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.     

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.     

The role of luxS in the fire blight pathogen Erwinia amylovora is limited to metabolism and does not involve quorum sensing

Erwinia amylovora is a gram-negative phytopathogen that causes fire blight of pome fruit and related members of the family Rosaceae. We sequenced the putative autoinducer-2 (AI-2) synthase gene luxS from E. amylovora. Diversity analysis indicated that this gene is extremely conserved among E. amylovora strains. Quorum sensing mediated by LuxS has been implicated in coordinated gene expression, growth, and virulence in other enterobacteria; however, our evidence suggests this is not the function in E. amylovora. Mutational analysis pointed to a role in colonization of apple blossoms, the primary infection court for fire blight, although little if any role in virulence on apple shoots and pear fruit was observed. Expression of key virulence genes hrpL and dspA/E was reduced in mutants of two E. amylovora strains. Stronger effects on gene expression were observed for metabolic genes involved in the activated methyl cycle with mutants having greater levels of expression. No quorum-sensing effect was observed in coculture experiments with wild-type and mutant strains either in vitro or in apple blossoms. Known receptors essential for AI-2 quorum sensing, the LuxPQ sensor kinase or the Lsr ABC-transporter, are absent in E. amylovora, further suggesting a primarily metabolic role for luxS in this bacterium.     

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.     

Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen

The disease-specific (dsp) gene dspA/E of Erwinia amylovora encodes an essential pathogenicity effector of 198 kDa, which is critical to the development of the devastating plant disease fire blight. A yeast two-hybrid assay and in vitro protein pull-down assay demonstrated that DspA/E interacts physically and specifically with four similar putative leucine-rich repeat (LRR) receptor-like serine/threonine kinases (RLK) from apple, an important host of E. amylovora. The genes encoding these four DspA/E-interacting proteins of Malus xdomestica (DIPM1 to 4) are conserved in all genera of hosts of E. amylovora tested. They also are conserved in all cultivars of apple tested that range in susceptibility to fire blight from highly susceptible to highly resistant. The four DIPMs have been characterized, and they are expressed constitutively in host plants. In silico analysis indicated that the DIPMs have similar sequence structure and resemble LRR RLKs from other organisms. Evidence is presented for direct physical interaction between DspA/E and the apple proteins encoded by the four identified clones, which may act as susceptibility factors and be essential to disease development. Knowledge of DIPMs and the interaction with DspA/E thus may facilitate understanding of fire blight development and lead to new approaches to control of disease.     

Inactivation of polycaprolactone depolymerase (cutinase) in Fusarium cultures by an extracellular protease

Cultures of Fusarium moniliforme grown on polycaprolactone (PCL) or on cutin as a sole source of carbon and energy had low levels of detectable PCL depolymerase (cutinase) activity in the supernatant medium. A small peak of depolymerase activity was observed after hyphal accumulation had ceased, but this activity soon declined. The low level of the peak of activity and its decline were attributable to proteolytic inactivation of the depolymerase. A decrease in the pH of cultures coincided with the appearance of protease activity in the supernatant at about the same time as the appearance of the transient peak of depolymerase activity. Addition of protease substrates (bovine serum albumin, casein) to the culture at this time caused a dramatic although temporary increase in PCL depolymerase activity. The same effect was seen for cultures of F. solani pisi. Use of a different buffer system for the medium prevented a drop in pH and resulted in higher and stable levels of PCL depolymerase activity. Received 1 July 1998/ Accepted in revised form 6 December 1998     

<Emphasis Type="Italic">Arabidopsis rd29A::DREB1A</Emphasis> enhances freezing tolerance in transgenic potato

The freezing tolerance of 38 independent transgenic potato lines derived from the cultivar Desiree was tested in vitro using plantlets. The lines were transgenic for the DREB1A gene under control of the rd29A promoter, both of which were derived from Arabidopsis thaliana. The level of damage caused by freezing varied significantly among the transgenic clones and a non-transgenic control (cv. Desiree). Phenotypic evaluation indicated that the variable responses to freezing were attributable to genotypic variation, but freezing tolerance was not dependent on the number of insertions. Northern blot analysis using a DREB1A cDNA probe revealed high levels of DREB1A expression among the transgenic clones during the initial cold exposure at 4°C (after 2 h) and in the early stages of freezing (−20°C, 1–10 min). Furthermore, a linear correlation was detected between the level of expression and the phenotypic response for all lines except D138. Thus, in the case of potato, a significant increase in freezing tolerance was observed in vitro on a small scale following the introduction of rd29A::DREB1A. Additional testing will show whether this strategy can be used for tolerance breeding in potato and to increase the freezing tolerance of other agriculturally important crops. Babak Behnam and Akira Kikuchi equally contributed for this work.     

Isolation of a Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterium from compost, and cloning and characterization of a gene encoding PHB depolymerase of Bacillus megaterium N-18-25-9

A Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterial strain was isolated from compost. This organism, identified as Bacillus megaterium N-18-25-9, produced a clearing zone on opaque NB-PHB agar, indicating the presence of extracellular PHB depolymerase. A PHB depolymerase gene, PhaZ(Bm), of B. megaterium N-18-25-9 was cloned and sequenced, and the recombinant gene product was purified from Escherichia coli. The N-terminal half region of PhaZ(Bm) shared significant homologies with a catalytic domain of other PHB depolymerases. Although the C-terminal half region of PhaZ(Bm) showed no significant similarity with those of other PHB depolymerases, that region was necessary for the PHB depolymerase activity. Therefore, this enzyme's domain structure is unique among extracellular PHB depolymerase domain structures. The addition of PHB to the medium led to a sixfold increase in PhaZ(Bm) mRNA, while the presence of glucose repressed PhaZ(Bm) expression. The maximum activity was observed at pH 9.0 at 65 degrees C.     

Reduction in bacterial ooze formation on immature fruitlets after preventive treatments of Fosethyl-Al against fire blight Erwinia amylovora

Fire blight, caused by the bacterium Erwinia amylovora (Burill Winslow et al.), is a very important bacterial disease on apple and pear orchards with devastating effects in some production area and in some years. Fire blight control consists in a whole strategy of measures that should start with control measures in and around the fruit tree nurseries. Only the use of Vacciplant (Laminarin), an inducer of the self-defence mechanism, is registered in Belgium since 2009. In other European countries Fosethyl-Al has been registered for fire blight control. Recently, research trials have been done at Pcfruit research station for several years on the activity of ALiette (fosethyl-Al) against fire blight. Fosethyl-Al, also a plant defence enhancing molecule, applied preventively 3 times at a dose of 3.75 kg/ha standard orchard (3 x 3000 g a.i./ha standard orchard), showed a reduction in the host susceptibility and decreased the disease development on artificial inoculated flower clusters and shoots. Also a clear reduction in the ooze droplet formation on artificially inoculated immature fruitlets has been observed with this molecule. This reduction in the bacterial ooze formation is considered as a very important factor in the spread of the disease in the orchard.     

Substrate specificity and contribution of the glycosyltransferase UGT71A15 to phloridzin biosynthesis

The dihydrochalcone phloridzin (phloretin 2′-O-glucoside) is the most abundant phenolic compound in apple trees (Malus × domestica) and was also discussed to have an influence on the pathogen defence by shifting the dihydrochalcone profile from the glucosides to the more active aglycones. The final step in the biosynthesis of phloridzin is the glycosylation of phloretin at position 2′. Three cDNA clones from apple encoding glycosyltransferases are available which are able to catalyze the reaction in vitro. We investigated the possible role of glycosyltransferase UGT71A15 in phloridzin biosynthesis. The recombinant enzyme showed broad substrate acceptance but highest activities were observed with flavonols. Specific activities and the kinetic data indicated that phloretin is not the preferred native substrate of the UGT71A15. However, an increase of the molar ratio phloridzin:phloretin was found in transgenic lines, indicating a physiological relevance of UGT71A15 in planta, although a decrease of the total amount of dihydrochalcones in the majority of the samples was found. Unexpectedly, the increase of the phloridzin:phloretin ratio was not reflected by an increase of the total glucosyltransferase activities. In contrast, the majority of transgenic plants showed a reduced glucosylating activity with both phloretin and quercetin as a substrate, but the observed activity changes in a given sample were not similar for the two substrates. An increased susceptibility of M. robusta against the fire blight causing bacterium E. amylovora as a result of UGT71A15 overexpression could not be observed. Overexpression of UGT71A15 in transgenic apple trees also did not lead to morphological changes.     

Variations in the molecular masses of the capsular exopolysaccharides amylovoran, pyrifolan and stewartan

Erwinia amylovora, causing fire blight of apple, pear and some ornamentals, Erwinia pyrifoliae, causing Asian pear blight, and Pantoea stewartii, causing Stewart's wilt of sweet maize, synthesize capsular extracellular polysaccharides (EPSs) with a high molecular mass. The EPSs are virulence factors and form viscous aggregates, which participate in clogging vessels of infected plants and causing wilting. The sizes of EPSs produced under different environmental growth conditions were determined by analysis with large pore HPLC columns. Their molecular mass of ca. 5 MDa, when isolated from agar plates, decreases to ca. 1 MDa for E. amylovora amylovoran from freeze-dried supernatants from liquid cultures and to 2 MDa for freeze-dried preparations of P. stewartii stewartan. Size changes were also found following growth in various other media and for different strains. Stewartan, amylovoran and E. pyrifoliae pyrifolan were also shown to be completely degraded by a bacteriophage EPS depolymerase.     

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.     

Yield,Yield‐related Traits and Response of Potato Clones to Late Blight Disease,in North‐Western Highlands of Ethiopia

Late blight disease of potato caused by Phytophthora infestans poses a significant threat to potato production in Ethiopia. The development of new high yielding genotypes with adequate late blight disease resistance will provide a strong component of an integrated management strategy for farmers. The objective of this study was to determine late blight resistance and yield of potato clones under field condition in north‐western Ethiopia. Twenty‐four clones (17 from the International Potato Centre B3C2 population and seven widely grown cultivars) were evaluated at three locations. The experiment was laid in a randomized complete block design with two replications. Late blight resistance and yield‐related traits were determined. Results showed that clones differ significantly for all traits across locations. The following five clones combine high to moderate resistance to late blight with high yields: 396029.250, 395017.229, 396004.263, 396034.103 and 395077.12. These clones are useful genetic resources for resistance breeding against late blight disease and for enhanced yields.     

Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering

 In order to test the possibility of enhancing the production of pharmaceutically valuable scopolamine in transgenic cultures, the 35S-h6h transgene that codes for the enzyme hyoscyamine-6β-hydroxylase (EC 1.14.11.11) was introduced into Hyoscyamus muticus L. strain Cairo (Egyptian henbane). This plant was chosen for its capability to produce very high amounts of tropane alkaloids (up to 6% of the dry weight in the leaves of mature plant). To our knowledge, this is the first time such a large population of transgenic cultures has been studied at the morphological, chemical and genetic levels. A great variation was observed in the tropane alkaloid production among the 43 positive transformants. The best clone (KB7) produced 17 mg/l scopolamine, which is over 100 times more than the control clones. However, conversion of hyoscyamine to scopolamine was still incomplete. The expression of h6h was found to be proportional to the scopolamine production, and was the main reason behind the variation in the scopolamine/hyoscyamine ratio in the hairy-root clones. These results indicate that H. muticus strain Cairo has a potential for even more enhanced scopolamine production with more efficient gene-expression systems. Received: 24 December 1998 / Accepted: 13 January 1999     

抗茎腐病转基因小麦新种质的筛选

为拓宽小麦茎腐病(又称茎基腐病)抗源种类,筛选抗茎腐病小麦新种质,对43份转TaPIMP1、AtNPR1和Gastrodianin基因小麦纯合株系,进行目的基因表达分析,以及茎腐病、纹枯病和赤霉病抗性鉴定。结果表明,转基因株系的目的基因均能正常表达;转基因株系间茎腐病抗性差异明显,24份转基因株系茎腐病抗性,比受体对照扬麦12显著提高;转基因株系茎腐病抗性与纹枯病抗性相关性显著,与赤霉病相关性不显著。结合农艺性状鉴定,筛选出5份抗茎腐病转基因株系,其中2份兼抗纹枯病和赤霉病,1份兼抗纹枯病,可作为长江中下游麦区茎腐病备用抗源。     

QTL mapping of fire blight resistance in apple

Fire blight caused by the bacterium Erwinia amylovora is a severe threat to apple and pear orchards worldwide. Apple varieties exhibit a wide range of relative susceptibility/tolerance to fire blight. Although, no monogenic resistance against fire blight has been identified yet, recent evidence indicates the existence of quantitative resistance. Potential sources of fire blight resistance include several wild Malus species and some apple cultivars. F1 progenies of ‘Fiesta’בDiscovery’ were inoculated with the Swiss strain Ea 610 and studied under controlled conditions to identify quantitative trait loci (QTLs) for fire blight resistance. Disease was evaluated at four time points after inoculation. Shoot lesion length and the area under disease progress curve (AUDPC) values were used for QTL analysis. One significant (LOD score of 7.5–8.1, p<0.001) QTL was identified on the linkage group 7 of ‘Fiesta’ (F7). The F7 QTL explained about 37.5–38.6% of the phenotypic variation.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.     

Fire blight resistance of pear genotypes from different European countries

Several old cultivars, and breeding clones of European pear Pyrus communis L. originating from Belgium, England, Sweden, and Switzerland were evaluated for their resistance/susceptibility to fire blight. Studies were carried out during three consecutive years 2007–2009 in the greenhouse of Warsaw University of Life Sciences, Poland. Strain 691 of Erwinia amylovora was used for artificial infection of plants. Genotypes included in this study considerably varied in their resistance to fire blight. The most resistant was the old English cultivar ‘Hessle’. The other two genotypes, i.e., ‘Gr?nna R?dp?ron’ originating from Sweden, and Pyrus communis FG 1606 from Switzerland were included in a group of low susceptible ones. The most susceptible were Cra Py H 18, Cra Py V 22 and Cra Py W 14 from Belgium.