 They're basically an environmentally friendly alternative to chemical pesticides and the active component is double-stranded RNA. So we synthesize this double-stranded RNA to be highly homologous to the pest or pathogen that we're targeting. So we apply the RNA to the plants, and then when the pest or pathogen comes to infect the plant, and in this case in this example it's a virus, RNA interference is initiated. So this is a natural cellular defense mechanism, which is highly conserved across new carriers, and basically involves double-stranded RNA being recognized as being foreign to the cell. And it's chopped up into small pieces of RNA, so these small interfering RNAs, which are then used. They're bound to protein complexes and they bind to any homologous RNA, in this case the virus RNA, but it could be fungal messenger RNA and target it for degradation. And this results in the plant being protected from disease. So the beauty of this approach is that it's highly specific. So we can use bioinformatics to make sure we select sequences that will be very specific to the pest or pathogen of interest, and we can make sure that no beneficial organisms are targeted. There's no chemical residues left on the produce. We don't need to integrate anything into the host genome, so it's non-GM. And the RNA can be stabilized on carriers such as Bioclay, which is the product we've developed at the University of Queensland. And Bioclay is where the RNA is bound to some clay nano sheets. And this protects the RNA from degradation. And so it extends the protection window from five to seven days up to almost a month. And so this technology is also active, it's not only active against viruses, but also against insect pests and fungal pathogens. And I've just shown a few examples from the literature here. So this first one is where they targeted the Colorado potato beetle, and they were targeting the actin gene. So when they applied double-stranded RNA, which is homologous to the beetles actin gene, there were some developmental problems. The beetles are very small. They didn't develop properly. When they just used GFP, double-stranded RNA, there were no problems and also nothing for the water control. So this is really showing that it's highly sequence specific. You can't just apply any RNA, it really has to match the sequence from the pestle pathogen. And these are some fungal examples here. So this top example is sclerotinia on canola. So these researchers tested three different RNAs, and you can see there was a reduction in lesion sizes. And here it was Fusarium, Grummania, and Lombali. Once again, when the Fusarium specific RNA was used, there was minimal lesion development compared with the control. This is betrider scenario. It also seems to work against this pathogen. So fruits, vegetables, flowers, they all showed reduced lesions when the betrider double-stranded RNA was applied. So we wanted to see whether this exogenous RNA interference or RNAi could be used as control against rust fungi. And we were particularly interested in myrtle rust, which actually arrived in Australia in 2010. And it's a huge problem because it infects more than 300 of our native metasis species. And this pathogen, these spores develop on the new growth and they're airborne so they can spread in the wind. And this pathogen has now pretty much spread across the whole east coast of Australia and into other parts of Australia. And it's not only a problem for natural environments, but also for the native plant industries. And so the work I'm going to be presenting here is the basis for Rebecca Degman's Honours thesis. And we've just submitted it for publication, so hopefully you'll see it come out very soon. And I've just listed all the authors involved. So it was a collaboration between UQ and DAF and also researchers from New Zealand. And so first of all, there had been some reports of RNAi against rust in the literature. So this exogenous RNAi had been successful in switchgrass rust and Asian soybean rust. And the transgenic version of this, so host-induced gene silencing, where a hairpin is transformed into the genomes of the plants, has been successful for wheat rust. So there was some sort of basis to start with. So what we did, well what, this is actually all allisteric targets work here. We searched through the genomes of rust from eight different families to look to see whether the RNA interference genes were conserved. So we looked for the DISA genes, the RDRs, and the Argonauts, which are all the fundamental components of the RNAi pathway. And we found that these genes were conserved, so there were copies of these genes across rusts in all of those eight families, including osteoplastinia CDI, which is the cause of agent of myrtle rust. So this led us to think that hopefully these genes were functional, and that these rusts would actually have functional RNAi pathways. So the next step was to see whether the rust spores could actually take up double-stranded RNA from the environment. Because the idea is that you spray plants with a double-stranded RNA, so we wanted to know whether rust spores that would blend on the surface of the plants would be able to take up that RNA, which would then trigger that RNAi pathway. So we labelled some double-stranded RNA, which are three nucleotides. So they fluoresce orange here, so we looked at the spores under the fluorescent microscope after they germinated. And what we found was that the uridiniospores, so these are the infectious spores, which you saw in that photo at the beginning, which make those yellow pastels, so they're yellow spores. They took up the RNA, and you could see it in the germ tubes, in the germinating spores. So this was seen for osteoplastinia CDI, as well as choleosporium plumeriae, which causes frangipani rust. Interestingly, the teliospores didn't seem to take up the RNA. We only saw it in the uridiniospores. But it did seem to be taken up at the very early stages of germination, so we can see it just when the germ tubes were starting to blot out. So the next step was to see whether we could actually initiate RNAi and get some sort of response in the rust fungi. So we used these two species again, osteoplastinia CDI and choleosporium plumeriae, and we were very lucky our collaborators in New Zealand had been making these artificial leaf substrates. So we were able to do all of these experiments on these substrates, which were great for microscopy, so we could get some really nice photos of the spores as they were germinating. So what we found is that, so we were targeting three essential genes in the rust, and what we found is that with each of these double-stranded RNAs targeting the three genes, there was a reduction in spore germination. And when the spores did germinate, they didn't develop the infection structures. So you can see here in the GFP control, the aphrosaurium is formed and there's even an infection peg that's developed. But in the RNA-treated spores, we only saw these sort of swellings here at the end of the germ tubes, so that was sort of as far as the infection structures got. And so that was the same for the choleosporium plumeriae. We saw some clear infection structures in the controls and not so much in the RNA-treated samples. We then wanted to see if we could, if we saw an effect in planter. So Beck did some detached leaf assays where she mixed the rust spores, and this is now myrtle rust. She mixed the spores with the double-stranded RNA and inoculated the leaves. And then we looked, we looked at the leaves after two weeks. And you could see that with the GFP, these pastures had developed as normal. But in the RNA-treated samples, when we targeted the specific rust genes, there was really a reduction in symptoms. So we saw very, very minimal symptoms. This is just one example, but we repeated it for quite a few of the genes. And we saw the same thing on whole plants, which was really exciting. So clear symptoms developed after two weeks on the GFP and the water controls. And then in the RNA treatments, we really saw very minimal development of symptoms. So those experiments were where we inoculated the plants with the mixture of the spores already premixed with the RNA. But then we repeated that experiment where at this time, we applied the RNA six days before we inoculated the plants with the spores. And really excitingly, we again saw very minimal symptoms develop in the RNA-sprayed plants. So that was really exciting. And so now we're doing more experiments. We want to see how long that protection will last. So can we apply the RNA at earlier time points, so like one, two weeks before inoculating? How stable is it on the plant? Is spraying the leaves the best way to deliver the RNA? Can we do trunk injections? And then we'll go into the field and see if we can get the same results. And we're working with these industry partners to do this. We've already started doing some of that work. So excitingly, we saw that we can deliver the RNA through the petioles of the plants. When we applied the RNA to the leaves, these are northern blots showing RNA from the leaves one week after we sprayed it. So the RNA was still there on the sprayed leaves from Syzygium Jambos, which was the model organism that we were using in all of our bioassays as well as lemon myrtle. But it hadn't moved in the plant. It wasn't present in any of the young leaves, which are the leaves that will get infected. However, when we soaked petioles in the RNA, we could detect it in the new growth. And so three different plants were tested for each. This is not the best blot, but there were some faint vents here, and we will be repeating that. But it does look like we can get the RNA into the plant if we need to. But it is stable on the leaves, at least for a week, and we'll be testing later time points. And just quickly, I'm really over time, but we are also trying to make some RNA constructs to target more than one rust fungus. So we've tested making constructs targeting all three of these rust fungi, and these were fungi where we had sequence information, and they were also all available at UQ, so we're able to harvest spores easily. And excitingly, we did still see the same effects on spore germination. So this graph is just comparing our original data shown in dark purple to the new constructs. So there was a bit of a loss in efficiency, but that was because now the RNA is not 100% homologous to myrtle rust. So this is a test on myrtle rust. But we will now be testing the other rusts to see how it works on them. So I'd just like to thank everyone again. This was mostly Beck's work, but to all the contributors and to our funders. And I'll take any questions. Thanks.