Chitin receptor CERK1 links salt stress and chitin-triggered innate immunity in Arabidopsis

Catherine Espinoza, Yan Liang, Gary Stacey

Summary

In nature, plants need to respond to multiple environmental stresses that require involvement and fine-tuning of different stress signaling pathways. Cross-tolerance in which plants pre-treated with chitin (a fungal microbe-associated molecular pattern) have improved salt tolerance was observed in Arabidopsis but is not well understood. Here, we show a unique link between chitin and salt signaling mediated by the chitin receptor CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1). Transcriptome analysis revealed that salt stress-induced genes are highly correlated with chitin-induced genes, while this was not observed with other microbe-associated molecular patterns (MAMP) or with other abiotic stresses. The cerk1 mutant was more susceptible to NaCl than wild type. cerk1 plants had an irregular increase of cytosolic calcium ([Ca2+]cyt) after NaCl treatment. Bimolecular fluorescence complementation (BiFC) and co-immuno precipitation experiments indicated that CERK1 physically interacts with ANNEXIN 1 (ANN1), which was reported to form a calcium-permeable channel that contributes to the NaCl-induced [Ca2+]cyt signal. In turn, ann1 mutants showed elevated chitin-induced rapid responses. In short, molecular components previously shown to function in chitin or salt signaling physically interact and intimately link the downstream responses to fungal attack and salt stress.

Link to the journal article

Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds (link to the full paper)

Minviluz G. Stacey*, Rebecca E. Cahoon , Hanh T. Nguyen, Yaya Cui, Shirley Sato, Cuong T. Nguyen, Nongnat Phoka, Kerry M. Clark, Yan Liang, Joe Forrester, Josef Batek, Phat Tien Do, David A. Sleper, Thomas E. Clemente, Edgar B. Cahoon, and Gary Stacey

Abstract

Soybean (Glycine max L.) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization (CGH) analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO) which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.

Protocol for nodulation assay in pouches

1. Sterilize pouches by placing them between wet paper towels then wrapping them in aluminum foil wrap. If you do not autoclave them moist, then they become quite brittle.
2. Sterilize soybean seeds, and germinate 2-3 days on wet paper or water agar. Place 3-5 seedlings per pouch; place seeds in the trough formed in the paper wick (there is a germination paper inside of the pouch and there are holes within the trough). For soybean, given the size of their roots, it helps to use a sterile needle or spatula to make holes in the paper trough for the roots. Note it is useful to select the seedlings so that they have straight roots…just makes it easier to insert them and they tend to grow better in the pouches. We plant seedlings since it allows us to better select the plants and also to eliminate any that may have fungus, etc. Fungus is one of the primary problems you have in pouch grown plants. If you like, you can just plant the seeds in the paper trough and this can work fine but this gives you less of an opportunity to select the best seedlings.

If you do use seeds, then we usually put 5 seeds per pouch as at least 1 of them will not germinate properly

3. Add 8-10 ml sterile H₂O or ½ strength plant nutrient solution(c/o nitrogen) to the pouch, wrap it in alufoil (to keep in dark) and place them into growth chamber (soybean conditions) for 3 days/dark (if you use seeds in step 2) or straight into the light, if you use seedlings. When we do a lot of these, then we have some old 45 rpm record racks (probably impossible to find now) and place one pouch in each slot with cardboard on each side. This eliminates the need for the alufoil and keeps the roots dark. Again, for larger scale use, we also use sterile soda straws (the ones you buy individually wrapped are supposedly sterile) and place one down one side of each pouch. This allows you to water the plants from the bottom up without removing them from the record racks. Important to use this bottom up approach to keep from washing contamination into the pouches by watering from the top.
4. Inoculate 3-days old seedlings, with rhizobia of OD₆₀₀~ 0.05-0.1 by spraying or pipetting. We make every attempt to make sure that the inoculant is applied to the roots. Note if you use seedlings then inoculation can be done at the time of sowing, otherwise, when the seeds have germinated.

Note: if you used seeds, not seedlings, then remove seedlings that are not properly germinated or seem to have fungi. Also, if the root is not going towards inside of the pouch, make them fit into hole (within the trough of the paper wick) and ensure that the roots are facing the bottom of the pouch.

5. Cover the pouch (downwards from the troughto ensure that the roots are covered and are in dark). Place the pouches into pouch-rack (paper racks, come with the pouches) or, in our case, the record racks.
6. Never let the paper inside the pouch get dry but it is also important not to over water. You can water with either dH₂O or with ½ plant nutrient solution, sometimes we alternate just to be sure we do not get salt buildup. Normally, plants will grow nicely in the pouches from 14-21 days without any problem but usually tend to yellow if you extend beyond this period (likely associated with drop in Ph). However, we normally grow plants to 28 days in pouches. Depending on overall conditions, you can see nodule formation within 12 days or so, sometimes sooner, sometimes a bit later.

The biggest issues with the pouches is that the pH will normally drop to quite low unless you make efforts to prevent this. For example, using a well buffered plant nutrient solution for watering. In the olden days, we even added solid calcium carbonate to the pouches, which would dissolve overtime and maintain pH but of course changes other parameters, such as ionic strength. Hence, keep an eye on how damp the plants are and water as needed. Check on the medium every day, medium needs to be replenished approx. every other day but more often as plants get bigger.

7. Important to check the pouches every day, sometimes twice as day, since water usage will vary significantly based on the size of the plants and other growth conditions (chamber humidity, light intensity, temperature, etc).

Pouches – cyg seed germination pouches (Mega-International of Minneapolis)

https://mega-international.com/

Straw – plastic drinking straws sanitary wrapped; 7¾” jumbo

Fahraeus medium (nothing special about this…you can use other nutrient solutions…indeed, something that is better buffered than Fahraeus is probably better)

• Prepare Fahraeus medium(Fahraeus, 1957) from concentrated stock solutions (autoclaved and stored at 4°C as follows):
• 5 mM MgSO4·H₂O
• 7 mM KH₂PO₄
• 8 mM Na₂HPO₄·2H₂O
• 50 μM Fe-EDTA [prepared using FeSO₄and disodium EDTA; alternatively use 20 mM ferric citrate (stored in the dark) to 50 μM final concentration]
• 1 μg/liter MnSO₄
• 1 μg/liter CuSO₄
• 1 μg/liter ZnSO₄
• 1 μg/liter H₃BO₃
• 1 μg/liter Na₂MoO₄
• Milli-Q-purified H₂O up to 1 liter
• Adjust pH to ∼5
• Autoclave

Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1

Cuong The Nguyen, Kiwamu Tanaka, Yangrong Cao, Sung-Hwan Cho, Dong Xu, Gary Stacey

Abstract

DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.

Click here to access the full paper

The International Society for Molecular Plant-Microbe Interactions (IS-MPMI) has awarded Beverly Agtuca a travel award  to attend the IS-MPMI XVII Congress, July 17-21, 2016 in Portland, Oregon, USA.

Awards up to $750 were used to pay for her registration, travel, and/or lodging expenses.  Award selection was based on the quality of the applicant’s science reflected in the research abstract, impact statement, and curriculum vitae.

Congratulations Beverly!

IS-MPMI 2016 – Awards Brochure

The 5th Annual Soybean Precision Genomics and Mutant Finder Workshops

August 3-4, 2016

University of Missouri – Columbia

The 5th annual Soybean Precision Genomics and Mutant Finder Workshops will be held on August 3-4, 2016, at the University of Missouri, Columbia Missouri.

Jesse Hall from the Francis Quadrangle, Missouri University

The workshops will feature:

1) Presentations on soybean mutant resources

2) Tutorials on cutting edge technologies used for:

• Soybean targeted mutagenesis,

• Gene editing,

• Gene silencing

• Other functional genomics applications.

3) Field demonstrations of mutant populations and opportunity to order seeds from selected lines.

Sponsors:

• United Soybean Board

• National Science Foundation.

Funding support:

Funding is available to help defray travel and lodging costs for participants. Please contact Bing Stacey (staceym@missouri.edu) to learn more about the event and request for travel support. A strong background in molecular biology is recommended.

MU Alumna Receives National Distinguished Dissertation Award
Research will open a new field of plant research

Dec. 04, 2015

Link to original story

Story Contact(s):
Nathan Hurst, hurstn@missouri.edu, 573-882-6217

By Amanda Narverud

COLUMBIA, Mo. – Mizzou alumna Jeongmin Choi has received the Council of Graduate Schools/ProQuest Distinguished Dissertation Award for her innovative research in plant sciences. The prestigious national award is given to an individual whose doctoral dissertation makes unusually significant contributions to its discipline. When Choi wrote her dissertation, she was a doctoral candidate in plant sciences from the MU College of Agriculture, Food and Natural Resources.

From left: Suzanne T. Ortega, CGS; Jeongmin Choi, winner, 2015 ProQuest Distinguished Dissertation Award; Marlene Coles, ProQuest

For her dissertation, Choi identified the first extracellular ATP receptor in plants. Her successful identification of the receptor has opened a new field of plant research that explores the interconnections among plant stress, pathogen defense and development.

ATP (adenosine 5’- triphosphate) is the main energy source inside cells and is considered to drive many life processes in plants, animals and humans. ATP is found in high concentrations inside cells. It also can be found secreted outside cells, which is referred to as extracellular ATP. When the molecule is found outside of a cell, it acts as a signaling molecule. ATP receptors are proteins localized on the cell surface to bind ATP from outside the cells and deliver the information inside the cells. Therefore, the identification of ATP receptors is essential in understanding of the cells signaling pathway. Until now, ATP receptors had been characterized in mammals, but receptors in plants had not been identified.

“The interesting part is that the newly identified plant receptor is different from animal receptors,” Choi said. “Although they sense the same molecule, they are actually quite different. The molecule can be released in response to various stress responses and also during development. We believe that the function of this molecule might relate as a danger signal, so that the plant knows that it is in danger.”

Now that the first plant receptor for extracellular ATP is known, it is likely that this receptor exists in a wide range of plant species. According to Choi, extracellular ATP has a role in many plant functions, but especially in how they perceive environmental stresses such as drought and insect feeding. According the Choi’s dissertation advisor, Gary Stacey, Curators Professor of Plant Sciences, Choi was a valued member of her Mizzou laboratory.

“As is so often the case, Jeongmin excelled not only as a researcher, but in many other ways,” Stacey said. “As a lab citizen, as a mentor to undergraduates in the lab, as a leader among her other graduate students and as a teacher, she was a great addition to our laboratory. It was a great pleasure to have her in our laboratory and we fully expect great things from her in the future.”

Choi was recognized in 2014 by the MU Office of Graduate Studies for her outstanding doctoral dissertation research. As the recipient of the MU Graduate Studies Distinguished Doctoral Dissertation Award, she was the first student to be nominated by the University of Missouri for the national Council of Graduate Schools (CGS)/ProQuest award.

“Mizzou has incredibly innovative graduate students who will become the leaders of tomorrow,” Leona Rubin, associate vice chancellor for Graduate Studies said. “One goal of the Office of Graduate Studies is to provide support for students and their academic units in submitting nominations that showcase and promote students and their accomplishments. I think it is telling that in the first year we made a concentrated effort to showcase our students nationally, a Mizzou graduate wins the national Distinguished Dissertation of the Year Award from the Council of Graduate Schools.”

Choi is currently working as a post-doctoral research associate at the University of Cambridge in the U.K. as a European Molecular Biology Organization (EMBO) fellow. EMBO supports post-doctoral research visits to laboratories throughout Europe.

Catherine Espinoza (Cathy) received her bachelor degree in biological sciences from the University of Federico Villarreal, Lima-Peru in 2001. Before coming to MU in 2008, she worked as a research assistant at the Division of Conservation and Characterization of Plant Genetic Resources, International Potato Center (CIP), Lima-Peru (2003-2008), and as a biotechnology and biodiversity consultant at the Economic Commission for Latin America and the Caribbean (ECLAC) and Andean Development Corporation (CAF), Lima-Peru (2003). Cathy joined the Stacey lab in 2013 and her dissertation was entitled “Molecular mechanisms of stress tolerance in plants”. We congratulate Cathy on her successful Ph.D. dissertation defense and wish her all the best for a new chapter in her life.

Cathy shares her happy moment after the defense with her Committee and the Stacey lab members

It would not be hard to understand why Homecoming at the University of Missouri may have an extra special meaning to Hli, Yia and See Yang.

As first-generation Americans and college students they share a common bond not only through their Mizzou pride but also through the embedded core values of family, hard work and agriculture that helped lead their family to find a new home in America.

Their parents, Chao and Toua Yang, came to the United States in 1992 when their first child, See, was two years old and her little brother, Yeng, was only a couple months old. The Yangs, former refugees of the Vietnam War, left their family, friends and the farming life behind in Laos in pursuit of better opportunity for See, Yeng and the four children that would later be born in the United States.

“Growing up I always spent time with my siblings, and back then I really didn’t know what my parents were doing, but now I do — they were working so hard to save up so they could give us a better life,” Hli said.

Humble roots

The family originally moved to Wisconsin where they worked blue collar jobs. The Yangs dreamed of raising several children on a farm, and when they had saved up enough money, they purchased a poultry farm in Wheaton, Mo., where the parents returned to their agricultural roots and continued to show their children the true meaning of hard work.

“When they first came to America, they did not have anything,” Yia said. “They started off with jobs like picking strawberries that were way below minimum wage, and now we have a pretty large-scale farm operation. So I would say that they are really hard working.”

Hli recalled the story of her dad rationing his earnings when he first came to the states. Her father would split $20 in various ways to afford a week’s worth of gas, water and food. The sacrifices her parents made to pave the way for better opportunities for their children taught Hli and her siblings the kind of ambition necessary to succeed in life.

“Everything about them, like their struggles and their hard work, I think that’s had a big impact on my siblings and me,” Hli said. “We were raised to understand just how hard success is and how hard you have to work for it. You don’t just get things handed to you.”

The Yangs have six children: See, Yeng, Chee, Hli, Yia and Meng. All of their children, aside from the Meng, 17, who still attends high school, are in pursuit of their career goals. See, 25, is a CAFNR alumna and attends law school at the University of Arkansas. Hli, 20, and Yia, 19, are CAFNR undergraduate students. Hli is a junior studying science and agricultural journalism; Yia is a sophomore in plant sciences. Yeng, 23, is in the Air Force, and Chee, 22, attends nursing school at Missouri Southern State University in Joplin, Mo.

Life on the farm had a profound impact on three of their children, who decided to attend programs within the University of Missouri’s College of Agriculture, Food and Natural Resources. See, Hli and Yia became involved in the Future Farmers of America at their high school where all of them held officer positions at the district level. After becoming Mizzou Tigers, the siblings became involved in CAFNR student organizations and now pursue career goals in agriculture.

Leading by example

See received her bachelor’s degree in agribusiness and her master’s degree in agriculture economics from the University of Missouri. She now attends law school at the University of Arkansas and plans to use her degrees to work for a firm or for a food or agricultural business as an in-house council.

It was See, the oldest sibling, who first became involved in FFA and other high school organizations, and was the first to attend school at Mizzou. See is also a former member of the CAFNR Ambassadors, a student group promoting the College, where Hli is now co-director and Yia is a member. Hli said her sister’s example had a profound impact on the performance of her and her siblings.

“See is very driven. She is always wanting to better her life and the lives of others. She is a very big family person, too. She loves coming home and seeing all of us. I honestly think that if she wouldn’t have joined FFA, we wouldn’t have joined. We have followed each other’s footsteps, and she is the one who led the way… She set the bar really high for the rest of us.”

While visiting See in Columbia on various family visits over the years, Yia became exposed to Mizzou and the opportunities available. Although schools like the University of Arkansas and Oklahoma State University were closer to his home, Yia had developed a strong connection the programs available at CAFNR. Without this insider knowledge provided by See’s experience at Mizzou, Yia probably would not have made the decision to attend school at MU.

“See is the big reason why the rest of us are where we are today,” Yia said. “She’s the oldest, and the first one to go to college. Probably without her coming to University of Missouri and being so involved in CAFNR, I don’t think I would have made the decision to come here.”

A network of support

In addition to being co-director of CAFNR Ambassadors, Hli was involved in CAFNR Student Council during her first two years at Mizzou. She wants to become a voice for agriculture in the media after she graduates. She has worked at KOMU and interned for RFT-TV and Farm Journal Media.

See and Hli developed a closer relationship with one another while living together during See’s last year at Mizzou, despite their five-year age difference. See enjoyed playing the role as confidant for Hli and watching her grow.

“We have a very close relationship,” See said. “Hli is a very ambitious girl, and I see a lot of myself in her. I am so amazed at what she does in ag journalism. I’ve seen some of her video productions and I’m impressed by how well she’s mastered all the programs.”

For Yia, it comforts him to know that his sister always has his back here at the University.

“Hli is a very caring sister,” Yia said. “I lived with her for the first semester of college. And I know she would do anything for me. If I ever need anything, I just give her a call. She plays that big sister role since it’s only me and her here at the university.”

Yia plans to attend graduate school after he graduates and wants to pursue a Ph.D. in plant sciences. Yia is also a member of CAFNR Ambassadors and is a member of the Alpha Gamma Rho fraternity.

“I’m really happy that Yia’s found his own way and am impressed by the way he excels at everything he does,” See said. “It makes me happy to see him shine so bright.”

Although Hli and Yia often butt heads because they have similar and competitive personalities, they enjoy working with one another in CAFNR Ambassadors.

“Because we are in CAFNR Ambassadors together, I see him once a week, at least. And I think that’s a really good thing,” Hli said. “Being in an organization with him, I get to see him and catch up with him… We both get to see each other mature, which is really funny.”

‘He’s my family’

With her family more than four hours away in southern-most Missouri, Hli is relieved to have at least one sibling close throughout her time at Mizzou.

“It’s nice to have at least one close family member around here, especially because everyone else is four hours away,” Hli said. “Whenever I need things to be taken care of, I go to him. I rely on him, and I know he hates it, but he’s my family.”

The Mizzou experience has created a closer bond between the three Yang siblings. Whenever Yia and Hli struggle with homework or other issues, they are able to go to See or one another for help.

“The three of us having shared that same Mizzou experience and it has elevated our relationship,” See said. “I’ve already taken a lot of the same classes they are taking now and have been involved with the same organizations. They can come to me and I’m able to give them valuable advice because I’ve been in their shoes before.”

Written by Emily Brehe – October 14, 2015

Original story here