Pamela Ronald, University of California, Davis

Pamela Ronald, University of California, Davis

October 13, 2019 0 By Stanley Isaacs


Thank you, Michael. It’s a great honor to
join you this morning and to participate in this very informative and exciting
symposium. As Michael and other speakers have mentioned, one of the greatest
challenges of our time is to feed the growing population without further
destroying the environment. One of the challenges facing agricultural
productions are the predicted effects of climate change. We think of climate
change as causing problems with droughts and heat. But the intergovernmental panel
on climate change has also pointed to the devastating effects of flooding.
See these boys walking through the rice field in Bangladesh.
Rice does grow in water. But if rice plants are completely submerged for more
than three days, most rice varieties will die. This is a very severe hardship in
many places in the world, such as Bangladesh, that rely on rice for two-thirds of their
calories and many people living on less than one dollar a day. Breeders, rice
breeders, have known for a long time – for 50 years – that there was an unusual
variety of rice that was found in Eastern India that had a very unusual
property. It could survive flooding for two weeks. And after the flooding was
gone, the plant could grow again. Now, this area shown in the red circle is where 25
percent of the world’s rice is grown and this region is flooded regularly. In the
last four years that floods have been increasing, likely due to the predicted
effects of climate change. In Bangladesh and India alone, four million tons of
rice – enough to feed 30 million people – is lost to flooding. Using conventional
breeding approaches, scientists tried to introduce this trait from this old
variety into modern varieties. But it’s a complex trait and the
resulting varieties were not acceptable to farmers. Likely because there was a
mixture of different genes that were brought in through the conventional
breeding process. A collaborator of mine David Mackill, who was at the University
of California-Davis with me and later moved to the International Rice Research
Institute, has studied the genetic basis of this trait, and we decided to try to
isolate the gene using a positional cloning approach. The idea was that if we
could isolate this gene, we could introduce it into locally adapted rice
varieties using either genetic engineering or marker-assisted breeding.
We were able to isolate this gene. We named it Submergence Tolerance Sub1A.
And this shows you an experiment carried out in my lab. On the left is a
control and on the right are two independently transformed Sub1A
transgenic lines. And you could see before the flood, they both look very
good. But 16 days after complete submergence, the controls are very sickly.
They have long leaves. The leaves have elongated quickly. They’re chlorotic and
two weeks after the flood, after the flood is removed, the control is dead.
Whereas the transgenic line survived. It was this experiment that allowed us to
confirm that Sub1A indeed was the gene encoding submergence tolerance. Now if
the project stopped here, it would have stayed in the laboratory and that would
not have benefited anybody. So my collaborator David Mackill took a
marker-assisted breeding approach to introduce this gene in two varieties
that were favored by farmers in India and Bangladesh. This shows you one of the
field trials at the International Rice Research Institute. And what it is is:
It’s a time-lapse video. So you’ll be able to see the difference between the
submergence tolerance variety and the control variety. So on the left
is the new variety containing the Sub1 gene. And you could see, both
varieties are growing quite well. And then a flood comes for 17 days, and you
can see the the growth after the flood. I like to show this slide, because it shows
the power of genetics. That a single gene can have the power to
enhance productivity. So in this field trial, the yields were threefold more
than the conventional variety after flooding. Dave took this a step
further. He carried out four years of field trials in Bangladesh. And these on
top, the team visited, in India and Bangladesh, the field sites a
few years ago. On the top, you can see Swarna-Sub1, which is the new variety
developed at the International Rice Research Institute. And on the left is
Swarna. This is a natural flooding situation. And on the bottom are some
examples in eastern India and Bangladesh. In farmers’ fields, the yields are three
to five fold higher after flooding. So this has been a very exciting and
successful project. This year it’s estimated that four million farmers are
growing submergence tolerance rice. So I want to turn to another topic. I want to
talk about engineering crops for resistance to disease. This is a very
severe disease of rice called xanthomonas oryzae pv. oryzae. It’s
call bacterial blight disease. You can see the bacteria oozing out of the leaf.
And not only plants are susceptible to disease, but so are flies. This is a fly
infected with aspergillus fumigatus. And humans. This is a baby that is highly
infected with a bacteria that causes menococcal sepsis when the bacteria
becomes invasive, it is a devastating disease. Now, research
over the past 15 years has shown that plants and animals use the same
mechanisms to resist disease. So I want to say that one way to control disease
is to spray chemicals. As we all know, some chemicals really serve to create a
better life. But some can be quite toxic. And especially in less developed
countries, where farmers don’t have the safety materials to use a
chemical safely, they can have severe consequences. Here’s a guy. He’s smiling
for the camera. No gloves, no mask. He spraying his foot. And in the less
developed world, it’s estimated that 300,000 people die every year from
pesticide poisonings. So there is a great need to develop alternatives, especially
in less developed countries. Gertev Khush, who is a emeritus professor at UC
Davis, who spent his career with the International Rice Research Institute
and won the World Food Prize several years ago for his work in Green
Revolution rice … he’s an excellent breeder. And he was in contact with
breeders about 30 years ago that had identified a wild species of rice that
had an unusual property. It was completely resistant to all races of the
bacterial pathogen. And you have to remember, at this time, even though
breeders had been using genes for resistance for a hundred years, no one
knew what these genes were. So we were very … I was very interested in this gene.
So I moved to Cornell and started to map this gene. And when I was at Davis, I
was able to isolate the gene called XA21. This is a typical experiment.
On the left are two leaves from a susceptible line, and on the right are the
genetically engineered lines expressing the resistance gene XA21. You can
see you have very robust resistance in the genetically engineered
lines. So what is XA21? XA21 is a receptor kinase. So it has a leucine-rich
repeat on the extracellular domain and a kinase in the intracellular domain.
This gave us a very nice model. We could imagine that the extracellular domain
detected a microbe, a molecule from the microbe, and that would signal the
intracellular kinase domain for defense signaling. The next year, the TOLL
receptor from flies was shown also to have a very important role in disease
resistance. And strikingly, it had a very similar structure, a leucine-rich repeat
domain, a transmembrane domain, and it associated with an intracellular kinase
domain. This intracellular domain has similarity with a gene isolated in
Barbara Baker’s lab called the N gene. And so at this point, it was a lot of
excitement in the field, because we could see that plants and animals use the same
domain structures to confer disease resistance. In 1998, the TLR4 receptor was
isolated in Bruce Beutler’s lab. And TLR4 was shown to be critical for the immune
response in mice. It had that exact same structure as the fly TOLL receptor. In
the year 2000, the arabidopsis FLS2 protein was isolated and it has the same
structure as the XA21 receptor kinase. So you could see the plant proteins are
a single protein, where the animal proteins associate with the kinase in
two separate molecules. TLR5 was isolated also from animals. And it
had the same structure as TOLL and TLR4. But very interestingly, it recognizes a very
similar ligand as FLS2. Both FLS2 and TLR4 recognize flagellin. We made a
another observation a few years ago. The kinome is
the biggest gene family in most organisms. In humans, there’s 500
kinases. In rice, there’s 1,500 kinases But a very small subset of those
kinases have this particular motif called the non arginine aspartate motif.
And it turns out that all the kinases associated with these receptors – often
called pattern-recognition receptors – carry the similar kinase domain. So
we think that these non-RD-kinases could be good targets for developing agonists
or antagonists to suppress or activate these responses. Now the conclusion is
that immune receptors and plants and animals are remarkably similar. It’s a
very exciting time, both in plant and animal biology, that we now know that the
mechanisms are so similar. In 2011, the Nobel Prize in Physiology and
Medicine went to Beutler and Hoffman for their very important discoveries in
animals. Discovering an entirely new immune assist immune system in animals. So
for many years, we’ve been trying to identify the microbial molecules that
activate XA21-mediated immunity and to identify the ceiling components that
transduce the response. I’ll just give you a brief summary. We’ve used many
different approaches: Yeast Two-hybrid screening, transcriptomics, forward
genetics. And what we’ve ended up with is a lot of data. And so it became very
challenging for us to integrate the data and pick those genes that were going to
be the most important in this response. So to help us with this project,
I turned to a computational biologist, Edward Marcotte and his postdoc Insuk
Lee, who’s now at Yonsei University. They had been developing computational
tools to predict gene function. So they developed a computational network called
RiceNet. And this is very exciting, because it
allows you to integrate diverse genetic information into one cohesive
predictable model. They gathered 50 million data points and they used 23
types of data from five different species. Because the innate immune
response is conserved between plants and animals, they were able to use data sets
from different organisms. From this, they were able to link 45% of
the total 40,000 rice genes. We recently developed a new version that
covers 80% of the rice genome. So these linkages are not necessarily
protein-protein interaction linkages. But these linkages are genes that are highly
predicted to function in the same biological pathway as the other genes in
the network. To make this kind of network useful, you need to query the network
with your genes of interest. So that’s what we did. We took 15 genes that we
knew were critical for XA21 mediated immunity and we queried the network.
What you can see here in red are the genes that have been validated
genetically to be important for the immune response. And in green are the
other genes that are predicted to be involved in the same biological process.
We are now slowly making our way through this data set. What we do then is
take the genes, we silence them and we overexpress them in rice plants. From
this, we were able to isolate, so far, three new regulators of XA21 mediated
immunity that we call ROX genes. They were predicted, highly predicted by
RiceNet to function in the immune response. Then we prioritized them
using some various plate assays. We used two hybrid systems to prioritize those that
we wanted to put into plants. Because still, rice at transformation, although a
simple technique, is still very time-consuming. And these, one of the
genes – ROX1 thiamine pyrophosphokinase – has some interesting properties.
Not only does it confer disease resistance, it enriches rice for
vitamin B synthesis. So this is a project we’re now working with the Gates
Foundation to try to engineer resistance to disease and enhance vitamin content.
Interesting, though: This gene or human orthologues have been implicated in some
types of human diseases. We’ve also isolated ROX2, which is a novel gene
that looks like it’s involved in methylation. Also, orthologues of this
gene have also been implicated in another human disease. And finally, ROX3
is a gene that’s highly conserved and involved in tumor proliferation. So
this type of network analysis cannot only identify genes important for rice
but as well as in other organisms. And I just want to quickly … we have
evidence that XA21 interacts with another receptor kinase, and we
believe that process is triggered and leading to … we’ve identified several
other genes in the pathway that activate this response. And we have evidence that
that the kinase moves into the nucleus. Okay. So, just to finish up, I want to
mention that we have a project funded by the National Science
Foundation to take RiceNet, prioritize 200 genes – and we’re aiming for genes that
have not before been characterized in plants or animals – and then move those
genes into wheat and barley, to see if we can translate the knowledge on the
immune response in rice to species that are more difficult to work with.
Wheat and barley. So finally, we are using the information on rice to try to
engineer resistance in other species. Banana is a very important staple food
source. It’s the fourth staple food crop. In East Africa, 100 million people rely
on banana for their staple food. Banana Xanthomonas Wilt
is a serious disease. Here is a banana cut in half. And you can
see the stem oozing. This attacks all banana varieties and there’s no known
method to control this disease right now. What farmers do if the field is infected:
They burn the field. So there’s a great need to develop methods to control this
disease. We decided on a simple approach to transfer the XA21 gene into banana.
This work was carried out by Leena Tripathi at the International Institute
of Tropical Agriculture in Africa. These are her recent results. This is the
control banana line infected with the disease, and these are two independently
transformed banana lines expressing XA21. You can see that there’s very robust
resistance to this disease by expression of the rice gene. So is this safe to eat?
I was asked to just briefly talk about the political situation. Obviously
putting a rice gene in banana is not something that’s going to happen in
nature. But is it safe to eat. Now, most of you are probably aware
that the scientific consensus is clear, after 2 billion acres and 20 years of
genetically engineered crops planted, that all the crops currently on the
market are safe to eat. There are clear environmental benefits – we could talk
about that later – and predicted health benefits. For example, golden rice,
expected to come out this year. Importantly, all processes of genetic
alteration, whether it’s conventional or genetically engineered, present some risk.
But the risks are similar between the two approaches. Each crop must be
evaluated on a case-by-case basis. And it’s not only your European Food Safety
Authority that has come to this conclusion, but every single
scientific society that has examined this issue. Still there are many
consumers that are worried about genetically engineered crops. And the
organic farming community is one of those communities that often questions
genetic engineering. This is my husband, Raoul Adamchak, on the farm at UC Davis.
He’s been an organic farmer for 35 years and he
teaches organic farming production and marketing. So you may think that a
geneticist and an organic farmer represent polar opposites of the
agricultural spectrum. Some people think we don’t even talk to each other. But we
do, and it’s not difficult, because we have the same goal, which is an
ecologically based system of agriculture. Still, over the years,
our friends and family and colleagues have asked us: Are genetically
engineered crops safe to eat? Can organic agriculture feed the world? So we
wrote this book to respond to those questions. And really, our idea is to
give readers a better idea of what geneticists do and a better idea of what
farmers do. And also, to distinguish between fact and fiction on the debate
on crop genetic engineering. To conclude, Raoul and I believe that the
discussions of agriculture must be focused on the context of the
environmental, economic and social impacts of agriculture, which are really
the three pillars of sustainable agriculture. Rather be than being
distracted by the process with which the seed is made, we need to really
think about how the new crops can enhance local food security and can
provide safe, abundant and nutritious food to consumers. And if we can allow
farmers to make a profit, if consumers can afford the food, and if we can reduce
the negative environmental impacts. So we believe that we need to incorporate both
the most modern forms of genetic seed improvement with agroecological
farming methods. So, thank you very much. I appreciate your time.