Source: MICHIGAN STATE UNIV submitted to
THE CONVERGENCE OF BIOTIC AND ABIOTIC STRESS RESPONSE SIGNALING IN PLANTS.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1014949
Grant No.
(N/A)
Project No.
MICL02529
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Feb 1, 2018
Project End Date
Jan 31, 2023
Grant Year
(N/A)
Project Director
Day, RO.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Plant, Soil and Microbial Science
Non Technical Summary
Plants are constantly exposed to stress, yet they have evolved numerous mechanisms to respond, adapt, and thrive under some of the most hostile conditions. In most cases, the ability to adapt to stress, whether from the environment (abiotic) or predators (biotic), simply requires a "reallocation of resources". For example, under conditions of drought, plants down-regulate growth and development, both being energy-expensive processes, in favor of minimizing water loss. Similarly, when plants are infected by pathogens, the primary goal is to restrict pathogen access to nutrients. This project seeks to understand how plants utilize basic physiological processes to respond to stress. To do this, the project will investigate how biotic stress signaling is activated and regulated, and moreover, how this signaling process is interlinked with general host physiology and plant health. The principal investigator hypothesizes that in a manner similar to humans, plants utilize mechanisms to integrate environmental and pathogen-related stress signaling responses. The goal of this project is to understand how abiotic and biotic stress signaling is co-regulated, and to develop a model of how plants sense the world around them, process these perceived stimuli, and ultimately, survive under a variety of stress conditions. The impact of this work links basic plant health to environment, threats from pathogens, and in total, food security.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2032420104050%
2122420104050%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
2420 - Noncrop plant research;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
Innate immune signaling in plants requires numerous preformed (e.g., structural barriers) and inducible (e.g., gene-for-gene) mechanisms to resist pathogen infection; often, these processes function in parallel. To this end, it is not surprising that many of these defense pathways share common, overlapping signaling networks with basic cellular processes. Indeed, it has been suggested that the plant defense response is "hard-wired" to many essential host processes as a mechanism to continuously survey the environment for biotic pressures. Based on our preliminary characterization of the functional similarity NDR1 shares with the well-characterized integrin node in mammalian cells, we propose to investigate the dynamic spatial and temporal engagement of NDR1 in stress signaling in Arabidopsis.The primary Aim of this proposal is to understand how plants perceive and respond to stress and how this response requires the function of basic physiological processes (e.g., osmotic pressure, PM-CW adhesion, stomata aperture) for the activation of defense signaling. Our preliminary data support this hypothesis and provide substantial proof-of-concept that required components of the plant defense machinery participate in critical host functions in the absence of pathogens. To date, an extensive mechanistic understanding of the basic function of "defense genes" in non-defense processes is poorly defined. Our ultimate goal is to define the processes and signaling cascades that link NDR1 function with abiotic/biotic stress signaling with the activation of innate immune signaling in plants.STRUCTURE: Define the biochemical, cellular and dynamic role of NDR1 in signaling by functionally characterizing the modular/domain function of NDR1. This will be accomplished via a multi-faceted approach of first determining the precise localization and orientation of NDR1, followed by elucidating the function of NDR1 following stress and defense signaling activation.FUNCTION: Elucidate the signaling mechanism(s) by which stress perception impacts the activation of defense signaling through NDR1 function. This will include investigating crosstalk among a suite of abiotic stresses previously described as associated with phytopathogen defense signaling in plants and the activation of PTI and ETI. Activation of both PTI and ETI signaling will be used to monitor the scope of NDR1-mediated responses in planta.Completion of the Aims presented herein will test the hypothesis that defense signaling components, in the absence of pathogens, play a critical cellular role in mediating basic host physiological responses. We posit that NDR1's primary function in defense signaling is to monitor and translate perturbations in host homeostasis to the initial activation and amplification of defense signaling.
Project Methods
AIM 1: Determine the biochemical and structural characteristics required for NDR1-mediated signaling.A. Orientation: NDR1 proximity to the CW. Laser confocal microscopy will be used to define the proximity of PM-localized GFP-NDR1 with the CW following induction of plasmolysis. Time points (0, 2, 5 and 10 minutes) will be assessed for GFP-NDR1 localization by laser scanning confocal microscopy after induction of plasmolysis. Cellulose will be counter-stained with Calcofluor white M2R to visualize the CW in relation to the PM and GFP-NDR1. NDR1-CW association following P. syringae infection. Eleven-day-old ndr1/GFP-NDR1 seedlings will be dip inoculated (108 cfu/ml) with Pst DC3000 (+/- Avr effector), and leaves will be sampled at 0, 6, 12, 24 and 48 hours post-inoculation; time points coincide with our previous observations of gene expression, onset of electrolyte leakage and the presence of disease symptoms.B. Dynamics of NDR1 following pathogen and stress perception: GPI cleavage and phospholipase sensitivity. Sensitivity to PLC will be determined by Triton X-114 partitioning following treatment with PLC. We hypothesize that cleavage of the lipid anchor will result in release of the C-terminus of NDR1 from the PM. A N-terminal deletion construct (i.e., NDR1-C) will be used to void the impact of N-terminus transmembrane anchoring. Herein, we will employ two approaches to assess sensitivity and release:a. Cleavage in vitro. Stable transgenic lines expressing N-terminal T7-epitope tagged NDR1-C (D amino acids 1-33) will be used. Leaf endomembrane fractions will be isolated as previously described and will be incubated with PLC. Release and solubilization will be monitored by Triton X-114 partitioning into the soluble phase (if cleaved) versus the insoluble phase (if not cleaved).b. Pathogen-induced cleavage in vivo. To assess NDR1 GPI anchor cleavage in planta, stable transgenic plants expressing the T7:NDR1-C construct (ndr1 mutant background) will be dip-inoculated (108 cfu/ml) with Pst DC3000 (+/- AvrRpt2, AvrB, AvrPphB; individually). Samples will be harvested at 0, 6 and 12 hours post-inoculation; leaf tissue (1 gram) will be harvested and intercellular (i.e., apoplast) fluids will be isolated as previously described.ii. Dimerization domain defined. Dimerization is a functional requirement for integrin activation 6. Our hypothesis is that dimerization, a mechanism required for activation and signaling through NDR1, occurs only after Pst DC3000 infection and is sustained throughout the infection and defense signaling process. Consistent with, and in support of our NDR1-integrin hypothesis, we recently demonstrated that NDR1 also self-associates in planta.AIM 2: Stress perception, host physiology and ndr1: a preamble to defense activation.A. Role of NDR1 in abiotic stresses and immune signaling. The experiments outlined below are designed to define the role of NDR1 in basic cellular processes executed both in the presence and absence of pathogen, and then to elucidate how the ndr1 mutation manifests into increased susceptibility and a loss in the activation of resistance signaling. a. Plasmolysis. During pathogen infection, plants engage multiple biochemical processes to control fluid dynamics to restrict pathogen access to nutrients. In total, this function is required to sustain cellular processes, including membrane dynamics and osmotic homeostasis of the cell 9. To assess the role of osmotic stability through monitoring the PM-CW adhesion, WT Col-0 and ndr1 mutant plants will be infected with Pst DC3000 by dip inoculation (108 cfu/ml).b. Drought. A study by Wright and Beattie demonstrated that Pst DC3000 growth is restricted in the host apoplast under reduced osmotic potential. More recently, Goel et al. demonstrated that the Pst effector HopAM1 enhances the growth of a weak pathogen in Arabidopsis, presumably through manipulation (i.e., enhancement) of ABA signaling.Evaluation of drought stress in ndr1. WT Col-0 and ndr1 mutant seedlings will be grown in trays (weighed and adjusted for initial moisture content) for 4 weeks under standard growth and watering conditions. After 4 weeks, plants will be subjected to two watering regimes: 1) control, where plants will be watered three times weekly, and 2) drought stress conditions, where plants will not be watered. In each regime, plants will be monitored and evaluated for 3 primary criteria at the start of each experiment.Bacterial growth and resistance during drought: Once RWC is stabilized at 40% (mild drought stress; control) and 20% (severe drought stress), plants will be dip-inoculated with Pst DC3000 (+/- Avr; 108 cfu/ml) and growth will be enumerated as previously described. Control plants (i.e., RWC = 80%) will be included in all analyses.ABA and the ndr1 mutant. First, WT Col-0 and ndr1 mutant seedlings will be grown under standard growth conditions. At four weeks post-germination, leaves from both genotypes will be harvested and used to compare the endogenous concentrations of ABA, according to the methods of Yasuda et al. In parallel, WT Col-0 and ndr1 seedlings will be grown on Murashige-Skoog (MS) media, and after 18 days, plants will be transferred to a hydroponic system containing 0 (control) or 400 mM of ABA).ii. Stomata response through NDR1. We have established that the ndr1 mutant has an altered guard cell aperture response, manifested presumably through an ABA-dependent mechanism. Based on the observations of Liu et al., as well as our own preliminary studies, we will determine the biochemical process(es) through which NDR1 functions to modulate stomata aperture. a. Guard cell aperture and dynamics. Induction of stomata opening will be performed by placing 4-5-week-old Arabidopsis plants in continuous light for 24 h under high humidity. Epidermal peels will be taken from the lower leaf surface and placed on microscope slides, individually, for each of the following treatments:Abscisic acid: 10 µM ABA will be applied to epidermal peels to monitor the stomata closure response; ABA treatment will serve as a baseline control to determine rate of response in WT Col-0 and the ndr1 mutant. Rapid closure (i.e., 60 min post-treatment) is typically observed in WT Col-0. Preliminary results show that in the ndr1 mutant, ABA-induced closure is delayed (ca. >1 h; 16).PAMP treatment: Our published data demonstrate an alteration in PTI signaling in the ndr1 mutant 3. To determine if PTI-induced stomata closure is modified in the ndr1 mutant, flg22 (1 µM) and elf26 (1 µM) will be applied, individually, to epidermal peels to monitor the closure response; this line of experiments will investigate the link between NDR1, PAMP perception and stomata aperture. WT Col-0 epidermal peels will be included as controls.Pathogen-induced closure and re-opening: Pst DC3000 induces closure of Arabidopsis stomata within 1 h of exposure; within 3 h, through the action of the strain specific toxin coronatine (a jasmonic acid mimic), re-opening of stomata is observed. We will examine the dynamics (rate and aperture over time) of closure and re-opening in WT Col-0 and the ndr1 mutant in the presence of Pst DC3000 and Pst DC3118, a coronatine deficient mutant. b. ATPase activity. Based on observed alterations in stomata aperture in the ndr1 mutant, coupled with NDR1 association with RIN4, PM H+-ATPase activity will be assessed as described in Liu et al., which demonstrated the effect of RIN4 over-expression on ATPase activity and subsequent stomata aperture.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:The target audience for this project is multi-fold. First, fundamental scientists in the broad area of climate, biotic/abiotic stress signaling, and more recently, broad topical areas in the space of GMO. Second, we have begun engaging with food production-related stakeholders. These have included growers, producers, Extension, and food processing scientists. Finally, we have actively engaged policy and education sectors; this relates to conversations related to GMO technologies, etc. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training and development for students and postdocs. Both postdoctoral scholars working on this project have moved on to permanent positions. How have the results been disseminated to communities of interest?Results have been disseminated through publication, attendance and presentations at scientific conferences, and via the following: 2021, Keynote Speaker, BioTalks, Hacettepe University, Food Engineering Department. Host: Graduate Students. 2021, Rajarata University, Sri Lanka. Faculty of Technology. Host: Dr. Dinum Perera. Virtual. 2020, Zhejiang University, China. College of Life Sciences. Host: Dr. Kun Jiang. 2020, Keynote speaker, The 1st International Conference On Plant Stress and Multi-Omics. Henan, China. 2020, Ankara University (Ankara, Turkey). Department of Biological Sciences. Host: Dr. Ilker Buyuk. Virtual. 96 attendees. 2020, U.S. National Science Foundation. BIO Directorate. 2020, Amity University, India. Food and Agriculture Foundation. Host: Dr. Nuntan Kaushik (Director General). 2020, Cornell University. Department of Plant Biology. Host: Jian Hua. Postponed (Fall 2021), COVID. Host: Jian Hua. 2020, University of Maryland, College Park. Plant Science and Landscape Architecture. Host: Yiping Qi. Postponed (Fall 2021), COVID. 2020, USA-Turkey AgriBiotech Symposium. US Embassy Sponsored. 2020, University of Maryland, Baltimore County. Dept. Biological Sciences. Host: Dr. Hua Lu. 2020, Michigan State University, Biochemistry and Molecular Biology. Host: Dr. Erich Grotewold. 2019, JSPS & T-PIRC Conference. Tokyo, Japan. Host: Dr. Hiroshi Ezura. 2019, The University of Tennessee. Genome Sciences (ORNL). Host: Postdocs. 2019, Hacettepe University. Ankara, Turkey. Host: Dr. Remziye Yilmaz. 2019, ?stanbul Ayd?n University. Istanbul, Turkey. Host: Prof. Dr. Dilek Heperkan. 2019, TEKFEN AGRI. Istanbul, Turkey. 2019, University of California, Davis. Department of Plant Biology, Davis, CA. Host: Graduate Students. 2019, University of Rajarata (Sri Lanka), Department of Biological Sciences. Host: Dr. Chathuranga Bamunuarachchige. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Summary Abiotic and biotic environments influence a myriad of plant-related processes, including growth, development, and the establishment and maintenance of interaction(s) with microbes. In the case of the latter, elevated temperature has been shown to be a key factor that underpins host resistance and pathogen virulence. In this study, we elucidate a role forArabidopsisNON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) by exploiting effector-triggered immunity to define the regulation of plant host immunity in response to both pathogen infection and elevated temperature. We generated time-series RNA sequencing data of WT Col-0, anNDR1overexpression line, andndr1andics1-2mutant plants under elevated temperature. Not surprisingly, theNDR1-overexpression line showed genotype-specific gene expression changes related to defense response and immune system function. The results described herein support a role for NDR1 in maintaining cell signaling during simultaneous exposure to elevated temperature and avirulent pathogen stressors. We havedescribeda role for NDR1 in plant immunity under heat stress conditions. We have identified a genotype specific cluster of highly expressed genes in the NDR1-overexpression line under heat stress. Most notably, unlike the temperature sensitive SA defense pathway gene ISOCHRISMATE SYNTHASE 1 (ICS1) (Huot et al., 2017), the NDR1-overexpression line stabilizes ETI-specific RPS2 mRNA accumulation at elevated temperature. Our findings suggest pathogen resistance at elevated temperature is mediated through crosstalk between NDR1 and RPS2, a mechanism that requires robust signaling of SA processes. In the current study, we sought to expand our understanding of the mechanisms that function at the nexus of heat stress response and immune signaling activation. To do this, we focused on the activation of a well-defined and genetically tractable immune signaling cascade, ETI. As presented herein, the data described point to the intersection of at least three immune signaling events (I. Transcriptional activation of immunity, II. Hormone response and signaling, and III. R Protein mediated immunity), each of which not only requires the function of NDR1 but is also impacted by the Pst type-III virulence effector AvrRpt2.?Through this approach, we identified two main clusters of DEGs that segregated based on genotype-independent expression, as well as those that were regulated in a genotype-specific manner under elevated temperature. Out of the two, the latter cluster was comprised of highly expressed genes in the NDR1-overexpression line. These genes were primarily related to GO terms like defense response and immune system function. Interestingly, we observed persistent stability in RPS2 mRNA at elevated temperatures in the NDR1-overexpression line. Based on this, we hypothesized that in the absence of pathogen infection, the overexpression of NDR1 leads to a preemptive transcriptional response to enhance resistance at elevated temperature. If true, the impetus for such a response would be the priming of immune signaling to protect plants during biotic stress in combination with abiotic stress exposure.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Saroopa P Samaradivakara, Huan Chen, Yi-Ju Lu, Pai Li, Yongsig Kim, Kenichi Tsuda, Akira Mine, Brad Day. 2022 Overexpression of NDR1 leads to pathogen resistance at elevated temperatures. New Phytol. Aug;235(3):1146-1162. doi: 10.1111/nph.18190. Epub 2022 May 21.


Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience for this reporting period was academia. To reach this audience, the following, invited, presentations were delivered: 2020, Zhejiang University, China. College of Life Sciences. Host: Kun Jiang. 2020, Virginia Tech University, Blacksburg, VA. Dept. Horticulture. Host: Bingyu Zhao. Postponed, COVID. 2020, U.S. National Science Foundation. BIO Directorate. 2020, University of Maryland, College Park. Plant Science and Landscape Architecture. Host: Yiping Qi. Postponed (Spring 2021), COVID. 2020, USA-Turkey AgriBiotech Symposium. US Embassy Sponsored. 2020, University of Maryland, Baltimore County. Dept. Biological Sciences. Host: Dr. Hua Lu. 2019, JSPS & T-PIRC Conference. Tokyo, Japan. Host: Dr. Hiroshi Ezura. 2019, The University of Tennessee. Genome Sciences (ORNL). Host: Postdocs. 2019, Hacettepe University. Ankara, Turkey. Host: Dr. Remziye Yilmaz. 2019, ?stanbul Ayd?n University. Istanbul, Turkey. Host: Prof. Dr. Dilek Heperkan. 2019, TEKFEN AGRI. Istanbul, Turkey. 2019, University of California, Davis. Department of Plant Biology, Davis, CA. Host: Graduate Students. 2019, University of Rajarata (Sri Lanka), Department of Biological Sciences. Host: Chathuranga Bamunuarachchige. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Postdoctoral researcher Dr. Saroopa Samaradivakara performed the described research, and in support of her professional development, attended a scientific conference to present her data. How have the results been disseminated to communities of interest?As described above, the research was disseminatedthrough invited seminars at ~12 venues. What do you plan to do during the next reporting period to accomplish the goals?Proposed Experiments (2020-2021) Central Hypothesis: NDR1 functions in response to drought stress, and in this role, is required for ABA-associated signaling processes. We posit that NDR1 functions as a key hub - a point of convergence - in ABA-associated signaling processes, responsible for balancing growth, defense, and survival. Our preliminary data support a role for NDR1 in regulating the growth/defense/survival response. Over the next year, we will address the following areas to address critical knowledge gaps in plant stress biology, as well as to test key aspects of our central hypothesis: Characterize the regulation of the abiotic (drought) signaling processes that require NDR1. Define the survive/thrive activity of NDR1. Characterization of a role for NDR1 in four important crop species to define the breadth of environmental factors that require NDR1 function for drought response signaling. SPECIFIC AIM 1: DEFINE THE PHYSIOLOGICAL ROLE OF NDR1 IN PLANT RESPONSE TO DROUGHT. Rationale: NDR1 is required for abiotic and biotic stress signaling. We posit that by modulating the expression of NDR1 (i.e., overexpression), we can dissect genotype and molecular mechanisms responsible for drought avoidance. Hypothesis: We hypothesize that the mechanism(s) driving the drought survival response through NDR1 lies in the function and regulation of ABA signaling/perception and stomatal-based processes. In this Aim, we will define the signaling mechanisms that mediate NDR1-specific response to stress. We have a collection of transgenic plants (soybean), generated my WCIC. The following will be used as criteria for selection of lines for analysis: ~20 lines (independent transformation events) will be used for all experiments proposed. Selection criteria will be guided by a suite of traits, including unstressed phenotype, yield, seed viability, and comparison to non-transformed parents; Further selection (i.e., narrowing) may be based on performance evaluation during initial drought screening; Selection will be guided based on sum of gene expression, yield, plant phenotypes (noted above, #2), comparison to non-transformed plants, performance under drought, etc. A. Molecular-genetic and physiological characterization of NDR1-overexpressing plants. Our work-plan includes the monitoring and evaluation of multiple, independent, transformation events from each of the crops. This approach will ensure that 1) we are selecting lines whose phenotypes can be associated with overexpression of NDR1, 2) we build a genetically and physiologically tractable system for further evaluation, and 3) we generate potential "gradients" in responses - i.e., some lines will invariably show higher levels of NDR1 transgene expression, some lower. We will use these to our advantage to enable us to correlate gene dose with response output. Once homozygous plants are identified (i.e., using antibiotic-based marker selection), individual lines will be evaluated as follows, using an 'omics-based approach to characterize the impact of transgene expression: PCR validation. PCR will be used to confirm the presence of the transgene, including the selection (i.e., antibiotic) marker. qPCR. Quantitative real-time PCR will be used to generate a comprehensive expression atlas of the transgene (i.e., NDR1). This approach will enable us to assign "no", "low", "medium", and "high" identifiers with regard to expression of NDR1. Native, untransformed, plants will be used as a baseline. Transformation events, regardless of expression level, will be moved forward. Expression levels will be recorded and noted. Protein expression. Western blot analysis will be performed on total protein (leaf) extracts to confirm the levels of expressed proteins (expression constructs consist of NDR1 fused to a HIS-epitope tag). Comparisons will be made with our native promoter and 35S-driven promoter Arabidopsis complementation lines, and qPCR (ii, above), to classify lines as low, medium, and high expression lines. Only events showing transgene protein expression will be moved forward. Localization. An examination of NDR1 protein localization will be conducted. We know that NDR1 is almost exclusively localized in the plasma membrane. We will establish localization early as to not incorrectly assign function and phenotype to mislocalization. Localization will be confirmed by 2-phase partitioning, using plasma membrane-enriched fractions, following western blot analysis. Hormone analysis. A quantitative analysis of the baseline hormone (ABA, SA, JA) content in the transgenic lines will be undertaken. We have experience in conducting these analyses, and phytohormone quantification will be done according to standard methods in the Day lab. B. Evaluation of drought response in NDR1-expressing lines. Our Preliminary Data demonstrate that Arabidopsis plants overexpressing AtNDR1 show enhanced tolerance to water stress (Figure 1, above). Here, we focus on the impact of overexpression of NDR1 in G. max (soybean). Plants will be grown in trays (pots weighed and adjusted/equalized for soil moisture content) for 4 weeks under standard growth and watering conditions. After 4 weeks, plants will be subjected to two watering regimes: 1) control, where plants will be watered three times weekly, and 2) drought stress conditions, where plants will not be watered. In each regime, plants will be monitored and quantitatively evaluated for 4 primary criteria at the start of the experiment. In brief, these include 1) soil water content, 2) RWC (relative water content), 3) hormone profile, and 4) mRNA accumulation of common marker genes for drought stress (e.g., RD29B; NCED3) This line of experimentation will be applied to all generated transgenic crops/lines to establish the level of drought stress achieved, as well as to correlate levels of previously observed resistance in WT Col-0, the ndr1 mutant, and the NDR1OE Arabidopsis line. C. NDR1 and stomatal guard cell response: Drought and the convergence of biotic signaling. To address the dual function and role of NDR1 in drought, and further, to establish the putative role of NDR1 as a point of convergence in abiotic and biotic signaling, we will use stomatal response as a marker for the impact of NDR1 on drought and immunity. Stomata aperture measurements will be performed using standard methods in the Day lab.?

Impacts
What was accomplished under these goals? We have demonstrated that the immune signaling component NDR1 (NON RACE-SPECIFIC DISEASE RESISTANCE 1) is required for the robust transcriptional activation of key ABA-associated stress signaling responses in plants, including those associated with both biotic and abiotic signaling mechanisms. Using a combination of physiological, genetic, and whole-transcriptome-based analyses, we demonstrate that a loss of NDR1 results in a block in stomatal-based mechanisms impacting the response to abiotic and biotic stress signaling. As a mechanism underpinning this, we demonstrate NDR1 associates with the plasma membrane-localized H+-ATPase, the function of which is to negatively regulate the activity of the guard cell-localized ATPase. Excitingly, we show that overexpression of NDR1 leads to enhanced drought tolerance, suggesting a role for NDR1 in the regulation of processes mediating cellular response - and tolerance - to drought signaling. In short, our current work in this area has demonstrated: The ndr1 mutant is susceptible to low water availability. To evaluate the physiological impact of water withholding and the onset of drought stress, we monitored electrolyte leakage and relative water content (RWC) over the timecourse of water withholding. As shown, from Day 12 until ~16 dps, all plant genotypes maintained a steady-state level of electrolytes and RWC (Figure 1C). However, by 17-21 dps, drought phenotypes became more pronounced, concomitant with enhanced electrolyte leakage and a significant loss in RWC. For example, at 18 dps, the ndr1 mutant showed an approximate 20% increase in electrolyte leakage compared to WT Col-0, and a ~50% increase in leakage compared to the NDR1 overexpression line. Similarly, the RWC in WT Col-0 and the ndr1 mutant dropped ~50% more than in the NDR1 overexpression line (i.e., ndr1/35S::NDR1), which maintained RWC of approximately 60% over the timecourse of water withholding. In total, these data suggest that NDR1 plays an important role in the response to low water availability and drought stress. The ndr1 mutant antagonizes ABA-mediated inhibition of seed germination. Abscisic acid production in the ndr1 mutant under conditions of drought. Abscisic acid biosynthesis and metabolic gene expression profiles are altered in the ndr1 mutant. The ndr1 mutant shows differential stomatal responses under abiotic and biotic stress elicitation. ABA- and flg22-induced stomata closure is blocked in the ndr1 mutant, whereas elf18-induced closure remains intact. ?

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Li, P., Lu, Y-J., Chen, H., and Day, B. (2020). Lifecycle of the plant immune response. Crit. Rev. Plant Biol. 39: 72-100. https://doi.org/10.1080/07352689.2020.1757829.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: Lian, Q., Zhao, X., Li, M., Dong, Z., Xu, Y., Wang, Y., Day, B.*, and Ma, Q.*. (2020). ShNPSN11, a vesicle-transport-related gene, confers resistance to Oidium neolycopersici in tomato. In Press, Biochem. J.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Baetsen-Young, A., Wei, C.M., VanBuren, R., and Day, B. (2020). Fusarium virguliforme transcriptional plasticity revealed by diverged host colonization. Plant Cell. 10. 1105/tpc. 19.00697.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Suzaki, T., Tsuda, M., Ezura, H., Day, B., and Miura, K. (2019). Agroinfiltration-based efficient transient protein expression in leguminous plants. Plant Biotech. 10.5511/plantbiotechnology.19.0220b.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Sun, G., Feng, C., Guo, J., Zhang, A., Wang, Y., Day, B*., and Ma, Q*. (2019). The tomato Arp2/3 complex is required for resistance to the powdery mildew fungus Oidium neolycopersici. Plant, Cell, Environ. 42:2664-2680. doi: 10.1111/pce.13569.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang, L., Paasch, B.C., Chen, J., Day, B., and He, S.Y. (2019). An important role of L-fucose biosynthesis and protein fucosylation genes in Arabidopsis immunity. New Phytol. doi:10.1111/nph.15639.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Dhar, N., Short, D.P.G., Mammo, B.E., Corrion, A.J., Wai, C.M., Anchieta, A., VanBuren, R., Day, B., Ajwa, H., Subbarao, K.V., and Klosterman, S. (2019). Gibberellic acid mediates accelerated development and early flowering in the Arabidopsis ndr1-1 mutant. Plant Sci. https://doi.org/10.1016/j.plantsci.2019.04.006.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: i, P. and Day, B. (2019). Battlefield Cytoskeleton: Cytoskeletal regulation and pathogen targeting of plant immunity. Mol Plant-Microbe Interact. doi.org/10.1094/MPMI-07-18-0195-FI.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Courville, K., Frantzeskakis, L., Haeger, N., Kellner, R., Day, B. Usadel, B., Gupta, Y., van Esse, P., Brachmann, A., Kemen, E., Feldbrugge, M., and G�hre, V. (2019). Smut infection of perennial hosts: the genome and the transcriptome of the Brassicaceae smut fungus Thecaphora thlaspeos reveal functionally conserved and novel effectors. New Phytol. 222:1474-1492.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Lokdarshi, A., Guan, J., Cho, S.K., Urquidi-Camacho, R.A., Leonard, M., Shimono, M., Day, B., and von Arnim, A.G. (2020). Light activates the translational regulatory GCN2 kinase through a pathway involving reactive oxygen species emanating from the chloroplast. Plant Cell. 32: 1161-1178.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: iang, K., Shimono, M., and Day, B. (2019). The guard cell actin cytoskeleton. In eLS, pp.1-8 10.1002/9780470015902.a0028039.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Academia is the primary target audience; however, the translational elements of the research does reach more applied personnel. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1 graduate student and 1 postdoc have been trained under the current project. How have the results been disseminated to communities of interest?Results have been disseminiated through the following presentations: 2019, The University of Tennessee, Knoxville. Plant Research Colloquium. 2019, The National Science Foundation. BIO Directorate. Host: Donal Monahan. 2019, Hacettepe University. Department of Food Engineering. Ankara, Turkey. Host: Dr. Remziye Yilmaz. 2019, Hacettepe University, Technology Development Zone. Host: Cem Unal, General Directorate 2019, ?stanbul Ayd?n University. Istanbul, Turkey. Host: Prof. Dr. Dilek Heperkan. 2019, TEKFEN AGRI. Istanbul, Turkey. 2019, Ankara Chamber of Commerce (ATO). Host: Ülkü Karaku?, Vice President of ATO. 2019, University of California, Davis. Plant Biology, Davis, CA. Host: Bo Liu. 2019, University of Rajarata (Sri Lanka), Biological Sciences. Host: Chathuranga Bamunuarachchige. 2018, University of North Texas, Department of Biological Sciences. Denton, TX. Host: Dr. Jyoti Shah. 2018, Tsukuba Global Science Week, University of Tsukuba. Tsukuba, Japan. Host: Dr. Hiroshi Ezura. 2018, Meiji University, Graduate School of Agriculture. Tokyo, Japan. Host: Dr. Hanae Kaku. 2018, Chinese Academy of Sciences - John Innes Center. Shanghai, China. Host: Dr. Xiufang Xin 2018, Nanjing Agriculture University. International Symposium on Agro-Environmental Quality. Nanjing, China. 2018, Nanjing Agriculture University. College of Plant Protection. Host: Dr. Yuanchao Wang. 2018, RIKEN (Yokohama). Center for Sustainable Resource Science (CSRS). Yokohama, Japan. Host: Dr. Kazuo Shinozaki. 2018, RIKEN (Wako). Center for Sustainable Resource Science (CSRS). Wako, Japan. Host: Dr. Takeshi Nakano. 2017, University of Delaware. College of Agriculture and Natural Resources. (Panelist). 2017, Hacettepe University. Ankara, Turkey. Host: Dr. Remziye Yilmaz 2017, RIKEN. Center for Sustainable Resource Science. Yokohama, Japan. Host: Dr. Kazuo Shinozaki. 2017, National Institute of Agroenvironmental Sciences. Tsukuba, Japan. 2017, Kumamoto University International Research Organization for Advanced Science & Technology (iROAST) Symposium (Advanced Green Bio). Host: Dr. Takumi Higaki. 2017, Meiji University. Department of Life Sciences. Tokyo, Japan. 2017, Tsukuba Global Science Week International Symposium. Tsukuba, Japan. Host: Dr. Hiroshi Ezura. What do you plan to do during the next reporting period to accomplish the goals?The next steps in this project are as follows: ENVIRONMENTAL RESPONSE AND FIELD TESTING. In this final Aim, we will determine the limits of the hypothesis: NDR1 mediates plant response, adaptation, and survival under conditions of multiple, simultaneous stresses. To accomplish the objectives of this Aim, we will extend our findings, above, and evaluate plant response to drought, as well as a suite of additional "involuntary" stressors, under appropriate field conditions where these crops are grown. These experiments will be led by our group, and completed in collaboration with the named collaborators of this proposal. For the purpose of this proposal, "Involuntary" responses are defined as response to pest, pathogen, soil nutrient, and temperature effects. NOTE: We are in the process of acquiring permission(s) to conduct the proposed field studies using genetically modified (i.e., transgene expression) plants. As part of this, we are working with USDA, the EPA (pathogen element), and the MI Department of Agriculture to obtain the necessary permits. For proposed growth chamber and greenhouse experiments, the required MSU-related and APHIS permits are in-hand. Our collaborators in Japan and Australia have the necessary permits and facilities to conduct field-release transgenic research. It will be necessary to work with USDA-APHIS and the relevant in-country agencies to obtain import and release permissions for the materials described herein. We anticipate having all of the required permitting in place by the end of Year 1 of the funding period. A. Field trial design and evaluation. Two field seasons at each location will be conducted. 1. Pre-season sampling. Soil organic matter, soil type, soil fertility, and soil pest population (Pythium, Phytophthora, nematode, and Fusarium virguliforme) will be evaluated at planting. This will enable us to determine what environmental variables are present that might impact observed drought, growth, development, and overall performance phenotypes for cowpea and soybean 2. Transgenic cowpea and soybean plants will be grown in designated, permitted, locations at the MSU Agronomy (Department of Plant, Soil and Microbial Sciences) farm and Kellogg Biological Station; KBS, at the Centre for Tropical Crops, and at the University of Tsukuba. At MSU, assistance for these experiments will be provided by the agronomy farm staff, as well as through ongoing collaboration with Dr. Addie Thompson (see attached letter of support). In brief, we will grow the crops, including non-transgenic "checks", using a randomized complete block design (RCBD), containing 20 four-foot plots in each field location (MSU Agronomy Farm and KBS). Soil moisture and plant biomass will be monitored at 10 time points, at weekly intervals over the growing season (May-September in East Lansing and Tsukuba, Japan; September-January in Australia). Outer plant rows will be destructively sampled, inner plant rows will be utilized for yield evaluations. Briefly, all soybean and cowpea lines will be grownusing recommended agronomic practices. For field experiments (cowpea and soybean), the experiment will be a randomized complete block design, with four blocks per field. Plots will be exposed to natural epidemics in the course of the growing season. Additionally, for induced drought conditions, samples will be grown under a rainout shelter at KBS. Parent lines (i.e., untransformed) will be used in border rows at field edges. Control (i.e., well-watered) plots will be managed by overhead irrigation. Plots will be rated weekly for response to drought and drought severity (47), growth, and photosynthetic (i.e., MultispeQ/PhotosynQ; see below) analysis, for a total of ten ratings, with yield data (September/October) taken on two rating dates. "Involuntary" responses, such as disease and pest, will be evaluated by the monitoring of symptom appearance, including chlorotic lesions, necrotic lesions, lesion size, lesion type, degree of stunting, and pest damage. Chlorosis, necrosis, and stunting ratings will be evaluated on a 0 to 9 scale based on percentage of symptomatic leaf area, and will be rated as the average percent of leaf area displaying each symptom. Overall growth, performance, yield, etc., will be compared to well-watered, and drought-exposed, transgenic lines and untransformed parents. Chlorophyll content, rates of photosynthesis, and gas exchange will be monitored using the MultispeQ/PhotosynQ instrument (described below). Two devices will be provided to each of the collaborators in Australia and Japan. 3. Transgenic tomato will be planted in a secured greenhouse setting at MSU (APHIS permitted) and Tsukuba (permitted), and plants will be evaluated for response to drought, using the aforementioned approaches. Evaluation of drought will be performed in a manner similar to the experiments described, above, for Arabidopsis. In short, transgenic tomato and wild-type Castlemart seeds will be sewn in individual 12-inch pots containing standard soil mix (e.g., Premier Pro-Mix BX); pots + soil weight will be adjusted across plantings. Seedlings will be flood-irrigated once every day for 10min. At 25-days post-emergences, plants will be randomly divided into four groups with 12 plants per line for each treatment. Initially, the experiment will include two treatments: 1) control, 26/20°C (day/night) with three times of irrigation every day; and 2) drought stress, 26/20°C (day/night) without irrigation. Treatments will last for up to 15 days. Soil moisture measurements will be taken daily over the course of the experiment. Leaves will be sampled for RWC. Temperature, humidity, and light conditions will be standardized at MSU and Tsukuba; multiple Onset HOBO monitors will be installed at each location to record environmental conditions. Chlorophyll content, rates of photosynthesis, and gas exchange will be monitored using a LiCOR 6800.?

Impacts
What was accomplished under these goals? NDR1 mediates stomata closure in response to diurnal cycling. The circadian clock regulates nearly all aspects of life, including organismal response to changes in development, environment, and abiotic and biotic stress response (19). Indeed, an abundance of data suggests that nearly 90% of the expressed plant genome is under the control of the circadian clock; much of this control is mediated by transcription factors, noted above, that also mediate dual response to abiotic and biotic stress (i.e., MYB; (20)). Of particular relevance to drought, numerous studies have demonstrated that a bidirectional mode of regulation exists between drought signaling, ABA metabolism, and the regulation of the clock (reviewed in (21)). In the case of biotic stress linkages to the clock, studies from the lab of Xinnian Dong also showed that biotic signaling and the accumulation of SA and JA are regulated by diurnal oscillations of the clock (22). These studies are significant, as they provide a foundation which describes how hormones - one of the key outputs of the clock - are regulated and tied to the function of stomatal gating and processes associated with stomatal function (e.g., drought, immunity). Recall: Stomata open/close is not only associated with growth and photosynthesis, but also immunity to a range of plant pathogenic organisms (23). In addition to the basic gating responses, stomatal opening is also dictated by the function and activity of the clock in well-watered plants: opening at dawn, closing at dusk. Thus, it is not surprising that the clock plays a key role in controlling multiple responses through stomatal function. In total, this phenomenon illustrates the importance of the circadian clock in plant adaptation to the environment, including responsiveness to hormonal cues which regulate response to endogenous and exogenous signals. Preliminary and Supporting Data: To determine the role of NDR1 in stomatal- and ABA-associated processes, we asked: What is the relationship between NDR1's role in hormone and stomatal processes as a function of the clock? To address this question, we collected images of stomata, as well as leaf samples for hormone analysis, from plants grown under a 12h light/12h dark diurnal cycle. As shown, we observed that WT Col-0 plants showed a stomatal open/close diurnal cycle, consistent with previously published studies. Interestingly, and as a likely basis for ndr1 enhanced drought sensitivity, the ndr1 mutant showed statistically significant increases in stomatal aperture over a 24h period, compared to WT Col-0. Conversely, overexpression of NDR1 showed a slowed response to opening at dawn (19h-21h Zeitgeber), and early closing at dusk (15h-17h Zeitgeber). Analysis of hormone accumulation in all 3 lines (WT Col-0, ndr1, and NDR1OE) revealed patterns of accumulation that are supportive of a role for NDR1 in stomatal function, and by extension, drought. Plant signaling and response to abiotic and biotic stress requires crosstalk between immunity and physiology. To dissect the interplay between recognition and response activation, a myriad of studies have investigated the shared signaling responses induced by abiotic and biotic stresses (15). These include the crosstalk between hormone perception (24, 25), the impact of environmental stress on host physiology and plant defense activation (26), as well as amplification (e.g., MAPK phosphorylation; (27)), and the induction of transcription (28). As highlighted in a recent review by Beattie, one of the primary environmental and cellular resources that underpins each of the above signaling processes is water (29). Manipulation of plant defense signaling by pathogens through modulation of fluid homeostasis is well-documented (29), and as a central component of this process, ABA accumulation in response to water stress is not only involved in processes that enhance drought tolerance in vegetative tissues (30), but also negatively impacts the activation of plant defense signaling (31). At a physiological level, this process is hypothesized to be regulated by the maintenance of water potential in the space surrounding the plant cell (i.e., apoplast); specifically, high water content in the apoplast suppresses the activation of plant immunity, while low water content restricts pathogen growth during immune activation (29). Preliminary and Supporting Data: In support of the experiments completed in 2017-2018, as well as predicted interactions in the field (proposed, future experiments, ca. 2019-2020), we evaluated the impact of NDR1 expression on pathogen resistance during drought exposure. In brief, we observed an increase in susceptibility to Pst DC3000-AvrRpt2 in the ndr1 mutant, compared to resistance in WT Col-0; this is likely due to AvrRpt2's impact on auxin (32). We did not observe a statistically significant increase in resistance as a function of overexpression of NDR1 (i.e., NDR1OE). While preliminary (n = 3), these data do not indicate an additive (i.e., drought tolerance + pathogen resistance) response associated with NDR1 overexpression. These experiments are currently being repeated, including against Pst expressing the effector HopAM1, which has been demonstrated to enhance in planta pathogen growth under conditions of drought (33). As outlined below, and as a minor component of this proposal (Aim 3), we will evaluate plant lines against bacterial and fungal pathogens, and pests (e.g., cabbage looper, nematode). ndr1 mutant and overexpression plants show altered ABA response signaling expression. As noted above, ABA plays a key role in both drought perception and defense signaling in plants (9). Based on our observations of increased electrolyte leakage following the induction of drought, we further investigated the impact of a loss of NDR1 - and overexpression of NDR1 - on the mRNA accumulation of key drought responsive markers. NOTE: As indicated above, we have recently completed a comprehensive RNA-seq experiment over a drought time course. To determine if the observed drought stress response in the ndr1 mutant was ABA dependent or independent, we monitored the expression of RD29B (ABA-dependent marker) and NCED3 (ABA biosynthesis) mRNAs by qRT-PCR. In the ndr1 mutant, RD29B mRNA was observed to be upregulated ~2000-fold at 21 dps (compared to well-watered plants), suggesting that ABA plays a role in mediating the drought response phenotype in the ndr1 mutant. In the overexpression line, NDROE, mRNA accumulation of both marker genes was essentially unchanged over the course of the experiment. As predicted, NCED3 expression was rapidly upregulated in WT Col-0 and the ndr1 mutant at the early stages of drought onset, with a later induction (i.e., 21 dps) in the NDROE line. These data support our hypothesis that NDR1OE functions to enable plants to avoid drought stress.?

Publications


    Progress 02/01/18 to 09/30/18

    Outputs
    Target Audience:Plant scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student training. Postdoctoral training. Meeting/conference attendance. Publications in process. How have the results been disseminated to communities of interest?2018, University of North Texas, Department of Biological Sciences. Denton, TX. 2018, Tsukuba Global Science Week, University of Tsukuba. Tsukuba, Japan. 2018, Meiji University, Graduate School of Agriculture. Tokyo, Japan. 2018, Chinese Academy of Sciences - John Innes Center. Shanghai, China. 2018, Nanjing Agriculture University. International Symposium on Agro-Environmental Quality. Nanjing, China. 2018, RIKEN (Yokohama). Center for Sustainable Resource Science (CSRS). Yokohama, Japan. 2018, RIKEN (Wako). Center for Sustainable Resource Science (CSRS). Wako, Japan. 2017, University of Delaware. College of Agriculture and Natural Resources. (panelist). 2017, Hacettepe University. Ankara, Turkey. 2017, RIKEN. Center for Sustainable Resource Science. Yokohama, Japan. 2017, National Institute of Agroenvironmental Sciences. Tsukuba, Japan. 2017, Kumamoto University International Research Organization for Advanced Science & Technology (iROAST) Symposium (Advanced Green Bio). 2017, Meiji University. Department of Life Sciences. Tokyo, Japan. 2017, Tsukuba Global Science Week (International Symposium). Tsukuba, Japan. 2017, Uman National University of Horticulture. Uman, Ukraine. 2017, Northwest A&F University, State Key Laboratory of Crop Stress Biology. Yangling, China. What do you plan to do during the next reporting period to accomplish the goals?PROPOSED RESEARCH DEFINE THE PHYSIOLOGICAL ROLE OF NDR1 IN PLANT RESPONSE TO DROUGHT. Rationale: NDR1 is required for abiotic and biotic stress signaling. We posit that by modulating the expression of NDR1 (i.e., overexpression), we can dissect genotype and molecular mechanisms responsible for drought avoidance. Hypothesis: We hypothesize that the mechanism(s) driving the drought survival response through NDR1 lies in the function and regulation of ABA signaling/perception and stomatal-based processes. In this Aim, we will define the signaling mechanisms that mediate NDR1-specific response to stress. We have a collection of transgenic plant construction activities underway; each are at various stages nearing completion. The following will be used as criteria for selection of lines for analysis: 2-3 lines/crop will be used for all experiments proposed. Selection criteria will be guided by a suite of traits, including unstressed phenotype, yield, seed viability, and comparison to non-transformed parents; Further selection (i.e., narrowing) may be based on performance evaluation during initial drought screening; Selection will be guided based on sum of gene expression, yield, plant phenotypes (noted above, #2), comparison to non-transformed plants, performance under drought, etc. Additional traits (e.g., nutrition content), as noted in Aim 3. A. Molecular-genetic and physiological characterization of NDR1-overexpressing plants. As briefly illustrated in Figure 8, our work-plan includes the monitoring and evaluation of multiple, independent, transformation events from each of the crops. This approach will ensure that 1) we are selecting lines whose phenotypes can be associated with overexpression of NDR1, 2) we build a genetically and physiologically tractable system for further evaluation (i.e., Aims 2 & 3), and 3) we generate potential "gradients" in responses - i.e., some lines will invariably show higher levels of NDR1 transgene expression, some lower. We will use these to our advantage to enable us to correlate gene dose with response output. Once homozygous plants are identified (i.e., using antibiotic-based marker selection), individual lines will be evaluated as follows, using an 'omics-based approach to characterize the impact of transgene expression: i. PCR validation. PCR will be used to confirm the presence of the transgene, including the selection (i.e., antibiotic) marker. qPCR analysis will be used to assess copy number. Only transformation events showing transgene insertion will be moved forward. ii. qPCR. Quantitative real-time PCR will be used to generate a comprehensive expression atlas of the transgene (i.e., NDR1). This approach will enable us to assign "no", "low", "medium", and "high" identifiers with regard to expression of NDR1. Native, untransformed, plants will be used as a baseline. NOTE: we are not complementing plant lines; rather, we are "stacking" constitutive expression of NDR1 "on top" of endogenous NDR1 (homolog/ortholog) expression. To distinguish transgene-expressed NDR1 from endogenous, we will use a combination of UTR primers (endogenous) and epitope tag primers (transgene). Transformation events, regardless of expression level, will be moved forward. Expression levels will be recorded and noted. iii. Protein expression. Western blot analysis will be performed on total protein (leaf) extracts to confirm the levels of expressed proteins (expression constructs consist of NDR1 fused to a HIS-epitope tag). Comparisons will be made with our native promoter and 35S-driven promoter Arabidopsis complementation lines, and qPCR (ii, above), to classify lines as low, medium, and high expression lines (3). Only events showing transgene protein expression will be moved forward. iv. Localization. An examination of NDR1 protein localization will be conducted. We know that NDR1 is almost exclusively localized in the plasma membrane (3, 6). We will establish localization early as to not incorrectly assign function and phenotype to mislocalization. Localization will be confirmed by 2-phase partitioning, using plasma membrane-enriched fractions, following western blot analysis. PD Day has extensive experience in this technique (6, 7, 35). v. Hormone analysis. A quantitative analysis of the baseline hormone (ABA, SA, JA) content in the transgenic lines will be undertaken. We have experience in conducting these analyses (e.g., Figures 3 & 4). Phytohormone quantification will be done according to previously published work by Velasquez et al. (36), with minor modifications. Abscisic acid (ABA)-d6 will be used as an internal standard. JA and SA standards are commercially available and are in-hand. Injections of plant extracts (10 ml per injection) will be separated on a fused core Ascentis Express C18 column (2.1x 100 mm, 2.7 mm) installed on an Acquity Ultra Performance Liquid Chromatography (UPLC) system. Resultant peak areas will be quantified based on standard curves generated from peak area ratios of analytes. Analytes will be quantified by converting peak area to phytohormone concentration (nM) per gram of dry weight of leaf tissue using a standard curve specific to each compound. B. Evaluation of drought response in NDR1-expressing lines. Our Preliminary Data demonstrate that Arabidopsis plants overexpressing AtNDR1 show enhanced tolerance to water stress. In support of our proposed approach, transgenic reciprocal overexpression of NDR1 from several non-model plants (e.g., coffee, citrus, Casuarina glauca) imparts enhanced resistance in each of the expressing lines (37-39). Here, we focus on the impact of overexpression of AtNDR1, BvNDR1, and SlNDR1 on drought tolerance. Plants will be grown in trays (pots weighed and adjusted/equalized for soil moisture content) for 4 weeks under standard growth and watering conditions. After 4 weeks, plants will be subjected to two watering regimes: 1) control, where plants will be watered three times weekly, and 2) drought stress conditions, where plants will not be watered. In each regime, plants will be monitored and quantitatively evaluated for 4 primary criteria at the start of the experiment. In brief, these include 1) soil water content, 2) RWC (40), 3) hormone profile (e.g., Figures 3 & 4), and 4) mRNA accumulation of common marker genes for drought stress (e.g., RD29B (41); NCED3 (42); e.g., Figure 6). This line of experimentation will be applied to all generated transgenic crops/lines to establish the level of drought stress achieved, as well as to correlate levels of previously observed resistance in WT Col-0, the ndr1 mutant, and the NDR1OE Arabidopsis line (e.g., Figure 2).

    Impacts
    What was accomplished under these goals? To determine the role of NDR1 in stomatal- and ABA-associated processes, we asked: What is the relationship between NDR1's role in hormone and stomatal processes as a function of the clock? To address this question, we collected images of stomata, as well as leaf samples for hormone analysis, from plants grown under a 12h light/12h dark diurnal cycle (Figure 4). As shown, we observed that WT Col-0 plants showed a stomatal open/close diurnal cycle, consistent with previously published studies. Interestingly, and as a likely basis for ndr1 enhanced drought sensitivity, the ndr1 mutant showed statistically significant increases in stomatal aperture over a 24h period, compared to WT Col-0. Conversely, overexpression of NDR1 showed a slowed response to opening at dawn (19h-21h Zeitgeber), and early closing at dusk (15h-17h Zeitgeber). Analysis of hormone accumulation in all 3 lines (WT Col-0, ndr1, and NDR1OE) revealed patterns of accumulation that are supportive of a role for NDR1 in stomatal function, and by extension, drought. In support of the experiments proposed in Aim 2, as well as predicted interactions in the field (Aim 3), we evaluated the impact of NDR1 expression on pathogen resistance during drought exposure. As shown in Figure 5, we observed an increase in susceptibility to Pst DC3000-AvrRpt2 in the ndr1 mutant, compared to resistance in WT Col-0; this is likely due to AvrRpt2's impact on auxin (32). We did not observe a statistically significant increase in resistance as a function of overexpression of NDR1 (i.e., NDR1OE). While preliminary (n = 3), these data do not indicate an additive (i.e., drought tolerance + pathogen resistance) response associated with NDR1 overexpression. These experiments are currently being repeated, including against Pst expressing the effector HopAM1, which has been demonstrated to enhance in planta pathogen growth under conditions of drought (33). As outlined below, and as a minor component of this proposal (Aim 3), we will evaluate plant lines against bacterial and fungal pathogens, and pests (e.g., cabbage looper, nematode). Based on our observation that ndr1 mutant plants exhibit enhanced sensitivity to drought, as well as undergo induced electrolyte leakage following Pst DC3000 infection (3), we quantified the aperture response in ndr1. As shown in Figure 7, guard cell dynamics and aperture are impacted in the ndr1 mutant plant during stress elicitation. Using ABA treatment as an inducer of stomatal closure (left), we observed that ndr1 mutant guard cells do not close, nor do they close in the presence of the PAMP flg22 (center). Interestingly, the PAMP elf18 can elicit closure (right), a process that we are separately investigating as a function of bifurcation of PTI signaling through NDR1. In 2017, Michigan State University and its co-inventors (Day, Santos, Knepper) were granted a patent "...describing a method for producing improved drought tolerance in plants....." (US9644213). The proposed research described herein represents a first key step in applying our findings towards defining the role of NDR1 in drought stress response.

    Publications