To examine the effect of growth under oxidative stress conditions, WT and mutant strains were grown on CM plates containing the oxidants hydrogen peroxide (H2O2, 30% in water; Fisher), 2-methyl-1,4-naphthoquinone (menadione; Acros Organics) or diamide [azodicarboxylic acid bis (N,N-dimethylamide) (Sigma)] solutions, at the concentrations shown, following Huang and colleagues [10] (link). Pictures were taken at 5 days post treatment.
To observe the accumulation of H2O2 at infection sites, infected rice leaf sheaths (48 hpi) were stained with 3, 3’-diaminobenzidine (DAB, Sigma) as described previously by Chi et al. [11] (link). Rice sheaths were incubated in 1 mg/ml DAB solution in the dark at room temperature for 8 hours and destained with ethanol: acetic acid solution (94:4 v/v) for 1 hr. Samples were excised and observed under a light microscope.
Mutant strains were tested for cell wall and osmotic stress conditions. Cell wall stress condition was performed by adding 100 ug/ml of Congo Red (Sigma) to solid complete media (CM). Osmotic stress conditions were applied using 0.5 M NaCl (Sigma) and 1 M Sorbitol (Sigma) in CM. Pictures were taken at 5 days post inoculation.
To observe the accumulation of H2O2 at infection sites, infected rice leaf sheaths (48 hpi) were stained with 3, 3’-diaminobenzidine (DAB, Sigma) as described previously by Chi et al. [11] (link). Rice sheaths were incubated in 1 mg/ml DAB solution in the dark at room temperature for 8 hours and destained with ethanol: acetic acid solution (94:4 v/v) for 1 hr. Samples were excised and observed under a light microscope.
Mutant strains were tested for cell wall and osmotic stress conditions. Cell wall stress condition was performed by adding 100 ug/ml of Congo Red (Sigma) to solid complete media (CM). Osmotic stress conditions were applied using 0.5 M NaCl (Sigma) and 1 M Sorbitol (Sigma) in CM. Pictures were taken at 5 days post inoculation.
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