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Research Projects

1. Using activation tagging to identify genes conferring resistance to feeding insects


Overview of activation tagging procedure

Poplars deploy an array of defense strategies against herbivores that can be grouped as chemical and physical defenses, direct and indirect defenses, constitutive and induced defenses, as well as local and systemic defenses (Philippe and Bohlmann, 2007). In herbaceous plant-herbivore defense systems, constitutive and induced defense mechanisms appear to be tightly regulated, permitting economy when active defense is not required, and presenting a shifting defense profile when herbivores are present (Kessler and Baldwin, 2002). It is therefore a priority to identify the gene regulatory networks and other feeding insect-induced changes that regulate defense responses. Recent work in multiple laboratories using transcriptomics and proteomics tools has identified extensive inventories of insect-induced poplar genes (Christopher et al., 2004; Ralph et al., 2006; Lawrence et al., 2006; Major and Constabel, 2006; Miranda et al., 2007; Ralph et al., 2008). However, the challenge remains to link these insect-induced changes in gene expression with altered resistance or tolerance to insect attack in poplar trees. Our laboratory is working to address this question through the analysis of activation tagged genetic mutants.

Until recently, large-scale mutant populations, which have served as critical resources for the discovery of genes controlling traits of agronomic interest, were not available for tree species. This impediment has recently been overcome through the use of activation tagging. With this method, one or more endogenous genes are "turned on" by the random insertion of an enhancer sequence near endogenous promoters such that the pattern of activated gene expression is the same as in wild-type plants, but the level of expression is enhanced. These gain-of-function mutants display different phenotypes from the wild-type lines and have proven effective for the discovery of genes controlling diverse plant functions including growth and development (Busov et al., 2003; Harrison et al., 2007). Using this approach, a population of 1,000+ independent gain-of-function poplar lines has recently been created by Dr. Sharon Regan at Queen's University in Kingston, Ontario, Canada that contain the cauliflower mosaic virus 35S promoter (CaMV) within the pSKI074 activation tagging T-DNA. This is the largest population of activation tagged poplar trees created to date. A screen of this population revealed that 2.4% (24 mutants) of poplar lines have a visible developmental abnormality (e.g., alterations in leaf and stem structure as well as overall structure; Harrison et al., 2007). Most of the phenotypes observed represent new phenotypes not previously identified in poplar, and in some cases, not in any other plant species. The broad range of developmental mutants identified in this pilot screen of the population confirms the tremendous value of this resource.

In collaboration with Dr. Regan, our lab has challenged this population with defoliating insect herbivores. We have used a combination of "choice" feeding and "no-choice" development bioassays to identify candidate herbivore resistant or sensitive mutants. This strategy has revealed a number of interesting mutants, for which we are currently working to 1) map the T-DNA insertion sites, 2) identify the nearby "activated" genes, and 3) conduct additionl molecular biochemistry experiments to acertain a role for the activated genes in mediating resistance to feeding insects.

We currently have several open positions for interested graduate students to work on this project.

"Choice" feeding assay showing insect feeding preference between two activation tagged trees Calculating leaf disk area using modified version of the GIMP software

 

References:

1. Philippe RN and Bohlmann J (2007) Poplar defense against insect herbivores. Canadian J. of Botany 85:1111-1126.

2. Kessler A and Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular analysis. Annual Review Plant Biology 53:299-328.

3. Christopher ME, Miranda M, Major IT and Constabel CP (2004) Gene expression profiling of systemically wound-induced defenses in hybrid poplar. Planta 219:936-947.

4. Ralph S , Oddy C, Cooper D, Yueh H, Jancsik S, Kolosova N, Philippe RN, Aeschliman D, White R, Huber D, Ritland CE, Benoit F, Rigby T, Nantel A, Butterfield YSN, Kirkpatrick R, Chun E, Liu J, Palmquist D, Wynhoven B, Stott J, Yang G, Barber S, Holt RA, Siddiqui A, Jones SJM, Marra MA, Ellis BE, Douglas CJ, Ritland K and Bohlmann J (2006) Genomics of hybrid poplar (Populus trichocarpa x deltoides ) interacting with forest tent caterpillars (Malacosoma disstria ): Normalized and full-length cDNA libraries, expressed sequence tags (ESTs), and a cDNA microarray for the study of insect-induced defenses in poplar. Molecular Ecology 15: 1275-1297.

5. Lawrence SD, Dervinis C, Novak N and Davis JM (2006) Wound and insect herbivory responsive genes in poplar. Biotechnology Letters 28:1493-1501.

6. Major IT and Constabel CP (2006) Molecular analysis of poplar defense against herbivory: comparison of wound- and insect elicitor-induced gene expression. New Phytologist 172:617-635.

7. Miranda M, Ralph SG, Mellway R, White R, Heath MC, Bohlmann J and Constabel CP (2007) The transcriptional response of hybrid poplar ( Populus trichocarpa x P. deltoides ) to infection by Melampsora medusae leaf rust involves induction of flavonoid pathway genes leading to the accumulation of proanthocyanidins. Molecular Plant-Microbe Interactions 20:816-831.

8. Ralph SG, Chun HJE, Cooper D, Kirkpatrick R, Kolosova N, Gunter L, Tuskan GA, Douglas CJ, Holt RA, Jones SJM, Marra MA and Bohlmann J (2008) Analysis of 4,664 high-quality sequence-finished poplar full-length cDNA clones and their utility for the discovery of genes responding to insect feeding. BMC Genomics 9:57.

9. Busov VB, Meilan R, Pearce DW, Ma C, Rood SB and Strauss SH (2003) Activation tagging of a dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that regulates tree stature. Plant Physiology 132:1283-1291.

10. Harrison EJ, Bush M, Plett JM, McPhee DP, Vitez R, O'Malley B, Sharma V, Bosnich W, Seguin A, MacKay J and Regan S (2007) Diverse developmental mutants revealed in an activation tagged population of poplar. Canadian J. of Botany 85:1071-1087.

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