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The bacterial disease fire blight is currently one of the most economically important plant diseases in pome fruit production worldwide. The disease is caused by the Gram-negative enterobacterium Erwinia amylovora, which was the first bacterium that has been identified as the causal agent of a plant disease (Winslow et al., 1920). E. amylovora was first observed in North America in the Hudson Valley of New York (Denning, 1794) and has since been decribed in more than 40 countries (Peil et al., 2009). The bacterium E. amylovora overwinters in cankers of infected trees and can be disseminated in spring of the next season by birds, insects, mites, spiders, humans, wind, water or mechanical equipment (Schroth et al., 1974). In plants, the bacteria cells are mostly localized in the xylem and intercellular spaces from where they disseminate downwards against the direction of the water flow (Bogs et al., 1998). Bacterial aggregation in the xylem causes a disruption of the vessel walls by changing the vessel pressure (Esau, 1965). Plugging of the vascular system by bacteria and capsular extracellular polysaccharide leads to wilting symptoms and necrosis of the plant tissue above the infection site (Van Alfen & Allard-Turner, 1979). Fire blight infections can cause severe economic losses in pome fruit production. In the United States, for example, the estimated annual costs as a result of fire blight infections are c. US$100 million (Norelli et al., 2003). In Switzerland, an amount of US$9 million of losses was reported between 1997 and 2000 (Hasler et al., 2002) and in Germany, losses of c. US$1.6 million were estimated for the Lake Constance region (southern Germany) after fire blight infections in 2007 (Scheer, 2009).
Disease management is possible with streptomycin or, less efficiently, with copper sprays; however, streptomycin resistance in E. amylovora occurs in the US and in other countries, including Canada, Israel, and New Zealand (McManus et al., 2002; McGhee et al., 2011). US apple growers spend c. US$2.8 million per year on antibiotic sprays (Gianessi et al., 2002). Streptomycin-containing products for fire blight control are not permitted in many European countries. Biological control is another possibility, but control can be variable and may not be effective in years with disease-conducive weather conditions (Johnson & Stockwell, 1998).
Planting of fire blight-resistant cultivars seems to be the most promising strategy, which is environmental and producer-friendly. Most of the apple cultivars in current production globally are highly susceptible to fire blight and thus justify resistance breeding to fire blight as a primary objective in many apple breeding programs. Donor genotypes for fire blight resistance in wild apple species have been described and a number of studies were performed with the aim of investigating the genetic basis of this trait. During the last decade, several quantitative trait loci (QTLs) for resistance to fire blight in different genetic backgrounds and in response to different strains of the pathogen were identified (Khan et al., 2012). For example, the QTL on linkage group 3 (LG 3) of Malus × robusta 5 (Mr5) is of particular interest for breeding (Gardiner et al., 2012). This QTL was stable during 14 yr of virulence screening, in different cross-bred populations and after inoculation with a number of different E. amylovora strains (Peil et al., 2007, 2008). In total, the QTL on LG 3 of Mr5 accounted for between 67 and 83% of the phenotypic variance, indicating the existence of one or a few major resistance genes in this genomic region. This assumption is supported by several publications, which described the existence of E. amylovora strains varying in virulence to Mr5 (Fazio et al., 2008) and strains overcoming the resistance of Mr5, (Norelli & Aldwinckle, 1986; Peil et al., 2011). Such diagnostic pathogen strains are very useful not only for future breeding programs with objectives of resistance gene pyramiding but also in studies of plant–pathogen interactions. Little is known about genes involved in the Mr5-E. amylovora host–pathogen interaction. Gardner et al. (1980) postulated a dominant resistant gene for Mr5 controlling resistance to fire blight and Peil et al. (2007) also assumed a major resistance gene.
Erwinia amylovora is known to encode a type III secretion system (T3SS), which secretes effector proteins such as DspA/E, Eop1, HopPtoCEA, HrpN, HrpW and others (Khan et al., 2012; Malnoy et al., 2012; McNally et al., 2012). One of these effectors is AvrRpt2EA, a homolog of the AvrRpt2 protein of Pseudomonas syringae (Zhao et al., 2006). AvrRpt2 activates the RPS2 resistance gene of Arabidopsis thaliana via the cleavage of RIN4, the guard of RPS2 (Axtell & Staskawicz, 2003; Mackey et al., 2003; Day et al., 2005).
Shoots of Mr5 were inoculated with a number of different E. amylovora wildtype strains and an avrRpt2EA mutant strain ZYRKD3-1 (Zhao et al., 2006) in order to investigate the role of AvrRpt2EA in the Mr5-E. amylovora host–pathogen interactions. Interestingly, four natural isolates and the avrRpt2EA mutant ZYRKD3-1 were found to overcome the resistance of Mr5, suggesting a contribution of avrRpt2EA to virulence. Sequencing of the avrRpt2EA gene of strains differing in virulence to Mr5 revealed a single nucleotide polymorphism (SNP), which resulted in a change in the amino acid sequence correlated with virulence. Artificial shoot inoculations with complemented mutant strains gave further evidence for the involvement and importance of the SNP in the avrRpt2EA gene in virulence to Mr5 and the probable existence of a gene-for-gene-relationship in the host–pathogen system Malus × robusta 5–E. amylovora.
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The crabapple species Malus × robusta (Carrière) Rehder (synonyms Malus microcarpa var. robusta Carrière, Pyrus baccata var. cerasifera Regel) is referred to as a hybrid between the two primary wild apple species M. prunifolia and M. baccata (Jefferson, 1970; Ignatov & Bodishevskaya, 2011). One of the most famous accessions of this species is selection M. × robusta No. 5 (Mr5), which was grown from seed obtained in 1927 from Russia through arrangements with the Arnold Arboretum (Jefferson, 1970). Mr5 has been described as tolerant to common viruses of eastern Canada, resistant to collar rot, woolly apple aphid (Watkins & Spangelo, 1970), powdery mildew (Wan & Fazio, 2011) and fire blight (Watkins, 1971; Van Der Zwet & Keil, 1974). Differential interaction between Mr5 and various E. amylovora strains were reported by Norelli & Aldwinckle (1986), who inoculated 25 apple cultivars, including Mr5, with E. amylovora strains Ea273 (Ea78 and Ea3051 in the author database) and Ea266 (Ea77, Ea396 and Ea3050 in the author database). Mr5 was resistant to Ea273 but susceptible to Ea266. The Canadian isolate Ea3049 could also overcome the resistance of Mr5 and resulted in 97% average shoot necrosis (Peil et al., 2011). In the present study, two additional E. amylovora isolates virulent to Mr5 have been identified, Ea400 and Ea110 (Figs 1, 6). The results obtained by Norelli & Aldwinckle (1986), Peil et al. (2011) and the present study demonstrate that the resistance of Mr5 to fire blight is highly strain-specific. Furthermore, it was shown that all E. amylovora strains able to overcome the resistance of Mr5 were highly virulent to susceptible and moderately resistant apple genotypes as well (Fig. 1).
In 2006, Zhao et al. identified an analog to the effector protein AvrRpt2 from P. syringae pv. tomato in the genome of E. amylovora and annotated it as AvrRpt2EA. Inoculation of immature pear fruits with an avrRpt2EA deletion mutant (ZYRKD3-1) resulted in reduction of disease symptoms, indicating that AvrRpt2EA is a potential virulence factor in the host–pathogen system pear–E. amylovora. To study the role of AvrRpt2EA in the Malus × robusta–E. amylovora host–pathogen relationship, the fire blight resistant wild apple genotypes M. baccata, M. fusca, Mr5 and the susceptible apple cv ‘Idared’ were inoculated with the wildtype strain Ea898 and ZYRKD3-1. Results obtained on ‘Idared’ could not support the hypothesis of Zhao et al. (2006), that avrRpt2EA seems to act as a virulence gene in susceptible cultivars, because the deletion mutant caused a similar number of symptoms as the wildtype strain. This indifferent reaction was confirmed by inoculation of the susceptible apple cvs ‘Royal Gala’ and ‘Pinova’, indicating that avrRpt2EA is not indispensable for a strong infection of apple cultivars. The results obtained on Mr5 were completely different. Whereas the wildtype strain Ea898 was not able to infect Mr5, the resistance of Mr5 was broken by the mutant strain which caused a percentage shoot necrosis of > 50% (Figs 2, 3). These results suggest that AvrRpt2EA acts as an avirulence factor in the host–pathogen relationship Mr5 and E. amylovora. Interestingly, the other two resistant wild apple accessions, M. baccata and M. fusca, showed resistance to both the wildtype and the mutant strain, suggesting a different mode of resistance for M. baccata and M. fusca.
The fact that the deletion of the avrRpt2EA gene of E. amylovora results in successful infection of Mr5 is a strong indication of a gene-for-gene relationship in the host–pathogen system Mr5–E. amylovora. In general, plants recognize pathogen effectors, also called avirulence proteins, by resistance proteins and activate a defense cascade. This mode of interaction was first described by Flor (1971) and later supplemented by the guard model (Van Der Biezen & Jones, 1998; Dangl & Jones, 2001), explaining indirect interactions where the avirulence gene is targeted/recognized by a guard. Effectors such as the AvrRpt2EA protein are translocated into plant cells by T3SSs (Collmer et al., 2002). Three T3SSs which are encoded by so-called pathogenicity islands (PAIs) are known in E. amylovora (He et al., 2004; Oh & Beer, 2005). The hypersensitive response and pathogenicity (Hrp) T3SS PAI1 is essential for virulence of fire blight (Bellemann & Geider, 1992; reviewed in Oh & Beer, 2005), whereas the PAI2 and PAI3 T3SSs are dispensable for virulence (Zhao et al., 2009). Besides avrRpt2EA, four more potential effector genes have been identified in the genome of E. amylovora: eop1 (orfB or eopB), eop3 (hopX1Ea), dspA/E and hopPtoCEa (McNally et al., 2012). DspA/E is known as essential pathogenicity factor, as mutants were not able to induce disease symptoms or to grow on host plants (Barny et al., 1990; Gaudriault et al., 1997; Bogdanove et al., 1998). Deletion mutants of eop1, eop3 and hopPtoCEa were also tested on immature pear fruits but did not differ in virulence from the wildtype strain (Zhao et al., 2005; Asselin et al., 2011).
The apparent central function of AvrRpt2EA in the defense mechanism of Mr5 gave cause for further investigations. For that reason, the avrRpt2EA gene of 22 E. amylovora strains (virulent and avirulent to Mr5) was sequenced and the amino acid sequences were deduced. Only one SNP was detected among the 22 sequences that resulted in an amino acid exchange at position 156 from cysteine to serine (C156S). SNP analysis showed that only five out of 75 strains encoded serine at position 156 (Tables 2, Fig. S1). Only strains containing serine on position 156 were able to overcome the resistance of Mr5, whereas strains with cysteine at position 156 were not. Therefore, both were considered as different alleles (C-allele and S-allele). The ability of cysteine to form disulfide bridges could result in a different tertiary structure for the two alleles, thereby modifying the recognition process in Mr5. Results obtained for the P. syringae AvrRpt2 protein in the A. thaliana–P. syringae host–pathogen system support the hypothesis that position 156 of the AvrRpt2EA protein seems to be important for avirulence activity (Lim & Kunkel, 2004).
ZYRKD3-1 was complemented with the C156 and the S156 avrRpt2EA allele driven by its own promoter to verify whether the C156S substitution affects the avirulence activity. The complementation with the C-allele should result in the recovery of resistance in Mr5 if there is a gene-for-gene relationship. On the other hand, ZYRKD3-1 complemented with the S-allele should overcome the resistance of Mr5. RT-PCR of the complemented strains showed that both alleles were expressed (Fig. S2). Virulence analysis was done with the wildtype strain Ea898 (carrying the C-allele), ZYRKD3-1, both complemented versions of ZYRKD3-1 and strains Ea110 and Ea3050 (both carrying the S-allele) on ‘Pinova’, M. baccata jackii, M. floribunda 821, M. baccata, M. fusca, Mr5, ‘Idared’ and ‘Royal Gala’. The mutant strain complemented with the C-allele was not virulent to Mr5, whereas the mutant strain complemented with the S-allele resulted in an average shoot necrosis of 70%, thus breaking down resistance. These results confirm a gene-for-gene relationship in the pathogen system Mr5 and E. amylovora.
The wild apple accessions of M. fusca, M. floribunda and M. baccata are highly resistant to all tested strains, indicating another resistance mechanism. M. baccata jackii has shown a similar pattern to that of Mr5, the mutant strain, as all strains carrying the S-allele were able to overcome the resistance of M. baccata jackii, indicating a similar mechanism. Since M. × robusta is a hybrid between M. prunifolia and M. baccata, this is not surprising.
The demonstrated gene-for-gene relationship and the high degree of similarity between the two effector proteins, AvrRpt2 from P. syringae pv. tomato and AvrRpt2EA from E. amylovora, support the hypothesis of a resistance mechanism in Mr5 similar to the one in A. thaliana. AvrRpt2 is activated in the plant host cytosol by ROC1, a cyclophilin, via prolyl isomerization (Coaker et al., 2006) and is able to cleave RIN4 (RPM1 interacting protein 4). RIN4 is physically associated with RPS2. The cleavage of RIN4 results in the activation of RPS2 (Resistance to P. syringae protein 2) and thereby in the activation of the pathogen defense of the plant (Mackey et al., 2002; Axtell & Staskawicz, 2003; Kim et al., 2005).
We were also able to show in a coexpression assay of both AvrRpt2EA alleles with A. thaliana RIN4 in N. benthamiana that the S-allele was capable of partial elimination of RIN4, whereas the C-allele had no effect on the abundance of the RPS2 target. Two alleles of the avrRpt2EA gene were transiently expressed via A. tumefaciens in N. benthamiana in order to test whether they are able to cause cell death. Similar to P. syringae AvrRpt2, both alleles of E. amylovora AvrRpt2EA elicited a cell death-like response in N. benthamiana. These data are in agreement with the hypothesis that, like that of previous studies (Lim & Kunkel, 2004; Chisholm et al., 2005), RIN4 is not the only target of AvrRpt2EA. Indeed, similar results were obtained by Lim & Kunkel (2004) and Axtell et al. (2003), who demonstrated that mutated avrRpt2 strains were also unable to eliminate RIN4 but still virulent to the host.
In addition to AvrRpt2, RIN4 is known to interact with the effectors AvrRpm1 and AvrB (Mackey et al., 2003), which in turn activate resistance through a second coiled coil-nucleotide-binding site-leucine rich repeat (CC-NBS-LRR) resistance gene, RPM1 (Grant et al., 1995). Thus, RIN4 appears to be a multi-functional target for the regulation and activation of gene-for-gene resistance to a variety of phytopathogenic effector proteins. A putative homolog of AtRIN4 was identified in the transcriptome of unchallenged Malus × robusta 5 (Fahrentrapp, 2012). Two nearly identical RIN4-like genes on the homologous apple chromosomes 5 and 10 have been detected in further investigations (Fig. 9). Furthermore, a putative fire blight resistance gene, FB_Mr5 from Mr5, located at the QTL on LG3 (Peil et al., 2007) was recently published, showing a certain similarity to RPS2 of A. thaliana (Fahrentrapp et al., 2012). This CNL (CC-NBS-LRR) gene was recently identified using a map-based cloning approach (Fahrentrapp et al., 2011). Parravicini et al. (2011) could identify two genes at the fire blight resistance locus of ‘Evereste’, showing high homology to the Pto/Prf complex, indicating a similar resistance mechanism.
This study gives a first indication of how fire blight resistance could function in the pathosystem Malus × robusta 5 and E. amylovora. We demonstrated that AvrRpt2EA, an analog to the effector protein AvrRpt2 from P. syringae pv. tomato, plays an important role in the resistance mechanism of Mr5 and that it is part of a gene-for-gene relationship. The existence of a homologous gene of RIN4 and an Rps2-like resistance gene in the genome of Mr5 assumes a similar resistance mechanism to the one in A. thaliana. This hypothesis is supported by the fact that AvrRpt2EA is able to cleave RIN4 in Arabidopsis. As AvrRpt2EA is not essential for virulence on Malus types other than Mr5, it is of interest that the respective gene is not found in other closely related Erwinia species. While avrRpt2EA is highly conserved among the E. amylovora analyzed here, and even those present in various Rubus isolates (see Table S1), no homolog is present even in closely related Erwinia species. While synteny of the respective genomic region is well conserved in E. amylovora, no similarity to the avrRpt2EA sequence is found in the pear blight pathogen E. pyrifoliae, and the nonpathogenic E. billingiae and E. tasmaniensis. It can be concluded that avrRpt2EA is a recently acquired gene in an evolutionary context.
Further investigations will reveal whether a cleavage of Mr5 Rin4 by the E. amylovora effector AvrRpt2EA is dependent on the respective allele. The functionality of the candidate resistance gene FB_Mr5 has to be proved in a complementation assay. The hypothesis of a similar system as described for A. thaliana–P. syringae could be confirmed by yeast two-hybrid experiments to examine the possible interactions of Fb_Mr5, MrRIN4 and AvrRpt2EA. Another interesting task would be to investigate the resistance mechanism of the two highly resistant wild species M. fusca and M. baccata, which are obviously different from the one of Mr5.
The resistance of the wild apple species Malus × robusta 5 (Mr5) to E. amylovora is highly strain-specific and can be broken only by a few strains originating from North America or the strain isolated from loquat in Israel. Furthermore, an avrRpt2EA deletion mutant, ZYRKD3-1 (Zhao et al., 2006), was able to overcome the resistance of Mr5, which gave cause for further investigations. Subsequently, 22 avrRpt2EA genes from different E. amylovora strains were sequenced and compared. Only one SNP has been identified which resulted in an exchange of cysteine to serine at position 156 in the amino acid sequence. The C-allele was responsible for the diverging resistance responses, because only strains carrying the S-allele were able to overcome the resistance of Mr5. Inoculation of Mr5 with the mutant ZYRKD3-1 complemented with the C- or S-allele resulted in receiving or breaking resistance and gave evidence of a gene-for-gene relationship. The defense mechanism appears to be similar to the one in A. thaliana triggered by the homolog effector AvrRpt2. In a disappearance assay, the S-allele of AvrRptEaA was able to cleave nearly all of the RIN4 of A. thaliana. Besides RIN4, an RPS2-like gene could also be identified in the genome of Mr5. In contrast to Arabidopsis (Cameron et al., 1994), the resistance found in Mr5 is not systemic.