TRANSCRIPTIONAL REGULATORY CASCADES MEDIATING UPREGULATION OF CYP6B GENES BY PLANT DEFENSE SIGNALS AND ALLELOCHEMICALS IN HELICOVERPA ZEA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0202584
• Project Start Date: Jan 1, 2005
• Project End Date: Sep 30, 2009
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824

Performing Department
ENTOMOLOGY

Non Technical Summary
Crop pests such as corn earworm are known to use plant defense toxin compounds and defense signaling molecules as cues to overproduce their detoxification enzymes such as cytochrome P450 monooxygenases, but how insects receive and transduce these cues chemicals is totally unknown. The purpose of this study is to reveal how insects receive and transduce plant defense toxic compounds and signaling molecules.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3110	1040	50%
211	3110	1070	25%
211	3110	1130	25%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology; 1130 - Entomology and acarology; 1070 - Ecology;

Keywords

allelochemicals

gene expression

protein synthesis

defense mechanisms

plant insect relations

transcription

gene regulation

signal transduction

insect physiology

insect genetics

helicoverpa

insecticides

insecticide resistance

insecticide resistant insects

nature of resistance

metabolic pathways

metabolic regulation

insect larvae

promoters (genetics)

detoxification

Goals / Objectives
In view of the magnitude of the management problems presented by this heliothine noctuid pest and polyphagous lepidopteran pests in general, new approaches based on a solid understanding of molecular mechanisms that allow these insects to exploit a wide variety of crop plants via their tripartite regulation system and to develop resistance to a broad spectrum of insecticides by their altered basal overexpression of P450 genes would be immense value in developing new tools and approaches for management. Elucidating the signaling pathways and involved components, transcription factors and ciselements of the tripartite regulation of CYP6B genes in H. zea will not only promote our understanding of signaling interactions between plant and insects, but also provide great insights into new avenue for controlling insects and designing new pesticides. New pesticides may be designed to block the signaling perception and signaling transduction in insects. Resolving the signaling pathway may also facilitate identification of mutated transcription factors that lead to the constitutive overexpression of P450 genes in resistant pests and thus provide great insights into new avenue for controlling resistant insects. The regulatory cascade revealed from this study will be likely informative for understanding of environment-dependent gene regulation in other herbivorous eukaryotes including humans. The methodologies deriving from such studies may be applicable to other eukaryotes in defining regulation of environment-dependent genes. To this end, I propose several objectives aimed at characterizing signaling steps, cis-elements, and transcription factors responsible for tripartite regulation of CYP6B genes. Specifically, I propose: A. To diagnosis the potential signaling steps and components in the regulatory cascade of H. zea CYP6B genes by in vivo analyses of the effects of protein synthesis inhibitors and specific kinase, phosphatases inhibitors on the basal and induced expression of these P450 genes; B. To clone the promoter sequences of these P450 genes by genomic library screening and /or reverse PCR strategy; C. To define JA-, SA- and allelochemical-response elements by bioinformatic approach, in vitro DNA mobility shift analysis, and luciferase reporter assay; D. To characterize the transcription factors that bind with JA-, SA- and allelochemical-response elements by yeast one-hybrid screening.

Project Methods
RTPCR blot analysis: Newly-emerged H. zea 5th larvae will be fed with inhibitors together with JA, SA or allelochemicals and RNA will be isolated. The RTPCR blot analysis described in Li et al (2002) will be employed to quantify the transcripts of CYP6B genes. Reverse PCR cloning: Genomic DNA isolated from single larvae will be completely digested with restriction enzymes with one cut close to the 5 prime end of the CYP6B gene. The digested genomic DNA will then be cyclized under dilute ligation conditions. The promoters of the CYP6B genes will then be amplified in an inverse-PCR reaction using diametrically opposed CYP6B-specific primers. Ten ul of amplification products will be electrophoresed and the largest size of band hybridized with 32P-labeled CYP6B cDNA will be cloned into pBluescript vector and sequenced. Genomic library construction and screening: If the inverse PCR fails to generate a reasonable size (1.5-2kb) of promoter, we will then construct a H. zea genomic library and screen it with the existing CYP6B DNA clones at high stringency. Positive clones identified will be mapped, subcloned, and sequenced. In vitro DNA mobility shift analysis: 32P-labeled putative elements and conserved fragment identified by bioinformatic approach will be incubated with induced and control H. zea midgut nuclear extracts and formation of sequence-specific complexes will be monitored by autoradiography. Luciferase reporter assay: The various length of segments containing critical elements will be fused to a Luciferase (Luc) reporter gene. The constructed CYP6B promoter-Luc fusions will be cotransfected along with a Drosophila 5C actin promoter:beta-galactosidase construct into Sf9 and Tn5 cells for monitoring Luc activity in the presence of single plant allelochemicals and signaling molecules. Yeast one-hybrid screening: At least three tandem repeats of each identified elements will be inserted into the reporter vector pHIS2 and the resulting CYP6B element-pHIS2 constructs will be recovered by transforming DH5alpha with CYP6B element-pHIS2 ligation mixture. RNA isolated from JA-, SA, or allelochemical-induced 5th instar larvae will be used to synthesis the first-strand cDNA using the CDS III oligo(dT) primer, which will be further amplified to produce double strand cDNA by long-distance PCR. The resulted double strand cDNA will be purified with a BD CHROMA SPIN TE-400 Column. Subsequently, a cDNA library (double strand cDNA), the Sma I-linearized GAL4 AD cloning vector pGADT7-Rec2, and a CYP6B element- pHIS2 construct will be used to cotransform yeast Y187. Positive one-hybrids can be identified immediately after cotransformation by plating the transformation mixture on medium that selects for the HIS3 nutritional reporter.

Progress 01/01/05 to 09/30/09

Outputs
OUTPUTS: Final report: 1. We have characterized three new P450 genes (CYP321A2, CYP9A12, and CYP9A14) from H. zea; 2. We have identified 12 novel transposons in six xenobiotic-metabolizing H. zea P450 genes; 3 We have cloned the promoter sequences of the 6 P450 genes; 4 We have defined the essential cis-acting elements for allelochemical induction of CYP321A1 in H. zea; 5 We have identified the putative positive and negative regulators for flavone and xanthotoxin regulation of CYP321A1; 6. We have defined the cis element mediate the premature polyadenylation of Hztransib in H. zea. DISSEMINATION: Four papers have been published. Two manuscripts have been submitted to journals and are currently under review. Three more manuscripts are in preparations. Some of the results were presented in various meetings or seminars. PARTICIPANTS: 1. Song Chen, postdoc. 2. Chunni Zhang, PhD student 3. Erxia Du, PhD student 4 Andy Wong, Research Technician. TARGET AUDIENCES: People working on plant-insect interactions, gene regulation, and transposons. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Characterization plant allelechemical-responsible cis elements and transcription factors will not only promote our understanding of signaling interactions between plants and insects, but will also provide new avenues for insecticide development and pest control. 2. Characterization of Hztransib, the first intact and active Transib transposons found in any organisms, has at least three important impacts. First, it can be used as a model element to dissect the transposition mechanism of Transib transposons. Second, it can be developed as a novel DNA vector that targets on GC-rich sequence. The current widely used vectors target on TA-rich sequence. Third, it may be developed as a gene drive for population replacement. 3. The six P450s can be used as targets for designing syngergists for controlling insecticide-resistant insects. 4. The positive and negative regulators in the allelochemical signaling pathway may be used as lead gene for RNAi control of H. zea and other pests.

Publications

• Li, X. & Ni, X. 2009. Deciphering the plant-insect phenotypic arms race. In Recent Advances in Entomological Research: From Molecular Biology to Pest Management, Liu, T-X. and Kang, L. (eds.), Higher Education Press, Beijing, China (in press).
• Chen, S. and Li, X. 2007. Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes. BMC Evolutionary B iology 7:46.
• Li, X., Schuler, M. A. and Berenbaum, M. R. 2007. Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Ann. Rev. Entomol. 52: 231-53.
• Chunni Zhang, Xiangxia Luo, Xinzhi Ni, Yalin Zhang and Xianchun Li. 2010. Functional characterization of cis-acting elements mediating flavone-inducible expression of CYP321A1. Journal of Cellular Biochemistry (submitted).
• Erxia Du, Xinzhi Ni, Huiyan, Zhao, and Xianchun Li. 2010. Natural history and intragenomic dynamics of Hztransib in Helicoverpa zea. Journal of Evolutionary Biology (Submitted).

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: OUTPUT: Progress made in this year: 1. We have constructed a systematic array of CYP321A1 promoter-PGL3 deletion/mutation constructs that progressively delete the CYP321A1 promoter sequence from its 5' or 3' end and mutate certain nucleotide in putative cis-elements; 2. We have idetified the basal, xanthotoxin-, and flavone-responsible cis-elements for regualtion of CYP321A1 expression by testing the promoter activities of those constructs; 3. We have found that the transposon inserted in the CYP321A1 promoter region marginally downregulate the expression of CYP321A1 through our luciferase reporter gene assay; 4. A pilot gel shift assay has been conducted to confirm if the identified xanthotoxin-responsible element produces sequence-specific DNA-nuclear protein complex; 5. We have fully the first intact Transib transposon from H. zea; and 6. We have conducted a transposon display to estimate the duplication and transposion activity of Hztransib in several Helicoverpa zea lab strains. DISSEMINATION: Some of results have been published and we are currently wrtiing three more manuscripts. Some of results were presented in various meetings or seminars. PARTICIPANTS: PARTICIPANTS: 1. Song Chen, postdoc. 2. Chunni Zhang, PhD student 3. Erxia Du, PhD student TARGET AUDIENCES: People work on plant-insect interactions, gene regulation, and transposons. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Elucidating the signaling pathways and components will not only promote our understanding of signaling interactions between plants and insects, but will also provide new avenues for insecticide development and pest control; 2. The characterized first intact Transib element Hztransib can be used as a model element to dissect the transposition mechanism of Transib transposons; 3. Hztranisb can be developed as a novel DNA vector that targets on GC-rich sequence. The current widely used vectors target on TA-rich sequence; 4. Hztransib could be a potential gene drive for population replacement.

Publications

• Chen, S. and Li, X. 2008. Molecular characterization of the first intact Transib transposon from Helicoverpa zea. Gene 408:51-63.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Progress made in this year: 1. We have tested the allelochemical inducibility of CYP321A2 and CYP321A1 by qRTPCR; 2. We have scanned two allels per each of six xenobiotic-metabolizing H. zea P450 loci (CYP6B8, CYP6B27, CYP321A1, CYP321A2, CYP9A12v3, and CYP9A14) for the presence of transposon insertions. A total of twelve novel transposons have been identified from the six H. zea P450 genes. 3. We have characterized the first intact Transib transposon from H. zea. 4. Several CYP321 promoter-PGL3 deletion constructs that remove introns or contain different putative cis-elements have been constructed. PARTICIPANTS: 1. Song Chen, postdoc. 2. Chunni Zhang, PhD student 3. Erxia Du, PhD student PROJECT MODIFICATIONS: Unexpected identification of twelve novel transoposons and of the first intact Transib does modify the goals of this project. We will continue elucidating the signaling pathways as planned. Meanwhile, we have two additional research objectives: 1) Demonstrate the mobility of Hztransib and develop a Hztransib-based DNA vector; and 2)Understand how the twelve transposons change the transcription/function of those P450 genes.

Impacts
1. Identification of twelve novel transposons in six xenobiotic-metabolizing H. zea P450 genes present for the first time evidence that transposons are selectively retained within or in close proximity to xenobiotic-metabolizing P450 genes. 2. Characterization of Hztransib, the first intact and active Transib transposons found in any organisms, has at least two important impacts. First, it can be used as a model element to dissect the transposition mechanism of Transib transposons. Second, it can be developed as a novel DNA vector that targets on GC-rich sequence. The current widely used vectors target on TA-rich sequence. 3. Elucidating the signaling pathways and components will not only promote our understanding of signaling interactions between plants and insects, but will also provide new avenues for insecticide development and pest control

Publications

• Song Chen and Xianchun Li. 2007. Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes. BMC Evolutionary Biology 2007, 4:46

Progress 01/01/06 to 12/31/06

Outputs
Progress made in this year: 1. We have demonstrated that plant defense signal and compound-responsible elements are located in the 2-3 kb upstrem flanking sequences obtained by luciferase reporter gene assay. 2. We have isolated three P450 genes (CYP321A2, CYP9A12, CYP9A14) from H. zea. 3. We have identified 6 novel transposable elements. Some of them may introduce cis-elements that increase/decrease the allelochemical inducibility of P450 genes. 4. We are now in the process of defining those cis-elements using multiple approaches.

Impacts
Elucidating the signaling pathways and components will not only promote our understanding of signaling interactions between plants and insects, but will also provide new avenues for insecticide development and pest control

Publications

• No publications reported this period

Progress 01/01/05 to 12/31/05

Outputs
Progress made in this year: 1.Determination of cell lines for promoter activity assays. We have confirmed, by cell induction and RT-PCR analysis, that the H. zea midgut and fatbody cell lines, established by Dr. Goodman of USDA, are capable of sensing and responding to plant defense signals and allelochemicals. Therefore, both cell lines are appropriate for testing the promoter activity of the H. zea P450 promoter :luciferase constructs and can be used to fish out the transcription factors later. 2.Cloning and characterization of the upstream flanking promoter sequences (2-3 kb) of CYP6B8, CYP6B27, and CYP321A1 by genome walking and sequencing the promoter sequences of the most inducible P450 genes by genomome walking. 3.Bioinformatic characterization of the putative signal and allelochemical-response elements in the promoter sequences of the P450 genes. 4.A pilot trial of the luciferase reporter assay system using the CYP321A1 promoter-luciferase PGL3 construct. 5.Cloning of additional H. zea P450 genes belong to the CYP9A and CYP321A subfamily. 6.The prevalence of transposons in the H .zea P450 genes

Impacts
Elucidating the signaling pathways and components will not only promote our understanding of signaling interactions between plants and insects, but will also provide new avenues for insecticide development and pest control

Publications

• Li, X., Schuler, M.A. and Berenbaum, M.R. 2007. Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics. Ann. Rev. Entomol. (in press)