genetics

#IAmScience: Ajay Gupta

July 1, 2022 By Penquite, Cara (MU-Student) in #IAmScience Tags: CRISPR, genetics, plant science, rice

By Cara Penquite | Bond LSC

#IAmScienceTemplate copy — Photo by Cara Penquite | Bond LSC

Ajay Gupta learned biology basics as a first year undergraduate on the bumpy bus ride from his small hometown to Punjab Agricultural University. Just a few hours’ ride, he made the most of his time before he returned home to help his family’s agricultural goods business.

Working extra hours in the margins of his time has become a habit for Gupta. Now a plant science first year Ph.D. student in the Bing Yang lab and Department of Plant Science and Technology Millikan Endowment recipient, Gupta starts his day around 9 a.m. and works on three plant science projects until night.

“My advisor has to sometimes push me out of the lab to go home,” Gupta said. “I have that fascination with science.”

Gupta5 — Gupta grows bacteria that carry recombinant DNA harboring CRISPR-Cas9 system. This DNA will be inserted into rice plant cells and the rice plant cells will duplicate with modified DNA. Photo by Cara Penquite | Bond LSC

For his Ph.D. thesis project, he studies how rice plants fight off bacterial leaf blight — a disease caused by the bacterium Xanthomonas oryzae. This disease can devastate rice fields in Asia and other areas that depend on the staple grain. His research focuses on genes that help plants resist the deadly disease.

“The second project, which is very close to my heart, … is the development of CRISPR genome editing toolkit,” Gupta said.

Gupta works to improve the gene-editing tool CRISPR-Cas9 in rice plants. Researchers currently modify chunks of DNA sequence with CRISPR, but Gupta’s goal is to modify the individual building blocks of DNA known as nucleotides. He simplifies the process to make gene editing easier for researchers with less CRISPR experience.

Gupta1 — Gupta shows three stages of growing genetically modified plants. The plants start as cells called calluses, infected by agrobacteria that inserts the modified DNA into the cell. The cell duplicates the modified DNA as it multiplies into small groups of cells that are all genetically modified. Photo by Cara Penquite | Bond LSC

His third project started with self-taught skills rooted in a side passion. Gupta contributes to a global bioinformatics project investigating bacterial resistance genes in rice. By understanding the genetic make-up of plant resistance to disease, the researchers may find innovative solutions to prevent crop infections. Gupta’s input involves assembling the genome of a type of rice that is resistant to the pathogen that causes bacterial blight in rice.

“It’s a global project where everyone across the globe who uses that variety [of rice] can take advantage of that genome,” Gupta said.

Most of Gupta’s time is spent in the lab now, but Gupta started studying biology by accident. As an undergraduate he decided to major in agricultural biotechnology thinking there would be more math involved.

Gupta2 — The genetically modified plants grow roots as the cluster of cells forms stalks of rice. Photo by Cara Penquite | Bond LSC

“It mentioned that it will have some math and some biology, and I knew nothing about biology,” Gupta said. “I said, ‘OK, it has math, I should go for it.’”

For the first year Gupta had six biology classes and one math class each semester.

“I said, ‘Oh my god, how am I going to survive,” Gupta said.

Gupta worked through basic biology courses in subjects like genetics, botany and zoology alongside his math courses. Then the math courses dropped off for the following years leaving Gupta’s schedule packed with biology classes.

“And then I started liking it,” Gupta said. “It seems more practical to me than math. I think if I would have gone to math I would have been in engineering, but I’m happy that I chose this path, and right now I’m working with plants which I love.”

Gupta4 — To verify that the plants contain the modified DNA, Gupta mixes the plants’ roots with chemicals. The roots turn blue in plants with modified DNA but remain their natural color if they do not contain modified DNA. Photo by Cara Penquite | Bond LSC

Gupta’s small high school in his agricultural hometown left out many biology basics, but without the finances to attend a better school in another city Gupta was left to catch up at the university level while also helping his older brother at home.

“It was very challenging for the first year. Our financial situation was not great, so I had to support myself for my studies,” Gupta said.

Gupta returned every day to work as an accountant at his family’s small agricultural goods store to earn money for his education and to help.

“My father passed away when I was 15, and me and my brother who was 18 at that time, took care of our business without having much experience. So, I had to ride the bus four hours every day to go there and help my brother during my first year in bachelor because we could not afford at that time to hire somebody” Gupta said.

Back on their feet by his second year, Gupta continued working on weekends throughout the rest of his studies, and he only separated from the business when he left for his masters’ studies halfway around the world at South Dakota State University.

Gupta initially stayed with friends in South Dakota to soften the transition to the United States. Although difficult to live an ocean away from family, Gupta maintains relationships with his family.

“I talk with my mom almost every day. That’s a kind of an Indian or Asian thing. We are very close to our parents,” Gupta said.

Gupta started CRISPR research while working on his master’s degree at South Dakota State University. He focused on modifying wheat genes to increase yield and diversity, but on the side he also found a passion for bioinformatics.

“Most of this was self-learning, so I just found things on the internet and found out how they work and then made my own protocols and now have the pipeline,” Gupta said.

After this taste of research, Gupta was hooked. He applied to continue his studies as a Ph.D. student and asked Bing Yang, a collaborator with the lab’s wheat genetics research, for a letter of recommendation.

Instead, Yang asked Gupta to join his lab.

Gupta accepted his offer to continue working on CRISPR alongside expanding to more basic research — a change of pace from his previous work in applied agriculture.

“I want to develop a mindset where I can work on [basic science] well where there is little or no information and then I can think on my own,” Gupta said.

Gupta is the primary bioinformatics researcher in his lab and helps with data analysis for several projects alongside his bioinformatics project. While he spends most of his time hard at work on a research bench — and can often be seen walking across the Bond LSC atrium to the greenhouses late into the evening and over holidays — he also enjoys talking with lab mates.

Gupta3 — In the Bond LSC rooftop greenhouses, Gupta grows the genetically modified rice plants at controlled conditions for optimum growth. Photo by Cara Penquite | Bond LSC

“Sometimes, post [research] talks, we sit together and then I discuss with them if they have some problem I can try to help, and I do the same as well. If I’m having some problem I talk to them,” Gupta said.

Gupta plans to stay in academia and continue research and teaching others. He enjoys mentoring other students and also works on outreach to high school students.

“I love to do those things and talk to students, and maybe in some ways help this university as well,” Gupta said.

Gupta holds on to the inquisitive spirit that drove him to spend hours studying basic biology on the bus despite now learning in advanced research facilities.

“I love to talk with people [to] find out what they’re doing and if I can learn something out of it,” Gupta said. “Because a lot of the time our knowledge is limited and when talking to people you can get something new.”

Protecting Plants: Researchers identify genes responsible for vital antimicrobial natural products

March 3, 2022 By Penquite, Cara (MU-Student) in Research Tags: CRISPR, genetics, research

CC_DNA_Pixabay — DNA is the genetic material that determines the characteristics of plants and animals. Using CRISPR gene editing, researchers altered the characteristics of rice plants. | Creative Commons Photo by Pixabay

By Cara Penquite | Bond LSC

A tickle in the throat, a stuffy nose, congestion . . . the tell-tale signs of a cold are familiar to most, and many know that with enough rest, the immune cells on standby in the human body will destroy any invaders. But what happens when plants get sick?

The Bing Yang Lab from the Bond Life Sciences Center at the University of Missouri studied this question with the Reuben Peters lab at Iowa State University by analyzing the purpose of gene clusters in rice. Along with the Zhaohu Li lab at China Agricultural University, the researchers — utilized CRISPR gene editing to determine the function of certain gene clusters in rice.

“I thought this could be something very important,” Yang said, “We can take out a huge amount of DNA to see if the gene cluster could play a role in this resistance [to disease].”

CRISPR – a gene editing tool – allows the researchers to change the DNA of the rice plants by removing sections of genetic code, known as gene clusters. By removing the cluster, scientists can see what breaks in the plant, thus identifying each cluster’s role in the plant’s health.

“These were what we termed biosynthetic gene clusters. They have unrelated genes that are close together, their physical proximity on a chromosome, and they all act in one single pathway to make a small family of natural products,” Peters said.

The scientists compared unedited rice plants with rice plants missing the gene clusters, and they determined that those without the gene clusters had decreased resistance to certain diseases. This was due to the decreased production of antimicrobial — or pathogen fighting — natural products.

The genes code for enzymes that create labdane-related diterpenoids — natural products responsible for protecting plants from infection, termed phytoalexins — a defense response.

The loss of these compounds made the plants less resistant to disease. While humans have mobile cells that can travel to pathogens through the immune system, plants rely on local chemical responses to fight off invaders.

“They’ll recognize components of the fungal cell wall, or they’ll interact with specific proteins from the fungus or the bacteria, [and] that triggers what we consider to be an immune response,” Peters said. “They have to rapidly make these compounds in what would otherwise be a general photosynthetic cell or just an epidermal cell on the surface of the leaf.”

By identifying which genes produce certain chemicals, the researchers have a greater understanding of the plant immune response, and they can manipulate the genetics of the plants to create stronger immune responses.

“We can fine tune those genes, which could increase the [protections] and make the plant more resistant to the pathogen,” Yang said.

Peters suggested since the scientists know which genes code for these proteins, they can make the proteins and create an antibiotic to prevent infections in crops and livestock.

“We do think that some of these compounds are going to be important for potential pharmaceutical activity,” Peters said. “Not necessarily the ones in rice, but related compounds, and that’s another reason we studied this.”

This research published online in New Phytologist October 16, 2021, under the title “Dissecting the labdane-related diterpenoid biosynthetic gene clusters in rice reveals directional cross-cluster phytotoxicity.”

Outlining Omicron: researchers determine key mutations in the latest COVID-19 variant

February 2, 2022 By Penquite, Cara (MU-Student) in Research Tags: computer science, COVID-19, genetics

Bond LSC and UNMC scientists explain mutations unique to the Omicron variant

Austin Spratt Protein Models — Austin Spratt, undergraduate mathematics student in the Kamlendra Singh lab, shows protein models of the Omicron spike protein and the receptor it attaches to when infecting cells. “The genetic codes are used to identify the mutations, and then we use the structure to see how it would change over time. It’s going to give us more information about new mutations that occur,” Spratt said. | Photo by Cara Penquite, Bond LSC

By Cara Penquite | Bond LSC

It took eight days for Kamlendra Singh and his team to crack the code behind the Omicron COVID-19 variant. Although usually characterized by months of waiting, the peer-review process matched their pace, publishing their research a week later.

“That’s how quick it went because the editors and the general [scientific community] recognize that it’s very timely and important research,” said the Bond Life Sciences Center principal investigator.

Discovering 46 genetic mutations in the Omicron variant, their research explains Omicron’s ability to efficiently infect cells and evade antibodies. Their explanation predicted Omicron’s possibility for increased transmissibility a couple weeks before the beginning of the late December surge of COVID-19 cases in the U.S. The work published online December 10, 2021, in the Journal of Autoimmunity.

“You will see a lot of infections now because the vaccine-induced immune responses are not able to control the [Omicron] infection,” said Siddappa Byrareddy, a collaborator on the project and Professor and Vice-Chair of Research in the Department of Pharmacology Experimental Neuroscience at the University of Nebraska Medical Center (UNMC).

Genetic mutations to the spike protein on the surface of the virus increase Omicron’s ability to infect cells and evade antibodies. In Omicron, the mutations allow for a stronger bond between the spike protein and the healthy cell. The spike protein acts as a key enable the virus to get inside the cell by attaching to receptors on the surface of healthy cells. Once inside, the virus hijacks the cell and uses it to create more copies of the virus.

“What happens is, these mutations are in the receptor-binding domain. Which means, when these two things come together, when the spike protein tries to attach to the ACE2 receptor, these mutations cause it to bind more. When that happens, we have increased transmissibility or increased infectivity,” said Saathvik Kannan, a Hickman High School sophomore, and lead author from the Singh lab involved in identifying the mutations.

The spike protein is also the primary point of contact for antibodies designed to neutralize the virus. Since antibodies are not universal, the immune system must be exposed to a virus, either through infection or vaccination, before creating antibodies to neutralize that virus. Omicron’s mutations in the spike protein make it different from other variants of COVID-19, so antibodies that can neutralize other variants seem to be less effective in preventing Omicron infection.

“Antibodies are very specific. Any one mutation can completely abolish the binding,” Singh said.

While antibodies from previous infection or vaccination may not prevent Omicron transmission, Byrareddy emphasized that they are still decreasing hospitalization.

“Even though the vaccines are not effective [against Omicron] transmission, they still help the immune response to fight against the virus. That’s why we don’t [see] a lot of hospitalization,” Byrareddy said.

One potential solution to mitigate Omicron’s effect would be to create an Omicron-specific vaccine. While a new vaccine could decrease Omicron transmission, Singh warns that it would not necessarily prevent the emergence of further variants. This is a particular concern with Omicron because of its many versions.

“We’re seeing different Omicron versions where you have the Omicron, but with a couple changes. Those couple changes are making a big impact.” said Austin Spratt, another Singh lab researcher and MU undergraduate studying mathematics involved in the project.

Spratt’s role in the project was to create computer simulations of the spike proteins to see models of the mutations.

“[Omicron] has bigger mutations and different flavor mutations [than previous variants], and there are starting to be subcategories of that,” Spratt said.

Singh and Byrareddy’s labs are taking their research in the direction of antivirals with the hope of creating a drug that could reduce COVID-19 symptoms. The antiviral would block receptors on the surface of healthy cells so that the virus’ spike protein will not fit anymore. Without a key to get into the cells, the virus would not take over the cells.

While antivirals would not cut out all variants, Spratt believes they could help the problem as they did with other pandemics such as the Spanish Flu.

“I think it’s possible that they create antivirals that will slowly but surely cut out variants over time,” Spratt said.

This research published in the Journal of Autoimmunity December 10, 2021, under the title “Omicron SARS-CoV-2 variant: Unique features and their impact on pre-existing antibodies.”

Genetic link in primate points to source of adaption and longevity in capuchins

March 2, 2021 By Lauren Hines in Research Tags: capuchins, DNA, genetics

Cebus Imitator — As part of an international collaboration, principal investigator Wes Warren helped study capuchins in Costa Rica. | Photo contributed by Amanda D Melin, Bond LSC.

By Lauren Hines | Bond LSC

Through monkey poop and three years of work researchers from all over the world sequenced the Panamanian white-faced capuchin genome for the first time and devised novel methods to sequence many more wild capuchin genomes.

These monkeys have large brains for their small size and can live past 50. Wes Warren — Bond Life Sciences Center principal investigator — helped sequence the genome to find more answers about their unique brain-to-body ratio and long lifespans as part of the study. This work published Feb. 16 in Proceedings of National Academy of Science looks deeper at the genetic connections to these adapted traits.

“Other species of nonhuman primates don’t have that long lifespan, so the capuchin is a nice model and a different group of nonhuman primates to study that question,” Warren said.

Warren first got involved in his previous job at Washington University in 2017 when Amanda Melin, former principal investigator at Washington University, approached him about building the capuchin genome.

His main role in the study was in directing the genome assembly and using this genetic blueprint to analyze different types of selection pressures that impacted the genome.

Selection pressures are environmental or social factors that make it harder for species to survive and reproduce. Yet, when a small percentage of these species do survive — possibly due to a certain gene — their populations can explode. Afterward, that particular gene becomes widespread across the new population.

“There’s a whole variety of selection pressures that are brought to bear on a species genome, and a lot of that is driven by the environment,” Warren said. “The beauty of this experiment is there are two very distinct populations of capuchins in Costa Rica.”

The study looked at two groups: one in the southern lowland evergreen rainforest where rainfall and fruit are plentiful and another in the northern lowland tropical dry forest where periodic droughts occur.

A gene that affects kidney and water maintenance in the body became a particular interest in their analysis. That could be necessary for survival for the northern population who faced a dry climate and needed to conserve water.

“We can speculate that [these genes are] associated with this adaptation,” Warren said. “We don’t know for sure, and we make that point in the paper. It’s more of a discussion about this is a possibility, and these are real signals of selection. We’re not sure exactly if we can directly correlate them with an adaptation to a dry, hot climate, but it certainly makes biological sense.”

Warren and the other researchers discovered the gene, among others, by lining up genetic codes of capuchins and other mammals to find matching sets of nucleotides. This allowed researchers to identify known and novel genes, like the ones related to brain size and longevity.

Mike Montague, who previously worked under Warren while they were at Washington University, believes the correlation between brain size and longevity is due to genetics.

Genomes between capuchins and other animals showed differences that seemed to be tied to brain development, size and metabolizing mechanisms. Large brains require lots of fuel, so an efficient system for processing sugars would also be needed.

“Having an understanding of these genes and why they’re different in capuchins might be linked to these larger genetic pathways, [which] allow the capuchin to have this large brain,” Montague said.

As for longevity, researchers identified some genes associated with DNA damage response, metabolism, cell cycle and insulin signaling. While damage to DNA is thought to be a major contributor to aging, aging-related genes also deal with growth and development. This means researchers can’t be sure if the identified genes correlate with longevity.

While some genes found are associated with human cognition and large brain size in humans, they can’t be directly linked to human genes without further examination.

Researchers wouldn’t have been able to identify these genes and sequence the genome without a new technique developed by Joe Orkin, the study’s lead author. Orkin’s new technique, FecalFACS, isolated the DNA in capuchin feces of the animal’s intestinal cells.

Since capuchins are an endangered species, scientists can’t kill or capture them for study. Instead, they had to analyze their feces. The trouble was researchers originally had to cipher between the DNA of the food the capuchins ate and the DNA of the capuchin in feces. Orkin’s new technique allows them to sort that information easily.

“We observe this really beautiful population structure to these capuchins that live in this particular area,” Warren said. “We could not have got that… this larger number really let us get a very accurate portrayal of the population structure and how related they are to each other.”

Now that the paper has been published, the study’s next steps include expanding the sample beyond the two populations and investigating the functions of the newly discovered genes in other animals.

“There’s just a host of different questions that you could ask, now that we have this one particular genome added to the collection of other genomes for different primates,” Montague said.

More information about the study can be found in, “The genomics of ecological flexibility, large brains, and long lives in capuchin monkeys revealed with fecalFACS.” The study was published on Feb. 16 in Proceedings of the National Academy of Sciences.

Finding hope by fixing a gene

July 21, 2016 By Roger Meissen in Research Tags: genetics, SMA

Lorson lab publishes research on a new therapeutic path to help treat spinal muscular atrophy

Erkan Osman shows iImages of neuro-muscular junctions. Osman, a post-doctoral fellow in Chris Lorson’s lab, co-authored research in the journal Molecular Therapy that details work in binding a synthetic nucleic acid to a normally useless motor neuron backup gene to help treat spinal muscular atrophy. | photo by Phillip Sitter, Bond LSC

By Phillip Sitter | Bond LSC

Imagine you are forced to jump out of an airplane.

Luckily, you find a parachute that even has a backup chute. You leap out of the plane and free-fall.

You pull the cord to open your parachute, but it doesn’t open. Don’t panic, though, you have a backup. But, you pull that cord and nothing happens. Now you face the reality of a death as firm and un-yielding as the ground rushing into your view.

This air disaster mirrors the mechanism and mortal threat posed for people born with the genetic problem that causes spinal muscular atrophy (SMA).

Chris Lorson’s lab at the Bond Life Sciences Center would like to change that situation by making an effective genetic backup to the defective gene that results in SMA. The journal Molecular Therapy, a publication of Nature, recently accepted their findings for publication.

The defect occurs in a specific gene called Survival Motor Neuron (SMN). If the SMN gene is defective because of mutation, this causes a deficiency of the SMN protein it is supposed to produce. Without this protein, the neurons that control muscle movement malfunction. Signals cease to stimulate muscles.

Muscles that are not stimulated atrophy, grow weak and waste away. At first this happens with the skeletal muscles, which leads to loss of motor function for simple activities like walking and swallowing. If it happens with the muscles that control breathing, you die.

News of the disease often presents a devastating prognosis. Infants have it worst; babies diagnosed with SMA only have a life expectancy of two to five years from birth.

Fortunately, our bodies have a sort of backup for the SMN gene, another one called SMN-2. But, like a useless backup parachute for an unlucky skydiver, SMN-2 isn’t actually very good at producing proteins of the quality needed to stave off SMA. It might just be a vestigial trait on its way down the evolutionary drain — it doesn’t even exist in the closest primate relatives of humans.

Discoveries in the Lorson lab look to make the SMN-2 gene an effective backup, and their recent publications indicate that this may be a viable possibility for future SMA treatments.

Christian Lorson studies the genes that cause SMA when they fail to adequately function. His team’s on a backup gene that greatly extended life expectancy in mouse studies. | photo by Phillip Sitter, Bond LSC

“What we’ve been working on in the lab is a potential therapeutic, and what it does, it’s a large small molecule that is called an antisense oligonucleotide, or ASO,” Lorson said. “And this is something that is essentially a synthetic piece of nucleic acid that is able to go in and bind to a specific sequence within a gene.”

Once bound to SMN-2, the ASO is designed to alter mRNA splicing, “essentially, the editing of a gene,” Lorson said. Speaking in terms akin to products leaving a factory, Lorson said that the attached ASO makes SMN-2 produce good quality proteins, the ones that it wasn’t able to produce before.

In other words, suddenly the backup protein-factory that was making poor-quality products is now pumping out top-of-the-line stuff that will work.

Previous research identified a strong ASO contender to experiment with, and Lorson said current research is about optimizing an ASO to extend survival times in mice with SMA — from just 13 days to five months after only one injection at birth.

Lorson stressed that his lab’s achievement doesn’t promise a fast cure for SMA. He said it is unlikely a single compound will address the full gambit of effects that people with SMA suffer, especially given that people can be identified as having SMA at any time from birth through later in life — often late onset SMA tends to be less severe than diagnosis as an infant.

There’s not yet any single compound treatment for SMA that has been approved by the Food and Drug Administration, Lorson said, so he cautions against getting hopes up of for a revolutionary treatment for SMA coming onto the market soon — “Near future but not tomorrow.”

He acknowledged, though, that “from a research perspective, things seem to be moving at lightning speed, but if you are a patient or a family member, things can never go fast enough, so I think there’s a realized sense of urgency, whether or not it’s for patients who don’t have the disease yet, are not born, or for patients who have had the disease for a decade and are wondering when their opportunity would come.”

Lorson’s work is funded in part by Cure SMA, FightSMA and the Gwendolyn Strong foundations. Erkan Osman, a post-doctoral fellow in Lorson’s lab and the first author on the most recent paper, won the emerging investigator award from FightSMA and Gwendolyn Strong in 2015.

Unmasking the unknown

March 1, 2016 By Justin L. Stewart in Research Tags: Arabidopsis, Bond Life Sciences Center, Dan Leuchtman, decoding science, genetics, Immune System, Mannie Liscum, mice, mizzou, MmSRFR1, plant science, research, SRFR1, Walter Gassmann

Scientists explore genetic similarities between plants and mice

University of Missouri PhD Candidate Daniel L. Leuchtman peers through an Arabidopsis plant. Leuchtman has been experimenting with replacing a gene in the plants immune system with a similar gene from mice. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

By Justin L. Stewart | MU Bond Life Sciences Center

Almost two-thirds of what makes a human a human and a fly a fly are the same, according to the NIH genome research institute.

If recent research at the University of Missouri’s Bond Life Sciences Center is verified, we’ll soon see that plants and mice aren’t all that different, either.

Dan Leuchtman studies a gene in Arabidopsis plants called SRFR1, or “Surfer One.” SRFR1 regulates plant immune systems and tell them when they are infected with diseases or illnesses. Leuchtman studies this model plant as a Ph.D. candidate at MU, splitting time between the labs of Walter Gassmann and Mannie Liscum.

His research involves breeding Arabidopsis plants missing the SRFR1 gene and then replacing it with the MmSRFR1 gene.

So, what is MmSRFR1? Leuchtman and company believe it’s the animal equivalent of SRFR1, though they aren’t fully aware of all of its’ functions.

“We’re actually one of the first groups to characterize it,” Leuchtman said.

Arabidopsis plants missing the SRFR1 gene struggle to grow at all, appearing vastly different from normal plants. Leuchtman says that a plant missing the SRFR1 gene is a mangled little ball of leaves curled in on itself. “It’s really strange looking.”

While his experiments haven’t created statuesque plants equal to those with natural SRFR1 genes present, the Arabidopsis plants with MmSRFR1 show a notable difference from those completely lacking SRFR1. Leuchtman says the plants with MmSRFR1 lie somewhere in between a normal plant and one lacking SRFR1.

“At its’ core, it’s more understanding fundamental biology. How do we work? How do organisms tick? How do you go from DNA in a little bag of salts to a walking, talking organism?” Leuchtman said. “The more you know about how an organism functions, the more opportunities you have to find something that makes an impact.”

Understanding spit

October 8, 2015 By Roger Meissen in Research 1,632 Comments Tags: Arabidopsis, genetics, nematodes, plant sciences, soybean

Scientists find how nematodes use key hormones to take over root cells

This Arabidopsis root shows how the beet cyst nematode activates cytokinin signaling in syncytium 10 days after infection. The root fluoresces green when the TCSn gene associated with cytokinin activation is turned on because it is fused with a jellyfish protein that acts as a reporter signal. Contributed by Carola De La Torre

Roger Meissen | Bond Life Sciences Center

This is a story about spit.

Not just any spit, but the saliva of cyst nematodes, a parasite that literally sucks away billions in profits from soybean and other crops every year.

Researchers are working to uncover exactly how these tiny worms trick plant root cells into feeding them for life.

A team at the University of Missouri Bond Life Sciences Center collaborated with scientists at the University of Bonn in Germany to discover genetic evidence that the parasite uses its own version of a key plant hormone and that of the plants to make root cells vulnerable to feeding. Their research recently appeared in Proceedings of the National Academy of Sciences.

Cytokinin is normally produced in plants, but these researchers determined that this growth hormone is also produced by nematode parasites that use it to take over plant root cells.

“While it’s well-known that certain bacteria and some fungi can produce and secrete cytokinin to cause disease, it’s not normal for an animal to do this,” said Melissa Mitchum, an MU plant scientist and co-author on the study. “This is the first study to demonstrate the ability of an animal to synthesize and secrete cytokinin for parasitism.”

Not Science Fiction

Reprogramming another organism might sound like a far out concept, but it’s a reality for plants susceptible to nematodes.

Cyst nematodes hatch from eggs laid in fields and quickly migrate to the roots of nearby plants. They inject nematode spit into a single host cell of soybean, beet and other crop roots.

“Imagine a hollow needle at the head of the nematode that the parasite uses to penetrate into the plant cell wall and secrete pathogenic proteins and hormone mimics,” said Carola De La Torre, a co-author of the study and plant sciences PhD student with Mitchum’s lab. “Nematodes use the spit to transform the host cell into a nutrient sink from which they feed on during their entire life cycle. This de novo differentiation process greatly depends on nematode–derived plant hormone mimics or manipulation of plant hormonal pathways caused by effector proteins present in the nematode spit.”

These effector proteins and other small molecules in their spit cause the root cell to forego normal processes and create a huge feeding site called a syncytium. In a short period of time, this causes hundreds of root cells to combine into a large nutrient storage unit that the nematode feeds from for its entire life.

Being able to convince a root cell to do the nematode’s bidding starts with a takeover of the plant host cell cycle — which regulates DNA replication and division. This implies that a plant hormone like cytokinin is involved, says Mitchum. Cytokinin normally regulates a plant’s shoot growth, leaf aging, and other cell processes.

Proving the relationship

While Mitchum’s lab had a hunch that cytokinin was key to this takeover, proving it took some creative science.

De La Torre and Demosthenis Chronis, a postdoctoral fellow MU at the Bond LSC depended on mutant Arabidopsis plants to explore the relationship. “One of the great things about using Arabidopsis as our host plant is the vast genetic resources of cytokinin and hormone mutants that are available through the scientific community,” De La Torre said.

She infected Arabidopsis that contained a reporter gene called TCSn/GFP with nematodes. This gene is associated with cytokinin responses within the plant cells and is fused with a jellyfish protein that glows green when turned on. So, De La Torre saw nematodes activated cytokinin responses in the plant early after infection when her plants emitted a green fluorescent glow under the microscope.

Next, she infected plants missing the majority of their cytokinin receptors with nematodes. Then she started counting nematodes present.

“After a careful evaluation of nematode infection, we observed less female nematodes developing in the receptor mutants compared to the wild type” De La Torre said. “The nematodes could not infect well, and that was a clear piece of evidence suggesting that cytokinin plays a main role in plant–nematode interactions.”

Another experiment looked at Arabidopsis containing a reporter gene called GUS that was fused to the regulatory sequences of the cytokinin receptor genes. All three cytokinin receptor genes were activated where the nematode was feeding.

A final experiment used a mutant that created an excess of an enzyme that degrades cytokinin, finding that a base level of plant cytokinin was also necessary for nematode growth.

“The simple statement is that the cytokinin receptors were activated in response to nematode infection and the mutants did not support growth and development of the nematodes,” Mitchum said. “This shows that if you take away the ability of the plant to recognize cytokinin the worms are unable to fully develop.”

An international collaboration

Mitchum’s team did not work alone.

The lab of Florian Grundler at Rheinische Friedrich-Wilhelms-University of Bonn, Germany, was also on a mission to uncover if genes in the nematode controlled cytokinin activation. They had identified a key gene in the beet cyst nematode that makes the cytokinin hormone. When they took away the ability of the nematode to secrete cytokinin certain cell cycle genes were not activated at the feeding site and the nematodes did not develop. Now we know that the nematode is also secreting cytokinin to modulate the pathways.

De La Torre took that information and found the same gene in the soybean cyst nematode.

Now, Mitchum’s team is trying to find how this key gene might work differently in other nematode types, like root-knot nematode as part of a new National Science Foundation grant. They hope this will help lead to better resistance in future crops.

“Understanding how the nematode modulates its host is going to help us exploit new technologies to engineer plants with enhanced resistance to this terribly devastating pathogen,” Mitchum said. “Technology is changing all the time, we’re gaining new tools constantly, so you never know when something new is going to allow us to do something specific at the site of nematode feeding that will lead to a breakthrough.”

Mitchum is a Bond LSC investigator and an associate professor of Plant Sciences in the College of Agriculture, Food and Natural Resources. The study “A Plant Parasitic Nematode Releases Cytokinin that Control Cell Division and Orchestrate Feeding-Site Formation in Host Plants” recently was published by the Proceedings of the National Academy of Sciences and was supported by the National Science Foundation (Grant #IOS-1456047 to Mitchum). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

Scientists uncover how caterpillars created condiments

June 22, 2015 By Roger Meissen in Research 18,234 Comments Tags: Bond LSC, Brassicales, cabbage butterfly, evolution, genetics, plant science

The next time you slather mustard on your hotdog or horseradish on your bun, thank caterpillars and brassica for that extra flavor.

While these condiments might be tasty to you, the mustard oils that create their flavors are the result of millions of years of plants playing defense against pests. But at the same time, clever insects like cabbage butterflies worked to counter these defenses, which then started an arms race between the plants and insects.

An international research team led by University of Missouri Bond Life Sciences Center researchers recently gained insight into a genetic basis for this co-evolution between butterflies and plants in Brassicales, an order of plants in the mustard family that includes cabbage, broccoli and kale.

Chris Pires | Image by Roger Meissen, Bond LSC

The team published these new insights online in Proceedings of the National Academy of Sciences (PNAS) in June.

“We found the genetic evidence for an arms race between plants like mustards, cabbage and broccoli and insects like cabbage butterflies,” said Chris Pires, an MU Bond Life Sciences Center researcher and associate professor of biological sciences in the College of Arts and Sciences. “These plants duplicated their genome and those multiple copies of genes evolved new traits like these chemical defenses and then cabbage butterflies responded by evolving new ways to fight against them.”

A biting taste

While you might like the zing in mustard, insects don’t.

Compounds, called glucosinolates, create these sharp flavors in plants to defend against caterpillars, butterflies and other pests. Brassicales species first evolved glucosinolate defenses around the KT Boundary — when dinosaurs went extinct — and eventually diversified to synthesize more than 120 different types of this compound.

For most insects, these glucosinolates prove toxic, but certain ones like the cabbage butterfly evolved ways to detoxify the compounds.

“Seeing the variation in the detoxification mechanisms among species and their gene copies gave us important evolutionary insights,” said Hanna Heidel-Fischer, a lead author on the study based at the Max Plank Institute for Chemical Ecology in Germany.

To look at these genetic differences, the team used 9 existing Brassicales genomes and also generated transcriptomes — the set of all RNA in a cell — across 14 Brassicales families. This allowed the team to map an evolutionary family tree of sorts over the millennia, seeing where major defense changes occurred. This family tree was compared with the family tree of 9 key species of Pieridae butterflies, which includes the cabbage butterfly.

Pires and his colleagues identified three significant evolutionary waves over 80 million years, where plants developed defenses and insects evolved counter tactics.

Pat Edger | Image by Roger Meissen, Bond LSC

“We found that the origin of brand-new chemicals in the plant arose through gene duplications that encode novel functions rather than single mutations,” said Pat Edger, a former MU post doc and lead author on the study. “Given sufficient amounts of time the insects repeatedly developed counter defenses and adaptations to these new plant defenses.”

This back-and-forth pressure resulted in the evolution of many more species of plants and butterflies than in other groups without glucosinolate pressures.

Proving an old concept

Co-evolution is not a new idea.

About 50 years ago two now-renowned biologists, Peter Raven and Paul Erhlich, introduced the idea of co-evolution to science. Using cabbage butterflies and Brassica plants as a prime example, the two published a landmark study in 1964 advancing the idea that two species can mutually influence the development and evolution of each other.

To explore the genetics of how this works, Pires’ lab partnered with Chris Wheat, professor of population genetics in the Department of Zoology at Stockholm University.

“Using Ehrlich and Raven’s principles and models, we looked at the evolutionary histories of these plants and butterflies side-by-side and discovered that major advances in the chemical defenses of the plants were followed by butterflies evolving counter-tactics that allowed them to keep eating these plants,” Wheat said.

Chris Pires and colleagues mapped the evolution of Brassicales and butterflies to find how each evolved to combat the defenses of the other. | Courtesy Chris Pires

This research provides striking support for the ideas of Ehrlich and Raven published 50 years ago.

“We looked at the patterns 50 years ago, and found conclusions that proved correct,” said Peter Raven, professor emeritus of the Missouri Botanical Garden and a former University of Missouri Curator. “The wonderful array of molecular and other analytical tools applied now under leadership of people like Chris Pires, provide verification and new insights that couldn’t even have been imagined then.”

Understanding more about how plants and insects co-evolve could one day lead to advances in crops.

“If we can harness the power of genetics and determine what causes these copies of genes, we could produce plants that are more pest-resistant to insects that are co-evolving with them—it could open different avenues for creating plants and food that are more efficiently grown,” said Pires.

Proceedings of the National Academy of Sciences (PNAS) published the study, “The butterfly plant arms-race escalated by gene and genome duplications,” in June. The National Science Foundation (PGRP 1202793), the Knut and Alice Wallenberg Foundation and the Academy of Finland provided the funding for this research.

SoyKB: Leading the convergence of wet and dry science in the era of Big Data

June 10, 2014 By Paige Blankenbuehler in Research Tags: big data, big data initiative, Bond Life Sciences, computer science, data, dry science, genetics, hightroughput data, mizzou, research, science, soybean, University of Missouri, wet science

Yaya Cui, an investigator in plant sciences at the Bond Life Sciences Center examines data on fast neuron soybean mutants that are represented on the SoyKB database.

The most puzzling scientific mysteries may be solved at the same machine you’re likely reading this sentence.

In the era of “Big Data” many significant scientific discoveries — the development of new drugs to fight diseases, strategies of agricultural breeding to solve world-hunger problems and figuring out why the world exists — are being made without ever stepping foot in a lab.

Developed by researchers at the Bond Life Sciences Center, SoyKB.org allows international researchers, scientists and farmers to chart the unknown territory of soybean genomics together — sometimes continents away from one another — through that data.

Digital solutions to real-world questions

As part of the Obama Administration’s $200 million “Big Data” Initiative, SoyKB (Soy Knowledge Base) was born.

The digital infrastructure changes the way researchers conduct their experiments dramatically, according to plant scientists like Gary Stacey, Bond LSC researcher, endowed professor of soybean biotechnology and professor of plant sciences and biochemistry.

“It’s very powerful,” Stacey said. “Humans can only look at so many lines in an excel spreadsheet — then it just kind of blurs. So we need these kinds of tools to be able to deal with this high-throughput data.”

The website, managed by Trupti Joshi, an assistant research professor in computer science at MU’s College of Engineering, enables researchers to develop important scientific questions and theories.

“There are people that during their entire career, don’t do any bench work or wet science, they just look at the data,” Stacey said.

The Gene Pathway Viewer available on SoyKB, shows different signaling pathways and points to the function of specific genes so that researchers can develop improvements for badly performing soybean lines.

“It’s much easier to grasp this whole data and narrow it down to basically what you want to focus on,” Joshi said.

A 3D-protein modeling tool lends itself especially to drug design. A pharmaceutical company could test the hypothesis and in some situations, the proposed drug turns out to yield the expected results — formulated solely by data analysis.

The Big Data initiative drives a blending of “wet science” — conducting experiments in the lab and gathering original data — and “dry science” — using computational methods.

Testament of the times?

“Oh, absolutely,” Joshi said.

Collaboration between the “wet” and “dry” sciences

Before SoyKB, data from numerous experiments would be gathered and disregarded, with only the desired results analyzed. The website makes it easy to dump all of the data gathered to then be repurposed by other researchers.

“With these kinds of databases now, all the data is put there so something that’s not valuable to me may be valuable to somebody else,” Stacey said,

Joshi said infrastructure like SoyKB is becoming more necessary in all realms of scientific discovery.

“(SoyKB) has turned out to be a very good public resource for the soybean community to cross reference that and check the details of their findings,” she said.

Computer science prevents researchers having to reinvent the wheel with their own digital platforms. SoyKB has a translational infrastructure with computational methods and tools that can be used for many disciplines like health sciences, animal sciences, physics and genetic research.

“I think there’s more and more need for these types of collaborations,” Joshi said. “It can be really difficult for biologists to handle the large scope of data by themselves and you really don’t want to spend time just dealing with files — You want to focus more on the biology, so these types of collaborations work really well.

It’s a win-win situation for everyone,” she said.

The success of SoyKB was perhaps catalyzed by Joshi. She adopted the website and the compilation of data in its infant stages as her PhD dissertation.

Joshi is unique because she has both a biology degree and a computer science background. Stacey said Joshi, who has “had a foot in each camp,” serves as an irreplaceable translator.

Most recently, the progress of SoyKB as part of the Big Data Initiative was presented at the International Conference on Bioinformatics and Biomedicine Dec. 2013 in Shanghai. The ongoing project is funded by NSF grants.