crystal-structure-n-terminal-domain-ryanodine-receptor-from-plutella

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from <em>Plutella xylostella</em>

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Fujian Agriculture and Forestry University

Ministry of Education, Fuzhou

We present here the de novo assembly and annotation of the transcriptome of Plutella xylostella (diamondback moth (DBM)), a widespread destructive pest of cruciferous plants, using short reads generated by Illumina sequencing from different developmental stages and insecticide-resistant strains. A total of 171,262 non-redundant sequences, denoted as unigenes, were obtained. They represented approximately 100-fold of all DBM mRNA and EST sequences in GenBank thus far. We identified 38,255 unigenes highly similar to the known functional protein-coding genes, most of which were annotated using gene ontology (GO) and orthologous groups of proteins (COG). Global profiling of differentially expressed unigenes revealed enriched GOs and biological pathways that were related to specific developmental stages and insecticide resistance. We also evaluated the resistance-related single nucleotide polymorphism (SNP) using this high-throughput genotyping method. The newly developed transcriptome will facilitate researches on the DBM developmental biology and insecticide resistance evolution, and ultimately provide better pest management systems.

Developmental and insecticide-resistant insights from the de novo assembled transcriptome of the diamondback moth, Plutella xylostella.

How an insect evolves to become a successful herbivore is of profound biological and practical importance. Herbivores are often adapted to feed on a specific group of evolutionarily and biochemically related host plants, but the genetic and molecular bases for adaptation to plant defense compounds remain poorly understood. We report the first whole-genome sequence of a basal lepidopteran species, Plutella xylostella, which contains 18,071 protein-coding and 1,412 unique genes with an expansion of gene families associated with perception and the detoxification of plant defense compounds. A recent expansion of retrotransposons near detoxification-related genes and a wider system used in the metabolism of plant defense compounds are shown to also be involved in the development of insecticide resistance. This work shows the genetic and molecular bases for the evolutionary success of this worldwide herbivore and offers wider insights into insect adaptation to plant feeding, as well as opening avenues for more sustainable pest management.

A heterozygous moth genome provides insights into herbivory and detoxification.

Insect midgut microbiota is important in host nutrition, development and immune response. Recent studies indicate possible links between insect gut microbiota and resistance to biological and chemical toxins. Studies of this phenomenon and symbionts in general have been hampered by difficulties in culture-based approach. In the present study, DNA sequencing was used to examine the midgut microbiota of diamondback moth (DBM), Plutella xylostella (L.), a destructive pest that attacks cruciferous crops worldwide. Its ability to develop resistance to many types of synthetic insecticide and even Bacillus thuringiensis toxins makes it an important species to study.

DNA sequencing reveals the midgut microbiota of diamondback moth, Plutella xylostella (L.) and a possible relationship with insecticide resistance.

The diamondback moth Genome Database (DBM-DB) is a central online repository for storing and integrating genomic data of diamondback moth (DBM), Plutella xylostella (L.). It provides comprehensive search tools and downloadable datasets for scientists to study comparative genomics, biological interpretation and gene annotation of this insect pest. DBM-DB contains assembled transcriptome datasets from multiple DBM strains and developmental stages, and the annotated genome of P. xylostella (version 2). We have also integrated publically available ESTs from NCBI and a putative gene set from a second DBM genome (KONAGbase) to enable users to compare different gene models. DBM-DB was developed with the capacity to incorporate future data resources, and will serve as a long-term and open-access database that can be conveniently used for research on the biology, distribution and evolution of DBM. This resource aims to help reduce the impact DBM has on agriculture using genomic and molecular tools. Database URL: http://iae.fafu.edu.cn/DBM/ 

DBM-DB: the diamondback moth genome database.

Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification. 

Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.).

Understanding how inbreeding affects fitness is biologically important for conservation and pest management. Despite being a worldwide pest of many economically important cruciferous crops, the influence of inbreeding on diamondback moth, Plutella xylostella (L.), populations is currently unknown. Using age-stage-specific life tables, we quantified the inbreeding effects on fitness-related traits and demographic parameters of P. xylostella. Egg hatching rate, survival and fecundity of the inbred line significantly declined compared to those of the outbred line over time. The inbred P. xylostella line showed significantly lower intrinsic rate of increase (r), net reproduction rate (R0), and finite increase rate (λ), and increasing generation time (T). Inbreeding effects vary with developmental stages and the fitness-related traits can be profoundly affected by the duration of inbreeding. Our work provides a foundation for further studies on molecular and genetic bases of the inbreeding depression for P. xylostella. 

Generation-based life table analysis reveals manifold effects of inbreeding on the population fitness in Plutella xylostella.

Movement of individuals through events, such as storms or crop transportation, may affect survival and distribution of insect pests, as well as population genetic structure at a regional scale. Understanding what factors contribute to gene flow in pest populations remains very important for sustainable pest management. The diamondback moth (Plutella xylostella) is an insect pest well known for its capacity of moving over short to long distances. Here, we used newly isolated microsatellite markers to analyze the genetic structure of nine populations across the Taiwan Strait of China (Taiwan and Fujian). A total of 12,152 simple sequence repeats (SSRs) were initially identified from the P. xylostella transcriptome (~94 Mb), with an average of 129 SSRs per Mb. Nine SSRs were validated to be polymorphic markers, and eight were used for this population genetic study. Our results showed that the P. xylostella populations could be divided into distinct two clusters, which is likely due to the year-round airflows in this region. A pattern of isolation by distance among the local populations within Fujian was found, and may be related to vegetable transportation. Considering the complexity of the P. xylostella population genetic structure from local and regional to global levels, we propose that developing ecologically sound strategies for managing this pest will require knowledge of the link between behavioral and population ecology and its genetic structure.

Genetic differentiation of the regional Plutella xylostella populations across the Taiwan Strait based on identification of microsatellite markers.

ATP-binding cassette (ABC) transporters are one of the major transmembrane protein families found in all organisms and play important roles in transporting a variety of compounds across intra and extra cellular membranes. In some species, ABC transporters may be involved in the detoxification of substances such as insecticides. The diamondback moth, Plutella xylostella (L.), a destructive pest of cruciferous crops worldwide, is an important species to study as it is resistant to many types of insecticides as well as biological control Bacillus thuringiensis toxins.

Characterization and expression profiling of ATP-binding cassette transporter genes in the diamondback moth, Plutella xylostella (L.).

Fourteen polymorphic microsatellite loci were isolated in this transcriptome-based data analysis for , which is an important larval parasitoid of the worldwide pest . A subsequent test was performed for a wild  population ( = 32) from Fuzhou, Fujian, southeastern China, to verify the effectiveness of the 14 microsatellite loci in future studies on  genetic diversity. The observed number of alleles ranged from two to six. The expected and observed heterozygosity ranged from 0.123 to 0.316 and from 0.141 to 0.281, respectively. The polymorphism information content () value ranged from 0.272 to 0.622. Potentially due to the substructure of the sampled population, three of the 14 microsatellite loci deviated from Hardy-Weinberg equilibrium (). Further, loci C6, C22, and C31 could be amplified in  and , suggesting the transferability of these three polymorphic loci to other species of Hymenoptera.

Isolation and Characterization of Microsatellite Loci for Cotesia plutellae (Hymenoptera: Braconidae).

Long non-coding RNAs (lncRNAs) are of particular interest because of their contributions to many biological processes. Here, we present the genome-wide identification and characterization of putative lncRNAs in a global insect pest, Plutella xylostella. A total of 8096 lncRNAs were identified and classified into three groups. The average length of exons in lncRNAs was longer than that in coding genes and the GC content was lower than that in mRNAs. Most lncRNAs were flanked by canonical splice sites, similar to mRNAs. Expression profiling identified 114 differentially expressed lncRNAs during the DBM development and found that majority were temporally specific. While the biological functions of lncRNAs remain uncharacterized, many are microRNA precursors or competing endogenous RNAs involved in micro-RNA regulatory pathways. This work provides a valuable resource for further studies on molecular bases for development of DBM and lay the foundation for discovery of lncRNA functions in P. xylostella.

Genome-wide identification and characterization of putative lncRNAs in the diamondback moth, Plutella xylostella (L.).

Transcription factors (TFs), which play a vital role in regulating gene expression, are prevalent in all organisms and characterization of them may provide important clues for understanding regulation in vivo. The present study reports a genome-wide investigation of TFs in the diamondback moth, Plutella xylostella (L.), a worldwide pest of crucifers. A total of 940 TFs distributed among 133 families were identified. Phylogenetic analysis of insect species showed that some of these families were found to have expanded during the evolution of P. xylostella or Lepidoptera. RNA-seq analysis showed that some of the TF families, such as zinc fingers, homeobox, bZIP, bHLH, and MADF_DNA_bdg genes, were highly expressed in certain tissues including midgut, salivary glands, fat body, and hemocytes, with an obvious sex-biased expression pattern. In addition, a number of TFs showed significant differences in expression between insecticide susceptible and resistant strains, suggesting that these TFs play a role in regulating genes related to insecticide resistance. Finally, we identified an expansion of the HOX cluster in Lepidoptera, which might be related to Lepidoptera-specific evolution. Knockout of this cluster using CRISPR/Cas9 showed that the egg cannot hatch, indicating that this cluster may be related to egg development and maturation. This is the first comprehensive study on identifying and characterizing TFs in P. xylostella. Our results suggest that some TF families are expanded in the P. xylostella genome, and these TFs may have important biological roles in growth, development, sexual dimorphism, and resistance to insecticides. The present work provides a solid foundation for understanding regulation via TFs in P. xylostella and insights into the evolution of the P. xylostella genome.

Genome-wide investigation of transcription factors provides insights into transcriptional regulation in Plutella xylostella.

Ryanodine receptors (RyRs) are large calcium-release channels located in sarcoplasmic reticulum membrane. They play a central role in excitation-contraction coupling of muscle cells. Three commercialized insecticides targeting pest RyRs generate worldwide sales over 2 billion U.S. dollars annually, but the structure of insect RyRs remains elusive, hindering our understanding of the mode of action of RyR-targeting insecticides and the development of insecticide resistance in pests. Here we present the crystal structure of RyR N-terminal domain (NTD) (residue 1-205) at 2.84 Å resolution from the diamondback moth (DBM), Plutella xylostella, a destructive pest devouring cruciferous crops all over the world. Similar to its mammalian homolog, DBM RyR NTD consists of a beta-trefoil folding motif and a flanking alpha helix. Interestingly, two regions in NTD interacting with neighboring domains showed distinguished conformations in DBM relative to mammalian RyRs. Using homology modeling and molecular dynamics simulation, we created a structural model of the N-terminal three domains, showing two unique binding pockets that could be targeted by potential species-specific insecticides. Thermal melt experiment showed that the stability of DBM RyR NTD was higher than mammalian RyRs, probably due to a stable intra-domain disulfide bond observed in the crystal structure. Previously DBM NTD was shown to be one of the two critical regions to interact with insecticide flubendiamide, but isothermal titration calorimetry experiments negated DBM NTD alone as a major binding site for flubendiamide.

Crystal structure of ryanodine receptor N-terminal domain from Plutella xylostella reveals two potential species-specific insecticide-targeting sites.

Calmodulin (CaM) is a Ca-binding protein that plays an important role in cellular Ca-signaling. CaM interacts with diverse downstream target proteins and regulates their functions in a Ca-dependent manner. CaM changes its conformation and hydrophobicity upon [Ca] change and consequently changes its interaction with CaM-binding domains from the targets. Based on these special properties of CaM, it was used as an affinity tag to develop a novel purification strategy by using it for two sequential orthogonal purification steps: 1) an affinity purification step, in which CaM-tag interacts with an immobilized CaM-binding domain; and 2) a hydrophobic interaction chromatography step, during which CaM binds to a phenyl sepharose column. In both steps, the CaM-tagged protein binds in the presence of Ca and unbinds in the presence of ethylenediaminetetraacetic acid (EDTA). An optional third step can be added to remove the CaM-tag if necessary. We used green fluorescent protein (GFP) as a test protein to demonstrate the effectiveness of the method. High yield and high purity of GFP with proper function was obtained using this novel strategy. We believe that this method can be applied to a wide range of protein targets for structural and functional studies.

Article	Year
Developmental and insecticide-resistant insights from the de novo assembled transcriptome of the diamondback moth, Plutella xylostella. Genomics\| PubMed ID: 22240003	2012
A heterozygous moth genome provides insights into herbivory and detoxification. Nature genetics\| PubMed ID: 23313953	2013
DNA sequencing reveals the midgut microbiota of diamondback moth, Plutella xylostella (L.) and a possible relationship with insecticide resistance. PloS one\| PubMed ID: 23894355	2013
DBM-DB: the diamondback moth genome database. Database : the journal of biological databases and curation\| PubMed ID: 24434032	2014
Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.). Scientific reports\| PubMed ID: 25752830	2015
Generation-based life table analysis reveals manifold effects of inbreeding on the population fitness in Plutella xylostella. Scientific reports\| PubMed ID: 26227337	2015
Genetic differentiation of the regional Plutella xylostella populations across the Taiwan Strait based on identification of microsatellite markers. Ecology and evolution\| PubMed ID: 26811762	2015
Characterization and expression profiling of ATP-binding cassette transporter genes in the diamondback moth, Plutella xylostella (L.). BMC genomics\| PubMed ID: 27678067	2016
Isolation and Characterization of Microsatellite Loci for Cotesia plutellae (Hymenoptera: Braconidae). Insects\| PubMed ID: 28632152	2017
Genome-wide identification and characterization of putative lncRNAs in the diamondback moth, Plutella xylostella (L.). Genomics\| PubMed ID: 28789862	2018
Genome-wide investigation of transcription factors provides insights into transcriptional regulation in Plutella xylostella. Molecular genetics and genomics : MGG\| PubMed ID: 29147778	2018
Crystal structure of ryanodine receptor N-terminal domain from Plutella xylostella reveals two potential species-specific insecticide-targeting sites. Insect biochemistry and molecular biology\| PubMed ID: 29191465	2018
A two-step purification strategy using calmodulin as an affinity tag. Journal of chromatography. A\| PubMed ID: 29499842	2018

Weiyi He

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