Pseudomonas syringae bacteria are causal agents of bacterial speck diseases on many crops and fruits in China and worldwide, leading to up to 80% yield loss in severe regions. P. syringae uses a type III secretion system (T3SS) to invade host plants. Conventional control methods such as various bactericide compounds, biological control strains and breeding approaches are not very effective due to the limitations of their uses. Therefore, the search for novel and potent anti-P. syringae therapy are urgently needed. Better understanding of the pathogenesis mechanism is a prerequisite for discovering such therapies. In bacteria, two-component systems (TCSs) sense environmental signals and rely on the phosphorylation of regulators to regulate downstream gene expression. Primarily, a canonical TCS is schematically composed of a sensor histidine kinase (HK) and its cognate response regulator (RR). Although studies on the pathogenicity of TCSs have been reported in many human and plant pathogens, the functions and regulatory mechanisms of most TCSs in P. syringae are still unclear. To the end, this thesis studied the regulatory mechanism and signals of the virulence related RhpRS TCS. Furthermore, through high-throughput sequencing, a genome-wide network that including the regulatory patterns of each TCS was established.

Our previous studies have demonstrated that a TCS, RhpRS, enables P. syringae to coordinate the T3SS gene expression, which depends on the phosphorylation state of response regulator RhpR under different environmental conditions. How RhpRS uses external signals and the phosphorylation state to exercise its regulatory functions remains unknown. In this thesis, we performed chromatin immunoprecipitation sequencing (ChIP-seq) assays to identify the specific binding sites of RhpR and RhpRD70A in either King’s B medium (KB, a T3SS-inhibiting medium) or minimal medium (MM, a T3SS-inducing medium). We identified 125 KB-dependent binding sites and 188 phosphorylation-dependent binding sites of RhpR. In KB, RhpR directly and positively regulated cytochrome c550 production (via ccmA) and alcohol dehydrogenase activity (via adhB) but negatively regulated anthranilate synthase activity (via trpG) and protease activity (via hemB). In addition, phosphorylated RhpR (RhpR-P) directly and negatively regulated the T3SS (via hrpR and hopR1), swimming motility (via flhA), c-di-GMP levels (via PSPPH_2590), and biofilm formation (via algD). It positively regulated twitching motility (via fimA) and lipopolysaccharide production (via PSPPH_2653). Our transcriptome sequencing (RNA-seq) analyses identified 474 and 840 new genes that were regulated by RhpR in KB and MM, respectively. We showed nutrient-rich conditions allowed RhpR to directly regulate multiple metabolic pathways of P. syringae and phosphorylation enabled RhpR to specifically control virulence and the cell envelope. The action of RhpRS switched between virulence and regulation of multiple metabolic pathways by tuning its phosphorylation and sensing environmental signals in KB, respectively.

Environmental signals are very important for bacteria to survive in changing environments. In the absence of RhpS, we found that the expressions of several genes were regulated by RhpR in KB but not in MM, indicating that the functions of RhpR were regulated by the external environment independent of RhpS. This result suggested that RhpR can be phosphorylated by another non-cognate sensor kinase in KB when rhpS is deleted. However, the presence of other non-cognate kinases that phosphorylate RhpR as well as the specific signal(s) that RhpS sensed still need to be explored. After screened a natural small molecule library, we found that a phenolic compound called 1,2,3,4,6-Pentagalloylglucose (PGG) has a significant activation effect on rhpR-lux reporter plasmid in vitro. Therefore, we showed that PGG may act as an external signal perceived by RhpS. On the other hand, phosphorylation experiments found that three kinases (PSPPH_3550, PSPPH_3736, PSPPH_5115) can phosphorylate RhpR in vitro. Compared to the rhpS deletion strain, the expression of hrpRS, hrpL, and T3SS effector genes were downregulated in rhpS/PSPPH_3550/3736/5115 quadruple deletion strain. These results suggest that these three kinases cooperatively phosphorylate RhpR in vivo. When the wild-type strain is grown in T3SS-inducing conditions (in planta or MM), RhpS preferably functions as a phosphatase that keeps RhpR in an unphosphorylated state, which allows the hrpRS-hrpL-T3SS cascade to be activated. When the wild-type strain is grown in the presence of PGG, RhpR is cooperatively phosphorylated by PSPPH_5115, PSPPH_3736, and PSPPH_3550, thus compromising the expression of T3SS genes.

In our previous studies, the regulatory patterns of T3SS master TCS RhpRS were revealed, implicating the vital roles of TCS in P. syringae pathogenicity and metabolic pathways. In fact, it is identified that more than 60 putative HKs and 70 RRs were shared among 3 most popular P. syringae strains (P. syringae pv. syringae B728a, pv. tomato DC3000 and pv. phaseolicola 1448A). Nonetheless, except for a few virulence related ones (such as RhpRS, CorRS, CvsRS, and GacAS etc.), the genomic regulon and functions of most TCSs are still elusive. In this study, RNA-seq were performed to detect the functions of each TCS under different environmental conditions. Five HK deletions (PSPPH_1568, PSPPH_2601, PSPPH_2606, PSPPH_, PSPPH_4451) shown reduced T3SS gene expression and attenuated pathogenicity on bean plant. The following co-expression analysis revealed the regulatory network for T3SS and surface attachment in P. syringae pathogenicity. Furthermore, ChIP-seq was performed to investigate binding pattern of RRs across the genome. To identify the crosstalk between P. syringae TCSs, we mapped an intricate network called ‘PSTCSome’ (Pseudomonas syringae two-component systems regulome) by combining RNA-seq and ChIP-seq of cluster TCSs.

To sum up, in this thesis, the pathogenicity molecular mechanism of RhpRS two-component system was explored. PGG, the signal of RhpRS, was found to suppress the expression of T3SS genes and compromise bacterial pathogenicity. Most importantly, this paper comprehensively analyzed the TCS network and revealed several important functional genes. Therefore, the development of antimicrobial agents or inhibitors against these key TCSs can lead to the discovery of novel drugs that target P. syringae.