Source: OHIO STATE UNIVERSITY submitted to NRP
DEFENSE SUPPRESSION IN MODEL AND CROP PLANTS THROUGH ISOFORM SPECIFIC TARGETING OF PP2A BY A CONSERVED FAMILY OF BACTERIAL EFFECTOR PROTEIN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1008483
Grant No.
2016-67013-24727
Cumulative Award Amt.
$499,970.00
Proposal No.
2015-06650
Multistate No.
(N/A)
Project Start Date
Dec 15, 2015
Project End Date
Sep 14, 2021
Grant Year
2016
Program Code
[A1121]- Plant Health and Production and Plant Products: Understanding Plant-Associated Microorganisms
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
Horticulture & Crop Science
Non Technical Summary
Plant diseases cause significant crop losses worldwide. The ability of plant-associated microbes to cause disease depends on virulence factors that manipulate host targets, often to alter host physiology, suppress host defenses, and/or promote disease symptoms. Thus, breeding, transgenic, or crop treatment strategies for combating plant disease will benefit from advanced fundamental knowledge about the mechanisms employed by virulence factors of pathogens and their targets in plants. The AvrE-family of virulence effectors are secreted from multiple genera of plant pathogenic bacteria into the cells of host plants, contributing to disease on a wide variety of crops ranging from monocots (e.g. maize) to dicots (e.g. tomato and bean) to fruit trees (e.g. apple and pear). Despite the widespread importance of this family of virulence factors, little is known about their mechanism of action. Preliminary data indicate that the AvrE-family effectors interact with a regulatory subunit of a protein phosphatase 2A (PP2A). PP2A plays key roles in the regulation of numerous plant processes, including responses to abiotic and biotic stress. We will describe the AvrE-PP2A interaction, determine its subcellular localization, and characterize the consequence on plant defense, including hormone and metabolic signals. Since the AvrE-family virulence effectors are widespread, the crop plants negatively affected by associated diseases are varied and numerous, and PP2A isoforms are key to a variety of plant responses to the environment, the potential long-range benefits to U.S. agriculture from applying the knowledge generated by this study will be broad and significant.
Animal Health Component
5%
Research Effort Categories
Basic
95%
Applied
5%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121510104050%
2122499104050%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1510 - Corn; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
1. Define the interactions between PP2A, AvrE-family T3Es and potential substrates. We will identify the AvrE-interacting subunits of PP2A, define the interacting domains, and apply the new knowledge to inhibit AvrE activity and identify putative substrates in planta.2. Define the subcellular localization of the AvrE-family effectors and PP2A subunits. Various in planta approaches will be used to localize the AvrE-family T3Es and B' subunits of PP2A, as well as how their localization is affected by their interaction.3. Define the role of PP2A isoforms in plant defense and the consequences of their targeting by AvrE-family T3Es. Examined potential roles of PP2A will include hypothesized effects on hormone signaling, demonstrated effects on maize phenylpropanoid metabolism, and hypothesized changes in PP2A activity.
Project Methods
To address the research questions, this project will employ a variety of molecular, genetic, biochemical, and cell biological methods. Molecular cloning and in vitro and in planta interaction assays will be used to identify and map interactions between AvrE-family T3Es and subunits of PP2A phosphatase complexes. Fluorescence microscopy and subcellular fractionation will be used to examine the localization of PP2A subunits and AvrE-family, including an examination of how those localizations are effected by AvrE-family T3E:PP2A interactions.Virus-induced gene silencing (VIGS) will be used to examine the role of specific maize genes for function of an AvrE-family T3E from a maize pathogen. Quantitative real-time PCR and chromatography combined with mass spectrometry will be used to examine the role of specific PP2A isoforms on accumulation of plant hormones and their consequence on plant transcription and metabolite accumulation, as well as how those responses are perturbed by the action of AvrE-family T3Es.

Progress 12/15/15 to 09/14/21

Outputs
Target Audience:One target audience for our efforts is the research community examining the plant-microbe interactions, particularly those focused on pathogenic microbes, virulence effectors produced by those microbes, and the molecular responses of host plants during disease and resistant interactions with microbes. A second target audience is plant breeders and biotechnologists for whom information about the targeting of PP2A by AvrEfamily type III effectors will guide efforts to breed or engineer disease resistant maize and other plants parasitized by the large family of bacteria deploying AvrE-family type III effectors. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities for 3 undergraduate students (Josh Jeyandran, Rena So, and Josh Yoder), 8 undergraduate students (Eliza Kallenbach, Ying Weng, Ian Gray, Emma Kraus, Taylor Klass, Connor Whitford, Dylan Lingerak, Je Cuan Choo), 7 graduate students (Priscila Rodriguez-Garcia, Lin Jin*, Eun Hyang Han*, Irene Gentzel*, Alex Turo*, Kelly Mikhail, and Reid Gohmann), 2 postdoctoral associates (Mingzeh Shen and Gayani Ekanayake) and one research associate (Laura Giese). Graduate students indicated by an asterisk (*) have completed their Ph.D. How have the results been disseminated to communities of interest?This project resulted in 8 oral presentations nationally, and 7 oral presentations and 1 poster presentation internationally. Additionally, the project has provided support contributing to 21 publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Aim 1: We defined the interaction between AvrE-family T3Es and specific subunits of PP2A (DOI:10.1371/journal.ppat).We demonstrated that multiple domains of the AvrE-family T3Es interact withPP2A subunits,but wereunsuccessful in our efforts to fine map the interacting domainsdue to the combined difficulties of the large size,in vitro insolubility, and in vitro toxicity of the AvrE-family T3Es. We built on our prior observation that WtsE, the AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii (Pss), perturbs maize metabolism (doi.org/10.1104/pp.114.253120) by showing thattargeting of PP2A by WtsE is critical to perturbing host metabolism(DOI:10.1371/journal.ppat). This identification of a linkage betweenAvrE-family T3Es and host metabolism led to a major breakthrough in our understanding of the function ofAvrE-family T3Es (see Aim 3 below). Aim 2: We demonstrated that the AvrE-family T3Es are localized to the plasma membrane (DOI:10.1371/journal.ppat). This work was corroborated by that from the laboratory of Sheng Yang He (doi.org/10.1104/pp.15.00547). Furthermore, we showed that the interaction of theAvrE-family T3Es with PP2Aoccurs at the plasma membrane(DOI:10.1371/journal.ppat). Additionally, this work revealed an interaction betweenthe AvrE-family T3Es and plasma membrane spanning receptor-like kinases (RLKs).Thus, the results of this aim point to manipulation of protein phosphorylation at the plasma membrane as a key mode of action of theAvrE-family T3Es. Aim 3: To facilitate the analysis of AvrE-family T3Es and their targets in host defense, we refined the method for measuring callose deposition (a cell wall-based defense response in Arabidopsis; DOI:10.1007/978-1-4939-6859-6_16). Using thismethod and other established methods, along with and lines of Arabidopsis with T-DNA knockouts of PP2A subunits targeted by AvrE1, which is theAvrE-family T3E of Pseudomonas syringae pv. tomato (Pst),we have demonstrated that these targets are critical to the virulence activity of AvrE1 (DOI:10.1371/journal.ppat). Additional work in progress will determine the signficance of PP2A and additional targets ofAvrE-family T3Es for their virulence activity in planta. These experiments will utilize homozyogous lines with T-DNA and CRISPR mutants of RLKs in Arabidopsis and CRISPR mutants of PP2A subunits and RLKs in maize that are in development.The development of CRISPR mutants in maize is an alternative approach to the use of VIGS described in the original proposal. We identified a virulence protein from a non-bacterial plant pathogen (an oomycete) with structural and functional similarity toAvrE-family T3Es, thus demonstrating that the functionality of AvrE-family T3Es extends even beyond their broad distribution across multiple genera of phytopathogenic bacteria(DOI:10.1371/journal.pone.0195559). We demonstrated that WtsE contributes to the virulence and mobility of Pss in the xylem of infected maize leaves (doi.org/10.1094/mpmi-05-19-0129-r). Perhaps most significantly, we have made significant progress in understanding the meachanism by which AvrE-family T3Es contribute to the virulence of bacterial pathogens inthe apoplast of infected leaves. First, weestablished a method for measuring apoplast hydration in and collecting apoplast contents from maize leaves (DOI:10.1104/pp.18.01076)and provided a framework for interpreting the metabolitecomposition of apoplast fluid and residualleaves (doi.org/10.3390/metabo10020052).Using these methods, we have demonstrated that Pss, dependent on WtsE, causes the accumulation of water and nutritive metabolites in the apoplast of infected maize leaves. Notably, this WtsE-dependent accumulation of water and nutrients in the apoplast and their dynamic assimilation by proliferating Pssoccurs during the biotrophic phase of infection, i.e., prior to disruption of host cell integrity.Given the broad distribution of AvrE-family effectors, this work highlights the importance of actively acquiring water and nutrients for the proliferation of Pss during its biotrophic phase of infection. This work will be published soon(Dynamic nutrient acquisition from a hydrated apoplast supports biotrophic proliferation of a bacterial pathogen of maize;Irene Gentzel, Laura Giese, Kelly Mikhail, Wanying Zhao, Jean-Christophe Cocuron, Ana Paula Alonso, and David Mackey; in revision for Cell Host & Microbe). To determine the generalizability of this finding, we have established a method forapoplast analysis in Arabiodpsis leaves(A method for quantitation of apoplast hydration in Arabidopsis leaves reveals water-soaking activity of effectors of Pseudomonas syringae during biotrophy;Gayani Ekanayake, Reid Gohmann, and David Mackey;in review at Scientific Reports) and are in the process ofdeterminingthe effect of AvrE1 on the water and metabolite composition ofthe apoplast ofArabidopsis leaves infected with Pst.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Redditt, T.J., E.H. Chung, H.Z. Karimi, N. Rodibaugh, Y. Zhang, J.C. Trinidad, J.H. Kim, Q. Zhou, M. Shen, J.L. Dangl, D. Mackey, R.W. Innes 2019. AvrRpm1 Functions as an ADP-Ribosyl Transferase to Modify NOI-domain Containing Proteins, Including Arabidopsis and Soybean RPM1-interacting Protein 4. Plant Cell, DOI:10.1105/tpc.19.00020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Castro-Moretti, F., I. Gentzel, D. Mackey, A.P. Alonso 2020. Metabolomics as an Emerging Tool for the Study of Plant-Pathogen Interactions. Metabolites, doi.org/10.3390/metabo10020052.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhao, Z., X. Yang, S. Lu, J. Fan, S. Opiyo, P. Yang, J. Mangold, D. Mackey, and Y. Xia 2020. Deciphering the Novel Role of AtMIN7 in Cuticle Formation and Defense against the Bacterial Pathogen Infection. Int J Mol Sci, doi.org/10.3390/ijms21155547
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Shen, M., C.J. Lim.,J. Park, J.E. Kim, D. Baek, J. Nam, S.Y. Lee, J.M. Pardo, W.Y. Kim, D. Mackey*, and D.J. Yun 2020. HOS15 is a transcriptional corepressor of NPR1-mediated gene activation of plant immunity. PNAS, doi.org/10.1073/pnas.2016049117
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Mackey, D., D.J. Yun, and J. Nam 2021. Proteasome-Dependent Degradation of RPM1 Desensitizes the RPM1-Mediated Hypersensitive Response. J Plant Biol, doi.org/10.1007/s12374-021-09296-4
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Chae, H.B., M.G. Kim, C.H. Kang, J.H. Park, E.S. Lee, S.U. Lee, Y.H. Chi, S.K. Paeng, S.B. Bae, S.D. Wi, B.W. Yun, W.Y. Kim, D.J. Yun, D. Mackey*, S.Y. Lee 2021. Redox sensor QSOX1 regulates plant immunity by targeting GSNOR to modulate ROS generation. Molecular Plant, doi.org/10.1016/j.molp.2021.05.004
  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Gentzel, I., L. Giese, K. Mikhail, W. Zhao, J.C. Cocuron, A.P. Alonso, D. Mackey 2021. Dynamic nutrient acquisition from a hydrated apoplast supports biotrophic proliferation of a bacterial pathogen of maize. In revision for Cell Host & Microbe.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Blakeslee, J.J., Murphy, A.S. (2016). Microscopic and Biochemical Visualization of Auxins in Plant Tissues". In "Methods in Molecular Biology: Environmental Responses in Plants," vol. 1398. Duque, P. ed. New York: Humana Press, Springer Science+Business Media. Pages 37-54. DOI 10.1007/978-1-4939-3356-3_5
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Gentzel, I., L. Giese, W. Zhao, A.P. Alonso, D. Mackey 2019. A simple method for measuring apoplast hydration and collecting apoplast contents. Plant Physiol. DOI:10.1104/pp.18.01076
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Doblas-Ib��ez, P., K. Deng, M.F. Vasquez, L. Giese, P.A. Cobine, J.M. Kolkman, H. King, T. Jamann, P. Balint-Kurti, L. De La Fuente, R.J. Nelson, D. Mackey, L.G. Smith 2019. Dominant, heritable resistance to Stewarts wilt in maize is associated with an enhanced vascular defense response to infection with P. stewartii. Mol Plant-Microbe Interact, doi.org/10.1094/mpmi-05-19-0129-r
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Jin, L., J.H. Ham, R. Hage, J. Soto-Hern�ndez, S.Y. Lee, S.M. Paek, D. Majerczak, M.G. Kim, C. Boone, D.L. Coplin, and D. Mackey 2016. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins. PLoS Pathogens. DOI:10.1371/journal.ppat.1005609: 1-33.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: EH Han, DP Petrella, JJ Blakeslee 2017 'Bending'models of halotropism: incorporating protein phosphatase 2A, ABCB transporters, and auxin metabolism. Journal of Experimental Botany 68:3071-3089.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Jin, L. and D. Mackey 2017. Measuring Callose Deposition, an Indicator of Cell Wall Reinforcement, during Bacterial Infection in Arabidopsis. Methods in Molecular Biology. vol. 1578. DOI:10.1007/978-1-4939-6859-6_16.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Uddin, M.N., S. Akhter, R. Chakraborty, J.H. Baek, J.-Y. Cha, S.J. Park, H. Kang, W.Y. Kim, S.Y. Lee, D. Mackey, M.G. Kim 2017. SDE5, a putative RNA export protein, participates in plant innate immunity through a flagellin-dependent signaling pathway in Arabidopsis. Scientific Reports. 7: 9859 | DOI:10.1038/s41598-017-07918-x
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Chakraborty, S. A.L. Hill, G. Shirsekar, A.J. Afzal, G.L. Wang, D. Mackey, P. Bonello 2016. Quantification of hydrogen peroxide in plant tissues using Amplex Red. Methods. DOI: 10.1016/j.ymeth.2016.07.016.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Eschen-Lippold, L., X. Jiang, J.M. Elmore, D. Mackey, L. Shan, G. Coaker, D. Scheel, J. Lee 2016. Bacterial AvrRpt2-like cysteine proteases block activation of the Arabidopsis mitogen activated protein kinases, MPK4 and MPK11. Plant Physiol. DOI:10.1104/pp.16.00336.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Geng, X., Shen, M., Kim, J.H. and D. Mackey 2016. The Pseudomonas syringae type III effectors AvrRpm1 and AvrRpt2 promote virulence dependent on the F-box protein COI1. Plant Cell Reports. 35: 921-932.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: DAO1 catalyzes temporal and tissue-specific oxidative inactivation of auxin in Arabidopsis thaliana doi: 10.1073/pnas.1604769113
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Quantification of Carbohydrates in Grape Tissues Using Capillary Zone Electrophoresis doi: 10.3389/fpls.2016.00818
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang, M., K. Zhang, H. Yan, W. Zhao, E. Lin, H. Shi, L. Rui J.J. Blakeslee, D. Mackey, D. Tang, Z. Wei, G.L. Wang 2018. The major leaf ferredoxin, Fd2, regulates plant innate immunity in Arabidopsis. Mol Plant Pathol. DOI:10.1111/mpp.12621
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Deb, D., D. Mackey, S. Opiyo, J.M. McDowell 2018. Application of alignment-free bioinformatics methods to identify functionally similar effector proteins from distantly related plant pathogen species. PLoS One. DOI:10.1371/journal.pone.0195559


Progress 12/15/19 to 12/14/20

Outputs
Target Audience:One target audience for our efforts is the research community examining the plant-microbe interactions, particularly those focused on pathogenic microbes, virulence effectors produced by those microbes, and the molecular responses of host plants during disease and resistant interactions with microbes. A second target audience is plant breeders and biotechnologists for whom information about the targeting of PP2A by AvrEfamily type III effectors will guide efforts to breed or engineer disease resistant maize and other plants parasitized by the large family of bacteria deploying AvrE-family type III effectors. Changes/Problems:Project completion was hampered by lab shutdown for more than half the year. What opportunities for training and professional development has the project provided?The project has supported the work of multiple undergraduate (8) and graduate (4) students. How have the results been disseminated to communities of interest?Dissemination during the first year of the pandemic was limited to publications. What do you plan to do during the next reporting period to accomplish the goals?To determine the source of metabolites that WtsE causes to accumulate, we will develop a detached leaf assay to determine if the nutrients are coming from a local of distal source. Our data indicate that the seed store is an ongoing source of protein at the time of our assays (six day old seedlings), thus making the distal source a viable hypothesis. However, the known targeting of maize kinases and phosphatases at the plasma membrane by WtsE, favors the model of mobilization from a local source. Also, we will use anassay for malate dehydrogenase activity to assess further the timing of loss of maize cell integrity relative to water and metabolite accumulation in the apoplast, as well asbacterial growth.

Impacts
What was accomplished under these goals? To observethe effect of WtsE delivery into maize cells in the absence of nutrient assimilation by proliferating Pantoea stewartii subsp. stewartii, we deployed the non-proliferating E. coli delivery system (EcDS). Timing of WtsE-induced apoplast hydration and loss of maize cell integrity were more rapid with the EcDS. Consistent with the hypothesis that assimilation by P. stewartii was obscuring the accumulation of nutritive metabolites in the apoplast, the levels of metabolites that accumulate in the apoplast following delivery of WtsE by EcDS were dramatically elevated to levels that could account for the assimilation by P. stewartii. These results support the conclusion that WtsE supports the dynamic mobilization of metabolites from plant cells and to P. stewartii during the biotrophic phase of infection.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Castro-Moretti, F., I. Gentzel, D. Mackey, A.P. Alonso 2020. Metabolomics as an Emerging Tool for the Study of Plant-Pathogen Interactions. Metabolites, doi.org/10.3390/metabo10020052.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhao, Z., X. Yang, S. Lu, J. Fan, S. Opiyo, P. Yang, J. Mangold, D. Mackey, and Y. Xia 2020. Deciphering the Novel Role of AtMIN7 in Cuticle Formation and Defense against the Bacterial Pathogen Infection. Int J Mol Sci, doi.org/10.3390/ijms21155547
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Shen, M., C.J. Lim.,J. Park, J.E. Kim, D. Baek, J. Nam, S.Y. Lee, J.M. Pardo, W.Y. Kim, D. Mackey*, and D.J. Yun 2020. HOS15 is a transcriptional corepressor of NPR1-mediated gene activation of plant immunity. PNAS, doi.org/10.1073/pnas.2016049117


Progress 12/15/18 to 12/14/19

Outputs
Target Audience:One target audience for our efforts is the research community examining the plant-microbe interactions, particularly those focused on pathogenic microbes, virulence effectors produced by those microbes, and the molecular responses of host plants during disease and resistant interactions with microbes. A second target audience is plant breeders and biotechnologists for whom information about the targeting of PP2A by AvrEfamily type III effectors will guide efforts to breed or engineer disease resistant maize and other plants parasitized by the large family of bacteria deploying AvrE-family type III effectors. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the work of multiple undergraduate (7) and graduate (4) students. How have the results been disseminated to communities of interest?Seminar -Liberation of water and nutrients from living leaf cells by a bacterial pathogen of maize. University of Missouri, Interdisciplinary Plant Group 37th Annual Symposium, May 2019, Columbia, Missouri. Seminar -Liberation of water and nutrients from living leaf cells by a bacterial pathogen of maize.Virginia Tech University, September 2019, Blacksburg, Virginia. Seminar -Liberation of water and nutrients from living leaf cells by a bacterial pathogen of maize.University of North Texas, September 2019, Denton, Texas. Seminar -Liberation of water and nutrients from living leaf cells by a bacterial pathogen of maize.The 5th International Gyeongsang National University Symposium, October 2019, Jinju, South Korea. What do you plan to do during the next reporting period to accomplish the goals?We plan to conduct two further analyses of the ability of WtsE to promote the accumulation of metabolites that are nutrients for P. stewartii in the apoplast of infected maize leaves during the biotrophic phase of infection. First, we will develop a detached leaf assay to determine if the nutrients are coming from a local of distal sources. Our data indicate that the seed store is an ongoing source of protein at the time of our assays (six day old seedlings), thus making the distal source a viable hypothesis. Second, we will use an E. coli delivery system for WtsE to measure the affect of WtsE on metabolite levels in the apoplast in the absence of significant bacterial assimilation. Our data on carbon and nitrogen assimilation by P. stewartii indicatea level assimilation expected to obscure the measurement of metabolites in the apoplast.Because the E. coli delivery system is non-proliferating, we anticpate greatly reduced assimilation and thus an unobscured view of the amount of metabolite that WtsE causes to accumulate in the apoplast.

Impacts
What was accomplished under these goals? We have completed studies linking AvrE-family type III effectors (T3Es)to PP2A and demonstrating the importance of these interactions for their virulence activity.Our work has continued to focus on Aim 3, and our novel observation that AvrE-family T3Es modifythe plant extracellular environment, including the accumulation of water (water-soaking) and metabolites. We have published a method for measuring apoplast hydration quantitatively and in advance of macroscopic water-soaking in maize. Using this method, we have precisely defined the timing with of water accumulation in the maize apoplast caused by delivery of the AvrE-family T3E from Pantoea stewartii supsp. stewartii. Cooupling this data with multiple methods (ion leakage, propridium iodide exclusion) to precisely definethe timing with which maize cells lose their integrity and a time series of bacterial growth in the apoplast space, we have defined a biotrophic phase of infection that is characterized by an increase in apoplast hydration. Targeted metabolomics has also been employed to characterize the composition of metabolites in the hydrated apoplast. WtsE causes a significant accumulation of amino acids (including predominantly those that P. stewartii can use as sources of nitrogen) in the apoplast during this biotrophic phase. These observations define a novel function for WtsE. The metabolites that accumulate can be used by the bacteria as nutrients. Notably, these changes occur prior to effector-induced losses of cell integrity. Thus, the acquisition of water and nutrients from living cells (i.e., during the biotrophic phase of infection) represents a brand new activity fo this important class of T3Es.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Gentzel, I., L. Giese, W. Zhao, A.P. Alonso, D. Mackey 2019. A simple method for measuring apoplast hydration and collecting apoplast contents. Plant Physiol. DOI:10.1104/pp.18.01076
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Doblas-Ib��ez, P., K. Deng, M.F. Vasquez, L. Giese, P.A. Cobine, J.M. Kolkman, H. King, T. Jamann, P. Balint-Kurti, L. De La Fuente, R.J. Nelson, D. Mackey, L.G. Smith 2019. Dominant, heritable resistance to Stewarts wilt in maize is associated with an enhanced vascular defense response to infection with P. stewartii. Mol Plant-Microbe Interact, doi.org/10.1094/mpmi-05-19-0129-r
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Redditt, T.J., E.H. Chung, H.Z. Karimi, N. Rodibaugh, Y. Zhang, J.C. Trinidad, J.H. Kim, Q. Zhou, M. Shen, J.L. Dangl, D. Mackey, R.W. Innes 2019. AvrRpm1 Functions as an ADP-Ribosyl Transferase to Modify NOI-domain Containing Proteins, Including Arabidopsis and Soybean RPM1-interacting Protein 4. Plant Cell, DOI:10.1105/tpc.19.00020


Progress 12/15/17 to 12/14/18

Outputs
Target Audience:One target audience for our efforts is the research community examining the plant-microbe interactions, particularly those focused on pathogenic microbes, virulence effectors produced by those microbes, and the molecular responses of host plants during disease and resistant interactions with microbes. A second target audience is plant breeders and biotechnologists for whom information about the targeting of PP2A by AvrEfamily type III effectors will guide efforts to breed or engineer disease resistant maize and other plants parasitized by the large family of bacteria deploying AvrE-family type III effectors. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the work of multiple undergraduate (6) and graduate (4) students. How have the results been disseminated to communities of interest?Seminar - WtsE, an AvrE-family Type III Effector, Converts the Maize Apoplast to an Environment Suitable for Proliferation of Pantoea Stewartii. The 4th International Gyeongsang National University Symposium, August 2017, Jinju, South Korea. Seminar - WtsE, an AvrE-family Type III Effector, Converts the Maize Apoplast to an Environment Suitable for Proliferation of Pantoea Stewartii. The 4th Beijing International Symposium on Moleculat Plant Pathology, August 2017, Beijing, China. Seminar - WtsE, a type III effector from Pantoea stewartii, perturbs metabolite levels in the apoplast of maize leaves to make nutrients available and possibly to drive water-soaking. NIFA Program Director's Meeting, December 2017, Washington, D.C. What do you plan to do during the next reporting period to accomplish the goals?We are in the process of establishing the role of PP2A isoforms targeted by AvrE-family T3Es in fundamental disease processes, specifically the ability of the T3Es to disrupt the partitioning of water and nutrients between living plant cells and the extracellular environment.

Impacts
What was accomplished under these goals? Our work has extended predominantly into Aim 3. We have completed studies linking AvrE-family T3Es to PP2A and demonstrating the importance of these interactions for their virulence activity. We have broken through into a new area of research by demonstrating the influence of AvrE1 on modifying the plant extracellular environment, including the accumulation of water (water-soaking) and metabolites. The metabolites that accumulate can be used by the bacteria as nutrients. Notably, these changes occur prior to effector-induced losses of cell integrity. Thus, the acquisition of water and nutrients from living cells (i.e., during the biotrophic phase of infection) represents a brand new activity fo this important class of T3Es.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang, M., K. Zhang, H. Yan, W. Zhao, E. Lin, H. Shi, L. Rui J.J. Blakeslee, D. Mackey, D. Tang, Z. Wei, G.L. Wang 2018. The major leaf ferredoxin, Fd2, regulates plant innate immunity in Arabidopsis. Mol Plant Pathol. DOI:10.1111/mpp.12621
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Deb, D., D. Mackey, S. Opiyo, J.M. McDowell 2018. Application of alignment-free bioinformatics methods to identify functionally similar effector proteins from distantly related plant pathogen species. PLoS One. DOI:10.1371/journal.pone.0195559


Progress 12/15/16 to 12/14/17

Outputs
Target Audience:One target audience for our efforts is the research community examining the plant-microbe interactions, particularly those focused on pathogenic microbes, virulence effectors produced by those microbes, and the molecular responses of host plants during disease and resistant interactions with microbes. A second target audience is plant breeders and biotechnologists for whom information about the targeting of PP2A by AvrE-family type III effectors will guide efforts to breed or engineer disease resistant maize and other plants parasitized by the large family of bacteria deploying AvrE-family type III effectors. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the work of multiple undergraduate (5) and graduate (4) students. How have the results been disseminated to communities of interest?Seminar - WtsE, an AvrE-family Type III Effector, Converts the Maize Apoplast to an Environment Suitable for Proliferation of Pantoea Stewartii. The 4th International Gyeongsang National University Symposium, August 2017, Jinju, South Korea. Seminar - WtsE, an AvrE-family Type III Effector, Converts the Maize Apoplast to an Environment Suitable for Proliferation of Pantoea Stewartii. The 4th Beijing International Symposium on Moleculat Plant Pathology, August 2017, Beijing, China. Seminar - WtsE, a type III effector from Pantoea stewartii, perturbs metabolite levels in the apoplast of maize leaves to make nutrients available and possibly to drive water-soaking. NIFA Program Director's Meeting, December 2017, Washington, D.C. What do you plan to do during the next reporting period to accomplish the goals?We will continue to create a high-resolution map of the interaction domains between AvrE-family T3Es and regulatory subunits of PP2A. We are developing a capillary electrophoresis-based assay able to quantify PP2A activity in plant extracts without the use of radioisotope, and capable of high-throughput measurement of PP2A activity. We are in the process of establishing the role of PP2A isoforms targeted by AvrE-family T3Es in fundamental disease processes, specifically the ability of the T3Es to disrupt the partitioning of water and nutrients between living plant cells and the extracellular environment..

Impacts
What was accomplished under these goals? The project seeks to further our understanding of the interaction of AvrE-family T3Es with PP2A in 3 ways: Aim 1 will define the interaction between AvrE-family T3Es and PP2A regulatory subunits and attempt to identify PP2A substrates in planta. So far, we have completed an initial assessment of the interacting fragments of AvrE1 with Arabidopsis B' regulatory subunits and of WtsE with maize B' regulatory subunits. Next, we will refine these studies to identify minimal domains required for the interaction. Those minimal domains will be expressed in planta and tested for their ability to disrupt the interaction and potentially block the virulence activity of the T3E. Aim 2 has defined the subcellular location of interaction between AvrE-family T3Es and PP2A. Each of the interacting partners, as well as the partners in association, has been localized to the plasma membrane of plant cells. Aim 3 will examine how targeting of PP2A by AvrE-family T3Es affects host cells, including host metabolism, partitioning of host metabolites into the apoplast, hormone signaling, transcriptional and cell wall-associated defense responses, and disease-associated cell death. We have already demonstrated that interaction of AvrE with PP2A is critical for the ability of WtsE to perturb secondary metabolism and cause cell death in maize. Current studies will extend these findings to monitor T3E-induced perturbations of primary metabolism, the apoplast metabolome, and hormone signaling.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: EH Han, DP Petrella, JJ Blakeslee 2017 'Bending'models of halotropism: incorporating protein phosphatase 2A, ABCB transporters, and auxin metabolism. Journal of Experimental Botany 68:3071-3089.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Jin, L. and D. Mackey 2017. Measuring Callose Deposition, an Indicator of Cell Wall Reinforcement, during Bacterial Infection in Arabidopsis. Methods in Molecular Biology. vol. 1578. DOI:10.1007/978-1-4939-6859-6_16.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Uddin, M.N., S. Akhter, R. Chakraborty, J.H. Baek, J.-Y. Cha, S.J. Park, H. Kang, W.Y. Kim, S.Y. Lee, D. Mackey, M.G. Kim 2017. SDE5, a putative RNA export protein, participates in plant innate immunity through a flagellin-dependent signaling pathway in Arabidopsis. Scientific Reports. 7: 9859 | DOI:10.1038/s41598-017-07918-x


Progress 12/15/15 to 12/14/16

Outputs
Target Audience:One target audience for our efforts is the research community examining the plant-microbe interactions, particularly those focused on pathogenic microbes, virulence effectors produced by those microbes, and the molecular responses of host plants during disease and resistant interactions with microbes. A second target audience is plant breeders and biotechnologists for whom information about the targeting of PP2A by AvrE-family type III effectors will guide efforts to breed or engineer disease resistant maize and other plants parasitized by the large family of bacteria deploying AvrE-family type III effectors. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the training of a graduate student (Eun-Hyang Han) who has worked with Dr. Blakeslee on development of the CE assay. Ms. Han is on schedule to complete her Ph.D. during the Spring semester of 2017. The project has also supported the work of undergraduate students in the Mackey (Ying Weng) and Blakeslee (Luke Miller) labs and a technician (Wanying Zhao) in the Mackey lab. The project also supported the rotation project of a Puerto Rican student (Priscila Hernandez) who joined the Mackey lab in March of 2017. How have the results been disseminated to communities of interest?Seminar: Blakeslee, JJ (2016). "Metabolic fingerprinting of plant stress responses." 17th International Workshop on Plant Membrane Biology, Annapolis, MD, USA. Seminar: Blakeslee, JJ (2016). "Defining the biochemical physiology of plant stress responses." Dept. of Biology seminar series, Oberlin College. Seminar: Blakeslee, JJ, (2016). "Metabolic eavesdropping: Intercepting the chemical signals of plant-microbe interactions." Plant Pathology Departmental seminar series, OSU. What do you plan to do during the next reporting period to accomplish the goals?Major areas of emphasis will be as follows: Conduct analyses to map interaction domains between AvrE-family effectors and PP2A B' subunits and to use this information to begin making plants expressing putative inhibitors of the interaction. Demonstrate the utility of the CE assay for analysis of cell and tissue samples and begin using the assay to examine the effect of AvrE-family effectors on the activity and sub-cellular localization of specific isoforms of PP2A. Establish maize VIGS against WtsE-interacting PP2A B' subunits and use these plant materials to determine the consequence of reducing the levels of specific PP2A isoforms on function of WtsE. Establish global patterns of changes in stress hormones caused by AvrE-family effectors.

Impacts
What was accomplished under these goals? For goal 1, we have defined the AvrE-interacting subunits of PP2A and these findings were published in Jin et al. 2016 PLoS Pathogens. We are in the process of cloning derivatives and conducting interaction assay to define the interacting domains. Applying that knowledge to inhibit AvrE activity in planta will commence once interaction domains of AvrE and PP2A B' subunits are more narrowly defined. For goal 2, we have made significant progress. The localization of fluorescent protein-tagged versions of both AvrE and of AvrE-targeted targeted and non-targeted PP2A B' subunits has been determined and these findings were published in Jin et al. 2016 PLoS Pathogens. Efforts to observe changes in localization of specific PP2A isoforms upon delivery of an AvrE-family effector using fluorescent protein-tagged PP2A B' subunits has been unsuccessful, likely due to the high levels of PP2A relative to the levels of AvrE-effector delivered into plant cells. For this reason, we are developing a capillary electrophoresis (CE) assay to measure PP2A activity. To date, we have synthesized small peptide PP2A substrates, and have developed protocols to: a. detect these substrates in yeast lysates (with a linear response curve); and b. separate these substrates based on the phosphorylation state of a single phosphoserine residue. The sensitivity and specificity of this assay will permit us to measure PP2A activity quantititatively. By combining this assay with sub-cellular fractionation of plant samples, we will assess the effect of AvrE-delivery on PP2A subcellular localization. Using plants expressing tagged PP2A B' subunits, we will have the ability to immunoprecipitate specific isoforms of PP2A holoenzyme complexes and thus ascertain both the activity level and sub-cellular localization of those specific isoforms. The CE assay, which will be used to measure levels of a peptide and its PP2A-mediated phosphorylated product, has been optimized to permit quantititive measurement of phosphorylated and non-phsophorylated peptides. Current efforts are focused on monitoring phosphorylation of a substrate peptide by PP2A samples prepared from yeast and plant cells. For goal 3, we have made progress on each of the sub-goals. Regarding hormone-signaling, we showed in a 2015 publication that WtsE (the AvrE-family type III effector from a maize pathogen) causes accumulation of salicylic acid (SA) in maize. More recently, using chemical inhibitors that distinguish between the two predominant SA-biosynthetic pathways, we have determined that the SA induced by WtsE accumulates via the cinnamic acid pathway rather than the isochorismate pathway often associated with biotic defense. To further extend our understanding of how AvrE-family effectors perturb plant hormone signaling, we are in the process of analyzing plant samples (both maize and Arabidopsis) that have been exposed to bacterial pathogens with or without their AvrE-faily effector for accumulation of a panel of plant stress and defense associated hormones. Our studies of phenylpropanoid metabolism have focused on WtsE-induced accumulation coumaroyl tyramine and coumaroyl tryptamine. We have demonstrated, and published in Jin et al. 2016 PLoS Pathogens, that inhibition of PP2A blocks the ability of WtsE to induce the high level accumulation of these compounds in maize. Our current focus is on developing VIGS reagents to target specific PP2A B' subunits in maize and use these tools to link AvrE-mediated effects on hormone and phenylpropanoid levels to specific isoforms PP2A.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins. PLoS Pathogens doi:10.1371/journal.ppat.1005609
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Quantification of hydrogen peroxide in plant tissues using Amplex Red. Methods doi.org/10.1016/j.ymeth.2016.07.016
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Bacterial AvrRpt2-like cysteine proteases block activation of the Arabidopsis mitogen activated protein kinases, MPK4 and MPK11 DOI:10.1104/pp.16.00336
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: The Pseudomonas syringae type III effectors AvrRpm1 and AvrRpt2 promote virulence dependent on the F-box protein COI1 DOI 10.1007/s00299-016-1932-z
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: DAO1 catalyzes temporal and tissue-specific oxidative inactivation of auxin in Arabidopsis thaliana doi: 10.1073/pnas.1604769113
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Quantification of Carbohydrates in Grape Tissues Using Capillary Zone Electrophoresis doi: 10.3389/fpls.2016.00818