Frontiers in Plant Science (Nov 2020)

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

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

DOI
https://doi.org/10.3389/fpls.2020.603693
Journal volume & issue
Vol. 11

Abstract

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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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