 Okay, right. Welcome to the digital world. So, in between, when I was at CSIRO, I had the opportunity to get into ANU through the ARC HECRO partnership. So, I really had an insight about how the university system was compared to the research organization. So, let's come into the subject. So, in which there are three types of breast disease. One is striped breast, caused by the pathogen, parxenia, stereochromia, almost to the CT side. So, the pathogen is called striped because the interaction of this particular pathogen is along the veins. So, that's why it clearly produces this stripy pattern. And this particular striped breast is more common in cooler climates. So, you won't imagine having more striped breast in 205S. Maybe it's a bit warm for striped breast, but it may or may not. So, mostly you can see in Australia, striped breast comes early in the season when it's very cooler. So, the second one is the leaf breast, caused by parxenia, triticinus. And it produces round to over shaped reddish pastils. And this leaf breast is very common, wherever wheat is grown, you can see leaf breast. And the third one is called stem breast, caused by parxenia, gramleys, parmospecies, triticin. So, as the name indicates, this particular fungus grows only on the stem. And it appears and it appears late in the season. So, whenever there is a very warm or close to the harvesting stage. So, rust has become a major threat to global food security because of the highly virulent stem and striped breast. So, one is the UG-99 race. So, one is the UG-99 race that emerged in the eastern part of Africa during 1998. So, to this race, more than 80% of the worldwide wheat culture has become susceptible. So, it's one of the major threat. So, that's where they formed this global, Bola Global Rust Initiative Consortium. And they bought more than 30 researchers around the globe to mitigate the spread of this common fungus. So, within a period of 10 years, it evolved more than 10 different lineage races and it spread down to South Africa and to Middle East. The other striking feature is the appearance of stem breast in Europe, where there is no sign of stem breast for the last 50 years. And one of the major issues is because stem breast has started adapting, adaptation to warm cooler climatic. Because normally stem breast is happening in the warm climatic condition, but those that appeared in the Europe, they were able to tolerate cool climatic conditions. And the overall worldwide sporadic incidence of stem breast because stem breast is a cooler climatic pathogen. However, the most recently known evolved races are able to withstand warm climatic conditions. So, it appeared in Africa, Australia, China, India, America and Europe. It's become a very, very big issue. So, coming to the Rust Management, there are two main strategies used. One is the Fungicide application. As you all know that Fungicide application is one of the most instant and immediate control. However, it's not a very welcoming control. For example, in Denmark, if you have two or three sprays of Fungicides on your wheat, your premium will be reduced. So, they want a wheat that has very less toxic chemical content. So, I think more soon they will be going to adopt this concept throughout the other places. Even in Australia, they are trying to see that if a farmer grows a wheat and it doesn't have any chemical residues, it has to have a high premium. That's why. Whereas on the other hand, genetic resistance, which refers to the innate ability of plants to resist pathogen attack, is one of the most sustainable and one of the most important strategies that has been followed worldwide right from the, like nearly more than 50 to 60 years. So, in the picture, you can see that the pathogen, the plants on the right hand side are more greener because they have resistant leaves. They can be able to resist the attack of the pathogen or they can work on the damage caused by the pathogen. Whereas on the plants on the right hand side is the one you can see it's almost killed because the pathogen, the plant cannot be able to tolerate the damage caused by the pathogen. Okay. So, in wheat, there are two types of rust resistance. One is sealing resistance. So, as the name indicates, the resistance is expressed right from the sealing state and it lasts throughout the plant growth stage. And they are more pathogen and strength specific. For example, SR-45 stem rust resistance can resist only against certain group of stem rust races. So, I will go back into detail about the function, why they are strength specific. And the other one is the adult plant resistance. So, as the name indicates, the resistance is expressed only at the later stages of the crop growth, only when the wheat plants are about to flower. So, there are two subtypes. One is the pathogen specific. So, that means, for example, YR-36 resistance, it can provide resistance only against strains of stripe rust. Whereas, we also have another group called multi-pathogen resistance, for example, LR-67 resistant gene, which can function against stem rust, leaf rust, stripe rust, and powdery milling. So, these adult plant resistance needs are very, very important because they are multi-pathogen resistant genes. And I will get back to you later about the function. So, before getting into the stem rust resistant gene function, I would like to play this video that will clearly explain how the sealing resistance function. Plants are under constant threat from invaders. This wheat crop stem is being attacked by a parasite. The parasite is called rust, although it is actually a type of fungus. The invading rust fungus has penetrated deep inside the plant tissue. As the invasion progresses, the fungus produces spores. These spores erupt from the surface of the plant. The spores spread, creating pustules, new sites of infection. Soon the plant's stem and leaves are covered in fungal growth. Carried by wind, the fungal spores can travel great distances, eventually reaching other potential hosts. This spore must now find the nutrients it requires to grow. Sending out the germ tube, it seeks an entry point into the plant. Newly formed penetration tube breaks inside the stem, and the fungus extends further into the plant. Once inside the stem, another structure called the haustorium is used to penetrate inside one of the plant's cells. The fungus can now take nutrients from within the plant. It also begins to secrete small protein molecules called effectors. This is a critical time for the plant. If it can detect the fungal effector proteins, it can try to stop the invasion. The plant has specialized resistance proteins which act like an immune system. The resistance proteins can bind to the fungal effector proteins. This binding event alerts the plant that an infection is taking place. Now that the parasite is detected, the infected cells are sacrificed, cutting off the energy supply to the invader. The fungus will eventually starve and the plant can continue to grow. Hope you enjoyed the video. So when the rust fungus infects the plants, it produces millions of molecules called effectors. So the plants have resistance proteins that can recognize some of these effector molecules. So they know that there is an enemy present there. Then they try to sacrifice the cell that is infected. Thereby, it will prevent the further infection or further spread of the fungus. Whereas on the other hand, pathogens try to evolve new strains with the modified effector molecules. So it may not be recognized by the resistant gene. Thereby, it can have a successful infection that leads to susceptible reaction. So there is an arm race going on between the host plant and the pathogen. The pathogen strives to evolve new effector molecules so that the plants won't be able to recognize. And at the same time, plants are trying to evolve new resistant genes that can try to recognize the modified effector molecule. So coming to the adult plant resistance, it changes the plant metabolic pathways. And one of the very good example is the sugar transporter protein. So in a normal wheat plant, this sugar transporter protein transports sugar molecules from outside the cell to inside so that the sugar molecule inside the cell will be taken by the host volume of the rust to proliferate. Whereas in case of resistant lines, the sugar transporter protein is a mutant form. It won't be able to transport sugar molecule from outside the inside. Thereby, there won't be enough sugar molecules inside the cell. Thereby, the rust fungus won't be able to proliferate as good as the susceptible line. So that the one of the main features why this adult plant resistance genes are more multi-pathogen or multi-strain resistant because of this peculiar character. So any pathogen that's where affected by the sugar block and sugar transport will be resistant. So coming to the source of genetic resistance, so we have been talking about different types of resistance, so where we can find. So one of the very important source is the ancestral species of cultivated or domesticated crops. For example, the two crop species in wheat, pasta and bread, it's a hybrid, natural hybrid. So first, the deployed wheat triticum mollococcus or triticum boiticum evolved to form the triticum uratum a diploid wheat species, which further hybridized with agelob spulchoid is to form the tritoploid with triticum turgidum. And triticum turgidum evolved to form the triticum turgidum durum, which we are currently growing to make our pasta. And at the same time, triticum turgidum dicocum further hybridized with another deployed grass called agelob storsi, which has a degenome to form the hexaploid wheat, which we currently grow to make our bread and other biscuits and other crops. So clearly what we can do is we can trans, we can, we can screen jump as some letter to this wild species and we can identify resistant things and they can be easily transformed into these two crops. So in our lab, we have been mainly focusing on triticum mollococcus and agelob storsi and we have grown a number of resistant things for us from these wild species and have transferred into cultivated wheat. So the other important source is the land races. So imagine 100 years back, we never had a very good breeding program like GRDC or anything. So farmers, they are smart breeders. So whenever they are growing crops, they try to have a very close eye on the crop and they try to identify lines that have superior performance for yield, for disease resistance, for drug tolerance, and they try to multiply those selected lines and they try to use that these generations after generations. They are smart breeders. So interestingly, families botanists like Watkins and Vavilov, they have explored like 30 to 28 countries like Vavilov, there is Vitis Grove and they have gone to door to door farmer's field and they have collected a lot of land race accessions. So at CSIRO, we have around 800 diverse wheat accessions collected by Watkins collections that has been collected during 1920 to 1930 and it has been collected from 32 different countries. And we also have access to Vavilov wheat collection, 300 diverse diverse accessions collected from 28 different countries from 1920 to 1990. So we have been exporting these germ plus and resources for new source of rust resistant. So now we found the two main sources. So what are the different methods we can use it for identifying the disease resistant genes? So one of the very common method is the map based cloning. So what we do is we try, we take the line that carries your gene of interest, cross with the susceptibility so that you can produce lot of recombinant lines, the line that has a chromosome crossover between the resistant and the susceptibility so that you can see a mixed match of chromosome segments. So in that way, you can identify a line that has mixed match of chromosomes. So when you find a marker or a DNA sequence that is closely linked to the resistance, then you are able to use it as a mark. So the next step is the genetic map. So wheat has 21 different chromosomes. So in order to identify which one of the chromosomes has your gene of interest and what are the different markers DNA polymers from that is present next to the gene, we need to do this genetic map. So we have to use those recombinant lines from this mapping population and you need to screen them with chromosomes specific markers. So in wheat, we have 90,000 sneak markers that are derived from all these 21 different chromosomes. So based on the resistance marker trait linkage analysis, you can identify what are the different markers that are present next to your R gene of interest. So once you map the R gene, the next step is physical mapping. Physical mapping is nothing but just zooming into your gene region of interest. Then you can identify what are the different genes present in this region, in this genomic region and what could be the best candidate for your resistance. So this physical map can be done previously when we have started in this mal cloning, we use back libraries because at that time we didn't have any good reference for wheat or any of the ancestors. But now we have a reference for wheat as well as for some of the cultivars as well. So once you develop the physical map and once you know the genes that are present in this region, the next step is screening candidate screening. So one of the very quickest way is to develop a mutant population. So what we do is we take the resistant line and treat them with mutagenizing agent like EMS or sodium azide and we try to knock out the resistant gene so that the line will become susceptible because there is a modification in the resistant gene. Then we use those lines to screen the candidate genes. So we know that whenever in the mutant lines, definitely the resistant gene should have a changes or should have a modification, that's where they lost the mutation. So therefore, for example, if you know that this is the gene, this particular gene will definitely be deleted or it will have some changes in those mutant lines. So the final step is the confirmation step. Once you know that this is the candidate, you try to amplify this candidate from the resistant line, the functional copy and then transform into a susceptible wheat cultivar and then you screen them with the rest. If the susceptible line carrying the gene from the resistant line shows resistant, then you know that this is the gene responsible for the resistance. So we use this protocol and it's like almost like a six, seven years journey. So we developed the physical map for the stem-res resistant gene SR23. So at the time, we didn't have a very good reference. So we have to do back-by-back screening and we used two back sources. One is from the Agilepsoxial and Aelite 78 and the other one is from Arius 18913. So in our analysis, we found a cluster of resistant gene analogs called MLA genes that has been mapped at PSR33 locals. So MLA stands for milieu-like resistant gene. It is one of the very large gene family that has been identified in Barney for milieu resistance. And this MLA gene belongs to the nucleotide binding leucine bridge lipid family, which is one of the very large gene family that has been known for test and disease resistance, not only in plants, but also in animals and also in humans as well. So it's one of the very well-known gene family that has been known to play a big role in plant defense. So next, in order to identify which one of these members is SR33, we screen all those candidate genes in the mutant population. So based on the presence and absence of markers, we can group those mutant population into three categories. The group one consists of mutant lines that has large deletions. So here you can see they are most of the markers from the regions that are deleted. And group two consists of one line that shows deletion in some of the genes. And group three consists of four lines that consistently had snippet changes only in one gene sequence, AET or GF1E, indicating it as could be the SR33 resistance. So before going into detail about the transformation, I would like to highlight the V-transformation unit at CSI. So before 2015, before the publication of the V-transformation protocol from Japan Tobacco Company, it's a very, very tedious process. You can't get more than one person satisfied. So you need to screen thousands and thousands of explants to find your transgenic line carrying your transgene. However, with this Japan Tobacco method, you are able to achieve more than 40%. For example, in our lab, using the Australian lead carriers, we were able to achieve up to 40% success rate. We have also established the protocol for durums, barley, and as well as kritikali. So in the last 20 years, the unit has processed more than 600 gene concepts and they have produced more than 12,000 transgenic lines. And we have also protocol where you can have without any selection markers. So using this pipeline, we introduced the AET or GF1E sequence into the stainless susceptible cultivar fielder. And in our rust test, all the transgenic lines that carried the full gene sequence showed the resistance response similar to the SR33 resistance compared to the non-transgenic line. So it clearly shows that AET or GF1E is the SR33 resistant gene. So clearly it's one of the very interesting study. If we have cloned SR33 long back, they may not have named as MLAs because they named this gene family as MLA because they thought that the genes belonging to this particular family only can confer resistance to mildew because in barley, there are 40 genes that were found too. So mildew resistance. And they also found one member in wheat, kritikam monococco. It also again produces resistor against mildew whereas 33 is the first known member of this gene family conferring resistance to a disease different from mildew. I know maybe you won't be happy why this. But it's a very interesting thing. We always see that it's just a narrow path. It never plans as to struggle against different multiple factors. So they have to have all mechanisms to change their way of action. They can't have genes for individual types because that's why we try to think and we blindly think that, oh this gene is only for this type. No. The plan has to struggle with multiple factors. So they need to find a way to fight against everything. So since SR33 is one of the first stemorous resistance cloned in wheat, even though we have more than 80 genes. And since it is effective against the UG19N race, it was one of the very good moments that we got our thing published in science as a cover page article. And that has also been highlighted in BBC news because of the UG19N outbreak. Because everybody was scared of what is going to happen. So following the discovery of SR33, we amplified the SR33 ortholog sequence from Rai which carries SR50 resistance and we found that SR50 gene is also an MLA gene family and it's a related member of SR30. So in our lab using this tedious map based cloning, we cloned three stemorous resistant SR33, SR50, SR46 and we also cloned adult plant resistant genes like LR34 and LR67 genes. So both the genes, LR34 is a ABC transporter and LR67 is a X-Rosugar transporter. So they both come for resistance against leaf rust, stem rust, stripe rust and polyamide disease. So that's where we have been doing a lot of experiments on these two genes. On cloning of all these genes has nearly taken from 8 to 12 years. And even though we have a lot of genes in our pipeline, still we couldn't able to find the genes even after 15 years. Like SR2, it's one of the very important stem-rest resistant genes that have been, I have used more than 100 years but still we are struggling around to find the causative gene responsible for the resistance. So why we think that map based, it looks like a very simple process when I was outlining the process but it's not a very simple process. One is we, it is made up of a very large genome around 17 gigahertz, five times bigger than a human genome and 40 times bigger than red. And the second thing, it is a polyplot. It has three homeologous chromosomes, 7A chromosomes, 7D and 7D. And the other bad thing is about more than, each of these genomes are more than 80 percent identical to each other. So whenever you are trying to design a marker, you have to make sure it is specific to the specific genome. So every time it takes at least three times more time than the other, like a deployed processor. And more than 80 percent is a repeat. As you know that when the plants evolve and they have a larger genome, it's not the gene content is evolving. It's just the repeat elements of the transpose that keeps on expanding. So more than 80 percent of the sequence are repeat. So it's really, really hard. And the other one, only 2 percent of the genome represent gene sequence, like a functional gene sequence. So it's really, really hard to find a gene within these two persons. And the final point is most of the resistant lines that we are doing is all wild species of land races. And they are not very much closely related to the reference genome or the pan genome. So you won't be able to identify the homolog gene sequence from using your reference. So however, cloning of SR-33 lead to a very simple gene cloning pipeline called mutarency, which stands for mutagenesis resistant gene enrichment sequencing techniques. So this particular technique is based on a very simple concept called targeted gene capital technique. So as I said you before, NLR is one of the very large family, more than 80 percent of the resistant gene identified for many disease in both plants and angles belongs to this NLR gene family. So what is the point of working on other genes? So we come up with an idea that, so you can use the fragments of NLR gene family and you can identify similar related fragments from the same species or different species. Then you analyze only the NLR-like sequence. So in this way, for example, in wheat we have 334,000 genes. Among the 334,000 genes, only 3,000 percent NLRs. So you are drastically reducing your complexity from 17 gigabytes to 0.01 gigabytes. So the whole strategy consists of this method. So the first step is mutagenesis. So you have to generate a loss of function muta. So you take your resistant line, treat them with EMS or sodium oxide, then try to knock out the resistant line so that you will be able to make a mutant population. Then the second step is a resistant gene enrichment step. So you take the total genomic DNA from this wild type as well as from the resistant line and break them into small fragments. And then you like it with adapters. And then you use NLR probes. So NLR probes are nothing but 120 baseware exon sequence of NLRs that are predicted from a reference genome. For example, we take the 3,000 NLR-like sequence from wheat and we break them into 50,000 probes, like 120 baseware probes. And these 120 baseware probes are designed in a way that it has 50 percent NLR-like adjacent probe so that you can develop a big quantity using those exon sequence. So we use these 15,000 probes and we have these probes attached with the step-down building. So step-down is a magnetic deep that you can pull down. So we use the PCR-based hybridization program and we use this NLR probe and we try to capture the fragments that are related to NLRs. So any fragment that has more than 80 percent identical with the NLR probes will be captured. So using the magnetic field you can elute the captured fragments. And then using the PCR primers that are designed specifically on the adapters, you can amplify the captured fragments to 20 to 40 times coverage so that you have a very good representation of the captured fragments. And then you try to sequence those captured and enriched fragments and based on the comparison between the voil type and the mutant line, you can identify your genome. So overall this process takes only two years, one year to deviate the mutant population, three months for the capture and sequencing and six months for the transformation and confirmation of the gene. So since we developed this pipeline using the SR33 material, since we already know that we clone the SR33 gene, nobody would believe right because you already know the gene sequence. So we want to validate using a normal gene. So we took the mutant lines of SR22, we had the six mutant lines and then we ran through the pipeline. Luckily we identified an NBSLR gene that consistently had sneak changes in all those six mutants. So next in order to confirm that this is the gene, we amplified the resistant gene from the resistant line and then we transformed and all the transgenic lines carrying the full gene sequence clearly showed the resistant response come back to the non-transgenic line. So people would think that okay, you just clone the gene, one gene is mere luck, so we still don't believe your method. So we went back with the second origin SR45 and luckily again we were able to identify NBSLR gene that consistently had sneak changes in all those six mutants and then in the transgenic test, we found all the transgenic lines that have the full gene sequence clearly showed the SR45 mediator resistance compared to the non-transgenic line and the line that had a truncated sequence. So then cloning of SR33 and SR45 lead to another modified technique called aggrandic which stands for association genetics resistant gene enrichment sequence. So this particular technique is just a one step forward of the mutagenesis resistant gene enrichment sequence. So instead of creating a artificial variation on a mutant population, here what we do is we take the naturally existing germ plasm and we group them into different groups like G1, G2, G3 based on their resistance response against multiple strains. So then you know that any group of land races that belongs to one particular group may have the same resistor gene or an allylic version of the same resistor gene. Then we do the capture and then we compare the sequence from each of the members of their group, different groups and based on the presence and absence of the mutation, we will be able to identify the gene of interest. It's just a combination of genome-wide association with the gene capture and sequence. So in addition to SR33 and SR45, we were able to clone two artificial genes SR46 and SRTA166 to using this method. And the other important method that we have been using heavily in our lab is mute chromosec, mutagenesis, chromosome isolation and sequencing. So as I said you before, wheat has 21 different chromosomes. But once you know that your gene of interest is present in one of those chromosomes, what you can do, you can specifically isolate that particular chromosome from the wild type as well as mutant and then you can sequence and then you can compare the sequence of those two and based on the presence and absence of mutation you can identify a gene of interest. So you can see that mute and seek, agranes seek, they are all based only on NLR, NLRs. So if your resistant gene is not an NLR, you won't be able to identify. So whereas this particular mute chromosec method can be able to identify any genes, whether it's an NLR, it's a kinase or it's a transport or anything. So that's where one of, so what we do is we normally take a resistant line, do the mute and seek, if you don't get a gene, we then go forward for the mute chromosec. So that's where we keep on going with the approach. So okay, now we have found that we have had technologies for rapid identification of resistance. So what is it for first? So normally in wheat breeding, if you want to cite a resistant line, what you have to do is you have to screen them by infecting with that particular type of strain. For example, if the Australian breeders want to screen their wheat lines against UG99, they have to go through a lot of SLI, like MPA, import permit, and then they have to send the seeds to Africa and then they have to screen against UG99 and then you have to get the line back and then you have to do the crossing. So it's a very tedious process. If you want to make your line ready for next year, you have to start with there. Whereas if you know the marker, if you know the gene sequence of the resistant line, you can design molecular markers based on the sequence. Then based on its presence and absence, you know that this is the line that carries your resistance. This will be a one day job. So next one is transfer of resistance from wild species to continental line. So as you have seen in our lab, we have been heavily focusing on wild species because wild species have very unique or normal resistance that are effective against broad range of approaches because the time and the place where those wild species are born, they might have had a very different type of pathogen strength, whereas right now with our current cultivar, we have a different type of pathogen strength. So they are also very much rich in genetic diversity for different traits. And one of the other problems in transferring genes from wild species to cultivated species is the transfer of undesirable traits. For example, a lot of wild species have grain shuttering phenotype. So we don't want those traits to be co-transformed when you are doing the conventional breeding. Whereas if you clone the gene, you'll be able to directly transform only the resistant gene sequence, whereas the other background will be same as the cultivated line. So since wheat and barley are infected with the same stem-based pathogen, and unfortunately more than 95% of the available wheat cultivars and wheat germ blossom are susceptible to UG-19N rates. So we were very hard to find any resistant gene effective against UG-19N in the barley. So what we did, we took the SR resistant gene that we clone from wheat and transformed into barley. And in our test, all those transgenic lines were found to show resistance against UG-19N and other stem-based. So clearly you can see once you identify the gene sequence, you can even transfer from different species, which were cross-incompatible, which you couldn't be able to cross in a conventional breeding product. So next, as I said before, this adult plant resistance is a very interesting gene. So in our lab, we have transformed LR-67 and LR-24 gene from wheat into barley, where it has given resistance to rust and foudium, into rice, it has given resistance against blast, into canola, it has given resistance to blackleg, and in sorghum against anthropolocontal. So as I said before, since this adult plant resistance changes the plant metabolic pathways, any pathogen that has been affected by the metabolic changes, they will be r-star, or they will be blocked from other infection. So the third important application is the gene cassettes. So in our lab, we were fortunate to be the first in the world to develop resistant gene cassettes for wheat. So what we do, we took different resistant genes from the wild species, and we state them as a single genetic unit block. So we developed a very large gene construct, and this gene construct will have different genes located side by side. So that when you are transforming these gene blocks, then when you are crossing this gene block, the line carrying this gene block into a conventional breeding line, all these genes will be going to the same genetic location. So there won't be much segregation. Whereas when you are doing a conventional breeding, each of these genes will be present in different chromosomes, and you need to do lots and lots of crossings to bring all those genes together in one line. Whereas in this method, you can have like a single genetic unit. It will keep going as a single block. And one of the very important thing is like, as I said before, pathogen tries to evolve virulence. When you have multiple genes together, the pathogen will try to take very, very long time to break down each and every single gene. And they have to accumulate all those mutations in one particular strain to overcome this particular line carrying multiple genes. So we have successfully tested these lines in the feedfields in USA. So the final take home is genetic resistance is one of the very ideal strategy for sustainable and effective disease management. And using the advancements in gene capture techniques, we have developed a targeted sequence capture and complexity direction techniques for rapid gene identification, which will enable us for the rapid identification of resistant genes and rapid generation of normal resistant lines through gene cassette and gene editing techniques. So finally, I would like to take, I would like to thank all my team members at CSRO, our collaborators at John Ender Center and Sainsbury Laboratory, UC Davis, USDA, Sydney USDA, New York State Queensland, and Department of Primary Industries. I can't imagine this work to be done without the whole hearted national and international collaborations. That's one of the very high strength of our group at CSRO. We are very, very open for anybody. And finally, I would like to thank, yes, without this big bag money, you can't do much, right? So the work I have been presented is mostly funded by GRDC, ARC, BGRA, and two Blitz funding. So finally, I would like to thank you all for making your time and coming through. Thank you. Thank you very much for this excellent presentation. I think we can stop the