Progress 10/01/02 to 09/30/08
Outputs
OUTPUTS: Our analysis of genes expressed in salivary glands of the pea aphid and encoding features typical of secreted proteins has allowed us to create an initial (and certainly incomplete) hypothetical salivary-gland secretome. The term "hypothetical" applies since direct experimental proof will be required to identify each protein, individually and directly, as a component of pea aphid saliva. But one has to have a hypothetical list of probable members before such direct evidence can be obtained. The list at this point contains oxidation-reduction enzymes and hydrolytic enzymes, both types having been predicted by earlier biochemical studies from other laboratories. Among the methods we have used to investigate Protein C002 is RNAi-based transcript knockdown. The method that worked best for us, in what was the first application of transcript-knockdown to any aphid, was injection of short double-stranded RNA molecules into the abdomen of adult aphids, with the result that the aphids injected with such RNA molecules targeted at the transcript encoding Protein C002 died very prematurely when placed on fava bean leaves. This will continue to be a powerful method in investigating the importance of other proteins of the salivary-gland secretome. PARTICIPANTS: Gerald R. Reeck - principle investigator; John C. Reese - growth of aphid colonies, electrophysiology experiments; Navdeep S. Mutti - molecular biology experiments; Joe Louis - electrophysiology experiments; Loretta K. Pappan - screening of cDNA libraries; Kirk Pappan - construction of cDNA libraries; Khurshida Begum - in situ hybridization, immunohistochemistry Ming-Shun Chen - construction of cDNA libraries; Yoonseong Park - in situ hybridization, immunohistochemistry; Neal Dittmer - sequencing of cDNA libraries; Jeremy Marshall - statistical analysis of data, protein evolution analysis; Doina Caragea - analysis of ESTs from cDNA libraries; Owain Edwards (CSIRO, Australia) - experimental design and consultation TARGET AUDIENCES: Other research investigaters and scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This is basic research. Therefore the outcomes are mainly publications (see below) and the impacts are on future research both in my laboratory and world wide. The work we have done will identify proteins of saliva for not only us to study but other investigators around the country and world. We have been contacted for information about Protein C002, for instance, by such investigators. But this protein is simply the first of many that we are identifying and studying in aphid saliva, so the impact on research in other laboratories should be continuing and substantial. In addition, the approach to transcript knockdown should prove valuable to other investigators. Indeed, one paper has already appeared using the basic approach that we adopted in our work.
Publications
- Navdeep S. Mutti, Yoonseong Park, John C. Reese and Gerald R. Reeck (2006) RNAi knockdown of a salivary transcript leading to lethality in the pea aphid, Acyrthosiphon pisum Journal of Insect Science. Online: 6.38.
- Navdeep S. Mutti, Joe Louis, Loretta K. Pappan, Kirk Pappan, Khurshida Begum, Ming-Shun Chen, Yoonseong Park, Neal Dittmer, Jeremy Marshall, John C. Reese, and Gerald R. Reeck (2008) A protein from the salivary glands of the pea aphid, Acyrthosiphon pisum, is essential in feeding on a host plant. PNAS. 105: 9965-9969
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We are working to identify proteins and enzymes of aphid saliva (particulary saliva of the model aphid, the pea aphid, Acyrthosiphon pisum), to assess the role or importance of individual salivary proteins and enzymes in the feeding of aphids on plant tissue. Using transcript knockdown, we have established that a protein we designate as C002 is essential for the pea aphid to feed on plant tissue, but not on artificial diet. Thus, this protein appears to be involved in some way in overcoming plant defense or natural barriers. We have now expressed the protein in E. coli and are beginning structural studies on it, in hopes that the three-dimensional structure might give insights into the protein's function, particularly its interaction with other compounds or proteins, presumably of plant origin. We have in addition discovered and studied the presence of the transcript for laccase-1 in the secretory cells of the salivary gland of the pea aphid. Interestingly, it is a different
subset of secretory cells that contain this transcript than the set of secretory cells that contains the C002 transcript, thus indicating that different secretory cells in the pea aphid salivary gland have different assignments in terms of the proteins that they synthesize and secrete. The function of the laccase is not known, and we are beginning a round of transcript-knockdown experiments to begin investigating this.
PARTICIPANTS: PI: Gerald Reeck; GRA: Navdeep S. Mutti
TARGET AUDIENCES: the scientific community, especially aphidologists
Impacts
This work should contribute to the development of methods for reducing reliance on pesticides in controlling aphid infestations of major crop plants such as wheat, sorghum and soybeans.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
We continue to focus on the development of transcript-knockdown using RNAi and on exploring the importance of a particular gene (c002) in the pea aphid (Acyrthosiphon pisum), and of its salivary transcript and its protein product. Adult parthenogenetic pea aphids are injected in the abdomen with siRNA that either matches the sequence of c002 or control (non-insect) gene. The most important finding in the last year is that although c002 transcript-knockdown greatly reduces lifespan on host-plant leaves, it has no effect on lifespan on an artificial liquid diet. We conclude that the C002 protein is not required for feeding, per se, but is instead needed for a productive interaction with the host plant. Electrical penetration graph analysis indicates highly aberrant and unsuccessful feeding on plant leaves by the c002-knockdown aphids, consistent with the RNAi-based evidence that C002 protein is important in feeding on plant tissue.
Impacts
This work should contribute to developing methods for reducing reliance on pesticides in controlling aphid infestations of major crop plants, such as wheat, sorghum and soybeans.
Publications
- Mutti N.S., Park Y., Reese J.C., Reeck G.R. 2006. RNAi knockdown of a salivary transcript leading to lethality in the pea aphid, Acyrthosiphon pisum. 7pp. Journal of Insect Science 6:38, available online: insectscience.org/6.38
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Progress 01/01/05 to 12/31/05
Outputs
We have developed RNA-interference (RNAi) in the pea aphid and used it to assess the importance of a transcript we call c002, which we previously obtained from a salivary gland cDNA library. We synthesize dsRNA from cloned c002 and inject the dsRNA into the body of adult aphids. As control we inject dsRNA that is unrelated to the aphid genome. We find profound effects of dsC002 injections, when the injected insects are placed on fava bean leaves. The effects include greatly reduced life span (an average of 3-4 days, compared to 10-12 days for insects given the control injection). Tentatively, we conclude that c002 protein is important in the pea aphid's attack on plant tissue.
Impacts
This is a long-term project to understand the biochemistry (especially enzyme action) in the attack of aphids on plant tissue. Since numerous aphids are pests of major crops, the work will contribute to developing better ways to control aphids and lessen aphid damage.
Publications
- Shen, Z.C., Pappan, K., Mutti, N.S., He, Q.J., Denton, M., Zhang, Y., Kanost, M., Reese, J.C. and Reeck, G.R. 2005. Pectinmethylesterase from the rice weevil, Sitophilus oryzae: cDNA isolation and sequencing, genetic origin, and expression of the recombinant enzyme. J. of Insect Sci. online publication: http://insectsci.org/5.21.
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Progress 01/01/04 to 12/31/04
Outputs
We have obtained full length cDNA clones for the most abundant transcript in our cDNA library from salivary glands. The protein encoded by the open reading frame consists of 199 amino acid residues, 17 of which are predicted to be an N-terminal signal peptide. The protein exists in two forms, apparently allelic variants, since the gene itself appears to be single-copy, based on Southern analysis. The cDNA clone encoding this enzyme was initially selected as having very weak pectinmethylesterase activity, but the sequence does not resemble that of known pectinmethylesterases. We are in the process of expressing the protein in cultured insect cells in order to begin assaying against possible polysaccharide structures.
Impacts
Proteins and enzymes of aphid saliva are potential targets for genetically engineered resistance. The fact that aphids are not affected by Bt toxin, means that such targets should be sought. In the long term, protein factors (inhibitors for instance) that interfere with the target enzymes could be effective in retarding aphid attack of plant tissue.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
We are working with cDNAs from libraries we have constructed from RNA from salivary glands and gut of the pea aphid (Acrythosiphon pisum). In each case, we have sequenced several hundred randomly selected cDNA clones. A finding of particular interest has been clones encoding cysteine proteinases. We have identified three separate sequences at this point and they occur in both salivary and gut libraries. In each case, this is somewhat unexpected. Presumably in salivary secretions, a cysteine proteinase would be involved in attacking plant tissue. In the gut, one would expect that such an enzyme would be a digestive enzyme, but this is a somewhat novel view since aphids have been considered to rely on amino acids from phloem tissue and therefore not have to rely on proteolysis. We are in the process of screening the library to obtain full length clones of the cysteine proteinases as well as expressing at least one of them in cultured Drosophila cells, in order to study
its enzymatic properties, obtain antibodies against it and search for inhibitors of it.
Impacts
Enzymes, including hydrolyases, including proteinases, are likely involved in the attack of aphids on plant tissue. They are, in that sense, potential targets for genetically engineering increased resistance in plants (by transforming plants with genes encoding inhibitors of such enzymes). This is the long term goal of this project.
Publications
- Fabrick, J. Behnke, C., Czapla, T., Bala, K. Rao, G., Kramer, K.J., and Reeck, G.R. 2003. Effects of a Potato Cysteine Proteinase Inhibitor on Midgut Proteolytic Enzyme Activity and Growth of the Southern Corn Rootworm, Diabrotica undecimpunctata (Coleoptera: Chrysomelidae). Insect Biochemistry and Molecular Biology 32: 405-415.
- Shen, Z., Denton, M., Mutti, N., Pappan, K., Kanost, M.R., Reese, J.C., and Reeck, G.R. 2003. Polygalacturonase from Sitophilus oryzae: Possible horizontal transfer of a pectinase gene from fungi to weevils. 9pp. Journal of Insect Science, 3:24, Available online: http://www.insectscience.org/3.24
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Progress 10/01/02 to 12/31/02
Outputs
We have created a new cDNA library using RNA from pea aphid (Acrythosiphon pisum)salivary glands dissected with an improved method. We will sequence several hundred clones, randomly selected from this library, for comparison to our previous library and identification of clones for further study. Analysis of clones from our previous salivary and gut libraries has revealed a surprisingly large fraction of clones that do not have clearly homologs in national databases. One particularly interesting such clone is the most heavily represented clone in our salivary cDNA library. Interestingly, it occurs as well in the gut library and contains a clear secretion signal. We continue to attempt to expression this protein in soluble form in E. coli mostly by altering the conditions of growth and expression. Several other clones, including ones encoding (putative) carbonic anhydrase, are common to the salivary and gut libraries. A phagemid-vector cDNA library created from RNA of
larval Hessian flies (Mayetiola destructor) has been created. Several hundred clones will be sequenced and analyzed by comparison of the sequences to national databases.
Impacts
Enzymes secreted by the salivary glands of the pea aphid and the Hessian fly are believed to be important in the attack of these insects on plant tissue. As such, the enzymes are potential targets for insect control by genetic engineering, an approach that could reduce reliance on chemical pesticides.
Publications
- No publications reported this period
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