Engineering Resistance to Bacterial Infection and Tolerance to Environmental Stresses

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00:00:07.23 Hello.
00:00:08.15 My name is Pam Ronald.
00:00:10.01 I am from the University of California, Davis.
00:00:13.01 Today, I'm gonna talk about plant biology research,
00:00:16.08 which is critical if we're going to
00:00:18.22 address one of the greatest challenges of our time:
00:00:21.05 how to feed the growing population
00:00:23.06 without further destroying the environment.
00:00:28.07 We know that the population is increasing
00:00:30.11 from a current 7 billion
00:00:32.24 to possibly 9 or 10 billion at the end of the century.
00:00:37.02 And we can't feed more people by plowing more land,
00:00:39.29 because most of the arable land has already been farmed.
00:00:42.28 This is a picture of a hillside in Ecuador.
00:00:46.01 And you can see farmers are farming
00:00:49.00 increasingly marginal lands,
00:00:50.23 such as these very steep hillsides.
00:00:53.15 Another issue is water.
00:00:55.22 Already, 70% of fresh water is used for agriculture,
00:00:59.21 and demands for water are increasing...
00:01:02.11 increasing over the next 50 years.
00:01:06.11 Plants are susceptible to diseases, as are humans.
00:01:10.02 And this is an image of a tobacco plant
00:01:12.16 that's infected with a very serious pathogen
00:01:15.24 that is also... a related pathogen
00:01:20.00 was responsible for the Irish potato famine,
00:01:22.09 which in 1850 caused the deaths...
00:01:24.28 contributed to the deaths of 1 million people
00:01:28.15 and caused another million people to emigrate.
00:01:31.27 This is a time-lapse photo
00:01:34.15 over a period of four days.
00:01:36.17 You can see how devastating plant diseases can be.
00:01:41.01 We can control diseases somewhat
00:01:43.15 by using pesticides.
00:01:46.25 But these pesticides...
00:01:49.03 some of them are toxic, and many of them
00:01:51.10 cannot be used safely.
00:01:53.20 So, this is a potato farmer in Peru.
00:01:55.22 And you can see that he is spraying his potato farm.
00:01:59.22 But he has no protective gloves;
00:02:01.27 he has no mask;
00:02:03.20 and as he poses for the camera he's spraying for his boot...
00:02:06.16 spraying his boot.
00:02:08.11 So, this is one of the reasons -- this lack of safety procedures --
00:02:11.23 that 300,000 deaths occur every year
00:02:15.08 due to pesticide poisonings.
00:02:17.06 And most of these deaths occur in the less developed countries.
00:02:22.17 In addition to pests and disease,
00:02:25.29 climate change is predicted to reduce agricultural production
00:02:29.14 in the future.
00:02:31.02 The Intergovernmental Panel on Climate Change
00:02:33.18 predicts that agricultural yields
00:02:36.06 will be reduced 2% per decade in the coming years.
00:02:39.26 And that is at a time
00:02:42.17 when we need to increase production instead of reduce production.
00:02:46.25 Environmental effects of climate change include droughts and heat,
00:02:51.03 but also flooding.
00:02:52.28 And we're going to see more and more floods
00:02:55.05 in areas where we don't want to have floods.
00:02:57.05 So, this is an example of boys walking through their family's rice field.
00:03:00.12 These rice fields can be flooded periodically,
00:03:04.22 and although rice does grow in water,
00:03:07.03 if rice is completely submerged,
00:03:09.25 most rice varieties will die within three days of flooding.
00:03:14.04 And this is particularly important in areas such as Bangladesh,
00:03:17.26 where people get two-thirds of their total calories from rice.
00:03:22.29 So, today I want to tell you the story of flood tolerant rice,
00:03:25.19 which is a collaborative project over the last 10 years.
00:03:31.02 25% of the world's rice is grown in flood-prone areas,
00:03:34.19 shown here in this red circle,
00:03:37.02 including eastern India and Bangladesh.
00:03:41.15 Now, in this area, just in Bangladesh and India alone,
00:03:46.18 4 million tons of rice -- enough to feed 30 million people --
00:03:50.19 is lost every year to floods.
00:03:54.11 About 50 years ago, there was an old rice variety that was discovered
00:03:58.12 that had an incredible property.
00:04:00.23 It could withstand two weeks of submergence.
00:04:03.29 That is, after the flood subsided, these...
00:04:08.00 this rice variety would survive the flood.
00:04:10.08 Now, breeders using conventional methods
00:04:14.02 tried to introduce this trait,
00:04:15.29 which is encoded by a gene or genes,
00:04:19.12 into varieties that were locally adapted
00:04:22.11 and favored by farmers.
00:04:24.04 However, the new varieties failed to...
00:04:27.10 did not carry the characteristics
00:04:30.12 that the farmers were looking for.
00:04:32.08 So, several years ago, a collaborator of mine, David Mackill, and I
00:04:35.27 decided to try to isolate the gene
00:04:38.22 encoding this important trait.
00:04:41.10 We were able to isolate a gene called Sub1A,
00:04:44.27 using a technique called positional cloning,
00:04:47.16 that confers submergence tolerance to rice varieties
00:04:51.01 that normally would die in a flood.
00:04:53.08 And you can see an example of this here.
00:04:55.03 On your left is the control rice strain before submergence.
00:04:58.22 And on the right are two genetically engineered varieties
00:05:02.09 carrying this single gene called Sub1A.
00:05:05.19 After 16 days of submergence,
00:05:08.28 you can see the control is...
00:05:10.29 the leaves are drooping.
00:05:12.19 They're yellowish -- chlorotic --
00:05:14.20 and elongated.
00:05:16.06 And the two cont... the two transgenic varieties look pretty good.
00:05:20.18 Now, this is what the plants looked like after 7 days of recovery.
00:05:24.18 So, the flood is removed
00:05:26.27 and the plants are given a chance to recover.
00:05:28.28 The control plant on the left has died,
00:05:31.13 whereas the two genetically engineered plants
00:05:34.09 carrying the Sub1A transcription factor
00:05:36.16 are completely healthy.
00:05:41.09 Now, my collaborators,
00:05:43.12 Julia Bailey-Serres, Takeshi Fukao, and Harkamal Walia,
00:05:49.17 have started to look at the molecular mechanism.
00:05:54.02 And the way Sub1A works is that
00:05:56.09 it sort of activates a hold-your-breath strategy.
00:05:59.04 And this allows the plant to conserve its energy
00:06:03.10 until after the flood subsides.
00:06:06.26 So, for example,
00:06:08.29 submergence is known to increase local concentrations of ethylene.
00:06:13.07 Now, ethylene then upregulates the Sub1A transcription factor,
00:06:18.12 which then induces ethanol fermentation genes.
00:06:23.12 Sub1A also suppresses alpha-expansins,
00:06:28.04 which are important in leaf elongation.
00:06:32.04 And Sub1A also suppresses
00:06:34.21 another related transcription factor we identified,
00:06:37.02 called Sub1C.
00:06:39.16 Normally, in the absence of Sub1A,
00:06:41.28 submergence would induce ethylene,
00:06:44.23 which would elevate gibberellic acid,
00:06:49.02 and that would turn on Sub1C.
00:06:51.15 And Sub1C is a positive activator of cell elongation and carbohydrate breakdown.
00:06:56.17 So, Sub1A is a key regulator of this process,
00:07:00.24 and essentially shuts down cell elongation
00:07:04.12 and carbohydrate breakdown.
00:07:06.26 And Harkamal Walia in my lab
00:07:09.07 has also been able to show that
00:07:11.22 upregulation of Sub1A also upregulates genes
00:07:14.24 that are important for biosynthesis of the hormone brassinosteroid.
00:07:19.20 And brassinosteroid then activates some very important genes,
00:07:25.01 including SLR1 and GA2 oxidase 7.
00:07:29.07 Now, GA2 oxidase 7 actually
00:07:33.01 contributes to the breakdown of gibberellic acid,
00:07:35.13 and SLR1 suppresses gibberellic acid-mediated processes.
00:07:40.06 So, this is another level of control...
00:07:43.24 of Sub1A controlling cell elongation and carbohydrate breakdown.
00:07:49.26 Now, if we had isolated this gene
00:07:53.07 and done some interesting biology,
00:07:55.07 that would have been great for basic plant biology.
00:07:57.20 However, the goal of this project always was
00:08:01.00 to try to develop a better rice variety
00:08:03.21 that can be used by farmers in Bangladesh and India
00:08:05.28 and other areas of the world.
00:08:07.22 And this is my collaborator, David Mackill,
00:08:10.07 who was at UC Davis
00:08:12.01 and then at the International Rice Research Institute.
00:08:14.18 And he led a team, including Abdel Ismail
00:08:17.11 and many other researchers,
00:08:19.20 to introduce Sub1A into various rice varieties
00:08:25.06 that are favored by farmers.
00:08:27.19 And in his approach,
00:08:29.14 he used what's called marker-assisted breeding.
00:08:31.01 So, in the next slide,
00:08:32.27 I will show you the effect of the introduction of the Sub1A gene
00:08:36.02 using marker-assisted breeding.
00:08:40.10 So, this is a time-lapse sequence
00:08:42.22 that you're going to look at.
00:08:44.24 So, it's a four-month time-lapse
00:08:46.17 that was filmed at the International Rice Research Institute.
00:08:49.13 And you can see the effect of the Sub1 gene
00:08:52.00 on a variety of rice called IR64
00:08:55.12 that was developed by David Mackill, Abdel Ismail, and their team.
00:09:01.19 So, what you see here on the left
00:09:04.14 is the new variety that was developed,
00:09:06.25 carrying the Sub1 gene,
00:09:08.19 and on the right is the control.
00:09:10.09 And here is an artificial flood
00:09:12.26 that was placed for 17 days.
00:09:15.05 And what's interesting is that you could see immediately
00:09:17.28 that the IR64 carrying the Sub1 gene
00:09:20.26 is highly tolerant of flooding,
00:09:23.05 whereas the control has very poor yield.
00:09:26.07 And I really like this video
00:09:29.23 because it shows the power of genetics,
00:09:32.15 the power of a single gene to transform
00:09:35.03 the yield of a very important staple food crop.
00:09:40.01 Dave and his team led four years of field trials,
00:09:44.05 and our team was fortunate
00:09:46.12 to visit the farmers in their field.
00:09:48.15 And over the last four years,
00:09:50.10 when these tests were done,
00:09:51.22 there was a lot of flooding every year.
00:09:53.09 So, these are conditions that were not induced in a field plot,
00:09:56.05 but were observed in farmer's fields.
00:09:59.06 So, you could see the farmers, on the left,
00:10:02.22 that planted the conventional variety, Swarna.
00:10:05.28 And on the right is the new variety,
00:10:08.02 carrying the Sub1 gene.
00:10:10.01 And what the farmers found was
00:10:12.02 a 3-5 fold yield increase in these new varieties.
00:10:17.24 We also had an opportunity to speak with
00:10:20.11 many of the farmers and their families.
00:10:22.25 And this is a woman explaining
00:10:25.20 what she thought of the new rice variety.
00:10:37.18 So, one advantage of this project
00:10:40.06 was that it was a fantastic collaboration,
00:10:42.12 not only between scientists in my laboratory at UC Davis
00:10:46.26 but also scientists at the International Rice Research Institute
00:10:50.02 and breeders at the Bangladesh Rice Research Institute,
00:10:54.07 and also other rice research institutes in Bangladesh and India.
00:11:01.02 I want to now turn to another critical issue in crop production,
00:11:06.14 which is disease.
00:11:08.04 And one of the goals of plant biologists
00:11:10.05 for many years -- over 100 years --
00:11:12.04 has been to develop...
00:11:15.26 to enhance the resistance of plants to disease.
00:11:20.21 Plants and animals are infected
00:11:22.27 by many different kinds of microbes.
00:11:24.20 So, for example,
00:11:26.13 here is a rice leaf that is infected
00:11:28.26 with a gram-negative bacteria called Xanthomonas oryzae pathovar oryzae.
00:11:33.27 You can see the bacteria, which has a yellow pigment,
00:11:36.20 oozing out of the rice leaf.
00:11:38.19 This is a dead fly that's infected with a fungal pathogen,
00:11:43.19 Aspergillus fumigatus.
00:11:45.10 And here is a baby that's infected
00:11:47.26 with a devastating disease
00:11:50.23 that when it becomes septic it can become invasive.
00:11:54.17 We now know, after 15 years of research,
00:11:57.10 that plants and animals
00:11:59.29 carry similar genes to resist these devastating diseases.
00:12:05.24 First, some history.
00:12:07.22 In 1845, late blight destroyed the Irish potato crop.
00:12:11.04 This serious disease caused the death of
00:12:14.12 a million Irish farmers and their families,
00:12:17.17 and contributed to a mass exodus to the United States
00:12:20.21 of another million people.
00:12:22.28 At the time, it was not known what caused this disease.
00:12:26.10 It was thought to be an act of God.
00:12:28.03 However, this disease triggered
00:12:30.15 the first serious investigation of diseases of plants and animals.
00:12:35.05 In 1853, Anton de Bary
00:12:37.16 demonstrated for the first time
00:12:40.04 that microbes can cause disease.
00:12:41.21 He was studying rust and smut fungi on cereals.
00:12:46.04 Louis Pasteur, who knew of Anton de Bary's work,
00:12:49.00 then showed that bacterial contamination
00:12:52.25 caused what he... caused what he called "diseases" of beer and wine.
00:12:58.00 And it wasn't until 1876 that Robert Koch
00:13:01.28 showed for the first time
00:13:04.12 that animals also are infected by microbes,
00:13:07.04 and these microbes can cause disease.
00:13:11.18 In 1905, Rowland Biffen used this new knowledge
00:13:17.10 on the genetic basis of disease
00:13:19.24 to develop the first varieties of plants that were resistant.
00:13:23.17 And he was studying cereals.
00:13:25.09 He was able to show that he could cross
00:13:27.14 a resistant wheat variety with a susceptible wheat variety,
00:13:30.03 and that that resistance would be inherited to the next generation.
00:13:34.03 Today, virtually everything we eat carries genes for resistance.
00:13:38.05 These resistance genes are critical to economic productivity,
00:13:42.06 and are the most safe and economically effective
00:13:46.09 method of controlling disease.
00:13:49.22 So, what are the genes that confer resistance to infection?
00:13:52.12 Over the last...
00:13:55.05 for the first 100 years after Rowland Biffen's discovery,
00:13:57.10 breeders were introducing these genes for resistance
00:14:00.07 without knowledge of really what they were or how they functioned.
00:14:03.27 And when I was a graduate student in the 1990s,
00:14:06.03 this was a really very important question,
00:14:08.15 to really understand the genetic basis of disease resistance.
00:14:13.10 And my research has primarily been
00:14:17.01 on the bacterial blight of rice.
00:14:18.16 You can see the yellow pigment
00:14:20.22 that this gram-negative bacteria forms in culture.
00:14:22.23 And this is a disease that occurs
00:14:25.14 in Western Africa and throughout South and Southeast Asia.
00:14:34.03 So, we began this work
00:14:36.11 with the help of Gurdev Khush,
00:14:37.28 who is now at the University of California, Davis.
00:14:41.18 But he spent most of his career
00:14:43.23 at the International Rice Research Institute.
00:14:46.00 He's considered one of the fathers of Green Revolution rice,
00:14:50.10 and he was awarded the World Food Prize
00:14:52.29 for his work in enhancing rice production
00:14:55.15 for farmers in very poor and less-developed countries.
00:15:00.08 Now, in collaboration with his colleagues,
00:15:03.18 he discovered a very unusual wild species of rice
00:15:06.19 called Oryza longistaminata.
00:15:09.10 Now, this wild species had an incredible property,
00:15:12.21 which conferred broad-spectrum resistance to virtually all strains of Xanthomonas
00:15:17.22 that were discovered in the field.
00:15:19.24 So, I was very inspired by this work,
00:15:22.05 and decided to try to isolate the gene.
00:15:25.14 We were successful,
00:15:27.08 again using a positional cloning strategy.
00:15:29.09 We isolated what was called the XA21 gene.
00:15:33.02 And you can see on your left the control rice variety,
00:15:38.16 called Taipei 309,
00:15:41.01 infected with the bacterial blight pathogen.
00:15:43.18 And what we do is simply dip scissors in a bacterial suspension,
00:15:47.01 clip off the top of the leaf,
00:15:49.19 and wait two weeks, and follow the lesion development.
00:15:52.10 And you can see on the right is the genetically engineered rice
00:15:56.17 carrying this gene, XA21,
00:15:58.27 from a wild rice species.
00:16:00.26 And this rice, now,
00:16:03.16 is completely resistant to disease.
00:16:06.21 So, what is this gene?
00:16:08.13 Well, it turns out that it's a receptor kinase.
00:16:11.02 So, it carries a leucine-rich repeat domain
00:16:15.05 on the outside of the cell,
00:16:17.13 a transmembrane domain,
00:16:19.12 and an intracellular kinase domain... kinase domain.
00:16:22.14 So, it's a receptor kinase
00:16:24.16 that senses the microbe outside the cell,
00:16:27.13 and then triggers a defense response inside the cell.
00:16:31.19 And this story became very interesting because
00:16:34.28 in 1996 the Toll receptor from Drosophila was isolated,
00:16:38.07 which had a very similar structure to XA21.
00:16:42.04 It had a leucine-rich repeat in the extracellular domain,
00:16:45.01 a transmembrane domain...
00:16:47.09 it had a small intracellular domain
00:16:50.06 called a Toll-Interleukin receptor domain.
00:16:52.22 And it associated with a kinase called Pelle.
00:16:57.16 In 199... oh, and I should mention
00:17:00.21 that this Toll-Interleukin receptor domain
00:17:02.29 is very similar to a domain that's present on a resistance gene in tobacco
00:17:08.07 called the N gene,
00:17:10.01 which you can see here.
00:17:11.17 It was discovered in 1994 in Barbara Baker's lab.
00:17:14.27 There was a nucleotide binding site, a leucine-rich repeat domain,
00:17:18.11 and a Toll-Interleukin receptor domain.
00:17:21.08 In 1998, TLR4 from mice
00:17:25.16 was isolated by Bruce Beutler’s lab.
00:17:27.19 It had an identical structure to the Toll receptor.
00:17:31.04 And you can see that in addition to the leucine-rich repeat domain
00:17:36.20 TLR4 also associates with an intracellular kinase called Rip1.
00:17:42.10 In the year 2000,
00:17:45.07 Arabidopsis FLS2 was isolated.
00:17:46.23 This is a model plant used for genetic studies in plant biology.
00:17:52.04 And it had an identical structure to XA21.
00:17:56.02 And in the year 2001,
00:17:58.15 the TLR5 receptor was isolated.
00:18:01.06 This is another mammalian receptor,
00:18:03.01 which not only has the same structure as TLR4 and Toll,
00:18:07.18 but it recognizes a very similar microbial molecule
00:18:11.26 as FLS2.
00:18:13.11 They both recognize a small peptide called flagellin.
00:18:18.15 So, we at this point we knew
00:18:21.25 that plants and animals use very similar strategies
00:18:24.05 for defending themselves against disease.
00:18:27.21 And another important point is that
00:18:31.04 although kinases are abundant
00:18:33.24 in both plant and animal genomes
00:18:36.02 -- there are about 500 in humans, about 1500 in rice --
00:18:40.06 there is a small subclass of these kinases,
00:18:44.06 called non-arginine-aspartate kinases,
00:18:47.17 that make up about 20% of the kinome in humans
00:18:51.18 and 25% in rice and Arabidopsis.
00:18:54.21 And it turns out that this class of kinases,
00:18:57.23 the non-RD kinases,
00:18:59.15 are critical for activation of the immune response
00:19:03.09 via these leucine-rich repeat-type receptors.
00:19:08.09 So, we know that immune receptors in plants and animals are remarkably similar.
00:19:14.15 And the discovery of these receptors
00:19:17.02 revealed a previously unknown mechanism of immunity
00:19:19.22 in both plants and animals,
00:19:21.16 and it was really a very exciting time
00:19:23.23 for both plant biologists and animal biologists
00:19:26.01 to see the merging of the fields of immunity.
00:19:32.17 So, I want to... that was a little history,
00:19:35.20 to bring you up to date to some of the types of things
00:19:37.23 we're doing to understand
00:19:40.00 the XA21-mediated immune response.
00:19:43.16 Now, we've used standard molecular techniques,
00:19:46.09 such as a yeast two-hybrid system,
00:19:48.11 which looks at protein-protein interaction.
00:19:50.13 We've done transcriptomics,
00:19:52.11 looking at changes in gene expression.
00:19:55.03 We can look in Arabidopsis or animals
00:19:58.05 and predict if similar proteins
00:20:02.10 transduce the rice defense response.
00:20:04.07 And we're carrying out several genetic screens
00:20:06.18 to identify other genes that are critical for this response.
00:20:10.23 As you can imagine,
00:20:12.25 these studies have led to a massive accumulation of data.
00:20:16.12 And we still have many gaps in our knowledge.
00:20:20.09 So, we needed help.
00:20:21.23 We turned to computational biologists
00:20:25.06 Edward Marcotte at the University of Texas, Austin,
00:20:29.01 and Insuk Lee, who is now at Yonsei University.
00:20:33.26 And they have been developing very interesting computational tools.
00:20:36.20 And they were able to develop a tool called
00:20:40.17 RiceNet, which collected 50 million data points
00:20:44.18 from 23 types of datasets and 5 different species.
00:20:49.12 And this allows us to identify genes
00:20:52.12 that function in the same biological pathway.
00:20:55.03 From this work, they were able to link
00:20:57.24 about 45% of the predicted 41,000 rice genes.
00:21:02.14 Now, what can you do with this type of model?
00:21:06.16 Well, what you can do is you can query it
00:21:09.01 with genes that you know are important for a response
00:21:11.15 and try to identify other genes
00:21:13.24 that function in the same pathway.
00:21:15.12 So, that's what we did.
00:21:16.23 We queried RiceNet
00:21:19.14 with genes that we knew were important for
00:21:22.00 the XA21-mediated response.
00:21:24.01 And those are the genes shown in red.
00:21:26.11 So, these are genes that we know, genetically,
00:21:28.22 are critical for the response.
00:21:30.16 And the balls, in green,
00:21:32.25 represent new genes that are predicted also
00:21:36.09 to be involved in this pathway.
00:21:38.22 This led to identification of hundreds of unknown genes,
00:21:43.17 and we can't analyze all of them.
00:21:45.22 So, we used a prioritization step
00:21:49.09 to try to figure out which ones we were most interested in.
00:21:53.11 So, what we did...
00:21:55.18 so, on the left, in green and white, are genes
00:21:58.24 that we had identified over the years that were important
00:22:01.08 for the XA21-mediated response.
00:22:03.13 And we decided that some of those network-predicted genes,
00:22:06.07 if they were truly important,
00:22:08.26 they might interact physically
00:22:12.28 with some of our other genes and proteins
00:22:15.10 that we had identified.
00:22:17.06 So, we carried out a pairwise protein-protein interaction test.
00:22:20.21 And we identified a set of about 12 genes,
00:22:23.21 shown here in yellow,
00:22:25.27 that encode proteins that interact
00:22:28.16 with some of our other very important proteins in the pathway.
00:22:32.26 And then we picked five of these to analyze genetically.
00:22:36.12 Because through genetic analysis,
00:22:38.19 by knocking out expression or overexpressing the gene,
00:22:42.04 you can try to understand if alteration in gene expression
00:22:47.13 affects the defense response.
00:22:50.00 And we've identified three new regulators
00:22:52.29 of XA21-mediated immunity
00:22:55.08 via this approach.
00:22:57.00 And so they were first predicted computationally by RiceNet;
00:23:01.17 and they were prioritized using a protein interaction assay;
00:23:05.21 and then they were validated in whole plants
00:23:08.14 using an Xoo infection assay.
00:23:12.14 Now, these genes are just...
00:23:15.19 we're just beginning to characterize them.
00:23:17.07 The first one is called ROX1,
00:23:20.06 which is a thiamine pyrophosphokinase,
00:23:23.04 which interestingly enough has also been implicated
00:23:26.04 in some types of human brain disease.
00:23:29.12 So, studies in rice can lead to the identification of genes
00:23:33.17 that may also be important
00:23:36.14 for controlling the disease response in humans.
00:23:40.23 Similarly, ROX2, which is a NOL1/NOP2 domain family protein
00:23:47.13 has been implicated in a childhood disease
00:23:49.28 called the Williams-Beuren syndrome.
00:23:53.19 And ROX3 is a nudC nuclear movement protein,
00:23:58.26 and orthologs in animals have been shown to be important
00:24:04.00 and involved in tumor proliferation.
00:24:09.04 So, where are we with XA21-mediated immunity?
00:24:12.05 We know a lot more than we knew 15 years ago,
00:24:15.14 but there's still a lot to be discovered.
00:24:18.00 So, we believe that XA21
00:24:21.19 is synthesized in the endoplasmic reticulum
00:24:24.29 with a chaperone called BiP3.
00:24:28.00 And we've recently shown that the XA21 receptor kinase
00:24:33.00 associates in vivo with another co-receptor kinase
00:24:36.19 called SERK2, which is critical for function
00:24:40.27 of this XA21-mediated response.
00:24:45.00 There's another protein called XA21 binding protein 24,
00:24:49.02 or Xb24,
00:24:50.19 which encodes an ATPase
00:24:53.11 that binds with XA21 in vivo
00:24:55.21 and promotes autophosphorylation
00:24:58.20 of the XA21 kinase domain.
00:25:00.11 Now, autophosphorylation on these particular amino acids
00:25:03.13 keeps XA21 in a resting state.
00:25:07.15 Upon pathogen infection,
00:25:09.25 the Xb24/XA21 complex releases,
00:25:14.04 and we then can activate the immune response
00:25:17.07 via several other proteins, such as Xb3
00:25:21.28 and a new protein called a receptor-like cytoplasmic kinase
00:25:27.18 that my collaborators Xuewei Chen and his lab have recently discovered.
00:25:33.05 And this is thought to trigger a MAP kinase cascade.
00:25:36.25 We've also identified a group of transcription factors
00:25:39.26 called WRKY transcription factors
00:25:42.04 that are similar to the mammalian Rel-related transcription factors
00:25:45.23 that are critical for regulating this response,
00:25:51.16 eventually leading to the immune response.
00:25:53.29 And this whole process can be negatively regulated
00:25:56.18 by a phosphatase called Xb15.
00:26:00.25 So, one of the important projects in my lab
00:26:02.28 is to try to identify the microbial molecule
00:26:05.20 that activates XA21-mediated immunity.
00:26:09.23 So, in other words, what is the micromolec...
00:26:12.05 microbial molecule detected by the rice XA21 receptor?
00:26:16.15 And for this, we've started to work on the bacterial...
00:26:20.01 to carry out bacterial genetics.
00:26:22.26 And the goal is to identify Xanthomonas genes
00:26:26.08 that are required for activation
00:26:28.29 of XA21-mediated immunity.
00:26:31.23 And we called these genes rax genes.
00:26:34.05 And now I'll briefly tell you about two groups of rax genes
00:26:37.07 that we've identified.
00:26:39.00 raxA, B, and C are genes predicted to control type 1 secretion.
00:26:44.27 And the genes raxST, raxP and Q
00:26:48.12 are genes predicted to control sulfation.
00:26:52.24 First of all, raxA and B are in a small operon.
00:26:55.28 And they encode predicted components
00:26:59.24 of a type 1 secretion system.
00:27:02.18 That is, they work together with another protein
00:27:06.07 to form a pore where molecules
00:27:08.18 are secreted from inside the bacterial cell
00:27:10.29 to outside the bacterial cell.
00:27:12.25 And raxB encodes a particular type of
00:27:15.27 ATP-binding cassette transporter
00:27:18.29 that has a C39 peptidase domain on the N-terminus,
00:27:23.19 which is predicted to cleave a leader sequence
00:27:26.28 from a small peptide and then,
00:27:30.04 after cleavage of that sequence, it's secreted outside the cell.
00:27:33.24 Now, these... RaxA and B orthologs in other species
00:27:38.17 are predicted to function with a third protein
00:27:42.29 called an outer membrane protein.
00:27:44.28 And through orthology search with E. coli,
00:27:47.19 we were able to identify such a protein in Xanthomonas.
00:27:51.04 When we make mutations in either raxA, B, or C,
00:27:55.19 the bacteria are impaired in their ability
00:27:57.28 to activate XA21-mediated immunity.
00:28:02.21 We've also identified genes predicted to control sulfation,
00:28:06.24 and those are called raxPQ and raxST.
00:28:09.26 And these genes work together
00:28:12.17 to add a sulfur residue to a substrate.
00:28:16.05 So, for example, raxP and Q
00:28:19.16 work together to encode an ATP sulfurylase.
00:28:23.03 And they convert sulfate to adenosine phosphosulfate.
00:28:27.29 And then raxQ encodes an APS kinase,
00:28:30.29 which converts APS to phosphoadenosine phosphosulfate.
00:28:35.23 Now, this molecule, PAPS, is critical for the...
00:28:41.05 as a substrate for the action of the sulfotransferase RaxST,
00:28:45.19 which can use the PAPS to catalyze sulfation of a target substrate.
00:28:53.05 So, our model now is that
00:28:56.09 a secreted, sulfated peptide
00:28:58.22 activates XA21-mediated immunity.
00:29:01.09 And that's shown here.
00:29:03.05 Again, RaxP and Q
00:29:05.27 work together to form phosphoadenosine phosphosulfate.
00:29:09.23 And that sulfate is utilized by the RaxST sulfotransferase
00:29:13.27 to transfer a sulfate to a predicted tyrosine residue
00:29:19.14 on an unknown substrate
00:29:22.04 that we're now trying to identify.
00:29:23.29 And that sulfated substrate
00:29:26.08 is secreted outside the bacterial cell
00:29:28.07 via the RaxABC type 1 secretion system.
00:29:32.13 And we're also very interested in this
00:29:34.29 because similar systems have been shown
00:29:37.14 to be very critical in agriculture.
00:29:39.08 So, for example, Sinorhizobium meliloti
00:29:42.13 is a gram-negative bacteria,
00:29:44.15 and it sulfates what's called the Nod factor,
00:29:47.24 which is a lipochitooligosaccharide
00:29:50.25 which is required for specific recognition by its host, alfalfa.
00:29:55.09 And this process establishes the ability of the microbe
00:30:00.16 to fix atmospheric nitrogen,
00:30:03.07 which is very critical for agricultural production.
00:30:07.05 We also know that in the HIV virus,
00:30:14.19 for attachment to human cells, it requires the CCR5 co-receptor.
00:30:18.12 And this co-receptor must be sulfated.
00:30:20.22 That's a key recognition of the HIV envelope glycoprotein.
00:30:26.16 And humans that don't have CCR5
00:30:29.14 are completely resistant to HIV.
00:30:32.22 And finally, we know in carrots
00:30:35.25 that a very small sulfated peptide called phytosulfokine
00:30:39.25 binds to carrot receptor kinase,
00:30:42.22 which controls cellular de-differentiation and proliferation.
00:30:46.22 So, we know, then, that sulfated molecules generally are directed outside the cell,
00:30:51.20 where they modulate cell-cell interactions.
00:30:55.18 So, a few years ago,
00:30:57.15 my laboratory established a collaboration
00:30:59.25 with Leena and Jaindra at Tripathi
00:31:02.03 at the International Institute of Tropical Agriculture in Kenya.
00:31:05.19 And we were very interested in trying to develop methods
00:31:09.04 to control disease of banana.
00:31:12.10 100 million people in East Africa
00:31:14.23 rely on banana as a staple food source.
00:31:19.23 And one of the devastating diseases of banana
00:31:22.27 is called banana Xanthomonas wilt.
00:31:25.14 And you can see here a cross-section of a banana,
00:31:29.14 and the bacteria losing out of the stem.
00:31:33.05 This bacterial infection attacks all the banana varieties.
00:31:37.14 There's no known genetic resistance.
00:31:39.26 And there is no way to control the disease.
00:31:43.06 Once the plant is infected, it leads to complete crop loss.
00:31:46.28 The methods to prevent infection
00:31:49.06 are very laborious and crude.
00:31:50.29 Farmers need to clip off the buds on the banana plants
00:31:55.02 and use very clean tools.
00:31:57.11 However, once the infection progresses,
00:31:59.27 the farmer has no choice but to either
00:32:04.10 burn the entire field or destroy the crop using an herbicide,
00:32:07.02 and start again.
00:32:08.16 So, scientists around the world are trying to develop
00:32:11.15 methods to control this disease.
00:32:14.06 Now, we discovered recently
00:32:17.19 that the bacterial genes in the rice pathogen that we were studying
00:32:22.29 are very similar to genes that are present in the banana pathogen,
00:32:25.18 called Xanthomonas campestris pathovar musacearum.
00:32:29.02 Now, this similarity of genes
00:32:32.23 suggested to us that the banana pathogen
00:32:35.04 may also produce a molecule
00:32:38.03 that's critical for activating the immune response
00:32:40.21 in the presence of the XA21 rice receptor.
00:32:44.13 So, we carried out a simple experiment.
00:32:48.07 Leena and Jaindra introduced the rice XA21 gene
00:32:52.25 into banana.
00:32:54.13 And they then took those bananas
00:32:58.10 and inoculated them with the bacteria that causes banana Xanthomonas wilt disease.
00:33:03.02 And they've recently shown that bananas...
00:33:06.16 this is the result of their experiments.
00:33:08.07 On the left, you can see a banana
00:33:10.16 that's completely susceptible to the disease.
00:33:13.02 And on the right are their genetically engineered bananas.
00:33:16.10 These are two independently transformed lines
00:33:18.12 -- and they've generated six lines --
00:33:21.11 that are completely resistant to disease.
00:33:25.14 So, we're very excited about these results,
00:33:27.25 that a simple experiment
00:33:29.26 -- putting a rice gene in a banana --
00:33:31.26 can have such a dramatic effect.
00:33:34.00 Now, if these experiments can be replicated in the field,
00:33:36.28 this will be, we believe,
00:33:39.12 a tremendous advance for farmers in Eastern Africa.
00:33:42.20 Now, putting a rice gene in a banana
00:33:46.03 has not been done before.
00:33:48.03 And many consumers question
00:33:51.05 whether tampering with nature in this way
00:33:53.14 can create some types of unintended consequences.
00:33:56.04 And so this question is something that
00:33:59.19 National Academies, scientific academies around the world,
00:34:02.28 have looked at over the last 20 years.
00:34:06.18 And over these 20 years,
00:34:09.01 we've accumulated a lot of scientific evidence and data.
00:34:13.02 So, I would like to just summarize the scientific consensus.
00:34:16.29 And this is based on 2 billion acres
00:34:19.24 of genetically engineered crops
00:34:21.18 and 20 years of these crops planted.
00:34:25.00 And the consensus is that the crops currently on the market
00:34:27.27 are safe to eat.
00:34:30.01 That there are clear environmental benefits,
00:34:32.15 and that's mainly relying on the studies of cotton, Bt cotton,
00:34:37.24 which has massively reduced sprayed insecticides.
00:34:42.11 And their predicted health benefits,
00:34:44.25 and that is because of golden rice,
00:34:47.07 which we can talk about later,
00:34:50.16 that's expected to be released later this year or next year,
00:34:52.22 which is enriched for vitamin A.
00:34:55.07 All processes of genetic alteration,
00:34:57.08 whether it's a conventional approach
00:34:59.28 or more modern approaches of marker-assisted breeding
00:35:02.11 and genetic engineering,
00:35:04.22 present some risk of unintended consequences.
00:35:07.00 So, there's not a situation where you have no risks.
00:35:10.00 But those risks must be weighed with the benefits.
00:35:13.13 We know that over-reliance on a single herbicide
00:35:15.28 will foster herbicide resistance in weeds.
00:35:18.14 So, there's big efforts around the globe
00:35:21.03 to try to develop integrated management for weed control.
00:35:25.09 We know that each genetically engineered crop
00:35:28.16 must be evaluated on a case-by-case basis.
00:35:31.12 There's no scientific reason
00:35:35.11 to group all genetically engineered crops together.
00:35:38.22 Every crop is different; every trait is different.
00:35:41.15 So, these are the conclusions of
00:35:44.20 the European Food Safety Authority,
00:35:46.15 which is the highest authority for food safety in Europe,
00:35:50.07 as well as the World Health Organization,
00:35:52.20 and the American Association for the Advancement of Science,
00:35:56.27 as well as every other scientific organization
00:36:00.05 that has examined genetically engineered crops.
00:36:04.08 And I'll show you some of these organizations here.
00:36:11.05 So, I'd like to conclude with a subject that's
00:36:13.25 important to my husband, Raul Adamchak, and myself,
00:36:16.26 which is, how is the best way to enhance sustainable agriculture?
00:36:20.05 So, he's been an organic farmer for 30 years.
00:36:22.26 And here he is at the University of California campus,
00:36:25.26 where he runs an organic farm
00:36:29.14 and a teaching program for students.
00:36:32.01 And many people think that a geneticist and an organic farmer
00:36:36.07 rest on opposite ends of an agricultural spectrum.
00:36:41.02 But we don't. Because we have... we have the same goal,
00:36:44.10 which is an ecologically based food system.
00:36:48.15 Still, many of our friends and family have asked us
00:36:51.26 if genetically engineered crops are safe to eat,
00:36:54.09 and whether organic farming can feed the world.
00:36:57.29 So, in response to those questions,
00:37:00.17 we wrote a book called Tomorrow's Table:
00:37:02.29 Organic Farming, Genetics, and the Future of Food.
00:37:05.24 And what we tried to do in this book is distinguish between fact and fiction
00:37:09.23 on the discussion on crop genetic engineering,
00:37:13.14 and also bring readers into the lives of
00:37:16.15 what an organic farmer does every day
00:37:18.23 as well as a geneticist.
00:37:21.16 And we believe that it's important to advance sustainable agriculture
00:37:25.14 using the most appropriate technologies.
00:37:28.02 And sustainable agriculture rests on three pillars:
00:37:32.06 the social, economic, and environmental consequences
00:37:35.07 of a particular crop production system.
00:37:38.08 Rather than focusing on how a seed was developed,
00:37:41.27 we need to ask important questions as to,
00:37:45.08 who benefits from a particular technology?,
00:37:47.24 what are the economic impacts on a farmer's ability
00:37:52.29 to sell their produce and produce their food and feed their families
00:37:56.26 and to enhance local food security?,
00:37:59.05 and importantly, what are the environmental effects
00:38:02.05 of each new technology or farming practice?
00:38:04.24 We need to reduce the amount of toxic inputs into the environment
00:38:09.29 -- and that includes some insecticides and some herbicides --
00:38:13.12 as well as conserving land and water,
00:38:17.05 and fostering soil fertility.
00:38:20.19 Thank you.
00:38:22.03 And I'd like to acknowledge several of my sponsors,
00:38:24.29 as well as a really dedicated and wonderful team
00:38:29.15 and collaborators.
00:38:31.04 There are many people in my laboratory that worked on isolation of the XA21 gene
00:38:35.06 and isolation of the Sub1 transcription factor.
00:38:38.21 That work would... was very...
00:38:41.17 David Mackill and his group were very critical for this work.
00:38:45.02 Yonsei University, as well as University of Texas, Austin,
00:38:51.15 my collaborators Insuk Lee and Edward Marcotte and their team,
00:38:55.06 were critical in developing the computational predictions.
00:38:58.10 And my collaborators at UC Riverside
00:39:00.29 had wonderful contributions to understanding the mechanism of Sub1 action.
00:39:05.19 And my recent collaborators,
00:39:08.15 Jaindra and Leena Tripathi at the International Institute of Tropical Agriculture,
00:39:12.06 have engineered banana to carry the rice XA21 resistance gene.
00:39:16.13 So, thank you very much for your time.

This Talk

Speaker: Pamela Ronald

Audience:

Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: September 2010

Talk Overview

Part 1: Sustainable Agricultural Practices: Organic Farming, Genetics, and the Future of Food

Ronald discusses one of the greatest challenges of our time: how to feed the growing population in the presence of disease and environmental stresses that threaten the world’s crops. Currently, twenty-five percent of the world’s rice is grown in flood-prone areas. Ronald and her colleagues characterized a gene, Sub1A, that confers tolerance to two weeks of flooding. They demonstrate that transferring Sub1A to a highly intolerant rice species is sufficient for the crop to tolerate submergence in water. Ronald shifts gears to discuss another gene, Xa21, that she and her colleagues discovered that controls the rice immune response. Ronald hypothesizes that Xa21 is activated by a sulfated peptides derived from the infecting bacteria.

Speaker Bio

Pamela Ronald

Pamela Ronald is Professor of Plant Pathology at the University of California, Davis, where she studies the role that genes play in a plant’s response to its environment. Her laboratory has genetically engineered rice for resistance to diseases and flooding, both of which are serious problems of rice crops in Asia and Africa. She also… Continue Reading