Fig. 4

The identified factors influencing the de-repression and suppression of T3SS regulation in terms of HrpRS-HrpL pathway in P. syringae. a HrpL transcription is regulated by different proteins that include AefR, PsrA (indirect positive activators), and CorR upstream binding agent (binds directly to HrpL and activates early transcription). HrpJ established a binding complex with hrpL promoter to prevent the formation of HrpRS complex, which creates a negative feedback loop to negatively regulate hrpL activity. Then, HrpS protein itself can stimulate the T3SS effector proteins. AlgU and HrpA induce the overexpression of the hrpRS locus in a direct manner. In GacSA TCS, phosphorylated-gacA binds indirectly to the IR (inverted repeats) region of hrpR and hrpL to induce their expression. CvsR has a direct regulatory effect on the activation of hrpRS, which is expressed in the presence of calcium ion Ca²⁺ as an external environmental stimulus and ultimately sustains the T3SS-dependent virulence in P. syringae. In a higher level of c-di-GMP, HrpR/HrpL expression is repressed. In addition, HrpV blockage is removed by HrpG activity and it frees the HrpS as well as HrpJ makes a complex with both (HrpJG/V) to allow the induction of the expression of T3SS. Thus, HrpG is the anti-repressor of an anti-activator HrpV. The bonding of HrpG with HrpF exerts a negative regulatory effect on T3SS by allowing the formation of the HrpRSVG circuit. P-RhpR independently activates T3SS effector genes like hopR1. The chaperone protein RhpC directs metalloprotease RhpP to the periplasm. In the absence of RhpC, RhpP decreases the expression of HrpL. Induction of hrpRS and hrpL transcription requires (p)ppGpp. b P. syringae cultured in Kings B (nutrient-rich condition, KB) resulting in suppression of its T3SS; hrpL gene allows HrpT to bind its promoter and motif which indicates an indirect regulation by HrpT as shown by dotted lines, The PRhpR starts to pile-up and its abundance leads to the suppression of hrpRS to check its low expression. HrpV influences the binding affinity of HrpS protein to prevent the oligomerization of HrpRS; HrpRS heterodimer destruction and negative regulation is described with red dotted arrow. Phosphorylation of RhpR is mediated by another TCS unit kinase RhpS, and helps in a sustained phosphorylated state. To achieve the phosphorylated RhpR abundance, another pathway is activated using its own promoter with an inverted repeat (IR) element, and their covalent bonding leads to producing more PRhpR. Unphosphorylated-RhpR has a positive regulatory effect on three genes a) functional accumulation of c-type cytochrome, b) regulate alcohol dehydrogenase synthesis, and c) positive modulation of hemB and negative effect on protease production in KB medium. RhpR also functions as a negative regulator of anthranilate synthase activity. On the other hand, phosphorylated RhpR inhibits swimming motility but shows a positive effect on the induction of twitching motility. Additionally, in vivo, P-RhpR functions as a negative regulator of the biofilm (algD) and c-di-GMP levels while improving lipopolysaccharide production in a positive manner. P-RhpR has a positive effect on LonB protein, which results in the breakdown of HrpR protein and prevention of HrpRS oligomerization. Gene orientations and scheme of events adapted with permission from Xie et al. (2019)