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Polygalacturonase-inhibiting protein (PGIP) from Japanese pear: possible involvement in resistance against scab
Physiological and Molecular Plant Pathology 63 (2003) 319–327 www.elsevier.com/locate/pmpp
Polygalacturonase-inhibiting protein (PGIP) from Japanese pear: possible involvement in resistance against scab Mohamed Faize, Tomoko Sugiyama, Lydia Faize, Hideo Ishii* National Institute for Agro-Environmental Sciences, 3-1-3 Tsukuba, Ibaraki 305-8604, Japan Accepted 29 March 2004
Abstract Venturia nashicola is the causal agent of scab, a fungal disease affecting Asian pears. The Japanese pear cv. ‘Kousui’ is highly susceptible to the race 1 of this fungus whereas the cv. ‘Kinchaku’ and the non-host European pear cv. ‘Flemish Beauty’ are resistant. The aim of this work is to investigate the role of polygalacturonase-inhibiting proteins (PGIPs) of pear during the interactions with V. nashicola leading to susceptibility or resistance. PGIP protein was detected from immature fruit of Kousui and Kinchaku. It showed a molecular mass of 42 kDa that shifted to 35 kDa after chemical deglycosylation. The gene pgip was amplified by PCR using genomic DNA and/or cDNA from young leaves of Kousui, Kinchaku, and European pear cvs. Flemish Beauty, ‘Bartlett’, and an Asian wild pear strain ‘Mamenashi 12’, then sequenced after sub-cloning. Some conserved variations were identified in the sequence indicating that gene family also exists in pgip of Japanese pear and confirmed by Southern blot analysis. The expression of PGIP was studied in scab-inoculated leaves of the susceptible Kousui and the resistant Kinchaku and Flemish Beauty. pgip Gene and its encoding protein were highly and rapidly activated in these resistant plants. In addition, PGIP extracts derived from Kinchaku and Flemish Beauty partially inhibited the activity of polygalacturonase (PG) from V. nashicola suggesting a possible role of PGIP in limiting fungal growth frequently observed in these resistant cultivars. q 2004 Elsevier Ltd. All rights reserved. Keywords: Polygalacturonase-inhibiting protein; Nucleotide sequence; Pear scab; Venturia nashicola; Japanese pear; European pear
1. Introduction Scab is an important disease of Asian pears such as Japanese pear (Pyrus pyrifolia var. culta) and Chinese pear (P. ussuriensis). So far examined, Venturia nashicola, the scab fungus of Asian pears, is pathogenic on Japanese and Chinese pears but not pathogenic on European pear like ‘Flemish Beauty’ [12,30]. Scab resistance of pear species and cultivars was assessed for their potential use in breeding programs and it was found that the non-commercial Japanese pear cv. ‘Kinchaku’ was resistant to V. nashicola [11]. In addition, Ishii et al. [13] found pathological specialization of V. nashicola and classified strains of this fungus into three races: race 1, pathogenic on the most popular but highly scab-susceptible Japanese pear cv. ‘Kousui’ but not on Abbreviations: PGIP, polygalacturonase-inhibiting protein; PG, polygalacturonase; LRR, leucine-rich repeat; LRPKm1, leucine-rich repeat receptor-like protein kinase; TFMS, trifluoromethanesulfonic acid. * Corresponding author. Tel./fax: þ 81-29-838-8307. E-mail addresses: [email protected] (H. Ishii), faizemohamed@ hotmail.com (M. Faize). 0885-5765/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2004.03.006
the Asian wild pear strain ‘Mamenashi 12’; race 2, pathogenic on Mamenashi 12 but not on Kousui; and race 3, pathogenic on both Mamenashi 12 and Kousui. Early and late stages of the infection behavior of the race 1 of V. nashicola were studied on the leaves of susceptible Kousui and the resistant cvs. Kinchaku and Flemish Beauty. Histological observations showed that early stages of infection were similar on the susceptible and the resistant cultivars, because V. nashicola penetrated only the cuticle layer and formed subcuticular hyphae on all cultivars [24]. The subcuticular hyphae never penetrated into the epidermal cells even 7 days after inoculation suggesting that V. nashicola produces and secretes pectin-degrading enzymes for utilizing nutrients. Subsequently, three endopolygalacturonases and an exopolygalacturonase produced by V. nashicola and V. pirina (the scab fungus of European pear), respectively, were purified and characterized recently [14]. However, during the late stage of infection, differences were observed between susceptible and resistant cultivars. Fungal cells appeared intact in susceptible Kousui, while in both resistant Kinchaku and Flemish
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Beauty fungal cell walls appeared decomposed, attenuated and hyphae collapsed, and general disruption of cellular membranes observed. Cell death of V. nashicola in these resistant cultivars implies the presence of some inhibition factors [24]. As a first approach to study on scab-resistance mechanism, we focused on polygalacturonase-inhibiting proteins (PGIPs). These proteins inhibit the activity of fungal polygaracturonase (PG), which is considered to be an important factor for pathogenesis [5]. PGIPs are glycoprotein present in the plant cell wall of vegetative as well as fruit tissues and genes encoding PGIPs have been cloned from various plants [4,6,8 – 10,15,16,18,19,27 – 29,31 – 33]. They belong to a super-family of leucine-rich repeat (LRR) protein and are considered to have important roles in plant – pathogen interactions. Several plant disease-resistance genes such as Cf-9, Cf-2 and Xa21 encode the member of the LRR protein family as well, and have high homology to PGIPs [5]. Furthermore, leucine-rich repeat receptor-like protein kinase (LRPKm1) that is likely to participate in defense-related signaling has been isolated by pgip probe in apple [17]. Although PGIPs are previously reported in apple and European pear fruit, there is no sufficient information concerning their role in resistance, specially those from leaves. This work aimed to detect PGIP protein and characterize pgip genes from fruit and leaves of Japanese pear. To help understanding the role of this protein in resistance against V. nashicola, the expression of PGIP protein as well as pgip gene was analyzed during susceptible and resistant interactions.
2. Materials and methods 2.1. Plant material, fungal strain and inoculation test The following pear trees were grown in a screen-house or an orchard in National Institute for Agro-Environmental Sciences: Japanese pear cvs. Kousui and Kinchaku, European pear cvs. Flemish Beauty and Bartlett, and the Asian wild pear strain Mamenashi 12. Fresh conidia collected directly from lesions on leaves of Kousui were suspended in distilled water, filtered through tissue paper and washed by centrifugation. Conidia were resuspended in 0.1% sucrose and concentrations were adjusted to ca. 5 £ 105 conidia ml21. Conidial suspensions were sprayed to young leaves of potted trees. After air-drying, the inoculated plants were incubated in a moist chamber at 20 8C for 48 h, transferred to a phytotron, and maintained at 25 8C. The inoculated leaves were detached from plants at intervals (0, 1, 2, 3, 5 and 7 days after inoculation) and stored at 2 80 8C until use. For RNA extraction 1 cm diameter leaf discs were cut from each leaf, snap frozen in liquid nitrogen and stored at 2 80 8C.
2.2. Protein extraction, chemical deglycosylation and Western blot analysis Proteins were extracted from immature fruit (3 – 5 cm in diameter) of Kousui and Kinchaku by using the modified method of Abu-Goukh et al. [1]. Fruit flesh was homogenized in extraction buffer [0.1 M sodium acetate, pH 6.0/1 M NaCl/1% (w/v) polyvinylpyrrolidone/ 0.2% (w/v) sodium bisulfite] with Physcotron (NITI-ON, Funabashi, Japan). The homogenate was stirred on ice for 1 h and centrifuged at 20,000 rpm for 20 min at 4 8C. The supernatant was passed through the filter paper No. 2 (ADVANTEC, Tokyo, Japan) and stored at 0 8C. The residue was resuspended in extraction buffer and stirred on ice for 1 h, centrifuged and filtered as described above. The obtained supernatant was combined and stored at 0 8C. The combined protein extracts were concentrated using Centricon Plus-20 (MILLIPORE, Bedford, USA) and Ultrafree0.5 (MILLIPORE). Protein preparation from leaves was carried out according to the method of Powell et al. [25]. Leaves were homogenized with a mortar and pestle in liquid nitrogen, the homogenate added with above-mentioned extraction buffer on ice and stirred overnight at 4 8C. The extraction mixture was centrifuged twice at 15,000 rpm for 30 min at 4 8C. The collected supernatant was added with 2-volumes of cooled acetone and centrifuged again at 10,000 rpm for 10 min at 4 8C. The pellet was collected, lyophilized and stored until use for Western blot analysis. Deglycosylation of protein was carried out according to the method of Edge et al. [7]. Lyophilized pear fruit protein (ca. 2.5 mg) was dissolved in 135 ml of trifluoromethanesulfonic acid (TFMS) and 15 ml of anisole in a 0.3 ml Reaction-Vial (Pierce, Rockford, USA) and kept on ice for 2 h. The reaction mixture was added to 3 ml of cooled pyridine/diethyl ether (1:9). The precipitated salts were collected by centrifugation (4000 rpm for 5 min at 4 8C), resuspended in 1 ml of 0.1 M NH4HCO3 and dialyzed against the same buffer. The resulting precipitated protein was pelleted by centrifugation (10,000 rpm for 10 min at 4 8C). Protein concentration was measured by the method of Bradford [3] using bovine serum albumin as a standard and 50 mg of protein loaded per lane on a gel. SDS-PAGE was performed using a ready-made 10% (w/v) acrylamide gel (ATTO, Tokyo, Japan) and separated proteins were transferred onto a polyvinyl difluoride membrane (PVDF Immobilon transfer membrane, pore size 0.45 mm, MILLIPORE). Polyclonal antibody raised against purified European pear PGIP [28] was used as a first antibody. Goat anti-rabbit antibody conjugated with horseradish peroxidase (Biomedical Technologies, Stoughton, USA) was used as a secondary antibody. Associated secondary antibodies were detected using ECL Western Blotting Detection Kit (Amersham Bioscience, Piskataway, USA) according to the manufacturer’s instructions.
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2.3. Fungal PG assay and inhibition with PGIP extracts The race 1 of V. nashicola was grown in potato dextrose broth (Difco, Detroit, USA) without pectin addition. After 45-day incubation at 20 8C mycelial mats were used for PG extraction as described by Isshiki et al. [14]. PG activity was measured by the 2-cyanoacetamide spectrophotometric method [2]. Polygalacturonic acid (1.5% w/v) was used as substrate, and reducing sugars released by PG were determined after 1 h of incubation at 37 8C in 50 mM acetate buffer, pH 5. Regression analysis showed that there was a linear increase in release of reducing sugars by V. nashicola PG from 0 to 90 min ðR2 ¼ 0:92Þ: These data enabled selection of 1 h time point, which was within the linear range of activity. At the end of the reaction, samples were diluted in ultra pure water (25-fold dilution). Five-hundred microliters of 2-cyanoacetamide (1%) and 1 ml of 1 M borate buffer, pH 9 were added to 500 ml of diluted samples, then the mixture was incubated at 100 8C for 10 min and absorbance measured at 274 nm. For the measurement of PGIP activity 100 ml of inhibitor (44 mg of proteins) extracted from inoculated pear leaves was pre-incubated with 100 ml of PG (0.188 U) for 10 min at 37 8C. The reaction was started after addition of 1 ml of substrate and incubation at 37 8C for 1 h. Controls with inhibitor boiled for 30 min were run simultaneously. The PGIP activity was determined as the percentage of PG activity inhibition. 2.4. PCR amplification and cloning of pear pgip genes Genomic DNA was isolated from pear leaves by using DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Oligonucleotides used as primers were synthesized on the basis of published sequence of a European pear pgip gene [28] and have the following sequences: primer 6 (50 -ACATCTCTCAGGCTCTCAACC-30 ) and primer 7 (50 -AAATTGCTGGCCAAATCTGCAG-30 ). PCR amplifications from 50 ng of pear genomic DNA were conducted on Perkin– Elmer Thermal Cycler (PERKIN ELMER Gene Amp System 2400, Norwalk, USA). Reaction conditions were as follows: 94 8C (5 min) for one cycle, then 94 (1 min), 58 (2 min) and 72 8C (2 min) for 35 cycles followed by a final extension at 72 8C (10 min). RNA was extracted from immature Kousui fruit and inoculated young leaves of Kousui and Flemish Beauty (2 days after inoculation) by using RNeasy Plant Mini Kit (QIAGEN). After DNase treatment with DNA-free (Ambion, Austin, USA), reverse transcription was performed according to the manufacturer’s instructions using RETRO Script Kit (Ambion). For RT-PCR, the primers PGIP-UniF (50 -CCACAAACCGCATCAACTCC-30 ) and PGIP-UniR (50 -GTAAACTCCACTGGGATACTCC-30 ) were designed using the Lasergene DNA Analysis Software (DNAStar, Madison, USA) and used for cloning and
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sequencing of cDNA. PCR amplification conditions were the same as described above. The PCR and RT-PCR products of pgip gene were purified and subcloned into pGEM-T Easy Vector (Promega, Madison, USA). Plasmid DNA was purified by using Wizard Plus SV Minipreps DNA Purification System (Promega). Subcloned PCR products were sequenced by the dideoxynucleotide method using the BigDye Terminator ver. 3.0 Kit (Applied Biosystems, Foster City, USA) from both strands. Multiple sequence alignment was obtained using the Lasergene Software package (DNAStar). 2.5. DNA gel blot analysis Genomic DNA isolated from leaves was digested with the indicated restriction enzymes, separated on an agarose gel (0.8%), depurinated, denatured, and transferred to a Hybond-N nylon membrane (Amersham) by capillary blotting. A 647 bp PCR fragment, amplified by the primers PGIP Uni-F and PGIP Uni-R was labeled using the DIG High Prime DNA Labeling and Detection Kit (Roche Applied Science, Penzberg, Germany) and used for hybridization. Pre-hybridization, hybridization and detection were carried out according to the manufacturer’s instructions. 2.6. pgip Gene expression analysis The expression of pgip gene was determined on inoculated leaves of potted trees (Kousui, Kinchaku, Mamenashi 12 and Flemish Beauty) by semi-quantitative RT-PCR using the designed primers KOSP-F (5 0 -ATCGTTTGGGCAGTTCATTG-3 0 ) and KOSP-R (50 ACTCCCGTAGATCTTATTGTGGTT-30 ). The amplification was limited to 26 cycles of 94 (30 s), 55 (30 s) and 65 8C (30 s), which was within the logarithmic range of amplification. Amplification of the translation elongation factor 1a (ef1-a, Mahe´ et al. [20]) was used to adjust initial amounts of RT-products of each sample in each PCR reaction. First, ef1-a amplification was carried out with degenerated primers (forward, 50 -ATTGTGGTCATTGGY CAYGT-30 and reverse, 50 -CCTATCTTGTAVA CATCCTG-30 ). These primers were previously designed, from multiple sequence alignment, to space an intron sequence [26] allowed us to check the absence of genomic DNA contamination. In a second reaction, comparative amplification was carried out with KOSP primers on equivalent amounts of total cDNA. Amplification products of pgip and ef1-a cDNA were separated on an agarose gel (1.2%), stained with ethidium bromide and finally analyzed using Bio-Rad’s FX Molecular Bioimager and Quantity One Quantitation Software (Nippon Bio-Rad, Tokyo, Japan). The ratio pgip/ef1-a was determined and plotted as fold increase above values measured at 0 d (immediately after inoculation and after checking that this value remained unchanged from that recorded in non-inoculated leaves).
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3. Results 3.1. Detection of PGIP protein from Japanese pear fruit Western blot analysis was performed with protein extracts obtained from immature fruit of cvs. Kousui and Kinchaku using a polyclonal antibody raised against European pear PGIP. One major band (approximately 42 kDa) was detected in samples from both cultivars. After chemical deglycosylation with TFMS, molecular mass changed to 35 kDa (Fig. 1). These results indicated that Japanese pear PGIP is a glycoprotein. 3.2. Cloning and sequence comparison of pgip genes isolated from different pear cultivars and strains In order to compare the sequence of pgip genes from different pear cultivars or strains, the analysis was carried out using Kousui, Kinchaku, Mamenashi 12, Flemish Beauty, and Bartlett. To minimize the possibility of DNA polymorphism due to the difference of samples, genomic DNA was extracted from a single leaf and the entire coding region of pgip gene amplified by PCR. Expected size (1057 bp) of PCR products were obtained and more than five clones from each cultivar or strain sequenced, then the pgip gene corresponding to the amino-acid positions 49 –292 sequenced from both strands and subjected to analysis (Fig. 2). The pgip sequences were highly conserved all over the clones (93.8 – 100% identity) and showed very high homology to the sequences of other pgip genes registered
Fig. 1. PGIP detected from immature fruit of Japanese pear. Crude proteins (50 mg per lane) from Kousui (lanes 1, 2) and Kinchaku (lanes 3, 4) were subjected to SDS-PAGE and PGIP detected by Western blotting with a polyclonal antibody raised against European pear PGIP. Proteins chemically deglycosylated with TFMS were shown on lanes 2 and 4.
in GenBank (Table 1). However, sequencing data revealed some variations, which seemed to be conserved among cultivars and strains (Table 2). Interestingly, these substituted amino-acids were almost identical to those in apple or European pear pgips (Fig. 2). Amplification of sequences, which are distinct, but possess high homology, may imply the presence of multiple pgip genes in a genomic DNA. To assess this hypothesis DNA isolated from leaves of Kousui, Kinchaku, Mamenashi 12 and (the European pear) Flemish Beauty was subjected to Southern blot analysis (Fig. 3). Eco RI and Hind III treatment yielded several hybridizing fragments in all cultivars and strains. These results suggest that the Asian pear genome has a family of pgip homologous genes. From multiple alignments of pgip gene sequences, we examined whether cultivar specific amino-acid substitutions could be found (Table 3). However, as pgip genes formed a multi-gene family it was difficult to identify the cultivar specificity. Some variations, detected at certain positions, were widely spread over cultivars. The gene sequences derived from cDNA in different pear organs (Kousui fruit and inoculated leaves of Kousui and Flemish Beauty) were also compared to those from genomic DNA. The pgip sequences had some variations at certain positions as same as genomic DNA-derived pgip (Table 2), suggesting that pgips are expressed in the organs. 3.3. Expression of pgip gene in different cultivars and strains inoculated with V. nashicola Northern blot analysis could not detect transcripts of pgip genes in total RNA isolated from healthy European pear [28]. However, semi-quantitative RT-PCR using primers KOSP-F and KOSP-R amplified a 278 bp pgip cDNA from pear leaves inoculated with the race 1 of V. nashicola. RTPCR analysis was performed in RNA extracted from young pear leaves inoculated with the fungus and harvested at 0, 1, 2, 3, 5 and 7 days after inoculation (Fig. 4). A transient activation was early noticed in the non-host resistant Flemish Beauty (1 and 2 days after inoculation). The same level of induction was also noticed in the host resistant Kinchaku, however kinetic of its activation was delayed and reached the highest level 3 days after inoculation. In inoculated leaves of the susceptible Kousui pgip gene expression was induced only weakly 3 days after inoculation but significantly lower than that induced in Kinchaku at the same period. In Mamenashi 12 no significant increase was noticed during the whole duration of the experiment. This experiment was repeated five times with similar results. 3.4. Expression of PGIP protein in different cultivars and strains inoculated with V. nashicola Western blot analysis was performed using proteins extracted from young pear leaves inoculated with the race 1
M. Faize et al. / Physiological and Molecular Plant Pathology 63 (2003) 319–327
Fig. 2. Comparison of deduced amino-acid sequences of PGIP. Consensus sequence of cloned pgip gene was obtained from Kousui (accession number AY333102), Kinchaku (AY333103), Mamenashi 12 (AY333104), Flemish Beauty (AY333105), and Bartlett in the current study. The sequences registered by other authors in GenBank were as follows: European pear (Pyrus communis, L09264), Japanese pear (P. pyrifolia, AB021791), and apple (Malus £ domestica, U77041). Amino-acid residues not identical are indicated in bold. The residue with an asterisk and a gray box in the consensus sequence indicates a position where an amino-acid substituted.
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Table 1 Comparison of sequence homology of pgip gene identified in this study with genes registered in GenBank
Table 3 Substitutions of amino-acid residues in pear PGIP Codon
Plant species
Pyrus pyrifolia P. communis Malus £ domestica
Cultivar
Kikusui Bartlett Golden Delicious
Accession no.
AB021791 L09264 U77041
Nucleotide identity (%) 93.5–98.1 94.7–99.6 95.1–98.0
Table 2 Variation in amino-acid residues deduced from the pgip gene sequences in pear Codon
123 165 170 173 185 218
Genomic DNA
cDNA
All cultivars
Kousui IF
Kousui IL
Flemish Beauty IL
S/F Q/E N/D/G H/R K/I/M G/T
S/F Q/E N/D/G H/R K/M G/T
S/F Q/E N/G H/R K/I G/T
F Q/E N/D H K G/T
117 185 202 233 236
Cultivar or strain Kousui
Kinchaku
Mamenashi 12
Flemish Beauty
Bartlett
K M/K/I N V G
K K/I N V G
K K/I N V V/G
N/K K/I N V G
K K/I S/N M/V G
The amino-acid residue in Italics indicates a substitution.
resistant Kinchaku at 3, 5 and 7 days after inoculation. It reached maximum intensity in inoculated leaf extracts of the non-host resistant Flemish Beauty at 3 and 5 days after inoculation. This experiment was performed twice with the similar pattern of results. No band, which matched
IF, immature fruit; IL, leaf inoculated with Venturia nashicola.
of V. nashicola and harvested at 0, 1, 2, 3, 5 and 7 days after inoculation. Results (Fig. 5) showed that despite the high quantity of protein loaded on the gel (50 mg) only a very weak 42-kDa band could be detected in leaf extracts from the susceptible Kousui. However, this band became clearly visible in extracts from inoculated leaves of the host
Fig. 3. Southern blot analysis of pgip gene in Asian and European pear genomic DNA. DNA from Kousui (lanes 1, 5), Kinchaku (lanes 2, 6), Mamenashi 12 (lanes 3, 7) and Flemish Beauty (lanes 4, 8) was digested with Eco RI or Hind III and hybridized with 647 bp digoxigenin-labeled pgip gene fragment. Sizes (kb) of the standard DNA fragments are indicated in the left.
Fig. 4. RT-PCR analysis of pgip gene expression in Asian and European pear leaves inoculated with the race 1 of Venturia nashicola. The size of the amplicon of pgip gene transcripts was 278 bp and that one of the ef1-a gene transcripts 700 bp. This latter was used as a constitutive control. Gels shown are the representatives of five replicates. Data points on graphs represent average fold increases (above that measured at 0 d) across five replicates ^ confidence interval ða ¼ 5%Þ:
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Fig. 5. Western blot analysis of PGIP protein accumulation in Asian and European pear leaves inoculated with the race 1 of Venturia nashicola. Crude proteins (50 mg per lane) from inoculated Kousui, Kinchaku and Flemish Beauty were subjected to SDS-PAGE and PGIP detected by Western blotting with a polyclonal antibody raised against European pear PGIP. Positive control (C, purified Kinchaku PGIP) is shown in the left and the right margins of the gel. The expected 42-kDa band is indicated by an arrow.
the 42-kDa protein, was detected in inoculated leaf extracts of Mamenashi 12 during the whole duration of the experiments (data not shown). 3.5. In vitro inhibition of fungal PG with PGIP from different cultivars and strains inoculated with V. nashicola PGIPs from inoculated leaf extracts of Kinchaku and Flemish Beauty (3 days after inoculation) showed relatively higher inhibition of V. nashicola PG when compared to PGIP activity of the susceptible Kousui or the strain Mamenashi 12 (9 and 12 versus 3 and 2%, on average, Fig. 6). This inhibition lasted until 5 days after inoculation and was achieved with 44 mg of protein but was never observed when only 20 mg of proteins from Kinchaku or Flemish Beauty were used. These results are consistent with our data from Western blot analysis in which PGIP protein was clearly detected 3 days after inoculation and after running with 50 mg of protein extracts from Kinchaku and Flemish Beauty (Fig. 5). The inhibition was also observed with extracts derived from these two resistant cultivars at 2 days after inoculation but higher protein amounts were needed (80 mg, not shown).
pear, PGIP has a molecular mass of 42 kDa that shifted to 35 kDa after chemical deglycosylation. We also attempted to isolate genes encoding PGIP from fruit and leaves of Japanese pear using genomic PCR and RT-PCR. The regions containing pgip genes in the genome of different pear species and cultivars as well as in apple are highly conserved. PCR amplification using genomic DNA identified different copies of the pgip gene, which was confirmed by Southern hybridization, indicating that pgip gene also exists as a gene family as reported in European pear [28] or in apple [33]. The alignment of deduced aminoacid sequences of PGIP showed differences among PGIPs, which consist of substitutions of a few amino-acids. The conservation of amino-acid substitutions over cultivars may suggest that these substitutions had occurred before cultivars were divided phylogenetically and maybe the major driving force for the evolution of pgip family. Such a variation was also found in pgip sequences derived from cDNA of leaves and fruits suggesting that they are not pseudo-genes and are expressed in the organs.
4. Discussion When young leaves of pear were inoculated with the race 1 of V. nashicola, death of subcuticular hypha cells was observed only in the host resistant Kinchaku and non-host resistant Flemish Beauty from 3 days after inoculation [24]. As V. nashicola inhabits pectin layers in pear cell walls of susceptible and resistant cultivars, and produces PG [14] it has been hypothesized that the presence of some inhibitors like PGIP could be involved in the resistance. To assess this hypothesis, we first attempted to detect PGIP protein from Japanese pear fruits. Our results showed that like in European pear [25,28] and apple [32], as well as Japanese
Fig. 6. Inhibition of PG derived from mycelia of Venturia nashicola race 1 with leaf extracts containing PGIP. 44 mg of proteins extracted from inoculated Kousui, Mamenashi 12, Kinchaku and Flemish Beauty 3 days after inoculation were mixed with fungal PG (0.188 U) and PGIP activity was assessed as the percentage of PG inhibition using the 2-cyanoacetamide method. Values are the mean from three separate reactions ^ confidence interval ða ¼ 5%Þ:
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In many plant species pgip gene family members are differentially regulated and have different functions [5]. Their expression is regulated during development and in response to several stimuli. In pear and apple the level of gene expression was higher in immature fruit than in mature fruit, the pgip transcripts were less abundant in flowers than in fruit and were not detectable from leaves [28]. Failure of detecting these transcripts from healthy leaves might be due to the technique used. For example, Northern blot analysis could not detect transcripts of pgip genes in RNA extracted from citrus and rough lemon [22,10] conversely to RT-PCR. Based on these observations we used a semi-quantitative RT-PCR to examine whether induction of pgip gene is always correlated with resistance against the race 1 of V. nashicola. Our results showed that it was possible to detect a fragment of this gene from leaves of several cultivars of pear and to study its expression. The expression of pgip gene was highly induced only in the two resistant cultivars Kinchaku and Flemish Beauty after inoculation with the race 1 of the pathogen. This observation suggested an involvement of PGIP in defense against V. nashicola. Accumulation of pgip transcripts has been shown to occur in different race –cultivar interactions between Colletotrichum lindemuthianum and bean [23]. The study reported intense accumulation of pgip transcripts in leaves, which was correlated with the hypersensitive response during incompatible interaction, while a more delayed increase, coincident with the onset of lesion formation, occurred in compatible interaction. In potato leaves, the level of PGIP protein increased during an incompatible interaction with Phytophthora infestans [18]. Our results from immuno-blotting revealed that PGIP protein was detected only in scab-inoculated leaves of the host resistant Kinchaku and the non-host resistant Flemish Beauty. Similarly to that reported by Stotz et al. [28] it was very difficult to clearly detect this protein from healthy leaves, despite the high amount of proteins loaded on the gel. PGIP activity has been also detected from scab-inoculated apple leaves [21], however, no positive correlation was found between PGIP content in the leaf and resistance against Venturia inaequalis. In this study, we examined whether PGIPs from inoculated leaves of pear cultivars were able to inhibit PG from the race 1 of V. nashicola. Although our results showed that only limited inhibition of PG could be achieved (9 –12% in the resistant cultivars Kinchaku and Flemish Beauty), they are consistent with our data from Western blot and RNA analyses. In addition, no significant inhibition was obtained from extract of the susceptible Kousui or the resistant strain Mamenashi 12 in which pgip gene expression was not induced and no PGIP protein was detected. Resistance of Mamenashi 12 was assessed recently through inoculation tests in the greenhouse and no histological data are yet available. The absence of PGIP induction in inoculated leaves of this strain may indicate that its resistance is not dependent on PGIP but rely on other mechanisms. The lowest rate of PG inhibition
obtained from extract of the two resistant cultivars Kinchaku and Flemish Beauty could be explained by the limited amounts of protein used in this study. As PG inhibition tests were performed with crude proteins, another explanation is that PGIP extracted from inoculated leaves are already ‘busy’ inhibiting co-extracted PG and so only a relatively low ‘residual’ activity was measured in the assay. It is also possible that there may be a mixture of V. nashicola PGs, some of which are not inhibited by pear PGIP. Taken together our data suggest the possible involvement of PGIP in host and non-host resistance of pear observed against V. nashicola. However, it is not excluded that the observed resistance is due, indirectly, to the induction of other defense responses. Indeed, an early induction of PGIP and a partial inhibition exhibited against fungal PG may lead to the accumulation of elicitor-like oligogalacturonides. This in turn may trigger other defense responses, enhancing then the ability of pear to resist fungal attacks. Work is now in progress to show if other molecular and biochemical mechanisms are involved in resistance along with PGIP. Mohamed Faize was supported by Japanese Society for Promotion of Science. We are grateful to Dr A.L.T. Powell, Department of Vegetable Crops, University of California, Davis, USA for her kind supply of an antibody. The authors also thank Dr P. Park, The Graduate School of Science and Technology, Kobe University, and Dr H.J. Cools, National Institute for Agro-Environmental Sciences, for useful discussion and technical guidance, respectively. Cooperation in the providing pear scions is greatly acknowledged from National Institute of Fruit Tree Science, Tottori Horticultural Experiment Station, and Akita Fruit Tree Experimental Station.
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M. Faize et al. / Physiological and Molecular Plant Pathology 63 (2003) 319–327 [8] Favaron F, D’Ovidio R, Purceddu E, Alghishi P. Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein. Planta 1994;195:80– 7. [9] Favaron F, Castiglioni C, D’Ovidio R, Alghishi P. Polygalacturonaseinhibiting proteins from Allium porrum and their role in plant tissue against fungal endopolygalacturonase. Physiol Mol Plant Pathol 1997; 50:403–17. [10] Gotoh Y, Nalumpang S, Isshiki A, Utsumi T, Gomi K, Yamamoto H, Akimitsu K. A cDNA encoding polygalacturonase inhibiting protein induced in citrus leaves by polygalacturonase of Alternaria citri. J Gen Plant Pathol 2002;68:57–61. [11] Ishii H, Udagawa H, Nishimoto S, Tsuda T, Nakashima H. Scab resistance in pear species and cultivars. Acta Phytopathol Entomol Hungarica 1992;27:293–8. [12] Ishii H, Yanase H. Venturia nashicola, the scab fungus of Japanese and Chinese pears: a species distinct from V. pirina. Mycol Res 2000; 104:755–9. [13] Ishii H, Watanabe H, Tanabe K. Venturia nashicola: pathological specialization on pears and control trial with resistance inducers. Acta Hortic 2002;587:613–21. [14] Isshiki A, Akimitsu K, Ishii H, Yamamoto H. Purification of polygalacturonases produced by the pear scab pathogens, Venturia nashicola and Venturia pirina. Physiol Mol Plant Pathol 2000;56: 263 –71. [15] James JT, Dubery IA. Inhibition of polygalacturonase from Verticillium dahliae by a polygalacturonase inhibiting protein from cotton. Phytochemistry 2001;57:149–56. [16] Johnston DJ, Ramanathan V, Williamson B. A protein from immature raspberry fruits which inhibits endopolygalacturonases from Botrytis cinerea and other microorganisms. J Exp Bot 1993;44:971– 6. [17] Komjanc M, Festi S, Rizzotti L, Cattivelli L, Cervone F, De Lorenzo G. A leucine-rich repeat receptor-like protein kinase (LRPKm1) gene is induced in Malus £ domestica by Venturia inaequalis infection and salicylic acid treatment. Plant Mol Biol 1999;40:945–57. [18] Machinandiarena MF, Olivieri FP, Daleo GR, Oliva CR. Isolation and characterization of a polygalacturonase-inhibiting protein from potato leaves: accumulation in response to salicylic acid, wounding and infection. Plant Physiol Biochem 2001;39:129–36. [19] Mahalingam R, Wang G, Knap HT. Polygalacturonase and polygalacturonase inhibitor protein: gene isolation and transcription in Glycine max–Heterodera glycines interactions. Mol Plant Microbe Interact 1999;12:490– 8. [20] Mahe´ A, Grisvard J, Dron M. Fungal and specific gene markers to follow the bean-anthracnose infection process and normalize the bean chitinase mRNA induction. Mol Plant Microbe Interact 1992;5: 242–8.
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[21] Mu¨ller M, Gessler C. A protein from apple leaves inhibits pectinolytic activity of Venturia inaequalis in vitro. In: Fritig B, Legrand M, editors. Mechanisms of plant defense responses. Dordrecht: Kluwer; 1993. p. 68–71. [22] Nalumpang S, Gotoh Y, Tsuboi H, Gomi K, Yamamoto H, Akimitsu K. Functional characterization of citrus polygalacturonase inhibiting protein. J Gen Plant Pathol 2002;68:118–27. [23] Nuss L, Mahe´ A, Clark AJ, Grisvard J, Dron M, Cervone F, De Lorenzo G. Differential accumulation of PGIP (polygalacturonaseinhibiting protein) mRNA in two near-isogenic lines of Phaseolus vulgaris L. upon infection with Colletotrichum lindemuthianum. Physiol Mol Plant Pathol 1996;48:83–9. [24] Park P, Ishii H, Adachi Y, Kanematsu S, Ieki H, Umemoto S. Infection behavior of Venturia nashicola, the cause of scab on Asian pears. Phytopathology 2000;90:1209–16. [25] Powell ALT, Van Kan J, Have AT, Visser J, Greve LC, Bennett AB, Labavitch JM. Transgenic expression of pear PGIP in tomato limits fungal colonization. Mol Plant Microbe Interact 2000;13:942–50. [26] Rosati C, Cadic A, Duron M, Renou JP, Simoneau P. Molecular cloning and expression analysis of dihydroflavanol 4-reductase gene in flower organs of Forsythia £ intermedia. Plant Mol Biol 1997;15: 303–11. [27] Simpson CG, MacRae E, Gardner RC. Cloning of a polygalacturonase inhibiting protein from kiwifruit (Actinidia deliciosa). Plant Physiol 1995;102:133 –8. [28] Stotz HU, Powell ALT, Damon SE, Greve LC, Bennett AB, Labavitch JM. Molecular characterization of a polygalacturonase inhibitor from Pyrus communis L. cv. Bartlett. Plant Physiol 1993;102:133– 8. [29] Stotz HU, Contos JJA, Powell ALT, Bennett AB, Labavitch JM. Structure and expression of an inhibitor of fungal polygalacturonases from tomato. Plant Mol Biol 1994;25:607–17. [30] Tanaka S, Yamamoto S. Studies on pear scab. II. Taxonomy of the causal fungus of Japanese pear scab. Ann Phytopathol Soc Jpn 1964; 29:128–36. [31] Toubart P, Desiderio A, Salvi G, Cervone F, Daroda L, De Lorenzo G. Cloning and characterization of the gene encoding the endopolygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L. Plant J 1992;2:367 –73. [32] Yao C, Conway WS, Sams CE. Purification and characterization of polygalacturonase-inhibiting protein from apple fruit. Phytopathology 1995;85:1373 –7. [33] Yao C, Conway WS, Ren R, Smith D, Ross GS, Sams CE. Gene encoding polygalacturonase inhibitor in apple fruit is developmentally regulated and activated by wounding and fungal infection. Plant Mol Biol 1999;39:1231– 41.
Polygalacturonase-inhibiting protein (PGIP) from Japanese pear: possible involvement in resistance against scab Mohamed Faize, Tomoko Sugiyama, Lydia Faize, Hideo Ishii* National Institute for Agro-Environmental Sciences, 3-1-3 Tsukuba, Ibaraki 305-8604, Japan Accepted 29 March 2004
Abstract Venturia nashicola is the causal agent of scab, a fungal disease affecting Asian pears. The Japanese pear cv. ‘Kousui’ is highly susceptible to the race 1 of this fungus whereas the cv. ‘Kinchaku’ and the non-host European pear cv. ‘Flemish Beauty’ are resistant. The aim of this work is to investigate the role of polygalacturonase-inhibiting proteins (PGIPs) of pear during the interactions with V. nashicola leading to susceptibility or resistance. PGIP protein was detected from immature fruit of Kousui and Kinchaku. It showed a molecular mass of 42 kDa that shifted to 35 kDa after chemical deglycosylation. The gene pgip was amplified by PCR using genomic DNA and/or cDNA from young leaves of Kousui, Kinchaku, and European pear cvs. Flemish Beauty, ‘Bartlett’, and an Asian wild pear strain ‘Mamenashi 12’, then sequenced after sub-cloning. Some conserved variations were identified in the sequence indicating that gene family also exists in pgip of Japanese pear and confirmed by Southern blot analysis. The expression of PGIP was studied in scab-inoculated leaves of the susceptible Kousui and the resistant Kinchaku and Flemish Beauty. pgip Gene and its encoding protein were highly and rapidly activated in these resistant plants. In addition, PGIP extracts derived from Kinchaku and Flemish Beauty partially inhibited the activity of polygalacturonase (PG) from V. nashicola suggesting a possible role of PGIP in limiting fungal growth frequently observed in these resistant cultivars. q 2004 Elsevier Ltd. All rights reserved. Keywords: Polygalacturonase-inhibiting protein; Nucleotide sequence; Pear scab; Venturia nashicola; Japanese pear; European pear
1. Introduction Scab is an important disease of Asian pears such as Japanese pear (Pyrus pyrifolia var. culta) and Chinese pear (P. ussuriensis). So far examined, Venturia nashicola, the scab fungus of Asian pears, is pathogenic on Japanese and Chinese pears but not pathogenic on European pear like ‘Flemish Beauty’ [12,30]. Scab resistance of pear species and cultivars was assessed for their potential use in breeding programs and it was found that the non-commercial Japanese pear cv. ‘Kinchaku’ was resistant to V. nashicola [11]. In addition, Ishii et al. [13] found pathological specialization of V. nashicola and classified strains of this fungus into three races: race 1, pathogenic on the most popular but highly scab-susceptible Japanese pear cv. ‘Kousui’ but not on Abbreviations: PGIP, polygalacturonase-inhibiting protein; PG, polygalacturonase; LRR, leucine-rich repeat; LRPKm1, leucine-rich repeat receptor-like protein kinase; TFMS, trifluoromethanesulfonic acid. * Corresponding author. Tel./fax: þ 81-29-838-8307. E-mail addresses: [email protected] (H. Ishii), faizemohamed@ hotmail.com (M. Faize). 0885-5765/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2004.03.006
the Asian wild pear strain ‘Mamenashi 12’; race 2, pathogenic on Mamenashi 12 but not on Kousui; and race 3, pathogenic on both Mamenashi 12 and Kousui. Early and late stages of the infection behavior of the race 1 of V. nashicola were studied on the leaves of susceptible Kousui and the resistant cvs. Kinchaku and Flemish Beauty. Histological observations showed that early stages of infection were similar on the susceptible and the resistant cultivars, because V. nashicola penetrated only the cuticle layer and formed subcuticular hyphae on all cultivars [24]. The subcuticular hyphae never penetrated into the epidermal cells even 7 days after inoculation suggesting that V. nashicola produces and secretes pectin-degrading enzymes for utilizing nutrients. Subsequently, three endopolygalacturonases and an exopolygalacturonase produced by V. nashicola and V. pirina (the scab fungus of European pear), respectively, were purified and characterized recently [14]. However, during the late stage of infection, differences were observed between susceptible and resistant cultivars. Fungal cells appeared intact in susceptible Kousui, while in both resistant Kinchaku and Flemish
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Beauty fungal cell walls appeared decomposed, attenuated and hyphae collapsed, and general disruption of cellular membranes observed. Cell death of V. nashicola in these resistant cultivars implies the presence of some inhibition factors [24]. As a first approach to study on scab-resistance mechanism, we focused on polygalacturonase-inhibiting proteins (PGIPs). These proteins inhibit the activity of fungal polygaracturonase (PG), which is considered to be an important factor for pathogenesis [5]. PGIPs are glycoprotein present in the plant cell wall of vegetative as well as fruit tissues and genes encoding PGIPs have been cloned from various plants [4,6,8 – 10,15,16,18,19,27 – 29,31 – 33]. They belong to a super-family of leucine-rich repeat (LRR) protein and are considered to have important roles in plant – pathogen interactions. Several plant disease-resistance genes such as Cf-9, Cf-2 and Xa21 encode the member of the LRR protein family as well, and have high homology to PGIPs [5]. Furthermore, leucine-rich repeat receptor-like protein kinase (LRPKm1) that is likely to participate in defense-related signaling has been isolated by pgip probe in apple [17]. Although PGIPs are previously reported in apple and European pear fruit, there is no sufficient information concerning their role in resistance, specially those from leaves. This work aimed to detect PGIP protein and characterize pgip genes from fruit and leaves of Japanese pear. To help understanding the role of this protein in resistance against V. nashicola, the expression of PGIP protein as well as pgip gene was analyzed during susceptible and resistant interactions.
2. Materials and methods 2.1. Plant material, fungal strain and inoculation test The following pear trees were grown in a screen-house or an orchard in National Institute for Agro-Environmental Sciences: Japanese pear cvs. Kousui and Kinchaku, European pear cvs. Flemish Beauty and Bartlett, and the Asian wild pear strain Mamenashi 12. Fresh conidia collected directly from lesions on leaves of Kousui were suspended in distilled water, filtered through tissue paper and washed by centrifugation. Conidia were resuspended in 0.1% sucrose and concentrations were adjusted to ca. 5 £ 105 conidia ml21. Conidial suspensions were sprayed to young leaves of potted trees. After air-drying, the inoculated plants were incubated in a moist chamber at 20 8C for 48 h, transferred to a phytotron, and maintained at 25 8C. The inoculated leaves were detached from plants at intervals (0, 1, 2, 3, 5 and 7 days after inoculation) and stored at 2 80 8C until use. For RNA extraction 1 cm diameter leaf discs were cut from each leaf, snap frozen in liquid nitrogen and stored at 2 80 8C.
2.2. Protein extraction, chemical deglycosylation and Western blot analysis Proteins were extracted from immature fruit (3 – 5 cm in diameter) of Kousui and Kinchaku by using the modified method of Abu-Goukh et al. [1]. Fruit flesh was homogenized in extraction buffer [0.1 M sodium acetate, pH 6.0/1 M NaCl/1% (w/v) polyvinylpyrrolidone/ 0.2% (w/v) sodium bisulfite] with Physcotron (NITI-ON, Funabashi, Japan). The homogenate was stirred on ice for 1 h and centrifuged at 20,000 rpm for 20 min at 4 8C. The supernatant was passed through the filter paper No. 2 (ADVANTEC, Tokyo, Japan) and stored at 0 8C. The residue was resuspended in extraction buffer and stirred on ice for 1 h, centrifuged and filtered as described above. The obtained supernatant was combined and stored at 0 8C. The combined protein extracts were concentrated using Centricon Plus-20 (MILLIPORE, Bedford, USA) and Ultrafree0.5 (MILLIPORE). Protein preparation from leaves was carried out according to the method of Powell et al. [25]. Leaves were homogenized with a mortar and pestle in liquid nitrogen, the homogenate added with above-mentioned extraction buffer on ice and stirred overnight at 4 8C. The extraction mixture was centrifuged twice at 15,000 rpm for 30 min at 4 8C. The collected supernatant was added with 2-volumes of cooled acetone and centrifuged again at 10,000 rpm for 10 min at 4 8C. The pellet was collected, lyophilized and stored until use for Western blot analysis. Deglycosylation of protein was carried out according to the method of Edge et al. [7]. Lyophilized pear fruit protein (ca. 2.5 mg) was dissolved in 135 ml of trifluoromethanesulfonic acid (TFMS) and 15 ml of anisole in a 0.3 ml Reaction-Vial (Pierce, Rockford, USA) and kept on ice for 2 h. The reaction mixture was added to 3 ml of cooled pyridine/diethyl ether (1:9). The precipitated salts were collected by centrifugation (4000 rpm for 5 min at 4 8C), resuspended in 1 ml of 0.1 M NH4HCO3 and dialyzed against the same buffer. The resulting precipitated protein was pelleted by centrifugation (10,000 rpm for 10 min at 4 8C). Protein concentration was measured by the method of Bradford [3] using bovine serum albumin as a standard and 50 mg of protein loaded per lane on a gel. SDS-PAGE was performed using a ready-made 10% (w/v) acrylamide gel (ATTO, Tokyo, Japan) and separated proteins were transferred onto a polyvinyl difluoride membrane (PVDF Immobilon transfer membrane, pore size 0.45 mm, MILLIPORE). Polyclonal antibody raised against purified European pear PGIP [28] was used as a first antibody. Goat anti-rabbit antibody conjugated with horseradish peroxidase (Biomedical Technologies, Stoughton, USA) was used as a secondary antibody. Associated secondary antibodies were detected using ECL Western Blotting Detection Kit (Amersham Bioscience, Piskataway, USA) according to the manufacturer’s instructions.
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2.3. Fungal PG assay and inhibition with PGIP extracts The race 1 of V. nashicola was grown in potato dextrose broth (Difco, Detroit, USA) without pectin addition. After 45-day incubation at 20 8C mycelial mats were used for PG extraction as described by Isshiki et al. [14]. PG activity was measured by the 2-cyanoacetamide spectrophotometric method [2]. Polygalacturonic acid (1.5% w/v) was used as substrate, and reducing sugars released by PG were determined after 1 h of incubation at 37 8C in 50 mM acetate buffer, pH 5. Regression analysis showed that there was a linear increase in release of reducing sugars by V. nashicola PG from 0 to 90 min ðR2 ¼ 0:92Þ: These data enabled selection of 1 h time point, which was within the linear range of activity. At the end of the reaction, samples were diluted in ultra pure water (25-fold dilution). Five-hundred microliters of 2-cyanoacetamide (1%) and 1 ml of 1 M borate buffer, pH 9 were added to 500 ml of diluted samples, then the mixture was incubated at 100 8C for 10 min and absorbance measured at 274 nm. For the measurement of PGIP activity 100 ml of inhibitor (44 mg of proteins) extracted from inoculated pear leaves was pre-incubated with 100 ml of PG (0.188 U) for 10 min at 37 8C. The reaction was started after addition of 1 ml of substrate and incubation at 37 8C for 1 h. Controls with inhibitor boiled for 30 min were run simultaneously. The PGIP activity was determined as the percentage of PG activity inhibition. 2.4. PCR amplification and cloning of pear pgip genes Genomic DNA was isolated from pear leaves by using DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Oligonucleotides used as primers were synthesized on the basis of published sequence of a European pear pgip gene [28] and have the following sequences: primer 6 (50 -ACATCTCTCAGGCTCTCAACC-30 ) and primer 7 (50 -AAATTGCTGGCCAAATCTGCAG-30 ). PCR amplifications from 50 ng of pear genomic DNA were conducted on Perkin– Elmer Thermal Cycler (PERKIN ELMER Gene Amp System 2400, Norwalk, USA). Reaction conditions were as follows: 94 8C (5 min) for one cycle, then 94 (1 min), 58 (2 min) and 72 8C (2 min) for 35 cycles followed by a final extension at 72 8C (10 min). RNA was extracted from immature Kousui fruit and inoculated young leaves of Kousui and Flemish Beauty (2 days after inoculation) by using RNeasy Plant Mini Kit (QIAGEN). After DNase treatment with DNA-free (Ambion, Austin, USA), reverse transcription was performed according to the manufacturer’s instructions using RETRO Script Kit (Ambion). For RT-PCR, the primers PGIP-UniF (50 -CCACAAACCGCATCAACTCC-30 ) and PGIP-UniR (50 -GTAAACTCCACTGGGATACTCC-30 ) were designed using the Lasergene DNA Analysis Software (DNAStar, Madison, USA) and used for cloning and
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sequencing of cDNA. PCR amplification conditions were the same as described above. The PCR and RT-PCR products of pgip gene were purified and subcloned into pGEM-T Easy Vector (Promega, Madison, USA). Plasmid DNA was purified by using Wizard Plus SV Minipreps DNA Purification System (Promega). Subcloned PCR products were sequenced by the dideoxynucleotide method using the BigDye Terminator ver. 3.0 Kit (Applied Biosystems, Foster City, USA) from both strands. Multiple sequence alignment was obtained using the Lasergene Software package (DNAStar). 2.5. DNA gel blot analysis Genomic DNA isolated from leaves was digested with the indicated restriction enzymes, separated on an agarose gel (0.8%), depurinated, denatured, and transferred to a Hybond-N nylon membrane (Amersham) by capillary blotting. A 647 bp PCR fragment, amplified by the primers PGIP Uni-F and PGIP Uni-R was labeled using the DIG High Prime DNA Labeling and Detection Kit (Roche Applied Science, Penzberg, Germany) and used for hybridization. Pre-hybridization, hybridization and detection were carried out according to the manufacturer’s instructions. 2.6. pgip Gene expression analysis The expression of pgip gene was determined on inoculated leaves of potted trees (Kousui, Kinchaku, Mamenashi 12 and Flemish Beauty) by semi-quantitative RT-PCR using the designed primers KOSP-F (5 0 -ATCGTTTGGGCAGTTCATTG-3 0 ) and KOSP-R (50 ACTCCCGTAGATCTTATTGTGGTT-30 ). The amplification was limited to 26 cycles of 94 (30 s), 55 (30 s) and 65 8C (30 s), which was within the logarithmic range of amplification. Amplification of the translation elongation factor 1a (ef1-a, Mahe´ et al. [20]) was used to adjust initial amounts of RT-products of each sample in each PCR reaction. First, ef1-a amplification was carried out with degenerated primers (forward, 50 -ATTGTGGTCATTGGY CAYGT-30 and reverse, 50 -CCTATCTTGTAVA CATCCTG-30 ). These primers were previously designed, from multiple sequence alignment, to space an intron sequence [26] allowed us to check the absence of genomic DNA contamination. In a second reaction, comparative amplification was carried out with KOSP primers on equivalent amounts of total cDNA. Amplification products of pgip and ef1-a cDNA were separated on an agarose gel (1.2%), stained with ethidium bromide and finally analyzed using Bio-Rad’s FX Molecular Bioimager and Quantity One Quantitation Software (Nippon Bio-Rad, Tokyo, Japan). The ratio pgip/ef1-a was determined and plotted as fold increase above values measured at 0 d (immediately after inoculation and after checking that this value remained unchanged from that recorded in non-inoculated leaves).
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3. Results 3.1. Detection of PGIP protein from Japanese pear fruit Western blot analysis was performed with protein extracts obtained from immature fruit of cvs. Kousui and Kinchaku using a polyclonal antibody raised against European pear PGIP. One major band (approximately 42 kDa) was detected in samples from both cultivars. After chemical deglycosylation with TFMS, molecular mass changed to 35 kDa (Fig. 1). These results indicated that Japanese pear PGIP is a glycoprotein. 3.2. Cloning and sequence comparison of pgip genes isolated from different pear cultivars and strains In order to compare the sequence of pgip genes from different pear cultivars or strains, the analysis was carried out using Kousui, Kinchaku, Mamenashi 12, Flemish Beauty, and Bartlett. To minimize the possibility of DNA polymorphism due to the difference of samples, genomic DNA was extracted from a single leaf and the entire coding region of pgip gene amplified by PCR. Expected size (1057 bp) of PCR products were obtained and more than five clones from each cultivar or strain sequenced, then the pgip gene corresponding to the amino-acid positions 49 –292 sequenced from both strands and subjected to analysis (Fig. 2). The pgip sequences were highly conserved all over the clones (93.8 – 100% identity) and showed very high homology to the sequences of other pgip genes registered
Fig. 1. PGIP detected from immature fruit of Japanese pear. Crude proteins (50 mg per lane) from Kousui (lanes 1, 2) and Kinchaku (lanes 3, 4) were subjected to SDS-PAGE and PGIP detected by Western blotting with a polyclonal antibody raised against European pear PGIP. Proteins chemically deglycosylated with TFMS were shown on lanes 2 and 4.
in GenBank (Table 1). However, sequencing data revealed some variations, which seemed to be conserved among cultivars and strains (Table 2). Interestingly, these substituted amino-acids were almost identical to those in apple or European pear pgips (Fig. 2). Amplification of sequences, which are distinct, but possess high homology, may imply the presence of multiple pgip genes in a genomic DNA. To assess this hypothesis DNA isolated from leaves of Kousui, Kinchaku, Mamenashi 12 and (the European pear) Flemish Beauty was subjected to Southern blot analysis (Fig. 3). Eco RI and Hind III treatment yielded several hybridizing fragments in all cultivars and strains. These results suggest that the Asian pear genome has a family of pgip homologous genes. From multiple alignments of pgip gene sequences, we examined whether cultivar specific amino-acid substitutions could be found (Table 3). However, as pgip genes formed a multi-gene family it was difficult to identify the cultivar specificity. Some variations, detected at certain positions, were widely spread over cultivars. The gene sequences derived from cDNA in different pear organs (Kousui fruit and inoculated leaves of Kousui and Flemish Beauty) were also compared to those from genomic DNA. The pgip sequences had some variations at certain positions as same as genomic DNA-derived pgip (Table 2), suggesting that pgips are expressed in the organs. 3.3. Expression of pgip gene in different cultivars and strains inoculated with V. nashicola Northern blot analysis could not detect transcripts of pgip genes in total RNA isolated from healthy European pear [28]. However, semi-quantitative RT-PCR using primers KOSP-F and KOSP-R amplified a 278 bp pgip cDNA from pear leaves inoculated with the race 1 of V. nashicola. RTPCR analysis was performed in RNA extracted from young pear leaves inoculated with the fungus and harvested at 0, 1, 2, 3, 5 and 7 days after inoculation (Fig. 4). A transient activation was early noticed in the non-host resistant Flemish Beauty (1 and 2 days after inoculation). The same level of induction was also noticed in the host resistant Kinchaku, however kinetic of its activation was delayed and reached the highest level 3 days after inoculation. In inoculated leaves of the susceptible Kousui pgip gene expression was induced only weakly 3 days after inoculation but significantly lower than that induced in Kinchaku at the same period. In Mamenashi 12 no significant increase was noticed during the whole duration of the experiment. This experiment was repeated five times with similar results. 3.4. Expression of PGIP protein in different cultivars and strains inoculated with V. nashicola Western blot analysis was performed using proteins extracted from young pear leaves inoculated with the race 1
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Fig. 2. Comparison of deduced amino-acid sequences of PGIP. Consensus sequence of cloned pgip gene was obtained from Kousui (accession number AY333102), Kinchaku (AY333103), Mamenashi 12 (AY333104), Flemish Beauty (AY333105), and Bartlett in the current study. The sequences registered by other authors in GenBank were as follows: European pear (Pyrus communis, L09264), Japanese pear (P. pyrifolia, AB021791), and apple (Malus £ domestica, U77041). Amino-acid residues not identical are indicated in bold. The residue with an asterisk and a gray box in the consensus sequence indicates a position where an amino-acid substituted.
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Table 1 Comparison of sequence homology of pgip gene identified in this study with genes registered in GenBank
Table 3 Substitutions of amino-acid residues in pear PGIP Codon
Plant species
Pyrus pyrifolia P. communis Malus £ domestica
Cultivar
Kikusui Bartlett Golden Delicious
Accession no.
AB021791 L09264 U77041
Nucleotide identity (%) 93.5–98.1 94.7–99.6 95.1–98.0
Table 2 Variation in amino-acid residues deduced from the pgip gene sequences in pear Codon
123 165 170 173 185 218
Genomic DNA
cDNA
All cultivars
Kousui IF
Kousui IL
Flemish Beauty IL
S/F Q/E N/D/G H/R K/I/M G/T
S/F Q/E N/D/G H/R K/M G/T
S/F Q/E N/G H/R K/I G/T
F Q/E N/D H K G/T
117 185 202 233 236
Cultivar or strain Kousui
Kinchaku
Mamenashi 12
Flemish Beauty
Bartlett
K M/K/I N V G
K K/I N V G
K K/I N V V/G
N/K K/I N V G
K K/I S/N M/V G
The amino-acid residue in Italics indicates a substitution.
resistant Kinchaku at 3, 5 and 7 days after inoculation. It reached maximum intensity in inoculated leaf extracts of the non-host resistant Flemish Beauty at 3 and 5 days after inoculation. This experiment was performed twice with the similar pattern of results. No band, which matched
IF, immature fruit; IL, leaf inoculated with Venturia nashicola.
of V. nashicola and harvested at 0, 1, 2, 3, 5 and 7 days after inoculation. Results (Fig. 5) showed that despite the high quantity of protein loaded on the gel (50 mg) only a very weak 42-kDa band could be detected in leaf extracts from the susceptible Kousui. However, this band became clearly visible in extracts from inoculated leaves of the host
Fig. 3. Southern blot analysis of pgip gene in Asian and European pear genomic DNA. DNA from Kousui (lanes 1, 5), Kinchaku (lanes 2, 6), Mamenashi 12 (lanes 3, 7) and Flemish Beauty (lanes 4, 8) was digested with Eco RI or Hind III and hybridized with 647 bp digoxigenin-labeled pgip gene fragment. Sizes (kb) of the standard DNA fragments are indicated in the left.
Fig. 4. RT-PCR analysis of pgip gene expression in Asian and European pear leaves inoculated with the race 1 of Venturia nashicola. The size of the amplicon of pgip gene transcripts was 278 bp and that one of the ef1-a gene transcripts 700 bp. This latter was used as a constitutive control. Gels shown are the representatives of five replicates. Data points on graphs represent average fold increases (above that measured at 0 d) across five replicates ^ confidence interval ða ¼ 5%Þ:
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Fig. 5. Western blot analysis of PGIP protein accumulation in Asian and European pear leaves inoculated with the race 1 of Venturia nashicola. Crude proteins (50 mg per lane) from inoculated Kousui, Kinchaku and Flemish Beauty were subjected to SDS-PAGE and PGIP detected by Western blotting with a polyclonal antibody raised against European pear PGIP. Positive control (C, purified Kinchaku PGIP) is shown in the left and the right margins of the gel. The expected 42-kDa band is indicated by an arrow.
the 42-kDa protein, was detected in inoculated leaf extracts of Mamenashi 12 during the whole duration of the experiments (data not shown). 3.5. In vitro inhibition of fungal PG with PGIP from different cultivars and strains inoculated with V. nashicola PGIPs from inoculated leaf extracts of Kinchaku and Flemish Beauty (3 days after inoculation) showed relatively higher inhibition of V. nashicola PG when compared to PGIP activity of the susceptible Kousui or the strain Mamenashi 12 (9 and 12 versus 3 and 2%, on average, Fig. 6). This inhibition lasted until 5 days after inoculation and was achieved with 44 mg of protein but was never observed when only 20 mg of proteins from Kinchaku or Flemish Beauty were used. These results are consistent with our data from Western blot analysis in which PGIP protein was clearly detected 3 days after inoculation and after running with 50 mg of protein extracts from Kinchaku and Flemish Beauty (Fig. 5). The inhibition was also observed with extracts derived from these two resistant cultivars at 2 days after inoculation but higher protein amounts were needed (80 mg, not shown).
pear, PGIP has a molecular mass of 42 kDa that shifted to 35 kDa after chemical deglycosylation. We also attempted to isolate genes encoding PGIP from fruit and leaves of Japanese pear using genomic PCR and RT-PCR. The regions containing pgip genes in the genome of different pear species and cultivars as well as in apple are highly conserved. PCR amplification using genomic DNA identified different copies of the pgip gene, which was confirmed by Southern hybridization, indicating that pgip gene also exists as a gene family as reported in European pear [28] or in apple [33]. The alignment of deduced aminoacid sequences of PGIP showed differences among PGIPs, which consist of substitutions of a few amino-acids. The conservation of amino-acid substitutions over cultivars may suggest that these substitutions had occurred before cultivars were divided phylogenetically and maybe the major driving force for the evolution of pgip family. Such a variation was also found in pgip sequences derived from cDNA of leaves and fruits suggesting that they are not pseudo-genes and are expressed in the organs.
4. Discussion When young leaves of pear were inoculated with the race 1 of V. nashicola, death of subcuticular hypha cells was observed only in the host resistant Kinchaku and non-host resistant Flemish Beauty from 3 days after inoculation [24]. As V. nashicola inhabits pectin layers in pear cell walls of susceptible and resistant cultivars, and produces PG [14] it has been hypothesized that the presence of some inhibitors like PGIP could be involved in the resistance. To assess this hypothesis, we first attempted to detect PGIP protein from Japanese pear fruits. Our results showed that like in European pear [25,28] and apple [32], as well as Japanese
Fig. 6. Inhibition of PG derived from mycelia of Venturia nashicola race 1 with leaf extracts containing PGIP. 44 mg of proteins extracted from inoculated Kousui, Mamenashi 12, Kinchaku and Flemish Beauty 3 days after inoculation were mixed with fungal PG (0.188 U) and PGIP activity was assessed as the percentage of PG inhibition using the 2-cyanoacetamide method. Values are the mean from three separate reactions ^ confidence interval ða ¼ 5%Þ:
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In many plant species pgip gene family members are differentially regulated and have different functions [5]. Their expression is regulated during development and in response to several stimuli. In pear and apple the level of gene expression was higher in immature fruit than in mature fruit, the pgip transcripts were less abundant in flowers than in fruit and were not detectable from leaves [28]. Failure of detecting these transcripts from healthy leaves might be due to the technique used. For example, Northern blot analysis could not detect transcripts of pgip genes in RNA extracted from citrus and rough lemon [22,10] conversely to RT-PCR. Based on these observations we used a semi-quantitative RT-PCR to examine whether induction of pgip gene is always correlated with resistance against the race 1 of V. nashicola. Our results showed that it was possible to detect a fragment of this gene from leaves of several cultivars of pear and to study its expression. The expression of pgip gene was highly induced only in the two resistant cultivars Kinchaku and Flemish Beauty after inoculation with the race 1 of the pathogen. This observation suggested an involvement of PGIP in defense against V. nashicola. Accumulation of pgip transcripts has been shown to occur in different race –cultivar interactions between Colletotrichum lindemuthianum and bean [23]. The study reported intense accumulation of pgip transcripts in leaves, which was correlated with the hypersensitive response during incompatible interaction, while a more delayed increase, coincident with the onset of lesion formation, occurred in compatible interaction. In potato leaves, the level of PGIP protein increased during an incompatible interaction with Phytophthora infestans [18]. Our results from immuno-blotting revealed that PGIP protein was detected only in scab-inoculated leaves of the host resistant Kinchaku and the non-host resistant Flemish Beauty. Similarly to that reported by Stotz et al. [28] it was very difficult to clearly detect this protein from healthy leaves, despite the high amount of proteins loaded on the gel. PGIP activity has been also detected from scab-inoculated apple leaves [21], however, no positive correlation was found between PGIP content in the leaf and resistance against Venturia inaequalis. In this study, we examined whether PGIPs from inoculated leaves of pear cultivars were able to inhibit PG from the race 1 of V. nashicola. Although our results showed that only limited inhibition of PG could be achieved (9 –12% in the resistant cultivars Kinchaku and Flemish Beauty), they are consistent with our data from Western blot and RNA analyses. In addition, no significant inhibition was obtained from extract of the susceptible Kousui or the resistant strain Mamenashi 12 in which pgip gene expression was not induced and no PGIP protein was detected. Resistance of Mamenashi 12 was assessed recently through inoculation tests in the greenhouse and no histological data are yet available. The absence of PGIP induction in inoculated leaves of this strain may indicate that its resistance is not dependent on PGIP but rely on other mechanisms. The lowest rate of PG inhibition
obtained from extract of the two resistant cultivars Kinchaku and Flemish Beauty could be explained by the limited amounts of protein used in this study. As PG inhibition tests were performed with crude proteins, another explanation is that PGIP extracted from inoculated leaves are already ‘busy’ inhibiting co-extracted PG and so only a relatively low ‘residual’ activity was measured in the assay. It is also possible that there may be a mixture of V. nashicola PGs, some of which are not inhibited by pear PGIP. Taken together our data suggest the possible involvement of PGIP in host and non-host resistance of pear observed against V. nashicola. However, it is not excluded that the observed resistance is due, indirectly, to the induction of other defense responses. Indeed, an early induction of PGIP and a partial inhibition exhibited against fungal PG may lead to the accumulation of elicitor-like oligogalacturonides. This in turn may trigger other defense responses, enhancing then the ability of pear to resist fungal attacks. Work is now in progress to show if other molecular and biochemical mechanisms are involved in resistance along with PGIP. Mohamed Faize was supported by Japanese Society for Promotion of Science. We are grateful to Dr A.L.T. Powell, Department of Vegetable Crops, University of California, Davis, USA for her kind supply of an antibody. The authors also thank Dr P. Park, The Graduate School of Science and Technology, Kobe University, and Dr H.J. Cools, National Institute for Agro-Environmental Sciences, for useful discussion and technical guidance, respectively. Cooperation in the providing pear scions is greatly acknowledged from National Institute of Fruit Tree Science, Tottori Horticultural Experiment Station, and Akita Fruit Tree Experimental Station.
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