**2. Citrus canker and DSF quorum sensing pathway in** *X. citri* **subsp.** *citri*

Bacterial citrus canker (BCC) is one of the major citrus diseases, almost all varieties of citrus crops are affected, and the severity of disease depends on bacterial species and weather conditions [7]. The etiological agent of BCC is the Gram-negative bacterium *X. citri* subsp. *citri* [8]. Nowadays, three types of BCC are recognized, which are: (i) citrus canker type A, also known as Asian citrus canker, is the most widespread disease. The BCC has a pronounced host range producing symptoms in: *Chrysopelea paradisi*, *C. aurantifolia*, *C. sinensis*, and *C. reticulata* (ii) Citrus canker type B is caused by the bacterium *Xanthomonas fuscans* subsp. *aurantifolii* type B (*XauB*) [9]. The symptomatology development is similar to citrus canker type A; however, because of the *XauB* slower growth rate, the symptoms spent more time to appear. Host range is limited to *C. limon*; however, *XauB* was rarely isolated from *C. sinensis* and *C. paradisi* [10]. Citrus canker type C is produced by *X. fuscans* subsp. *aurantifolii* type C (*XauC*). The symptomatology is similar to citrus canker type A; nevertheless, its host range is restricted to *C. aurantifolia* [9].

Pathognomonic symptoms of BCC type A are the raised corky and spongy lessons surrounded by a water-soaked margin, which are present in leaves and fruits. This lesion results from the hypertrophy and hyperplasia of mesophilic cells. This cell division disorder is induced by the bacterial effector from family AvrBs3/PthA [11].

*Quorum Quenching Bacteria: An Approach for Phytopathogens Control in Citrus Cultivars DOI: http://dx.doi.org/10.5772/intechopen.107902*

The bacterium *Xcc* is outfitted with a vast arsenal of organelles responsible for the pathogenic traits in citrus host. The main known are: bacterial attachment, antagonism, effector production, quorum sensing regulation, and biofilm formation. For an in-depth review, please refer to Caicedo and Villamizar [12].

#### **2.1 DSF quorum sensing pathway in Xcc**

*Xcc* bacteria have a quorum sensing system whose auto-inducer molecule (AI) is a short-chain fatty acid belonging to DSF (diffusible signal factor) family. The DSF auto-inducer family modulates the expression of virulence and pathogenicity in several pathogenic bacteria to plants and humans [13]. The DSF molecules display a *cys* unsaturated double bond at position two as a distinguished feature in the family. The DSF family are cis-2-unsaturated fatty acids (**Figure 1**), the cis-11-methyl-2-dodecenoic acid was the auto-inducer molecule characterized to be responsible for the signaling processes in *Xcc* [15].

The discovery of the DSF signaling molecule came within a genomic study that seeks to identify a gene cluster termed *rpf* (regulation of pathogenicity factor) *rpfb*- in the bacterium *Xanthomonas campestris* pv. *campestris*. Researchers found that mutation of components of this gene cluster drives the decrease in extracellular enzyme production and exopolysaccharide as well as reduction in pathogenicity in plant susceptible [16]. Later works established the participation of these genes in the coding of elements belonging to quorum sensing communication system involved in the synthesis and perception of the DSF signal molecule [17]. The gen *rpf*F encodes an enzyme, amino acids sequence of which is related to enoyl CoA hydratase; this enzyme is responsible for DSF synthesis. The gen *rpf*B encodes and Acyl CoA ligase, which participates to a lesser extent in the synthesis of DSF auto-inducer [16].

DSF perception and signal transduction are encoded by an *rpfGHC* gene operon. *rpfC* encodes the receptor RpfC protein. This protein has a transmembrane domain, which is involved in the perception of DSF auto-inducer and the cytoplasmic domains: His-Kinase A (phosphoacceptor), His-Kinase-like ATPase, REC domain (receiver

#### **Figure 1.**

*(Left) Structure of the main molecules from DSF auto-inducers family and some related putative auto-inducers. (Right) Related molecules with signaling activity. Notice that these molecules lack the cys unsaturated double bond at position two. (Adapted from Ryan and Dow [14]).*

#### **Figure 2.**

*At low cell population condition, RpfF the DSF synthase remains bound to REC domain from RpfC sensor, which maintains compact conformation. At high cell population condition, the DSF binds to RpfC sensor domain and induces a conformational change releasing the synthase RpfF, thus triggering an autophosphorylation and phosphorelay and the subsequent phosphotransfer to the REC domain of response regulator RpfG. The phosphorylation of RpfG, it triggers the activation of RpfG as a cyclic di-GMP phosphodiesterase reducing the level of cyclic di-GMP and releasing Clp that promotes the synthesis of extracellular enzymes and EPS (take it from Caicedo et al. [18]).*

domain), and finally, the HTP domain histidine phosphotransfer (**Figure 2**). The gene *rpfG* encodes for response regulator protein, this protein include two domains: (i) an REC domain, which receives the phosphate of HTP domain from the RpfC; and (ii) the HD- GYP, which displays phosphodiesterase activity responsible for degradation of second messenger cyclic di-GMP. At physiological levels of cyclic di-GMP, the transcriptional activator cAMP-receptor-like-protein Clp remains bound to second messenger cyclic di-GMP. Consequently, reduction of cyclic di-GMP via DSF in *Xcc* leads to the release of Clp, thus allowing Clp to activate the expression of several gene and proteins involved in virulence such as: extracellular enzyme production, EPS production, biofilm formation, motility, iron uptake [19]. Furthermore, DSF signaling also positively controls the expression of *clp* gene, which suggests a supplementary regulatory complexity [14]. Summarizing, the perception of auto-inducer molecule DSF in *Xcc* by the receptor RpfC triggers the expression of its virulome. For all aforementioned, the disruption of this quorum sensing communication system could turn into a valuable tool for reducing citrus canker severity.
