Progress 12/01/04 to 11/20/08
Outputs
OUTPUTS: Activities: This proposal consisted of laboratory research on the effects on aphids of Pea enation virus coat protein fused to various toxins. This activity generated new knowledge and trained three postdocs. Events: In the past year, results were presented at the 41st Annual Meeting of the Society for Invertebrate Pathology, Warwick, UK, August 2008; the Second Iowa State University Aphid Research Symposium, Ames, IA, January 2009; and the 8th International Symposium on Aphids, Catania, Italy, June 2009. Dissemination: In addition to presenting the new knowledge at the above meetings, two papers were published and another one will be submitted soon. This work has also been presented to the Iowa Soybean Association and the Iowa State University Plant Sciences Institute which now jointly fund the PDs to perform research on aphid viruses and the soybean aphid. Thus knowledge from this work is being translated to applied research by a commodity group. PARTICIPANTS: W. Allen Miller, PD; Bryony C. Bonning, co-PD; Sivakumar Swaminathan, postdoc; Zhaohui Wang, postdoc; Liljana Georgievska, postdoc. Collaborator: Glenn King, University of Queensland. TARGET AUDIENCES: Scientists including virologists and entomologists, agricultural commodity groups, agricultural biotechnology companies. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our goal has been to test the possibility of using the coat protein (CP) and parts of the read through domain (RTD) of Pea enation mosaic virus (PEMV) as a delivery vehicle of intrahemocoelic aphicidal proteins to kill aphids. We showed that expression of a fusion protein (CP-P-GFP) consisting of the CP, the proline-rich (P) amino end of the RTD (immediately downstream of the CP), and green fluorescent protein (GFP) in a baculovirus vector allowed the GFP to be delivered to the interior of the pea aphid (Acyrthosiphon pisum) feeding on this protein. This uptake of CP-P-GFP from the gut into the hemocoel required the presence of the CP-P domain at its amino terminus. Previously, transient expression in Nicotiana benthamiana of the fusion of CP-P to either the scorpion AaIT toxin, or a membrane degrading enzyme (Scath-L), did not result in toxicity to the aphid although in planta expression could not be determined. Change in knowledge: We showed that the spider venom peptide alone, Hv1a, is not toxic when fed to aphids. However, membrane feeding assays on the CP-P-Hv1a fusion protein, and transient expression of CP-P-Hv1a in plant leaves caused greater aphid mortality than did the negative control proteins. The negative control, CP-P-Hv1am contained two amino acid changes in the pharmacophore of Hv1a known to ablate its toxic activity. Three times as many pea aphids feeding on a sucrose solution containing CP-P-Hv1a died, compared to those feeding on nonfunctional CP-P-Hv1am or plain sucrose. CP-P-Hv1a was expressed transiently in N. benthamiana leaves by Agroinoculation. Mortality of Myzus persicae aphids fed on leaves expressing CP-P-Hv1a was 76%, which is significantly higher than the 35-50% mortality of aphids fed on leaves expressing various negative controls (p<0.05, ANOVA). The background mortality is relatively high on negative controls because N. benthamiana is naturally toxic to most aphids, including M. persicae (a major pest). (Pea aphids do not feed on N. benthamiana.) Change in actions: Funding expired at this point. We are in a good position to obtain additional funds which we will seek to determine mortality of CP-P-Hv1a when expressed uniformly in stably transformed plants, using a species such as Arabidopsis that is not toxic to M. persicae or other aphid species. Aim I originally involved expressing CP-toxin fusions from replicating PEMV RNA. While investigating sequences required for gene expression from PEMV RNA2 we discovered a new kind of RNA sequence in the 3' untranslated region that confers efficient cap-independent translation of the viral RNA. The element was mapped, essential bases identified, and the secondary structure determined. Its mechanism is being studied as part of a separately funded project in Miller's lab. This knowledge could be valuable for high level expression of any protein from PEMV RNA, including anti-aphid proteins.
Publications
- Sivakumar S, Wang Z, Harrison RL, Liu S, Miller WA, Bonning BC (2009) Baculovirus-expressed virus-like particles of Pea enation mosaic virus vary in size and encapsidate baculovirus mRNAs. Virus Research 139, 54-63.
- Liu S, Sivakumar S, Wang Z, Bonning BC, Miller WA (2009) The readthrough domain of pea enation mosaic virus coat protein is not essential for virus stability in the hemolymph of the pea aphid. Arch Virol 164, 469-479.
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Progress 12/01/06 to 11/30/07
Outputs
OUTPUTS: Activities: This proposal consisted of laboratory research on the effects on aphids of Pea enation virus coat protein fused to various toxins. This activity generated new knowledge and trained two postdocs. Events: The results were presented at the following conferences. Entomological Society of America meeting, Indianapolis, December 2006; The First Iowa State University Aphid Research Symposium, Ames, IA, January 2007; Annual Meeting of the Iowa Soybean Association, Ames, IA February 2007; RNA Society Meeting, Madison, June 2007; American Society for Virology Meeting, Corvallis, OR, July 2007; American Phytopathological Society Meeting, San Diego, August 2007. Services: Some of the knowledge was presented by Miller in consulting with an agricultural biotechnology company. Dissemination: This knowledge gained here was presented to (i) the Iowa Soybean Association which, as a result, funded the PDs to perform research on the soybean aphid and aphid viruses; (ii) a major ag
biotech company which, as a result, is likely to fund both PDs to perform research on aphid viruses as aphid control agents and, in a separate project, fund PD Miller to investigate the translation control elements of viruses including PEMV, as tools for enhancing plant virus expression vectors; (iii) the Iowa State University Plant Sciences Institute which is now funding both PDs to perform research on the soybean aphid. Thus this knowledge from this work is being translated to applied research by a commodity group and a company.
PARTICIPANTS: Individuals: W. Allen Miller, PD; Bryony C. Bonning, co-PD; Sivakumar Swaminathan, postdoc; Zhaohui Wang, postdoc. Collaborator: Glenn King, University of Queensland.
TARGET AUDIENCES: Scientists including virologists and entomologists, agricultural commodity groups, agricultural biotechnology companies.
Impacts
Our goal is to test the possibility of using the coat protein (CP) and possibly parts of the read through domain (RTD) of Pea enation mosaic virus (PEMV) as a delivery vehicle of intrahemocoelic aphicidal proteins to kill aphids. In previous years, we successfully expressed a fusion of the CP, the proline-rich amino end of the RTD, and GFP (CP-P-GFP) in a baculovirus vector and showed that indeed the CP-P-GFP was delivered to the interior of the aphid owing to the presence of the CP-P domain. Change in knowledge: subesequent experiments using CP-P fused to either AaIT toxin (from scorpions), or a membrane-degrading enzyme, Scath-L, showed no obvious aphid toxicity. As a control we found that the pure peptides, without fusions, were toxic upon injection in aphids. This had not been reported previously. Efforts to purify AaIT and ScathL in an active form using E. coli (Origami2) and/or baculovirus expression were unsuccessful due to structural considerations (AaIT) or pH
sensitivity (ScathL). Change in actions: Thus, we switched to a different toxin, the insect-specific omega-atracotoxin Hv1a from funnel web spiders. This small (37 aa) peptide is highly stable and toxic to many insects. We obtained a clone of the Hv1a gene and antisera from Dr. Glenn King, University of Queensland. CP-P-Hv1a fusion protein was expressed in E. coli and purified. Feeding assays were inconclusive. The same construct was introduced into an Agrobacterium expression vector or into the PEMV expression vector and expressed in Nicotiana benthamiana plants by agroinfiltration or agroinfection, respectively. Unfortunately, upon feeding of aphids, no increase in aphid death above background was observed. We know that the agroinfiltration and agroinfection systems work in our hands because using the same vectors and methods we expressed GFP to easily detectable levels in plants. It is possible that Agrobacterium-expressed CP-P-Hv1a did not kill aphids because it was not uniformly
expressed in the plants, or the expression level is simply not high enough. We expect that Hv1a folds correctly despite being fused to another protein because GFP was functional in the same fusion context, and Hv1a is highly stable, structured and self-crosslinked via disulfide bonds, and has been shown previously to tolerate fusions. In a second line of research, we identified sequences in PEMV RNA2 that control gene expression. This is important because Aim I involves expressing CP-toxin fusions from replicating PEMV RNA. Change in knowledge: As a result, we discovered a sequence in the 3' untranslated region (3' UTR) that allows highly efficient cap-independent translation (protein synthesis) by the viral RNA. The element was mapped, essential bases identified, and the secondary structure determined. This knowledge could be valuable for high level expression of any protein from PEMV, not just anti-aphid proteins.
Publications
- US Patent No.7,312,080, "Plant Resistance to Insect Pests Mediated by Viral Proteins" issued December 25, 2007. Inventors: Dr. Wyatt Allen Miller and Dr. Bryony C. Bonning. http://patft.uspto.gov/netacgi/nph-Parser ?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm &r=1&f= G&l=50&s1=7312080.PN.&OS=PN/7312080&RS=PN/7312080
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Progress 12/01/05 to 12/01/06
Outputs
Our objective is to test the possibility of using the coat protein (CP) of Pea enation mosaic virus (PEMV) as a delivery vehicle of intrahemocoelic aphicidal proteins such as the insect basement membrane-degrading protease, ScathL, and the insect specific neurotoxin, AaIT to kill aphids. First we expressed the CP in insect cell line Sf21 (by Bac-to-Bac Baculovirus Expression System) and found that it formed irregular virus like particles (VLPs) 13-30 nm in diameter. Further we found by northern blot hybridization that the mRNA transcript of CP was packaged inside the VLPs. Membrane feeding assays were carried with the purified CP VLPs and the entry of VLP in the hemocoel was detected by RT-PCR detection of CP mRNA using the total RNA isolated from the hemolymph. This result shows that the VLP could undergo transcytosis into aphid hemocoel without the other structural protein of PEMV, the read through domain (RTD). This supports our hypothesis to use the CP as a
vehicle to deliver the intrahemocoelic aphicidal fusion proteins in the hemocoel. To visualize delivery of the fusion proteins to the aphid hemolymph, we fused enhanced green fluorescent protein (EGFP) to the CP. Baculovirus expression of CP-EGFP in the insect cells yielded insoluble aggregates and inclusion bodies rather than the expected VLPs. In another construct, we included the viral sequence immediately downstream of the CP comprising a proline-rich (P) domain of the RTD. Presence of this P domain between CP and EGFP domains resulted in soluble CP-P-EGFP protein without VLPs or aggregation. Membrane feeding assays were carried out and fluorescence microscopic observation revealed that the non-VLP form of CP-P-EGFP could enter the aphid hemocoel. The CP-P-EGFP fluorescent protein accumulated in the pericardial cells in the aphid. This requires passage through the hemocoel. In contrast, the EGFP protein alone did not enter the hemocoel upon feeding. These results demonstrate that
the non-VLP form of CP can transport large fused peptides such as EGFP into the aphid hemocoel by crossing the gut epithelial cells. These results support the possibility that the CP may work as a carrier to deliver intrahemocoelic aphicidal proteins like ScathL, AaIT, or others. Cloning and baculovirus expression of CP-intrahemocoelic insecticidal fusion proteins and testing their utility to kill the aphids is in progress. In a different approach, we made constructs for expression in plants with CP-P-EGFP and CP-EGFP under the 35S promoter. After agroinfiltration of Nicotiana benthamiana, both CP-GFP and CP-P-GFP were expressed, but the CP-P-GFP expression level was higher than CP-GFP as detected by UV light examination and western blot. We then substituted GFP with either ScathL or AaIT to express CP-P-ScathL and CP-P-AaIT fusions by agroinfiltration. Western blots confirmed the expression of both fusion proteins in plants. However, initial aphid feeding experiments on these plants
did not revealed significant aphicidal activity. Experiments are underway to optimize expression and feeding conditions and to test other fusion constructs.
Impacts
Ultimately this approach may be used for delivery of insect toxins into the aphid hemocoel for a highly target-specific, efficacious means of reducing aphid populations. We will also gain knowledge about gene expression and replication mechanisms of RNA viruses, which may be useful to control or exploit viruses for many purposes. Use of these tools to construct transgenic, aphid resistant crops will reduce pesticide use thus benefit the environment; it will also reduce input expenses for the farmer.
Publications
- Liu S, Bonning BC, Miller WA (2006) A simple wax-embedding method for isolation of hemolymph for detection of luteoviruses in the hemocoel. Journal of Virological Methods 132, 174-180.
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Progress 12/01/04 to 11/30/05
Outputs
One aim is to fuse green fluorescent protein (GFP) to the coat protein (CP) of Pea enation mosaic virus (PEMV) to allow us to literally see if PEMV can deliver fusion proteins to the aphid hemolymph. We made the crucial observation to support the feasibility of this approach that the natural PEMV mutant (delta RNA1- PEMV) without its full length RTD is stable in the pea aphid hemocoel. This result indicates that the RTD is not required for virus movement into the hemocoel. We attempted to fuse GFP to the read-through domain (RTD) of the CP. Translation of the RTD requires programmed read-through of the CP open reading frame (ORF) stop codon. Many constructs were made with GFP fused to CP and portions of the RTD. In some cases we were able to detect read-through translation of the RTD-GFP fusion by in vitro translation of a transcript representing the viral subgenomic RNA from which these genes are expressed. However, the signals required for read-through are in the
RTD ORF, so they had been replaced in many of the GFP fusion constructs. To allow read-through in these cases, we inserted the short, well-characterized read-through sequence of Tobacco mosaic virus (TMV) at the CP stop codon. Surprisingly no readthrough was detected. Thus, the TMV read-through signal may not be as simple as has been reported. We will perform studies to map the PEMV sequences required for read-through. Plants were inoculated mechanically with various constructs but GFP expression was not detected, even when virus infection was evident based on disease symptoms and viral RNA accumulation (northern blots). Interestingly, the engineered PEMV, and even wild type aphid-transmissible version of PEMV, recombined rapidly upon passaging in plants. It is difficult to maintain aphid transmissibility of PEMV as much of the RTD is deleted by recombination during mechanical transmission. A hot spot for recombination was identified between two GC-rich sites in the RTD. This will be
mutated to prevent undesired deletion of engineered sequences, and to investigate the mechanism of recombination, which should shed light on RNA virus evolution in general, and specifically on the origins of aphid transmissibility. A second aim was to express PEMV CP-GFP fusions at high levels in insect cells using s baculovirus vector. Expression of recombinant protein of the correct size, and fluorescence of GFP when fused to CP in baculovirus-infected insect cells was confirmed. The fusion protein CP-GFP was expressed in the nucleus in aggregates, rather than as virus-like particles (VLP). For production of VLPs, insect cells have been co-infected with virus expressing CP and CP-GFP fusions simultaneously in different ratios, and will be examined by transmission electron microscopy. To facilitate purification of VLPs, a His tag was added to the amino-terminus of CP and CP-GFP. Constructs were also made with a proline-rich region between CP and GFP, which occurs naturally between CP
and the body of the RTD ORF. Bioassays of purified recombinant proteins will address whether VLP assembly is required for uptake of the fusion proteins into the aphid hemocoel.
Impacts
Ultimately this approach may be used for delivery of insect toxins into the aphid hemocoel for a highly target-specific, efficacious means of reducing aphid populations. We will also gain knowledge about gene expression and replication mechanisms of RNA viruses, which may be useful to control or exploit viruses for many purposes. Use of these tools to construct transgenic, aphid resistant crops will reduce pesticide use thus benefit the environment; it will also reduce input expenses for the farmer.
Publications
- No publications reported this period
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