Source: PENNSYLVANIA STATE UNIVERSITY submitted to
PLANT DETECTION OF HERBIVORE FEEDING CUES: A COMPARATIVE APPROACH TOWARDS UNDERSTANDING HOST EVASION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0225018
Grant No.
2011-67013-30352
Project No.
PEN04434
Proposal No.
2010-03720
Multistate No.
(N/A)
Program Code
A1111
Project Start Date
Apr 1, 2011
Project End Date
Mar 31, 2016
Grant Year
2011
Project Director
Felton, G. W.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
Entomology
Non Technical Summary
When insects feed on crop plants they release saliva or other secretions that may inadvertently mobilize plant defenses against the insect attackers. Plants may "recognize" these cues found in secretions or excrement that are left behind during their feeding. These cues may then "vaccinate" the plant to protect it against future attack. The identification of these cues may aid in the development of new pest management strategies and/or by guiding the development of resistant crops against insect pests. These new approaches may help reduce the use of synthetic pesticides and thus help protect the environment from the undesirable effects of pesticide use. We will also study how some insects are stealthy in their feeding habits and thus avoid mobilizing plant defenses. This will help us understand how some insects can achieve pest status. Training the next generation of scientists will be an important aspect of this project. We will also incorporate the scientific training and mentoring of undergraduate students from minority serving institutions as part our project.
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
2113110113050%
2061460106025%
2061510106025%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants; 206 - Basic Plant Biology;

Subject Of Investigation
1510 - Corn; 3110 - Insects; 1460 - Tomato;

Field Of Science
1130 - Entomology and acarology; 1060 - Biology (whole systems);
Goals / Objectives
When herbivores initiate feeding on a host plant, they present "feeding cues" that the plant perceives and uses to rapidly mobilize induced defenses in response to the attack. We test two hypotheses: 1/ Generalist herbivores partially avoid detection by minimizing their display of elicitors; and 2/ Specialist herbivores use their secretions to actively suppress plant responses. The model system includes two of the most important crops worldwide (maize, tomato) and important Lepidopteran and Coleopteran pests. The following objectives will be tested: 1/ Compare plant defense responses to salivary secretions and regurgitant/oral secretions of generalist and specialist herbivores; 2/ Compare the amount of regurgitant deposited during feeding by specialist and generalist species; 3/ Determine if the frass from generalist and specialist herbivores (that feed in enclosed plant tissues) affects plant defense responses; and 4/Characterize the active components of secretions from several key species. This study will provide fundamental knowledge on how herbivores evade host defenses or avoid detection by their host plants. We will extend the known repertoire of herbivore effectors and compare herbivore offense strategies in species with differing lifestyles. Identification of effectors and their specific modes of action will provide the basis for designing more resistant cultivars through modern breeding techniques or through transgenic approaches. Mentoring of undergraduate and graduate students will be an important part of the project.
Project Methods
To test the hypotheses, we will use a combination of surgical, pharmacological, biochemical and molecular approaches. During Years 1 and 2, the major activities will include characterization of plant responses to herbivore secretions. In Years 3 through 5, quantification of regurgitation and characterization of herbivore effectors will be the major activity conducted. In last year we will complete full analyses of data and write publications. As part of this project, we enlist the assistance of Dr. Ketia Shumaker and undergraduate students from the University of Western Alabama (UWA), a minority serving institution in Livingston Alabama. During the second summer of the project, Dr. Shumaker will visit Penn State for one month to learn about the project and help conduct experiments. When she returns to UWA, she will teach students about the program's area priorities and begin recruiting. In summers of year 3 and 4, Ketia will select students (2 per/year) to spend 8 weeks in the laboratories of Drs. Felton and Luthe. Each student will be given a specific project to complete during that time which will require them to become familiar with RNA techniques, confocal microscopy, etc. They will be expected to attend joint weekly lab meetings between our two groups.

Progress 04/01/11 to 03/31/16

Outputs
Target Audience:Efforts: Research related to this project were presented at meetings of the Entomological Society of America, International Society of Chemical Ecology, and the Plant and Animal Genome Conference. Part of the research on this project was incorporated into lessons for the International Insect Chemical Ecology short course which was presented at Penn State University in June 2014. Students from more than 15 countries including the continents of South America and Africa attended the short course. This course was taught in Sweden in June 2015 Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project involved the training of one postdoc Joe Louis who now is a tenure track professor at the University of Nebraska. Ph. students who completed their programs include Donglan Tian who graduated and is senior scientist at Bayer and Seung Ho Chung who is a postdoc at Cornell University. Flor Acevedo and Loren Rivera are completing their degrees and will graduate in 2016/2017. Undergraduates included Morgan Elston who is employed as a lab technician and Morgan Brittany who is a senior now at Auburn University. How have the results been disseminated to communities of interest?Several of our papers received considerable press coverage which resulted in both popular press stories and radio interviews. We have made several papers including the PNAS and Plant Physiology papers open access and we have published several other papers in open access journals. Numerous invited talks were given in national and international arenas. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Essentially all of the goals were met and exceeded. We published a total of 17 papers from this effort and have at least a couple additional in preparation. Our findings from Objectives 1 and 2 have been addressed in prior reports and several publications contain the results of these studies. Essentially, we have not found that the strategies used by specialist vs. generalist herbivores differed, but most systems were idiosyncratic. To address Objective 3, we compared the effects of frass from caterpillars that feed in enclosed plant organs (such as fruits or whorls) where it remains in close contact with the herbivore damaged tissues with those of frass from caterpillars whose frass does not remain in contact with the damaged tissues. We measured frass-induced plant defenses in maize, rice, cabbage and tomato by chewing herbivores such as European corn borer (ECB), fall armyworm (FAW), cabbage looper (CL) and tomato fruit worm (TFW). We observed that caterpillar frass-induced plant defenses are specific to each host-herbivore system and the type of induced defense response depends on the composition of the frass deposited on the plant, the plant organ where it is deposited and the species of insect. Our results show that generally in host-herbivore systems where frass accumulates in inclosed host organs for extended periods of time such as TFW feeding in tomato fruits, frass from the herbivore triggers pathogen defenses. On the other hand, frass induced herbivore defenses in host-herbivore systems where the frass does not remain in contact with the wounded tissue (e.g. FAW feeding on rice leaves or TFW feeding on tomato leaves defenses). These results suggest that frass-induced defenses are specific to host-herbivore systems and are dependent on feeding habit of the herbivore on the plant, the host tissue on which the frass is deposited and the composition of the frass. These results have been submitted to the Journal of Chemical Ecology and the manuscript is currently being revised for resubmission to that journal. We also conducted a more detailed examination of the role of FAW frass on the elaboration of maize defense responses. To our surprise, the short term application of frass (<24 hr) induced an attenuated anti-herbivore defense, but longer term exposure (24 to 96 hr) shifted the defense pattern to anti-pathogen defenses that were accompanied by increases in salicylic acid levels, increased accumulation of transcripts for an antifungal gene (Pr5) and improved resistance to the maize pathogen Cochlioblus heterostropus. We also determined that the effect was caused by the protein fraction in the frass extract. The results of this study were published by Ray et al. (13) in the Journal of Chemical Ecology and featured on the cover of the September 2015 issue (http://link.springer.com/journal/10886/41/9/page/1). The article was also mentioned by Science (http://www.sciencemag.org/news/2015/09/how-caterpillars-use-their-feces-fool-corn) and several other science news outlets. To address Objective 4, we attempted to identify proteins from frass and/or oral secretions that enhance or suppress induced defenses in maize and tomato. As shown by Chung et al. 2013 (3, 4), the salivary secretions of the Colorado Potato Beetle contain bacteria that suppress tomato defenses. This article was extensively publicized and has been cited 89 times to date. In another study, Louis et al. (10) demonstrated that glucose oxidase (GOX) in the saliva of the ECB induces defenses in tomato, but not maize. It was shown that the glucose concentration in maize leaves were insufficient to support GOX activity and consequently some other ECB salivary factor(s) may be responsible for defense induction in maize. Salivary GOX from several different noctuids also plays a role in the activation herbivore defenses in tomato (15). Another active protein factor, an ATPase was found in Helicoverpa zea saliva (17). Unlike GOX, salivary ATPase suppresses jasmonic acid-induced defenses (17). In addition to the identification of active protein components in saliva, we have identified two components in FAW frass that suppress herbivore and activate the pathogen defense pathway in maize (12). The active components are two maize chitinases that are induced by FAW feeding, consumed by FAW and excreted back on the plant where they induce pathogen salicylic acid defense pathway. This paper (12) was featured on the cover the May 2016 issue of Plant Physiology (http://www.plantphysiol.org/content/171/1.cover-expansion). All in all, we believe that the objectives of the award have been achieved and that we have been very productive with 17 peer-reviewed publications resulting from the funding.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Louis J, Basu S, Varsani S, Castano-Duque L, Jiang V, Williams WP, Felton GW, Luthe DS: Ethylene contributes to maize insect resistance1-mediated maize defense against the phloem sap-sucking corn leaf aphid. Plant Physiology 2015, 169:313-324.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ray S, Gaffor I, Acevedo FE, Helms A, Chuang W-P, Tooker J, Felton GW, Luthe DS: Maize Plants Recognize Herbivore-Associated Cues from Caterpillar Frass. Journal of Chemical Ecology 2015, 41:781-792.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Dussourd DE, Peiffer M, Felton GW: Chew and spit: tree-feeding notodontid caterpillars anoint girdles with saliva. Arthropod-Plant Interactions 2016, 10:143-150.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ray S, Alves PC, Ahmad I, Gaffoor I, Acevedo FE, Peiffer M, Jin S, Han Y, Shakeel S, Felton GW: Turnabout is fair play: Herbivory-induced plant chitinases excreted in fall armyworm frass suppress herbivore defenses in maize. Plant Physiology 2016:pp. 01854.02015.


Progress 04/01/14 to 03/31/15

Outputs
Target Audience: Include scientists, graduate students, and undergraduate Efforts: Research related to this project were presented at meetings of the Entomological Society of America, International Society of Chemical Ecology, and the Plant and Animal Genome Conference. Part of the research on this project was incorporated into lessons for the International Insect Chemical Ecology short course which was presented at Penn State University in June 2014. Students from more than 15 countries including the continents of South America and Africa attended the short course. This course will again be taught in Sweden in June 2015. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Three students have worked on various components of this project: Syamajit Ray (Plant Biology); Flor Acevedo (Entomology), and Loren Rivera Vega (Entomology). Each has presented their findings at multiple meetings including the Entomologial Society of America, American Society of Plant Biology, and International Society of Chemical Ecology. How have the results been disseminated to communities of interest? The primary dissemenation has occurred through refereed journal articles and the professional society meetings previously mentioned. What do you plan to do during the next reporting period to accomplish the goals? Primary focus will be placed on finalizing experiments with recombinant proteins from frass including chitinase and insect intestinal mucin. Experiments with salivary HzPLC are being completed for maize and then journal article will be compelted and submitted.

Impacts
What was accomplished under these goals? Objectives 1 and 2 have largely been completed and have appeared in a series of journal articles over the past couple of years. Most of the effort in this past year has focused on Objectives 3 & 4. Pertaining to Objective 3:Fall armyworms(FAW, Spodoptera frugiperda), a major threat to maize (Zea mays), are voraciouseaters and deposit copious amounts of frass in the enclosed whorl tissue surroundingtheir feeding site where it remains for long periods of time. FAW frass is composed ofmolecules derived from the host plant, the insect itself and associated microbes andhence provides abundant cues that may alter plant defense responses. We observedthat proteins from FAW frass initially induced wound-responsive defense genes inmaize; however, a pathogenesis-related (pr) defense gene was induced as the timeafter application increased. Elicitation of pathogen defenses by frass proteins wascorrelated with increased herbivore performance and reduced fungal pathogenperformance over time. These responses differ from the typical plant response to oralsecretions of the FAW. The results pave the way for identification of proteinmolecule(s) from the excretion of an herbivore that elicits pathogen defense responseswhile attenuating herbivore defenses in plants. We have also completed studies in tomato with the tomato fruitworm Helicoverpa zea and shown that tomato fruit responds to caterpillar frass. Pertaining to Objective 4: We have identified two components of FAW frass which contribute to eliciting plant defenses. First, an ingested maize chitinase is stable within the gut of the insect and when excreted can elicit pathogen defenses. We have verified this with recombinant protein and treatment of plant tissue. This protein inhibits the growth of the insect when ingested, but in turn the insect deposits this on wounded tissues, which suppresses induced resistance. Another protein or component of insect intestinal mucin elicits antiherbivore defenses in maize and represents a herbivore-associated molecular pattern or HAMP. Few HAMPs have been identified and the mucin would represent one of the most evolutionary conserved HAMPs ever reported. We have identified another important effector or HAMP in caterpillar saliva: phospholipase C (PLC). Application of recombinant PLC from H. zea suppresses defenses in tomato, but may trigger defenses in maize. Salivary PLC appears to be widespread among caterpillar species.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Flor E. Acevedo, Loren J. Rivera-Vega, Seung Ho Chung, Swayamjit Ray, and Gary W. Felton. 2015. Cues from Chewing Insectsthe Intersection of DAMPs, HAMPs, MAMPs and Effectors. Current Opinion in Plant Biology (revision submitted).
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Swayamajit Ray, Iffa Gifford, Flor Acevedo, Anjel Helms Wen-Po Chuang, John Tooker, Gary W. Felton, and Dawn S. Luthe. 2015. Maize Plants Recognize Herbivore-Associated Cues from Caterpillar Frass. Journal of Chemical Ecology (accepted)
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: S. Paudel, E. G. Rajotte, G. W. Felton. 2014. Benefits and costs of tomato seed treatment with plant defense elicitors for insect resistance. Arthropod Plant Interactions 8:539-545


Progress 04/01/13 to 03/31/14

Outputs
Target Audience: The target audience was industry (Monsanto, Bayer) and academic entomologists, plant biologists, and ecologists. The PIs and participants presented talks to industry and professional societies such as the Entomological Society of America, International Organization of Biological Control, and the American Society of Plant Biologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Dr. Joe Louis completed his postdoc and began a tenure track faculty position at the University of Nebraska, Department of Entomology in 2013. He has published two papers thus far from his research at Penn State. Seung Ho Chung completed his Ph.D. in 2013 working on this project. He published 4 papers from his Ph.D. work including one as first author for PNAS. He is currently a postdoc in my laboratory but will be moving to Cornell University to work on a postdoc with Dr. Angela Douglas. Donglan Tian completed her Ph.D. in 2012 and is employed as a research scientist with Bayer Corp. in Davis, CA. Swayamjat Ray has completed hsi candidacy and comprehensive exams. His paper on frass induced defenses is in revision for the New Phytologist. He was an invited speaker at the 2013 Society of Plant Biologists annual meeting in Rohsoe Island. Loren Rivera Vega has completed her candidacy and comprehensive exams for her Ph.D. and has presented her research at the annual ESA meeting. She will be conducting a 60month study abroad experience in Germany working with Dr. Nicole van Dam on a related project with crucifers. Flor Edith Acevedo has also completed her candidacy and comprehensive exams for the Ph.D., has presented her research at the annual ESA meeting, and is a co-author on the FAW paper in MMPI. Morgan Elston (sophomore at the Univesity of West Alabama) did an internship in the Felton lab in summer 2013. She worked on beetles and oral secretions and presented her research at the annual undergraduate summer exhibition at Penn State Hershey Medical School. She also completed a two week course in molecular biology at Penn State University. Morgan was awarded a university SROP award and has returned to the Felton lab for summer 2014. Brittany Harry (sophomore at the University of West Alabama) did an internship in the Luthe lab in summer 2013. She worked on caterpillar saliva and the induction of defenses in maize. She also completed a two week course in molecular biology at Penn State University.Brittany was awarded a university SROP award and has returned to theLuthe lab for summer 2014. How have the results been disseminated to communities of interest? In addition to the publication outlets, our research received signficant press coverage for the 2013 Proceedings of National Academy article. This article has had more than 9150 downloads since Sept. 2013, was covered in the popular press, received four endorsements by Faculty 1000, and was a featured story in Nature ReviewsMicrobiology 11:740-741. doi:10.1038/nrmicro3142. The article by Christina Tobin Kåhrström was entitled Bacterial Decoy Skews Defenses. The MMPI article published in Molecular Plant-Microbe Interactions was a featured choice of the month by the Editor for May 2014: http://www.apsnet.org/publications/APSjournalsEditorsPicks/Pages/mpmi.aspx Drs. Luthe and Felton presented 3 invited talks at the annual meeting of the Entomological Society of America in 2013 and two invited talks at the IOBC Meeting on Induced Resistancein Avignon France in June 2013. Several students and postdoc (Joe Louis) also presented invited talks at a variety of national and international meetings. What do you plan to do during the next reporting period to accomplish the goals? The primary focus on the next reporting period will related to objectives three and four. We are expanding the frass studies to include different host plants and lepidopteran species. We will provide further identification of the active components from the Colorado potato beetle oral secretions and are attempting to identify components of FAW frass and saliva responsible for mediating induced defenses in maize.

Impacts
What was accomplished under these goals? Objectives 1 and 2: We have published several papers on the oral secretions of insects including the fall armyworm (FAW) (Spodoptera frugiperda) (Molecular Plant Microbe Interactions, Plant Signaling & Behavior), the European Corn Borer (ECB) (Ostrinia nubilalis) (New Phytologist), and the Colorado potato beetle (CPB)(Leptinotarsa decemlineata)(Proceedings of the National Academy of Science; Plant Signaling & Behavior). We have found that the saliva ofgeneralist caterpillars induce defenses in maize and tomato, whereas they also secreted minimal amounts of regurgitant. In general the caterpillars species Manduca sexta, Helicoverpa zea, Spodoptera eridania, Trichoplusia ni, ECB, and FAW secrete very small amounts of regurgitant during feeding. In fact, in most feeding bouts they avoid regurgitation altogether. Alternatively, beetles (CPB, western corn rootworm) typically secrete relatively large amounts of regurgitant that at least in the case of CPB, contains a plethora of bacteria. In the case of CPB, its oral secretions strongly supress induced defenses in tomato. Objective 3: Considerable progress has been made on this objective. We observed that proteins from FAW frass initially induced wound responsive defense genes in maize; however, pathogenesis-related (pr) defense genes were induced as the time after application increased. Elicitation of pathogen defenses by frass proteins was correlated with simultaneous increase of salicylic acid and suppression of jasmonic acid accumulation in leaves, as well as increased performance of herbivores on the plant and reduced fungal pathogen performance over time. These results pave the way for identification of a proteinaceous effector molecule from the excretion of an herbivore that mediates herbivore defense suppression in plants. The first manuscript from this work is currently being revised for the New Phytologist. Objective 4: We are characterizing the active oral components for several species of herbivores. We reported in our 2013 PNAS paper, that several specific bacteria (Pseudomonas, Enterobacter, Stenotrophomonas)in the oral secretions of the Colorado potato beetle are responsible for supressing induced defenses in tomato. We also identified flagellin protein from Pseudomonas as one of the suppressing factors from the bacteria. Following protein fractionation, we are in the process of idnetifying one or two additional bacterial proteins that may be responsible. We have also cloned and expressed a lepidopteranphospholipase C (PLC)in E. colithat may be an important factor in mediating induced defenses in tomato. PLC is found in the salivary glands of all caterpillars we have examined thus far. In order to faciltate identification of active components, we have completed salivary proteomes for Helicoverpa zea, Trichoplusia ni, and S. frugiperda. We have also completed an RNAseq analysis of salivary glands from T. ni.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Louis, J., Peiffer, M., Ray, S., Luthe, D.S., and Felton, G.W. (2013). Host-specific salivary elicitor (s) of European corn borer induce defenses in tomato and maize. New Phytologist 199, 66-73.
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Louis, J., Luthe, D.S., and Felton, G.W. (2013). Salivary signals of European corn borer induce indirect defenses in tomato. Plant Signaling & Behavior 8, e27318.
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Ho Chung, S., Rosa, C., Hoover, K., Luthe, D.S., and Felton, G.W. (2013). Colorado potato beetle manipulates plant defenses in local and systemic leaves. Plant Signaling & Behavior 8, e27592.
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Chung, S.H., Rosa, C., Scully, E.D., Peiffer, M., Tooker, J.F., Hoover, K., Luthe, D.S., and Felton, G.W. (2013). Herbivore exploits orally secreted bacteria to suppress plant defenses. Proceedings of the National Academy of Sciences 110, 15728-15733.
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Tian, D., Peiffer, M., De Moraes, C.M., and Felton, G.W. (2014). Roles of ethylene and jasmonic acid in systemic induced defense in tomato (Solanum lycopersicum) against Helicoverpa zea. Planta 239, 577-589.
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Chuang, W.-P., Ray, S., Acevedo, F.E., Peiffer, M., Felton, G., and Luthe, D.S. (2014). Herbivore cues from the fall armyworm (Spodoptera frugiperda) larvae trigger direct defenses in maize. Molecular Plant-Microbe Interactions 27, 461-470.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Felton, G.W., Chung, S.H., Gloria, M., Estrada Hernandez, J.L., Peiffer, M., and Tian, D. (2014). Herbivore Oral Secretions are the First Line of Protection Against Plant Induced Defences. Annual Plant Reviews volume 47: Insect-Plant Interactions, 37-76.


Progress 04/01/12 to 03/31/13

Outputs
Target Audience: Target audience includes entomologists, ecologists, and plant biologists studying plant-herbivore interactions. Also, from a more applied research aspect, plant breeders and IPM specialistcould be included in the potential target audience. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has involved a training of a postdoctoral scholar, Joe Louis, who has published one paper this year and has presented his work at the Entomological Society of America, University of Nebraska, and Monsanto Corporation. Several students have contributted to this project including Seung Ho Chung, Donglan Tian, Flor Acevado, Loren Rivera and Swayamjit Ray. Of special note is that Joe Louis and Seung Ho Chung have co-organized a symposium for the 2013 Annual Meeting of the Entomological Society of America. Also, Swayamjit Ray was selected as a mini-symposium speaker for the annual meeting of American Society of Plant Biologists. Addtionally, this summer two undergraduates from the University of West Alabama (a minority serving institution) are devloping research projects in our laboratories and are being provided additional classroom training in molecular biology. How have the results been disseminated to communities of interest? Yes, several papers have been published, talks have been given at annual meetings of the Entomolgical Society of America, American Society of Plant Biologists, Monsanto Coporation, and the IOBC. Additionally several gene sequences have been (and will be) depostied in NCBI and made available to the scientific community. What do you plan to do during the next reporting period to accomplish the goals? We are continuing to identify specific components of salivary secretions, frass,and regurgitant that are important mediators of plant defenses. We are making good progress on this front through a combined set of techniques including protein purification, proteomics, gene cloning, and protein expression.

Impacts
What was accomplished under these goals? We have made important progress in achieving many of the goals of the project. Under objective 1, we determined that caterpillar species infrequently secrete minute amounts of regurgitant during feeding bouts. we have exmined this in several caterpillar species including Helicoverpa zea, Manduca sexta, Spodoptera frugiperda, Spodoptera eridania, Trichoplusia ni, and Ostinia nubilalis. The host plant (tomato vs. maize) does not seem to have a major impact on regurgitation. Overall for most species, the regurgitant component of oral secretions has little impact on direct defenses in maize or in tomato for most species examined. In contrast, the saliva of caterpillars appears to play a dominant role in mediating induced direct defenses in tomato and maize. The saliva of H. zea Ostrinia nubilalis contains active elicitors of defenses in some host plants, of which glucose oxidase appears to play an important role in elicitation in tomato, but not in maize. By contrast the saliva of T. ni, S. eridania, and S. frugiperda appears to suppress direct defenses in tomato. Under objective 2, there does not appear to be a relationship between the amounts of regurgitant secreted and theirhost range. However, there is a stark contrast between coleopteran vs. lepidopteran herbivores. Coleopterans including Leptinotarsa decemlineata typically secrete copious amounts of regurgitant during every feeding bout. Under objective 3, we have determined that frass from several caterpillar species can elicit direct defenses in maize and in tomato. We have determined that several protein components in the frass are likely responsible for elicitation and are in the progress of dtermining the identity of these components. Under objective 4, we have determined that salivary glucose oxidase (GOX)is a major eliciting component in the saliva of H. zea and O. nublilalis when they feed on tomato. However, GOX is insufficient to explain the induction we observe in maize. We are in the progress of identifying additional salivary components that may contribute to elicitation. To date, we have cloned and sequenced a salivary phospholipase that may a significant role in eliciting plant defenses. This has been an overlooked factor due to the lability of the enzyme in saliva collections. Due to its lability and instability it is not identifiable in saliva collections unless they are collected fresh and analyzed immediately.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Louis, J., M. Peiffer, S. Ray, D.S. Luthe, and G.W. Felton, Host-specific salivary elicitor(s) of European corn borer induce defenses in tomato and maize. New Phytologist, 2013. Early View
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Kim, J. and G.W. Felton, Priming of antiherbivore defensive responses in plants. Insect Science, 2013. 20(3): p. 273-285.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Tian, D., M. Peiffer, E. Shoemaker, J. Tooker, E. Haubruge, F. Francis, D.S. Luthe, and G.W. Felton, Salivary glucose oxidase from caterpillars mediates the induction of rapid and delayed-induced defenses in the tomato plant. Plos One, 2012. 7(4): p. e36168.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Tian, D., J. Tooker, M. Peiffer, S.H. Chung, and G.W. Felton, Role of trichomes in defense against herbivores: comparison of herbivore response to woolly and hairless trichome mutants in tomato (Solanum lycopersicum). Planta, 2012. 236(4): p. 1053-1066.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Rasmann, S., M. De Vos, C.L. Casteel, D. Tian, R. Halitschke, J.Y. Sun, A.A. Agrawal, G.W. Felton, and G. Jander, Herbivory in the previous generation primes plants for enhanced insect resistance. Plant Physiology, 2012. 158: p. 854-863.


Progress 04/01/11 to 03/31/12

Outputs
OUTPUTS: Research from this project has been disseminated in seminars at Monsanto, University of California, Davis, and the Entomological Society of America Annual Meeting. The research has also been presented and discussed at the Annual Insect Chemical Ecology Workshop in Sweden. Research was presented and discussed in 2 lectures for a graduate course in Entomology (Frontiers in Insect Science) at Penn State. PARTICIPANTS: Dr. Joe Louis has been working on the project and has presented findings in local workshops at Penn State, attended the Annual Meeting of the Entomological Society of America and wrote a postdoctoral grant for LSB. Micehlle Peiffer has presented research at the Entomological Society of America meeting. Seung Ho Chung will present research at the upcoming Entomological Society Meeting. Ray Swayamjit will present his findings at the American Society of Plant Biologists meeting. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We investigated if tomato responds specifically to two specialists: Colorado potato beetle (CPB) and tobacco hornworm (THW). Tomato did not induce different defense genes in response to CPB or THW but accumulated more transcripts for some genes after damage by THW feeding compared to damage by CPB feeding. Application of oral secretion (OS) from THW to wounded tomato plants increased transcripts compared to controls, but oral secretion from CPB decreased defense transcripts. These data suggest distinct quantitative responses to two different specialist herbivores. Herbivore OS are responsible for these species-specific responses. We also found that saliva from several generalist caterpillars including Helicoverpa zea, Spodoptera exigua, Heliothis virescens and Ostrinia nubilalis induce defense genes such as proteinase inhibitor 2 (Pin2) in tomato. Transcripts encoding genes associated with the JA pathway were not affected by saliva. Saliva from the specialist THW did not induce defenses. In a separate study, the effect of fall armyworm (Spodoptera frugiperda) OS on the maize (Zea mays) defense response was examined. Analyses indicated that only minute amounts of OS were deposited on the maize leaf during feeding and that is application to leaves failed to induce the expression of herbivore defenses. Saliva induced defense genes including those from the jasmonate pathway and those involved in direct defenses. Similar results indicate that saliva from European corn borer also elicits maize defenses. We found that frass proteins from S. frugiperda significantly elicit jasmonate-regulated defenses in maize. We extracted frass protein from maize fed larvae in PBS and then used a sterile 0.24 micron filter to obtain a sterile filtrate. The frass protein significantly enhanced the expression of defense genes and we have determined that major activity is present in the >10Kd protein fraction. Further work is underway to identify the protein components. We have identied the active components of oral secretions from CPB. Quite unexpectedly we discovered that the source of the active component was microbial. We demonstrate that oral bacteria secreted by larvae of Colorado potato beetles (CPBs) suppress plant defenses by negative crosstalk between jasmonic acid and salicylic acid signaling pathways. Three microbial symbionts, the genera Stenotrophomonas, Pseudomonas, and Enterobacter, isolated from OS of the larvae are responsible for defense suppression. CPBs benefit from down-regulated plant defenses through enhanced larval growth. Our findings indicate that the herbivore exploits oral bacteria as a decoy and thus plants incorrectly perceive the threat as microbial defenses of its host. We do not have evidence that active components of caterpillar secretions are of microbial origin. We have identified glucose oxidase as the active component of saliva of the corn earworm, beet armyworm, fall armyworm and European corn borer that elicits defenses in tomato. In contrast, saliva from the fall armyworm and European corn borer also elicit defenses in maize, but the active component is not glucose oxidase but is likely another protein.

Publications

  • Tian, D., M. Peiffer, E. Shoemaker, J. Tooker, E. Haubruge, F. Francis, D. S. Luthe, and G. W. Felton. 2012. Salivary glucose oxidase from caterpillars mediates the induction of rapid and delayed-induced defenses in the tomato plant. Plos One volume 7, issue 4, p. e36168.
  • Chung, S. H. and G. W. Felton. 2011. Specificity of induced resistance in tomato against specialist Lepidopteran and Coleopteran species. Journal of Chemical Ecology 37(4):378-386.
  • Kim, J., H. Quaghebeur, and G. W. Felton. 2011. Reiterative and interruptive signaling in induced plant resistance to chewing insects. Phytochemistry 72(13):1624-1634.
  • Leroy, P. D., A. Sabri, S. Heuskin, P. Thonart, G. Lognay, F. J. Verheggen, F. Francis, Y. Brostaux, G. W. Felton, and E. Haubruge. 2011. Microorganisms from aphid honeydew attract and enhance the efficacy of natural enemies. Nat. Commun. 2, Article number:348.