Mechanisms of infection used by Xanthomonas axonopodis pv. citri in

citrus canker disease

N. Gottig1, B.S. Garavaglia

1,2, C.G. Garofalo

1, T. Zimaro

1, G.G. Sgro

1, F.A. Ficarra

1, G. Dunger

1, L.D.

Daurelio1, L. Thomas

3, C. Gehring

3, E.G. Orellano

1 and J. Ottado

1

1 Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones

Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario,

Argentina 2 Consejo de Investigaciones, Universidad Nacional de Rosario, Rosario, Argentina 3 Computational Bioscience Research Centre, King Abdullah University of Science and Technology, Thuwal, Kingdom of

Saudi Arabia

The citrus canker causing bacteria Xanthomonas axonopodis pv. citri uses several mechanisms to colonize its host. Among

them, attachment to the host surface by specific proteins named adhesins is important to the progress of the disease. Once

the bacteria is attached to the host it delivers pathogenicity effector proteins inside the plant cell through the type III

protein secretion system that modulate the plant basal defense response for the benefit of the pathogen. As a later step in

pathogenesis, X. axonopodis pv. citri must form biofilms which are matrix-enclosed bacterial populations that adhere to

each other with the involving of adhesins and the exopolysaccharide xanthan. Besides these basic mechanisms this bacteria

uses also a plant natriuretic peptide-like protein to regulate host homeostasis and in that way improves host photosynthesis

leading to a more healthy tissue during the infection that suit X. axonopodis pv. citri biotrophic lifestyle. These bacterial

strategies in citrus canker are discussed.

Keywords Xanthomonas; citrus canker

1. Introduction

Citrus canker caused by X. axonopodis pv. citri (Xac) affects citrus plants worldwide. The disease is endemic in

Southeast Asia and has been spread to most citrus producing areas of the world. In South America it was first

discovered in Brazil and then found in many other countries as Argentina that is a major producer of oranges, lemons,

tangerines and grapefruits, and where the disease has been considered endemic since 1970. This devastating disease

affects most commercial citrus cultivars, resulting in significant economic losses mainly due to its effect on fruits,

which are no longer suitable for commercializing as fresh fruits in international markets. Measures for controlling citrus

canker include eradication of infected plants and construction of windbreaks that greatly increase production costs [1].

The bacteria infect leaves, stems and fruits and enter mainly by stomata and wounds. The infection is visualized as

circular spots in leaves abaxial surface. Then, the bacteria colonize the apoplast and, the leaf epidermis is broken due to

cell hyperplasia induced by the pathogen (Fig. 1). In leaves, stems and fruits the lesions raise and then darken and

thicken showing the characteristic raised necrotic corky lesion. Once the pathogen propagates in lesions and in the

presence of moisture on them, bacteria can be dispersed to new growth and to other plants [2].

The genome of X. axonopodis pv. citri has been completely sequenced and gives insights into the importance of

genes and gene clusters governing strategic mechanisms of pathogenesis [3]. Some relevant genes identified in Xac are

the ones coding for bacterial adhesion and surface structural elements, protein secretion systems, toxins, plant cell-wall

degrading enzymes and the ones involved in the response to the oxidative environment bacteria encounter in the

attacked tissue [4]. To better understand the molecular mechanisms underlying disease development, the role of several

of these genes was characterized providing new information about citrus canker disease.

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Fig. 1 Citrus canker disease cycle. The bacteria infect leaves, stems and fruits. The bacteria presumably persist as epiphytes on the

plant surface (1) before they enter the plant mainly by stomata and wounds (2). The infection is visualized as circular spots on leaf

abaxial surfaces. The bacteria then colonize the apoplast (3) and the lesions become raised, darken and thicken resulting in the

characteristic canker (4). The leaf epidermis is disrupted as a result of cell hyperplasia induced by the pathogen (5) allowing the

released of bacteria that are dispersed by wind and rain initiating a new cycle.

2. Bacterial attachment mediated by adhesin proteins

Bacterial attachment is a crucial early step in the pathogenicity process. Adhesins are specialized surface exposed

proteins that mediate bacterial adhesion. Adhesins from animal bacterial pathogens have been widely studied and are

secreted by type V protein secretion system. This system stands out by its apparent simplicity and comprises two

distinct pathways, the autotransporter and the two-partner secretion (TPS) pathways [5]. Both, the autotransporter and

TPS systems translocate large proteins or protein domains, mostly adhesins and hemolysins, and have been identified in

many bacterial genera, including human, animal, and plant pathogens. TPS systems are composed of two proteins, the

transported protein named TpsA and the specific transporter TpsB in the outer membrane [6]. TpsA proteins share a

highly conserved N-proximal region of approximately 250 residues essential for secretion called the ‘TPS domain’ [7;8]

that is secreted through the channel-forming outer membrane porin-like protein TpsB [6].

The most studied filamentous hemagglutinin (FHA) protein of the whooping cough agent Bordetella pertussis is a

230-kDa adhesin, named FhaB, that is secreted by the other TPS partner FhaC, standing for TpsA and TpsB,

respectively [9]. Although adhesins from animal pathogens have been deeply studied, there are only few examples in

their counterparts in plant pathogens. Erwinia chrysanthemi, the pathogen that causes soft-rot diseases on a wide variety

of plants, has a gene codifying for the 3,850-aa protein homolog to FhaB from B. pertussis. Mutants in this gene

showed reduced ability to attach and to form aggregates on Nicotiana clevelandii leaves and thus reduced virulence on

its host plant [10]. In other study the role of hemagglutinin genes from Xylella fastidiosa, the causal agent of several

diseases of citrus, grapevines and peach among others, was investigated. Random mutations of X. fastidiosa that

produced hypervirulent strains with more severe symptoms and earlier grapevine death than did vines infected with the

wild type strain showed insertion in hemagglutinin codifying genes, suggesting that hemagglutinins mediate contact

between bacterial cells impairing X. fastidiosa movement in the plant xylem and thus reducing virulence [11].

X. axonopodis pv. citri has in its genome at least one homolog to a TPS type V secretion system to secrete a FhaB-

like hemagglutinin protein that codifies for a 4,753-aa protein named XacFhaB. The gene present upstream of

XacFhaB, named XacFhaC codifies for a putative TpsB partner secretion of XacFhaB protein. XacFhaB has a predicted

signal peptide followed by a conserved TPS domain. It is notable that all residues that were found to be determinant for

secretion in B. pertussis TPS domain are also present in XacFhaB TPS domain [12]. When mutants of Xac in XacFhaB

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are inoculated on leaf surface they exhibit a general lack of adhesion as could be observed by staining the adhered

bacteria with the crystal violet stain (Fig. 2a). This also leads to a less number of cankers compared with the wild type

bacteria (Fig. 2b). The lack of XacFhaB gene also impairs bacteria-to-bacteria attachment; this mutant displays more

dispersed lesions with smaller and less number of cankers than the wild type Xac strain, also when infiltrated in citrus

leaves (Fig. 2c). Moreover, XacFhaB mutants are affected in bacterial aggregation. Contrarily, mutants in the

transporter protein XacFhaC showed an intermediate virulence phenotype resembling wild type infection, suggesting

that XacFhaB could be secreted also by another partner different from XacFhaC (Fig. 2). In summary, this non-fimbrial

adhesin protein is crucial for the initial stages of the infection process that allows attachment to the plant tissue [13].

Fig. 2 Effects of adhesin and its transporter protein in virulence and bacterial attachment on citrus leaves. a) X. axonopodis pv. citri

wild-type (XacWT) and knocked out strains ∆XacFhaB and ∆XacFhaC adhesion on abaxial leaves surfaces analyzed by crystal

violet stain on a citrus leaf. b) Xac wild-type and mutants strains were inoculated at 104 cfu/ml into the intercellular spaces of fully

expanded orange leaves. A representative leaf is shown 7 days after inoculation. c) Quantification of canker number in orange leaves

after one month of spray inoculation with XacWT, ∆XacFhaB and ∆XacFhaC strains at 109 cfu/ml. Bars are the means of 20 leaves

assayed and error bars are standard deviations, the results are representative of three independent experiments.

3. Type III protein secretion system in citrus canker

Plants are colonized by pathogens that are able to penetrate host surface or through wounds or natural openings like

stomata. Once inside the host, pathogens must cross the cell wall to reach the plasma membrane where they encounter

extracellular receptors that recognize PAMPs (pathogen associated molecular patterns). This recognition initiates

PAMPs mediated immunity that stops the infection. However, pathogens have evolved to remove this immunity by

interfering with the recognition in the plasma membrane or by secreting effector proteins into the cytosol of the plant

cell that alter the expression of resistance responses. Once the pathogens acquired the ability to suppress primary

defense, plants have developed a more specialized mechanism for detecting pathogens which involves direct or indirect

recognition of microbial proteins by resistance proteins (R). Activation of R protein-mediated resistance also suppresses

microbial growth but not before the pathogen may have the opportunity for limited proliferation [14]. These results

challenge the gene-for-gene hypothesis [15] in which disease resistance occurs only with the simultaneous presence of

R gene resistance in the plant and corresponding avirulence gene (Avr) in the pathogen, suggesting that a direct Avr-R

interaction activates the resistance. However, despite many efforts, only in a few cases the physical interaction between

Avr and R proteins has been observed. Because many Avr are known to increase the virulence of the pathogen in

susceptible plants, an alternative model has been proposed in which the Avr protein is able to interact and modify a

target specific to the plant to promote disease in the absence of the corresponding R protein. The R protein function

would be to "guard" or protect these targets and activate the resistance signalling pathways when these plant targets are

modified by a specific Avr [14].

Accordingly, phytopathogenic bacteria, as well as animal pathogenic bacteria, colonize their hosts through the

secretion of virulence effector proteins. Type III protein secretion system is essential for pathogenicity or avirulence of

bacterial pathogens. The cluster codifying for type III protein secretion system is named hrp (for HR (hypersensitive

response) and pathogenicity) because it is indispensable for the trafficking of HR elicitors or pathogenic factors to the

plant cell [16, 17]. HR is observed in resistant plants challenged with a pathogen as well as a consequence of non host

resistance in non host plants [18]. This response is associated with cell death at the initial site of infection that results in

rapid localized necrosis that initially involves the activation of preexisting compounds, followed by induction of plant

defense mechanisms. This is accompanied by the production of reactive oxygen species leading to plant cell death, and

consequently, elimination of the pathogen [19].

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Fig. 3 Schematic representation of Xanthomonas axonopodis pv. citri type III protein secretion system and its interaction with the

plant cell.

The hrp clusters from several phytopathogenic bacteria have been deeply studied. The first one to be characterized

was that of Pseudomonas syringae pv. phaseolicola [20] while for Xanthomonas, the first one was from X. campestris

pv. campestris [21]. Then, the hrp clusters from X. campestris pv. vesicatoria, X. oryzae pv. oryzae and X. axonopodis

pv. glycines have been sequenced and characterized [17, 22, 23]. The broadly studied hrp cluster from X. campestris pv.

vesicatoria spans a region of 23 kb and contains six operons hrpA to hrpF. Nine out of the twenty two proteins encoded

by the cluster are conserved between plant and animal pathogens, are named hrc (hrp conserved), and constitute the

core of the type III translocon. Structurally, type III protein secretion system is composed by a flagellum -the Hrp pilus

in phytopathogens- and a basal body that crosses the two bacterial membranes and ensures the stabilization of the whole

structure. This complex structure allows the establishment of the bacterial attachment to the host cell membrane to

assure the translocation of effector proteins to the interior of the plant cell (Fig. 3) [24, 25]

In Xac several effector proteins have been predicted to be secreted by this system [26] and among them is the well

characterized PthA, a member of the avrBs3 gene family which has been first identified as the elicitor of the symptoms

of citrus canker [27]. Hrp cluster is critical for the development of the disease. Xac mutants in the hrp cluster, one in the

gene that codifies for HrcN, the ATPase that may be providing the energy for the transport system or for the assembly

of the secretion apparatus; another in an operon bearing genes that codify for proteins of the core of the translocon

(HrcQ, HrcR, and HrcS); and the last one in a gene that codifies for a pore-forming protein that mediates translocation

of effector proteins into the plant cell (HrpF), showed no virulence when infiltrated in citrus leaves, consistently with

the crucial role of this system in the pathogenesis [28]. In addition to the compatible interactions produced in citrus

canker, Xac induces non host resistance with HR in cotton, tobacco, tomato, bean, and pepper and a functional hrp

cluster is required for the induction of HR on these non host plants, since the above described mutants produced no HR

lesions in these plants [28].

4. Xac EPS in epiphytic survival and biofilm formation

A Xanthomonas genus characteristic is the synthesis and secretion of an exopolysaccharide (EPS), xanthan, mainly in

the late logarithmic and stationary growth phases. Xanthan is highly regulated by rpf (regulation of pathogenicity

factors) genes [29]. The compound is synthesized, polimerized and secreted by the products of the gum cluster [30] and

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it is structurally composed of a β-1,4-linked D-glucose backbone with trisaccharide side-chains containing mannose-(β-

1,4)-glucuronic acid-(β-1,2)-mannose attached to alternate glucose residues in the backbone by α-1,3 linkages [31], and

mannose residues that are acetylated and pyruvylated at specific sites. The gum clusters in Xac and X. campestris pv.

campestris encompass 12 open reading frames (gumB to gumM). The first reaction in xanthan biosynthesis is catalized

by GumD that provides the glucosyl-1-phosphate transferase activity required for assembling glucose pyrophosphate

polyprenol [32]. Spite of most of the studies focused on the roles of Xanthomonas that show reduced virulence in

xanthan deficient mutants [32-34], in the case of Xac, mutants deficient in xanthan (∆gumD) remained pathogenic for

citrus plants to the same extent as wild-type bacteria, demonstrating that in this particular interaction the EPS does not

modulate the virulence of the pathogen [35]. However, gumD mutants displayed impaired survival under oxidative

stress at the stationary phase, as well as epiphytic survival on citrus leaves. These results suggest that xanthan does not

play an essential role in citrus canker at the initial stages of infection but facilitates the maintenance of bacteria on the

host plant, possibly improving the colonization of distant tissue [35].

A bacterial biofilm is defined as a structured community of bacterial cells enclosed in a self-produced polymeric

matrix and adherent to an inert or living surface. In bacteria like Pseudomonas, this matrix is thought to be comprised of

EPS, DNA, and protein and the main proteins identified in this matrix were adhesin proteins [36] (Fig. 4). Interestingly

in Xac, both molecules: the XacFhaB adhesin and the EPS are involved in biofilm formation. A XacFhaB deletion

mutant impaired in biofilm formation overproduces xanthan, suggesting a mechanism of counteracting the lack of

biofilm. Morever, EPS in concert with adhesins are responsible for biofilm formation, suggesting that xanthan may be

maintained in the extracellular matrix in an adhesin-dependent manner [13]. In conclusion, these results support the fact

that XacFhaB adhesin and the exopolysacharide xanthan both are involved in the later stages of citrus canker by

enhancing bacteria-bacteria interactions necessary to form Xac biofilms and also improving the epiphytic survival on

the host tissue and thereby facilitating the colonization of more distant tissues. These molecules are also implicated in

bacteria motility leading to larger cankers in infected citrus leaves [13].

Fig. 4 Stages in Xac biofilm formation. Confocal microscopic analysis of GFP expressing Xac in different biofilm stages of

multicellular organization, that represent planktonic cells, attachment, microcolony and macrocolony formation.

5. Xac uses a host-acquired gene as a virulence strategy

Xac, but no other phytopathogen or bacteria, has a plant natriuretic peptide (PNP)-like gene (XacPNP) [37] that is

expressed during Xac infection [38]. Natriuretic peptides (NPs) have been identified in many vertebrates and are

commonly associated with organs involved in cardiac and osmoregulatory homeostasis and have been shown to play a

critical role in the regulation of salt and water balance [39]. In higher plants, PNPs elicit a number of responses that

contribute to the regulation of homeostasis and growth [40]. PNPs act via rapid and transient increases in cellular cGMP

levels [41] and promote tissue specific ion movements [42], increases in net water uptake into cells as well as stomatal

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opening [43-45]. PNPs are up-regulated under conditions of osmotic stress [46] and K+ starvation [47] and have been

localized in conductive tissue [48]. Furthermore, PNPs have been identified in the apoplastic proteome and biologically

active PNP was isolated from xylem exudates [48].

XacPNP, shares significant sequence similarity and identical domain organization with PNPs and recombinant

XacPNP can cause plant physiological responses such as stomatal opening [38]. Canker lesions produced in leaves

infected with a XacPNP deletion mutant are more necrotic than those with the wild type bacteria and the highly necrotic

tissue led to earlier bacterial cell death in the mutant infection, suggesting that the plant-like bacterial PNP enables the

plant pathogen to modify host responses in order to create conditions favourable to its own survival (Fig. 5) [38,49].

Proteomics studies on citrus leaves infected with Xac showed a decrease in the expression of sugar-regulated

photosynthetic proteins such as Rubisco and Rubisco activase and also of ATP synthase and an increase in NADH

dehydrogenase diagnostic for a reduction in photosynthetic efficiency during citrus canker and an increase of proteins

related to plant defense against the pathogen [50]. This indicates that during pathogen attack the biosynthesis of

defense-related compounds is a priority for the plant while other (e.g. growth related) cellular activities are reduced thus

permitting a reduction in photosynthetic rates until pathogenic growth has been halted [51]. Such a reduction in

photosynthesis may starve biotrophic pathogens of nutrients thereby benefitting the plant. Further, the analysis of the

host proteome after infection with a XacPNP deletion mutant strain (∆XacPNP) demonstrated that the main difference

between infections with Xac wild type and ∆XacPNP was in proteins with a key role in carbon metabolism and

photosynthesis showing that ∆XacPNP caused a considerably bigger reduction in the expression of photosynthesis

genes [50]. Consistent with this the application of recombinant XacPNP to leaves increases the expression levels of

these photosynthetic proteins [52], while in both cases photosynthesis efficiency measured by chlorophyll fluorescence

assays was dependent on the presence of a functional PNP [50, 52, 53]. These results suggest that Xac has acquired and

adapted this plant protein and mimics its function to maintain host conditions better suited to its biotrophic lifestyle and

doing so by directly or indirectly modulating and/or sustaining chloroplast function (Fig. 5). In summary, XacPNP

probably overcomes host defense responses by counteracting shutting down of photosynthesis and this is the first report

in which primary metabolism is manipulated by a plant-like molecule operating in a pathogen.

Fig. 5 Model of XacPNP action in citrus canker. Typical citrus leaves lesions with ∆XacPNP (left) and Xac wild type (right) are

observed in the bottom images where differences in tissue necrosis are shown. On the leaf is a proposed model for PNP-dependent

cellular processes. Solid arrows indicate experimentally confirmed functions and dashed arrows indicate proposed mechanisms.

6. Concluding remarks

In plant-pathogen interactions, pathogens aim to overcome host defense responses while plants employ a battery of

responses to limit pathogen growth and thus disease. In this “arms race” between hosts and pathogens, phytopathogens

have evolved different molecular mechanisms that mediate adhesion to the host cell, colonization and dissemination and

also different strategies to prolong their survival in the host.

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In citrus canker as well as in other plant-pathogen interactions, bacteria attachment to the host surface is determinant

for the invasion of the tissue and depends on specific adhesins that are anchored in the bacterial outer membrane. Once

attached to the host, Xac delivers pathogenicity effector proteins inside the plant cell trough the type III secretion

system that modulate the plant basal defense response for the benefit of the pathogen. In order to prolong its survival in

the plant, Xac must form biofilms that allow further colonization, process in which XacFhaB adhesin and EPS are

involved. Finally, Xac has a new strategy to manipulate host responses that involves XacPNP, a gene probably acquired

in a horizontal gene transfer event, that modulates host homeostasis through mimicking the role of PNPs, improving

host photosynthesis and thus leading to a more healthy tissue during the infection. This regulation of the host status

serves to suit Xac biotrophic lifestyle and to prolong Xac survival. In conclusion, Xac has several different molecular

mechanisms to infect citrus plants being an interestingly system to study plant-pathogen interactions. The knowledge in

citrus canker pathogen-host interaction is critically needed in order to provide new tools for developing sustainable and

economically viable control measures. Moreover, understanding the bacterial mechanisms by which plant pathogens

attack their hosts is central to the study of plant pathology.

Acknowledgements The support by Argentine Federal Government (ANPCyT PICT2006-00678 to J.O.) is gratefully acknowledged.

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