JXB Advance Access originally published online on December 22, 2006
Journal of Experimental Botany 2007 58(3):733-741; doi:10.1093/jxb/erl249
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RESEARCH PAPER |
Identification of differentially expressed genes in Malus domestica after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the phyllosphere
1Leibniz University of Hannover, Institute of Botany, Herrenhäuserstr. 2, D-30419 Hannover, Germany
2Embrapa Clima Temperado, BR 392 Km 78 CP-403, CEP 96001-970 Pelotas/RS, Brasil
* To whom correspondence should be addressed. E-mail: achim.gau{at}botanik.uni-hannover.de
Received 9 October 2006; Accepted 25 October 2006
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Abstract |
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Biological control of plant diseases by the application of antagonistic micro-organisms to the plant phyllosphere is only marginally understood. Suppression subtractive hybridization (SSH) was used for the identification of genes expressed after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the phyllosphere of the apple scab-susceptible cultivar Malus domestica cv. Holsteiner Cox. In total, 157 expressed sequence tag (EST) clones were obtained. The sequencing of 113 ESTs which have a significantly elevated transcript level and the comparison of the obtained sequences with databases revealed similarities to different classes of pathogenesis-related proteins, for example, RNase-like PR10 protein and endochitinase, or similarities to proteins expressed under stress conditions that could have a protective function, for example, a germin-like protein, glutathione S-transferase, thioredoxin-like proteins, and heat shock proteins. In addition, several transcripts were identified that code for proteins which have a crucial role at different stages of pathogen recognition and in signalling pathways or an as yet unknown function in plant defence. The results show that a number of transcripts encoding proteins/enzymes which are known to be up-regulated after pathogen infection are also up-regulated after the application of a non-pathogenic bacterium to a M. domestica cultivar. The expression of these proteins might increase the plant resistance towards pathogen infection and damage.
Key words: Biological control, pathogenesis-related proteins, suppression subtractive hybridization, systemic acquired resistance
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Introduction |
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The interaction of root-colonizing bacteria with the rhizosphere of the host plant has been characterized in previous studies (van Loon et al., 1998; Pieterse et al., 2001; Gozzo, 2003). The application of these bacteria to the rhizosphere can cause an induction of plant defence genes and priming against pathogen attack (Conrath et al., 2002). In contrast, only a few details are known about the influence of non-pathogenic microorganisms in the phyllosphere of a host plant. A special case represents the interaction in which non-pathogenic bacteria improve the growth or defence of plants against pathogens such as fungi. In general, the phyllosphere of plants is a biocoenosis of different non-pathogenic micro-organisms, such as bacteria and fungi including yeasts, that colonize the host plant without causing significant morphological changes in the appearance of the plant (Beattie and Lindow, 1995; Burr et al., 1996). Some of these organisms have the potential to act as antagonists against some plant pathogens, such as fungi. Despite numerous investigations and practical approaches, the biological control of plant diseases by antagonist treatment as a natural replacement for fungicides in the phyllosphere is only marginally understood (Boland and Kuykendall, 1997; Barbosa, 1998; Elad et al., 2001). This limited knowledge explains the failures of alternative methods using natural antagonists instead of excess fungicides (Myers, 2000). The biological control of pathogenic micro-organisms has a great potential to reduce or to abolish the chemical treatment of crop plants with pesticides. Due to the performance, the acceptance of this alternative approach is still low.
A previous study showed that the non-pathogenic bacterium Pseudomonas fluorescens Bk3 can suppress the conidial germination of the pathogen Venturia inaequalis (Fiss, 2001) and that it can reduce in vitro the mycelium growth (Singh et al., 2004). Apart from the direct suppression of fungal growth, the application of the non-pathogenic bacterium P. fluorescens Bk3 to the phyllosphere of Malus domestica cv. Holsteiner Cox also revealed the induction of a number of pathogenesis-related (PR) proteins in the intercellular washing fluid obtained from the apoplast of leaves (Kürkcüoglu et al., 2004). These results show that non-pathogenic bacteria from the phyllosphere can also cause the induction of PR proteins, as previously shown by root-colonizing or plant growth-promoting bacteria (Maurhofer et al., 1994; Zhang et al., 1998, 2002). In the case of the non-pathogenic bacteria, this induction of PR proteins does not lead to any visible morphological alterations and detectable symptoms, but seems to enable the plant to defend itself better against damage by a pathogenic micro-organism.
The aim of this study is to analyse the inducible plant protection machinery of M. domestica cv. Hosteiner Cox after the application of a non-pathogenic bacterium to the phyllosphere on a broader scale. This analysis was performed by utilizing the suppression subtractive hybridization (SSH) procedure (Diatchenko et al., 1996, 1999) for comparing the transcript level of young leaves from the apple scab-susceptible cultivar M. domestica cv. Holsteiner Cox before and after the application of the non-pathogenic bacterium P. fluorescens Bk3, which is characterized as an antagonist of the fungus V. inaequalis, to the leaf surface.
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Materials and methods |
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Bacterial strain and growth
In an orchard that was cultivated without using chemicals throughout a period of 15 years, P. fluorescens Bk3 had been isolated from the leaf surface of M. domestica cv. Holsteiner Cox. This strain had shown an in vitro antagonistic effect to V. inaequalis and was kindly provided by G Auling (Kucheryava et al., 1999). The strain was grown in Luria broth medium (LB) at 28 °C.
Plant propagation, inoculation of Malus domestica with bacteria, and determination of CFU
Malus domestica cv. Holsteiner Cox was cultivated as described by Gau et al. (2002). These sterile and genetically identical plants were transferred to rooting media containing 1x Murashige and Skoog medium including vitamins, 3% sucrose, 1.5 µM indolebutyric acid, and 0.7% plant agar. Four weeks after the transplantation to rooting media, five plants with a leaf fresh weight of 
0.7–1.3 g were sprayed with 1 ml of an overnight culture of P. fluorescens Bk3 (2x109 cells) in distilled H2O on both sides of the leaves. The control plants were treated with the same amount of H2O. Subsequently, the plants were cultivated under a light/dark cycle of 12/12 h at 24 °C.
For the determination of colony-forming units (cfu), 1 g of plant material was rinsed in 50 ml of 0.9% NaCl solution. Aliquots of the washing solution were distributed on LB-agar plates and incubated for 24 h at 27 °C.
Cultivation of Venturia inaequalis and inoculation of Malus domestica cv. Holsteiner Cox
An isolate of V. inaequalis, isolated from a leaf of M. domestica cv. Elstar in Biologische Bundesanstalt (Dossenheim, Germany) and designated as strain no. 15, was grown as previously reported by Parker et al. (1995) on potato dextrose agar (PDA) Petri dishes covered by a cellophane membrane. For the inoculation of plants with V. inaequalis, conidia were harvested 7 d after propagation on PDA Petri dishes, and five sterile in vitro-propagated plants were inoculated by spraying 1 ml of conidia suspension (containing 1x105 conidia in water). Subsequently, the inoculated plants were stored in a growth chamber at 24 °C under a light/dark cycle of 12/12 h. Control plants were sprayed with 1 ml of distilled water. The samples were harvested 24, 48, and 72 h after inoculation, and total RNA was isolated from leaves as described below.
RNA isolation
Total RNA was isolated with slight modifications from the apple cultivar Holsteiner Cox as described (Barlow et al., 1963; von Gromoff et al., 1989). Samples were taken immediately or 24 h after inoculation with the antagonistic bacteria. For the isolation of total RNA, 0.3 g of leaf material from five plants was used. The leaves were ground in liquid nitrogen. Subsequently, 0.75 ml of lysis buffer (100 mM TRIS–HCl pH 8.0, 600 mM NaCl, 20 mM EDTA, 4% SDS) and 0.75 ml of phenol/chloroform/isoamyl alcohol (PCI) (25:24:1 by vol.) were added. After shaking for 20 min, and a further 20 min centrifugation step, 0.75 ml of PCI was added to the supernatant and centrifuged for a further 15 min. After adding 0.75 vol. of 8 M LiCl to the supernatant, the RNA was precipitated overnight at 4 °C. The pellet obtained after centrifugation was dissolved in 0.5 ml of distilled sterile water and precipitated by incubation for 1 h at –20 °C after addition of 0.5 ml of 3 M Na acetate pH 5.2 and 0.7 ml of pre-cooled 96% ethanol. After washing the pellet in 0.5 ml of 70% ethanol (–20 °C) and centrifuging, it was dried, dissolved in 0.1 ml of distilled sterile water, and stored at –70 °C.
SSH library construction
The library was constructed using the polymerase chain reaction (PCR) Select cDNA Subtraction kit (Clontech, Palo Alto, CA, USA). Briefly, cDNA was obtained from total RNA using the SMART cDNA synthesis kit (Clontech) with an oligo(dT) primer, which allowed the amplification of the nuclear-encoded mRNA population contained in each sample. The cDNA populations of the tester (after application of the antagonist) and the driver (mock inoculation) were digested with the restriction enzyme RsaI (Gibco, USA) to obtain short blunt-ended fragments. The tester pool was then divided into two populations, of which the first was ligated to adaptor 1 and the second to adaptor 2R, provided with the kit. Each tester pool was hybridized separately with excess driver cDNA, and finally mixed together for a second subtractive hybridization. The fragments differentially expressed in the tester were then amplified in two PCRs, according to the manufacturer's recommendations. The fragments obtained were subsequently cloned into the pGEM-T Vector (Promega, Mannheim, Germany), which was used for the transformation of Escherichia coli XL-1 blue (Stratagene, La Jolla, CA, USA).
Reverse northern blot analysis
32P-labelled cDNA was synthesized from the first-strand cDNA obtained by SSH reactions (Smart kit, Clontech) by using the tester and the driver cDNAs, in two different reactions. For labelling, 2 µl of cDNA from each cultivar was added to a reaction containing 2 µl of the polymerase of the kit, 2 ml of 5' PCR primer II A (10 µM), 2 µl of dNTPs (10 µM from dATP, dTTP, and dGTP, and 0.05 µM dCTP), and 5 µl of [
-32P]dCTP (activity >3.000 Ci mol–1, 10 µCi ml–1). The labelled cDNA was obtained in 20 PCR cycles (an initial denaturing cycle of 1 min at 95 °C; 20 cycles of denaturation at 95 °C for 15 s, annealing at 65 °C for 30 s and elongation at 68 °C for 6 min). Radioactive labelled cDNA was purified on Sephadex G50 columns.
The cDNA fragments obtained in the subtraction were directly amplified from the colonies by PCR (an initial denaturing cycle of 3 min at 94 °C; 35 cycles of denaturation at 94 °C for 30 s, annealing at 45 °C for 30 s, and elongation at 72 °C for 1 min), using the T7 and the M13 reverse primers, which were able to bind in the pGEM-T vector.
Aliquots of 1 µl of the PCR were transferred to two nylon membranes according to the Gibco dot blot system. The cDNAs were cross-linked to the membrane by short-wavelength UV radiation (Sambrook et al., 1989). The membranes were pre-hybridized for 2 h with the prehybridization buffer (20x Denhardt; 5x SSPE, 0.2% SDS, 0.2 mg ml–1 salmon sperm) and subsequently incubated overnight at 60 °C in hybridization buffer (pre-hybridization buffer containing the labelled cDNA). Afterwards the membranes were washed twice with buffer (2x SSC, 0.2% SDS) for 15 min at room temperature and twice with buffer (1x SSC, 0.2% SDS) for 15 min at 60 °C. Hybridization signals were detected by using Phosphoimager plates (Raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany) and quantified by the TINA software package.
The fragments that hybridized only with the tester-labelled cDNA or showed at least 3-fold higher signals on these membranes compared with the signals on the membrane hybridized with the driver-labelled cDNA were sent for sequencing.
Another dot blot was performed to confirm the result with independent probe material, which was not obtained directly from the SSH procedure.
32P-labelled cDNA was synthesized from the first-strand cDNA obtained from new plant material treated for 24 h with water as control and with plants treated for 24 h with P. fluorescens Bk3 as antagonist. For labelling, 100 µg of cDNA was used with the Deca Label DNA Labeling Kit (Fermentas, Germany). The membranes were treated with selected clones obtained by SSH in the same way as described before.
Fragment sequencing and sequence evaluation
ESTs were sequenced using the M13 forward primer in the Innovation Technology Transfer (University of Bielefeld, Germany). The sequences were compared with databases using the BLASTs algorithm with sequences in the NCBI database.
Semi-quantitative determination of transcript levels by RT–PCR
The total RNA was treated with DNase and subsequently converted to cDNA by Moloney murine leukaemia virus (MMLV) reverse transcriptase as recommended by the manufacturer (Clontech). The cDNA (1 µl) was used directly for the PCR with 1 U of Taq polymerase (Sigma) in the presence of 200 µM dNTPs and 1 µM of the respective primers (Table 1). Amplification was carried out for 3 min at 94 °C followed by 35 cycles (30 s at 94 °C, primer-specific annealing temperature 46 °C for 1 min and elongation for 1.5 min at 72 °C). The final extension was performed at 72 °C for 3 min. Amplification products were directly separated in a 1% agarose gel and analysed by the TINA software package (Raytest Isotopenmeßgeräte GmbH).
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Real-time PCR analyses
The expression level of selected genes was determined with a real-time PCR of the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Scoresby, Victoria, Australia) using the two-step QuantiTectTM SYBR® Green PCR Kit (Qiagen, Hilden, Germany). The PCR quantification was performed according to the manufacturer's user manual with small modifications. The reaction volume of each PCR tube was reduced to 25 µl instead of 50 µl.
Total RNA was isolated from leaves of sterile cultured plants of M. domestica 24 h after treatment with the non-pathogenic bacterium P. fluorescens Bk3. For the inoculation of five sterile and identical plants of M. domestica cv. Holsteiner Cox, 1 ml of a bacterial suspension containing 1x109 cells ml–1 (suspended in distilled H2O) was sprayed onto the lower and upper leaf surface. The mock inoculation was performed with 1 ml of distilled H2O.
Total RNA was treated with DNase and subsequently converted to cDNA by MMLV reverse transcriptase as recommended by the manufacturer (Fermentas GmbH, Germany).
The real-time PCR transcript quantification was performed with 11 genes and the housekeeping gene ß-actin as endogenous control. Amplification was carried out with 32 ng of cDNA per each PCR under the following conditions: one initial activation step of the HotStar Taq DNA polymerase followed by 35 cycles [15 s at 94 °C (denaturation), 30 s at 50 °C (annealing), 30 s at 72 °C (extension)] and 0.4 µM of the respective primers (Table 1). All primer sequences were deduced from M. domestica. Real-time PCR results were analysed using the 
–Ct method.
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Results |
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A previous study using SDS–PAGE and Esi-Q-TOF MS (Kürkcüoglu et al., 2004) has revealed that the application of the non-pathogenic bacterium P. fluorescens Bk3 to the phyllosphere of M. domestica cv. Holsteiner Cox induces a number of PR proteins, such as a chitinase, ß-1,3-glucanase, hevein-like protein, RNase-like protein, and thaumatin-like protein in the apoplast of the treated plant.
Inoculation of M. domestica cv. Holsteiner Cox with P. fluorescens Bk3
For the identification of additionally expressed genes in young leaves of the apple scab-susceptible M. domestica cv. Holsteiner Cox, an SSH of total leaf extract was performed after the application of the non-pathogenic P. fluorescens Bk3 to the phyllosphere. The sterile cultured plants of M. domestica cv. Holstener Cox were inoculated with 2x109 cells of P. fluorescens Bk3. The determination of the cfu of P. fluorescens Bk3 revealed that in the course of 24 h after application of bacteria to the phyllosphere, the cfu had increased from 1x109 to 2x1010 cells. The cDNA library contains the differentially expressed genes which have been induced 24 h after the inoculation with the bacterial antagonist P. fluorescens Bk3.
Classification of identified EST clones
In total, 157 EST clones were obtained and analysed by reverse northern dot blot hybridization (data not shown). After selection of 112 clones which have a significantly higher expression level than the control plants, the clones were subsequently sequenced. The obtained EST sequences were identified by similarity search (BLASTN and BLASTX) in different databases (non-redundant sequences in NCBI, EST sequences in NCBI, potato and rice EST in database TIGR, and Populus trichocarpa database). The results are listed in Table 2.
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The sequenced EST clones can be divided into the following six classes: PR and more general plant defence proteins (22%), proteins or enzymes expressed under stress (14%), components of signal transduction and transcription factors (14%), nucleic acid metabolism-related proteins (26%), proteins not belonging to one of these classes (18%), and proteins of unknown function (6%).
These are a chitinase class III and an RNase-like PR-10b protein which were also found in the previous study. The SSH analysis revealed that, in addition to the above transcripts, a number of transcripts encoding proteins or enzymes that have a more general function in plant defence became apparent. These are an ADP-ribosylation factor, a cytochrome P450 which is up-regulated during the early defence reaction (Nakane et al., 2003), a 2-oxoglutarate-Fe(II) oxygenase/flavonol synthase, an O-methyltransferase (which is supposed to have a function in cell wall improvement), a proteasome subunit -type 4 (which has a function in protein degradation), an Avr9/Cf-9 rapidly elicited protein 284 (possibly showing similarities to a phosphatase), and two proteins related to allergen-type proteins homologous to some PR proteins.
Moreover, a number of transcripts up-regulated encoding proteins or enzymes which have a function in reducing oxidative or more general stress were also included. A thioredoxin-like protein, glutathione S-transferase, a germin-like protein, and three heat shock proteins also belong to this category.
As expected, regulatory proteins were also up-regulated as well as a substantial number of transcripts, which encode proteins with similarity to known transcription factors, or to proteins having a function in signal transduction pathways. Three members of this group are: a zinc finger transcription factor, a phosphoprotein phosphatase, and a 1-aminocyclopropane-1-carboxylate oxidase, the key enzyme in the ethylene biosynthetic pathway.
Somewhat surprising was the high number of up-regulated transcripts encoding proteins related to nucleic acid metabolism (66 clones out of 112 clones). This seems to imply that the translationary machinery becomes significantly modified.
Among those proteins and enzymes which do not belong to the above four classes, a NADP-specific isocitrate dehydrogenase and an enolase were up-regulated, thus implying an alteration in carbon metabolism. Moreover, transcripts encoding mitochondrial carrier protein and phosphate transporter were elevated.
Determination of transcript level of selected genes of M. domestica cv. Holsteiner Cox after inoculation with V. inaequalis or P. fluorescens Bk3
As numerous genes were identified after inoculation of the non-pathogenic bacterium P. fluorescens Bk3, which belong to the group of PR proteins and plant defence genes, a comparison on the transcript level after inoculation with the fungus V. inaequalis, the causal agent of apple scab, and the non-pathogenic bacterium P. fluorescens Bk3 was performed. The evaluation of the transcript level by reverse transcription–PCR (RT–PCR) is presented in Table 3 and reveals that the ß-1,3-glucanase as well as the ribosomal ly200 protein were up-regulated by both organisms, whereas the thaumatin-like protein is only affected by the application of P. fluorescens Bk3. Contrary to expectations, the thaumatin-like protein is only influenced by application of P. fluorescens Bk3 24 h after application and not by the fungal pathogen V. inaequalis. Moreover, the down-regulation of metallothionin 3 was observed as an early response to the application of these two counteracting organisms to the plant.
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Dot blot hybridization and quantification of selected transcripts by real-time PCR
Dot blot hybridization of selected up-regulated cDNA clones confirmed that the majority of identified transcripts had an elevated transcript level 24 h after application of the non-pathogenic P. fluorescens Bk3 to the plant phyllosphere in comparison with the mock-inoculated apple tissue. The up-regulation of most transcripts in relation to the ß-actin standard varied in the range of 0.3–21.6 (Table 2).
To verify the difference in some of the transcripts being more highly expressed in the P. fluorecens Bk3-treated M. domestica cv. Holsteiner Cox, a quantitative real-time PCR was performed (Table 4). As a template, total RNA from young and healthy leaves grown under sterile conditions from cv. Holsteiner Cox was used. For control purposes, the expression level of the highly conserved ß-actin from M. domestica which has a nearly equal expression level was used in treated and untreated apple cultivars. As expected from the SSH results (Table 2), the transcript levels of the P. fluorescens Bk3-treated leaves compared with the mock-inoculated leaves were higher for all analysed transcripts. The highest relative expression levels were determined for the proteasome subunit -type 4 and the Avr9/Cf-9 rapidly elicited protein 284, which is a putative phosphatase (Table 4).
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Discussion |
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The results clearly show that application of the non-pathogenic P. fluorescens Bk3 to the phyllosphere of M. domestica cv. Holsteiner Cox leads to the up-regulation of at least 50 different transcripts in the leaves. Among these are a number of transcripts which encode PR proteins and proteins which have a function in the more general plant defence. As expected from previous investigations (Kürkcüoglu et al., 2004), some of these were already detected on the protein level when the apoplastic fluid was investigated after treatment. Among these are a chitinase and RNase-like PR-10b which recently proved to be up-regulated after treatment of apple leaves with ethylene, generating ethephon, as well as salicylic acid (Poupard et al., 2003). It was previously shown that in the V. inaequalis-resistant M. domestica cv. Remo a number of PR proteins (ß-1,3-glucanase, thaumatin-like protein, osmotin-like protein, cysteine protease, and PR1 protein) are constitutively up-regulated, in contrast to M. domestica cv. Elstar where these transcripts/proteins were only induced after infection with the fungus (Gau et al., 2004). Thus, the results presented here as well as those previously presented underline the hypothesis that bacterial antagonists in the phyllosphere of plants, causing no visible morphological alterations of the plant, can initiate the transcriptional activation of plant defence genes. The induction of such transcripts leading to synthesis of PR protein, which in part are transported into the apoplast, are likely to prepare the plant against a pathogen attack.
Thus, the constitutive expression of PR proteins or the induction of PR proteins by the application of the non-pathogenic P. fluorescens Bk3 could elevate the resistance of M. domestica cv. Holsteiner Cox against pathogens.
In the very early stages of the plant defence, reactive oxygen species (ROS) are formed as a means to prevent or inhibit pathogen infection. This oxidative burst requires a plasma membrane-located NADPH oxidoreductase that generates hydrogen peroxide, which can destroy the infected tissue and avoids the spreading and multiplication of pathogens. In this context, it is also relevant to mention the identification of a transcript with similarity to the auxin-binding protein ABP20 precursor that also has a significant sequence similarity to germin-like proteins. Germin or germin-like proteins convert oxalacetate to hydrogen peroxide (Patnaik and Khurana, 2001; Lane, 2002). Therefore, the germin-like proteins might also contribute to the plant defence machanism. On the other hand, it is important that the uninfected tissues are protected from damage by ROS. Therefore, it is not surprising that the results of the experiments undertaken reveal that a substantial number of transcripts that encode enzymes or proteins, which have a function in the adaptation process to oxidative or more general stress, are up-regulated after the application of the non-pathogenic bacterium to keep the fungus under control. As a consequence of this molecular response, the plant is prepared for a successful defence against pathogens.
Besides the well-characterized proteins with a function in plant defence, more recently, a few additional ones have been described. Amongst these are phospholipid-derived molecules with a function as novel second messengers in signal transduction pathways for plant defence (Laxalt and Munnik, 2002). The phosphoprotein phosphatase identified as being up-regulated upon P. fluorescens Bk3 application might have a function in a signalling pathway.
In summary, application of the non-pathogenic bacterium P. fluorescens Bk3 to the phyllosphere of M. domestica cv. Holsteiner Cox leads to the up-regulation of a large number of transcripts. Several of these transcripts encode proteins/enzymes that are also implicated during infection with the pathogen V. inaequalis (Gau et al., 2004). Thus, it can be concluded that the expression of these proteins, initiated by the non-pathogenic bacterium, possibly helps to cope against infection with a pathogen. The molecular mechanism of signal perception of P. fluorescens Bk3 by the host plant is still elusive. A possible candidate for an elicitor of the plant defence reaction could be flagellin that is released by P. fluorescens Bk3 (Singh et al., 2004) and which is well known as a signal molecule (Zipfel et al., 2004).
It is tempting to assume that P. fluorescens Bk3 is a potent antagonist against the fungal plant pathogen V. inaequalis and can be used for the biological control of plant diseases under field conditions. Preliminary results revealed a reduction of infection by V. inaequalis of up to 80%.
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Acknowledgements |
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SK expresses her gratitude to the Deutsche Bundesstiftung Umwelt for financial support and JD to CAPES (Brazil) for providing a PhD scholarship. The financial support and many helpful discussions with Professor Dr K Kloppstech are gratefully acknowledged. We are also grateful to A Fröhlich for critical reading of the manuscript.
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Abbreviations |
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EST, expressed sequence tag; PDA, potato dextrose agar; PR, pathogenesis related; ROS, reactive oxygen species; RT–PCR, reverse tanscription–polymerase chain reaction; SSH, suppression subtractive hybridization.
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