Progress 12/04/15 to 09/30/20
Outputs
Target Audience:This basic research project expands our understanding in plant immunity, specifically how plants recognize pathogen associated molecular patterns and trigger an innate immune response. Therefore, the target audience for this research project is mainly other scientists, both in academia and the agriculture biotechnology industry, because the proteins and genes under investigation have long-term agricultural applications. Agribusiness enterprises would be greatly interested in the outcomes of this research because it may be used to either engineer crop plants that would naturally defend against potential pathogens or set the foundation to develop novel treatments to control plant pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This five-year proposal involved the mentoring and training of many graduate and undergraduate students and postdoctoral researchers. Specifically, five graduate students (Melissa Matthews, Nicholas Hurlburt, Alex Thuy-Boun, Xander Wilcox and Samantha Hartanto) and a postdoc (Vandana Gaded) worked on these AES supported projects. There were numerous one-on-one discussions and training with these students. I mentored and trained my students in all aspects of research including safety, reproducibility, ethical and responsible conduct of research. I also train students in professional development of scientific writing. I encourage and support my students to apply for fellowships, which usually includes writing a research proposal. Additionally, I ask graduate students and postdocs to write the first draft of manuscripts to be published, where they are first author. I also champion my students and postdocs to present their research at scientific conferences at the local, national and international level. Additionally, this five-year project also helped support and train 13 undergraduate students in my lab that worked on various aspects of this AES proposal. Of the 13 undergraduate students, 9 were females, and 2 come from underrepresented groups. Most of my undergraduates participate in the UC-Davis campus-wide research symposium, where they present their research in scientific talks or poster sessions. Additionally, my students have presented their research at the ABRCMS conference in Indianapolis, IN (2018) and Anaheim, CA (2019). I have also helped train and mentor high school FFA and 4-H students by serving as judge for the regional UC-Davis Agriscience fair FFA science competition and the FFA public speaking competition. In addition to judging the students, it was very rewarding to talk with the students about their projects and how the students can improve on the science fair projects if selected to advance to State competition or to improve their understanding of the science if they participate in the science fair competition next year. Additionally, I served as a judge for the California State Agriscience Fair competition by judging and reviewing proposals at the Advanced level in the area of PLANT SYSTEMS submitted from students throughout the state of California. This judging helps select students to advance to the National competition. How have the results been disseminated to communities of interest?The results of this proposal were disseminated through a number of public channels that include publications in scientific journals and lectures at universities, scientific conferences, and K-12 schools. For publications published this year in peer-reviewed journals, see the list above. Additionally, this change in knowledge has been disseminated through workshops and conferences. For example, this work was presented at the Gordon Research Conference in Hong Kong in June of 2019. Additionally, my graduate students presented the results locally at UC-Davis through departmental retreats, campus symposiums, as well as at the national level at the Chemical Biology in the Bay Area at Berkeley and San Francisco and other scientific society national meetings. Additionally, some of my undergraduate students have presented their research at the ABRCMS (Annual Biomedical Research Conference for Minority Students) conference in Indianapolis, IN (2018) and Anaheim, CA (2019) thus enhancing public understanding and interest in the research supported by the USDA National Institute of Food and Agriculture. Finally, my collaborators have also presented results of this work at international conferences on plant pathology. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The growing population places a significant burden on crop plants to supply world's increasing demand for food and energy. Therefore, the future necessitates an ever-increasing pressure for the efficient production of crop plants. Currently, global crop losses from plant pathogens amount to several hundred billion dollars annually. Current measures used to control plant pests and pathogens are costly and rely on methods that can aversely affect the environment and human health (e.g., toxic pesticide applications). However, plants do possess an innate immunity to fight against many pathogens, but little is known about the biochemistry and molecular details by which plants recognize the pathogens or how the plant triggers an immune response. Therefore, food security and the success of California agriculture depend on understanding this important innate immunity pathway. The conditional changes or impacts of this project can lead to novel methods to fight plant pathogens or developing plants that are able better defend themselves against infiltrations by pathogens. The long-term conditional changes that will impact future generations include; increasing crop yields, which will lead to improved food security, improved management of land use, and protected and conserved soil quality. Additionally, this fundamental research can increase the preparedness and resilience to climate change, which can facilitate potentially new pathogens not currently seen in today's climate. The objectives for the original proposal were to create a knowledge change in our basic fundamental understanding of the plant immune system, and how plants respond and defend themselves from attacks from pathogens. The proposed project has two broad goals; first to understand the structural basis of function of a fungal pathogen effector and how plants recognize it; second, understand the structural basis of plant kinases involved in triggering the innate immune response when plants are infected by a pathogen. We proposed to use the structural biology techniques, specifically X-ray crystallography, to determine the atomic resolution structure of many of the key players in this important plant immune pathway. Obtaining these detailed high-resolution structures provides unparallel insight into better understand how they function at the molecular level. This fundamental knowledge change can lead to novel designs of non-toxic proteins or peptides that can help plants fight off pathogens. Additionally, this basic research can lead to engineering plants that might be able to naturally defend against common pathogens, resulting in better food security and land management. Significant accomplishments were achieved in this five-year project. The lab was able to structurally and biochemically characterize two plant fungal pathogen effectors. These fungal proteins, or avirulence proteins, are expressed in response to one pathway of the plant's innate immune response. Often plants will express chitinases, that breakdown chitin, the main coating in fungal and insect pathogens. In this "biochemical warfare" some fungi express a protein that binds to and provides a proactive barrier against the plant chitinases. But as a countermeasure, plants recognize this protein and elicits another counter-defense measure by initiating a hypersensitive response to help fight off the fungal attack. Our lab, together with the Stergiopoulos lab (UCD, CA&ES, Department of Plant Pathology), determined the structure of these fungal effector proteins, both by themself and bound to chitin. These results lead to hypotheses on the molecular function of specific amino acids. This was tested by subsequent biochemical and biophysical experiments. We discovered that regions of the protein that are important for binding chitin and how the plant recognizes the fungal protein (by cell-surface receptors) are mutually exclusive. These results were the first time the molecular details of action were observed and understood of these fungal pathogen proteins, and how they interact with the plant receptor. The second main goal of this proposal was to better understand the molecular basis for function of plant kinases that relay the signal to initiate a plant innate immune response after the plant recognizes an attack by a plant pathogen. Before this research was initiated, nothing was known about the molecular details of this BIK1 kinase and how it transduced signals between the cell surface and launching the immune pathway within the plant cell. Understanding how the kinase responds and binds to different cellular components can lead to development of unique ways to control the plant immune response, to help fight off pests and pathogens before significant crop loss can happen. We determined the crystal structure of the plant kinase BIK1, which resulted in a knowledge change. Unexpectantly, the molecular structure revealed this kinase may be regulated by another binding partner much like cyclins bind and activate the cyclin-dependent kinases. This is because the structure revealed that helix alpha-C, which contains catalytically important Glu51, must shift into an active conformation upon binding another protein suggesting that our BIK1 structure may be in an inactive conformation and may interact with other cellular factor(s) to push the helix into an active conformation. More importantly, the structure revealed a unique phosphorylation loop that is not observed in other kinases, this loop contains a serine and threonine residue (S89/T90) that we show is important for a unique function of BIK1 never before seen. This change in fundamental knowledge revealed that BIK1 is involved in the phytohormone jasmonic acid (JA) pathway, which was totally unexpected. Finally, during this funding time period, the lab also initiated a project that can be applied to help plants and livestock animals increase yields or introduce desirable traits (including combating pathogens, or tolerating environmental climate changes). We are investigating using an RNA-editing enzyme to treat genetic disorders or introducing traits by altering the mRNA producing changes in protein products. Additionally, we are also investigating repurposing this RNA editing enzyme to genetically change the DNA of targeted genes producing a desired trait in a plant or livestock animal. Overall, the results of this project lay the groundwork for more long-term benefits to future generations in food security and improved management of land use. This project has increased the basic fundamental knowledge, which was lacking, in understanding the molecular mechanisms of many players in these important pathways. The findings of this project were disseminated through peer-reviewed publications, public databases and public lectures so other agribusinesses or academic labs can apply our findings to benefit future generations.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Li, Y., Li, R., Yu, H., Sheng, X., Wang, J., Fisher, A. J., Chen, X. (2020). Enterococcus faecalis ?12-mannosidase (EfMan-I): an efficient catalyst for glycoprotein N-glycan modification. FEBS Letters. 594:439-451.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Matthews, M. M., McArthur, J. B., Li, Y., Yu, H., Chen, X., Fisher, A. J. (2020). Catalytic cycle of Neisseria meningitidis CMP-sialic acid synthetase illustrated by high-resolution protein crystallography. Biochemistry. 59:3157-3168.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Thuy-Boun, A. S., Thomas, J. M., Grajo, H. L., Palumbo, C. M., Park, S., Nguyen, L. T., Fisher, A. J., and Beal, P. A. (2020). Asymmetric Dimerization of Adenosine Deaminase acting on RNA Facilitates Substrate Recognition. Nucleic Acids Res. 48:7958-7972.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Hurlburt, N. K., Guan, J., Ong, H., Yu, H., Chen, X., and Fisher, A. J. (2020). Structural characterization of a non-hydrolyzing UDP-GlcNAc 2-epimerase from Neisseria meningitidis serogroup A. Acta Crystallogr. F Struct. Biol. Commun. 76:557-567.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Feng, Y., Hua, X., Shen, Q., Matthews, M., Zhang, Y., Fisher, A. J., Lyu, X., and Yang, R. (2020) Insight into the potential factors influencing the catalytic direction in cellobiose 2-epimerase by crystallization and mutagenesis, Acta Crystallogr. D Struct. Biol. 76:1104-1113.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Moreno, M. V., Rockwell, N. C., Mora, M., Fisher, A. J., and Lagarias, J. C. (2020). A far-red cyanobacteriochrome lineage specific for verdins. Proc. Natl. Acad. Sci. U.S.A., 117:27962-27970.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Unger, E. K., Keller J. P., Altermatt, M., Liang, R., Yao, Z., Sun, J., Matsui, A., Dong, C. E., Jaffe, D. A., Hartanto, S., Mizuno, G. O., Borden, P. M., Shivange, A. V., Sinning, S., Carlin, J., Banala, S., Cameron, L. P., Olson, D. E., Temple-Lang, D., Rudnick, G., Marvin J., Lavis, L. D., Fisher, A. J., Alvarez, V. A., Prescher, J. A., Yarov-Yarovoy, V., Gradinaru, V., Looker L. L., TIan, L. (2020) Directed evolution of a selective and sensitive serotonin biosensor via machine learning. Cell, 183:1986-2002.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Yu, Q., Anderson, D. E., Kaur, R., Fisher, A. J., and Ames, J. B. (2021). Crystal Structure of Calmodulin Bound to the Cardiac Ryanodine Receptor (RyR2) at Residues: Phe4246 to Val4270, Reveals a Fifth Calcium Binding Site. Biochemistry, Submitted.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Doherty, E., Wilcox, X., van Sint Fiet, L., Kemmel, C., Turunen, J., Klein, B., Tantillo, D., Fisher, A. J., Beal, P. A. (2021). Rational Design of RNA Editing Guide Strands: Cytidine Analogs at the Orphan Position. J. Am. Chem. Soc., Submitted.
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:This basic research project expands our understanding in plant immunity, therefore its target audience is mainly other scientists, both in academia and the biotech industry, because the proteins and genes under investigation have long-term agricultural applications. Agribusiness enterprises would be greatly interested in the outcomes of this research because it may be used to either; engineer crop plants that would naturally defend against potential pathogens, or set the foundation to develop novel treatments to control plant pathogens. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?These projects involve the mentoring and training of many graduate and undergraduate students and postdoctoral researchers. Specifically, my graduate students Xander Wilcox and Samantha Hartanto and postdoc Vandana Gaded worked on these AES supported projects. There were numerous one-on-one discussions and training with these students. I mentored and trained my students in all aspects of research including ethical and responsible conduct of research. I also train students in professional development of scientific writing. I ask students to apply for fellowships, which includes writing a research proposal. Additionally, I ask graduate students and postdocs to write the first draft of manuscripts to be published, where they are first author. I also encourage my students and postdocs to present their research at scientific conferences at the local, national and international level. Additionally, I've helped train undergraduate students in my lab, which include: Jessamine Dandan, Macy Ha, Kimberly Maldonado, Abdulsamad Mohammed, and Cao Nguyen. Most of my undergraduates participate in the UC-Davis campus-wide research symposium, where they present their research in scientific talks or poster sessions. Additionally, my students have presented their research at the ABRCMS conference in Indianapolis, IN (2018) and Anaheim, CA (2019). I have also helped train some FFA and 4-H students by serving as judge for the regional UC-Davis Agriscience State Finals Competition. In addition to judging the students, it was very rewarding to talk with the students about their projects and how the students can improve on the science fair projects if selected to advance to State competition or to improve their understanding of the science if they participate in the science fair competition next year. Additionally, I served as a judge for the California State Agriscience Fair competition by judging and reviewing proposals at the Advanced level in the area of PLANT SYSTEMS. This judging helped select student to advance to the National competition in Indiana. How have the results been disseminated to communities of interest?The results of the projects described above were disseminated through publications in scientific journals and scientific lectures both at universities and scientific conferences. For publications, see the list above. Additionally, this knowledge has been disseminated through workshops and conferences. For example, I presented work on plant pathogen Avr4 interacting with carbohydrates at the Gordon Research Conference in Hong Kong in June of 2019. Additionally, my students presented work locally at UC-Davis through departmental retreats, and more nationally at the Chemical Biology in the Bay Area at UC-San Francisco. Additionally, my collaborators have present this work at international conferences. What do you plan to do during the next reporting period to accomplish the goals?For the coming year, we will still try to crystallize other fungal effectors such as Avr4.2 and Ecp2 to determine the structural basis for function and plant interaction. We will also focus on better developing ADARs as base-editors of DNA to improve selectivity, precision and reduce off-target edits. This will be accomplished by investigating larger fragments of ADAR that include the dsRNA binding domains. We will also find ways to create DNA/RNA hybrids that can serve as a substrate for ADAR DNA editing. One avenue we are pursuing is using locked nucleic acids (LNAs) that may be able to displace a strand of DNA to create a "D-loop" where one strand of DNA is hybridized to LNA (or RNA), which can serve as an RNA substrate. We will also continue our collaboration with a group in China working on the structure of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus to better understand the mechanism of cis-enediol intermediate in epimerization and hydride shift in isomerization of AGE superfamily. Finally, we will also continue exploring enzymes involved in carbohydrate processing and synthesis, and determining how the enzymes function at the atomic level and can be engineered to create novel carbohydrates and oligosaccharides for biotechnology applications.
Impacts
What was accomplished under these goals?
The growing global population will place a significant burden on the utilization of crop plants to supply world's insatiable demand for food and energy. Therefore, the future necessitates an ever-increasing pressure for the efficient production of crop plants. Currently, global crop losses from plant pathogens and climate change amount to several hundred billion dollars annually. Prevailing measures used to control plant pests and pathogens are costly and rely on methods with potentially adverse effects on the environment and human health. However, plants do possess an innate immunity to fight against many pathogens, but little is known about the molecular detains by which plants recognize the pathogens or how the plant triggers an immune response. Therefore, global food security and the success of California agriculture depend on understanding this important innate immunity pathway. Recognizing how the immunity pathway functions can lead to developing plants that are more resistant or better equipped to fight off attacks from pathogens. One long-term goal of this project is to understand the molecular details of how plants recognize pathogens and elicit a defensive response. Another long-term goal is to develop efficient ways to alter or modify the plant genome, to introduce or correct genomic changes that can result in plants with resistance to pathogens and/or better suited to grow and produce crops in the changing climate conditions. These results can lead to plants that are able better defend themselves against infiltrations by pathogens or changing growing conditions, thus increasing crop yields, which will lead to global food security. Last year our lab focused on understanding the structural bases of how plants perceive plant pathogens and how plants transmit the signal intracellularly that a pathogen is present to initiate a plant response. This funding period, the lab has focused on genome and RNA editing, which would be of great benefit to modify plants with genes required to combat infiltration by pests, or introduce changes or properties into plant to increase tolerance. Recently, the laboratory determined the crystal structure of the enzyme Adenosine Deaminase acting on dsRNA (ADAR) bound to dsRNA. This enzyme deaminates adenosine at select sites of the RNA creating the nucleotide inosine, which behaves like guanosine in Watson-Crick base pairing. This can change the meaning of codons in mRNA that when translated into protein, will produce a modified protein, that differs from that expected based on the DNA sequence. This structure led us to hypothesize that ADARs cannot base-edit adenosines in dsDNA because it only can bind to A-form duplex nucleic acid, as seen in dsRNA. However, when a single strand of RNA is base-paired to a single strand of DNA, this DNA/RNA hybrid does indeed wind into a structure very similar to A-form double helix. We have shown that this DNA/RNA hybrid can not only bind to ADAR, but ADAR will actually deaminate adenosine nucleotides in the DNA strand. This technology can alter the genomic material of plant creating novel properties, or reversing single point mutations where a G is mutated to A (ADAR can reverse the A back to a G in the DNA). Another interesting property of ADAR is to exploit the ability of its natural function of editing RNA. This has the ability of making similar A to G changes in mRNA thus avoiding making permanent changes in the genomic DNA at specific genes (creating a genetically modified organism). One drawback applying ADAR to modify RNA is the possibility of unwanted off-targe RNA edits. Since ADARs work on dsRNA, a oligonucleotide and be administered to the cell, when an abasic site opposite the targeted A on the RNA needed to be changed. This RNA duplex can only be edited by a mutant ADAR, that we generated where the base-flipping residue glutamate is changed to a larger bulkier residue like tyrosine or tryptophan. This year we published these results, where we tested the ability of ADAR to only edit adenosines opposite abasic sites in RNA. This was published in: Cell Chem. Biol. 26:269-277 (2019). Additionally, we have published two additional papers investigating the structural basis for carbohydrate synthesis and modification. These papers, while not directly related to initial project do have many implications in plant immunity, because many pathogens like fungus and insect contain carbohydrate cell-surface molecules, that are recognized by plant, and initiates an innate immune response. One of these papers is in press (Li, Y., Li, R., Yu, H., Sheng, X., Wang, J., Fisher, A. J., Chen, X. (2019). Enterococcus faecalis α1-2-mannosidase (EfMan-I): an efficient catalyst for glycoprotein N-glycan modification. FEBS Letters. In Press.), and the other paper is out for review (Matthews, M. M., McArthur, J. B., Li, Y., Yu, H., Chen, X., Fisher, A. J. (2019). Catalytic cycle of Neisseria meningitidis CMP-sialic acid synthetase illustrated by high-resolution protein crystallography. Submitted to Biochemistry.).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Monteleone. L. R., Matthews, M. M., Palumbo, C. M., Thomas, J. M., Zheng, Y., Chiang, Y., Fisher, A. J., and Beal, P. A. (2019). A Bump-Hole Approach for Directed RNA Editing. Cell Chem. Biol. 26:269-277.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Li, Y., Li, R., Yu, H., Sheng, X., Wang, J., Fisher, A. J., Chen, X. (2019). Enterococcus faecalis ?12-mannosidase (EfMan-I): an efficient catalyst for glycoprotein N-glycan modification. FEBS Letters. In Press.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Matthews, M. M., McArthur, J. B., Li, Y., Yu, H., Chen, X., Fisher, A. J. (2019). Catalytic cycle of Neisseria meningitidis CMP-sialic acid synthetase illustrated by high-resolution protein crystallography. Biochemistry.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:This basic research project enlarges our understanding in plant immunity, therefore its target audience is mainly other scientists, both academically and commercially, because the proteins under investigation have long-term agricultural applications. Agribusiness enterprises would be greatly interested in the outcomes of this research because it may be used to either; engineer crop plants that would naturally defend against potential pathogens, or set the foundation to develop novel treatments to control plant pathogens. These accomplishments will result in benefits to humanity in the long-term. The target audience also includes students at all levels. All of the proposed work will be carried out by undergraduate and graduate students, and the lab often participates in high school student outreach programs where the K-12 students participate in the proposed research project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?These projects involve the mentoring and training of many graduate and undergraduate students. Specifically, my graduate students Nicholas Hurlburt, Xander Wilcox, and Samantha Hartanto worked on these AES supported projects. There were numerous one-on-one discussions and training with these students. Additionally, I've helped train graduate students in my collaborator labs, which include Neeraj Lal (whom I share two co-author manuscripts with), and the postdoc Li Chen from another collaborator lab (whom I share two co-authorships with). A few undergraduates have also worked on these projects including: Madeline Mumbleau (who co-authored a paper), Zoe Aspitz, Kimberly Maldonado, and Chi Pham. How have the results been disseminated to communities of interest?The results of the projects described above were disseminated through publications in scientific journals and scientific lectures both at universities and scientific conferences. For publications, see the list above. Additionally, this knowledge has been disseminated through workshops and conferences. For example, my student Nicholas Hurlburt, presented the Avr4 work locally at UC-Davis through departmental retreats, and more nationally at the Chemical Biology in the Bay Area at UC-Berkeley. Additionally, my collaborators have present this work at international conferences. Finally, the PI also makes yearly visits to a local fourth grade science class where the very basic results from this data are disseminated through a science demonstration presentation. What do you plan to do during the next reporting period to accomplish the goals?For the fungal effector project, we will continue to help elucidate the structural basis for activity by determining the atomic resolution structure of other fungal effectors such as Avr4.2 and Ecp2. We have already made significant progress along these goals. For the Kinase signal transduction project we are working on identifying other plant proteins that may bind to and activate the BIK1 kinases in a similar fashion as cyclin activated the cyclin-dependent kinases. This is due to fact the the BIK1 structure we determined appears to be in the inactive state based on structural similarities to other kinases, and the "activation" helix is shifted in our structure like the inactive cyclin-dependent kinases found in mammals. Details for both goals can be found in the original proposal.
Impacts
What was accomplished under these goals?
The growing global population will place a significant burden on the utilization of crop plants to supply world's insatiable demand for food and energy. Therefore, the future necessitates an ever-increasing pressure for the efficient production of crop plants. Currently, global crop losses from plant pathogens amount to several hundred billion dollars annually. Prevailing measures used to control plant pests and pathogens are costly and rely on methods with potentially adverse effects on the environment and human health. However, plants do possess an innate immunity to fight against many pathogens, but little is known about the molecular detains by which plants recognize the pathogens or how the plant triggers an immune response. Therefore, global food security and the success of California agriculture depend on understanding this important innate immunity pathway. Recognizing how the immunity pathway functions can lead to developing plants that are more resistant or better equipped to fight off attacks from pathogens. The long-term goal of this project is to understand the molecular details of how plants recognize pathogens and elicit a defensive response. These results can lead to plants that are able better defend themselves against infiltrations by pathogens, thus increasing crop yields, which will lead to global food security. One major goal of the project was to understand how the fungal pathogen Cladosporium fulvum (Cf) is perceived by the plant, and how the fungal pathogen inhibits the plant innate immune system. In this past year major research and output products have been accomplished resulting in a change in knowledge. Namely, we published a very important paper on the crystal structure of Cf Avirulence factor-4 (Cf-Avr4). This structure was determined with chitin fragments bound to the protein. These seminal results elucidated, for the first time, the structural basis on how Avr4 binds to chitin, its primary function in protecting fungal plant pathogens from degradation from plant chitinases. Plants often express chitinases in a first-line response to infection from pathogens such as fungi. This Avr4-chitin structure revealed that Avr4 binds to chitin in a way that was not predicted from binding models from earlier publications resulting in a change in knowledge. This structure revealed that two Avr4 proteins sandwich two chitin oligosaccharides, where each Avr4 protein makes extensive contacts along the entire length of the chitin hexa-saccharide, but two Avr4 proteins do not contact each other. Additionally, this publication presents strong evidence that amino acid residues important for chitin binding are not required for plant detection, because mutating these residues affected chitin binding, but not the hypersensitive response from the plant. These results are published this past year: (PLOS Pathogens, 14:e1007263). In the coming year, we will focus on other fungal effectors like Avr4.2 and Ecp2. Another major goal of this project was to investigate the mechanism of plant innate immunity signal transduction. After the plant Pattern Recognition Receptors (PRR) binds to the Pathogen associated molecular patterns (PAMPs), an immune response is initiated. This signal is transduced a series of kinases. Little is know on the structural basis for signal transduction and how this is regulated by phosphorylation by other kinases. In a paper reported this past year, my lab in collaboration with Dinesh-Kumar's lab (Plant Biology, UC-Davis), determined the structure of BIK1 kinase, an important kinase involved in plant immunity. This structure revealed a unique loop that is not seen in other mammalian and plant kinases, and that phosphorylation of the loop regulates the phytohormone jasmonic acid (JA), something no one has ever seen or modeled before resulting in a change in knowledge. These results were published this past year: (Cell & Host Microbe 23:485-497).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Lal, N. K., Nagalakshmi ,U., Hurlburt , N. K., Flores, R., Bak, A., Sone, P., Ma, X., Song, G., Walley, J., Shan, L., He, P., Casteel, C., Fisher, A. J., Dinesh-Kumar, S. P. (2018). The Receptor-like Cytoplasmic Kinase BIK1 Localizes to the Nucleus and Regulates Defense Hormone Expression during Plant Innate Immunity. Cell & Host Microbe 23:485-497.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
McArthur, J. B., Yu, H., Tasnima, N., Lee, C. M., Fisher, A. J, and Chen, X. (2018). ?26-Neosialidase: A sialyltransferase mutant as a sialyl linkage-specific sialidase. ACS Chemical Biology 13:1228-1234.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Fisher, A. J., and Beal, P. A. (2018). Structural basis for eukaryotic mRNA modification. Curr. Opin. Struct. Biol. 53:59-68.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Hurlburt, N. K., Chen, L.-H., Stergiopoulos, I., and Fisher, A. J. (2018). Structure of the Cladosporium fulvum Avr4 effector in complex with (GlcNAc)6 reveals the ligand-binding mechanism and uncouples its intrinsic function from recognition by the Cf-4 resistance protein. PLOS Pathogens, 14:e1007263.
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audience for this basic research project, which expands our knowledge in plant immunity, is mostly other scientists, both academically and commercially, because the proteins under investigation have long-term potential biotechnology and agricultural applications. Agribusiness enterprises would be greatly interested in the outcomes of this research because it may be used to either; engineer crop plants that would naturally defend against potential pathogens, or set the foundation to develop novel treatments to control plant pathogens. These accomplishments will result in benefits to humanity in the long-term. The target audience also includes students at all levels. All of the proposed work will be carried out by undergraduate and graduate students, and the lab often participates in high school student outreach programs where the K-12 students participate in the proposed research project. Finally the PI of the proposal also gives informal outreach demonstrations to local grade schools and High School students in FFA. In all these student activities, the students gain knowledge of the use of plant immunity and how it may be used to benefit humankind. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?These projects involve the mentoring and training of many graduate and undergraduate students. Specifically, my graduate students Melissa Matthews and Nicholas Hurlburt worked on these AES supported projects. There were numerous one-on-one discussions and training with these students. Additionally, I've helped train graduate students in my collaborative labs that include: Neeraj Lal (who I share a co-author paper), and the postdoc Li Chen from a collaborator lab. A few undergraduates have also worked on these projects including: Madeline Mumbleau (who co-authored a paper), and Zoe Aspitz. How have the results been disseminated to communities of interest?The results of the projects described above were disseminated through publications in scientific journals and scientific lectures both at universities and scientific conferences. For example, my students Nicholas Hurlburt, presented the Avr4 work at the Chemical Biology in the Bay Area at UC-Berkeley. Additionally, the PI also makes yearly visits to a local fourth grade science class where the very basic results from this data are disseminated through a science demonstration presentation. What do you plan to do during the next reporting period to accomplish the goals?For the Avr4 project we plan to determine the structural basis of another recently identified fungal effector, Ecp2. The function of this fungal protein is unknown, but may bind to chitin too. We will also continue to more fully analyze the structural feature of Avr4 from a couple different sources. For the BIK1 project, we plan on identifying a potential protein binding partner and determine the crystal structure of BIK1 bound to this partner in the active conformation. The structure also identified a couple residues on this helix alpha-C that may be phosphorylated, which may be involved in the regulation of BIK1 activation. We plan on making mutations of these residues that mimic phosphorylation my mutating the serine and threonine to glutamate residues. Additionally, mutating them to alanine would block any potential phosphorylation. These mutants may help identify a phenotype and potentially the binding partner.
Impacts
What was accomplished under these goals?
One major goal of the project was to understand how the fungal pathogen Cladosporium fulvum is perceived by the plant, and how the fungal pathogen inhibits the plant innate immune system. Previously it was determined that the fungus excretes an avirulence protein, Avr4, that binds and protects the chitin layer coating the fungal cell wall. This protects the fungus from plant chitinases that would "digest" the cell wall, making the fungus more vulnerable to death, and stopping the spread of the fungal disease. This past year we determined the crystal structure of Avr4 bound to a chitin hexasaccharide. This is the first known structure of an avirulence protein bound to a carbohydrate effector. We were able to precisely determine the atomic details on how the fungal protein binds to chitin. This structure lead to additional hypotheses on determining the importance of individual amino acid residues in function of chitin binding. This lead to new experiments of mutating individual residues and testing the effect on chitin binding. The results of this research resulted in change in fundamental knowledge of understanding how this family of proteins binds to carbohydrates. Our results revealed that previous hypotheses from many other labs were incorrect in their understanding of how this avirulence factor functions. We show that the protein dimerizes upon binding chitin, and this dimerization is mediated by chitin. However, unexpectedly the two proteins of the dimer do not contact each other, the dimerization is solely mediated by each protein binding to two layered oligosaccharides. These results were written up and submitted to the journal: Proceedings of the National Academy of Science (PNAS) and is currently under review. The second main project of this research proposal is to better understand the fundamental details of how plant cells communicate and initiate the plant innate immune response pathway upon recognizing pathogen effectors such as PAMPs (pathogen-associated molecular patterns). Once a plant cell binds the PAMP through the membrane-bound immune pattern recognition receptor, the kinase cascade is triggered. This results ultimately in the activation of the innate immune pathway, through transcription activators. We recently determined the crystal structure of the plant kinase BIK1, which is involved in this innate signal kinase cascade. The structure revealed this kinase may be regulated by another binding partner much like cyclins bind and activate the cyclin-dependent kinases (CDKs). This is because the structure revealed that helix alpha-C lies in an orientation compared to other inactive CDKs, where it is shifted away from the active site. This helix, which corresponds to the "PSTAIRE helix" in cyclin-dependent kinase (CDKs) contains catalytically important Glu51, which shifts into an active conformation upon binding cyclin suggesting that our BIK1 structure may be in an inactive conformation and may interact with other cellular factor(s) to push the helix into an active conformation. More importantly, the structure revealed a unique phosphorylation loop that is not observed in other kinases, this loop contains a serine and threonine residue (S89/T90) that we show is important for a unique function of BIK1 never before seen. This change in fundamental knowledge revealed that BIK1 is involved in the phytohormone jasmonic acid (JA) pathway, which was totally unexpected. These results were also written up and submitted to the journal, Cell Host & Microbe, which is currently under review.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Suter, S. R., Ball-Jones, A., Mumbleau, M. M., Valenzuela, R., Ibarra-Soza, J., Owens, H., Fisher, A. J., and Beal P. A. (2017). Controlling miRNA-like Off-target Effects of an siRNA with Nucleobase Modifications. Org. Biomol. Chem. 15:10029-10036.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Hurlburt, N. K., Chen, L.-H., Stergiopoulos, I., and Fisher, A. J. (2017). Structure of the Cladosporium fulvum Avr4 effector in complex with chitin uncouples the ligand-binding function from Cf-4 recognition. Submitted to PNAS.
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Progress 12/04/15 to 09/30/16
Outputs
Target Audience:The main target audience for the proposed research, which expands our knowledge in plant immunity, is other scientists, both academically and commercially, because the proteins under investigation have potential biotechnology and agricultural applications. Agribusiness enterprises would be interested in the outcomes of the proposed research because it may be used to either; engineer crop plants that would naturally defend against potential pathogens, or develop novel treatments to control plant pathogens. These accomplishments will result in benefits to humanity in the long-term. The target audience also includes students at all levels. All of the proposed work will be carried out by undergraduate and graduate students, and the lab often participates in high school student outreach programs where the K-12 students participate in the proposed research project. Finally the PI of the proposal also gives informal outreach demonstrations to local grade schools. In all student activities, the students gain knowledge of the use of plant immunity and how it may be used to benefit humankind. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Both of these projects involve the mentoring and training of many graduate and undergraduate students. Specifically, the graduate students Melissa Matthews and Nicholas Hurlburt worked on these AES supported projects. A few undergraduates have also worked on these projects including: Danielle Dubian, Jiahui Lu, and Rucha Mahadik. How have the results been disseminated to communities of interest?The outcomes of both projects were disseminated through publications in scientific journals and scientific lectures both at universities and scientific conferences. For example, my student Nicholas Hurlburt, presented the Avr4 work at the NAIST Bio International Student Workshop in Osaka Japan, as well as the Chemical Biology in the Bay Area at UC-Berkeley. Additionally, the PI also makes yearly visits to a local fourth grade science class where the very basic results from this data are disseminated through a science demonstration presentation. What do you plan to do during the next reporting period to accomplish the goals?For the Avr4 project, we will continue to characterize the structural basis of function by investigating the effects of mutants, both in vitro and in plant model systems. We will publish the structure of Avr4 bound to chitin. We will also continue our efforts on crystallizing the plant receptor Cf4, which binds Avr4 from the fungus, and initiates an immune response. For the plant kinase project, we propose to do additional structures in different phosphorylated states, since the BIK1 itself is phosphorylated by upstream kinases. These different phosphorylation events may alter the structure to allow binding with partners. Also, we will aim to determine the structure with ATP analogs bound, as well as complex structures with other binding partners like other kinases. We will also test the kinase efficiency of BIK1 at different phosphorylation sites.
Impacts
What was accomplished under these goals?
The overall goal of this hatch proposal is to investigate the structural basis for function of many key proteins, receptors, effectors and enzymes involved in plant innate immunity. The project achieved some important accomplishments this past year. First we were able to structurally characterize the fungal avirulence effector protein Avr4, which was published in The Plant Cell. This past year we have been able to determine the structure of Avr4 bound to chitin. We are currently determining the structural basis for function by making site-directed mutants and testing for activity. I expect these experiments will be complete in the coming year and published. Another major accomplishment achieved in the past year deals with enzymes transducing the innate immune signal within the cell. Recently we crystallized the plant kinase BIK1 and have published this result. We have also determined the crystal structure, which lead to many hypotheses on plant innate immunity signaling. These have been tested by creating site-directed mutants and studying their effect both in vitro and in Arabidopsis. These results have determined an unexpected finding, which we have submitted to the journal Nature. The reviews are mostly positive and we are currently running subsequent experiments in response to the reviewers. Once complete in the coming month, we will re-submit to Nature.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Matthews, M. M., Thomas, J. M., Zheng, Y., Tran, K., Phelps, K. J., Scott, A. I., Havel, J., Fisher, A. J., and Beal, P. A. (2016). Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nat. Struct. Mol. Biol. 23:426-433.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Kohler, A. C., Chen, L.-H., Hurlburt, N., Salvucci, A., Schwessinger, B., Fisher, A. J., and Stergiopoulos, I. (2016). Structural analysis of an Avr4 effector ortholog offers insight into chitin- binding and recognition by the Cf-4 receptor. The Plant Cell 28:1945-1965.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Lal, N. K., Fisher, A. J., and Dinesh-Kumar, S. P. (2016). Arabidopsis receptor-like cytoplasmic kinase BIK1: purification, crystallization and X-ray diffraction analysis. Acta Crystallographica Section F 72:738-742.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Fisher, A. J., and Beal, P. A. (2016). Effects of Aicardi-Gouti�res Syndrome Mutations Predicted from ADAR-RNA Structures. RNA Biol. In Press.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Lal, N. K., Hurlburt, N, Nagalakshmi, U., Flores, R., Sone, P., Casteel, C., Fisher, A. J., and Dinesh- Kumar, S. P. (2016). EFR phosphorylates BIK1 at S89/T90 to regulate jasmonic acid during bacterial immunity. Submitted to Nature
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