Team:CoBRA/LabNotebook

Hi Stephanie,
Apologies for the delay in responding to your email. We sent out a request to our TRIA members to see if anyone would be interested in mentoring your iGEM team in this project. I was disappointed that no one was available to mentor your team on this. I am probably not the most appropriate person to guide your group, but I would be willing to take a stab at it.

I have spent quite a lot of time thinking about synthetic biology products that TRIA would be interested in, and how we could provide guidance. My lab is not really in the business of biotechnology, so this is a stretch for me. After some thought, I think that there are two avenues that the Cochrane students could take.

The first avenue would be more aligned with TRIA research: here, your students could try to develop a system that synthesizes different mountain pine beetle pheromones or tree chemicals that can cause changes in beetle behaviour that could help in controlling the outbreak. An example of a commercially available beetle pheromone is verbenone, which is emitted by beetles to signal that an attacked tree is fully occupied, and that incoming beetles should find another tree. Similarly, maybe there is the potential to synthetically create a blend of tree chemicals that could be used as a repellent (or as a trap). I have cc’d two TRIA researchers on this email who have expertise in this area; if you wanted to follow up on these ideas, I would hope to convince them to provide some mentorship on this project.

The second avenue is quite far from TRIA research: here, your students could use mountain pine beetle killed timber (infected with the mountain pine beetle fungal associates) as a feedstock to create new products from this wood. Given that the fungus is degrading the wood, the wood composition will be different than conventional softwood, and potentially could be used to generate different extractives, such as for the biofuel industry. This would be fundamentally exciting, but is very far from what I do. I have a colleague on campus who may be able to help you on this project, if your team was interested in this avenue.

Apologies again for the delayed response. Please let me know if you would like to proceed with one of these projects (or if you’ve found another mentor), and we can decide next steps.

Best wishes,

Janice
Associate Professor
Department of Biological Sciences
CW405 Biological Sciences Building
University of Alberta

Entry #5: Mountain Pine Beetle

http://www.nrcan.gc.ca/forests/insects-diseases/13397

This site has the scientific name of the mountain pine beetle (Dendroctonus ponderosae), as well as its' life history and how it attacks and damages the tree.

http://www.publicaffairs.ubc.ca/2011/01/24/genome-of-blue-stain-fungus-evolved-to-bypass-tree-defense-in-mountain-pine-beetle-epidemic-ubc-research/

"Now, researchers from UBC and the BC Cancer Agency’s Genome Sciences Centre have conducted a detailed genome analysis and identified genes in Grosmannia clavigera that are responsible for the fungus’s ability to bypass the lodgepole pine’s natural fungicide – and use it as a carbon source for fungal growth."

http://www.ncbi.nlm.nih.gov/pubmed/20727970

"The first aggregation pheromone components from bark beetles were identified in 1966 as a mixture of ipsdienol, ipsenol and verbenol. Since then, a number of additional components have been identified as both aggregation and anti-aggregation pheromones, with many of them being monoterpenoids or derived from monoterpenoids."

Scientific name: Grosmannia clavigera

Kingdom: Fungi

Phylum: Ascomycota

Class: Sordariomycetes

Order: Ophiostomatales

Family: Ophiostomataceae/p>

Genus: Grosmannia

Species: G. clavigera

http://www.pc.gc.ca/docs/v-g/dpp-mpb/sec3/dpp-mpb3a.aspx

“Mountain pine beetle carry fungus spores near their mouthparts”

“the chewing action empties the spores into the tissues of the tree”

“After the beetle introduces the fungi to the tree, *mycelia grow rapidly into the sapwood, slowing the movement of fluids and disrupting the tree’s defences against the invading beetles”

• *Mycelia is the vegetative part of a fungus that grows in thread-like structures

• Mycelia grows in the phloem and blocks the flow of fluids, which would block the flow of sap. This sap would trap the beetles and kill them if the fungus was not present

Guy that researched blue stain fungus genome and identified genes that are able to convert natural tree fungicide into food source:

Joerg Bohlmann (EMAILED)

Michael Smith Laboratories

http://www.cfs.nrcan.gc.ca/bookstore_pdfs/28144.pdf

• The fungus bypass the trees natural defences due to a genetic change that allows them to turn these defenses into a carbon based food source.

• Mountain Pine Beetles serve as a vector for three different types of blue stain fungus, Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum

• Based on the fungi’s isolated location (Alberta, BC, Colorado, California, etc.) different fungal isolates immerge with varying temperature tolerances and optimal growing conditions.

• “The precise role of these fungi is not known, but they are thought to help the beetle overwhelm the defences of the host tree… and provide necessary nutrition throughout the lifecycle of the beetle”

• Grosmannia clavigera is the more aggressive of the three fungi on most host trees.

http://www.pnas.org/content/108/6/2504.short

• We establish that Gc is heterothallic, and report evidence for repeat-induced point mutation. We report insights, from genome and transcriptome analyses, into how Gc tolerates conifer-defense chemicals, including oleoresin terpenoids, as they colonize a host tree. RNA-seq data indicate that terpenoids induce a substantial antimicrobial stress in Gc, and suggest that the fungus may detoxify these chemicals by using them as a carbon source. *Terpenoid treatment strongly activated a ∼100-kb region of the Gc genome that contains a set of genes that may be important for detoxification of these host-defense chemicals. This work is a major step toward understanding the biological interactions between the tripartite MPB/fungus/forest system.

*Terpenoid - Any of a large class of organic compounds including terpenes, diterpenes, and sesquiterpenes. They have unsaturated molecules composed of linked isoprene units, generally having the formula (C 5H 8)

Entry #11: Random thoughts and ideas

Female pine beetles would release pheromones (aggregation pheromone) once in the tree to attract male beetles for reproduction. They are also capable of releasing pheromones (anti-aggregation pheromones) that contradict these pheromones that give the message to other beetles that the tree is already occupied. These pheromones only have a small area around the beetle where they are actually effective. I was thinking if there is any way to take the anti-aggregation pheromone and to neutralize it with the aggregation pheromone. if we neutralize this pheromone will this stop the male beetle from finding the female beetle? Or will the time it will take for the two beetles to meet just be slowed down and give time for the trees natural defenses to kick in and kill the female beetle? Obviously this will take some more research but thought I should just write my thoughts down before they slipped away.

You want to have the chitinase turned on when the fungus is introduced into the tree, so that its growth is compromised. So maybe there is a fungal gene that is strongly upregulated by something about the tree, like monoterpenes? If Joerg's group (or Colette's group) has identified one or more of these genes (which I'm pretty sure they have), then you can identify the promoter for this gene from the G. clavigera genome sequence. This can be used to create the construct that you would use to create the transgenic fungus, which then could be introduced into the tree (or at least, for proof of concept, onto culture plates that contain the monoterpenes) and see if you can turn on the gene. If the gene gets turned on, then you can see what happens to the fungal cell wall.

From Magda Pop - in an earlier gmail - which could be what Dr Cooke is speaking to
You could use a (pinII) promoter which is induced by monoterpenes (stress tree chemicals) to control a chi gene => when the tree gives off monoterpenes, the (pinII) promoter is activated to produce chitinase enzyme, which degrades the chitin from the cell wall of the fungus ***Note: the pinII promoter might be available in Vancouver (http://www.researchgate.net/publication/6167104_Testing_of_a_heterologous_wound-_and_insect-inducible_promoter_for_functional_genomics_studies_in_conifer_defense )

Or instead of pinII, use a stress/terpenoid-induced promoter that dr Bohlmann recommends?

From Patrick Wu in response to Dr Cook

If we engineer the fungus directly, however, I'm not too certain if the fungus will be willing to keep those genes as it reproduces. Suicidal organisms tend not to be considered evolutionary fit, so those genes might simply disappear in one or two generations...

But we mentioned a different organism during our brainstorming session, Agrobacterium tumefaciens. This is an organism that usually infects plants and can introduce DNA into them (which is how scientists can transform algae--for cheap!). What can be done, if we can't express the chitinase in E. coli, is to instead grow Agrobacterium that can introduce the chitinase genes into the fungus. From there, the fungus can create the chitinase inside itself.

There seems to be at least a few papers on Agrobacterium being able to introduce DNA into blue stain fungus. I've attached one here.

Blue Stain Fungus Transformation.pdf

The only issue with this idea is accidental transformation of the trees along with the fungus, since the bacterium was originally a plant pathogen. That might be something to explore further... hm.

Entry #19 Building From Latest Consideration - February 19, 2014