genetics

Finding hope by fixing a gene

Lorson lab publishes research on a new therapeutic path to help treat spinal muscular atrophy
By Phillip Sitter | MU Bond Life Sciences Center

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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

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.

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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

Scientists explore genetic similarities between plants and mice

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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.

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A series of Arabidopsis plants show the differences between the plants, from left, without SRFR1, with MmSRFR1 and with SRFR1. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

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.

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University of Missouri PhD Candidate Daniel L. Leuchtman poses for a portrait in a Bond Life Sciences Center greenhouse. Leuchtman has been experimenting with replacing a gene in Arabidopsis plants immune system with a similar gene from mice. | Photograph by Justin L. Stewart/MU Bond Life Sciences Center

“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

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

Roger Meissen | Bond Life Sciences Center
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. (N=nematode; S=Syncytium). Contributed by Carola De La Torre

This Arabidopsis root shows how the beet cyst nematode activates cytokinin signaling in the 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. (N=nematode; S=Syncytium). Contributed by Carola De La Torre

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.

Melissa Mitchum

Melissa Mitchum

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.

Carola De La Torre

Carola De La Torre

“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

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

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

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

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

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.

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.