(A) Gene expression analyses with X. oryzae pv. oryzae wild-type and PhoP mutant strains. RT-PCR was carried out with PXO99 and PXO99P strains cultured at high and low Mg²⁺ and Ca²⁺ concentrations. The cells were cultured in NB medium for 3 days, washed three times with sterilized and deionized water, transferred into M9 minimal medium containing different concentrations (10 mM or 10 μM) of Mg²⁺ and Ca²⁺ ions, and cultured for 2 days at 28°C. cDNA was synthesized from 5 μg of total RNA extracted from the cells, and 1 μl of the synthesized cDNA in each cell strain was used for PCR. This experiment was repeated three times each with three independent replicates. (B) hrpG, hrpA, and hrpX expression analysis using semiquantitative real-time PCR. Wild-type PXO99 and PXO99P mutant strains were cultured as described above for panel A. Five micrograms of total RNA extracted from cells cultured in each condition was subjected to one-step real-time PCR using nonspecific binding chemistry (SYBR green). Standard curves for each gene or rRNA, hrpG, hrpA, and hrpX to quantify transcripts of each gene in each condition were established by the threshold cycle values obtained against the known concentration of 10-fold serially diluted genes (rRNA, hrpG, hrpA, and hrpX) ranging from 10⁵ to 0.001 pg per milliliter. Quantitative determination for each gene was obtained through equations computed from standard curves of each gene, and the data present relative copy numbers of each gene to the copy number of rRNA in each condition. The error bars correspond to the standard deviations from two independent replicates carried out with two sets of primers for the hrpG gene and a set of primers for the hrpA and hrpX genes.