# Innovation and Application of the Type III Secretion System Inhibitors in Plant Pathogenic Bacteria
## 2 Regulation of T3 SS in _D. dadantii_
### Bacterial Second Messengers Regulate T3 SS  
_D. dadantii_ can infect a wide range of host plants and has the ability to survive in soil and groundwater [36; 37]. In the past decade, several regulators have been identified to be involved in the regulation of T3 SS, implying a sophisticated regulatory network that allows _D. dadantii_ to control its virulence gene expression to adapt to various environmental conditions. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a ubiquitous bacterial second messenger in most major bacterial phyla, is one of the most critical and well-studied nodes of this network [38; 39]. The metabolism of c-di-GMP is dependent on two kinds of enzymes: The GGDEF domain-containing diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP from two molecules of guanosine-5'-triphosphate (GTP) and the EAL or HD-GYP domain-containing phosphodiesterase (PDE) enzymes that degrade c-di-GMP to 5'-phosphoguanylyl-(3'-5')-guanosine or two molecules of guanosine monophosphate (GMP), respectively [40; 41; 42; 43; 44]. Figure 1: Model of the type III secretion system (T3 SS) regulation and modes of action of T3 SS inhibitors in _Dickey dadantii_. The expression of T3 SS master regulator HrpL is transcriptionally regulated by the HrpX/HrpY-HrpS-RpoN pathway and post-transcriptionally regulated by the GacS/GacA-RsmB-RsmA pathway. Several transcriptional regulators, including FluDC, SlyA, PecS, PecT, and RpoS, regulate the expression of T3 SS genes via targeting multiply key components in the T3 SS regulatory pathways. Hfo and its dependent sRNA ArcZ form a feed-forward signaling cascade that positively control the expression of RsmB. PNPage degrades RsmB sRNA and is also required for the stability of _rpoN_ mRNA. c-di-GMP signaling is involved in the FluDC-mediated RpoN regulation and the Hfo-mediated RsmB regulation. T3 SS injects bacterial effectors into the plant cells to suppress plant immune responses. On the other hand, plants secrete a variety of phenolic compounds that are able to regulate the T3 SS in bacteria. For _D. dadantii_, two plant phenolic compounds, _o_-cumaric acid (OCA) and _t_-cinnamic acid (TCA), upregulate T3 SS gene expression via the GacS/GacA-RsmB-RsmA-HrpL pathway. _Trans_-4-hydroxycinnamohdroxamic acid (TS103), a plant phenolic compound derivative, represses the expression of T3 SS via the HrpX/HrpY-HrpS-RpoN-HrpL pathway. \(\bot\) represents negative control; \(\rightarrow\) represents positive control. The presence of GGDEF and/or EAL domain protein-encoding genes is abundant in many Gram-negative bacteria. For example, _Escherichia coli_ K-12 contains 29 genes, _Vibrio cholerae_ contains 53, and _E. amylovora_ contains 8 [45; 46; 47; 48]. In _D. adandtii_, genomic analysis has identified twelve _gcp_ (GGDEF domain-containing protein), four _ecp_ (EAL domain-containing protein), and two _egcp_ (EAL-GGDEF domain-containing protein) genes. Interestingly, genes encoding the HD-GYP domain-containing protein have not yet been annotated. Exploratory studies by [PERSON] and colleagues demonstrated that two PDEs, EGcpB and EcpC, positively regulate the T3 SS gene expression, swimming motility, and the production of spectate lyase (Pel), one of the major plant cell wall degrading enzymes [37; 49], whereas negatively regulate biofilm formation, suggesting a pleiotropic effect of c-di-GMP in controlling _D. adandtii_ pathogenesis [50; 51]. These observations are also in agreement with reports from other bacteria, suggesting that bacterial second messenger c-di-GMP plays an essential role in promoting the transition from motile to sessile lifestyles [39; 52]. [PERSON] and colleagues further demonstrated that deletions of _egcpB_ or _ecpC_ cause elevated intracellular c-di-GMP levels, which in turn post-transcriptionally repress the expression of _rpoN_ (Figure 1) [50]. Why _D. adandtii_ utilizes two functionally redundant enzymes to modulate the same biological target remains unknown. To elucidate the function of DGCs in _D. adandtii_, [PERSON] and [PERSON] et al.