Making a natural genetic engineer even better
From Victoria Conrad
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Hi all! I'm excited to talk to you today about genetic engineering in plants and the system we've come up with at the forest biotech lab here at the College of Forestry to remove pathogenic genes after gene transfer.
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Gene editing in plants, which is the direct modification of their DNA, is a key tool for plant science and biotechnology. The direct modification of plant DNA at the cellular level and then the regeneration of whole plants with modified DNA from those cells are the key tools for plant science and biotechnology.
Unfortunately, gene editing is challenging or impossible in the large majority of plant species including woody plants and trees, the focus in our lab here. Luckily, plant pathogens have evolved alongside plants to become uniquely equipped to infect plant cells and integrate their DNA into the plant genome.
Agrobacterium can, therefore, be used as a "middleman" for gene delivery and is the most widely used method in difficult plant species.
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The Agrobacteria of interest to this project, Rhizobium rhizogenes, is the causative agent of "hairy root" disease--which you can see here on the right--and induces and out-of-control hairy root production in infected plants by delivering a set of genes known by their abbreviation R-O-L or "rol." Hairy roots are valuable as a propagation tool because of their prolific growth, but at the end of the day, Rhizobium is a pathogen, so the presence of its genes does disturb plant development.
The Agrobacteria of interest to this project, Rhizobium rhizogenes, is the causative agent of "hairy root" disease--which you can see here on the right--and induces and out-of-control hairy root production in infected plants by delivering a set of genes known by their abbreviation R-O-L or "rol." Hairy roots are valuable as a propagation tool because of their prolific growth, but at the end of the day, Rhizobium is a pathogen, so the presence of its genes does disturb plant development.
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So the question is, to enable the use of this powerful gene transfer system while allowing the production of normal plants, can we engineer hairy root genes for removal after they've done their job?
So the question is, to enable the use of this powerful gene transfer system while allowing the production of normal plants, can we engineer hairy root genes for removal after they've done their job?
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This is our custom set of genes that we assembled, which we call a construct. The way we designed our system depends on us bookending our construct with two special locations called lox sites.
This is our custom set of genes that we assembled, which we call a construct. The way we designed our system depends on us bookending our construct with two special locations called lox sites.
We also included several other components, including Cas9 for gene editing, the hairy root genes, "reporter" genes that cause cells to glow or become colored, and a heat-inducible control element that encourages the formation of shoots and starts gene removal after a heat shock treatment.
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Our gene removal method, otherwise known as excision, works through the Cre/lox recombinase system--which allows for control over gene deletion by utilizing Cre recombinase, an enzyme that acts on these lox sites. When we activate our heat-inducible genes, the Cre recombinase recognizes the two lox sites, cuts the DNA right there, and after DNA repair, the result is one circular piece of DNA with one of the lox sites and the other lox site still in the plant genome. We call this single lox site a "footprint" because it is all that is left of our construct after its visit. This footprint is non-coding DNA, which means it won't disturb the plant's growth and this circular piece containing the construct does not stick around.
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Our gene removal method, otherwise known as excision, works through the Cre/lox recombinase system--which allows for control over gene deletion by utilizing Cre recombinase, an enzyme that acts on these lox sites. When we activate our heat-inducible genes, the Cre recombinase recognizes the two lox sites, cuts the DNA right there, and after DNA repair, the result is one circular piece of DNA with one of the lox sites and the other lox site still in the plant genome. We call this single lox site a "footprint" because it is all that is left of our construct after its visit. This footprint is non-coding DNA, which means it won't disturb the plant's growth and this circular piece containing the construct does not stick around.
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In order to determine if this gene removal has actually occurred after heat shock, we used several visual reporters. One is pigment visible to the naked eye, which produces this great magenta color seen on this root here. It was really helpful for quick screening. We also have two fluorescent markers, one also red and one green, on opposite sides of the transgene--so the presence of the combination of them creating this orange or any one of these visual markers, indicated that the transgene or even a part of it was still present. Seeing no markers at all tells us that the transgene was removed because the footprint has no visual reporter genes on it.
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For the experiment, we cultured our Agrobacterium with leaf discs and waited for the ruby-colored roots to grow. Once we saw that we had roots with modified DNA, we took just those root segments and heat-treated them at 37 degrees Celsius (which is almost 100 degrees F) for several hours per day and for different treatment lengths. We then counted up shoots at the end of each treatment to figure out regeneration rates and found excision rates on a DNA-level using PCR.
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The genes that promote shoot growth and excision are all activated by heat shock, and we can see here that a little bit of heat shock compared to nothing may stress the plants as we see the proportion start to decline. Then, we found that the longest heat shock of 14 days improved regeneration the most, which shows us that Mild heat shock stressed the plants, but as soon as these regeneration genes are able to really activate, growth just takes right off. To determine our editing rate, we looked at the DNA level. We used PCR to detect the presence of different components of the construct we made or to detect the small footprint left over after excision and have found that we had a really good editing rate of about three-quarters of replicates being transgenic, and then about half of those replicates successfully excised their transgene.
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The goal was to produce edited plants and they're almost all edited. Our next steps are to optimize antibiotics in our media and to investigate the relative importance of the different shoot development components we used.
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With that, thank you so much for listening; thank you to everyone in the lab who has helped me with this project, and I look forward to answering your questions in our discussion board and seeing all the other projects!
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