T3SS-dependent differential modulations of the jasmonic acid pathway in susceptible and resistant genotypes of Malus spp. challenged with Erwinia amylovora.

Fire blight is a bacterial disease of Maloideae caused by Erwinia amylovora (Ea). This necrogenic enterobacterium uses a type III secretion system (T3SS) to inject type III effectors into the plant cells to cause disease on its susceptible hosts, including economically important crops like apple and pear. The expressions of marker genes of the salicylic acid (SA) and jasmonic acid (JA) defense regulation pathways were monitored by RT-qPCR in leaves of two apple genotypes, one susceptible and one resistant, challenged with a wild type strain, a T3SS-deficient strain or water. The transcriptional data taken together with hormone level measurements indicated that the SA pathway was similarly induced in both apple genotypes during infection by Ea. On the contrary, the data clearly showed a strong T3SS-dependent down-regulation of the JA pathway in leaves of the susceptible genotype but not in those of the resistant one. Accordingly, methyl-jasmonate treated susceptible plants displayed an increased resistance to Ea. Bacterial mutant analysis indicated that JA manipulation by Ea mainly relies on the type III effector DspA/E. Taken together, our data suggest that the T3SS-dependent down-regulation of the JA pathway is a critical step in the infection process of Malus spp. by Ea.

Dihydrochalcones: Implication in resistance to oxidative stress and bioactivities against advanced glycation end-products and vasoconstriction.

Flavonoids are a group of polyphenol compounds with known antioxidant activities. Among them, dihydrochalcones are mainly found in apple leaves (Malus domestica). Glycosylated dihydrochalcones were previously found in large amounts in leaves of two genotypes of Malus with contrasting resistance to fire blight, a bacterial disease caused by Erwinia amylovora. In the present study we demonstrate that soluble polyphenol patterns comprised phloridzin alone or in combination with two additional dihydrochalcones, identified as sieboldin and trilobatin. Presence of sieboldin in young leaves correlated well with a high 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. Moreover, these leaves displayed enhanced tolerance to paraquat, a photooxidative-stress generating herbicide. Interestingly, phloridzin had a high activity in the oxygen radical absorbance capacity (ORAC) assay, but its presence alone in leaves did not correlate with tolerance to paraquat. In order to further characterise the activity of these compounds, we tested their ability to prevent oxidative-dependent formation of advanced glycation end-products (AGEs) and phenylephrine-induced contraction of isolated rat mesenteric arteries. The antioxidant capacity of sieboldin was clearly demonstrated by showing that this compound (i) prevented vasoconstriction and (ii) inhibited AGEs formation. Both assays provided interesting information concerning a potential use of sieboldin as a therapeutic. Hence, our results strongly argue for a bioactivity of dihydrochalcones as functional antioxidants in the resistance of Malus leaves to oxidative stress. In addition, we demonstrate for the first time that sieboldin is a powerful multipotent antioxidant, effective in preventing physiopathological processes. Further work should aim at demonstrating the potential use of this compound as a therapeutic in treating free radical-involving diseases.

Erwinia amylovora modifies phenolic profiles of susceptible and resistant apple through its type III secretion system.

Fire blight is a disease affecting Maloideae caused by the necrogenic bacterium Erwinia amylovora, which requires the type III protein secretion system (TTSS) for pathogenicity. Profiles of methanol-extractable leaf phenolics of two apple (Malus x domestica) genotypes with contrasting susceptibility to this disease were analyzed by HPLC after infection. Some qualitative differences were recorded between the constitutive compositions of the two genotypes but in both of them dihydrochalcones accounted for more than 90% of total phenolics. Principal component analysis separated leaves inoculated with a virulent wild-type strain from those inoculated with a non-pathogenic TTSS-defective mutant or with water. The changes in levels of the various groups of phenolics in response to the virulent bacterium were similar between the two genotypes, with a significant decrease of dihydrochalcones and a significant increase of hydroxycinnamate derivatives. Differences between genotypes were, however, recorded in amplitude and kinetic of variation in these groups. Occurrence of oxidation and polymerization reactions is proposed, based on the browning process of infected tissues, but whether some by-products act in defense as toxic compounds remain to be tested. Among direct antibacterial constitutive compounds present in apple leaves, the dihydrochalcone phloretin only was found at levels close to lethal concentrations in both genotypes. However, E. amylovora exhibited the ability to stabilize this compound at sublethal levels even in the resistant apple, rejecting the hypothesis of its involvement in the resistance of this genotype.

The alternative sigma factor HrpL negatively modulates the flagellar system in the phytopathogenic bacterium Erwinia amylovora under hrp-inducing conditions.

In this work we present evidence of an opposite regulation in the phytopathogenic bacteria Erwinia amylovora between the virulence-associated Type III secretion system (TTSS) and the flagellar system. Using loss-of-function mutants we show that motility enhanced the virulence of wild-type bacteria relative to a nonmotile mutant when sprayed on apple seedlings with unwounded leaves. Then we demonstrated through analyses of motility, flagellin export and visualization of flagellar filament that HrpL, the positive key regulator of the TTSS, also down-regulates the flagellar system. Such a dual regulation mediated by an alternative sigma factor of the TTSS appears to be a level of regulation between virulence and motility not yet described among Proteobacteria.

Involvement of three pathogenicity factors of Erwinia amylovora in the oxidative stress associated with compatible interaction in pear.

Erwinia amylovora, the causal agent of fire blight of Maloideae, induces in its susceptible host plants an oxidative burst as does an incompatible pathogen. In this paper we present evidence that the elicitation of this phenomenon is the result of the combined action of two Hrp effectors of the bacteria, HrpN and DspA. We also confirmed that desferrioxamine, the siderophore of E. amylovora, is necessary for the bacteria to tolerate high levels of hydrogen peroxide. Two other pathogenicity factors of the bacteria, the HrpW effector and the capsule, do not seem to play any role in the elicitation of the oxidative burst nor in the protection of the bacteria.

Modulation of defense responses of Malus spp. during compatible and incompatible interactions with Erwinia amylovora.

Erwinia amylovora is the causal agent of fire blight, a disease affecting members of subfamily Maloideae. In order to analyze mechanisms leading to compatible or incompatible interactions, early plant molecular events were investigated in two genotypes of Malus with contrasting susceptibility to fire blight, after confrontation with either E. amylovora or the incompatible tobacco pathogen Pseudomonas syringae pv. tabaci. Many defense mechanisms, including generation of an oxidative burst and accumulation of pathogenesis-related proteins, were elicited in both resistant and susceptible genotypes by the two pathogens at similar rates and according to an equivalent time course. This elicitation was linked with the functional hypersensitive reaction and pathogenicity (hrp) cluster of E. amylovora, because an hrp secretion mutant did not induce such responses. However, a delayed induction of several genes of various branch pathways of the phenylpropanoid metabolism was recorded in tissues of the susceptible genotype challenged with the wild-type strain of E. amylovora, whereas these genes were quickly induced in every other plant-bacteria interaction, including interactions with the hrp secretion mutant. This suggests the existence of hrp-independent elicitors of defense in the fire blight pathogen as well as hrp-dependant mechanisms of suppression of these nonspecific inductions.

Rapid Diagnosis of Pseudomonas syringae pv. papulans, the Causal Agent of Blister Spot of Apple, by Polymerase Chain Reaction Using Specifically Designed hrpL Gene Primers.

ABSTRACT The identification and detection of Pseudomonas syringae pv. papulans, the causal agent of blister spot of apple, on apple leaves and fruit was achieved by polymerase chain reaction amplification of a specific DNA fragment of the hrpL sequence. The consensus primers hrpL(1) and hrpL(2) were designed based on the alignment of pseudomonad hrpL gene sequences available in nucleic acid data banks. This primer set produced a 631-bp amplicon from 37 of the 57 pseudomonads strains tested. These strains belonged to genomospecies 1 and 2, as described by Gardan et al. (8). The amplicon obtained from 30 of these strains was digested with eight restriction enzymes. Three different restriction patterns were produced from strains belonging to genomospecies 1, resulting in A1 and A2 patterns, while strains belonging to genomospecies 2 were characterized by a B pattern. Patterns A1 and A2 differed at only two sites, a Bsp 143I site located at nucleotide 360 and a MseI site located at nucleotides 22-24. Group A2 consisted solely of P. syringae pv. papulans strains. The hrpL gene in P. syringae pv. papulans strain CFBP3323 was sequenced. Two primer sets, Pap1/Pap2 and Pap1/Pap3, were designed and tested for specificity to P. syringae pv. papulans. These primers amplified expected fragments of 242 and 303 bp, respectively. Pap1/Pap2 amplified a fragment only with P. syringae pv. papulans DNA, while Pap1/Pap3 amplified all tested strains belonging to genomospecies 1. A diagnostic procedure using the Pap1/Pap2 primer set was successful for the detection of P. syringae pv. papulans in diseased fruit and artificially inoculated leaves.

The DspB/F protein of Erwinia amylovora is a type III secretion chaperone ensuring efficient intrabacterial production of the Hrp-secreted DspA/E pathogenicity factor.

Summary Erwinia amylovora is a Gram-negative bacterium responsible for fire blight, a necrotic disease affecting plants of the Rosaceae family. E. amylovora virulence is dependent on a functional type III secretion system. To date, four proteins have been shown to travel through this secretion system: HrpN, HrpW, HrpA, and DspA/E. Next to dspA/E, dspB/F encodes a small acidic protein sharing features similar to those of type III secretion chaperones described in animal systems. Here, we show that DspA/E was not secreted in a dspB/F background, while other known type III secreted proteins (HrpN, HrpW, and HrpA) remained secreted to wild-type level. A functional copy of dspB/F introduced in trans in a dspB/F background restored DspA/E secretion. Further analysis showed that DspA/E was not detected in a dspB/F background. This effect was post-transcriptional as the expression of a dspA/E::uidA transcriptional fusion was not reduced in a dspB/F background. Affinity blot analysis experiments demonstrated that DspB/F specifically interacts with DspA/E in vitro. Therefore, DspB/F acts as a specific DspA/E chaperone and DspB/F prevents intrabacterial DspA/E degradation. dspB/F mutants were found to retain some pathogenicity to pear seedlings. This phenotype contrasts with the non-pathogenic phenotype of the dspA/E mutant and suggests that the DspA/E protein still transits through the type III secretion machinery in a dspB/F mutant even though it is not detected in vitro.

Molecular Detection and Differentiation of Erwinia pyrifoliae and Host Range Analysis of the Asian Pear Pathogen.

The recently described pathogen Erwinia pyrifoliae, isolated from Nashi pear fruit trees in Korea, resembles the fire blight pathogen Erwinia amylovora in some of its properties. The two pathogens were classified into different species by DNA hybridization kinetics and microbiological criteria. From the nucleotide sequences of the 16S rRNA and the internal transcribed spacer (ITS) region as well as extracellular polysaccharide (EPS)-encoding genes, polymerase chain reaction (PCR) primers were designed that specifically detect E. pyrifoliae but not the fire blight pathogen Erwinia amylovora, and these primers were also applied to identify E. pyrifoliae in necrotic plant material. The genomes of several strains were digested with the restriction enzyme SpeI, and the DNA fragments were analyzed by pulsed-field gel electrophoresis (PFGE). Three groups of patterns could be distinguished for the isolated E. pyrifoliae strains, all different from various E. amylovora strains, which produce a relatively homogeneous PFGE pattern after SpeI digests. Typical fire blight host plants were assayed in a growth chamber or an experimental field for their susceptibility to E. pyrifoliae. A strong preference was found for pear varieties, whereas apple, cotoneaster, hawthorn, or raspberry rarely produced necrotic symptoms. E. pyrifoliae was readily detected in samples from pear orchards in South Korea during 1995 to 1998; however, the Asian pear pathogen was not recovered in necrotic plant tissue from 1999 and 2000.

Fire blight protection with avirulent mutants of Erwinia amylovora.

Fire blight is a necrotic disease caused by the bacterium Erwinia amyiovora, which affects pears, apples and ornamentals including Crataegus, Pyracantha, and Cotoneaster. The disease can be only partially controlled, through the use of resistant genotypes, cultural measures and antibacterial compounds, thus other methods must be investigated. It has long been established that avirulent isolates of the pathogen can control the disease, under experimental conditions. However, field use of avirulent isolates is not acceptable because of their unknown genetic stability. The protective ability under controlled conditions of genetically characterized avirulent insertion mutants of E. amylovora was examined. A bioassay on apple seedlings was used for the determination of the protective ability of 34 insertion mutants (hrp, dsp, ams). Some protective effect could be observed with most of the mutants tested and was dependent on the avirulent/virulent inoculum ratio as well as on the level of virulence of the pathogen; a minimal concentration of the avirulent mutant was necessary to give a significant level of protection. An early competition between avirulent and virulent strains for putative infection sites might be involved. For six of the mutants tested, the protective ability was particularly high and might be related to the alteration of regulatory functions of hrp genes. Results obtained with Ams- and Ams- Hrp- mutants suggested that the bacterial exopolysaccharide might play a role in the protection.