## 5 Downstream Targets and Regulatory Pathways Controlled by HrpG and HrpX  
HrpG and HrpX are master regulators of _Xanthomonas_ T3 SS and T3E. HrpG-mediated expression of most of T3 SS and T3 Es genes is dependent on _lrrpX_, which is directly regulated by HrpG [35, 36]. HrpX induces transcription by directly binding to plant-inducible promoter (PIP) box, a DNA motif of TTCGB-N15/N8-TTCGB found in _cis_ element of five of the six structural T3 SS operons and multiple T3 Es [22]. The DNA-binding motif of HrpG remained elusive for years. A recent study [39] utilized ChIP-seq analysis to identify HrpG\({}_{Xc}\)-bound promoter sequences. The study found that HrpG physically interacted with 186 promoter elements. Promoter analysis identified a common DNA motif of ATT(C/T)(C/T)(G/C/A)(T/A)T in the bound promoters and demonstrated direct binding _in vitro_. It should be noted that the study failed to demonstrate a robust correlation between the HrpG bound promoters and transcriptional induction of these targets by HrpG [39]. For instance, _lrrpX_, which is hypothesized to be one of the main direct targets of HrpG, was not among the 186 promoters. Functional studies found that accumulation of active HrpG and HrpX proteins is the main factor that controls the expression of the T3 SS, and constitutive expression of either of these regulators results in the induction of the T3 SS and T3E in non-inducing media, hypervirulence upon inoculation of susceptible plants and enhanced immune responses in resistant plants [3]. It was recently suggested that the regulatory network of the T3 SS harbors an internal positive feedback loop in _X. oryzae_ pv. _oryzicola (Xoc)_, in which _lrrpX_ expression is regulated by T3 SS protein HrcT [48] (Figure 2). It is unknown whether this mechanism is unique to this species or it is conserved among _Xanthomonas_ spp. High throughput transcriptional profiling analyses of the HrpG regulon and/or the HrpX regulon were conducted in _Xeu_, _Xci_, and _X. campestris_ pv. _raphani (Xcr)_[36, 37, 49, 50]. Two independent transcriptome studies (conducted using microarray and RNA-seq) were done with _hrpG_ and _hrpX_ deletion strains of _Xcci_ grown in plant-mimicking media [37, 49]. Transcriptomic studies of _Xeu_ and _Xcr_ that were grown in rich media compared the wild-type strain with a strain harboring the constitutively active HrpG E44K variant (_lrrpG*_) using cDNA-AFLP [36] and RNA-seq [50], respectively. T3 SS and T3E coding genes were found to be positively regulated by HrpX or HrpG in all four studies [36, 37, 49, 50] (Figure 2). The T3 SS gene _hrc_C and multiple T3E genes that lack plant-inducible promoter (PIP) box in their promoters were induced by HrpX and HrpG as well. However, the expression of several T3 Es was not HrpG/HrpX dependent. In particular, HrpG and HrpX did not affect the expression of all four transcription activator-like effectors (TALEs) coding genes in _Xcci_[37, 49]. Unlike most other T3 Es, which were identified to be associated with suppression of host immune responses, TALEs were found to control more diverse factors such as sugar efflux and cell identity [51]. It is possible that TALEs are regulated independently of the rest of the T3 Es because of their unique functions. In addition to T3 SS-associated genes, the four transcriptome analyses identified that HrpG or HrpX positively regulates a large number of genes encoding secreted hydrolases, such as endoglucanas, proteases, esterases, and pectinases [12, 36, 37, 49, 50] (Figure 2). Unlike the T3 SS, the operons that encode structural components of either of the two T2 SS identified in most _Xanthomonas_ spp. were not regulated by HrpG or HrpX. Interestingly, several studies reported that HrpG or HrpX negatively regulates secreted protease activity even though a large number of genes encoding secreted peptidases are positively regulated by the two regulators [36, 52, 53]. The inconsistencies between the reported transcriptional regulations and functional activity need to be addressed.