Progress 03/01/12 to 02/28/16
Outputs
Target Audience:Scientists and plant breeders who are interested using RNA interference (RNAi) to control phloem-feeding insects are the main target audience of this research. New pest control opportunities will be opened up by research on specific osmoregulatory gene targets that can be used to inhibit growth of aphids, whiteflies and psyllids. Additionally, through writing a review article and presenting insect-targeted RNAi in in summer programs for high school teachers and in the Successful Farming Radio Magazine we have reached a broader audience, including farmers who will eventually benefit from improved transgenic pest control methods. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three postdocs, Xiaowei Yang, Xiangfeng Jing, and Vered Tzin, received research training through this project. In addition to research training in the laboratory, major professional development activities during the project period included: Jing attended a PERL class at the Computational Biology Services Unit at Cornell University to receive training in bioinformatics. Tzin was invited to five faculty job interviews in 2014-2016, resulting in three offers of faculty positions. She will assume an assistant professor position at Ben Gurion University in October, 2016. Jander assisted Tzin with her job applications, writing a research plan, and preparing for the seminar presentations. Tzin applied for and received travel grants from the American Society of Plant Biologists (ASPB) to present her research at the 2014 annual meeting in Portland and the 2016 annual meeting in Austin. Jing was offered, and accepted, a faculty position at Northwest A&F University, China in September 2015. How have the results been disseminated to communities of interest?Communication with other scientists: In addition to scientific publications, the results of this research have been presented in the form of talks at meetings and universities. Xiangfeng Jing presented USDA-funded research in seminars at the Entomological Society of America 2013 Annual Meeting, Huazhong Agricultural University, Wuhan, China Department of Entomology, Northwest A&F University, Shaanxi, and China Department of Entomology, Shandong University, China. Vered Tzin presented the results of her research in talks at the Volcani Center, Haifa University, Hebrew University of Jerusalem, Ben Gurion University, the ASPB Annual Meeting, and the northeast regional ASPB Meeting. Angela Dougles presented USDA funded work in seminars at University of Western Australia, CSIRO Perth, Australia, and CSIRO Canberra, Australia Angela Douglas co-organized a section symposium at the November 2012 Annual meeting of the Entomological Society of America on ''RNAi: The Power, the Promise and the Frustration'' Georg Jander presented results of TRV silencing in seminars at UC Davis, the Max Planck Institute for Chemical Ecology, the University of Copenhagen, UC Riverside, and the University of Michigan. Angela Douglas presented results from this research at: 2015: Entomological Society of America Annual Meeting, PBT Section Symposium: Water and Ion Homeostasis-Role of Aquaporins and Other Channel Proteins Minneapolis, MN 2013: Entomological Society of America Annual Meeting, Program Symposium How New Technologies and Interdisciplinary Approaches are Transforming our Understanding of Complex Biological Interactions Advancing public understanding: In 2014 Jander discussed targeted disruption of aphid genes in an interview for the Successful Farming Radio Magazine, which airs on radio stations in the central United States. Jander discussed RNAi for control of phloem-feeding insects in workshops for high school science teachers at the Boyce Thompson Institute in 2015 and 2016. Jing and Douglas introduced the concepts of RNAi and its potential for the control of insect pests to the general public at the annual Open Day (Insectapalooza) of the Department of Entomology, What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Silencing of Osmoregulatory genes The goal of this project was to develop transgenic control approaches that target three agricultural pest species: Myzus persicae (green peach aphid), Bemisia tabaci (whitefly), and Bactericera cockerelli (potato psyllid). Three classes of genes with osmoregulatory functions were chosen as targets: sucrase-transglucosidase, sugar transporters, and aquaporins. Target genes in M. persicae, Be. tabaci, and Ba. cockerelli were identified by mining insect genomic and transcriptomic datasets for genes orthologous to empirically-tested osmoregulatory genes of the pea aphid, Acyrthosiphon pisum. Insects feeding on plants with Tobacco rattle virus (TRV)-mediated RNAi against the target genes exhibited elevated hemolymph osmotic pressure (a predicted effect of perturbed osmotic function) and some reduction in performance. In particular decreased offspring production and adult size in M. persicae and increased mortality in B. cockerelli were associated with up to 50% reduction in mean expression of the target genes. The effects were particularly pronounced for insects treated with RNAi against multiple osmoregulatory genes, i.e. combinatorial RNAi, suggesting that the partial silencing of multiple genes with related roles can yield greater functional impairment than RNAi against a single gene. These results demonstrate the potential of RNAi against osmoregulatory genes, but further advances to improve the efficacy of RNAi in phloem-feeding insects are required to achieve effective pest control. RNAi experiments with Be. tabaci were less conclusive. Given the feeding habits of Be. tabaci larvae, it was difficult to conduct experiments with plants that were transiently expressing whitefly-targeted RNAi constructs in TRV or Agrobacterium tumefaciens T-DNA. It may be necessary to conduct further experiments with stable transgenic plants that are designed to cause osmoregulatory collapse in Be. tabaci through RNAi. RNAi of other aphid genes As a negative control for the experiments with RNAi of osmoregulatory genes, two genes in M. persicae carotenoid biosynthesis, carotene desaturase (Tor) and carotenoid cyclase-carotenoid synthase (CarRP) were silenced. As not all aphids produce carotenoids, it was predicted that there would be no negative effects from silencing these genes. However, a 50% reduction in expression of these genes reduced M. persicae reproduction by 25%, a bigger effect than that which was seen with RNAi of osmoregulatory genes in M. persicae. There were no synergistic effects from silencing Tor and CarRP simultaneously, suggesting that these genes act in the same pathway leading to the production of a metabolite that is important for aphid reproduction and/or survival. In related research that was partially funded by this award, the expression of M. persicae salivary gene was silenced. Silencing expression of a particular M. persicae salivary gene, Mp55, using RNA interferences reduced aphid reproduction on Nicotiana tabacum, Arabidopsis thaliana, and Nicotiana benthamiana. In contrast, overexpressing Mp55 in plants, increased aphid reproduction, and resulted in lower accumulation of 4-methoxyindol-3-ylmethylglucosinolate, callose, and hydrogen peroxide in response to aphid feeding. Mp55-overexpressing plants also were more attractive for aphids in choice assays. Together, these results demonstrate a role for Mp55, a protein with as-yet unknown molecular function, in the interaction of M. persicae with its host plants. Aphid and whitefly genome sequencing The lack of available genome sequences has been a limitation in the design of RNAi constructs for the control of hemipteran pests. However, with the rapid drop in the cost of genome sequencing, this will issue will be resolved soon. Assembly of Be. tabaci and M. persicae genome sequences was completed in 2016. Members of the Douglas and Jander labs assisted in the annotation of the genome sequences and are listed as co-authors on the Be. tabaci (Douglas and Jander) and M. persicae (Jander) manuscripts, which are currently in review.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Chen W, Hasegawa DK, Kliot A, Kaur N, Valle Pinheiro P, Luan J, Zheng Y, Liu W, Sun H, Xu Y, Luo Y, Kruse A, Yang X, Elimelech M, Fisher TW, MacCoss M, Johnson R, Nelson DR, Cohen E, Hunter WB, Brown JK, Jander G, Cilia M, Douglas AE, Ghanim M, Simmons AM, Wintermantel WM, Ling KS, and Fei Z (2016) The genome of whitefly Bemisia tabaci, an important plant pest and virus, in review.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Mathers TC, Chen Y, Kaihakottil G, Ledeai F, Mugford ST, Baa-Puyoulet P, Bretaudeau2,4,5, Bernardo A, Colella CS, Collin O, Dalmay T, Derrien T, Feng H, Gabald�n T, Jordan A, Julca I,
Kettles GJ, Kowitwanich K, Lavenier D, Lenzi P, Lopez-Gomollon S, Loska D, Mapleson D, Maumus F, Moxon S, Price DRG, Sugio A, van Munster M, Uzest M, Waite D, Jander G,15, Tagu D, Wilson ACC, van Oosterhout C, Swarbreck D, and Hogenhout SA (2016) A clonally reproducing aphid colonizes diverse host plants by unidirectionally regulating lineage-expanded gene clusters., in review.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Jing XF, White TA, Luan J, Jiao C, Fei Z and Douglas AE, 2016. Evolutionary conservation of candidate osmoregulation genes in plant phloem-sap feeding insects. Insect Molecular Biology 25, 251-8. http://dx.doi.org/10.1111/imb.12215
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Scott JG, Michel K, Bartholomay LC, Siegfried BD, Hunter WB, Smagghe G, Zhu KY, Douglas AE. (2013) Towards the elements of successful insect RNAi. J Insect Physiol 12:1212-21. doi: 10.1016/j.jinsphys.2013.08.014.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Elzinga DA, De Vos M, Jander G. (2014) Suppression of plant defenses by a Myzus persicae (green peach aphid) salivary effector protein. Mol Plant Microbe Interact. 27:747-56. doi: 10.1094/MPMI-01-14-0018-R.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Tzin V, Yang X, Jing X, Zhang K, Jander G, Douglas AE. (2015) RNA interference against gut osmoregulatory genes in phloem-feeding insects. J Insect Physiol. 79:105-12. doi: 10.1016/j.jinsphys.2015.06.006.
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Progress 03/01/13 to 02/28/14
Outputs
Target Audience: During this reporting period, the target audience has consisted of other scientists who have heard the results of this research presented in the form of seminars and posters at meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two postdocs, Xiangfeng Jing and Vered Tzin, received research training through this project. In addition to research training in the laboratory, major activities of the past year include: Jing attended a PERL class at the Computational Biology Services Unit at Cornell University to receive training in bioinformatics. Tzin was invited for a faculty job interview at the Volcani Center in Israel and recently received a travel grant from the American Society of Plant Biologists (ASPB) to present her research from this project at the ASPB Annual Meeting in Portland, Oregon, in July. Jander assisted Tzin with her job application, preparing for the seminar presentation, and applying for the ASPB travel grant. How have the results been disseminated to communities of interest? Communication with other scientists: Georg Jander presented results of TRV silencing in seminars at UC Davis, the Max Planck Institute for Chemical Ecology, and the University of Copenhagen. Xiangfeng Jing presented USDA-funded research in seminars at the Entomological Society of America 2013 Annual Meeting, Huazhong Agricultural University, Wuhan, China Department of Entomology, Northwest A&F University, Shaanxi, and China Department of Entomology, Shandong University, China. Angela Dougles presented USDA funded work in seminars at University of Western Australia, CSIRO Perth, Australia, and CSIRO Canberra, Australia Angela Douglas co-organized a section symposium at the November 2012 Annual meeting of the Entomological Society of America on ‘‘RNAi: The Power, the Promise and the Frustration’’ Advancing public understanding: Jander discussed targeted disruption of aphid genes in an interview for the Successful Farming Radio Magazine, which airs on radio stations in the central United States. What do you plan to do during the next reporting period to accomplish the goals? To date, gene silencing efforts have been directed primarily at Myzus persicae. In the coming year, this research will be expanded to include a greater emphasis on silencing the expression of osmoregulatory gene expression in Bemisia tabaci and Bactericera cockerelli.
Impacts
What was accomplished under these goals?
Objective 1: To identify candidate osmoregulatory genes in the gut of the peach potato aphid, Myzus persicae by global expression analysis. This objective was accomplished during the previous reporting period. There is nothing new to report. Objective-2: Identification of the genes underpinning osmoregulation in two non-aphid hemipteran pests. Taking advantage of recent published transcriptomes for both Bemisia tabaci and Bactericera cockerelli, we have (1) used various bioinformatics tools to search the sugar osmoregulation genes and (2) verified that these genes are authentic and enriched in the gut by PCR, Sanger sequencing and qPCR of cDNA samples. This research has enabled us to compare the selected genes to homologs in other model organisms to identify conserved domains and motifs for use in RNAi. For our bioinformatics analyses of the whitefly, Be. tabaci MEAM1, we reassembled gut RNAseq data kindly supplied by Professor Liu, Zhengjiang University. Our analysis of the psyllid Ba. cockerelli used published transcriptome data. Using Baysian and maximum parsimony methods, we have identified one aquaporin and two sucrases in Be. tabaci and four aquaporin and six sucrase gene candidates for Ba. cockerelli, validated the genes by PCR with sequencing, tested for expression in the gut by qPCR, and verified the conserved domain and motifs. The parallel analysis of sugar transporters has revealed >30 candidate transporters of the PFAM family PF00083 in each of the insects. Orthologs of the transporters mediating sugar transport in the aphid gut are currently being identified and validated by combined bioinformatics and empirical analyses. A manuscript on the phylogenetic analysis of osmoregulatory genes in phloem-feeding insects is in preparation. Objective-3: Construction of transgenic plants with RNAi that depress expression of insect genes required for osmoregulation resulting in osmotic collapse and premature death of the insect pest. Aphid genes contibuting to osmoregulation (sucrase-transglucosidase, sugar transporters, and aquaporins) were targeted for expression silencing using tobacco rattle virus (TRV) constructs in Nicotiana benthamiana and Nicotiana tabacum. Reduced expression of the target genes was verified by quantitative PCR. In the experiments on the aphid Myzus persicae, we quantified the osmotic dysfunction of in planta RNAi against sucrase and aquaporin on expression of the target genes by measuring osmotic pressure in heamolymph. Results indicated that average osmotic pressure of insects feeding on aquaporin and sucrase dsRNA expressing plants is slightly higher than those on control plants. In control experiments, expression of carotenoid biosynthesis genes was targeted by RNA interference using TRV constructs. Reduced expression of carotenoid biosynthesis genes suggests that the RNA interference signal spreads through the aphids and is not only present in the gut of the insects. As not all isolates of Myzus persicae produce carotenoids, it was presumed that these genes are non-essential. Unexpectedly, aphids with silenced carotenoid biosynthesis genes grew less well than control aphids. A review article on the application of RNAi to insects has been published. Scott JG, Michel K, Bartholomay LC, Siegfried BD, Hunter WB, Smagghe G, Zhu KY, Douglas AE. (2013) Towards the elements of successful insect RNAi, J Insect Physiol 12:1212-21. doi: 10.1016/j.jinsphys.2013.08.014. A manuscript, in which the TRV expression silencing method was applied for targeted reduction of M. persicae salivary gene expression has been accepted for publication. Elzinga DA, de Vos M, Jander G (2014) Suppression of plant defenses by a Myzus persicae (green peach aphid) salivary effector protein, Mol Plant Microbe Interact [Epub ahead of print]. A manuscript describing method of aphid gene expression silencing using TRV is being modified for resubmission.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Scott JG, Michel K, Bartholomay LC, Siegfried BD, Hunter WB, Smagghe G, Zhu KY, Douglas AE. (2013) Towards the elements of successful insect RNAi. J Insect Physiol 12:1212-21. doi: 10.1016/j.jinsphys.2013.08.014.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Elzinga DA, de Vos M, Jander G (2014) Suppression of plant defenses by a Myzus persicae (green peach aphid) salivary effector protein, Mol Plant Microbe Interact [Epub ahead of print].
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Urizarna Espa�a1 M, Tzin V, Villaplana L, Mingot, A, Jander G, Lopez-Moya JJ (2014) Silencing aphid gene expression using a plant-pathogenic virus vector, in review
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Progress 03/01/12 to 02/28/13
Outputs
OUTPUTS: Two draft sequences of the Myzus persicae (green peach aphid) genome have been assembled in collaboration with researchers at Rothamsted Research in the United Kingdom and the French National Institute for Agricultural Research (INRA). Both sequences are publicly available for BLAST searches at AphidBase, http://tools.genouest.org/tools/myzus/login. The two sets of sequences are from two previously studied aphid lineages, a North American M. persicae strain, G006 (Ramsey et al, 2007, BMC Genomics 8:423 doi:10.1186/1471-2164-8-423), and a British strain, Clone O (Fenton et al, 2010, Ecological Entomology, 35:S131-S146 DOI: 10.1111/j.1365-2311.2009.01150.x), respectively. Once further assembly and corrections have been made, these genome sequences also will be submitted to GenBank. Genome assembly of the Buchnera aphidicola endosymbiont bacteria of M. persicae (Buchnera Mp) has been completed. Endosymbionts from four strains of M. persicae were sequenced, two from tobacco-adapted aphid lineages and two from non-tobacco-adapted lineages. The four bacterial genome sequences have been submitted to GenBank and also will be included in BuchneraBase (www.buchnera.org), a comparative database of insect bacterial endosymbiont genome sequences. PARTICIPANTS: Project Participants are working at the Boyce Thompson Institute (Georg Jander and Dezi Elzinga) and the University of Miami (Alex Wilson, Zhijie Jiang, Derek Jones Sawsan Khuri, Nicholas Tsinoremas, and Tania Wyss). Collaborators are working at INRA (Denis Tagu and Fabrice Legai), Rothamsted Research (Saskia Hogenhout and Lin Field), the James Hutton Institute (Brian Fenton), and the National University of Ireland Maynooth (James Carolan). Students that are being trained through the project include Dezi Elzinga, Derek Jones, and Tania Wyss. TARGET AUDIENCES: Aphid researchers are the primary target audience for the most recent results from this project. Both the Myzus persicae genome sequence and new methods for gene silencing will accelerate research on this important agricultural pest. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Myzus persicae genome sequences, which have been made publicly available through this project, will be of use to aphid researchers around the world. It will be possible to rapidly identify M. persicae homologs of genes from other organisms. M. persicae genomic sequences will facilitate research on insecticide resistance, host plant utilization, and other aspects of this agriculturally important pest species. To facilitate investigation of aphid gene function, a new gene silencing approach was developed. This RNA interference (RNAi) approach, which is based on expressing aphid-targeted RNA in tobacco rattle virus, makes it possible to reduce gene expression in plant-feeding aphids. By allowing rapid functional analysis of M. persicae genes, the new RNAi method will be useful for many aphid researchers. In the current project, the gene silencing approach was used to reduce expression of three aphid salivary proteins, showing that they are required for efficient feeding of M. persicae on plants. Antibodies have been generated against two of these functionally important aphid salivary proteins. Use of these antibodies will make it possible to localize salivary proteins, both within the aphids and in the plants. Pull-down experiments will help to determine what plant proteins the aphid salivary proteins interact with, thereby helping to determine their as yet unknown functions in plant-aphid interactions. Assembly of Buchnera aphidicola endosymbiont (Buchnera Mp) genomes from four lineages of M. persicae showed not differences in gene content. Two of these M. persicae lineages were tobacco adapted. Although some prior studies demonstrated that tobacco-adapted lineages are genetically distinct, there were no differences in the gene content of the Buchera Mp endosymbionts. Thus, it is unlikely that these bacteria contribute to the ability of some M. persicae strains to grow particularly well on tobacco. Comparisons to Buchnera from other aphid species suggest that certain genes in Buchnera Mp may facilitate feeding on a broad range of host plants.
Publications
- No publications reported this period
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