# Regulation of the _Pseudomonas syringae_ Type III Secretion System by Host Environment Signals
## 2 Environmental Regulation of the _P. syringae_ T3 SS
### Dynamics of T3 SS Deployment within the Host Plant Environment  
Multiple distinct approaches have been taken to assess the deployment of the T3 SS by _P. syringae_ during infection. In early work, pre-treatment of _P. syringae_ with RNA or protein synthesis inhibitors prior to infection prevented _P. syringae_ from eliciting T3 SS effector-dependent plant defenses, providing foundational evidence that the expression of T3 SS encoding genes is necessary at early stages of infection [100]. In planta expression of T3 SS genes was also assessed using promoter reporter fusions and the quantification of T3 SS-encoding transcripts [79; 53]. These studies revealed that T3 SS genes are rapidly induced within the initial hours of plant infection, with transcript accumulation detectable as early as two hours post-inoculation into host leaves [100]. Transcript levels of _hrpR_ accumulated more rapidly than other T3 SS genes during host infection, consistent with the role of HrpR as an upstream regulator of the T3 SS cascade [42; 53]. More recently, various global transcriptomic analyses of _P. syringae_ within the plant environment have similarly observed the induction of T3 SS gene expression during the initial hours of infection [101; 102; 103]. Interestingly, a general decline in T3 SS expression in _P. syringae_ has been observed after the initial 24 h of host infection, suggesting complex temporal regulation may occur [93].  
T3 SS deployment by _P. syringae_ may also vary between distinct plant microenvironments inhabited during infection. A transcriptomic analysis of _P. syringae_ pv. _synfage_ B72 sa cells extracted from either the leaf surface or apoplast of bean plants indicated that the T3 SS was expressed to a greater extent within apoplastic sites of host leaves [102]. While the underlying cause of this difference is not known, cuticle wax components were shown to inhibit T3 SS expression by both _P. syringae_ pv. _phaseolicola_ NPS3121 and _P. syringae_ pv. _tomato DC3000 and therefore could contribute to this phenotype [79]. Within the plant leaf apoplast, _P. syringae_ exhibited maximal T3 SS expression within the substomatal chamber, which is typically the first location colonized by infectious bacteria [79]. Although T3 SS genes were expressed by _P. syringae_ on the leaf surface, the role of the T3 SS in epiphychit fitness remains ambiguous. In a genome-wide fitness profiling study in _P. syringae_ pv. _synfage_ B72 sa, genes encoding the T3 SS provided a greater fitness benefit within the foliar apoplast than on the surface of host plants [104]. However, in other work the T3 SS pro moted epiphylytic survival of _P. syringae_ pv. _syngae_ B728a on host leaf surfaces, with T3 SS effectors HopZ3 and HopAA1 specifically enhancing epiphylytic bacterial growth [105; 106].  
Within the host apoplast, _P. syringae_ reversibly differentiates into subpopulations of high- and low-T3 SS expressing cells [107; 108]. This phenotypic differentiation required the HrpVG negative regulatory circuit, and was enhanced by the HrpA1 T3 SS plus protein, potentially due to its role as a positive auto-activator of the T3 SS [107; 108]. Bistable (stable high and low) levels of T3 SS gene expression have been noted in a variety of Gram-negative bacterial pathogens [109; 110], though the biological significance of this phenomenon is not fully understood [66; 107]. Given that the production and maintenance of the T3 SS is energetically costly [72], low-T3 SS expressing \"cheater\" cells may be free to devote their energetic resources to multiplication or other cellular processes, promoting the overall resiliency and growth potential of the pathogen population [110]. The consequences of this behavior for _P. syringae_ virulence, as well as the specific mechanism(s) by which T3 SS bistability is initiated and maintained in the host environment, have yet to be fully characterized.