Figure 7.

The ‘‐930’ locus upstream of the hrpR gene is involved in transcriptional regulation of hrpR/S and its integrity is required for the expression and secretion of Type III secretion system (T3SS) and effectors (T3Es). (1) The pathogenicity‐related ‘‐930’ locus is involved in the transcription of hrpR/S, but not hrpH. We constructed three mutants of the low‐virulent strain M227 by expressing each of the three fragments, hrpH‐NCS, NCS alone and NCS‐hrpR/S, cloned from M228. However, only M227 expressing NCS‐hrpR/S _M228 demonstrated significantly increased pathogenicity. (2) The transcriptional patterns of T3SS and effector genes in M228 and the mutant M228gfp in the hrp‐inducing condition. The nutrient‐rich King’s B (KB) medium and HDM medium were hrp‐repressing and hrp‐de‐repressing, respectively. Bacteria were cultured in KB at the designated ‘0 h’ and subsequently transferred into HDM medium at a final concentration of 0.02 OD₆₀₀. Quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR) was performed in a Bio‐Rad IQ5 thermal cycler using SYBR Green reagent. Three replicates were performed for each sample. The results shown are the mean and standard deviation, and the relative expression ratios were compared amongst samples for each gene (Duncan’s multiple range test, P < 0.05). Experiments were repeated at least twice with similar results. (3) M227 secreted less Type III pilus, translocator, harpins and effectors in hrp‐inducing conditions. The values in the heat‐map are log₂ (protein_ratio_in_M227/ protein_ ratio_in_M228). The purple, green and red fonts indicate the T3SS structure or helper proteins, chaperons and effectors, respectively. M228 is the high‐virulent Psa strain, and in M228gfp an 841 bp gfp fragment was artificially inserted at the ‘‐930’ locus.