JXB Advance Access published online on April 4, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern044
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Silencing of acidic pathogenesis-related PR-1 genes increases extracellular β-(1
3)-glucanase activity at the onset of tobacco defence reactions
1UMR 1301 Interactions Biotiques et Santé Végétale, INRA-Université Nice-SophiaAntipolis-CNRS, F-06903 Sophia Antipolis Cedex, France
2Department of Plant Physiology, Institute of Molecular Biology and Physiology, The University of Copenhagen, DK-1353 Copenhagen, Denmark
To whom correspondence should be addressed. E-mail: galiana{at}sophia.inra.fr
Received 12 November 2007; Revised 8 January 2008 Accepted 21 January 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The class 1 pathogenesis-related (PR) proteins are thought to be involved in plant defence responses, but their molecular functions are unknown. The function of PR-1 was investigated in tobacco by generating stable PR-1a-silenced lines in which other acidic PR-1 genes (PR-1b and PR-1c) were silenced. Plants lacking extracellular PR-1s were more susceptible than wild-type plants to the oomycete Phytophthora parasitica but displayed unaffected systemic acquired resistance and developmental resistance to this pathogen. Treatment with salicylic acid up-regulates the PR-1g gene, encoding a basic protein of the PR-1 family, in PR-1-deficient tobacco, indicating that PR-1 expression may repress that of PR-1g. This shows that acidic PR-1s are dispensable for expression of salicylic acid-dependent acquired resistances against P. parasitica and may reveal a functional overlap in tobacco defence or a functional redundancy in the PR-1 gene family. The data also show that there is a specific increase in apoplastic β-(1
3)-glucanase activity and a decrease in β-(13)-glucan deposition in PR-1-silenced lines following activation of defence reactions. Complementation of the silencing by apoplastic treatment with a recombinant PR-1a protein largely restores the wild-type β-(13)-glucanase activity and callose phenotype. Taken together with the immunolocalization of PR-1a to sites of β-(13)-glucan deposition in wild-type plants, these results are indicative of a function for PR-1a in regulation of enzymatic activity of extracellular β-(13)-glucanases.
Key words:
ARR, callose, β-(13)-glucanase, PR-1a, RNAi, SAR
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Class 1 pathogenesis-related (PR) proteins are defence factors ubiquitously synthesized by plants in response to pathogen infections (Van Loon and Van Strien, 1999). They are produced following the recognition of pathogen-derived molecules by host plant cells and the activation of transduction pathways. Together with other defence proteins, such as β-(1
3)-glucanases, chitinases, and secondary metabolism enzymes including phytoalexin biosynthetic enzymes, they may contribute, directly or indirectly, to resistance to pathogen attack. Some members of the PR-1 family are secreted in response to infection, in particular as part of the local hypersensitive response (HR) to an avirulent strain of pathogen, a response which involves cell death and which confers resistance to the specific pathogen concerned. The accumulation of these proteins in the extracellular space is also the major quantitative change observed in distal, uninfected plant tissues, which may develop systemic acquired resistance (SAR) to unrelated virulent pathogens after the development of an HR (Ross, 1961; Kuc, 1982). In tobacco, it is only the subset of acidic PR-1 proteins (PR-1a, PR-1b, and PR-1c) that is secreted. The plant signalling networks governing PR-1 gene regulation during SAR are well understood (Ryals et al., 1996; Dong, 2001). Salicylic acid (SA) is a positive component (Malamy et al., 1990; Metraux et al., 1990; Rasmussen et al., 1991; Ward et al., 1991a; Gaffney et al., 1993; Delaney et al., 1994) inducing expression of PR-1a, PR-1b, and PR-1c genes in Nicotiana and of PR-1 in Arabidopsis, in a co-ordinate manner, with other plant defence genes of classes 2 and 5 (Uknes et al., 1992; Maleck et al., 2000; Petersen et al., 2000). PR-1 genes are also regulated by developmental signals. During floral development, PR-1 proteins are expressed in tobacco floral tissues (Lotan et al., 1989; Uknes et al., 1993) and in healthy leaves (Fraser, 1981; Uknes et al., 1993; Hugot et al., 1999, 2004), indicating an involvement not only in defence, but also in development. Little is known about the plant signalling networks governing PR-1 gene regulation at late developmental stages. In healthy leaves of flowering tobacco, PR-1a gene activation, like SAR, requires SA and is correlated with the expression of developmental resistance (or age-related resistance, ARR) to Phytophthora parasitica (Hugot et al., 2004; Develey-Rivière, 2007).
Plant PR-1 proteins occur in both basic and acidic isoforms, with no consistent amino acid sequence differences between the two subclasses (Van Loon and Van Strien, 1999). In Nicotiana tabacum, acidic PR-1 isoforms predominate and are found in the extracellular spaces, in xylem elements, and in the vacuoles of the crystal idioblasts of tobacco mosaic virus-infected leaves (Dixon et al., 1991; Buchel and Linthorst, 1999). The basic PR-1 isoforms are targeted mainly to the vacuole (Eyal et al., 1993; Sessa et al., 1995; Buchel and Linthorst, 1999). The tobacco PR-1 genes have been shown to be induced via separate signal transduction pathways involving SA, jasmonic acid (JA), and/or ethylene. A JA-dependent pathway and/or an ethylene-dependent pathway was found to induce the expression of (some) basic PR-1 genes strongly, whereas an SA-dependent pathway was found to activate primarily the expression of acidic PR-1 genes (Ward et al., 1991a; Eyal et al., 1992; Niki et al., 1998). These activations are mutually antagonistic (Niki et al., 1998), reflecting the cross-talk inhibition of SA- and JA-dependent defence pathways in plants (Rojo et al., 2003).
The mode of action of plant PR-1 proteins remains largely unknown, and no enzymatic activity has yet been demonstrated for these proteins. PR-1 proteins are very widespread in angiosperms, and equivalent proteins have been found in yeasts, insects, and vertebrates (Van Loon and Van Strien, 1999). Sequence comparisons may provide information about the function of plant PR-1 proteins. The weak inhibition of trypsin by P25TI, a human protein presenting homologies with plant PR-1 proteins, could provide a clue to the activity of the plant proteins, but these proteins have never been reported to inhibit proteases (Yamakawa et al., 1998). A protease from the venom of Conus textile, Tex31, also displays similarity to members of the PR-1 protein superfamily (Milne et al., 2003). A human protein present in glioma, GliPR (for ‘glioma pathogenesis-related protein’), is 35% identical to the tomato P14a protein (Fernandez et al., 1997; Szyperski et al., 1998). Comparison of the structures of these two proteins led to the identification of two conserved histidine residues exposed at the surface of the two proteins, making these two residues the main candidates for a role in any active site. These residues might correspond to the active site of an RNase, but no RNase activity has been detected (Szyperski et al., 1998).
The functional significance of these genes in defence is poorly understood and the function of proteins of the PR-1 family remains unclear. Alexander et al. (1993) provided the first indirect indication of antimicrobial activity for a member of this family, by showing that transgenic lines of tobacco constitutively expressing the PR-1a gene were more tolerant to P. parasitica and Peronospora tabacina than the wild type. Niderman et al. (1995) demonstrated direct anti-oomycete activity of the PR-1 proteins purified from tomato both in vitro (inhibition of zoospore germination) and in vivo (reduction of the leaf area invaded by Phytophthora infestans). Differences in activity have been observed between the acidic PR-1 proteins of tobacco (PR-1a and PR-1b) and the basic P14c from tomato and PR-1g from tobacco, with the basic proteins being more effective. PR-1a is the most abundant PR protein in infected tobacco tissues. This secreted protein displays only weak direct activity, suggesting that it may have a more important indirect function, responsible for the resistance of transgenic lines expressing the PR-1a gene constitutively (Alexander et al., 1993). The role of PR-1 proteins was investigated by silencing tobacco PR-1 genes encoding acidic isoforms through constitutive expression of hairpin PR-1a RNAs. This manuscript reports the phenotype of dominant negative mutants generated by RNA interference (RNAi).
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant growth conditions, chemical application, and inoculation
Experiments were performed with N. tabacum cv. Xanthi nc. tobacco plants. Plants were grown in a growth chamber at 24 °C, with a 16 h photoperiod, and a light intensity of 100 µE m–2 s–1. Susceptibility and SAR establishment were assessed in 7–8-week-old tobacco plants. SAR was induced by elicitin application (Bonnet et al., 1996): plants were decapitated and their stems treated with 20 µl of water or a 5 µM aqueous solution of cryptogein. ARR establishment was assessed in 7–8-week-old or 14–15-week-old tobacco plants, susceptible or resistant, respectively, to P. parasitica (Hugot et al., 1999).
Treatments with SA (1 mM) and water (as control) were performed on detached leaves of 5–7-week-old plants by petiole absorption for 36 h. Methyl jasmonate [45 µM in 0.1% (v/v) ethanol] and 0.1% (v/v) ethanol were applied by spraying the surface of detached leaves.
Inoculations with P. parasitica were performed by infiltrating leaf parenchyma tissue with a 50 µl suspension containing 100 zoospores (Hugot et al., 1999). In each experiment, at least four consecutive leaves received two infiltrations of zoospore suspension each. Susceptibility and resistance were evaluated by measuring the areas over which disease symptoms were observed, various numbers of days after inoculation, for each leaf. The development of disease symptoms is strictly correlated with the development of the oomycete (Galiana et al., 1997). The spread of disease symptoms was therefore also used to evaluate the development of the oomycete in the plant.
All experiments were performed with at least three replicates of plants or leaves.
Transgene constructs and Nicotiana transformation
Lines of transgenic plants for RNAi of PR-1a (X12737
[GenBank]
) were constructed as follows. A gene-specific cDNA fragment (nucleotides 1085–1309) was amplified by PCR, using the following primers: 5'-CGGGATCCATTTAAATACCTTTGACCTGGGACGA-3' (forward, BamHI and SwaI sites underlined) and 5'- GGACTAGTGGCGCGCCGTGTCCACACACCTGTCC-3' (reverse, SpeI and AscI sites underlined). The cDNA fragment was first inserted into the pFGC1008 vector (http://ag.arizona.edu/chromatin/fgc1008.html, ABRC), between the SwaI and AscI sites, in the antisense orientation. The sense fragment was then inserted between the BamHI and SpeI sites. These constructs were introduced into Agrobacterium tumefaciens strain GV3101. Leaf discs of N. tabacum cv. Xanthi nc. plants were infected with the bacterial strain. They were incubated on MS agar media for 3 d and then on MS agar media containing hygromycin (40 µg l–1) and carbenicillin (400 µg l–1), to favour callus growth. A single shoot was regenerated from each leaf disc, and heterozygous plants were transferred to soil, with plants then placed under normal growth conditions (24 °C with a 16 h photoperiod and a light intensity of 100 µE m–2 s–1). Silencing was characterized in heterozygous transgenic plants. Seed resulting from self-pollination of the regenerated transformants was scored for hygromycin resistance on MS medium. Homozygous transgenic lines and wild-type plants were used for phenotypic and resistance analyses.
Production and purification of the recombinant PR-1a protein
The pPIC9 vector (Invitrogen, Groningen, The Netherlands) allows the induction of gene expression by methanol in Pichia pastoris and secretion of the resulting recombinant protein in response to the Saccharomyces cerevisiae 
-factor secretion signal. The PR-1a gene was cloned in-frame with the initiation codon of the signal sequence by synthesizing the DNA fragment containing the N. tabacum PR-1a coding sequence (nucleotides 1020–1438) by PCR, using the purified pPR-1a plasmid (Ward et al., 1991a) as a template, with the sense and antisense primers 5'-GTATCTCTCGAGAAAAGAGAGGCTGAAGCTCCAAAATTCTCAACA-3' and 5'-CTTAAGAATTCCAATTAGTATGGACTTTCGC-3', respectively. The pPIC9-PR1a plasmid was constructed using the XhoI and EcoRI restriction sites, with confirmation by sequencing.
The yeast P. pastoris strain GS115 (Invitrogen) was transformed with pPIC9-PR-1a plasmid linearized by digestion with SacI, according to the manufacturer's instructions. Yeast clones were selected and the efficiency of PR-1a secretion was analysed by immunoblotting. PR-1a was purified from the culture filtrate by column chromatography. The supernatant was concentrated by tangential-flow ultrafiltration, using an Amicon miniplate bioconcentrator (Millipore, St Quentin Yvelines, France), and dialysed against water. It was then loaded onto a Macro-Prep HQ anion-exchange support (Bio-Rad, Hercules, CA, USA) equilibrated with 10 mM Na2HPO4/NaH2PO4, pH 6. The protein was eluted with increasing concentrations of NaCl (0.25, 0.5, and 1 M) in the same buffer. The PR-1a protein-containing fraction (0.1 M NaCl) was applied to a reverse-phase column (Vidal C415-20 µm, Interchim, Montluçon, France) equilibrated with 10 mM Na2HPO4/NaH2PO4, pH 6. The column was washed with 20% (v/v) CH3CN, 0.1% (v/v) trifluoroacetic acid (TFA), and the protein was eluted with increasing concentrations of aqueous CH3CN (20, 30, 40, 50, and 60%), in the presence of 0.1% (v/v) TFA. The CH3CN was evaporated off and the protein preparation was dialysed against water. The resulting purified PR-1a protein was then lyophilized. During the purification procedure, all fractions were analysed by HPLC to identify the PR-1a-containing fractions.
For functional complementation assays, leaves of siP1a were first petiole-treated with 1 mM SA for 24 h, then leaf-infiltrated with different concentration of recombinant PR-1a or bovine serum albumin (BSA; Sigma). In each experiment, four leaves from four plants (same rank) were infiltrated twice: the right part of each leaf was infiltrated with water in the presence of PR-1a; the left part was infiltrated in the presence of BSA at the same concentration. Intercellular fluids were extracted as described below 24 h after leaf infiltration for enzyme activity determination.
Real-time PCR analysis
The expression of genes in leaf tissues was analysed by real-time quantitative PCR (RT-Q-PCR), using the fluorescent intercalating dye SYBR-Green, in a DNA Engine Opticon®2 (MJ Research, Bio-ad, Hercules, CA, USA). Total RNA was isolated for three independent replicates, using TRIzol reagent (Invitrogen Gmbh, Karlsruhe, Germany). Reverse transcriptase reactions were performed with the iScript cDNA Synthesis kit from Bio-Rad, with 10 µg of total RNA in a volume of 20 µl, according to the manufacturer's instructions. The reaction mixtures were set up at the same time for all samples and diluted 50-fold before PCR analysis. The cDNAs produced were used as templates in real-time PCRs with gene-specific primers (see next paragraph), using the qPCRTM Mastermix Plus for SybrTM Green I (Eurogentec, Belgium) according to the manufacturer's instructions. PCR amplification was carried out as follows: 40 cycles of DNA denaturation at 95 °C for 15 s, annealing at 58 °C for 30 s, and elongation at 72 °C for 30 s, with three replicates for each sample analysed. Fluorescence was evaluated at the end of each 30 s elongation step. Data were treated with Opticon 3.1 software, provided by MJ Research. The C(t), defined as the PCR cycle at which reporter fluorescence increased significantly, was used as an estimation of the initial copy number of the target gene. A tobacco actin gene (Nt-ACT9) was used as an endogenous control, to normalize the relative level of target gene expression by calculating 
C(t), the difference in Ct between the control Nt-ACT9 products and the target gene products. The difference in target gene expression between a standard control and a sample was estimated by calculating 2–Ct (Ct = Ct sample–Ct standard). Two main comparisons were carried out: one in which the standard control was the unsilenced transformed line and the tested samples were the silenced transformed lines, and another in which the standard controls were the lines treated with water (or 0.1% ethanol) and the tested samples were the lines treated with SA.
The following RT-Q-PCR primer sequences were used: for PR-1a, PR-1b, and PR-1c (Cutt et al., 1988), a forward 5'-GGATGCCCATAACACAGCTC-3' and a reverse 5'-GCTAGGTTTTCGCCGTATTG-3 primer; for PR-1g (Payne et al., 1989), a forward 5'-TCCCTCAACTTAACGCTGCT-3' and a reverse 5'-GCTTCGAACCCTAGCACAAC-3' primer; for PR-2 (Ward et al., 1991b), a forward 5'-AGCAGCATCAGGGTTGCAAGA-3' and a reverse 5'-GGCCAGCCACTTTCAGATAC-3' primer; for Nt-ACT9, an actin gene expressed in leaves (Thangavelu et al., 1993), a forward 5'-AGGGTTTGCTGGAGATGATG-3' and reverse 5'-CGGGTTAAGAGGTGCTTCAG-3' primer.
Immunohistological microscopy
After the application of a chemical or inoculation, the leaf pieces were washed, dehydrated in a series of ethanol solutions, and embedded in plastic (Technovit 7100; Kuizer, Wehrheim, Germany). Sections (5 µm) of leaf tissue were obtained with a microtome (Leica, Germany).
For the histological detection of callose, fixed tissues were stained for 30 min with aniline blue (0.003% in 100 mM K3PO4 pH 8.6, Biosupplies Australia, Parkville, Australia). The sections were then washed and fluorescence assessed under UV light excitation.
For immunofluorescence analysis, leaf sections were treated with a blocking solution containing 10% normal donkey serum and 3% BSA in phosphate-buffered saline (PBS), pH 7.2, for 30 min, in a humidified chamber. Sections were then incubated in solutions made up in PBS pH 7.2 supplemented with 3% BSA and were washed in this buffer. Sections were first incubated for 1 h at room temperature with the primary antibody, diluted 1:100 for the rabbit anti-PR1a (also cross-reacting with PR-1b and -1c), anti-PR-2, or anti-PR-5, and 1:20 for the mouse anti-β-(13)-glucan antibody (Biosupplies Australia). Sections were washed three times and incubated for 1 h at room temperature with the appropriate conjugated secondary antibody diluted 1:100 for the Fluoprobes 546 or 488 anti-rabbit IgG antibody, and for the Fluoprobes 488 anti-mouse IgG antibody. Samples were rinsed three times, mounted in 80:20 PBS/glycerol or in Vectashield (Vector Laboratories Inc., USA) and examined under an Axioplan fluorescence microscope (Carl Zeiss MicroImaging, Inc., Germany) equipped with appropriate filters.
SDS–polyacrylamide gel electrophoresis and immunoblot analysis
Intercellular fluids were prepared from leaves treated with SA for 36 h as described by Hammond-Kosak (1992). Twenty half-leaves were taken, rinsed with water, and vacuum-infiltrated with 50 mM TRIS-HCl (pH 7). Then they were dried and rolled into 20 ml syringe barrels which were placed in centrifuge tubes before centrifugation at 800 g for 10 min. Intercellular fluids were collected at the bottom of the tube.
Protein extracts (2 µg) were subjected to 15% SDS–PAGE. The gels were silver-stained, as described by Blum et al. (1987). Alternatively, proteins were transferred electrophoretically from SDS–polyacrylamide gels onto nitrocellulose membranes (Amersham). The membranes were incubated with purified rabbit IgG antibodies against acidic PR-1 proteins (Abad et al., 1989), PR-2, or PR-5 proteins (Kauffmann et al., 1990). They were then incubated with goat peroxidase-conjugated IgG against rabbit immunoglobulins (Amersham). The bound antibodies were detected by enhanced chemiluminescence (ECL; Amersham), according to the kit manufacturer's instructions.
Generation and probing of cell wall polymer microarrays
A range of 50 cell wall glycans were spotted as arrays onto nitrocellulose membranes using split pins in a microarray robot (MicroGrid II, Genomic Solutions, Ann Arbor, MI, USA). The arrays included representative glycans from the three major cell wall polymer classes, cellulose hemicellulose and pectin as well as proteoglycans, β(13)-glucan and β(13),(14)-glucan (Megazyme, Wicklow, Ireland). Each sample was printed at two concentrations (0.2 mg ml–1 and 0.04 mg ml–1) and in two replicates. Arrays were probed with antibodies as follows: membranes were blocked with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.7 mM KH2PO4, pH. 7.5) containing 5% (v/w) fat-free milk powder for 1 h at room temperature. After washing for 2 min in PBS, the membranes were incubated in purified rabbit IgG antibodies against PR1 proteins (diluted in PBS, 1:100), or with a mouse anti-β(13)-glucan antibody (diluted in PBS, 1:50) for 1 h at room temperature. Membranes were then washed three times in PBS and incubated for 1 h with the appropriate Fluoprobes 488-conjugated secondary anti-rabbit or anti-mouse IgG antibody (diluted in PBS, 1:100), respectively. After washing, the bound antibodies were detected using an FLA-3000 phosphorimager (Fuji) equipped with an SHG laser source with a wavelength of 473 nm (Filter Y520).
Enzyme activity determination
Spectrophotometric assays were used to determine the β-(13)-glucanase, chitinase, peroxidase, and RNase activities of the intercellular fluids of the various lines. Protein extracts [20 µl containing 1 µg of protein for β-(13)-glucanase or RNase activities, 2 µg of protein for chitinase, and 0.2 µg of protein for peroxidase activity] were incubated at 24 °C for various periods of time, in 1 ml of 50 mM sodium acetate–acetic acid buffer (pH 5.6). The reaction mixture was supplemented with 5 mg ml–1 laminarin azure (Sigma) as the β-(13)-glucanase substrate, with 5 mg ml–1 chitin azure (Sigma) as the chitinase substrate, with 0.1 mg ml–1 o-phenylenediamine dihydrochloride (Sigma) and 0.02% H2O2 as peroxidase substrates, or with 0.4 µg ml–1 of RNA from the yeast Torulopsis utilis (Sigma) as an RNase substrate. For the estimation of β-(13)-glucanase activity, after 2 h of incubation the reaction was stopped by adding 1 ml of ice-cold ethanol to 0.4 ml of reaction mixture. The mixture was then placed on ice for 2 h and centrifuged for 5 min at 15 000 g, for recovery of the supernatant. Soluble, dyed fragments released from laminarin azure were quantified colorimetrically, at 590 nm. For chitinase assay, after 24 h of incubation samples were cooled and spun for 10 min. The absorbance of the supernatant solution was measured at 550 nm. For the evaluation of peroxidase and RNase activities, the kinetics of enzymatic reactions were determined from 0 to 1 h by absorbance measurement of the assay mixtures at 440 nm and 260 nm, respectively. All assays were performed with four replicates, and blanks lacking intercellular fluids were also prepared.
Zymogram plates were also used to assess hydrolytic activity. Enzymatic activity was detected on agar substrate plates containing 1.5% agar (w/v) in 50 mM sodium acetate–acetic acid buffer (pH 5.6), supplemented with either 10 mg ml–1 laminarin from Laminaria digitata for β-(13)-glucanase activity, 5 mg ml–1 chitin from crab shells for chitinase activity, 5 mg ml–1 carboxymethylcellulose for cellulose activity, 0.5 mg ml–1 4-chloro-1-naphthol and 0.02% H2O2 for peroxidase activity, or 10 mg ml–1 gelatin for protease activity. Intercellular fluids (containing 1 µg of protein, in a volume adjusted to 20 µl) were deposited on the plate surface and incubated for 18 h at 24 °C. For the detection of endoglucanase, chitinase, or cellulase activity, plates were stained with 0.1% (w/v) Congo Red (Sigma) for 10 min, bleached with 1 M NaCl, and photographed on a transilluminator. For the detection of proteolytic activity, plates were immersed in saturated (NH4)2SO4 for 10 min and photographed against a dark background.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Molecular characterization of PR-1a-silenced plants
For the functional analysis of PR-1a, the PR-1a cDNA (X12737 [GenBank] , nucleotides 1084–1309) was used for post-transcriptional gene silencing (PTGS) with the pFGC1008 vector to silence the PR-1a gene in N. tabacum. Transgenic tobacco plants producing a hairpin loop PR-1a RNA under control of the strong promoter cauliflower mosaic virus (CaMV) 35S were generated. Of the 41 independent primary transformants, 24 randomly chosen plants were tested to confirm that the levels of PR-1a in intercellular fluids extracted from leaves treated with 1 mM SA were low. SA treatment can be used to mimic SAR in plants and is correlated with the strong induction of extracellular PR-1, PR-2, and PR-5 protein accumulation. SDS–PAGE and silver staining showed that the profile of proteins from plants transformed for PR-1a silencing was almost identical to that of control transformed plants (Fig. 1A). Thus, transformation did not induce a non-specific response triggering the differential accumulation of a number of extracellular proteins. However, there was one marked difference, the absence of a polypeptide with an apparent molecular mass of 17 kDa, corresponding to PR-1a. Immunoblot analyses with antibodies against acidic PR-1s confirmed that production or secretion of acidic PR-1s was abolished in the independent PR-1a-silenced plants analysed (Fig. 1B). Immunoblots with antibodies against PR-2 and PR-5 showed that these two proteins accumulated in large amounts in intercellular fluids devoid of PR-1a, indicating that SA treatment was effective. Of the 24 plants analysed for PR-1a silencing, 17 (71%) displayed specific silencing. Three transgenic lines—siP1a, siP1b, and siP1c—generated from independent leaf discs, were chosen. For further analyses, they were compared with the wild-type line (WT) or with a control line (C) generated by transformation with the empty pFGC1008 vector.
|
The level of silencing was determined for these three lines by RT-Q-PCR. The primers using for the RT-Q-PCR perfectly matching the three acidic PR-1 genes (PR-1a, PR-1b, and PR-1c), As expected, expression of acidic PR-1 genes was significantly increased after SA treatment compared with water treatment (270-fold induction) in control lines. When siP1a, siP1b, and siP1c were analysed following treatment with water, the abundance of PR-1a, PR-1b, and PR-1c mRNAs was so low than no threshold cycle (Ct) values could be determined (Fig. 2A). A comparison of Ct values between the SA-treated control line and the SA-treated silenced lines showed that PR-1 mRNAs were 151x, 240x, and 148x more abundant in the control line than in the siP1a, siP1b, and siP1c lines, respectively (Fig. 2B). Thus, a rate of silencing at least equal to 99.5% was observed, and this result was confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA) analysis (data not shown).
|
RT-Q-PCR and immunoblot analyses did not lead to the detection of acidic PR-1gene expression (Fig. 2) or protein accumulation (Fig. 1B) in silenced plants. Based on these results and given the high level of similarity between sequences corresponding to acidic PR-1 isoforms—95% at the mRNA level (Cornelissen et al., 1987)—it can be assumed that all acidic PR-1 genes (PR-1a, PR-1b, and PR-1c) were silenced in PR-1a-silenced plants. Thus the following results are presented in relation to silencing of acidic PR-1 genes rather than to PR-1a silencing.
Seed segregation for hygromycin resistance was consistent with a single insertion of the transgene in each of the three lines. PR-1a-silenced tobacco plants develop normally, indicating that the constitutive expression of PR-1a dsRNA had no visible pleiotropic effects. However, these plants flower late (3.4–4.5 d later compared with control lines, data not shown), suggesting that acidic PR-1 genes may promote flowering, although they are not functionally required for it to occur.
PR-1 silencing increases susceptibility to P. parasitica, but does not impair SAR or ARR
The impact of PR-1 silencing on susceptibility, on SAR, and on ARR to the agent of black shank disease, the oomycete P. parasitica, was then analysed.
The impact of silencing on susceptibility was assessed by inoculating the leaves of vegetative plants (8 weeks old) with a suspension of zoospores of P. parasitica (100 zoospores/infiltration). Susceptibility was estimated by determining the leaf area displaying disease symptoms 2 d and 3 d after inoculation. In the three silenced lines (siP1a, siP1b, and siP1c) disease symptoms extended over a larger area than in control unsilenced plants (Fig. 3A), consistent with the findings of Alexander et al. (1993), who previously showed an increase in the tolerance to oomycetes of transgenic plants constitutively expressing PR-1a.
|
SAR was induced by applying 5 µM cryptogein—a proteinaceous elicitor that induces the HR and SAR in tobacco, notably against P. parasitica (Bonnet et al., 1996)—to the stem. Forty-eight hours after the cryptogein treatment of vegetative plants, non-necrotic leaf areas were inoculated with a suspension of zoospores of P. parasitica (Galiana et al., 1997). SAR was quantified by measuring the percentage of protection conferred by cryptogein treatment. Three days after inoculation, this percentage reached 91% and 100% for wild-type and control transformed plants, respectively, whereas it reached 100, 91, and 92% for siP1a, siP1b, and siP1c, respectively (Fig. 3B). These results indicated that acidic PR-1 gene silencing did not affect cryptogein-induced SAR against P. parasitica. As shown in Fig. 3C, immunoblot analyses confirmed the absence of acidic PR1 proteins in intercellular fluids from cryptogein-treated silenced plants.
As for SAR, the induction in leaves of an ARR against P. parasitica is associated with PR-1a expression during floral development. The influence of PR-1 silencing on this form of resistance was investigated by inoculating the leaves of adult tobacco plants (14–15-week-old tobacco plants) with P. parasitica zoospores and assessing the decrease in infection efficiency and the restriction of fungal hyphal growth, the two main characteristics of mature plants expressing this type of resistance (Hugot et al., 1999, 2004). In these experiments, adult silenced tobacco plants displayed levels of resistance similar to those of control lines. The kinetics of disease development in silenced plants were similar to those in non-silenced plants (Fig. 3D). A slight reduction of disease progression could be noted at 144 h after inoculation, but the differences between control and silenced plants were not statistically significant. Silencing also did not affect the control of infection efficiency (data not shown). Immunoblot analyses confirmed that PR-1 silencing was effective in adult plants (Fig. 3E). Thus, the loss of PR-1 impaired neither ARR nor SAR against P. parasitica. These data are consistent with the absence of acidic PR-1 being compensated by a diversity of defence reactions activated during acquired resistance or by redundancy in the PR-1 gene family, with another protein substituting for acidic PR-1s.
The silencing of genes for acidic PR-1 isoforms leads to the up-regulation of PR-1g gene expression after SA treatment
To investigate whether the expression of any other member of the PR-1 gene family was affected by acidic PR-1 gene silencing, the levels of different PR-1 and PR-2 mRNAs were compared between control plants and silenced plants. After SA treatment, total RNA was isolated, and the relative amounts of PR-1 mRNAs were determined by RT-Q-PCR. The accumulation of PR-1g transcripts encoding a basic PR-1 protein (Niderman et al., 1995), and displaying no more than 69% identity to PR-1 mRNAs encoding acidic isoforms, was analysed. First, RT-PCR analyses on SA-treated leaf tissues showed, as previously described for basic PR1 mRNAs (Ward et al., 1991a), that PR-1g mRNAs were present in small amounts in both the control line and the wild type. PR-1g was also clearly expressed in the siP1a and siP1b lines (Fig. 4A). Thus, when acidic PR-1 genes were silenced, PR-1g was not. PR-1g mRNAs seemed to accumulate more strongly in silenced lines than in control lines, suggesting that when acidic PR-1 genes were silenced, PR-1g was up-regulated. The induction of PR-1g expression after SA treatment was quantified by real-time PCR analyses. A 1.44-fold repression in the control line, and 2.7-, 1.9-, and 7.6-fold induction in siP1a, siP1b, and siP1c, respectively, were observed in response to SA treatment and compared with water treatment (Fig. 4B). Thus PR-1g was up-regulated in response to SA in silenced lines. This up-regulation could be specific at least when compared with PR-2, because PR-2 mRNA levels were similar in siP1a and the control line (Fig. 4C). These results suggest that PR-1g expression is normally suppressed by acidic PR-1 gene expression. They also indicate that the expression of the PR-2 gene, encoding a β-(13)-glucanase and co-ordinately up-regulated with PR-1a in wild-type plants, is not affected by PR-1 silencing.
|
The loss of apoplastic PR-1 increases extracellular β-(1
3)-glucanase activityThe molecular function of PR-1 in plants is unknown. PR-1 function was investigated by screening extracellular enzymatic activities for any modification in PR-1-silenced lines. Different apoplastic enzymatic activities (cellulase, RNase, protease, and peroxidase) were analysed, including β-(1
3)-glucanase and chitinase induced by SA treatment in tobacco and known to increase following the triggering of plant defences. The influence of PR-1 silencing on these enzymatic activities was assessed by means of assays on intercellular fluids from the SA-treated leaves of control and silenced lines. The cellulase activity remained very weak in the extracts of all lines and could not be detected in these experiments. RNase, protease, peroxidase, and chitinase activities were similar in the four tested lines: siP1a, siP1b, siP1c, and the control (Fig. 5). Only β-(13)-glucanase activity differed significantly between silenced and control lines. β-(13)-Glucanase activity was higher in the three silenced lines, with 2.34-, 1.59-, and 1.97-fold induction, respectively, for siP1a, siP1b, and siP1c, with respect to the control line (P <0.005). Similar results were obtained whether laminarin azure was used as soluble substrate or laminarin was used as an immobilized substrate for the analysis of β-(13)-glucanase activity in intercellular fluids by zymography. Thus, and based on the different enzymatic activities tested, PR-1 silencing specifically increased β-(13)-glucanase activity, suggesting a role for acidic PR-1 in the negative regulation of β-(13)-glucanase activity.
|
The interference of PR-1a with β-(1
3)-glucanase activity by directly interacting with β-(13)-glucanase was investigated by far-western blotting to identify proteins secreted after the induction of SAR and able to bind the recombinant purified PR-1a (10–50 µg ml–1). Analyses of intercellular fluids from the SA-treated leaves of the control and siP1a revealed a major spot including putative PR-1a-binding proteins with an apparent molecular weight of 45 kDa. However, exploration of protein spots by trypsin digestion and tandem mass spectroscopy (MS/MS) did not lead to the identification of peptide sequences matching known β-(13)-glucanases (data not shown). The possible interaction between PR-1a and the extracellular β-(13)-glucanase encoded by PR-2 was also assessed by immunoprecipitation. Indeed, the coordinated up-regulation of the PR-1 and PR-2 genes is one of the most spectacular transcriptional events associated with the activation of plant defence reactions (Uknes et al., 1992; Maleck et al., 2000; Petersen et al., 2000; Hugot et al., 2004). This co-ordinated up-regulation suggests that there may be a functional link between these two gene families in plants. However, in the conditions used here, with intercellular fluids prepared from SA-treated Xanthi leaves, no co-immunoprecipitation of acidic PR-1s with PR-2 was observed, whatever the antibody used for immunoprecipitation (antibody against acidic PR-1 or PR-2) or immunoblotting after SDS–PAGE (antibody against PR-2 or acidic PR-1). These results suggest an absence of physical interaction between these two proteins.
Co-localization of PR-1 with β-(13)-glucans
The localization of PR-1s with the β-(13) glucan deposits occurring with activation of plant defences was established by immunohistochemical microscopy. The leaves of Xanthi nc. plants were infiltrated with cryptogein (1 µM) for 36 h and transverse semi-thin sections were assayed for the simultaneous localization of β-(13)-glucans and acidic PR-1, PR-2, or PR-5, three proteins produced in large amounts during the onset of HR and SAR. As shown in Fig. 6A, PR-1a staining was observed as fluorescent aggregates decorating the cell wall of parenchyma and xylem-associated cells. Double staining with a monoclonal antibody against β-(13)-glucans revealed that PR-1s were present in the cell wall, close to apoplastic microregions enriched in β-(13)-glucans (Fig. 6A, B). The co-localization of PR-1s and β-(13)-glucans was not systematic, but was observed regularly, at a frequency similar to that for co-localization of the β-(13)-glucanase PR-2 and its putative substrate (Fig. 6C, D). PR-5 proteins were never co-localized with β-(13)-glucans (Fig. 6E, F). In order to establish that the anti-PR-1a antibody did not bind to cell wall glycans (and β-linked glucans in particular) the antibody was used to probe cell wall glycan arrays (Moller et al., 2007) that contained β-(13)-glucan, β-(13),(14)-glucan, as well as 48 other cell wall glycans. The anti-PR-1a antibody did not bind to any of the glycans on the array, and the lack of binding to β-(13)-glucan is shown in Fig. 6A (lower insert). In contrast, the antibody against β-(13)-glucans did bind to β-(13)-glucan, as shown in Fig. 6B for Pachyman β-1,3-glucan (lower insert), and not to other glycans on the array (data not shown). These results demonstrate that the co-localization of PR-1s and β-(13)-glucan was not due to unspecific recognition of β-(13)-glucan by anti-PR-1a antibody.
|
PR-1 gene silencing reduces callose deposition after cryptogein treatment
Callose [containing β-(1
3)-linked glucan] deposition is one the characteristics of defence reactions associated with HR (Stone and Clarke, 1992). The involvement of PR-1s in callose deposition during defence responses was investigated by localizing the acidic proteins and β-(13)-glucan polymers in cryptogein-treated leaf tissues from control and PR-1-silenced lines. Immunolocalization with a monoclonal antibody against β-(13) glucans showed staining decorating the cell wall of parenchyma and xylem-associated cells in wild-type (Fig. 7A) and control transformed lines (data not shown), but not in the siP1a line (Fig. 7B). The same was true of cryptogein-treated leaf tissues from siP1b and siP1c (data not shown). Callose deposition was visualized by aniline blue staining in the wild type (Fig. 7C). The aniline blue staining was clearly weaker in the three silenced lines as illustrated in Fig. 7D for siP1a. The in situ analysis was confirmed by determining levels of NaOH-soluble callose, which was undetectable in extracts from silenced lines and abundant in controls (Fig. 7E). These results indicate that acidic PR-1 proteins can be positively involved in callose deposition.
|
Functional complementation of PR-1 silencing with a tobacco PR-1a recombinant protein
To determine unambiguously if the increased extracellular β-(1
3)-glucanase activity phenotype was truly linked to the silencing of acidic PR-1 genes and not to off-target effects (Ma et al., 2006; Xu et al., 2006), a functional complementation assay of silenced lines was developed by delivery of a PR-1a recombinant protein directly in the apoplast by infiltration. To do that, PR-1a was expressed in P. pastoris and then the recombinant protein was purified. The N. tabacum sequence required for coding the mature secreted PR-1a (residue Gln31 to Tyr168) was cloned in-frame with the signal sequence of the -factor which has been demonstrated to be very efficient in P. pastoris. After induction of PR-1a gene expression, SDS–PAGE and immunoblot analysis were coupled with a two-step column chromatography strategy to purify PR-1a from yeast culture with an efficiency of 40 mg l–1.
To evaluate the influence of the recombinant PR-1a on the extracellular enzymatic activities, leaves of siP1a were first petiole-treated with 1 mM SA for 24 h, then leaf-infiltrated with different concentrations (ranging from 0 mM to 0.25 mM) of PR-1a for 24 h, before extraction of intercellular fluids. This range of concentrations has been established after determination of the concentration of endogenous PR-1 in intercellular fluid of wild-type SA-treated leaves (0.02–0.04 mM) using an ELISA. In these conditions, the phenotype associated with PR-1 silencing is suppressed by apoplastic management of PR-1a. Indeed, β-(13)-glucanase activity (Fig. 8A) but not peroxidase activity (Fig. 8B) gradually and significantly decreased whereas the PR-1a concentration increased (Fig. 8C). Thus a specific dose-dependent effect of PR-1a on negative regulation of β-(13)-glucanase activity was observed.
|
The influence of the recombinant PR-1a on callose deposition was then assessed. Plants were first stem-treated with 5 µM cryptogein for 24 h, then leaf-infiltrated with 0.05 mM PR-1a or BSA into necrosis adjacent tissues for 24 h before visualization of callose using aniline blue or the callose-specific monoclonal antibody. The PR-1a treatment obviously led to a more important callose deposition than the BSA treatment. In the three silenced lines, the PR-1a treatment restores callose deposition as illustrated in Fig. 8D–G for siP1a. Thus, the leaf tissues of silenced plants treated with the PR-1a recombinant protein display a wild-type phenotype, establishing that the callose and the β-(1
3)-glucanase phenotype is indeed due to reduced expression of PR-1. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The PR-1 gene family includes vacuolar or extracellular proteins, all of unknown molecular function, produced in response to pathogen infections and developmental stimuli. Transgenic tobacco plants expressing a hairpin loop PR-1a RNA under control of the 35S promoter in the Xanthi nc. background were created to analyse the biological function of PR-1 proteins.
Cross-talk between the regulation of genes for acidic and basic PR-1 proteins
The tobacco genome contains 16 genes (Cornelissen et al., 1987) likely to encode either acidic PR-1 proteins such as PR-1a (Pfitzner et al., 1987), PR-1b (Ohshima et al., 1990a), and PR-1c (Ohshima et al., 1990b), or basic PR-1 proteins (Cornelissen et al., 1987; Payne et al., 1989; Eyal et al., 1992; Niderman et al., 1995). The present data show that, in PR-1-silenced plants, the three genes encoding acidic PR-1s and known to be expressed are silenced. This result is consistent with the very high level of identity of the corresponding mRNAs, reaching 90%, and the perfect base-pairing RNA interference mechanism (Baulcombe, 2004). Sequence identity between the genes encoding acidic and basic PR-1s does not exceed 70%. For PR-1a and PR-1g (only, among the basic ones, characterized as being expressed), coding sequence identity is 69%. An alignment of these two sequences revealed a single stretch of 23 nucleotides displaying complete sequence identity (from nucleotides 340 to 368 in the PR-1g coding sequence, accession number X14065), which could be targeted by siRNA generated by interference between hairpin loop PR-1a RNAs and PR-1a mRNAs. The accumulation of PR-1g transcripts in PR-1-silenced lines shows that this stretch is not sufficient for interference with basic PR-1 mRNAs. These findings are consistent with previous suggestions that PTGS is effective against genes displaying at least 80–90% nucleotide sequence identity to the inserted transgene.
A finding of this study is that PR-1g mRNA accumulates in larger amounts in acidic PR-1-silenced lines than in control plants after SA treatment. This observation is based on a very limited study, and a transcriptome analysis of these pathways is required to clarify the overall changes in gene expression in the PR-1-silenced plants. However, our results suggest that one or several mRNAs for acidic PR-1 or the proteins they encode may negatively regulate, either directly or indirectly, the accumulation of PR-1g mRNAs. Basic PR-1 genes are strongly induced via a JA-dependent pathway, whereas the expression of acidic PR-1 genes is SA dependent (Seo et al., 1995). The activation of PR-1a via an SA-dependent pathway is antagonistic to the activation of the PRB-1b gene (encoding a basic PR-1 isoform) expression via a JA pathway (Niki et al., 1998), illustrating the mutual antagonism between SA- and JA-dependent defence pathways in plants (Rojo et al., 2003). The influence of PR-1a expression on JA-dependent PR-1g expression was investigated by RT-Q-PCR. The abundance of PR-1g mRNAs was determined from leaves treated with 1 mM SA for 24 h, and then treated with 45 µM MeJA for an additional 24 h. No difference was found between the control and the silenced lines (data not shown), indicating that PR-1 gene silencing may influence SA-dependent, but not JA-dependent PR-1g gene expression. Thus, in wild-type tobacco, acidic PR-1 genes may repress SA-dependent, but not JA-dependent PR-1g gene expression.
The role of PR-1 proteins in regulating β-(13)-glucanase activity and callose deposition
PR-1-silenced lines displayed enhanced apoplastic β-(13)-glucanase activity after SA treatment. This increase was specific, as shown by comparison with other extracellular activities tested, among which chitinase activity is known to be up-regulated after activation of the SA pathway following the triggering of plant defence reactions. This analysis thus provides the first evidence that proteins of the PR-1 family may regulate β-(13)-glucanase activity. The demonstration of complete and specific silencing of acidic PR-1 taken together with the suppression of the associated phenotype by apoplastic management of PR-1a indicates that PR-1 negatively regulates these apoplastic enzymes. However, as PR-1g gene expression was up-regulated concomitantly with PR-1 silencing, the possibility that PR-1g would positively regulate one or several β-(13)-glucanase(s) cannot be completely excluded. Although this up-regulation was small, it is also possible that PR-1g is a β-(13)-glucanase, and that its release and accumulation in the apoplast increase enzymatic activity in PR-1-silenced plants. However, no such sequence similarity or enzymatic activity has been found for PR-1g and any member of the PR-1 family in plants (Van Loon and Van Strien, 1999). The mechanism by which acidic PR-1s may repress apoplastic β-(13)-glucanase activity after SA induction is unknown. The fact that transcript abundance of the β-(13)-glucanase PR-2 is not enhanced in PR-1-silenced lines (Fig. 4) suggests a post-transcriptional regulation because the massive PR-2 mRNA accumulation is a major transcriptional regulation event associated with PR-1a gene expression following pathogen recognition when the SA pathway is activated. Far-western blotting and immunoprecipitation analysis did not lead to the identification of known β-(13)-glucanases interacting with PR-1a, in particular PR-2 which is co-ordinately up-regulated and secreted with PR-1a suggesting a functional link. The finding that acidic PR-1s were deposited at the cell wall, at the sites at which β-(13)-glucans are detected in the wild type (Fig. 6A, B), suggests that PR-1s negatively regulate β-(13)-glucanases through their glucan-binding or modifying properties.
The regulation mechanism of callose deposition during biotic stress is poorly understood despite the identification of callose synthases required for callose formation in Arabidopsis (Jacobs et al., 2003; Nishimura et al., 2003) or of GLU 1, a β-(13)-glucanase important for callose metabolism, in tobacco (Beffa et al., 1996; Iglesias and Meins, 2000). The finding that the increase of β-(13)-glucanase activity correlates with the reduction of callose apposition in PR-1-silenced lines sheds new light on callose deposition. It is indicative for this mechanism of a positive regulation by PR-1 and of a negative regulation by PR-1-regulated β-(13)-glucanases. The identification of PR-1-targeted β-(13)-glucanases in plants will help to define the enzymatic machinery driving the balance between callose synthesis and degradation occurring during a plant–pathogen interaction.
The role of PR-1 in tobacco defences
Consistent with the findings of Alexander et al. (1993) for constitutive PR-1a expression in tobacco, plants lacking acidic PR-1s were more susceptible to the oomycete P. parasitica. However, infection experiments were inconclusive concerning the possible effects of this protein on expression of SAR and ARR against P. parasitica. Indeed, PR-1-silenced plants display no impairment in the expression of these two forms of acquired resistance. Thus, either PR-1s are not required for SAR and ARR or PR-1 function is redundant and may be compensated when affected. SAR (Maleck et al., 2000) and ARR (Hugot et al., 2004) are established through the expression of a set of functions required in plant defences. The diversity, overlapping, or redundancy of these functions may compensate for the deleterious effects of the loss of one such function, PR-1 in this case. This is illustrated by the higher extracellular β-(13)-glucanase activity in PR-1 gene-silenced plants than in control plants after SA treatment. The resulting enhanced release of putative elicitor-active carbohydrates from P. parasitica cell wall by β-(13)-endoglucanase (Okinaka et al., 1995) could lead to reinforcement of host defence responses. In Medicago truncatula, silencing of PR-10-like proteins is associated with the induction of a new set of PR proteins upon infection with Aphanomyces euteiches, as the two thaumatin-like proteins (PR-5b), which normally are repressed due to PR-10 expression. The antagonistic induction of other PR proteins is supposed to cause an increased resistance of M. truncatula upon an A. euteiches in vitro infection (Colditz et al., 2007). Furthermore, although all the acidic PR-1 genes were silenced, the basic PR-1 genes were not. Indeed, PR-1g gene expression was 2–7 times higher in silenced lines than in control lines treated with SA. Thus, the lack of acidic PR-1 function may be compensated by the recruitment of a basic PR-1 function. Evidence for molecular redundancy or defence diversity, leading to a lack of effect on SAR and ARR in plants lacking PR-1s, should be provided by the generation of plants in which genes of both the PR-1 and PR-2 families are silenced and plants in which both acidic and basic PR-1 genes are silenced.
Hypotheses can be put forward to explain the apparent discrepancy between the increase in susceptibility and the absence of change in SAR and ARR in the PR-1-silenced lines. The main difference between susceptible plants and plants expressing acquired resistance is, of course, the activation of plant defence systems in resistant plants. In PR-1-silenced plants without the induction of defence mechanisms by exogenous or developmental stimuli, susceptible plants cannot perceive the lack of a defence function before pathogen attack. The pathogen may therefore develop because of the alteration of basal resistance against P. parasitica and before plants have time to compensate for the absence of acidic PR-1 and to activate their defence systems fully, so as to limit the disease. In contrast, in SAR or ARR, in which defence pathways have already been activated, compensation mechanisms must be activated before pathogen challenge.
|
Acknowledgements |
|---|
We would like to thank Catherine Etienne and Gilles Arbiol for technical assistance, and Janice de Almeida-Engler for kindly helping us with immunohistological microscopy. M-PR was supported by a fellowship from INRA and the Association pour la Recherche sur les Nicotianées.
|
Footnotes |
|---|
* Present address: Departamento de Biotecnología-UPM, E.T.S. Ingenieros Agrónomos, Avda. Complutense, E-28040 Madrid, Spain.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Abad A, Cardin L, Poupet A, Ponchet M. Comparison of pathogenesis related ‘b1’ protein determination obtained by ELISA and HPLC techniques. Journal of Phytopathology (1989) 124:175–188.
Alexander D, Goodman RM, Gut-Rella M, et al. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proceedings of the National Academy of Sciences, USA (1993) 90:7327–7331.
Baulcombe D. RNA silencing in plants. Nature (2004) 431:356–363.[CrossRef][Medline]
Beffa RS, Hofer RM, Thomas M, Meins F Jr. Decreased susceptibility to viral disease of [beta]-1,3-glucanase-deficient plants generated by antisense transformation. The Plant Cell (1996) 8:1001–1011.[Abstract]
Blum H, Beier H, Gross HJ. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis (1987) 8:93–99.[CrossRef][Web of Science]
Bonnet P, Bourdon E, Ponchet M, Blein JP, Ricci P. Acquired resistance triggered by elicitins in tobacco and other plants. European Journal of Plant Pathology (1996) 102:181–192.[CrossRef][Web of Science]
Buchel AS, Linthorst HJM. PR-1: a group of plant proteins induced upon pathogen infection. In: Pathogenesis-related proteins in plants—Datta SK, Muthukrishnan S, eds. (1999) Boca Raton, FL: CRC Press.
Colditz F, Niehaus K, Krajinski F. Silencing of PR-10-like proteins in Medicago truncatula results in an antagonistic induction of other PR proteins and in an increased tolerance upon infection with the oomycete Aphanomyces euteiches. Planta (2007) 226:57–71.[CrossRef][Web of Science][Medline]
Cornelissen BJC, Horowitz J, van Kan JAL, Goldberg RB, Bol FJ. Structure of tobacco genes encoding pathogenesis-related proteins from the PR-1 group. Nucleic Acids Research (1987) 15:6799–6811.
Cutt JR, Dixon DC, Carr JP, Klessig DF. Isolation and nucleotide sequence of cDNA clones for the pathogenesis-related proteins PR1a, PR1b and PR1c of Nicotiana tabacum cv. Xanthi nc induced by TMV infection. Nucleic Acids Research (1988) 16:9861.
Delaney TP, Uknes S, Vernooij B, et al. A central role of salicylic acid in plant disease resistance. Science (1994) 266:1247–1250.
Develey-Rivière MP, Galiana E. Resistance to pathogens and host development stage: a multifaceted relationship within the plant kingdom. New Phytologist (2007) 175:405–416.[CrossRef][Web of Science][Medline]
Dixon DC, Cutt JR, Klessig FD. Differential targeting of the tobacco PR-1 pathogenesis-related proteins to the extracellular space and vacuoles of crystal idioblasts. EMBO Journal (1991) 10:1317–1324.[Web of Science][Medline]
Dong X. Genetic dissection of systemic acquired resistance. Current Opinion in Plant Biology (2001) 4:309–314.[CrossRef][Web of Science][Medline]
Eyal Y, Meller Y, Lev-Yadun S, Fluhr R. A basic-type PR-1 promoter directs ethylene responsiveness, vascular and abscission zone-specific expression. The Plant Journal (1993) 4:225–234.[CrossRef][Web of Science][Medline]
Eyal Y, Sagee O, Fluhr R. Dark-induced accumulation of a basic pathogenesis-related (PR-1) transcript and a light requirement for its induction by ethylene. Plant Molecular Biology (1992) 19:589–599.[CrossRef][Web of Science][Medline]
Fernandez C, Szyperski T, Bruyere T, Ramage P, Mosinger E, Wuthrich K. NMR solution structure of the pathogenesis-related protein P14a. Journal of Molecular Biology (1997) 266:576–593.[CrossRef][Web of Science][Medline]
Fraser RSS. Evidence for the occurence of the ‘pathogenesis-related’ proteins in the leaves of healthy tobacco during flowering. Physiological Plant Pathology (1981) 19:69–76.[Web of Science]
Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E, Kessmann H, Ryals J. Requirement of salicylic acid for the induction of systemic acquired resistance. Science (1993) 261:754–756.
Galiana E, Bonnet P, Conrod S, Keller H, Panabieres F, Ponchet M, Poupet A, Ricci P. RNase activity prevents the growth of a fungal pathogen in tobacco leaves and increases upon induction of systemic acquired resistance with elicitin. Plant Physiology (1997) 115:1557–1567.[Abstract]
Hammond-Kosak KE. Preparation and analysis of intercellular fluid. In: Molecular plant pathology: a practical approach—Gurr SJ, McPherson MJ, Bowles DJ, eds. (1992) Volume II. Oxford: Oxford University Press. 21–27.
Hugot K, Aime S, Conrod S, Poupet A, Galiana E. Developmental regulated mechanisms affect the ability of a fungal pathogen to infect and colonize tobacco leaves. The Plant Journal (1999) 20:163–170.[CrossRef][Web of Science][Medline]
Hugot K, Riviere MP, Moreilhon C, Dayem MA, Cozzitorto J, Arbiol G, Barbry P, Weiss C, Galiana E. Coordinated regulation of genes for secretion in tobacco at late developmental stages: association with resistance against oomycetes. Plant Physiology (2004) 134:858–870.
Iglesias VA, Meins F Jr. Movement of plant viruses is delayed in a beta-1,3-glucanase-deficient mutant showing a reduced plasmodesmatal size exclusion limit and enhanced callose deposition. The Plant Journal (2000) 21:157–166.[CrossRef][Web of Science][Medline]
Jacobs AK, Lipka V, Burton RA, Panstruga R, Strizhov N, Schulze-Lefert P, Fincher GB. An Arabidopsis callose synthase, GSL5, is required for wound and papillary callose formation. The Plant Cell (2003) 15:2503–2513.
Kauffmann S, Legrand M, Fritig B. Isolation and characterization of six pathogenesis-related (PR) proteins of Samsun NN tobacco. Plant Molecular Biology (1990) 14:381–390.[CrossRef][Web of Science][Medline]
Kuc J. Induced immunity to plant disease. BioScience (1982) 32:854–860.[CrossRef][Web of Science]
Lotan T, Ori N, Fluhr R. Pathogenesis-related proteins are developmentally regulated in tobacco flowers. The Plant Cell (1989) 1:881–887.
Ma Y, Creanga A, Lum L, Beachy P. Prevalence of off-target effects in Drosophila RNA interference screens. Nature (2006) 443:359–363.[CrossRef][Medline]
Malamy J, Carr JP, Klessig DF, Raskin I. Salicylic acid: a likely endogenous signal in the resistance response of tobacco to viral infection. Science (1990) 250:1002–1004.
Maleck K, Levine A, Eulgem T, Morgan A, Schmidn J, Lawton KA, Dangl JL, Dietrich RA. The transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nature Genetics (2000) 26:403–410.[CrossRef][Web of Science][Medline]
Metraux JP, Signer H, Ryals J, Ward E, Wyss-Benz M, Gaudin J, Raschdorf K, Schmid E, Blum W, Inverardi B. Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science (1990) 250:1004–1006.
Milne TJ, Abbenante G, Tyndall JD, Halliday J, Lewis RJ. Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily. Journal of Biological Chemistry (2003) 278:31105–31110.
Moller I, Sørensen I, Bernal AJ, et al. High-throughput mapping of cell wall polymers within and between plants using novel microarrays. The Plant Journal (2007) 50:1118–1128.[CrossRef][Web of Science][Medline]
Niderman T, Genetet I, Bruyere T, Gees R, Stintzi A, Legrand M, Fritig B, Mosinger E. Pathogenesis-related PR-1 proteins are antifungal. Isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans. Plant Physiology (1995) 108:17–27.[Abstract]
Niki T, Mitsuhara I, Seo S, Ohtsubo N, Ohashi Y. Antagonistic effect of salicylic acid and jasmonic acid on the expression of pathogenesis-related (PR) protein genes in wounded mature tobacco leaves. Plant and Cell Physiology (1998) 39:500–507.
Nishimura MT, Stein M, Hou BH, Vogel JP, Edwards H, Somerville SC. Loss of a callose synthase results in salicylic acid-dependent disease resistance. Science (2003) 301:969–972.
Ohshima M, Harada N, Matsuoka M, Ohashi Y. The nucleotide sequence of pathogenesis-related (PR) 1b protein gene of tobacco. Nucleic Acids Research (1990a) 18:181.
Ohshima M, Harada N, Matsuoka M, Ohashi Y. The nucleotide sequence of pathogenesis-related (PR) 1c protein gene of tobacco. Nucleic Acids Research (1990b) 18:182.
Okinaka Y, Mimori K, Takeo K, Kitamura S, Takeuchi Y, Yamaoka N, Yoshikawa M. A structural model for the mechanisms of elicitor release from fungal cell walls by plant beta-1,3-endoglucanase. Plant Physiology (1995) 109:839–845.[Abstract]
Payne G, Middlesteadt W, Desai N, Williams S, Dincher S, Carnes M, Ryals J. Isolation and sequence of a genomic clone encoding the basic form of pathogenesis related protein 1 from Nicotiana tabacum. Plant Molecular Biology (1989) 12:595–596.[CrossRef][Web of Science]
Petersen M, Brodersen P, Naested H, et al. Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance. Cell (2000) 103:1111–1120.[CrossRef][Web of Science][Medline]
Pfitzner UM, Goodman HM. Isolation and characterization of cDNA clones encoding pathogenesis-related proteins from tobacco mosaic virus infected tobacco plants. Nucleic Acids Research (1987) 15:4449–4465.
Rasmussen JB, Hammerschmidt R, Zook MN. Systemic induction of salicylic acid accumulation in cucumber after inoculation with Pseudomonas syringae pv. syringae. Plant Physiology (1991) 97:1342–1347.
Rojo E, Solano R, Sanchez-Serrano JJ. Interactions between signaling compounds involved in plant defense. Journal of Plant Growth Regulation (2003) 22:82–98.
Ross AF. Systemic acquired resistance induced by localized virus infections in plants. Virology (1961) 14:340–358.[CrossRef][Web of Science][Medline]
Ryals J, Neuenschwander UH, Willits MG, Molina A, Steiner HY, Hunt MD. Systemic acquired resistance. The Plant Cell (1996) 8:1809–1819.[CrossRef][Web of Science][Medline]
Seo S, Okamoto M, Seto H, Ishizuka K, Sano H, Ohashi Y. Tobacco MAP kinase: a possible mediator in wound signal transduction pathways. Science (1995) 270:1988–1992.
Sessa G, Yang XQ, Raz V, Eyal Y, Fluhr R. Dark induction and subcellular localization of the pathogenesis-related PRB-1b protein. Plant Molecular Biology (1995) 28:537–547.[CrossRef][Web of Science][Medline]
Stone AS, Clarke AE. (1,3)-β-glucans in plant host–pathogen interactions. In: Chemistry and biology of (1,3)-β-glucans (1992) Bundoora, Australia: La Trobe University Press. 491–512.
Szyperski T, Fernandez C, Mumenthaler C, Wuthrich K. Structure comparison of human glioma pathogenesis-related protein GliPR and the plant pathogenesis-related protein P14a indicates a functional link between the human immune system and a plant defense system. Proceedings of the National Academy of Sciences, USA (1998) 95:2262–2266.
Thangavelu M, Belostotsky D, Bevan MW, Flavell RB, Rogers HJ, Lonsdale DM. Partial characterization of the Nicotiana tabacum actin gene family: evidence for pollen-specific expression of one of the gene family members. Molecular and General Genetics (1993) 240:290–295.[CrossRef]
Uknes S, Mauch-Mani B, Moyer M, Potter S, Williams S, Dincher S, Chandler D, Slusarenko A, Ward E, Ryals J. Acquired resistance in Arabidopsis. The Plant Cell (1992) 4:645–656.
Uknes S, Dincher S, Friedrich L, Negrotto D, Williams S, Thompson-Taylor H, Potter S, Ward E, Ryals J. Regulation of pathogenesis-related protein-1a gene expression in tobacco. The Plant Cell (1993) 5:159–169.[Abstract]
Van Loon LC, Van Strien EA. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology (1999) 55:85–97.[CrossRef][Web of Science]
Ward ER, Payne GB, Moyer MB, et al. Differential regulation of beta-1,3-glucanase messenger RNAs in response to pathogen infection. Plant Physiology (1991b) 96:390–397.
Ward E, Uknes S, Williams SC, Dincher SS, Wiederhold DL, Alexander DC, Ahl-Goy P, Metraux JP, Ryals J. Coordinate gene activity in response to agents that induce systemic acquired resistance. The Plant Cell (1991a) 3:1085–1094.
Xu P, Zhang Y, Kang L, Roossinck MJ, Mysore KS. Computational estimation and experimental verification of off-target silencing during posttranscriptional gene silencing in plants. Plant Physiology (2006) 142:429–440.
Yamakawa T, Miyata S, Ogawa N, Koshikawa N, Yasumitsu H, Kanamori T, Miyazaki K. cDNA cloning of a novel trypsin inhibitor with similarity to pathogenesis-related proteins, and its frequent expression in human brain cancer cells. Biochimica et Biophysica Acta (1998) 1395:202–208.[Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  G. M. Gibbs, K. Roelants, and M. K. O'Bryan The CAP Superfamily: Cysteine-Rich Secretory Proteins, Antigen 5, and Pathogenesis-Related 1 Proteins--Roles in Reproduction, Cancer, and Immune Defense Endocr. Rev., December 1, 2008; 29(7): 865 - 897. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||