**3.2 The pathogenic mechanism of** *Candidatus* **Liberibacter**

*Candidatus* Liberibacter species are gram-negative, phloem-restricted bacterium associated with the pernicious disease of citrus HLB. Although *Candidatus* Liberibacters have been cultivated in artificial media, traditional molecular and genetic analyses have been difficult to perform owing to declining viability in culture [46, 48]. This difficulty has significantly limited efforts to comprehend the mechanisms of Liberibacter virulence. To date, most insights into the mechanisms of Liberibacter pathogenesis have been acquired through genomic analyses of Liberibacter sequences, host plant transcriptomic, proteomic, and metabolomic data associated with Liberibacter infection, and studies involving surrogates such as *Sinorhizobium* and *E. coli*, and expression in planta [15]. Evidence suggests that Liberibacters species associated with HLB live solely within the phloem tissues of host citrus plants [15]. Las bacterium resides inside a sieve tube and companion cells [47, 49]. The relatively consistent symptomology among various symptom-expressing hosts is one of the hallmarks of diseases caused by Liberibacter species [15].

### *3.2.1 Liberibacter secretion system and effector protein*

The secretome of a pathogenic bacterium represents an array of molecules that play offensive roles during colonization, among which effectors are an important class of proteins capable of suppressing defense and/or manipulating host physiology [50, 51]. Interestingly, Las contain type I secretion systems (T1SSs) and a complete general secretory pathway (Sec), but lack other secretion systems (T2SS and T3SS) [15, 52], which play a significant role in extracellular pathogenic attacks on plant and animal host [16].

Liberibacter genome analyses found a complete T1SSs system in Las and Laf, but not in Lam [15]. Genes encode for serralysin and hemolysin; a T1SSs effector protein has been identified in Las and Laf genomes [53]. Serralysin is a metalloprotease secreted by gram-negative bacteria to inactivate peptides and antimicrobial proteins produced by the host plant. Las bacterium might use serralysin to degrade antimicrobial proteins in the host as its defense mechanism. This degraded protein is used for growth and metabolism by the Las bacterium as a carbon and nitrogen nutrient [16]. On the other hand, the hemolysin gene has been identified in all sequenced Liberibacters, which play an essential role in bacteria survival in the host plant. Lasproduced hemolysin triggers ion leakage and water molecules from the host cell that lead to host cell apoptosis [16, 54].

The Secretary pathway (Sec) or Sec-translocon facilitates these effector proteins' transports outside the cytoplasm membrane vital for bacterial viability. The Sec machinery also secretes essential virulence factors in some plant-pathogenic bacteria [15]. *Candidatus* Liberibacter species have a general secretory pathway, which may lead to the secretion of effector proteins [55]. Since *Candidatus* Liberibacter species are phloem-resided bacteria, there is an inference that the bacteria secrete effector proteins directly into the cytoplasm of the host cells and modulate their physiology [56]. The effector protein CLIBASIA\_05315 was located in transgenic citrus chloroplasts, resulting in leaf chlorosis and plant growth retardation [57]. Several research

*Devious Phloem Intruder* Candidatus *Liberibacter Species Causing Huanglongbing: History… DOI: http://dx.doi.org/10.5772/intechopen.105089*

groups are currently focusing on identifying and characterizing the effector proteins of *Candidatus* Liberibacter species, and it is expected that we will have an improved view of this pathogenic mechanism of bacteria in a few years.

#### *3.2.2 Lipopolysaccharides*

Lipopolysaccharides (LPS), also known as endotoxin, are critical components derived from the outer membranes of gram-negative bacteria consisting of lipid A, an oligosaccharide core, and an O-antigen. LPSs are involved in outer membrane functions that are crucial for bacterial growth, survival against antimicrobial chemicals, and virulence, particularly within a host-parasite interaction. Lipid A is highly conserved, then the oligosaccharide core and O-antigen [15, 16]. LPSs are classical activators of defense responses in plants during plant-pathogen interaction [58]. Las bacteria use gene encoding active salicylate hydroxylase (SahA) to degrade salicylic acid (SA) and suppress plant defense mechanism. Intriguingly, the *SahA* gene is highly expressed *in planta*, while it is not expressed in psyllid vectors [56]. Las impedes SA-mediated defense responses in the phloem using its SA hydroxylase and maintains significant bacterial titer in citrus HLB disease progression over several years before the tree irrevocably declines. It is yet to be determined whether LPSs of Liberibacter cause callose accumulation in the phloem.

#### *3.2.3 Flagella*

The bacterial flagellum organelle, an intricate multiprotein essential for its rotational propulsion, promotes host colonization through adherence and induces plant immune modulation [15]. Las flagella have been reported to trigger host plant defense *in planta* as a pathogen-associated molecular pattern (PAMP) [59]. Microscopic studies found that flagella have not been observed in the *Candidatus* Liberibacter species that reside in the phloem in HLB-infected samples [11]. Despite the small size of the genome, genes associated with flagella biosynthesis have been identified in the sequenced Liberibacter genome [15, 16, 51, 52]. The genes *fliF*, *flgI*, and *flgD* expressed in flagellar assembly and the *motB* gene associated with the motor function were overexpressed *in planta*.

The *flbT*, an essential flagellin regulatory protein that acts as a regulatory checkpoint for flagellin gene expression, is found in the Las bacterial genome, whereas it is not in the Lam genome. The absence of *flbT* in the Lam genome results in no PAMP activation *in planta* [60]. Conversely, *flgL*, *flgK*, and *fliE* were overexpressed in psyllid [61].

#### *3.2.4 Prophages*

Several pathogenic bacteria harbor prophages or phage remnants integrated into their genome, encoding lysogenic genes that are proven or suspected virulence factors [59]. Las- and Lam-sequenced genome contains two potential prophages, Type 1 represents prophage SC1, and Type 2 represents prophage SC2. SC1 involved in the lytic cycle of forming phage particles. SC2 was implicated in the lysogenic conversion of Las pathogenesis [60, 62]. Type 3 prophage (P-JXGC-3) was identified in Las samples collected from Southern China. This prophage carries another bacterial defense system, such as a restriction-modification system (RM system) [63]. This RM system is fortified with endonucleases, which cleaves invading DNA that protects

host DNA by altering specific sequences [64]. Type 1 and Type 2 prophages were not detected in the Las strain from Southern China. It is not clear whether these strains contain prophages or have unknown prophages. There are no comprehensive studies to describe the Las prophage repertoire [65]. Among strains observed in an extensive survey of Las isolates in China, it was typical for Las to have a single prophage, with Guangdong isolates harboring mainly the type 2 prophage, whereas isolates from Yunnan are dominated by the type 1 prophage [65]. The Las strain genome from Japan does not contain prophages [56]. Among the Las whole-genome sequences recently reported from different geographic areas around the globe, eight Las genomes contain extensive prophage sequences [63]. A survey of prophage prevalence in southern China revealed active prophage-phage interactions in the Las bacterial strains [63]. The exact function of the RM system has yet to be experimentally determined in Type 3 prophages. However, the lack of a prophage in many Las strains does not relate to the lack of HLB symptoms because Ishi-1 and the Guangdong isolates, which do not contain any prophages, induce similar HLB symptoms as isolates containing prophages [54, 65]. Overall, this evidence suggests that prophages contribute to bacterial virulence but are not required for Las pathogenicity.

### **3.3 Phloem dysfunction of HLB-affected citrus**

Las bacteria reside within phloem and colonize sieve tubes [15, 16, 66]. Phloem dysfunction is a primary modification due to hyperactive differentiation of vascular cambium and hypertrophy of parenchyma cells surrounding the necrotic phloem pocket that may determine the development of HLB symptoms [32, 67]. HLBassociated Liberibacter secretes virulence factor and Sec-dependent effectors (SDEs) into phloem that stimulates HLB symptoms by interfering with either phloem or companion cell protein and genes of the host [15]. The secreted SDEs and virulence factors may interact with plastids, mitochondria, vacuole, and endoplasmic reticulum in the host phloem and target host genes and proteins to promote pathogen growth and disease development and suppress host immune responses [15]. Eventually, it leads to phloem malfunction in the host plant due to the Liberibacter virulence factors and SDE effects on sieve tubes and companion cells, which provide protein and transcripts to the sieve elements. Necrotic phloem was found in the HLB-infected plants due to starch (**Figure 1**) and callose deposition [32]. Callose accumulation was observed in sieve plates of Las-infected citrus [67]. Phloem dysfunction is generally associated with phloem sieve elements plugged with extensive deposition of callose and phloem protein 2 [67, 68], followed by phloem cell wall distortion and sieve element collapse [69]. Subsequently, photoassimilate transport was significantly blocked due to necrotic phloem [15, 16, 66, 68], which might result in substantial quantities of starch particles in almost all living cells of aerial parts, including phloem parenchyma and the sieve tube elements [32, 70]. The excessive accumulation of starch and zinc deficiency in chloroplast disrupts the thylakoid resulting in nonuniform loss of chlorophyll that triggers noticeable blotchy mottle appearance in the HLB-infected leaves [40, 70, 71]. The anatomical transverse section of HLB-infected leaf midrib exhibited phloem collapse with cell wall distortion and thickening in Valencia sweet orange and SB siblings [72]. In addition, hyperactive vascular cambium regenerates new phloem in the HLB-infected trees, consisting of assemblies of sieve elements, companion cells, and phloem parenchyma cells, but lacks phloemic fibers [72].

In addition to anatomical changes, several metabolic imbalances and genetic reprogramming are noticed in HLB-affected plants [57, 66]. Salicylic acid and

*Devious Phloem Intruder* Candidatus *Liberibacter Species Causing Huanglongbing: History… DOI: http://dx.doi.org/10.5772/intechopen.105089*

**Figure 1.** *SEM micrographs of transverse section of healthy and HLB-infected citrus petiole. A and B. Healthy plant; C and D. HLB-infected plants.*

downstream signaling play a key role in provoking plant defense mechanisms against biotrophic pathogens [73, 74]. Wang and Trivedi postulated that a protein with potential salicylate hydroxylase activity might convert salicylic acid into catechol [75]. Salicylic acid pathway depression was observed in HLB-susceptible citrus plants [76]. Based on the *Candidatus* Liberibacter and plant interactions mechanism literature, we suggest the pathogenic mechanism of *Candidatus* Liberibacter species associated with citrus HLB in the following model (**Figure 2**).
