AUTHOR=Carril Pablo , da Silva Anabela Bernardes , Tenreiro Rogério , Cruz Cristina 

TITLE=An Optimized in situ Quantification Method of Leaf H2O2 Unveils Interaction Dynamics of Pathogenic and Beneficial Bacteria in Wheat

JOURNAL=Frontiers in Plant Science

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.00889

DOI=10.3389/fpls.2020.00889

ISSN=1664-462X

ABSTRACT=<p>Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) functions as an important signaling molecule in plants during biotic interactions. However, the extent to which H<sub>2</sub>O<sub>2</sub> accumulates during these interactions and its implications in the development of disease symptoms is unclear. In this work, we provide a step-by-step optimized protocol for <italic>in situ</italic> quantification of relative H<sub>2</sub>O<sub>2</sub> concentrations in wheat leaves infected with the pathogenic bacterium <italic>Pseudomonas syringae</italic> pv. <italic>atrofaciens</italic> (<italic>Psa</italic>), either alone or in the presence of the beneficial bacterium <italic>Herbaspirillum seropedicae</italic> (RAM10). This protocol involved the use of 3-3′diaminobenzidine (DAB) staining method combined with image processing to conduct deconvolution and downstream analysis of the digitalized leaf image. The application of a linear regression model allowed to relate the intensity of the pixels resulting from DAB staining with a given concentration of H<sub>2</sub>O<sub>2</sub>. Decreasing H<sub>2</sub>O<sub>2</sub> accumulation patterns were detected at increasing distances from the site of pathogen infection, and H<sub>2</sub>O<sub>2</sub> concentrations were different depending on the bacterial combinations tested. Notably, <italic>Psa</italic>-challenged plants in presence of RAM10 accumulated less H<sub>2</sub>O<sub>2</sub> in the leaf and showed reduced necrotic symptoms, pointing to a potential role of RAM10 in reducing pathogen-triggered H<sub>2</sub>O<sub>2</sub> levels in young wheat plants.</p>