Quantitative Hormone Signaling Output Analyses of Arabidopsis thaliana Interactions With Virulent and Avirulent Hyaloperonospora arabidopsidis Isolates at Single-Cell Resolution.

Hassan Ghareeb,Michael Pound,Olena Tetyuk,Cornelia Herrfurth,Ivo Feussner,Volker Lipka,Katharina Hanika,Mohamed El-Sayed

doi:10.3389/fpls.2020.603693

Hassan Ghareeb, Michael Pound + Show 6 more

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https://doi.org/10.3389/fpls.2020.603693

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The phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are central regulators of biotic and abiotic stress responses in Arabidopsis thaliana. Here, we generated modular fluorescent protein-based reporter lines termed COLORFUL-PR1pro, -VSP2pro, and -PDF1.2apro. These feature hormone-controlled nucleus-targeted transcriptional output sensors and the simultaneous constitutive expression of spectrally separated nuclear reference and plasma membrane-localized reporters. This set-up allowed the study of cell-type specific hormone activities, cellular viability and microbial invasion. Moreover, we developed a software-supported high-throughput confocal microscopy imaging protocol for output quantification to resolve the spatio-temporal dynamics of respective hormonal signaling activities at single-cell resolution. Proof-of-principle analyses in A. thaliana leaves revealed distinguished hormone sensitivities in mesophyll, epidermal pavement and stomatal guard cells, suggesting cell type-specific regulatory protein activities. In plant-microbe interaction studies, we found that virulent and avirulent Hyaloperonospora arabidopsidis (Hpa) isolates exhibit different invasion dynamics and induce spatio-temporally distinct hormonal activity signatures. On the cellular level, these hormone-controlled reporter signatures demarcate the nascent sites of Hpa entry and progression, and highlight initiation, transduction and local containment of immune signals.

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The phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) represent fundamental components of the plant immune signaling network (Robert-Seilaniantz et al, 2011)
To reveal the spatial dynamics of SA-dependent signaling processes at single-cell resolution, we developed a confocal laser scanning microscopy (CLSM)-based reporter system in A. thaliana plants
The three reporter modules employ the spectrally separable and monomeric fluorescent proteins VENUS, mKATE2 and EGFP (Zhang et al, 1996; Nagai et al, 2002; Shcherbo et al, 2009), whose expression are controlled by the SA-responsive promoter of the PATHOGENESIS-RELATED GENE 1 (PR1) gene (Cao et al, 1994; Mou et al, 2003), the constitutive POLYUBIQUITIN 10 (UBQ10) gene promoter and the constitutive Cauliflower Mosaic Virus 35S RNA (CaMV35S) promoter, respectively

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The phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) represent fundamental components of the plant immune signaling network (Robert-Seilaniantz et al, 2011). Whilst JA and ET have been demonstrated to synergistically regulate defense against necrotrophic pathogens (Zhu, 2014), the marker gene-defined JA and JA/ET signaling pathways can act antagonistically upon each other (Lorenzo et al, 2004), and are suppressed by SA (Van der Does et al, 2013). These findings underpin the complex crosstalk between these hormone signals and their importance for coordinating and fine-tuning defense against different types of pathogens (Thomma et al, 1998; Robert-Seilaniantz et al, 2011)

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