Our latest publication, S-acylation of P2K1 mediates extracellular ATP-induced immune signaling in Arabidopsis, has just been published in Nature Communications. 

Abstract:

S-acylation is a reversible protein post-translational modification mediated by protein S-acyltransferases (PATs). How S-acylation regulates plant innate immunity is our main concern. Here, we show that the plant immune receptor P2K1 (DORN1, LecRK-I.9; extracellular ATP receptor) directly interacts with and phosphorylates Arabidopsis PAT5 and PAT9 to stimulate their S-acyltransferase activity. This leads, in a time-dependent manner, to greater S-acylation of P2K1, which dampens the immune response. pat5 and pat9 mutants have an elevated extracellular ATP-induced immune response, limited bacterial invasion, increased phosphorylation and decreased degradation of P2K1 during immune signaling. Mutation of S-acylated cysteine residues in P2K1 results in a similar phenotype. Our study reveals that S-acylation effects the temporal dynamics of P2K1 receptor activity, through autophosphorylation and protein degradation, suggesting an important role for this modification in regulating the ability of plants in respond to external stimuli.

Read the full article here.

 

By Becca Wolf | Bond LSC

When one hears of a magician, they think of a man that pulls a rabbit out of a top hat or ‘cuts’ people in half. Magicians have a lot of tricks up their sleeve.

People do not think of scientists as magicians, yet they still perform wonderous things.

“I heard a quote one time that says science and technology is kind of like magic,” Jordan Brungardt said. “For somebody that doesn’t know what is happening, experiments look like magic if they’re performed well. Think of cell phones allowing us to talk to people across the world. For somebody from a few decades ago, that would seem like magic.”

For Brungardt, a post-doctoral fellow in the Gary Stacey lab at Bond Life Sciences Center, this magic of sorts makes research exciting.

“When you get sequencing information back in the lab after going weeks without actually seeing something and it lines up with what you’re predicting, it’s like Christmas,” Brungardt said.

In the Stacey lab, Brungardt studies the genes involved in early nodule development in soybeans.

He looks at the interaction between rhizobia, a species of bacteria, and their plant hosts, legumes. His work aims to explore how the plants allow access to their inner roots through infection thread development.

“They undergo an intimate interaction that is really unique,” Brungardt said. “It’s pretty cool from that aspect, and the infection thread is an important part of the interaction.”

Brungardt currently works with Jaehyo Song, a postdoctoral student in the Stacey lab.

“He is a very careful person, and experimenting together helps prevent any mistakes,” Song said. “I can also see that the experiment is progressing step by step because we work very diligently.”

The two have been working together for a few months and have found their collaboration to be very beneficial.

“Even when a simple problem occurred in the experiment, he did not ignore it,” Song said. “He found the exact cause of the problem through previous research and eventually solved it. Through this, I could see that his problem-solving skills were very good.”

Brungardt is able to dodge problems that arise in his research easily.

“There’s a lot of different facets of my research,” Brungardt said. “If I get stuck somewhere, I can make headway somewhere else, which allows me to take my time on the process that I’m stuck on and come back to it later.”

Brungardt completed his undergraduate degree at Fort Hays State University in Kansas and his master’s degree at Wichita State University. That wasn’t his initial plan, though.

“I knew I wanted to do something with soybeans coming out of my undergrad and, to be honest, I actually interviewed with Dr. Stacy, who is my mentor right now, but it didn’t work out,” Brungardt said. “So, it was really nice coming back and getting a job here, because that’s what I initially wanted to do.”

Even though Brungardt did not initially come to Mizzou, he remained focused on it as the end goal. He ended up getting a job as a microbiologist where he produced Rhizobia for farmers to use to improve their crop production.

“My Ph.D. dealt with microbial interaction with plants, so it still had some loose application for what I’m doing now,” Brungardt said. “While my path wasn’t straight, it never strayed from what I wanted to do. I liked that I still did things that were relevant to what I wanted to do.

Brungardt arrived at Bond LSC last June and has adjusted nicely. His research is similar to what he has done previously, just in a new building in a new lab. He is happy to finally be able to completely focus on his research.

“I just graduated with my Ph.D. last year so there’s no more homework or classes for me. It’s a little bit of a change,” Brungardt said. “It’s simpler, I don’t have to break up my workday to go to class and then come back and work, and then switch up to something again. As a student I was doing several things at once and had other things in the back of my mind. Now I can just focus on my research.”

Since Brungardt can devote all of his time to his research now, he expects to see a lot more magical moments in the lab.

Available at: https://decodingscience.missouri.edu/2021/03/26/iamscience-jordan-brungardt/

Position 1: Seeking candidates skilled in plant genetics/ molecular biology to join an NSF funded project to apply high resolution methods (single cell) to investigating the mechanisms of legume-rhizobium nodulation. Candidate will join an excellent, multidisciplinary, collaborative group using cutting edge, single-cell methods to explore metabolomic, proteomic and transcriptomic responses of the plant to rhizobial infection. See website for the long list of publications that have arisen from this research.

Position 2: A postdoctoral position is available for a NIH/NSF-funded project to explore the role of purinergic signaling in in plants. Purinergic signaling, although not extensively studied in plants, affects a wide range of plant processes, including those associated with the plant response to abiotic and biotic stress. The project will involve further studies of the mechanisms by which the P2K1 (DORN1) and P2K2 receptors recognize extracellular ATP and induces downstream signaling, as well as characterization of various Arabidopsis mutants defective in purinergic signaling. The work involves a variety of functional genomic and biochemical methods to elucidate the molecular mechanisms involved in plant purinergic signaling. Experience with modern molecular and biochemical methods (e.g., those involved in protein isolation, protein characterization and protein complex formation are essential).

Both positions come with a competitive salary (beginning at $47,500), fringe benefits including health insurance, retirement, and travel support to meetings are available for this position.

Application Instructions: Please email a cover letter outlining your suitability for the position, list of references with contact information and recent CV to Gary Stacey at staceyg@missouri.edu. 

The University of Missouri is an equal opportunity employer. 

Our PhD student, Bruna Montes Luz, presented her work “Tissue-specific translatome analysis unveils new genetic players in the soybean-Bradyrhizobium interaction” at the MU Plant Research Symposium 2021. She was awarded a prize for third best lightning talk! 

Check out her presentation below

One of the projects in the lab is aimed at developing a novel infrared super resolution fluorescence imaging technique to investigate important receptor-ligand binding events in plant cells. This is done in collaboration with researchers from University of Maryland, Baltimore County and from EMSL (Environmental Molecular Sciences Laboratory). 

Dr. Rosenzweig, thank you for sharing the presentation.

 Postdoctoral position, Purinergic signaling in plants, available immediately, University of Missouri 

Description: A postdoctoral position is available for a NIH/NSF-funded project to explore the role of purinergic signaling in in plants. Purinergic signaling, although not extensively studied in plants, affects a wide range of plant processes, including those associated with the plant response to abiotic and biotic stress. The project will involve further studies of the mechanisms by which the P2K1 (DORN1) and P2K2 receptors recognize extracellular ATP and induces downstream signaling, as well as characterization of various Arabidopsis mutants defective in purinergic signaling. The work involves a variety of functional genomic and biochemical methods to elucidate the molecular mechanisms involved in plant purinergic signaling. Experience with modern molecular and biochemical methods (e.g., those involved in protein isolation, protein characterization and protein complex formation are essential). Candidates that do not have a strong track record of achievement and who do not have the requisite biochemical skills are strongly discouraged from applying. 

The successful candidate will join a team of other postdoctoral associates, graduate students and undergraduates exploring plant purinergic signaling in the laboratory. Prior experience in advanced methods in biochemistry is essential for the project. Prior experience in any of the following areas: protein biochemistry, proteomics, protein-protein interactions, protein covalent modification, site-directed mutagenesis, protein expression, mass spectrometry and bioinformatics is highly desirable. Applicants should possess a Ph.D. degree in Biochemistry, Biology or Molecular Biology and have a strong interest in plant cellular signaling. Excellent oral and written communication skills and the ability to work well in a collaborative research environment are essential. The successful candidate is expected to have demonstrated an impressive record of achievement and excellence. 

Competitive salary (beginning at $47,500), fringe benefits including health insurance, retirement, and travel support to meetings are available for this position. Funding for this project is renewable for a period of three years based on performance. 

Application Instructions: Please email a cover letter outlining your suitability for the position, list of references with contact information and recent CV to Gary Stacey at staceyg@missouri.edu. 

The University of Missouri is an equal opportunity employer. 

 

Purinergic signaling in plants: 

ATP is a ubiquitous compound in all living cells; it not only provides the energy to drive many biochemical reactions, but also functions in signal transduction as a substrate for kinases, adenylate cyclases, etc. However, ATP was also shown to be an essential signaling agent outside of cells, where it is referred to as extracellular ATP (eATP). An extensive literature exists in animals implicating eATP in numerous cellular processes, including neurotransmission, immune responses, cell growth, and cell death. Initial observations of effects of eATP in animals were met with considerable skepticism. However, this changed when the plasma membrane-localized receptors, purinoceptors of the P2X and P2Y classes, were identified and shown to mediate the effects of eATP. 

In contrast, relatively little has been done to examine the role of eATP in plants. However, over the past several years, eATP has been implicated in a variety of plant processes, including root-hair growth, stress responses, gravitropism, cell viability, pathogen responses and thigmotropism. A significant break through in the study of purinergic signaling (eATP response) in plants was our identification of the first, plant eATP receptor (Choi et al., 2014). DORN1 is a lectin receptor-like kinase and, therefore, identifies a new family of purinoreceptors (P2K). 

The laboratory is continuing our characterization of P2K1 (DORN1) and other proteins involved in eATP recognition, as well as exploring the role that eATP signaling plays in plant growth and development. Our findings clearly implicate purinergic signaling in a variety of key plant processes suggesting that eATP is just as important and interesting in plants as it is in animals (including humans). 

Selected, publications from the lab on this topic: 

Pham et al. 2020. P2K2 is a second plant receptor for extracellular ATP and contributes to innate immunity. Plant Physiol. 183 (3) 1364-1375; DOI: 10.1104/pp.19.01265 

Matthus et al. 2019. DORN1/P2K1 and purino-calcium signalling in plants; making waves with extracellular ATP. Ann. Bot. https://doi.org/10.17863/CAM.42662 

Chen et al. 2019. S-Acylation of plant immune receptors mediates immune signaling in plasma membrane nanodomains BioRxiv https://doi.org/10.1101/720482 270. 

Chen et al. (2017) Extracellular ATP elicits DORN1-mediated RBOHD phosphorylation to regulate stomatal aperture. Nature Commun. 8: 2265. doi:10.1038/s41467-017-02340-3255. 

Tripathi et al. (2017) Extracellular ATP acts on jasmonate signaling to reinforce plant defense. Plant Physiol. 176: 511–523. https://doi.org/10.1104/pp.17.01477 

Cao et al. (2014) Extracellular ATP is a central signaling molecule in plant stress responses. Curr. Op. Plant Biol. 20: 82-87. https://doi.org/10.1016/j.pbi.2014.04.009 

Choi et al. (2014) Identification of a plant receptor for extracellular ATP. Science Vol. 343 no. 6168 pp. 290-294. DOI: 10.1126/science.343.6168.290 

 

By Becca Wolf | Bond LSC

Technology advancements have always driven scientific discoveries in order to perform in depth research, but that has never been more true today.

“A couple of decades ago it was perfectly fine to be an engineer and a biologist and live in your own world,” David Mendoza said. “But as science has advanced, we depend more on mathematics and computer sciences now.”

Mendoza, principal investigator at Bond Life Sciences Center and associate professor of plant sciences, created a program to help develop those skill in the next generation of scientists.

Bioinformatics in Plant Sciences (BIPS) was born in 2016. The undergraduate program pairs plant science or biology majors with computer science or engineering majors. Through research, field trips, and journal clubs, undergraduate students learn how to collaborate on projects and how the two fields help each other.

While Mendoza is the PI who initiated BIPS through a NSF grant, students from all labs are welcome in this program. More recently, a second NSF grant in collaboration with Gary Stacey has allowed the program to expand. BIPS identifies labs that have projects with both computer and biology components for students to work on. Students can also come to BIPS with a project already in mind.

Graduate Student Mentors

BIPS is run by students, for students, with a handful of graduate student mentors and undergraduate students in the program.

The graduate student mentors run weekly meetings, invite in guest speakers, and organize the journal clubs, which is where students review scientific publications and discuss them. Graduate mentors, Marianne Slaten and Nick Dietz, have enjoyed their time and responsibilities at BIPS.

“We make sure they’re on track and don’t get stuck. We want them to become well-rounded researchers,” Slaten said. “It’s a really novel opportunity to jumpstart the next generation of researchers.”

While BIPS cannot go on a field trip this year or have in-person speakers, Slaten and Dietz have come up with alternatives to keep undergrads engaged.

“We’ve been doing workshops showing different bioinformatics tools that can be used to address different research questions,” Dietz said. “We’re also having them do journal club, which gives them a better understanding of the literature surrounding what they are studying. We’re trying to normalize the experience as much as we can, even though everything’s virtual right now.”

Undergraduates

Undergrads are put into research teams and begin working.

Many projects start with a biology student cataloguing physical plant traits and a computer science or engineering student creates a way to take images of the plants and organize data and information.

“Some of the files you work with are so big you can’t even open them in Excel, so there’s always room for computational people that really know how to harness all that data,” Slaten said. “The files are just so big that your computer crashes. It takes additional skills to know how to deal with that data.”

While a majority of their projects focus on phenotyping, BIPS is looking to branch out into other areas.

“A big problem in biology right now is big data, which you can’t get through that if you don’t have computer science,” said Maddy Creach, a junior from Walter Gassmann’s lab at Bond LSC. “It’s interesting to see the way computer scientists look at problems and that has definitely made me a better researcher, especially since I know 1,000% more about computer science than I did when I started.”

Creach has found that her two years in BIPS helps her think about new ways to think about research.

“A big thing that I have gotten out of BIPS is how to manage my own project,” Creach said. “It’s on me to communicate with my partner and get stuff done and put work towards it, because no one else going to tell me to do it.”

In addition to working on their research and participating in journal club, the undergraduate students are expected to make a poster each year to present their research either at Life Sciences Week or at the Undergraduate Research Forum. Since the pandemic has limited or canceled these events, the students now submit a video of their work online for others to watch.

BIPS’ Impact

Building research and collaboration skills has helped everyone involved.

“I’m learning as much from them as I’m teaching them, so that’s been really awesome,” Slaten said.

Students also hone their communication skills.

“I didn’t realize being a good mentor is a skill set, it’s something you can cultivate over time,” Dietz said. “Oftentimes as students, we think of mentors as either good or bad, you either have it or you don’t. But it’s actually a skill that you can develop over time and I’ve picked that up since I’ve been helping run the program.”

But all remember it’s a learning process.

“Everyone’s really fun and easygoing, no one is judgmental because there is a range in expertise,” Creach said. “There are no dumb questions.”

BIPS has helped many students become well-rounded in their research abilities and is always looking for more students.

“The world is a big place that is moving at a fast pace, and if they don’t get on the train, they are going to miss it,” Mendoza said. “It’s that simple. There’s so much happening in real time, that if you don’t learn how to integrate technology into your research, you’re going lag behind.”

Original post available at: https://decodingscience.missouri.edu/2021/02/03/bips-bringing-plant-science-and-engineering-together/?fbclid=IwAR1_fbZgB046GfIKIZxntX5ShzH6LH56J8x4HOMYaa2w-1KiNnbV1K7mL5Y

Dr. Gary Stacey has been recognized as one of CAFNR’s Drivers of Distinction, alongside other 9 faculty members. 

The honor was given to the faculty members who had the highest total shared credit research expenditures over the past 10 years (2011-20).

To check out all the faculty members who were recognized, access the CAFNR website.

 

By Becca Wolf | Bond LSC

Patience is a virtue, at least it is for Bing Stacey.

Stacey recently completed a project that took her a total of eight years. It took her five years to develop a fast neutron mutant population and it took an additional three years to screen the population to identify a mutant that showed increase soybean seed size and then identifying the causative gene.

This gene, GmKIX8-1, and the seed size QTL, qSW17-1, can potentially be exploited for increasing yield in soybeans. Being able to alter these to increase seed size is important to improve the economic traits of soybeans such as yield and seed quality.

“The most important thing for farmers is the yield,” said Stacey, assistant professor of plant sciences at Bond Life Sciences Center. “For farmers, they plant soybeans, and the value or the profit they get is based on yield.”

There are two components of yield: seed size and seed quality. Stacey went to work finding what genes contribute to these components.

Mutants are very important for gene study because they have a new DNA sequence for genes. While it sounds like a sci-fi term, mutants are just plants manipulated using chemicals, radiations, or genetic modifications to change their characteristic traits in comparison to the norm. And the DNA changes to the GmKIX8-1 gene Stacey found is exactly the mutation she was looking for.

When she began this research, there were not many mutants for soybeans regarding seed size, so she had to develop her own mutant population using fast neutron irradiation.

After taking several years to develop this population, Stacey eventually found several mutants showing altered seed traits, including one showing increased seed size.

“So, we have the mutant, and then we utilized a fast and cost-effective way to genotype for changes in the DNA sequence of the mutant that is associated with the increased seed size. The genotyping method is called Comparative Genome Hybridization (CGH) which can be completed within one week and specifically detects missing DNA sequences in the mutant genome,” Stacey said.

Next, Stacey used CRISPR/Cas9 mutagenesis, which is a very powerful way to cut out specific DNA sequences in targeted genes in an organism. Before CRISPR, plant scientists could only create random mutations in plants. For example, using processes involving mutagenic chemicals and radiations, which meant one had to look at a very large number of mutants to screen for induced changes in the DNA sequence of specific genes.

Using fast neutron mutagenesis which creates deletions in bases, combined with CRISPR/Cas9, Stacey was able to characterize the role of the GmKIX8-1gene in controlling seed size in soybeans.

“In a way, we got lucky because there is a QTL overlapping GmKIX8-1,” Stacey said. “And once you know the genetics behind a trait, then it is possible to define a possible mechanism of how the gene works, for example, how it makes soybean plants produce bigger seeds.” GmKIX8-1 controls the ability of cells to multiply. It also increases the size of the organs and the seeds of soybeans, which is exactly what Stacey was looking for.

An additional new and important thing Stacey found in her work was that GmKIX8-1 controls seed size, but not leaf size, in a dosage-dependent manner indicating that this gene is a major player in regulating seed size but not leaf size.

“This gene dosage effect on seed size and the identification of GmKIX8-1 as the causative gene behind a major seed size QTL are the novel aspects of this work,” Stacey said.

These novel aspects have been rewarding for Stacey.

“I’m quite happy, and it’s more fulfilling discovering something novel, something that hasn’t been reported before in other plants, instead of confirming what has been already reported,” Stacey said, “For example, based on what is already well-known in maize, or in rice, or in Arabidopsis, I can ask the question, ‘Does this gene also work in soybean?’ Answering this question is still worthwhile to me because maybe the gene will not work at all, or maybe it works in a different way, and that would also be novel, but to discover new genes or new explanations on how a gene works is more fulfilling.”

For more information on Bing Stacey’s work, check out the September 2020 edition of the New Phytologist Foundation article, “GmKIX8‐1 regulates organ size in soybean and is the causative gene for the major seed weight QTL qSw17‐1”

 

Original post available at: https://decodingscience.missouri.edu/2020/11/02/seed-size-matters-searching-for-a-gene-to-make-a-bigger-soybean/

• 

By Lauren Hines | Bond LSC

Until the 1990s, the presence and significance of extracellular ATP, a nucleotide that normally provides energy to a cell, in animal cells was highly contested for decades. Now, the Gary Stacey lab at Bond Life Sciences Center is using that history lesson to explore ATP’s role in plant cells.

Let’s say you have $100 in your pocket. As you’re walking down the street, you throw dollar bills over your shoulder. Sounds ridiculous, right?

Much like these dollar bills, ATP is the currency of cells. When the idea of animal cells dispensing ATP outside the cell to trigger danger signals came out in the 1970s, the scientific community thought it was wasteful and therefore the idea was ridiculed.

“The idea of throwing money over your shoulder is a very good analogy because that’s a good way to influence people,” said Stacey, a principal investigator at Bond LSC. “Give them a financial incentive. It’s the same thing with ATP. The idea is that this is the currency of the cell, but they’re putting it outside the cell in order to signal and influence interactions with their environment or other cells.”

This process of ATP receptors on the cellular membrane bonding with molecules to send signals and communicate between cells is called purinergic signaling.

The term was coined by retired neurobiologist Geoffrey Burnstock in the 1970s when he discovered it in animals; however, Burnstock’s findings were met with skepticism. It wasn’t until 20 years later when the first ATP receptor in animal cells was discovered that his idea gained acceptance.

Today, animal cell purinergic signaling has been used to make drugs that target diseases and develop drug therapies. As for purinergic signaling in plant cells, the research is just beginning. Much like what Burnstock faced, this new research is met with skepticism.

“It was clear to me that if we really wanted to establish this so that people would believe it, we had to get the receptor,” Stacey said.

In 2014, the Stacey lab reported its first ATP receptor called P2K1. Earlier this year, they found another one called P2K2.

“I think more and more people are working on purinergic signaling in plants,” Stacey said. “I think we will see a big explosion of people working in this area. Just as is the case with the animal field, the plant field is really going to take off now that we actually know what the receptors are.”

Currently, the Stacey lab is trying to understand how these receptors work. Research scientist Sung-Hwan Cho from the Stacey lab took over the project regarding P2K1.

“The ATP study in animals is just half a piece of the puzzle,” Cho said. “You need to find the other half of the piece. For 40 years, many labs have already studied this, but in plants, there’s just been a few.”

Research done by the Stacey lab has shown that although not much is known about purinergic signaling in plants compared to in animals, purinergic signaling can impact plant growth, development, defense against diseases and stress tolerance. Cho is currently studying how P2K1 impacts certain metabolic pathways in plants.

In another experiment done by the lab, they found that mutants without the ATP receptor that experienced stress did not grow smaller while plants that did have the receptor and experienced stress grew smaller.

“That says the purinergic signaling is what’s mediating the stress response,” Stacey said. “That is affecting the development of the plant in response to stress… So, if we understand this better then maybe we can make plants that are more drought and stress-tolerant and grow better.”

Original post available at: https://decodingscience.missouri.edu/2020/10/26/learning-from-history/