Pathogenicity and infection strategies of the fire blight pathogen Erwinia amylovora in Rosaceae : State of the art

Plants are host to a large amount of pathogenic bacteria. Fire blight, caused by the bacterium Erwinia amylovora, is an important disease in Rosaceae. Pathogenicity of E. amylovora is greatly influenced by the production of exopolysaccharides, such as amylovoran, and the use of the type III secretion system, which enables bacteria to penetrate host tissue and cause disease. When infection takes place, plants have to rely on the ability of each cell to recognize the pathogen and the signals emanating from the infection site in order to generate several defence mechanisms. These mechanisms consist of physical barriers and the production of antimicrobial components, both in a preformed and an inducible manner. Inducible defence responses are activated upon the recognition of elicitor molecules by plant cell receptors, either derived from invading microorganisms or from pathogen-induced degradation of plant tissue. This recognition event triggers a signal transduction cascade, leading to a range of defence responses [reactive oxygen species (ROS), plant hormones, secondary metabolites, ...] and redeployment of cellular energy in a fast, efficient and multiresponsive manner, which prevents further pathogen ingress. This review highlights the research that has been performed during recent years regarding this specific plant– pathogen interaction between Erwinia amylovora and Rosaceae, with a special emphasis on the pathogenicity and the infection strategy of E. amylovora and the possible defence mechanisms of the plant against this disease.


Introduction
Fire blight, caused by the Gram-negative bacterium Erwinia amylovora, represents an enormous threat to fruit cultivation in many parts of the world, as it can infect most plants of the Rosaceae, both ornamental and economic cultivars, among which apple and pear are important hosts.It is a complex disease which passes its entire cycle in close association with the host plant, where it is able to infect fruit, leaf, shoot and flower tissue.Infected plant parts will first appear water soaked, next they will turn dark green, wilt and finally turn brownish to black.In all cases, sticky, amber-like ooze drops, composed of viable bacteria in a polysaccharide matrix might be formed on the blighted plant parts.Fire blight is difficult to control, as it is able to rapidly spread in the plant and effective control methods still are lacking.Every year, infected apple and pear orchards are reduced in size because of quarantine-related measures.In Europe, fire blight is considered an increasing problem as higher temperatures, breeding of cultivars on susceptible rootstocks and the introduction of susceptible cultivars could enlarge the risk of infection in the near future (Deckers & Schoofs, 2008).

Pathogenicity and virulence of E. amylovora
Pathogenicity and virulence of the pathogen E. amylovora depend on different factors.Besides the production of the siderophore desferrioxamine for the acquisition of iron molecules from the host tissue (Dellagi et al., 1998;Expert, 1999;Smits & Duffy, 2011) and the presence of other virulence factors such as metalloproteases (Zhang et al., 1999), the presence of plasmids (Llop et al., 2011(Llop et al., , 2012;;McGhee & Jones, 2000;Mohammadi, 2010), two-component signal transduction systems (Wang et al., 2009;Zhao et al., 2009) and histone-like proteins (Hildebrand et al., 2006) are also important factors in pathogenesis.However, probably the most essential reasons for differences in virulence between different strains of E. amylovora are due to a variation in synthesis of exopolysaccharides and the mechanism of the type III secretion system (T3SS) and associated proteins.

Exopolysaccharides
Exopolysaccharides (EPS) have been suggested to play a key role in bypassing the plant defence system, in disturbing and obstructing the vascular system of the plant and in protecting the bacteria against water and nutrient loss during dry conditions (Denny, 1995;Ordax et al., 2010).
One of these EPS is amylovoran, which is the main constituent of bacterial ooze.Amylovoran is a polymer of a pentasaccharide repeating unit that generally consists of four galactose residues and one glucuronic acid residue (Maes et al., 2001;Nimtz et al., 1996).The molecular size of amylovoran is influenced by several environmental conditions and cell-metabolism-related factors (Schollmeyer et al., 2012).E. amylovora strains that do not have the capacity to produce amylovoran are non-pathogenic and are unable to spread in plant vessels (Bellemann & Geider, 1992).Another EPS that is synthesized by E. amylovora is levan.Lack of levan synthesis can result in a slow development of symptoms in the host plant (Geier & Geider, 1993).
The amylovoran synthesis (ams) gene cluster involved in the biosynthesis of amylovoran produces 12 ams-encoded gene products (AmsA to AmsL).AmsC, AmsH and AmsL are believed to be involved in oligosaccharide transport and assembly whereas AmsA possesses a tyrosine kinase activity.AmsB, AmsD, AmsE, AmsG, AmsJ and AmsK proteins appear to play part in annealing the different galactose, glucuronic acid and pyruvyl subunits to the lipid carrier in order to form an amylovoran unit.AmsF may process newly synthesized repeating units and/or be involved in their polymerization by adding them to an existing amylovoran chain.Finally, AmsI seems to have a distinct function in recycling of the diphosphorylated lipid carrier after release of the synthesized repeating unit (Bugert & Geider, 1997;Eastgate, 2000;Langlotz et al., 2011).
Recently Koczan et al. (2009) discovered that EPS of E. amylovora are also involved in biofilm formation, which enables the bacteria to attach to several surfaces and each other (Koczan et al., 2011;Ramey et al., 2004).Studies of Pseudomonas aeruginosa have revealed that the process of biofilm formation can be divided into five distinct phases including reversible attachment, irreversible attachment, maturation 1, maturation 2 and detachment in the dispersion phase (Sauer et al., 2002).Biofilm infections appear to be very persistent and it has been shown that bacteria that are able to compose a biofilm can be up to 1000-fold more resistant to antibiotic treatment than their planktonic counterparts (Gander & Gilbert, 1997).Koczan et al. (2009) have suggested that biofilm formation plays an important role in pathogenesis of E. amylovora, as their study showed that amylovoran is necessary for biofilm formation and that levan contributes to this biofilm formation.They also confirmed the results of Maes et al. (2001), in which it was shown that the quantity of amylovoran produced by individual E. amylovora strains is correlated with the degree of virulence.
The mechanistic details behind biofilm formation remain largely unknown but it is suggested that they are formed in response to environmental triggers (Davey & O'toole, 2000) and quorum sensing (QS) signals (Sauer et al., 2002).Although research has shown that Erwiniae species produce two types of QS molecules namely N-acylhomoserine lactones and autoinducer-2 type signalling molecules (Barnard & Salmond, 2007;Molina et al., 2005), Rezzonico & Duffy (2008) suggested a non-quorumsensing role for the autoinducer-2 luxS gene due to a lack of genomic evidence for autoinducer-2 receptors.

Type III secretion system
Another important factor in pathogenicity is confined by the action of the T3SS.Gram-negative phytopathogenic bacteria such as E. amylovora utilize this evolutionarily conserved secretion system to export and deliver effector proteins into the cytosol of host plant cells through a piluslike structure, which forms the central core element of the T3SS.The needle complex is composed of a large, cylindrically shaped macromolecular complex organized into a series of ring-like structures with inner rings, outer rings and a neck structure.It is embedded in the inner and outer membrane of the bacteria, while spanning the periplasmic membrane and extending into the extracellular environment with a needle filament (Alfano & Collmer, 2004;Block et al., 2008;Bu ¨ttner & Bonas, 2006;Bu ¨ttner & He, 2009;Cornelis & Van Gijsegem, 2000;Gala ´n & Wolf-Watz, 2006;Grant et al., 2006;He et al., 2004;Hueck, 1998;Jin et al., 2001;Loquet et al., 2012;McCann & Guttman, 2008;Mudgett, 2005;Schraidt & Marlovits, 2011).
The T3SS of plant-pathogenic bacteria is mainly made out of Hrc proteins, encoded by hrp-conserved (hrc) genes among plant-pathogenic bacteria and Hrp proteins, encoded by hypersensitive response and pathogenicity (hrp) genes.In E. amylovora, hrc and hrp genes are clustered in a pathogenicity island which contains four regions, i.e. a hrp/hrc region, a Hrc effectors and elicitors region, a Hrp-associated enzymes region and an island transfer region (Oh & Beer, 2005).The key regulatory gene is hrpL, which encodes the extracytoplasmic function of s- factor HrpL, which in turn recognizes conserved sequence motifs (hrp boxes) located in promoters of hrp secretion genes and of genes encoding secreted proteins (McNally et al., 2012;Oh et al., 2005;Pester et al., 2012).
To date, 12 proteins have been found that are secreted via this T3SS (Nissinen et al., 2007).Four of them (Eop1, Eop3, Eop4 and DspA/E) have clear similarity to known effectors.The 200 kDa disease factor DspA/E for example, which is homologous to the type III effector AvrE discovered in soybean after inoculation with Pseudomonas syringae pv.tomato, is required for pathogenicity in several strains of E. amylovora and interacts with the intracellular domains of host plant receptor kinases and preferredoxin (Boureau et al., 2006;Meng et al., 2006;Nissinen et al., 2007;Oh et al., 2007Oh et al., , 2010;;Triplett et al., 2009) secretion of DspA/E requires a type III chaperone DspB/F, which is a small acidic protein that binds to its cognate secreted protein (Gaudriault et al., 2002;Triplett et al., 2010).Five proteins belong to the helper protein class, namely Eop2, HrpK, HrpN, HrpW and HrpJ.Both Eop2 and HrpK have clear similarities to proteins in P. syringae, but their functions still remain elusive (Nissinen et al., 2007).HrpN and HrpW instead are harpins.These proteins are glycine-rich, lack cysteine and are involved in inducing the hypersensitive response in non-host plants.Unlike HrpW, HrpN is required for full virulence in plants (Kim & Beer, 1998;Reboutier et al., 2007;Sinn et al., 2008;Wei et al., 1992) and plays an important role in the translocation of DspA/E (Bocsanczy et al., 2008).HrpJ has been postulated to act as an essential extracellular chaperone to prevent aggregation of harpins in the apoplast, and thus facilitate translocation of effector proteins into the host cells (Nissinen et al., 2007).The three remaining proteins that are secreted via the T3SS are HrpA, TraF and FlgL.HrpA is an essential structural protein of the type III secretion pilus, TraF is involved in pilus formation and FlgL is similar to a flagellar hookfilament junction protein (Nissinen et al., 2007).
Further signalling of the plant in response to E. amylovora When a bacterial interaction with a plant occurs, there are intrinsically two levels of the plant immune system (Jones & Dangl, 2006;Robert-Seilaniantz et al., 2007).The first level is performed by the action of multiple transmembrane pattern recognition receptors (PRRs) belonging to either the receptor-like kinase or receptor-like protein families.PRRs bear structural similarities to animal Toll-like receptors (He et al., 2007;McDowell & Simon, 2008;Segonzac & Zipfel, 2011) and respond to multiple cellsurface components of Gram-negative bacteria, including lipopolysaccharide, a major constituent of the outer membrane (Dow et al., 2000;Gerber et al., 2004;Meyer et al., 2001;Newman et al., 2002), and flagellin, the protein subunit of the flagellum (Asai et al., 2002;Takeuchi et al., 2003).The second level of the plant immune system acts largely inside the cell, using the polymorphic nucleotide binding-leucine-rich repeats protein products encoded by plant-derived Resistance (R) genes to counter pathogen secreted effectors [Avirulence (Avr) proteins].Avr proteins are considered factors that contribute to host infection, although the biochemical function of most Avr proteins remains unidentified.However, in those cases when Avr factors are recognized by resistant host plants through direct or indirect interaction with their complementary Rgene-encoded protein counterparts, they act as specific elicitors of plant defence rather than as virulence factors.When this genetic interaction takes place, a defence response is triggered and gene-for-gene resistance is established (Abramovitch & Martin, 2004;Belkhadir et al., 2004;Bent & Mackey, 2007;Lahaye & Bonas, 2001;Mansfield, 2009;Martin et al., 2003;McDowell & Simon, 2006;White et al., 2000).Contrary to several other plantbacteria interactions (Kunkel et al., 1993;Ronald et al., 1992;Tai et al., 1999;Tsiamis et al., 2000), until now no related avirulence gene and corresponding plant resistance gene have been reported in the pathosystem of E. amylovora and Rosaceous plants.
Nevertheless, much research has been performed concerning the signalling pathways in planta and the different modes of protection after inoculation with E. amylovora.Sarowar et al. (2011) for instance obtained a total of 3500 genes involved in metabolism, signal transduction and stress response, which were significantly modulated in fireblight-infected blossoms of the apple cultivar 'Gala' and which indicates that several pathways are affected as a result of a successful infection with E. amylovora.Probably one of the earliest responses of a plant to fire blight is a rapid increase of reactive oxygen species (ROS), followed by the production of several secondary metabolites, plant hormones and components of other defence-related pathways, of which some are discussed here more in detail and are represented in Fig. 1.

ROS and the generation of an oxidative burst
ROS are normally only produced as side-products of some general pathways such as photosynthesis (Krieger-Liszkay, 2005).They are generated by various enzymic activities of which the best studied are NADPH oxidases.However, during an incompatible reaction, an increased production of ROS and a hypersensitive response can be observed.Interestingly, in the case of the compatible interaction between E. amylovora and a host plant, E. amylovora is perceived by this host plant as an incompatible pathogen, which results in the generation of ROS by the plant.These bursts of ROS seem to be paradoxically necessary for a successful bacterial colonization (Venisse et al., 2001).The oxidative burst is elicited by HrpN proteins in non-host plants (Baker et al., 1993;Chang & Nick, 2012;Desikan et al., 1998;Livaja et al., 2008) and by both HrpN and DspA in host plants (Venisse et al., 2003).Furthermore, it is believed that the bacterial exopolysaccharides protect E. amylovora against the toxic effects of ROS since a noncapsular mutant of E. amylovora induced locally the same responses as the wild-type but was unable to further colonize the host plant (Venisse et al., 2001).
As a response of this increase in ROS, the concomitant activity of some antioxidative enzymes and redox metabolites is often reported in pear and apple, according to the necessity to carry out ROS detoxification to less toxic compounds (Faize et al., 1999 Venisse et al., 2001Venisse et al., , 2002Venisse et al., , 2003;;Viljevac et al., 2009).Specific balances in antioxidative genes countering these ROS could explain why ontogenesis-related differences exist between cultivars regarding fire blight susceptibility (Vrancken et al., 2012).

Secondary metabolites
Another phenomenon that could be observed as a result of a successful inoculation is the change in levels of phenylpropanoid-flavonoid pathway derived compounds with important groups such as flavonoids, phenolamines and lignin (Fischer et al., 2007;Pfeiffer et al., 2006;Treutter, 2001Treutter, , 2010)).The general phenylpropanoid metabolism generates an enormous array of secondary metabolites based on the few intermediates of the shikimate pathway as the core unit.The phenylpropanoid-flavonoid pathway is typified by an enormous complexity, which is caused by a large number of branches and branchpoints and many end products.Hence, the pathway has the ability to produce certain end products via different branches by combining the function of large superfamilies of reductases, oxygenases, ligases and transferases.The overall result is an organ-and developmentally specific pattern of metabolites, characteristic for each plant species.Jensen et al. (2012) suggested that the expression of the phenylpropanoid pathway as a whole might be one of the many predictors of fire blight resistance.Burse et al. (2004) already showed that E. amylovora has the ability to protect itself against secondary metabolites in apple because of the internal efflux pump AcrAB, indicating the possible involvement of these metabolites in plant defence.Depending on the cultivar, the bacterial strain, the inoculation method and the time after inoculation, different results are reported throughout the literature regarding the phenylpropanoid-flavonoid pathway.For instance, both Venisse et al. (2002) and Milcevicova et al. (2010) found that most of the phenylpropanoid-flavonoid related enzymes investigated were repressed in some apple cultivars after inoculation with a specific fire blight strain, whereas Sklodowska et al. (2011) and Pontais et al. (2008) demonstrated that the level of some hydroxycinnamate derivatives was significantly augmented in both resistant and sensitive apple cultivars.Moreover, phloretin was found at a bacteriotoxic concentration in both genotypes, but E. amylovora exhibited the ability to stabilize this compound at sublethal levels (Pontais et al., 2008).de Bernonville et al. (2011) proposed that the constitutive phenolic composition of two apple cultivars 'Evereste' and 'MM106' is not responsible for their contrasted differences in susceptibility to fire blight.
In pear, Gunen et al. (2005) reported a higher content of arbutin in resistant cultivars, while sensitive cultivars obtained a higher level of chlorogenic acid.Research done by our lab showed that the transcription patterns of two key genes, anthocyanidin reductase (ANR) and chalcone synthase (CHS), related to this phenylpropanoid-flavonoid pathway, considerately increased in E. amylovora-inoculated mature leaves compared with the control and mockinoculated mature leaves of the pear cultivar 'Conference', with the strongest reaction 48 h after inoculation (Vrancken et al., 2012).These effects of E. amylovora were also visualized in histological sections, and confirmed by HPLC, as epicatechin, which is produced from cyanidin via ANR, increased 72 h after inoculation in E. amylovora-inoculated mature leaf tissue.The rise in CHS is in agreement with the work of Baldo et al. (2010) who used cDNA-amplified fragment length polymorphism analysis to demonstrate a sudden rise of CHS in the fire-blight-susceptible apple root stock M26 after inoculation with E. amylovora.
Although the real function of these secondary compounds in a fire blight-pome fruit interaction is still not clear, it has been reported many times in the literature that phenolic components have direct antioxidant properties which are even better than those of vitamins and ascorbic acid, for instance (Agati & Tattini, 2010;Feucht et al., 1996;Gould, 2004).Moreover, they share the ability to influence cell signalling by downregulating pro-oxidant enzymes such as NADPH oxidases and lipoxygenases, by altering the phosphorylation state of target molecules or by chelating transition metals that mask pro-oxidant actions of reactive nitrogen and oxygen species, both in plants (Treutter, 2005) as in human and mammalian tissue (Fraga & Oteiza, 2011;Williams et al., 2004).However, because of a lack of convincing spatiotemporal correlations with the flavonoid oxidation products, the widely accepted antioxidant function of flavonoids in plants is still a matter of debate (Herna ´ndez et al., 2009).Furthermore, flavonoids have also been described as having antibacterial, antitoxin, antiviral and/or antifungal activities (Ardi et al., 1998;Friedman, 2007;Treutter, 2005) or being involved in creating a structural defence, as research in other plant-pathogenic interactions revealed ultrastructural modifications with incorporated flavonoids, middle lamellae or callose-rich papillae to obstruct further progress of different pathogens (Dai et al., 1996;Loureiro et al., 2012;Nicaise et al., 2009;Soylu, 2006).Probably, a combination of all these factors could affect susceptibility to E. amylovora.

Plant hormones
Jasmonic acid (JA), salicylic acid (SA) and ethylene are three distinct plant hormones which also interfere during microbial attack.Both the SA and JA defence pathways are mutually antagonistic (Chisholm et al., 2006;Robert-Seilaniantz et al., 2007), which has also been shown for the pathosystem E. amylovora-Malus.Ethylene also seems to have a big part in the response of the plant after mechanical wounding and after pathogen attack.The group of Spinelli et al. (2011) measured ethylene production in both E. amylovora-inoculated and mock-inoculated apple plants, reaching a peak approximately five hours after inoculation.However, in mockinoculated plants, this ethylene burst was much lower and faded away after six hours.Next to ethylene, the production of other volatiles such as 2,3-butanediole, isoprene-ozone and 3-hexenal were also detected in the E. amylovora-inoculated plants (Spinelli et al., 2011).Whether this rise in ethylene and other volatiles is involved in a possible plant defence mechanism is still not known for this pathosystem.

Pathogenesis-related (PR) proteins
Pathogenesis-related proteins of plants are divided into more than 15 subfamilies and have been defined as hostoriginating proteins with direct antimicrobial activity that are induced only in response to a pathogen attack or related event.Induction of PR proteins has been found in many plant species belonging to different families, suggesting a general role of these proteins in adaptation to biotic stress conditions.
Not much scientific research has been published concerning PR proteins in woody fruit perennials after infection with E. amylovora.Only a systemic upregulation of the PR-5 (Bonasera et al., 2006a;Venisse et al., 2002), PR-2 (Bonasera et al., 2006a;Heyens et al., 2006), PR-8 (Bonasera et al., 2006a) and PR-10 (Mayer et al., 2011) families was reported in infected tissues.The PR-1 gene family seems not to be involved (Bonasera et al., 2006a;Pester et al., 2012).Furthermore, overexpression of the MpNPR1 gene confers activation of PR-2, PR-5 and PR-8 in Malus 6 domestica (Malnoy et al., 2007).Although some of these PR proteins exhibit potential in vitro antimicrobial activities and their accumulation in the plant is related to plant resistance responses, a direct functional role in defence could not be demonstrated for all (Sels et al., 2008;Van Loon & Van Strien, 1999).

Phytoalexins
Phytoalexins are low-molecular-mass secondary metabolites with antimicrobial activity, which are synthesized de novo after biotic and abiotic stress and occur in a wide variety of chemical structures and in different plant species.The biosynthesis of most phytoalexins, the regulatory networks involved in their induction by biotic and abiotic stress and the molecular mechanism behind their cytotoxicity remain largely unknown (Ahuja et al., 2012;Chizzali & Beerhues, 2012).In both Malus 6 domestica 'Holsteiner Cox' and Pyrus communis 'Conference', the phytoalexin group of the biphenyls and dibenzofurans were detected in the transition between healthy and diseased tissue of the stem after a fire blight infection.In leaves, no phytoalexins could be measured (Chizzali et al., 2012a(Chizzali et al., , 2012b;;Hu ¨ttner et al., 2010).Probably, both the outermembrane protein TolC and the AcrAB transport system in E. amylovora play important roles as protein complexes that are capable in offering resistance to phytoalexins (Al-Karablieh et al., 2009;Burse et al., 2004).
Remarkably, the flavan-4-ol luteoforol, which is the unstable and highly reactive precursor of luteoliflavan, is induced in pome fruits after treatment with the growth regulator prohexiadone-Ca and shows phytoalexin-like properties against E. amylovora and other pathogens (Flachowsky et al., 2012;Halbwirth et al., 2003;Spinelli et al., 2005).

Photosynthesis
Infection of apple by E. amylovora results in a decrease of photosynthetic activity, suggesting an inhibition of photosystem I and/or II (Bonasera et al., 2006b).Similarly, changes in the chlorophyll fluorescence of E. amylovorachallenged apple leaves are observed prior to the development of disease symptoms.Both Heyens & Valcke (2006) and Baldo et al. (2010) noticed an induction of some photosynthetic genes during a Malus-E.amylovora interaction.Research by Singh et al. (2010) suggested that FIBRILLIN4, which is associated with photosystem II, could also play a part in fire blight infections, as the disease is more expressed in the knockdown mutant.

Conclusion
Despite the many efforts that have been put forward concerning the study of the pathogenicity and the infection strategy of E. amylovora and the possible defence mechanisms of the plant against this disease, this economically important pathosystem remains largely unexplored and/or is far from well understood.Little is known about endstage disease, latent infections, survival away from the host, interaction with other microbial organisms and secondary bloom infections.Furthermore, regarding the infection process, the function and presence of avirulence genes, the amount of pathogenicity factors and the mechanism of the T3SS system remains poorly understood.The recent sequencing and annotation of the complete genome of E. amylovora CFBP1430 by Smits et al. (2010) is a welcome tool in revealing novel insights into the genome, which will surely lead to increased understanding of the virulence, host range and ecological behaviour of these bacteria on their host plants in the near future.
However, knowing the bacteria is not enough, as it is essential to study the plant as well.The availability of molecular markers and genetic mapping of fruit crops would allow identification of major resistance genes and disease-specific loci.An excellent review about candidate resistance genes and the application of genomics to improve fire blight management has been written by Malnoy et al. (2012).
In conclusion, gaining insight into infection strategies by E. amylovora and defence mechanisms of the host plant is crucial in obtaining a fire-blight-free environment.
Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11On: Tue, 25 Jun 2019 14:39:40 Milcevicova et al. (2010)andMilcevicova et al. (2010)reported a significant accumulation of total SA in different apple cultivars after infection with E. amylovora; de Bernonville et al. (2012) also demonstrated a downregulation of JA levels in a susceptible cultivar of apple.Accordingly, treatment of these susceptible plants with methyljasmonate increases the resistance of these plants against E. amylovora, indicating that the downregulation of the JA pathway is a critical step in the infection process.