Publications by authors named "Xi'en Chen"

11 Publications

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Mutation of P-element somatic inhibitor induces male sterility in the diamondback moth, Plutella xylostella.

Pest Manag Sci 2021 Apr 11. Epub 2021 Apr 11.

CAS Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences/Institute of Plant Physiology and Ecology, Shanghai, China.

Background: Genetic manipulation of sex determination pathways in insects provides the basis for a broad range of strategies to benefit agricultural security and human health. The P-element somatic inhibitor (PSI) protein, an exon splicing silencer that promotes male-specific splicing of dsx, plays a critical role in male sexual differentiation and development. The functions of PSI have been characterized in the lepidopteran model species Bombyx mori. However, the molecular mechanism and functions of PSI in Plutella xylostella, a worldwide agricultural pest and taxonomically basal species, are still unknown.

Results: Here we identified PxPSI transcripts and analyzed their spatiotemporal expression pattern in P. xylostella. Multiple sequence alignment revealed that PxPSI contains four KH domains and is highly conserved in lepidopterans. We used the CRISPR-Cas9 system to generate mutations of the PxPSI genomic locus. Disruptions of PxPSI caused male-specific defects in internal and external genitals. In addition, we detected female-specific Pxdsx transcripts in PxPSI male mutants. Mutations also caused changes in expression of several sex-biased genes and induced male sterility.

Conclusion: Our study demonstrates that PxPSI plays a key role in male sex determination in P. xylostella and suggests a potential molecular target for genetic-based pest management in lepidopteran pests.
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http://dx.doi.org/10.1002/ps.6413DOI Listing
April 2021

Mutation of doublesex induces sex-specific sterility of the diamondback moth Plutella xylostella.

Insect Biochem Mol Biol 2019 09 3;112:103180. Epub 2019 Jul 3.

CAS Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai, 200032, China. Electronic address:

DOUBLESEX (DSX): the downstream gene in the insect sex determination pathway, plays a critical role in sexual differentiation and development. The functions of dsx have been characterized in several model insect species. However, the molecular mechanism and functions of sex determination of dsx in Plutella xylostella, an agricultural pest, are still unknown. In present study, we identified a male-specific and three female-specific Pxdsx transcripts in P. xylostella. Phylogenetic analyses and multiple sequence alignment revealed that Pxdsx is highly conserved in lepidopterans. The CRISPR/Cas9 technology was used to induce mutations in the male-specific isoform, the female-specific isoform, and common regions of Pxdsx. Disruptions of Pxdsx sex-specific isoforms caused sex-specific defects in external genitals and partial sexual reversal. In addition, we found that female specific transcripts were detected in Pxdsx male mutants and male-specific transcripts were detected in Pxdsx female mutants. Mutations also caused changes in expression of several sex-biased genes and induced sex-specific sterility. This study demonstrates that Pxdsx plays a key role in sex determination of P. xylostella and suggests novel genetic control approaches for the management of P. xylostella.
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http://dx.doi.org/10.1016/j.ibmb.2019.103180DOI Listing
September 2019

Identification of Key Residues Associated with the Interaction between Plutella xylostella Sigma-Class Glutathione S-Transferase and the Inhibitor S-Hexyl Glutathione.

J Agric Food Chem 2018 Oct 20;66(39):10169-10178. Epub 2018 Sep 20.

Key Laboratory of Plant Protection Resources & Pest Management of the Ministry of Education, College of Plant Protection , Northwest A&F University , Yangling , Shaanxi 712100 , China.

Glutathione S-transferases (GSTs) are important detoxification enzymes involved in the development of metabolic resistance in Plutella xylostella. Uncovering the interactions between representative PxGSTs and the inhibitor S-hexyl glutathione (GTX), helps in the development of effective PxGST inhibitors for resistance management. As the PxGST most severely inhibited by GTX, PxGSTσ (sigma-class PxGST) adopts the canonical fold of insect GSTs. The formation of the PxGSTσ-GTX complex is mainly driven by H-bond and hydrophobic interactions derived from the side chains of favorable residues. Of the residues composing the active site of PxGSTσ, Lys43 and Arg99 are two hot spots, first reported in the binding of GSH derivatives to GSTs. Such differences indicate the metabolism discrimination of different insect GSTs. Unfavorable interactions between the PxGSTσ active site and GTX are depicted as well. The research guides the discovery and optimization of PxGSTσ inhibitors.
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http://dx.doi.org/10.1021/acs.jafc.8b03967DOI Listing
October 2018

Identification of yellow gene family in Agrotis ipsilon and functional analysis of Aiyellow-y by CRISPR/Cas9.

Insect Biochem Mol Biol 2018 03 11;94:1-9. Epub 2018 Jan 11.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

The yellow gene family has been identified in several model insects, but yellow genes were poorly identified in non-model insects and the functions of yellow genes are largely unknown. In this study, we identified seven yellow genes in an important agricultural pest Agrotis ipsilon. Each gene encodes a protein containing a major royal jelly domain. Phylogenetic analysis defined these genes as yellow-y, -b, -b2, -c, -d, -e, and -h, respectively. The A. ipsilon yellow genes yellow-b, -b2, and -c were stably expressed in all developmental stages and tissues analyzed, whereas the other four yellow genes had unique expression patterns, suggesting distinct physiological roles of each gene. Using the CRISPR/Cas9 system, we successfully disrupted yellow-y in A. ipsilon and obtained G insects with somatic mutations. Unlike the black of wild-type newly hatched larvae and of adults, the mutants were yellow, although in the pupal stage mutant coloration did not differ from wild-type coloration. This phenotype was inherited by G offspring. The G mutants did not show any growth deficiency compared with control insects; however, a dehydration-like phenotype was observed in newly hatched G larvae from sibling crossed mutants. Our results indicate that A. ipsilon yellow-y gene plays a role in body pigmentation and also might function in waterproofing.
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http://dx.doi.org/10.1016/j.ibmb.2018.01.002DOI Listing
March 2018

Identification and characterization of NADPH-dependent cytochrome P450 reductase gene and cytochrome b₅ gene from Plutella xylostella: possible involvement in resistance to beta-cypermethrin.

Gene 2015 Mar 27;558(2):208-14. Epub 2014 Dec 27.

Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China. Electronic address:

NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides.
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http://dx.doi.org/10.1016/j.gene.2014.12.053DOI Listing
March 2015

Identification and characterisation of multiple glutathione S-transferase genes from the diamondback moth, Plutella xylostella.

Pest Manag Sci 2015 Apr 10;71(4):592-600. Epub 2014 Sep 10.

Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, China.

Background: The diamondback moth (DBM), Plutella xylostella, is one of the most harmful insect pests on crucifer crops worldwide. In this study, 19 cDNAs encoding glutathione S-transferases (GSTs) were identified from the genomic and transcriptomic database for DBM (KONAGAbase) and further characterized.

Results: Phylogenetic analysis showed that the 19 GSTs were classified into six different cytosolic classes, including four in delta, six in epsilon, three in omega, two in sigma, one in theta and one in zeta. Two GSTs were unclassified. RT-PCR analysis revealed that most GST genes were expressed in all developmental stages, with higher expression in the larval stages. Six DBM GSTs were expressed at the highest levels in the midgut tissue. Twelve purified recombinant GSTs showed varied enzymatic properties towards 1-chloro-2,4-dinitrobenzene and glutathione, whereas rPxGSTo2, rPxGSTz1 and rPxGSTu2 had no activity. Real-time quantitative PCR revealed that expression levels of the 19 DBM GST genes were varied and changed after exposure to acephate, indoxacarb, beta-cypermethrin and spinosad. PxGSTd3 was significantly overexpressed, while PxGSTe3 and PxGSTs2 were significantly downregulated by all four insecticide exposures.

Conclusion: The changes in DBM GST gene expression levels exposed to different insecticides indicate that they may play individual roles in tolerance to insecticides and xenobiotics.
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http://dx.doi.org/10.1002/ps.3884DOI Listing
April 2015

Identification of multiple small heat-shock protein genes in Plutella xylostella (L.) and their expression profiles in response to abiotic stresses.

Cell Stress Chaperones 2015 Jan 20;20(1):23-35. Epub 2014 Jun 20.

Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, College of Plant Protection, Northwest A & F University, Yangling, Shaanxi, China.

We identify and characterize 14 small heat-shock protein (sHSP) genes from the diamondback moth (DBM), Plutella xylostella (L.), a destructive pest. Phylogenetic analyses indicate that, except for sHSP18.8 and sHSP19.22, the other 12 DBM sHSPs belong to five known insect sHSP groups. Developmental expression analysis revealed that most sHSPs peaked in the pupal and adult stages. The transcripts of sHSPs display tissue specificity with two exhibiting constitutive expression in four tested tissues. Expression of sHSP18.8 in fourth instar larvae is not induced by the tested abiotic stressors, and unless sHSP21.8 is not sensitive to thermal stress, 12 sHSPs are significantly up-regulated. The messenger RNA (mRNA) levels of all sHSPs are reduced under oxidative stress. Food deprivation leads to significant down-regulation of three sHSPs. The majority of sHSPs show expression variation to various heavy metals, whereas mRNA abundances of sHSP22.1 and sHSP 28.9 are reduced by four heavy metals. The responses of sHSPs to indoxacarb and cantharidin are varied. Beta-cypermethrin and chlorfenapyr exposure results in an increase of 13 sHSP transcripts and a reduction of 12 sHSP transcripts, respectively. These results show that different sHSPs might play distinct roles in the development and regulation of physiological activities, as well as in response to various abiotic stresses of DBM.
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http://dx.doi.org/10.1007/s12192-014-0522-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255244PMC
January 2015

Characterization of protein phosphatase 5 from three lepidopteran insects: Helicoverpa armigera, Mythimna separata and Plutella xylostella.

PLoS One 2014 13;9(5):e97437. Epub 2014 May 13.

Key Laboratory of Plant Protection Resources & Pest Management of the Ministry of Education, Northwest A&F University, Yangling, Shaanxi, China.

Protein phosphatase 5 (PP5), a unique member of serine/threonine phosphatases, regulates a variety of biological processes. We obtained full-length PP5 cDNAs from three lepidopteran insects, Helicoverpa armigera, Mythimna separata and Plutella xylostella, encoding predicted proteins of 490 (55.98 kDa), 490 (55.82 kDa) and 491 (56.07 kDa) amino acids, respectively. These sequences shared a high identity with other insect PP5s and contained the TPR (tetratricopeptide repeat) domains at N-terminal regions and highly conserved C-terminal catalytic domains. Tissue- and stage-specific expression pattern analyses revealed these three PP5 genes were constitutively expressed in all stages and in tested tissues with predominant transcription occurring at the egg and adult stages. Activities of Escherichia coli-produced recombinant PP5 proteins could be enhanced by almost 2-fold by a known PP5 activator: arachidonic acid. Kinetic parameters of three recombinant proteins against substrate pNPP were similar both in the absence or presence of arachidonic acid. Protein phosphatases inhibitors, okadaic acid, cantharidin, and endothall strongly impeded the activities of the three recombinant PP5 proteins, as well as exerted an inhibitory effect on crude protein phosphatases extractions from these three insects. In summary, lepidopteran PP5s share similar characteristics and are all sensitive to the protein phosphatases inhibitors. Our results also imply protein phosphatase inhibitors might be used in the management of lepidopteran pests.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097437PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019573PMC
June 2015

Identification and biochemical characterization of protein phosphatase 5 from the cantharidin-producing blister beetle, Epicauta chinensis.

Int J Mol Sci 2013 Dec 16;14(12):24501-13. Epub 2013 Dec 16.

Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, Northwest A&F University, Yangling 712100, China.

Protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5) was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat) motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5) was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate) and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin.
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http://dx.doi.org/10.3390/ijms141224501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3876124PMC
December 2013

Cantharidin impedes the activity of protein serine/threonine phosphatase in Plutella xylostella.

Mol Biosyst 2014 Feb;10(2):240-50

Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, China.

Cantharidin, a natural toxin produced by the blister beetle, was reported to be toxic to some pests, but the mechanism of its toxicity in insects remains undefined. We found that cantharidin exerted in vivo and in vitro inhibitory effects on protein serine/threonine phosphatases (PSPs) of Plutella xylostella. Five PSP genes, PP1, PP2A, PP4, PP5, and PP6, were cloned from P. xylostella. Phosphatase domain alignment showed a high similarity. Recombinant PxPP5 (rPxPP5) was expressed in Escherichia coli and purified. Cantharidin and its 11 analogs were used to perform the rPxPP5 activity inhibition assay in vitro. Cantharidin strongly inhibited rPxPP5 activity competitively, with an IC50 of 0.38 μM. All analogs also showed inhibitory activity, with an IC50 of 7.42-538.38 μM. The rank of IC50 values was found to be consistent with their toxicities in P. xylostella larvae with a correlation coefficient (R(2)) of 0.87. 3D models of the phosphatase domains of these five PxPSPs were constructed which superimposed well indicating a high structural similarity demonstrating that the chemicals used may be inhibitors of the other four PxPSPs. Binding model analysis of cantharidin and its analogs which interacted with PxPP5 showed that the cantharidin-derived moiety was anchored to the active site, explaining their inhibitory effect on rPxPP5 in vitro. Results of binding free energy calculations are also well in line with their inhibition effects on rPxPP5, with a correlation coefficient (R(2)) of 0.72. In light of the above results we argue that protein serine/threonine phosphatases are the targets of cantharidin and its analogs acting on P. xylostella.
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http://dx.doi.org/10.1039/c3mb70410fDOI Listing
February 2014

Molecular cloning and characterization of the calcineurin subunit A from Plutella xylostella.

Int J Mol Sci 2013 Oct 15;14(10):20692-703. Epub 2013 Oct 15.

Key Laboratory of Plant Protection Resources & Pest Management of Ministry of Education, Northwest A&F University, Yangling 712100, Shaanxi, China.

Calcineurin (or PP2B) has been reported to be involved in an array of physiological process in insects, and the calcineurin subunit A (CNA) plays a central role in calcineurin activity. We cloned the CNA gene from Plutella xylostella (PxCNA). This gene contains an ORF of 1488 bp that encodes a 495 amino acid protein, showing 98%, and 80% identities to the CNA of Bombyx mori, and humans respectively. The full-length of PxCNA and its catalytic domain (CNA(1-341), defined as PxCNα) were both expressed in Escherichia coli. Purified recombinant PxCNA displayed no phosphatase activity, whereas recombinant PxCNα showed high phosphatase activity with a Km of 4.6 mM and a kcat of 0.66 S(-1) against pNPP. It could be activated at different degrees by Mn2+, Ni2+, Mg2+, and Ca2+. The optimum reaction pH was about 7.5 and the optimum reaction temperature was around 45 °C. An in vitro inhibition assay showed that okadaic acid (OA) and cantharidin (CTD) competitively inhibited recombinant PxCNα activity with the IC50 values of 8.95 μM and 77.64 μM, respectively. However, unlike previous reports, pyrethroid insecticides were unable to inhibit recombinant PxCNα, indicating that the P. xylostella calcineurin appears not to be sensitive to class II pyrethroid insecticides.
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http://dx.doi.org/10.3390/ijms141020692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821638PMC
October 2013