## 3 HrpG and HrpX Are Key Virulence Regulators in _Xanthomonas_ spp.  
The ability to cause disease in host plants in _hrp_-dependent _Xanthomonas_ spp. is dependent on two transcriptional regulators--HrpX and HrpG [3]. HrpX was first identified in the early 1990s in a transposon screen in _X. campestris_ pv. _campestris_ (_Xcc_) [28] and soon after was found to have similar functions in other _Xanthomonas_ spp. [29; 30]. The _hrpX_ gene encodes a ~476 amino acids (AA) (sizes may vary between species, mostly due to alternative prediction of the start codon) AraC-type bacterial transcriptional regulator [31]. Disruption of _hrpX_ in _Xanthomonas_ bacteria that harbor T3 SS completely abolished the ability to cause disease, colonize the plant host, or induce HR in resistant or non-host plants in a similar manner to disruption of structural components of the T3 SS [3]. Further analyses demonstrated that transcriptional regulation of the six T3 SS transcriptional operons and multiple T3 Es is dependent on HrpX [22; 30; 32; 33; 34], thus establishing HrpX as a master regulator of _hrp_-dependent protein secretion.  
HrpG was identified in a screen for virulence-associated mutants using N-methyl-N\({}^{\prime}\)-nitro-N-nitrosopainidine mutagenesis in _Xeu_[35]. Similarly to _hrpX_, inactivation or deletion of _hrpG_ eliminates the ability of the bacteria to induce HR in resistant or non-host plants, or cause disease and colonize host plants [35; 36; 37]. _hrpG_ inactivation mutants also display a significant reduction in the transcription of T3 SS and T3 Es coding genes and _hrpX_ itself [35; 36; 37] (Figure 2). _In vitro_ studies showed that HrpG directly binds to the promoter region of _hrpX_ and induces the transcription of _hrpX_, indicating that _hrpX_ is a direct target of HrpG [38; 39] (Figure 2). _hrpG_ encodes a ~260-267 AA orphan OmpR family transcription regulator [35]. While HrpG structure was not experimentally determined, the mechanistic function was characterized by several key point mutations that affected its activity. Most OmpR regulators are part of a two-component system and are encoded in an operon or in the proximity of a histidine kinase that activates OmpR protein by phosphorylation [40]. HrpG harbors a conserved aspartate residue at position 60 (of _Xcci_ and _Xeu_ HrpG) that serves as a phosphorylation site; however, no histidine kinase gene is encoded in proximity to _hrpG_. HrpG D60N substitution mutant of _Xcci_ is not phosphorylated in _hrp_-inducing conditions and cannot complement the _hrpG_ mutant [41], suggesting that HrpG is activated through phosphorylation by an unknown kinase. The mutation that resulted in R210C substitution in _Xcci_ HrpG produced a dominantly negative inactive variant [38]. This mutant variant was not able to complement the _hrpG_ inactivation mutant and reduced the expression levels of HrpG associated genes and effector delivery when introduced into wild-type _Xcci_[38]. _In vitro_ assays identified that this HrpG variant can still bind to the _hrpX_ promoter but is unable to induce the transcription of _hrpX_[38]. Random mutagenesis conducted in _Xeu_ hrpG identified three constitutively active variants (corresponding to  
Figure 1: Genetic organization of the hypersensitive reaction and pathogenicity (_hrp_) gene cluster and the _hrpG/hrpX_ coding regions in _Xanthomonas_ spp. Schematic representation of the gene clusters of group 2 xanthomonas _X. citri_ subsp. _citri_ (_Xcci_, strain 306), _X. evesiatoria_ (_Xeu_, strain 85-10), _X. oryzae_ pv. _coyage_ (_Xoo_, strain KACC10331), and _X. campestris_ pv. _campestris_ (_Xcc_, strain 8004), and group 1 Xanthomonas _X. translucens_ pv. _undulosa_ (_Xtu_, strain 4699). The location of the indicated regions within their respective genomes is stated.  
AA substitution of E44K, H194R, D199N), which induced the expression of _hrp_ genes in non-inducing media and caused enhanced HR in resistant plants [42]. The mechanism that enables the constitutive activity by these variants remains elusive.