Background

The plant cell wall represents one of the main obstacles to the colonization of the plant tissue by microbial pathogens. To surmount this barrier, most fungal pathogens produce cell wall degrading enzymes (CWDEs), among which endo-polygalacturonases (PGs; EC 3.2.1.15) are secreted at very early stages of the infection process [1]. PGs cleave the α-(1-4) linkages between D-galacturonic acid residues in homogalacturonan, causing cell separation and maceration of host tissue. To counteract the activity of PGs, plants possess cell wall glycoproteins, called polygalacturonase-inhibiting proteins (PGIPs), the importance of which in defence has been demonstrated in different plant species [2]-[12].

Like the products of many resistance genes, PGIPs belong to the subclass of proteins containing leucine-rich repeats (LRRs) of the extracytoplasmic type (LxxLxLxxNxLT/SGxIPxxLxxLxx) [13]. The LRR domain of PGIP is typically formed by 10 imperfect LRRs of 24 residues each and is responsible for the molecular interaction with PGs. The LRRs are organized to form two β-sheets, one of which (sheet B1) occupies the concave inner side of the molecule and contains residues crucial for PG recognition [14].

To counteract the many PGs produced by fungal pathogens, plants have evolved a variety of PGIPs with different specificities. Variability is present also within each plant species, since PGIPs are encoded by gene families, comprising 2 members in Arabidopsis thaliana [3] up to 16 in Brassica napus [15]. A clear example of intra-specific variation in the inhibition properties against fungal and insect PGs has been reported for members of the bean (Phaseolus vulgaris L.) pgip family [16]. Variation among different family members extends also to the regulation of their expression [17].

The soybean (Glycine max [L.] Merr.) pgip family is composed by at least four genes forming two clusters, one containing Gmpgip1 and Gmpgip2, separated by about 3 kbp, and the other containing Gmpgip3 and Gmpgip4, separated by a maximum distance of 60 Kbp [18]. These findings are particularly interesting because soybean is a well-established paleopolyploid plant species. Consequently, the characterization of the full complement of the soybean pgip family could provide important information about the forces driving the evolution of this gene family.

In this study, we have characterized the complete set of pgip genes in the soybean genotype Williams 82 and demonstrated the existence of two pgip loci. The study was extended to other legume species by characterizing a region of about 140 Kb, comprising the single pgip locus of bean, and the pgip regions in the recently released genomes of Medicago truncatula Gaertn. [19] and Cicer arietinum L. [20]. Comparative analysis between the pgip regions of these species suggests that the legume pgip family follows the birth-and-death model of evolution.

Results

Characterization of soybean BAC clones and isolation of two novel Gmpgipgenes

Seven BAC clones, previously isolated from a soybean BAC library using a pgip probe, were analysed with primers specific for Gmpgip1, Gmpgip2, Gmpgip3 and Gmpgip4 [18]. None of the BAC clones contained all four pgip sequences together. Three of them (95O4, 85 M15, 28B18) contained both Gmpgip1 and Gmpgip2 and two (26I2, 6 F5) contained both Gmpgip3 and Gmpgip4. The remaining two BAC clones (1 F11, 62 K14) did not produce a clear amplicon with none of the Gmpgip primer combinations; therefore, they were not analyzed further. The size of the insert contained in each BAC clone, determined by pulsed-field gel electrophoresis (PFGE) following NotI digestion, varied between about 50 Kb and 190 Kb (Additional files 1 and 2). Fingerprinting of the BAC clones following HindIII digestion showed overlapping profiles for those containing Gmpgip1 and Gmpgip2 (95O4, 85 M15, 28B18), and those containing Gmpgip3 and Gmpgip4 (26I2, 6 F5) (Additional file 1). BAC end sequencing and shotgun subcloning and sequencing identified two novel Gmpgip genes, one in the clone 85 M15 (Gmpgip5), the other in the clone 26I2 (Gmpgip7). Gmpgip5 was at the terminal end of 85 M15 and was partial; its complete sequence was obtained by PCR performed on genomic DNA. The coding regions of Gmpgip5 and Gmpgip7 contain uninterrupted open reading frames (ORFs) of 1008 and 1011 bp, respectively, including the stop codon. The predicted amino acid sequence of these two ORFs showed the typical PGIP structure, comprising a 21 amino acid signal peptide for secretion in the apoplast, 10 leucine rich repeats (LRRs) of about 24 amino acids each and eight cysteine residues, four each at N- and C-terminal part of the protein (Additional file 3).

Since fingerprint analysis indicated the lack of overlapping portions between the two sets of BAC clones, we hypothesized the existence of two pgip loci, one including Gmpgip1, Gmpgip2 and Gmpgip5, as identified in the BAC clone 85 M15 and the other one including Gmpgip3, Gmpgip4 and Gmpgip7, as identified in the BAC clone 26I2. The recent availability of the soybean genome sequence [21] allowed us to confirm the existence of two pgip loci and to define the spatial distribution of the Gmpgip genes (see below).

Transcript accumulation and in vitro inhibition assays of Gmpgip5 and Gmpgip7

We have previously reported variation in the expression pattern of Gmpgip genes (Gmpgip1, Gmpgip2, Gmpgip3 and Gmpgip4) following infection of soybean hypocotyls with the necrotrophic fungal pathogen Sclerotinia sclerotiorum [18]. In this work, we extended the study to Gmpgip5 and Gmpgip7. qRT-PCR analysis showed that Gmpgip5 and Gmpip7 are expressed in soybean hypocotyls and are induced following the infection with S. sclerotiorum. The basal transcript levels of both Gmpgip5 and Gmpgip7 are much lower than that of Gmpgip3, used as control because it is the most highly expressed Gmpgip gene in soybean hypocotyls [18]; moreover, basal expression of Gmpgip7 was higher than that of Gmpgip5 (Table 1). Expression of Gmpgip5 decreases during the first 24 hour post infection (hpi) with S. sclerotiorum, to greatly increase by more than 1000 fold at 48 hpi, the last time point analyzed, when the tissue is almost completely macerated (Table 2). Upon infection, expression of Gmpgip7 shows a moderate increase during the first 24 hpi to reach high levels at 48 hpi (Table 2). Gmpgip3 showed different kinetics of transcript accumulation, with a maximum of five fold increase at 24 hpi and no further increase at 48 h (Table 2).

Table 1 Basal expression of Gmpgip5 and Gmpgip7 compared with Gmpgip3 in soybean hypocotyls prior pathogen infection
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Table 2 Time-course expression analysis a of Gmpgip genes in etiolated soybean hypocotyls infected with S . sclerotiorum
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Database searches confirmed the presence of expressed sequence tags (ESTs) corresponding to Gmpgip5 and Gmpgip7 and showed variation of their expression during development, with Gmpgip5 ESTs present in hypocotyls and pods and Gmpgip7 ESTs present in roots and stem (Additional file 4). ESTs for the remaining Gmpgip genes were also identified, with Gmpgip3 being the most represented in soybean tissues (Additional file 4). In particular, about twice more transcripts have been found in the hypocotyl for this gene compared to Gmpgip5, confirming the higher level of expression of Gmpgip3 shown by the qRT-PCR results (Table 1).

In order to verify the inhibition activities of Gmpgip5 and Gmpgip7 against fungal PGs, we have expressed these genes in Nicotiana benthamiana using a vector based on potato virus X (PVX; [22]). Western blot analyses on total protein extracts using an antibody raised against the bean PGIP showed the accumulation of GmPGIP7 and GmPGIP3, which was used as positive control (Additional file 5). On the contrary, no immunodecoration signal was detected in extracts prepared from control plants (non-inoculated or inoculated with the empty vector) (Additional file 5) or from plants infected with the PVX-Gmpgip5 construct (data not shown). Despite considerable effort, all PVX-based attempts to express GmPGIP5 failed.

Total protein extracts of N. benthamiana leaves infected with the PVX-Gmpgip7 were then used to test the inhibitory activity of GmPGIP7 against the fungal PGs of S. sclerotiorum, Fusarium graminearum, Colletotrichum acutatum and Aspergillus niger (data not shown). No inhibition was observed; on the contrary, all the PGs examined were inhibited by GmPGIP3, used as control (Additional file 5).

The soybean pgipgenes are organized in two distinct loci

Sequence comparison of the Gmpgip genes contained within the BAC clones and the assembled soybean genome sequence allowed us to confirm the presence of two pgip loci and to determine the arrangement of the Gmpgip genes within each of them. Each cluster of pgip genes spans a region of similar length (about 18 Kb) on chromosome 5 (in order, Gmpgip5, Gmpgip2 and Gmpgip1) and chromosome 8 (Gmpgip7, Gmpgip3 and Gmpgip4). All genes are transcribed in the same direction (Figure 1). In addition to these transcribed pgip genes, two and one remnant sequences were found on chromosome 5 and 8, respectively. One of the remnants in the locus on chromosome 5 [Gmpgip*(1)] is heavily fragmented, whereas the other two, on chromosomes 5 [Gmpgip*(2)] and 8 (Gmpgip*) encode 65 residues of the C-terminal region and 220 residues of the N- terminal and middle regions, respectively (Additional file 6).

Figure 1
figure 1

Genomic organization of the pgip gene family in soybean cv. Williams 82. Schematic representation of the arrangement of the Gmpgip genes in the two loci of the soybean genome. Regions covered by the BAC clones 85 M15 and 26I2 on chromosomes 5 and 8, respectively, are indicated. On the basis of the soybean genome database, 85 M15 starts and ends at 31,481,533 bp and 31,579,062 bp, respectively, whereas 26I2 starts and ends at 5,980,885 and 6,108,916, respectively. Numbers between the genes represent distances in kb as determined on the soybean genome database. Arrows indicate the direction of the coding region from ATG to stop codon. Boxes are not in scale. Chr, chromosome.

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Nucleotide sequences of regions the two soybean pgip loci were compared by a Bl2seq analysis, showing that the regions flanking the pgip loci are well conserved in nucleotide sequence and collinear in gene order and orientation (Figure 2). The main exception to this collinearity is represented by two major gaps in the alignment, due to LTR retrotransposon insertions in the locus on chromosome 5 (Figure 2). Notably, the region containing the Gmpgip copies shows the most exceptions to the sequence collinearity, as no clear diagonal is visible in this region and alignments are limited to the coding regions of different pgip members (Figure 2).

Figure 2
figure 2

Bl2seq alignment analysis of the genomic regions encompassing the two pgip loci of G. max . Bl2seq analysis was performed between the pgip region (˜96 Kb) in chromosome 5 and that (˜75 Kb) in chromosome 8. Regions flanking the pgip loci are collinear in gene order, orientation as well as nucleotide sequence. *, remnant; Chr, Chromosome.

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In the comparison between loci, only Gmpgip1 and Gmpgip3, on the one side, and Gmpgip2 and Gmpgip7, on the other side , share similarity in the 3′ regions, limited to the proximal 200 bp sequences. The 5′ regions of the different Gmpgip genes are also strongly divergent and sequence divergence in these regions is reflected also in the composition of cis-elements. Sequence analysis. limited to known cis-acting elements regulating genes involved in the defence response, showed that all six Gmpgip genes contain sequences sharing identity with these elements; differing, however, in types and numbers. For example Gmpgip3 contains the highest number of W-box elements, whereas it lacks sequences matching BIHD1OS2 elements (Additional file 7).

Structural analysis of the bean pgiplocus

For the characterization of the bean pgip locus, the bean BAC clones 129 F4 and 10G1 spanning the bean pgip locus were isolated from a genomic library prepared from BAT93 genotype. The 129 F4 and 10G1 clones contain an insert of about 37150 bp and 107473 bp, respectively, with an overlapping segment of 5201 bp [16]. These clones were completely sequenced allowing the characterization of a total of 139420 bp, with the pgip region spanning about 50 Kb. This region represents the only pgip locus present in the bean genome and contains four intronless Pvpgip genes (Pvpgip1Bat, Pvpgip2Bat, Pvpgip3Bat and Pvpgip4Bat) transcribed in the same direction [16].

The assembled BAC sequence of P. vulgaris BAT93 was mapped in two different genomic regions of the recently available genome sequence of P. vulgaris accession G19833. A major portion of the assembled BAT93 sequence (1..126624) was mapped on chromosome 2 [complement (36019507..36152120)] and shown to contain several annotated genes (from Phvul.002G200800.1 to Phvul.002G201900.1), whereas the remaining 13 Kb were mapped on chromosome 1 (49570303..49583535) and did not contain annotated genes. Three hypotheses can be made to explain this discrepancy: 1) the BAC 10G1 that contains the two regions is chimeric, i.e. two independent portions of BAT93 genome were cloned in the same BAC; 2) an assembly error, involving the analyzed regions, is present in the whole genome sequence, and 3) a translocation took place that differentiated the P. vulgaris BAT3 and G19833 accessions. Since the shorter region accounts only for about 10% of the entire BAC assembled sequence and shows a potentially different origin from the main BAC sequence assembly, we excluded it from subsequent analyses.

In order to analyze the structure of the region containing the bean pgip genes, a Blast2seq analysis (sequence aligned with itself), limited to the first 62 Kb of the assembled sequence that contain the four bean pgip genes, was performed. In addition to the diagonal alignment, several short alignments are present (Additional file 8). These include two retrotransposable elements (positions around 10 Kb and 40 Kb), which showed off-diagonal alignment of their Long Tandem Repeats (LTRs), as tipically shown by LTR retrotransposon ends (Additional file 8). The four pgip genes align to each other, and alignment extending beyond the coding regions was observed only between PvBpgip1 and PvBpgip2 and between PvBpgip3 and PvBpgip4 (Additional file 8). Conversely, the alignment between pgip pairs PvBpgip1/PvBpgip3, PvBpgip2/PvBpgip3, PvBpgip1/PvBpgip4, and PvBpgip2/PvBpgip4 is limited to the coding regions.

Comparison of pgiploci in four Fabaceae species

The assembled bean sequence (1..126624) containing the cluster of pgip genes was compared with the soybean genome and with the recent genome sequence releases of M. truncatula [19] and C. arietinum [20]. Like in bean, the genomes of M. truncatula and C. arietinum contain only one pgip locus. The pgip locus of M. truncatula spans a region of about 25 Kb containing a cluster of two genes with uninterrupted open reading frames (MTR_119s0023, Mtpgip1; MTR_119s0021, Mtpgip2), one possible pseudogene, annotated with an intron of 40 bp to restore the correct open reading frame (MTR_119s0017, Mtpgip3), and one remnant corresponding to a sequence encoding a C-terminal 53 residue PGIP fragment. The products encoded by Mtpgip1 Mtpgip2 and Mtpgip3 are shown in Additional file 9. They all contain a signal peptide for secretion to the apoplast, the typical 10 LRRs and eight cysteine residues, four each in N- and C-terminal portion of the protein; MtPGIP1 contains an additional cysteine in the C-terminal region (Additional file 9). Similarly, the pgip locus of C. arietinum spans a region of about 30 K and contains two pgip genes (Additional file 10), one of which (LOC101504619, Capgip2) is interrupted by a fragment of about 17 Kb in the middle of the coding region. The putative protein encoded by Capgip1 (LOC101505245) also contains the typical PGIP features described above (Additional file 10). Taken together, these sequence analyses highlight that the typical PGIP structure is strongly conserved among and within all these pgip families. This conservation is accompanied, as expected, by the typical variation within LRRs composing each protein, and LRRs of different proteins.

Sequence comparison between the flanking regions of the pgip clusters in all four species, showed few genes and a very well conserved order, with few exceptions. Of the ten bean genes flanking the pgip cluster, only Pv_202000 and Pv_201300 are not conserved in all four legume species (Figure 3). Moreover, Pv_201200 and Pv_201100 exist as duplicated genes only in the bean genome, and Pv 202100 was lost in soybean chromosome 8 (Figure 3).

Figure 3
figure 3

Schematic representation of genomic regions containing the pgip loci in Fabaceae species. Genomic regions containing the pgip loci were analysed for their shared synteny. Each block-arrow represents a predicted gene and the direction of the coding region from ATG to stop codon. Colored block-arrows are genes with a homolog. The gray block-arrow indicates a gene with no homolog. Genomic position from the left to the right element: P. vulgaris chromosome 2 (36,203,534 to 36,041,993 bp), G. max chromosome 5 (31,445,987 to 31,542,227 bp), G. max chromosome 8 (59,75,212 to 60,52,779 bp), M. truncatula (unplaced genomic scaffold 119) and C. aretinum (14,308,467, complement to 14,227,083 bp). *, remnant; Chr, chromosome.

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PGIP protein sequences from these four Fabaceae species (P. vulgaris, G. max, M. truncatula, and C. arietinum) were aligned by MUSCLE and a phylogenetic tree was constructed by a Maximum likelihood approach (PhyML). As shown in Figure 4, the unrooted tree revealed that: i) M. truncatula and C. arietinum pgip copies are in species-specific clusters, suggesting that copy amplification took place after the divergence of species; ii) the M. truncatula and C. arietinum pgip clusters are close to each other and form a very well supported cluster; iii) the G. max and P. vulgaris pgip members are in a different cluster, consistent with the general taxonomic relationships of these members of the Fabaceae. Furthermore, G. max and P. vulgaris genes are distributed in three main clusters: a) a cluster including PvBPGIP1, PvBPGIP2 and GmPGIP3; b) a cluster including PvBPGIP3 and PvBPGIP4 and c) a cluster containing the remaining G. max genes.

Figure 4
figure 4

Phylogenetic tree showing the relationship among different PGIPs from Fabaceae species. The unrooted tree was constructed by a Maximum likelihood approach (PhyML) using the deduced amino acid sequences. Gm, Glycine max; PvB, Phaseolus vulgaris; Mt, Medicago truncatula; Ca, Cicer aretinum; Chr, Chromosome.

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Discussion

In this work, we have demonstrated that the full complement of the soybean pgip family is composed of six transcribed genes located in two different loci in the sub-terminal and terminal regions of chromosomes 5 and 8, respectively. Each pgip locus contains both complete and disrupted coding regions indicating that a pseudogenization (“death”) process is active in the family. As assumed for NBS-LRR R-genes [23], the clusters of recently duplicated pgip copies should provide a reservoir of genetic variation from which novel pgip genes can evolve. The resemblance to NBS-LRR R-genes is further supported by previous findings showing that variation between pgip genes of different species or copies within a gene family is mainly due to single substitutions within the LRR domain and in particular in the xxLxLxx solvent-exposed region [15],[16],[18],[24]. Like in R-genes, models of codon evolution suggest the presence, in the solvent-exposed region of PGIP, of sites under positive selection [25]-[27], and functional analysis demonstrated that single substitutions or a short deletion within this region can cause changes in the inhibition properties of PGIP against fungal PGs [16],[28]-[33].

An interesting feature of the soybean pgip loci, which are included in a larger duplicated region, is the high sequence conservation of the regions surrounding the pgip clusters that contrasts with the variability in the intergenic regions between pgip genes. Differences in the regions external to the pgip gene clusters are limited to small indels and to the presence of two transposable elements only in the region of chromosome 8. This conserved organization is typical of the paleopolyploid soybean genome, which underwent two rounds of Whole Genome Duplication (WGD) [34],[35]. Recent analysis of the complete soybean genome sequence has revealed that indeed this is composed to a large extent by blocks of duplicated genes [21]; however. before the availability of the complete assembled soybean genome, data had indicated that the soybean genome was a mosaic of alternating homeologous regions retaining high sequence conservation and regions showing very low conservation [35].

In the homeologous regions containing the pgip clusters, the striking conservation is interrupted only by the remarkable sequence variability in the intergenic regions between the pgip genes. This low sequence conservation includes the proximal 5′ flanking regions, suggesting a differential regulation of the different pgip genes. Indeed, the six pgip genes show variation in cis-acting elements known to regulate defense response genes, and their expression patterns following pathogen infection show clear differences. The two novel pgip genes, Gmpgip5 and Gmpgip7, are poorly expressed in soybean hypocotyls but are strongly induced at late stages of infection with the fungal pathogen S. sclerotiorum. This expression pattern is similar to that of Gmpgip2 and differs from that of Gmpgip1, Gmpgip3 and Gmpgip4, which all show a more prompt induction following S. sclerotiorum infection [18]. Diversification of gene regulation following fungal pathogen infections or stress stimuli has been reported for other pgip gene families, including those of Arabidopsis [3], bean [16] and B. napus [15], and suggests adaptation against stresses. However, this possibility in soybean is still poorly supported at the protein level, since, at present, inhibition activity against fungal PGs has been shown in vitro only for the product encoded by Gmpgip3, which is also the most expressed soybean family member [18]. In fact, although GmPGIP1, GmPGIP2 and GmPGIP4 [18] and now also GmPGIP7 were expressed in N. benthamiana using PVX as a vector, they did not show any inhibitory activity. The lack of in vitro inhibition activity of GmPGIPs does not exclude the possibility that they can inhibit PGs from different sources not yet examined, or that only the in planta environment provides a suitable context to support the interaction with PGs. This last possibility has been suggested by Joubert et al. [6] who found a reduction of symptoms caused by the activity of Botrytis cinerea BcPG2 on plant tissue when co-infiltrated with Vitis vinifera VvPGIP1, although no interaction between VvPGIP1 and BcPG2 was detected in vitro. Moreover, as suggested previously [18], the lack of inhibition activity by GmPGIP1, GmPGIP2 GmPGIP4 and now GmPGIP7 towards fungal PGs may reflect a different physiological role in planta. This possibility is supported by several observations in different plant species. For example, OsFOR1, a rice protein possessing PG inhibiting capabilities, affects the formation and/or maintenance of floral organ primordia in rice [36]; levels of PGIP1 determine the timing of radicle protrusion in Arabidopsis [37], and Vvpgip1 can affect gene expression and cell wall structure in transgenic tobacco plants [38],[39].

The genomic regions encompassing the pgip genes of soybean were also compared to the corresponding region of bean, which shares a very close phylogenetic relationship with soybean. The pgip gene family of the bean genotype BAT93, which comprises four clustered paralogs, has been previously characterized [16]. We have now extended the analysis to the bean BAT93 sequences flanking the pgip cluster and found a very strong conservation in the distribution of the genes compared to duplicated soybean regions encompassing the pgip loci. Of the ten bean genes flanking the pgip cluster, seven were conserved in both soybean chromosomes 5 and 8. Pv_202100 was lost only on soybean chromosome 8, and the duplicated bean genes Pv_201200 and Pv_201100 exist as a single copy in both soybean chromosome 5 and 8.

Conservation of the genomic pgip region is also evident in the more distantly related legume species M. truncatula and C. arietinum, whose assembled genomes have been recently released [19],[20]. Both species possess a single pgip locus, with a cluster organization of the paralogs, and regions flanking the pgip array that maintain the strongly conserved distribution of the genes observed in the soybean and bean. Of the ten bean genes that surround the pgip cluster, seven are conserved in all four legume species. The finding that most of the pgip genes are organized in species-specific phylogenetic clusters indicates that the pgip copies within each cluster were independently formed after speciation. An exception to this observation is represented by GmPGIP3, PvPGIP1 and PvPGIP2. In fact, the soybean and bean PGIPs form separated clusters in maximum likelihood-based gene trees and, within the bean cluster, the four PvPGIPs form two well separated groups. However, as previously highlighted [16],[18], the soybean GmPGIP3 groups with PvPGIP1 and PvPGIP2, suggesting that the duplication originating the ancestors of PvPGIP1/PvPGIP2 and PvPGIP3/PvPGIP4 took place before the separation of Glycine and Phaseolus lineages. In this context, it is noteworthy that the high sequence conservation of PvPGIP2 extends across a range of P. vulgaris germplasm and other Phaseolus species, suggesting an adaptive significance [30].

Conclusions

The paleopolyploid soybean genome contains two pgip loci comprised in large and highly conserved duplicated regions, likely originating from WGD. The region encompassing the pgip locus is also conserved in bean, M. truncatula and C. arietinum. The genomic features of these legume pgip families, which include inferred recent duplications and pseudogenization of pgip copies, suggest that the forces driving the evolution of pgip genes follow the birth-and-death model, similarly to that proposed for the evolution of NBS-LRR-type R genes [23].

Methods

Plant material and infection experiments

Soybean seeds (G. max [L.] Merr. cv. Williams 82) were germinated by placing them on previously sterilized and soaked paper towels which were then rolled and incubated for five days in the dark at 24°C.

The B-24 isolate of S. sclerotiorum (Lib. De Bary) was grown for three days at 24°C on potato dextrose agar to obtain mycelium for the inoculation of soybean seedlings.

Infection experiments were performed by inoculating the middle region of etiolated soybean hypocotyls with actively growing mycelium of S. sclerotiorum as described by Favaron et al. [40]. Soybean seedlings were placed horizontally on plastic trays. Roots were covered with a layer of moist paper towel. Plants were inoculated by placing small plugs (5 × 2 mm) of mycelium-colonized agar, cut from marginal zones of actively growing colonies, along the middle region of hypocotyls. Control soybean seedlings were mock inoculated with sterile agar medium. After incubated at 24°C in the dark hypocotyl segments (approximately 5 mm) cut transversally with a razor blade exactly below the agar plugs were collected at 0, 8, 16, 24, and 48 h after inoculation, frozen in liquid nitrogen and stored at –80°C for subsequent analyses. Lesion of the tissue increased during time and at 48 hpi it affected most part of the hypocotyls as reported by Favaron et al. [40]. Two independent infection experiments were performed.

RNA extraction and RT-PCR analysis

Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen, Italy) according to manufacturer’s instructions. RNA concentration was determined both spectrophotometrically and by densitometric analysis of rRNA fragment following agarose gel electrophoresis. QuantiTect® Reverse Transcription Kit (Qiagen) was used to remove genomic DNA contamination and to synthesize cDNA. Elimination of genomic DNA from cDNA preparation was verified by PCR with primers aligned in different exons for gene Translation elongation factor (GmELF1A) and Glucose-6-phosphate dehydrogenase (GmG6PD) as described by Miranda et al. [41].

The quantitative real-time PCR experiments were performed using the iCycler (Bio-Rad, Italy) and using master mix iQTMSYBER Green Supermix (BioRad, Italy), containing the SYBR Green I DNA binding dye. Each reaction was made in triplicate. Primers were designed using Primer 3 software (http://fokker.wi.mit.edu/primer3/, [42]) on the basis of the Gmpgip genes and have the following sequences (sense and antisense, respectively): GmPGIP3-3 F 5′-ACCCCAACCCTAATCGGTCA-3′ and GmPGIP3-3R 5′-AGGTGATTCCGACGAGATTG-3′ for Gmpgip3; GmPGIP5-1 F 5′-ACCGGACTCCTTCGGCTACTTCC-3′ and GmPGIP5-1R 5′- TGTTTCCCAGATACATGTGCC-3′ for Gmpgip5; GmPGIP7-1 F 5′- TAAGGGTGTCAAAGACCTTGTT-3′and GmPGIP7-1R 5′- CACTTGTTATGAGCGTACAGC-3′ for Gmpgip7; GmELF1A-F 5′-GACCTTCTTCGTTTCTCGCA-3′ and GmELF1A-R 5′-GAACCTCTCAATCACACGC-3′ for GmELF1A [41]. Total reaction volume was 20 μl and included 10 μl (2×) master mix, 100 ng of cDNA, 0.5 μl (10 μM) of each forward and reverse primers and volume was adjusted with water. The PCR reaction conditions were: one cycle at 50°C for 2 min, 94°C for 15 min, then 40 cycles at 95°C for 15 sec, 60°C for 50 sec and 72°C for 50 sec. Primer specificity was confirmed by nucleotide sequencing (MWG, Germany) of amplicon. The Ct values of target genes (Gmpgip3, Gmpgip5 and Gmpgip7) and reference gene (GmELF1A) were used for further relative expression analysis by using the 2-ΔΔCT method [43]. Relative induction level was relative to the corresponding non infected sample at each time point analyzed. Calculation and statistical analyses were performed by Gene Expression Macro™ Version 1.1 (Bio-Rad, Italy). The qRT-PCR experiments included three replicas for each sample in two different biological replicas. PCR efficiency (ε) was calculated for each gene from the slope of linear-regression of the threshold cycle versus log dilution serial of the cDNA according to equation ε = (10^(-1/slope)-1)*100.

PVX-mediated expression of GmPGIP5 and GmPGIP7, and immunoblotting

The coding region of Gmpgip5 and Gmpgip7 was amplified by PCR with primers including restriction sites for ClaI and SalI or NruI at the 5′ and 3′ ends, respectively. The amplified fragments were double digested with ClaI and SalI or NruI and cloned into corresponding sites of the pPVX201 expression vector. The plasmids obtained were used to inoculate N. benthamiana plants using 30 μg of plasmid DNA per plant as described by Baulcombe et al. [22]. Transiently expressed GmPGIP7 was extracted from leaves of N. benthamiana plants infected with single PVX-Pgip constructs or with the empty vector. Leaves were homogenized in 1 M NaCl (2 ml/g), incubated with gentle shaking for 1 h at 4°C, and centrifuged 20 min at 10,000 g. Supernatant was filtered through Miracloth (Calchem, USA), centrifuged to remove debris and stored at -20°C. Protein concentration was determined with the Coomassie Plus™ (Bradford) assay kit (Pierce, Rockford, IL, USA). SDS-PAGE and immunoblotting were performed as previously described [18]. Polyclonal antibodies raised against bean PGIP were used for immunoblotting experiments.

Fungal growth, PG preparation and enzymatic assays

Fungal growth and PG preparation were performed as previously described for A. niger [44], S. sclerotiorum isolate B-24 [45], and C. acutatum isolate SHK788 [16]. F. graminearum isolate 3827 [46]. Inhibitory activity of PGIP was performed as previously described by D’Ovidio et al. [18]. PG activity was expressed as reducing units (RU). One RU was defined as the amount of enzyme required to release reducing groups at 1 μmol min-1 using D-galacturonic acid as standard.

Screening of genomic libraries and sequencing

The Bacterial artificial chromosome (BAC) library (prepared from G. max cv. Williams 82) was purchased from the Clemson University Genomics Institute (CUGI; Clemson, SC, USA). Screening and sequencing of BAC clone was performed as previously described by D’Ovidio et al. [18]. The bean BAC clones 129 F4 and 10G1 spanning the bean pgip locus were isolated from 16.603 recombinant clones of a genomic library prepared from BAT93 genotype [16].

Sequencing reactions were performed using the “ABI PRISM dye terminator cycle sequencing ready reaction” kit and DNA sequences were determined with the semiautomatic ABI PRISM 310 sequencer (Applied Biosystem, Monza, Italy). Sequences were also determined through the MWG-BIOTECH AG (Ebersberg, Germany) and PRIMM Srl. Sequencing Services (Milano, Italy).

Nucleic acid manipulation and amino sequence analysis

Nucleic acid manipulation, PCR, and cloning were performed according to the standard procedures [47]. DNAMAN software (Lynnon, BioSoft, Quebec, Canada) was used for nucleotide and amino acid sequence analyses. Signal peptide for GmPGIP5 and GmPGIP7 was predicted using http://wolfpsort.org/ [48]. The 5′ flanking region of Gmpgip genes was scanned for presence of the cis-elements using PLACE (http://www.dna.affrc.go.jp/PLACE/signalscan.html), a database of plant Cis-acting regulatory DNA elements [49].

Phylogenetic analysis

BLASTp analysis was performed by using bean PGIP sequences to identify the already annotated PGIP genes and detect the occurrence of partial PGIP sequences (remnants) in G. max, M. truncatula and C. arietinum genomes.

The PGIP protein sequences from the four Fabaceae species (P. vulgaris, G. max, M. truncatula, and C. arietinum) were aligned by MUltiple Sequence Comparison by Log- Expectation (MUSCLE) and used for reconstructing phylogenetic tree. This was performed on web interface www.phylogeny.fr [50], using the PhyML software based on the Maximum likelihood principle.

Comparative analysis

The assembled sequences of the BAC clones 129 F4 and 10G1 (accession number HG964426) from the P. vulgaris genotype BAT93 were mapped in the P. vulgaris genome sequence by Bl2seq using the www.phytozome.org web interface.

Protein sequences of the genes spanning from Phvul.002G200900.1 to Phvul.002G202200.1 on the bean genome sequence were used as query in Bl2seq to detect most similar sequences in the sequenced genomes of G. max, C. arietinum and M. truncatula.

Additional files