El-Sayed, Mohamed

English

Plants are concurrently challenged by various invaders that can severely affect their development and productivity. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET)-dependent defense pathways are generally regarded as the major defense-related phytohormones. However, accumulating evidence suggests that abscisic acid (ABA) also functions as a modulator of plant innate immunity. Moreover, it is well established that pathogen attack modifies hormone-homeostasis in the host leading to activation or suppression of immune responses. However, the spatio-temporal cellular activities of SA, JA, ET and ABA in defense have so far not been fully understood. 
To enable quantitative measurement of SA, JA, ET and ABA signaling outputs at the single-cell level, a set of multi-modular hormone-signaling reporters termed “COLORFUL-SA, -JA, -JA/ET and -ABA, respectively, were developed. These COLORFUL-reporters, together with a software-supported high-throughput imaging protocol for output quantification, were used to resolve the spatio-temporal dynamics of respective hormonal signaling activities in Arabidopsis leaves. Functional characterization was performed using exogenous hormone treatments and mutant analyses which confirmed specificity, sensitivity and rapid responsiveness of these COLORFUL-biosensors. Distinct cell and tissue type-specific signaling patterns, which are consistent with previously described spatial roles of these hormones in Arabidopsis were observed. Moreover, the COLORFUL-reporter lines were utilized to resolve the kinetics of hormone signaling and hormone crosstalk in Arabidopsis at interaction sites with the biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 and Emwa1 strains, which represent virulent and avirulent isolates, respectively. Thus, this study pioneered the spatial dissection of plant immune responses at the initial site of invasion and allowed mapping of the respective hormone signaling activities at an unprecedented single-cell resolution. 
In the conducted comparative plant-microbe interaction studies, the virulent and avirulent oomycete isolates exhibited remarkably different invasion dynamics, which correlated with spatiotemporally distinct hormone signatures. At cellular resolution, these hormone-specific reporter signatures demarcate pathogen entry and progression and highlight initiation, transduction and local containment of immune signals. The avirulent isolate Emwa1 significantly triggered SA responses in the cells that are in direct contact with the invaded cells (adjacent cells), suggesting that these may contribute to effector-triggered immunity (ETI) mediated by the Col-0 RPP4 resistance gene. Moreover, during incompatible interactions with Emwa1, a drastic suppression of JA and JA/ET signaling was observed in the adjacent cells, whereas a pronounced activation of the JA/ET signaling pathway was observed in the adjacent cells during compatible interactions with Noco2. Thus, in compatible interactions, JA/ET-dependent signaling may play a role in antagonizing the SA response-associated with ETI in the adjacent cells. Furthermore, ABA signaling was activated in invaded cells during both compatible and incompatible interactions and showed a similar activation pattern to the SA responses triggered in Noco2-haustoriated cells. 
In order to test whether other unrelated biotrophic pathogens activate a similar pattern of ABA and SA responses, the ascomycete fungal pathogen Golovinomyces orontii was used. Interestingly, a cross-kingdom-conserved induction of a cellularly confined activation of ABA- but not of SA-signaling was observed at Arabidopsis interaction sites. Mutant analyses supported the hypothesis that ABA functions as an important and common susceptibility factor for both biotrophic pathogens. To further corroborate this hypothesis, the ABA biosynthesis mutants aba1-101 and aba2-1 were challenged with Noco2 and Golovinomyces orontii which showed significantly lower spore counts relative to the wildtype Col-0. To further investigate the cell-specific contribution of ABA signalosome components to susceptibility, the sporulation of Noco2 and Golovinomyces orontii was tested on different ABA signaling mutants. The enhanced disease resistance of the double loss-of-function mutants of the positive ABA regulators SnRK2D and SnRK2I suggested a functional role of these kinases on ABA-dependent susceptibility. Individual and double mutants of SnRK2D and SnRK2I were crossed with COLORFUL-ABA to test the correlation of the enhanced disease resistance in the snrk2d snrk2i double mutant to ABA signaling activities. The significant reduction in ABA signaling activity in these lines provided additional proof that SnRK2D and SnRK2I are redundantly involved in upregulation of the ABA signaling cascade by virulent biotrophic pathogens.
In addition, two recent studies were performed in our lab to characterize the sporulation of the aforementioned pathogens on different Arabidopsis Type 2C protein phosphatase (PP2C) loss-of-function mutants. PP2Cs are negative regulators of the ABA signaling cascade. The PP2CA knockout mutant pp2ca-1 showed enhanced disease resistance to Noco2 and Golovinomyces orontii in contrast to the ahg3-1 missense mutant of the same gene and wildtype Col-0 (Lübbers 2018; Schliekmann 2017). These two mutants were crossed with the COLORFUL-SA and COLORFUL-ABA to evaluate their contribution to the regulation of SA and ABA signaling cascades. Notably, pp2ca-1 exhibits significantly induced ABA and SA signaling activities during interaction with H. arabidopsidis Noco2 relative to Arabidopsis wildtype and ahg3-1. These results may be explained by a physical interaction between PP2CA and SA as recently reported by Manohar et al. (2017) for the PP2C proteins PP2C-D4/PP2C6, ABI1 and ABI2. In the future, the COLORFUL reporter system developed and established in this study will allow further disentanglement of the complexities of basal and R-gene mediated resistance during different plant-microbe interactions in more detail and at cellular resolution.2021-06-2

Georg-August-University Göttingen

Cell-specific phytohormone responses mapped by the COLORFUL-biosensors during plant-microbe interactions

https://ediss.uni-goettingen.de/bitstream/handle/21.11130/00-1735-0000-0005-1455-5/PhD%20thesis-08.2020_Mohamed%20El-Sayed.pdf?sequence=1

Cell-specific phytohormone responses mapped by the COLORFUL-biosensors during plant-microbe interactions

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Georg-August-University Göttingen