1Centro de Citricultura Sylvio Moreira, Instituto Agronômico de Campinas, Caixa Postal 04, CEP 13490-970, Cordeirópolis - SP, Brazil
2Dept. de Produção Vegetal, FCA/Unesp/Botucatu, Brazil
Abstract
The complete sequencing of the Xylella fastidiosa genome was the first project of this kind to focus on a plant pathogen. The choice of this bacteria was tightly associated with its importance as the causal agent of citrus variegated chlorosis (CVC). Adopting a sequencing strategy based on shotgun and cosmids, the project allowed a 10 fold coverage of the genome. The specific mechanisms of pathogenicity are not yet clear while the annotation of the genome took in consideration several hypothesis. Based on the current pathogenicity hypothesis, the genes were sorted into different categories with special focus on those associated with attachment to bacterial cells and different hosts, as well as genes related to the adaptation capacity to the xylem conditions. Genes previously found only in animal pathogens were also observed in X. fastidiosa, suggesting that they could share some of the pathogenicity mechanisms.
Introduction
Xylella fastidiosa was the first plant pathogen to have its full genome sequenced (Simpson et al., 2000). In a cooperative effort of 32 laboratories in the ONSA (Organization for Nucleotide and Sequence Analysis) network supported by FAPESP this important pathogen for citrus, grape, plum and other species was chosen to be a model for genomic studies in Brazil. After the sequencing of 23 complete bacterial genomes, this project pointed out the need for acceleration of such studies in plant pathology.
Known for its prolific growth and broad range of hosts, X. fastidiosa became a serious problem in Brazil when it was definitively associated with citrus variegated chlorosis (CVC), one the most destructive citrus diseases in the world (Rossetti et al., 1990). As an agent of other important diseases, such as Pierce's disease specific to grape, this bacterium has been a difficult to control due to its restricted growth in the xylem vessels, its ability to survive within tissues used for vegetative propagation, and the efficient vector for dispersion plant to plant in the orchard.
This review will point out the most relevant aspects associated with its pathogenicity. Although specific mechanisms of pathogenicity are not yet clear, the annotation of the genome took in consideration several hypothesis. These hypothesis could explain why the bacteria are able to attach to surfaces, i.e, its ability to interact with surfaces and to spread within the plant, specifically in the xylem causing disease symptoms. All the genes mentioned in the text are listed in Table 1. Detailed annotation of the complete genome can be found in the Xylella database at NCBI or at University of Campinas (http://onsona.lbi.ic.unicamp.br/xf/). Aspects associated with disease epidemiology, interaction with vectors, hosts and other information can be found in more specific reviews (Purcell and Frazier, 1985; Hopkins, 1989; Hopkins, 1995).
Hypothesis of Pathogenicity
As a xylem-limited bacterium X. fastidiosa has developed survival mechanisms for living in adverse conditions including water stream turbulence at negative pressure, and low availability of nutrients, such as nitrogen and carbon sources. These conditions, associated with the bacteria production of toxins and ability to spread within the plant, appear to be correlated with its pathogenicity (Hopkins, 1995). The bacteria seem to be able to interact with surfaces (xylem wall and other bacterium cells), uptake nutrients efficiently from the xylem sap, and produce compounds (toxins or enzymes) capable of active interaction with the plant tissues. The most accepted hypotheses of pathogenicity are based on the following mechanisms:
Occlusion of the Xylem Vessel
Scanning electron images show bacteria embedded within an amorphous extra-cellular matrix, probably consisting of extracellular polysaccharides (EPS) attached to the xylem wall or the cibarium of the vector (Brlansky et al., 1983; Chagas et al., 1992). The development of water stress in affected plants, specifically citrus trees, suggests the occurrence of xylem blockage (Hopkins, 1989, 1995; Goodwin et al., 1988; Machado et al., 1994). EPS, fimbriae, and adhesins are involved in the adhesion of bacteria-bacteria, bacteria-plant. These are essential in building up aggregates that cause occlusion in the xylem (Hopkins, 1995). The aggregates of colonies within the xylem not only promote obstructions, but also allow the colonization of the xylem up and down stream. In addition, the occlusions prohibit the free movement of nutrients in the sap and function as a filter, concentrating available nutrients for the bacteria.
Nutritional Competition
The bacterium lives in the xylem vessel obtaining all nutritional requirements from the sap, whose composition changes according to the age of the plant and the environmental conditions. This causes a competition for nutrients between the bacteria and the plant that can give rise to nutritional disturbances in the leaves, branches and fruit. It should be pointed out that the typical symptoms of iron, copper, magnesium and manganese deficiency in citrus trees are similar to those caused by X. fastidiosa. The bacterium efficiently uptakes carbon and nitrogen compounds and inorganic compounds essential for its metabolism from the xylem sap. Organic acids, aminoacids, amines, ammonium, nitrate, sulphate, cations (Mg, Ca), minor elements (Fe, B, Zn, Mn, etc), and plant regulators are the most prevalent compounds in the xylem sap.
Intervessel Migration
Although several species of sharpshooter leafhoper vectors (Cicadelideae) seem to transmit the bacteria efficiently, not all the vessels are uniformly infected. On the other hand, the bacteria seem to have the capacity to migrate laterally and in certain cases, such as Pierce´s disease and plum leaf scald, the uniformity required for pathogenicity may be explained by intervessel migration. Both multiplication / colonization and migration within the vessel have been related to pathogenicity (Hopkins, 1985). The production of exo-enzymes such as cellulases and pectinases, allows colonization of lateral vessels after degradation of pit membranes and may also represent an additional way of obtaining small carbohydrates molecules.
Production of Toxins
Symptoms resembling disturbances caused by exogenous toxins or disruption in plant regulators are visible in the leaves and fruits of several species infected with X. fastidiosa. EPS, such as xanthan gum and toxins such as bacteriocins and RTX are produced by several Gram negative bacteria. These may disrupt cells and organelles inducing the development of symptoms such as chlorosis, or affecting growth and development of the tree. It is reasonable to suppose that such toxins especially those from bacteria living within xylem vessel could affect tissues and organs above the colonization area.
Interactions with Surfaces
Adhesion and persistence of the pathogen, in spite of host defense factors, could be the key to pathogenicity during development of the disease (Costerton et al., 1981). Extracellular polysaccharides (EPSs) and adhesins (pili and non-pili formation) in plant bacteria have been related to their survival, colonization and adhesion to host tissues.
Besides electrostatic forces generated by the charged surfaces of the bacteria and the plant cell wall, the habitats of X. fastidiosa (the xylem vessel and the foregut system of the vectors) are characterized by high turbulence and low nutrient concentration. Thus, the colonization of such specific habitats is probably mediated by an efficient mechanism of attachment to other bacteria and to the surfaces of the hosts. According to Hopkins (1989; 1995), extracellular strands such as fibrous microfibrils, fimbriae, and EPS, are considered to be the major component of the dense matrix of X. fastidiosa within the vessels (Brlansky et al., 1983). This matrix was also verified in xylem vessels of citrus trees with CVC symptoms (Chagas et al., 1992).
EPS
Extracellular polysaccharides are normally produced by species of Erwinia, Pseudomonas and Xanthomonas (Denny, 1995), causing plant diseases such as wilt by blocking xylem vessels. This polymer is also required for colonization and enhances the pathogen survival within host tissues by acting as a protection mechanism against environmental stress (Király et al., 1997).
Gum gene cluster similar to the xantham gum of Xanthomonas campestris pv campestris (Xcc) (Becker et al., 1998) was identified in the X. fastidiosa genome, but lacking the genes gumI, gumL and gumG (Figure 1). The absence of gumI (glycosyltransferase V) should not allow the incorporation of a terminal mannose to the polymer. Consequently, the addition of pyruvate by the ketal-pyruvate transferase enzyme (gumL) and acetyl group (gumG - acetyl transferase II enzyme) should also not occur. Mutations in gumI produce a less viscous polytetrameric gum (Leigh and Coplin, 1992).
Pili and Non-pili Structures
Bacterial adhesion to host tissues is usually mediated by adhesins occurring on the cell surface. These adhesins can either be assembled in structures like pili or fimbriae, or directly associated with the microbial cell surface, so called non-pili adhesins (Soto and Hultgreen, 1999). The genes responsible for the assembly of such structures and their importance in human and animal pathosystems are well-characterized (Soto and Hultgreen, 1999). In contrast, the importance of fimbrial structures to the plant pathogenic process is not yet well determined (Romantschuk, 1992). The functions of type-4 pili structures that are more related to pathogenicity are adhesion to host cell surface, adhesion bacteria-bacteria and twitching motility, a form of surface translocation of bacteria (Hahn, 1997; Kirov et al., 1999).
X. fastidiosa contains ORFs encoding pili and non-pili adhesins. Out of more than 30 genes involved in the type-4 fimbriae synthesis, 26 were detected in its genome (Table 1). Among them are pilE, which encodes the pilin-like protein, and the pilS and pilR genes that control the transcription of fimbrial subunits (Hobbs et al., 1993). Moreover, genes that act in response to some environmental signal regulating pilus biosynthesis and movement of bacteria (pilG, H, I, and J) (Alm and Mattick, 1997) are also present.
Alginate, an exopolysaccharide that causes cells to become mucoid, is another determinant virulence factor in P. aeruginosa (Schurr et al., 1994), and P. seringae pv seringae (Yu et al., 1999). The alginate gene algR encodes a non-pili adhesin similar to both the hsf gene product of H. influenzae (St Geme, 1996), and to the uspA1 gene product of Moraxella catarrhalis (Cope et al., 1999). Homologues to these genes are present in X. fastidiosa and encode adhesins, non-pili structures that are directly associated with the bacterial cell surface. Filamentous hemagglutinins, structures related to bacterial adhesion, were identified in the X. fastidiosa genome. Three genes (pspA) with homology to hemagglutinin-like structures from Neisseria meningitidis were observed. This was the first time that a hemagglutinin was identified in a phytopathogenic bacteria.
Plant Cell Wall Degradation
Like many plant pathogens, X. fastidiosa seems to produce an array of enzymes capable of hydrolyzing components of plant cell wall. The importance of such enzymes in pathogenicity has been reported (Hopkins 1985, 1995). The extracellular enzymes are not only important for nutrition but they also play an essential role in lateral vessel to vessel movement since they are responsible for degradation of pit membranes. The ability of the bacteria to move through the vessels has been pointed out as a key element in pathogenicity. Some avirulent strains of Pierce´s Disease (PD) grow well in culture, but are not able to move through the xylem (Hopkins, 1985). In the genome of X. fastidiosa several genes coding for cell wall degradation enzymes were identified.
Cellulases
Microbial cellulose degradation usually involves enzymes of two major types: ENGs (endo-1,4-b-glucanases) and EXGs (cellobiohydrolases). ENGs cleave internal b-1,4-glucosidic bonds, whereas EXGs cut the dissaccharide cellobiose from the non-reducing end of the cellulose polymer chain. These enzymes show different types of synergy when hydrolyzing crystalline cellulose (Henrissat et al., 1985; Meinke et al., 1994). X. fastidiosa has three ORFs similar to ENGs and one to EXG.
Endo-1,4-b-Glucanase
Two ORFs with strong similarity to genes encoding extracellular endoglucanases (engXCA) of Xcc were found in X. fastidiosa. However, the X. fastidiosa protein shows a Glu/Ser rich region instead of Thr/Pro (Knowles et al., 1987), which could be a potential site for proteolysis. A third ORF encoding ENG is similar to egl of Ralstonia solanacearum. The egl mutant produced at least 200-fold less endoglucanase than the wild-type strain suggesting that other glucanases are produced at very low levels. This EGL-deficient strain was significantly less virulent than the wild-type strain on tomato plants (Roberts et al., 1988).
Cellobiohydrolases
A similar ORF of exocellobiohydrolase A (cbhA) of Cellulomonas fimi was found. The structure and activity of the cbhA catalytic domain are closely related to those of CBH II, an exocellobiohydrolase from the fungus Thichoderma reesei a key enzyme in the hydrolysis of crystaline cellulose. CbhA was the first enzyme of this kind to be characterized in bacteria (Meinke et al., 1994; Wood and Garcia-Campayo, 1990).
Pectinases
A wide range of fungal and bacterial plant pathogens produce pectolytic enzymes (pectate lyase, pectin lyase, and exopolygalacturonate lyase) that cleave by b-elimination. On the other hand, polygalacturonase and exo-poly-a-D-galacturonosidase cleave by hydrolysis (Collmer and Kenn, 1986). In the genome of X. fastidiosa, an ORF similar to the polygalacturonase genes of different organisms was found. The greatest similarity was found with the gene pglA (polygalacturonase) of R. solaneacearum (Huang and Shell, 1990). However, in this ORF of X. fastidiosa there is a frameshift suggesting it is nonfunctional.
Glycoside Hydrolase
An ORF showing partial homology with the xylA gene of Ruminococcus flavefaciens is also present in the X. fastidiosa genome. It codes for the family 3 glycosyl hydrolase (Henrissat et al., 1989). BLASTp searches using this protein resulted in partial hits with several b-xylosidases and b-glucosidases from different organisms including bacteria and fungi (La Grange et al., 1997; Goyal and Eveleigh, 1996).
Regulator of EPS and Extracellular Enzymes
The synthesis of extracellular enzymes and EPS in Xcc is subject to co-ordinated regulation by a cluster of genes called rpf (regulator of pathogenicity factors) (Dow et al., 2000). The biosynthetic regulation of extracellular enzymes and EPS in X. fastidiosa may occur in a way similar to Xcc, since several of these genes were detected. ORFs similar to rpfA and rpfB are found in close proximity to one another in the X. fastidiosa genome, but they are not arranged in cluster as in the Xcc genome. The rpfA gene codes for a bifuncional protein which acts as an aconitase at high iron levels and at low iron concentration works as a regulatory protein (Wilson et al., 1998). The rpfB and rpfF genes are required for a small molecule-mediated pathway, these genes represent a mechanism for regulating virulence factor synthesis in response to physiological or environmental changes (Barber et al., 1997). An ORF similar to rpfF is present in X. fastidiosa, and as in Xcc, it is adjacent to rpfC and other genes present in the cluster (Figure 2). The rpfC, rpfG and rpfH genes encode components of a "two-component" phosphorelay system (Barber et al., 1997) which is involved in regulating gene expression in response to environmental stimuli (Tang et al., 1991). The rpfG gene was detected in the X. fastidiosa genome downstream of rpfC but there is no ORF similar to rpfH. More recently eight other genes belonging to the rpf cluster of Xcc were identified (Dow et al., 2000). Two of these genes, recJ and greA, have functions in recombination and transcriptional elongation, respectively. Mutation in rpfD had minor effects on the production of extracellular enzymes and EPS. Mutation of rpfE leads to a reduction in the levels of endoglucanase, protease, and EPS, but there is an increase in polygalacturonate lyase. Mutation in orf4 had no effect on polygalacturonate lyase, but reduced the levels of protease and endoglucanase. However, orf1, 2, and 3 mutations did not affect the synthesis of extracellular enzymes or EPS. In the genome of X. fastidiosa, ORFs similar to rpfD, orf1, 2, 3, and 4 were not found. Nevertheless, ORFs highly similar to recJ, rpfE, and greA were detected in cluster. The absence of some genes may be related to absence of some extracellular enzymes or components of the EPS biosynthesis machinery of Xylella.
Biosynthesis of Toxins and Antibiotics
Although the production of toxins and/or antibiotics can not always be associated with pathogenicity, their presence increases virulence and severity of symptoms. Toxins can be important in the development of plant diseases, mainly for the establishment of the pathogen within the plant and development of symptoms, like chlorosis and necrosis.
Hemolysin
Hemolysin, a toxic protein produced and secreted by E. coli is among the cytolytic, structurally homologous proteic bacterial toxins known as RTX (repeats in toxin) toxins (Trent et al., 1998). RTX cytolysins are a family of calcium-dependent, pore-forming, secreted toxins found in a variety of gram-negative bacteria encoded in the gene cluster hlyCABD. X. fastidiosa may also synthesize hemolysin-like toxins that can act against other endophytic microorganisms in the xylem. In its genome there is one ORF homologous to the gene that codes for the hemolysin III protein of Bacillus cereus. Three other ORFs show homology to different hemolysin-type calcium binding proteins (128.4 kDa -25 gly-rich repeats, 138.9 kDa and 173.0 kDa -43 Gly-rich repeats) of the Neisseria meningitidis iron-regulated protein FRPC (also known as cytotoxin RTX homolog FrpC). Xylella also has homologous ORFs to hlyC, hlyD and hlyA of E. coli. However the homologue of hlyB, the gene responsible for the toxin secretion was not found suggesting that hlyD may have this function. As in E. coli, only the ORFs homologous to hlyC and hlyD are in a cluster.
Bacteriocin
X. fastidiosa also synthesizes a bacteriocin-like protein. In general, bacteriocins are ribosomally synthesized antimicrobial polypeptides (Schripsema et al., 1996), whose production involves several genes (Nes et al., 1996). The bacteriocin found in X. fastidiosa is similar to the one found in Rizobium leguminosarum, a RTX protein similar to hemolysin and leukotoxin (Oresnik et al., 1999).
Two copies of cvaC encoding a colicin-like precursor similar to the E. coli colicin V precursor were found in a cluster. Colicin V belongs to a family of small peptide bacteriocins produced by E.coli and other closely related bacteria (Havarstein et al., 1994). The secretion of colicin V in E. coli requires the products of two linked genes cvaA (a secretion protein) and cvaB (for peptide tranport) both have homologous in X. fastidiosa. X. fastidiosa also has a similar ORF to tolC encoding for an outer membrane protein, which is probably involved in the transport of a hemolysin-like protein that is similar to the ABC-transporter dependent secretion of E. coli.
Polyketides constitute a huge family of structurally diverse natural products including those with antibiotic and antiparasitic activities. The biosynthesis of polyketides is similar to the assembly of fatty acids but unlike a fatty acid, a polyketide synthase (PKS) can make additional choices in the starting and extending groups. X. fastidiosa appears to have a polyketide synthase, a key enzyme involved in the production of antibiotics. Two ORFs were found similar to peptide synthase which is involved in the biosynthesis of peptide antibiotics.
Adaptation to the Strees Conditions
Proteolytic Enzymes
Several ORFs related to protein degradation were found in the X. fastidiosa genome. However, the analysis of the sequences does not give a hint as to whether or not the proteases are involved in pathogenesis. There is only one ORF, htrA (high temperature resistance) whose product is highly similar to proteins found in pathogenic organisms. htrA is a periplasmic serine protease essential for cell survival at high temperatures (Lipinska et al. 1989, 1990). A proposed function for this protein is to remove denatured protein that could be toxic for the cells (Lipinska et al., 1990). Skórko-Glonek et al. (1999) showed this protein importance for defense against oxidative stress. It should be pointed out that one the most efficient mechanisms of defense in plant is the production of reactive oxygen species (ROS) (Wojtaszek, 1997; Dat et al., 2000). htrA is also a protein induced by ferrous sulfate and cumene hydroperoxide. X. fastidiosa has two genes (htrA and mucD (mucilage) that belong to this family which is not an uncommon feature since some other organisms like E. coli, P. aeruginosa, and B. abortus have at least two of such elements in their genomes. Interestingly, algW (another homologue of htrA) and mucD were shown to affect the expression of the mucoid phenotype in P. aeruginosa (Boucher et al., 1996). Alginate is responsible for this phenotype and has been related to persistence of the pathogen in patients. It acts leading to resistance to free radical released by macrophages (Simpson et al., 1989). These genes may be important factors for adapting to such an environment.
Oxidative Stress
The xylem-limited bacteria X. fastidiosa can be exposed to active oxygen produced both by the plant and by other endophytic microorganisms living in the xylem vessels. To overcome detoxification and prevent damage caused by reactive oxygen the bacteria might require an efficient protection mechanism. Enzymes involved in the detoxification or protection of oxidative stress include superoxide dismutases, catalases, enzymes involved in DNA and protein repair, and transcription factors involved in the transduction and regulation of genes (Loprasert et al., 1996; Farr and Kogoma, 1991; Bauer et al., 1999). Exposure to peroxide stimulus activates OxyR which drives the expression of several genes including heat-shock proteins, catalase, alkyl hydroperoxidase reductase, and glutathione reductase a protein associated with H2O2 cellular resistance. The signal for induction of the SoxR regulon is not known but the most regulated genes are sodA (superoxide dismutase), nfo (Endonuclease IV) and zwf (glicose-6-P dehydrogenase) all of them with roles in the antioxidant defense. The SoxR system can also affect the expression of porins in the outer membrane (ompF) these are associated with multiple antibiotic resistance.
Genes of the oxyR (H2O2) and soxR (superoxide response) regulons are present in the genome of X. fastidiosa these are involved directly in cell detoxification and adaptation to adverse conditions. Genes were found that code for catalase, superoxide dismutase, alkyl hydroperoxide reductase, glutathione reductase, and an organic hydroperoxide resistance protein which is similar to several other bacteria. Genes coding for enzymes involved in the repair of DNA, membrane and proteins against damage caused by oxygen radicals were also detected. OxyR, a transcriptional regulator factor that controls the expression of H2O2 inducible proteins was found with greater similarity to Xcc than E. coli or S. typhimurium (Farr and Kogoma, 1991). soxR, as known in E. coli and S. typhimurium was not found.
Osmotic Stress
Within the xylem X. fastidiosa survives in a highly diluted solution which includes organic (aminoacids, amines, plant regulators, organic acids, etc.) and inorganic compounds (macro and micronutrients) uptaken by roots. Since the composition and flux in the xylem are strictly associated to environmental factors (water potential in the system air-leaves-roots, composition of the soil solution) and uptake capacity of the roots, representative changes occur in the sap composition during the day. The bacteria should have osmosensing adaptive mechanisms to respond both passively and actively to these changes. As pointed out by Wood (1999), more than specific sensors, osmoregulars are devices that implement the response of an organism to a changing environmental osmolarity.
Among genes related to osmoregulars (Wood, 1999) the X. fastidiosa genome contains mdoH, mdoG (membrane derived oligosacharide), porin (oprO), and an outer membrane protein (mopB) all of which well evaluated in several bacteria. Although many genes of several bacteria have been associated with osmoresponse, this adaptation capacity certainly should involve complex processes typical to each microorganism and its habitat. Therefore, more functional studies should be done with X. fastidiosa for a better characterization of such genes.
Iron Metabolism
In the X. fastidiosa genome there are at least 67 ORFs involved in iron metabolism. The mechanism of iron acquisition in plant pathogens seems to be based mainly on the production of siderophores. When there is low internal iron level the cytosolic ferric uptake regulatory protein (Fur) product of the fur gene (Hantke, 1984) acts as an iron responsive transcriptional repressor (Crosa, 1997; Neilands, 1995). At low iron levels in the cell, Fur has lower affinity for the operator of the sequences in the promoters of siderophore biosynthesis or transport genes. As the iron concentration raises more ferrous ion binds to Fur protein enhancing Fur binding to the operator (De Lorenzo et al., 1988; De Lorenzo et al., 1987). Thus, there is a decrease in transcription of Fur-regulated genes and consequently a lower production of siderophores. Less iron is chelated and the rate of iron assimilation decreases.
X. fastidiosa appears to have a Fur similar to X. campestris pv vesicatoria ferric uptake regulator but the production of siderophores in X. fastidiosa is not yet clear. The only ORF related to siderophores synthesis found in X. fastidiosa encodes a protein similar to factors involved in the secretion of pyroverdine, a siderophore of P. aeruginosa. Another mechanism for iron uptake in bacteria is through the TonB, ExbB, ExbD1 system. X. fastidiosa has ORFs homologous to tonB, exbB, exbD1, exbD2 in a cluster that are highly similar to the genes of Xcc (Wiggerich et al., 1997).
Detoxification and Drug Efflux
X. fastidiosa seems to have at least two different mechanisms for drug inactivation: Multidrug Resistance (MDR) and Specific Drug Resistance (SDR), both of which are well known in bacteria and can afford protection against toxic compounds. The bacteria may also have drug efflux mechanisms to release and transport those compounds. The secondary drug transporters comprise the largest group of known drug extrusion systems in bacteria, some of which are involved in multidrug resistance, whereas others mediate efflux with a high specificity. The secondary drug transporters can be subdivided into three groups: the Major Facilitator Superfamily (MFS) of transporters; the Resistance, Nodulation and cell Division (RND) family of membrane proteins; and the family of Small Multidrug Resistance (SMR) transporters. In Xylella fastidiosa there are at least two of these groups, MFS and RND.
Other detoxification proteins were found in the Xylella fastidiosa genome. ORFs encoding proteins specific for toluene and tetracycline resistance, penicillin tolerance and inactivation of b-lactam antibiotics are also present in the genome. This arsenal makes this bacterium a strong competitor in a very harsh environment.
Not Detected Genes
Common sense would predict phytopathogenic bacteria to have a limited range of hosts. In general, the hosts are limited to members of a single species or genus. Avirulence (avr) genes present in the pathogen encode factors which interact with matching resistance (R) proteins in the host (for recent reviews see Bonas and Van den Ackerveken, 1999 and Martin, 1999). Bacterial avirulence genes that have been characterized fall into two groups: those that resemble avrBs3 of Xanthomonas campestris pv. vesicatoria, and those that resemble avr gene of Pseudomonas syringae, together with some gene of X. campestris (other pathovars) and Pseudomonas solanacearum. This subdivision involves differences in both structural and functional characteristics (Vivian and Gibbon, 1997). There are growing evidences that Avr proteins have a primary function in virulence, even though the HR (hypersensitive)-triggering effects of the Avr-R interactions are epistatic over these virulence functions. It is still unknown how Avr proteins promote parasitism but support for such a primary role comes from observations that their place of action is within host cells (Alfano and Collmer, 1997). The Type III secretion system is responsible for delivering these proteins to the interior of the host cells. Its components are encoded by genes called hrp (hypersensitive response and pathogenicity) or hrc (hrp conserved) that are present both in plant and animal pathogens. In the Xylella fastidiosa genome no homology with known avr, hrp or hrc was found. The absence of such genes suggests that the pathogenicity of Xylella fastidiosa is not related to the presence of Avr proteins and therefore, it does not depend on a type III secretion machinery for the transport of such proteins. As mentioned earlier in this review, Xylella does not penetrate the plant actively, the cells are rather inoculated by the vector straight into the xylem of the plant so it does not need the structures that are important in the first interactions with the host.
Other Genes Potentially Associated with Pathogenicity
Genes associated with virulence were also identified in the Xylella genome. Copies of xrvA (Xanthomonas regulator of virulence) and vap (virulence associated proteins) genes of Dichelobacter nodosus. vapA encodes an antitoxin similar to HigA (host inhibition of growth) from the killer plasmid Rts1 (Tian et al., 1996). An ORF upstream of vapA codes for a toxin similar to HigB from the same plasmid, it was then termed toxA (toxin) (Bloomfield et al., 1997). In this system, only cells carrying vapA survive in presence of ToxA. There is also a homologue of higB upstream of vapA in Xylella fastidiosa, suggesting the same functionality. It is interesting to note that these genes are located in one of the phages present in this Xylella genome.
Conclusions
The genome project of X. fastidiosa was a milestone in the plant pathology. The choice for this bacteria as the first plant pathogen to be sequenced, was tightly associated to its importance as the causal agent of citrus variegated chlorosis which became a real threat for the world´s citrus industry. Unlike other completely sequenced bacteria, very little is known about the biology of X. fastidiosa. Studies on physiology and pathogenicity of the bacteria were challenged by its fastidious growth, and its specificity to xylem tissues of woody plants, probably associated to the vector specificity. Therefore, an open question is how the knowledge of the genome could help us better understand the biology of this microorganism, specially its pathogenicity.
As a fastidious bacteria living exclusively within xylem vessels, and surviving well in the foregut system of the vectors, X. fastidiosa seems to have no mechanism of interaction cell to cell involving specific virulence or pathogenicity genes, like many other pathogens. Its fastidious growth that can not be changed even by cultivation in rich medium, may reflect adaptation to the xylem conditions of low nutrient concentration, negative pressure, and high turbulence. On the other hand, this bacterium has a wide range of hosts, suggesting either that it also has a general mechanism of pathogenicity, or that these mechanisms can not be identified among those already known.
The capacity of the insects to transmit the bacteria plant to plant together with the survival aptitude of the bacteria within the insect, suggest that the bacteria should also have more interaction mechanisms with the vector than supposed before. This bacterium interacts efficiently with both plants and insects. Its capability to interact with surfaces building biofilms and aggregates makes it similar to animal pathogens.
Although the size of a genome can not be correlated with the specialization of an organism, it should be pointed out that X. fastidiosa has a small genome compared to phylogenetically related bacteria, like E. coli and Xanthomonas spp. and even though around 53% of its genes still do not have a known function. In the future, the information generated by completely sequencing other genomes together with the functional genome projects will provide a better understanding of the complex functioning of these organisms.
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