Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC‐NBS‐LRR resistance gene of Malus × robusta 5

The fire blight susceptible apple cultivar Malus × domestica Borkh. cv. ‘Gala’ was transformed with the candidate fire blight resistance gene FB_MR5 originating from the crab apple accession Malus × robusta 5 (Mr5). A total of five different transgenic lines were obtained. All transgenic lines were shown to be stably transformed and originate from different transgenic events. The transgenic lines express the FB_MR5 either driven by the constitutive CaMV 35S promoter and the ocs terminator or by its native promoter and terminator sequences. Phenotyping experiments were performed with Mr5‐virulent and Mr5‐avirulent strains of Erwinia amylovora, the causal agent of fire blight. Significantly less disease symptoms were detected on transgenic lines after inoculation with two different Mr5‐avirulent E. amylovora strains, while significantly more shoot necrosis was observed after inoculation with the Mr5‐virulent mutant strain ZYRKD3_1. The results of these experiments demonstrated the ability of a single gene isolated from the native gene pool of apple to protect a susceptible cultivar from fire blight. Furthermore, this gene is confirmed to be the resistance determinant of Mr5 as the transformed lines undergo the same gene‐for‐gene interaction in the host–pathogen relationship Mr5–E. amylovora.     

In vitro pollen functionality of attacin-transgenic “Royal Gala” apple plants and apples transformed with 1-aminocyclopropane-1-carboxylic acid synthase (ACS)-antisense vector

Abstract

To assess pollen functionality of transgenic apple trees, in vitro pollen germination and tube growth were evaluated. Flowers of transgenic “Royal Gala” apple lines containing attacin E gene to confer resistance to fire blight (Erwinia amylovora), or antisense 1-aminocyclopropane-1-carboxylic acid synthase (ACS) construct to improve fruit storage life, were collected, and pollen was harvested. Amongst the 19 transgenic lines, pollen from three lines transformed with an ACS-antisense vector consistently had significantly lower germination rate compared to “Royal Gala”; however, no correlation between ACS level in fruit and pollen germination rate was observed. Western blots showed that the amounts of the lytic protein, attacin, varied in pollen of the four attacin-transgenic lines sampled. There was no significant correlation between attacin level in the pollen and pollen germination rate or pollen tube growth. The addition of boric acid to the germination buffer enhanced germination in attacin-transgenic lines, as well as in lines down-regulated in ethylene synthesis and control “Royal Gala”. This initial study suggests that the majority of transgenic lines tested do not differ from the control “Royal Gala” in pollen germination, and that attacin or down-regulation of ethylene does not influence in vitro pollen functionality.     

Expression of bovine lactoferrin cDNA confers resistance to Erwinia amylovora in transgenic pear

The siderophore produced by Erwinia amylovora, the causal agent of fire blight of Maloideae, is one of the virulence factors of this bacterium. The production of siderophores enables E. amylovora to overcome the conditions of iron limitation met in plant tissue, and may also protect the bacteria against active oxygen species produced through the Fenton reaction. In this paper, we have examined the ability of an iron chelator protein, encoded by the bovine lactoferrin gene, to reduce fire blight susceptibility in pear (Pyrus communis L.). Transgenic pear clones expressing this gene controlled by the CaMV35S promoter were produced by Agrobacterium tumefaciens mediated transformation. Transformants were analysed by RT-PCR and western blot to determine lactoferrin expression levels. Most transgenic clones demonstrated significant reduction of susceptibility to fire blight in vitro and in the greenhouse when inoculated by E. amylovora. These transgenic clones also showed a significant reduction of symptoms when inoculated with two other pear bacterial pathogens : Pseudomonas syringae pv. syringae and Agrobacterium tumefaciens. Moreover, we have shown that this increase in bacterial resistance was correlated with an increase in root ferric reductase level activity and leaf iron content. Despite negative effects on the growth of a few clones, our results indicate the potential of lactoferrin gene transformation to protect pear from fire blight through increased iron chelation.     

Phenotypic and genetic diversity of Erwinia amylovora: the causal agent of fire blight

Erwinia amylovora is a polyphagous bacterium causing fire blight on apple, pear and over 130 other plant species belonging mainly to the Rosaceae family. Although E. amylovora is regarded as a very homogenous species, the particular strains can differ in pathogenic ability as far as their host range is concerned (e.g. those originating from Rubus or Maloidae plants) as well as by the extent of the disease they cause. It was found that strains originating from North America are generally more genetically heterogeneous than those from Europe. Diversity of E. amylovora is also related to streptomycin resistance as a result of its application to control of fire blight. The level of genetic heterogeneity of E. amylovora is so low (comparative genome analysis revealed a similarity of over 99% for the two genomes tested) that standard DNA-based techniques fail in detection of intra-species variability. Amplified fragment length polymorphism was found to be most useful for differentiation of strains of fire blight causal agent as well as techniques ensuing release of pan-genome sequences of two E. amylovora strains: multi-locus variable number of tandem repeats analysis and clustered regularly interspaced short palindrome repeats.     

Expression of a bacterial effector,harpin N,causes increased resistance to fire blight in <Emphasis Type="Italic">Pyrus communis</Emphasis>

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. In order to create novel mechanisms for fire blight resistance in pear, we have generated transgenic pears expressing the elicitor harpin N_ea from Erwinia amylovora under the control of the constitutive promoter CaMV35S. The transient expression of hrpN _Ea in pear cells did not provoke any apparent damage. Therefore, stable constitutive expression of hrpN _Ea was studied in seventeen transgenic clones of the very susceptible cultivar “Passe Crassane.” Most transgenic clones displayed significant reduction of susceptibility to fire blight in vitro when inoculated by E. amylovora, which was positively correlated to their degree of expression of the transgene hrpN _Ea . These results indicate that ectopic expression of a bacterial elicitor such as harpin N_ea is a promising way to improve pear resistance to fire blight.     

Identification of genes differentially expressed during interaction of resistant and susceptible apple cultivars (Malus × domestica) with Erwinia amylovora

Background  

The necrogenic enterobacterium, Erwinia amylovora is the causal agent of the fire blight (FB) disease in many Rosaceaespecies, including apple and pear. During the infection process, the bacteria induce an oxidative stress response with kinetics similar to those induced in an incompatible bacteria-plant interaction. No resistance mechanism to E. amylovora in host plants has yet been characterized, recent work has identified some molecular events which occur in resistant and/or susceptible host interaction with E. amylovora: In order to understand the mechanisms that characterize responses to FB, differentially expressed genes were identified by cDNA-AFLP analysis in resistant and susceptible apple genotypes after inoculation with E. amylovora.     

Anti-attacin polyclonal antibody from an in vitro derived antigen used for immunoblot to quantify attacin expressed in transgenic apple

A gene encoding attacin E, an inducible antibacterial protein from Hyalophora cecropia pupae, was cloned into the pRSETB Escherichia coli expression vector under the control of the T7 promoter. The resulting vector, pRSETBAtt, produced a fusion protein in E. coli JM109 of attacin with an N-terminal peptide containing six histidine residues in tandem. Fusion attacin was purified from cell lysates (6–9 mg l^–1) by Ni²⁺-Sepharose affinity chromatography. Purified attacin protein was used as antigen to produce polyclonal antibody to detect attacin expressed in transgenic apple. Antibody capture immunoassay and immunoblot assays indicated that polyclonal antisera derived from fusion attacin had specific immunoreaction against attacins in the hemolymph of immunized pupae and attacin expressed in transgenic apple lines similar to native attacin antisera. Attacin expressed in transgenic apple could be quantified using immunoblot assays with the fusion attacin polyclonal antibody.     

Survival and Possible Spread of Erwinia amylovora and Related Plant-Pathogenic Bacteria Exposed to Environmental Stress Conditions

The fire blight pathogen Erwinia amylovora was assayed for survival under unfavourable conditions such as on nitrocellulose filters, in non‐host plants as well as in inoculated mature apples and in infested apple stem sections. In a sterile dry environment, an E. amylovora EPS (exopolysaccharide) mutant, and to a lesser extent its parental wild‐type strain decreased within 3 weeks to a low titre. However, under moist conditions the decrease of viable cells occurred only partially for both strains. Very low cell titres were recovered after application of E. amylovora onto the surface of tobacco leaves, whereas infiltration into the leaves produced lesions (hypersensitive response, HR), in which the bacteria survived in significant amounts. A similar effect was found for the necrotic zones of HR in tobacco leaves caused by E. pyrifoliae, by Pseudomonas syringae pathovars and HR‐deficient E. amylovora mutants or mutants deficient in EPS synthesis and disease‐specific genes. During 7 years of storage, the viability of E. amylovora in wood sections from fire blight‐infested apple trees declined to a low titre. In tissue of mature apples, E. amylovora cells slowly dispersed and could still be recovered after several weeks of storage at room temperature. A minimal risk of accidental dissemination of E. amylovora apart from infested host plants can experimentally not be excluded, but other data confirm a very low incidence of any long distance distribution.     

Hypersensitive response and acyl‐homoserine lactone production of the fire blight antagonists Erwinia tasmaniensis and Erwinia billingiae

Fire blight caused by the Gram‐negative bacterium Erwinia amylovora can be controlled by antagonistic microorganisms. We characterized epiphytic bacteria isolated from healthy apple and pear trees in Australia, named Erwinia tasmaniensis, and the epiphytic bacterium Erwinia billingiae from England for physiological properties, interaction with plants and interference with growth of E. amylovora. They reduced symptom formation by the fire blight pathogen on immature pears and the colonization of apple flowers. In contrast to E. billingiae, E. tasmaniensis strains induced a hypersensitive response in tobacco leaves and synthesized levan in the presence of sucrose. With consensus primers deduced from lsc as well as hrpL, hrcC and hrcR of the hrp region of E. amylovora and of related bacteria, these genes were successfully amplified from E. tasmaniensis DNA and alignment of the encoded proteins to other Erwinia species supported a role for environmental fitness of the epiphytic bacterium. Unlike E. tasmaniensis, the epiphytic bacterium E. billingiae produced an acyl‐homoserine lactone for bacterial cell‐to‐cell communication. Their competition with the growth of E. amylovora may be involved in controlling fire blight.     

Screening for fire blight resistance in apple (Malus pumila) using excised leaf assays from in vitro and in vivo grown material

The apple cultivars Greensleeves and Novole and somaclones derived from Greensleeves were tested for resistance to fire blight (Erwinia amylovora) using excised leaf bioassays. Cv. Greensleeves was consistently susceptible to fire blight in in vitro, and in glasshouse and excised leaf bioassays whilst cv. Novole was resistant. Greensleeves somaclones, identified as having intermediate levels of fire blight resistance in glasshouse and in vitro screening tests, also showed intermediate levels of resistance in excised leaf bioassays. The length of leaves and the inoculum concentration employed affected the severity of symptoms observed. Excised leaf bioassays were unsuitable as tests for fire blight resistance using field grown material.     

Identification of a major QTL together with several minor additive or epistatic QTLs for resistance to fire blight in apple in two related progenies

Although fire blight, caused by the bacterium Erwinia amylovora, is one of the most destructive diseases of apple (Malus × domestica) worldwide, no major, qualitative gene for resistance to this disease has been identified to date in apple. We conducted a quantitative trait locus (QTL) analysis in two F₁ progenies derived from crosses between the cultivars Fiesta and either Discovery or Prima. Both progenies were inoculated in the greenhouse with the same strain of E. amylovora, and the length of necrosis was scored 7 days and 14 days after inoculation. Additive QTLs were identified using the mapqtl software, and digenic epistatic interactions, which are an indication of putative epistatic QTLs, were detected by two-way analyses of variance. A major QTL explaining 34.3–46.6% of the phenotypic variation was identified on linkage group (LG) 7 of Fiesta in both progenies at the same genetic position. Four minor QTLs were also identified on LGs 3, 12 and 13. In addition, several significant digenic interactions were identified in both progenies. These results confirm the complex polygenic nature of resistance to fire blight in the progenies studied and also reveal the existence of a major QTL on LG7 that is stable in two distinct genetic backgrounds. This QTL could be a valuable target in marker-assisted selection to obtain new, fire blight-resistant apple cultivars and forms a starting point for discovering the function of the genes underlying such QTLs involved in fire blight control.     

A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmids

Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents.     

Evidence of Greater Competitive Fitness of Erwinia amylovora over E. pyrifoliae in Korean Isolates

Erwinia amylovora and E. pyrifoliae are the causative agents of destructive diseases in both apple and pear trees viz. fire blight and black shoot blight, respectively. Since the introduction of fire blight in Korea in 2015, the occurrence of both pathogens has been independently reported. The co-incidence of these diseases is highly probable given the co-existence of their pathogenic bacteria in the same trees or orchards in a city/district. Hence, this study evaluated whether both diseases occurred in neighboring orchards and whether they occurred together in a single orchard. The competition and virulence of the two pathogens was compared using growth rates in vitro and in planta. Importantly, E amylovora showed significantly higher colony numbers than E. pyrifoliae when they were co-cultured in liquid media and co-inoculated into immature apple fruits and seedlings. In a comparison of the usage of major carbon sources, which are abundant in immature apple fruits and seedlings, E. amylovora also showed better growth rates than E. pyrifoliae. In virulence assays, including motility and a hypersensitive response (HR), E. amylovora demonstrated a larger diameter of travel from the inoculation site than E. pyrifoliae in both swarming and swimming motilities. E. amylovora elicited a HR in tobacco leaves when diluted from 1:1 to 1:16 but E. pyrifoliae does not elicit a HR when diluted at 1:16. Therefore, E. amylovora was concluded to have a greater competitive fitness than E. pyrifoliae.     

Growth inhibition of Erwinia amylovora and related Erwinia species by neutralized short-chain fatty acids

Short-chain fatty acids (SCFAs) are used to preserve food and could be a tool for control of fire blight caused by Erwinia amylovora on apple, pear and related rosaceous plants. Neutralized acids were added to buffered growth media at 0.5–75 mM and tested at pHs ranging from 6.8 to 5.5. Particularly at low pH, SCFAs with a chain length exceeding that of acetic acid such as propionic acid were effective growth inhibitors of E. amylovora possibly due to uptake of free acid and its intracellular accumulation. We also observed high inhibition with monochloroacetic acid. An E. billingiae strain was as sensitive to the acids as E. amylovora or E. tasmaniensis. Fire blight symptoms on pear slices were reduced when the slices were pretreated with neutralized propionic acid. Propionic acid is well water soluble and could be applied in orchards as a control agent for fire blight.     

Effects of codon modification on human BMP2 gene expression in tobacco plants

Bone morphogenetic protein 2 (BMP2) has great potential in therapeutic applications. We are working on generating transgenic plants as a bioreactor to produce BMP2. We have studied the effects of codon optimization on the expression of human BMP2 (hBMP2) in tobacco plants. Three modified hBMP2 genes were transformed into tobacco under the control of either cauliflower mosaic virus 35S (CaMV35S) promoter or double-CaMV35S promoter plus alfalfa mosaic virus (AMV) enhancer. The fused β-glucuronidase (GUS) reporter gene was used to facilitate the assay of protein expression. The results indicated that codon optimization could increase the protein expression level obviously under CaMV35S promoter. However, under relatively stronger initiation condition (double-CaMV35S promoter plus AMV enhancer), only the gene with the lowest degree of codon optimization could increase the protein expression level. Our findings suggest that the action of codon optimization may be influenced by the factors of promoter strength and A+T content in tobacco plants.     

QTL mapping of fire blight resistance in Malus ×robusta 5 after inoculation with different strains of Erwinia amylovora

Fire blight caused by Erwinia amylovora is one of the most disastrous diseases in apple production. Whereas most apple cultivars are susceptible to fire blight, several wild apple species accessions like Malus ×robusta 5 (Mr5) bear significant resistance. The resistance of Mr5 is mainly inherited by a major quantitative trait locus (QTL) on linkage group 3. QTL mapping was performed after inoculation of the population 04208 (Idared × Mr5) using strains differing in their virulence to Mr5. The QTL mapping approach demonstrated that the major QTL on linkage group 3 could be confirmed after inoculation with strains non-virulent to Mr5. In contrast, the major QTL disappeared after inoculation with strains virulent to Mr5. Only after inoculation with the resistance breaking strain Ea 3049 was a minor QTL with a LOD >3 found on linkage group 3. Additionally, several minor QTLs were detected on linkage groups 5, 7, 11 and 14 of Mr5 after inoculation with virulent strains able to overcome the major resistance QTL of Mr5. Their usefulness for further breeding activities will be discussed. The strain-specific results obtained in the present study provide further evidence for the existence of gene-for-gene relationships in the host–pathogen system Mr5–E. amylovora. Of the newly discovered minor QTLs, the one detected on LG7 contributes significantly to fire blight resistance in the presence of the major QTL, independently of the strain used.