ...family is now recognized as one of the largest transporter families in all kingdoms of life. The majority of ABC proteins function as primary-active transporters that bind and hydrolyze ATP while transporting a large diversity of substrates across lipid membranes. Although extremely well studied in vertebrates for their role in drug resistance, less is known about the role of this family in the transport of endogenous and exogenous substances in arthropods. The ABC families of five insect species, a crustacean and a chelicerate have been annotated in some detail. We conducted a thorough phylogenetic analysis of the seven arthropod and human ABC protein subfamilies, to infer orthologous relationships that might suggest conserved function. Most orthologous relationships were found in the ABCB half transporter, ABCD, ABCE and ABCF subfamilies, but specific expansions within species and lineages are frequently observed and discussed. We next surveyed the role of ABC transporters in the transport of xenobiotics/plant allelochemicals and their involvement in insecticide resistance. The involvement of ABC transporters in xenobiotic resistance in arthropods is historically not well documented, but an increasing number of studies using unbiased differential gene expression analysis now points to their importance. We give an overview of methods that can be used to link ABC transporters to resistance. ABC proteins have also recently been implicated in the mode of action and resistance to Bt toxins in Lepidoptera. Given the enormous interest in Bt toxicology in transgenic crops, such findings will provide an impetus to further reveal the role of ABC transporters in arthropods.
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•A phylogenetic reconstruction of arthropod and human ABC proteins.•Species-specific and lineage-specific ABC gene expansions were uncovered.•Orthologous relationships suggest conserved function.•The role of ABC transporters in insecticide/allelochemical transport and resistance is surveyed.
The P450 family (CYP genes) of arthropods encodes diverse enzymes involved in the metabolism of foreign compounds and in essential endocrine or ecophysiological functions. The P450 sequences (CYPome) ...from 40 arthropod species were manually curated, including 31 complete CYPomes, and a maximum likelihood phylogeny of nearly 3000 sequences is presented. Arthropod CYPomes are assembled from members of six CYP clans of variable size, the CYP2, CYP3, CYP4 and mitochondrial clans, as well as the CYP20 and CYP16 clans that are not found in Neoptera. CYPome sizes vary from two dozen genes in some parasitic species to over 200 in species as diverse as collembolans or ticks. CYPomes are comprised of few CYP families with many genes and many CYP families with few genes, and this distribution is the result of dynamic birth and death processes. Lineage-specific expansions or blooms are found throughout the phylogeny and often result in genomic clusters that appear to form a reservoir of catalytic diversity maintained as heritable units. Among the many P450s with physiological functions, six CYP families are involved in ecdysteroid metabolism. However, five so-called Halloween genes are not universally represented and do not constitute the unique pathway of ecdysteroid biosynthesis. The diversity of arthropod CYPomes has only partially been uncovered to date and many P450s with physiological functions regulating the synthesis and degradation of endogenous signal molecules (including ecdysteroids) and semiochemicals (including pheromones and defense chemicals) remain to be discovered. Sequence diversity of arthropod P450s is extreme, and P450 sequences lacking the universally conserved Cys ligand to the heme have evolved several times. A better understanding of P450 evolution is needed to discern the relative contributions of stochastic processes and adaptive processes in shaping the size and diversity of CYPomes.
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•Phylogeny of nearly 3000 sequences from 40 arthropod CYPomes.•Six CYP clans represented in arthropods.•Few CYP families with many genes and many CYP families with few genes.•Current paradigm not adequate to explain ecdysteroid biosynthesis.•Multiple origins of P450s lacking the conserved Cys ligand to the heme.
The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The ...extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo.
Pesticide resistance represents a clear and trackable case of adaptive evolution with a strong societal impact. Understanding the factors associated with the evolution and spread of resistance is ...imperative to develop sustainable crop management strategies. The two‐spotted spider mite Tetranychus urticae, a major crop pest with worldwide distribution and a polyphagous lifestyle, has evolved resistance to most classes of pesticides. Tetranychus urticae exists as either a green‐ or a red‐coloured morph. However, the extent of genetic divergence and reproductive compatibility vary across populations of these colour morphs, complicating their taxonomic resolution at the species level. Here, we studied patterns of genetic differentiation and barriers to gene flow within and between morphs of T. urticae in order to understand the factors that influence the spread of resistance mutations across its populations. We derived multiple iso‐female lines from Tetranychus populations collected from agricultural crops. We generated genomic and morphological data, characterized their bacterial communities and performed controlled crosses. Despite morphological similarities, we found large genomic differentiation between the morphs. This pattern was reflected in the incomplete, but strong postzygotic incompatibility in crosses between colour morphs, while crosses within morphs from different geographical locations were largely compatible. In addition, our results suggest recent/on‐going gene flow between green‐coloured T. urticae and T. turkestani. By screening the sequences of 10 resistance genes, we found evidence for multiple independent origins and for single evolutionary origins of target‐site resistance mutations. Our results indicate that target‐site mutations mostly evolve independently in populations on different geographical locations, and that these mutations can spread due to incomplete barriers to gene flow within and between populations.
•The different classes of mutations associated with resistance are discussed.•A survey of mutations in each class is provided.•Point mutations in target sites are still abundantly described.•Gene ...duplications, amplifications and gene disruptions are increasingly important.•Mutations affecting gene regulation are frequent but difficult to describe with precision.
The recent accumulation of molecular studies on mutations in insects, ticks and mites conferring resistance to insecticides, acaricides and biopesticides is reviewed. Resistance is traditionally classified by physiological and biochemical criteria, such as target-site insensitivity and metabolic resistance. However, mutations are discrete molecular changes that differ in their intrinsic frequency, effects on gene dosage and fitness consequences. These attributes in turn impact the population genetics of resistance and resistance management strategies, thus calling for a molecular genetic classification. Mutations in structural genes remain the most abundantly described, mostly in genes coding for target proteins. These provide the most compelling examples of parallel mutations in response to selection. Mutations causing upregulation and downregulation of genes, both in cis (in the gene itself) and in trans (in regulatory processes) remain difficult to characterize precisely. Gene duplications and gene disruption are increasingly reported. Gene disruption appears prevalent in the case of multiple, hetero-oligomeric or redundant targets.
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