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. 2006 Sep;98(3):555–564. doi: 10.1093/aob/mcl149

Cytological and Other Aspects of Pathogenesis-related Gene Expression in Tomato Plants Grown on a Suppressive Compost

NEKTARIOS KAVROULAKIS 1,*, KALLIOPE K PAPADOPOULOU 1,†, SPYRIDON NTOUGIAS 1, GEORGIOS I ZERVAKIS 1, CONSTANTINOS EHALIOTIS 2
PMCID: PMC2803568  PMID: 16877456

Abstract

• Background and Aims Recent studies have shown that certain composts may trigger indirect defence mechanisms by sensitizing the plant to create an increased state of resistance, similar to systemic acquired resistance. In this study, the capacity of a disease-suppressive compost to alter the expression pattern of certain pathogenesis-related (PR) genes in the root system of tomato plants (Solanum lycopersicum) provided the opportunity to study their cellular expression pattern and to investigate putative roles of these genes in the mechanisms of plant defence.

• Methods Employing the reverse transcription–polymerase chain reaction (RT–PCR) and in situ RNA:RNA hybridization techniques, the accumulation and distribution of the transcripts of the differentially expressed PR genes were examined in plants grown on compost and compared with those of control plants grown on peat.

• Key Results Elevated levels of expression of the pathogenesis-related genes PR-1, PR-5 and P69/PR-7 were detected in the roots of tomato plants grown on the compost. A clearly distinguished spatial induction pattern was observed for these PR genes: PR-1 transcripts were almost exclusively detected in the pericycle cells surrounding the root stele of the main and lateral roots; PR-5 transcripts were present in the phloem of the root and stem tissues; and the accumulation and distribution of PR-7 transcripts was detected in discrete groups of cells that appeared sporadically in both the parenchyma and vascular system of the root, suggesting that the gene is not expressed in a tissue-specific manner. In addition, a novel cDNA clone was isolated (P69G), which probably encodes a new tomato P69 isoform.

• Conclusions This study provides evidence that a supressive compost is able to elicit consistent and increased expression of certain PR genes in the roots of tomato plants, even in the absence of any pathogen. The in situ localization studies reveal expression patterns which are in accordance with the presence of protein or with the putative roles of the respective encoded proteins. The expression of the PR genes may be triggered by the microflora of the compost or could be associated with abiotic factors of the compost.

Keywords: Compost, induced resistance, Solanum lycopersicum, PR-1, PR-5, P69, pathogenesis-related (PR) proteins, tomato

INTRODUCTION

Numerous studies have demonstrated the effectiveness of agricultural residue-derived compost mixtures in providing protection against various plant diseases, especially against soil-borne pathogens (Hoitink et al., 1996; De Ceuster and Hoitink, 1999). Disease suppression induced by composts includes the reduction of both disease incidence and severity. Most of the research was focused on elucidating the mechanism of soil-borne pathogen suppression and suggests several potential types of interactions between composts or the compost microflora and pathogens: competition for nutrients (McKellar and Nelson, 2003), antifungal production (Singh et al., 2003), parasitism and cell wall enzymatic hydrolysis (Kwok et al., 1987; Gorodecki and Hadar, 1990). Fewer studies on the influence of compost mixtures on foliar pathogens (Vallad et al., 2003; Khan et al., 2004; Kavroulakis et al., 2005) indicate that composts may also trigger indirect defence mechanisms by sensitizing the plant to create an increased state of resistance, similar to systemic acquired resistance (SAR). This compost-mediated induced resistance appears to correlate with the activation of plant defence-related genes and subsequent accumulation of several proteins such as peroxidases, β-1,3-glucanases and pathogenesis-related 1 (PR-1) (Zhang et al., 1998; Kavroulakis et al., 2005).

Induced resistance is a state of enhanced defensive capacity, triggered by specific contact stimuli, whereby the plant's active defences are potentiated against subsequent pathogen challenge. The resistance responses are usually systemic, but localized types also exist, and are effective against a broad range of pathogens. Induced resistance can basically be triggered by exposure of plants to virulent, avirulent or non-pathogenic microbes, or artificially by various chemical agents.

The induction of PR proteins in various plant tissues is one of the major biochemical and molecular events when plants are subjected to infections with pathogens such as viroids, viruses, bacteria and fungi (Van Loon, 1997). Induction is achieved through many signalling pathway elements, including different receptor components or chemical elicitors such as salicylic acid (SA), ethylene, jasmonic acid and systemin (Ward et al., 1991; Xu et al., 1994; Maleck et al., 2000; Campos et al., 2002). The PR proteins are thought to play an important role in induced resistance (SAR). Furthermore, colonization of plants with some rhizobacteria also leads to induced systemic resistance (ISR) and protection of plants against pathogens. The capacity of antagonistic bacteria has been correlated in some cases with alterations in the expression pattern of the PR genes (Park and Kloepper, 2000; Ramamoorthy et al., 2002; Viswanathan et al., 2003). However, ISR may be induced without the concomitant expression of PR genes (Pieterse et al., 2001, 2003; Verhagen et al., 2004). Expression of PR genes may also occur in ‘primed’ plants. Priming refers to a latent state, in which PR genes are not expressed in response to an inducing stimulus but this situation leads to an earlier and stronger expression once the plant reacts to a challenging pathogen, as compared with a non-induced plant (Conrath et al., 2001; Heil and Bostock, 2002; Verhagen et al., 2004). PR proteins are classified into 14 distinct families and include both basic and acidic isoforms (Van Loon and Van Strien, 1999).

In this study, elevated levels of expression are reported of PR-1, PR5 and PR-7 (P69) genes in the root tissues of tomato plants grown on a compost derived from grape marc and extracted olive press cake (GM-EPC). In a previous study, a transgenic β-glucuronidase (GUS)-expressing strain of the fungal root pathogen Fusarium oxysporum f.sp. radicis-lycopersici was used to demonstrate the enhanced defensive capacity conferred to the plants by the compost. Furthermore, the foliar pathogen Septoria lycopersici was used to assess the induction of systemic resistance in plants growing on compost as compared with plants growing on peat (Kavroulakis et al., 2005). The capacity of this compost to alter the expression pattern of certain PR genes in the root system of tomato plants provided the opportunity to study their cellular localization and suggest putative roles for these genes in the mechanisms of plant defence, without having to deal with the inherent complexities of the pathological condition of diseased tissues.

MATERIALS AND METHODS

Plant material and growth conditions

Tomato seeds (Solanum lycopersicum ‘ACE 55’) were surface sterilized in 2·5 % NaOCl for 5 min, followed by extensive rinsing in sterilized water. Seeds were planted in 300 cm3 pots. Sphagnum peat [Sunu Kura, Seda joint-Stock company amended with an NPK fertilizer (20-20-20) to a total concentration of 0·8 g L−1 (referred to hereafter as peat)] was used as potting mix in all control experiments. Alternatively, a mixture of peat moss and compost mix derived from GM-EPC (1 : 1 w/w) was used (referred to hereafter as compost). The preparation, chemical characteristics and suppressive properties against plant pathogens of the compost were described previously (Ntougias et al., 2003; Kavroulakis et al., 2005). The pots were placed in a growth chamber at 25 °C with a 16 h photoperiod at 65 % relative humidity. The plants were watered to the initial weight on alternate days and with a balanced nutrient solution, including micronutrients, once a week. Under these growth conditions, the plants grew equally well and no nutrient deficiencies were detected, at least macroscopically.

Isolation of total plant RNA and RT–PCR assay, cloning and sequence analysis of the cDNAs

Roots from tomato plants, grown on compost or peat for 1 or 4 weeks, were harvested and ground in liquid nitrogen. Three pots with five plants each were used for each treatment. Samples comprised of all 15 plants were immediately frozen in liquid nitrogen, and then stored at −80 °C until use. Repeated reverse transcription–polymerase chain reaction (RT–PCR) assessment of gene expression was performed. The experiment was conducted twice. The time points of 1 and 4 weeks were chosen based on previous findings (Kavroulakis et al., 2005) that a plant's response to both root and foliar pathogens, mediated by the compost used, is established by this time. Furthermore, comparative analysis between PR gene expression in the root tissues (this study) and the leaves (Kavroulakis et al., 2005) was feasible.

For total RNA isolation, a Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) was used. Prior to RT–PCR, the total RNA samples were treated with DNase I (Roche, Mannheim, Germany) for 10 min and quantified by spectrophotometry and agarose gel electrophoresis. Total RNA (0·5 µg) was used for first-strand cDNA synthesis using Extend Reverse Transcriptase (Roche). Reverse transcription was carried out using random hexanucleotides (Amersham Biosciences, Freiburg, Germany) at 37 °C for 1 h. The single-stranded DNA mixture was used as template in PCRs. The sequences of gene-specific primer pairs used are presented in Table 1. The PCRs were performed in 10 mm Tris–HCl buffer pH 8·3, 50 mm KCl, 1·5 mm MgCl2, 100 μm dNTPs, 50 ng of each primer and 1 U of DyNAzyme EXT DNA polymerase (Finnzymes Oy, Espoo, Finland). Amplifications were performed in a PTC-200 thermal cycler (MJ Research Inc., Waltham, MA, USA) with a denaturation step of 3 min at 94 °C, followed by 30–35 cycles of 30 s at 94 °C, 30 s at each Tm and 1 min at 72 °C. Optimal numbers of cycles corresponding to the exponential phase of the reaction were determined for each primer pair. Contamination with genomic DNA was ruled out by omitting reverse transcriptase in duplicate samples. The primer pair for the glyceraldehyde phosphate dehydrogenase (gapdh) gene was used as an internal control.

Table 1.

Sequences of gene-specific primers used in RT–PCR analysis

Gene family Specific class Accession no. 5′ primer 3′ primer References
PR1 PR1b, basic PR1 AJ011520 5′-CCAAGACTATCTTGCGGTTC-3′; Tm: 57.3 °C 5′-GAACCTAAGCCACGATACCA-3′; Tm: 57.3 °C Van Kan et al. (1992)
PR2 PR2a, acidic glucanase M80604 5′-TATAGCCGTTGGAAACGAAG-3′; Tm: 55.3 °C 5′-TGATACTTTGGCCTCTGGTC-3′; Tm: 57.3 °C Van Kan et al. (1992)
PR2b, basic glucanase M80608 5′-CAACTTGCCATCACATTCTG-3′; Tm: 55.3 °C 5′-CCAAAATGCTTCTCAAGCTC-3′; Tm: 55.3 °C Van Kan et al. (1992)
PR3 Chitinase 3, acidic Z15141 5′-CAATTCGTTTCCAGGTTTTG-3′; Tm: 53.2 °C 5′-ACTTTCCGCTGCAGTATTTG-3′; Tm: 55.3 °C Danhash et al. (1993)
Chitinase 9, basic Z15140 5′-AATTGTCAGAGCCAGTGTCC-3′; Tm: 57.3 °C 5′-TCCAAAAGACCTCTGATTGC-3′; Tm: 55.3 °C Danhash et al. (1993)
PR5 Osmotin-like PR5 AY093593 5′-AATTGCAATTTTAATGGTGC-3′; Tm: 49.1 °C 5′-TAGCAGACCGTTTAAGATGC-3′; Tm: 55.3 °C Rep et al. (2002)
PR7 P69A, subtulisin-like Y17275 5′-AACTGCAGAACAAGTGAAGG-3′; Tm: 55.3 °C 5′-AAC GTGATTGTAGCAACAGG-3′; Tm: 55.3 °C Tornero et al. (1996)
gapdh Glyceraldehyde-3-phosphate-dehydrogenase M64114 5′-GAAATGCATCTTGCACTACCAACTGTCTTGC-3′; Tm: 67.6 °C 5′-CTGTGAGTAACCCCATTCATTATCATACCAAGC-3′; Tm: 64.1 °C Shih et al. (1992)
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PCR products were separated on 1 % agarose gels in 1× TAE buffer, extracted using QIAquick gel extraction columns (Qiagen, Germany), cloned into the pGEM-T easy vector (Promega, Madison, WI, USA), and then both strands were sequenced. The nucleotide and amino acids sequences were aligned using the CLUSTAL W and BlastX algorithms.

Tissue processing for microscopy

Root and stem samples from plants, grown on compost or peat for 4 weeks, were immediately immersed in 4 % paraformaldeyde/0·25 % glutaraldehyde in 10 mm phosphate buffer (pH 6·8) at 4 °C overnight. They were subsequently dehydrated in an ethanol series, cleaned in an ethanol/xylene series and embedded in Paraplast (Sigma-Aldrich Inc., St Louis, MO, USA). Thin sections (8 µm), cut from the embedded material, were mounted on glass slides. The sections were subsequently hydrated and stained with 1 % aqueous toluidine blue before microscopic observations using bright-field optics or 0·5 % safranin for the in situ hybridization analysis.

In situ hybridization

Antisense and sense riboprobes were obtained from linearized pGEM-T easy plasmid clones by in vitro transcription using the T7 and SP6 RNA polymerase. The RNA probes were labelled using digoxygenin-11-UTPs (DIG-11-UTPs) according to the Roche manual. The probes were partially alkaline degraded to an average length of 150 nucleotides. Sections were hydrated and treated with 1 µg mL−1 proteinase K (Roche) in 100 mm Tris–HCl (pH 7·5)–50 mm EDTA for 30 min at 37 °C. Slides were incubated in 0·1 m triethanolamine and 0·25 % (v/v) acetic anhydride for 10 min and rinsed in 2× SSC for 5 min. Following dehydration in an ethanol series, sections were hybridized overnight at 42 °C in 50 % formamide, 300 mm NaCl, 1 mm EDTA, 0·02 % polyvinyl-pyrrolidone, 0·025 % bovine serum albumin (BSA), 10 % dextran sulfate, 60 mm dithiothreitol (DTT) and RNA probe at a concentration of 1 µg mL−1. The samples were then washed four times for 10 min each, in 4× SSC containing 5 mm DTT, and treated with 50 µg mL−1 RNase A (Roche) in 500 mm NaCl and 1 mm EDTA solution supplemented with 5 mm DTT for 30 min at 37 °C. Sections were washed again in 500 mm NaCl and 1 mm EDTA at 37 °C four times for 15 min each and finally in 2× SSC for 30 min at room temperature. Hybridization signals were detected using anti-DIG conjugated with alkaline phosphatase in conjunction with the detection system of 5-bromo-3-chloro-3-indolylyphosphate/nitroblue tetrazolium (BCIP/NBT).

RESULTS

Expression analysis of PR genes in tomato roots grown on the suppressive compost

The differential expression analysis of the PR genes examined was initially determined by gene-specific RT–PCR analysis. In vitro synthesized single-stranded cDNAs from RNA samples isolated from tomato roots grown on compost and peat, in the absence of pathogen, were assessed using sets of specifically designed primers (Table 1), which enabled the amplification of genes representing the PR1, PR2, PR3, PR5 and PR7 families of PR proteins.

PR1 and PR5 mRNAs accumulated to detectable levels in roots grown on compost 4 weeks after tomato planting, while no expression was detected in roots of tomato plants grown on peat (Fig. 1). Furthermore, an obvious increase in the expression of the PR7 (P69) gene was induced in plants grown on compost as compared with plants grown on peat. None of the other PR genes examined (i.e. genes encoding basic and acidic glucanases, and basic and acidic chitinases) were induced in higher levels by the presence of the compost compared with control. The experiment was conducted twice with similar results.

Fig. 1.

Fig. 1.

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RT–PCR analysis of PR gene expression in tomato roots grown either on peat (p) or on compost (c). Root samples were collected from plants 1 week and 1 month post-seeding. Host genes assessed encode a basic PR1, basic and acidic β-1,3-glucanase (GLUB and GLUA), basic and acidic chitinase (CHI3 and CHI9), an osmotin-like PR5 and endoproteinase P69 (PR7). As a control, the gapdh gene was used.

These data imply that the compost triggers in the root of tomato plants an altered expression of genes associated with the defence response, even in the absence of pathogens.

Isolation of cDNA clones coding for PR genes expressed in tomato roots grown on the suppressive compost

RT–PCR fragments encoding PR1, PR5 and P69 proteins, expressed in tomato roots grown on compost (designated as LePR1, LePR5 and P69G, respectively), were isolated and their nucleotide sequences were determined. All isolated and sequenced LePR-1 clones were identical to the one used for primer design (P4; VanKan et al., 1992) and 95–96 % identical at the nucleotide level to a basic PR1 isoform (PR1b; Tornero et al., 1997). A second amplified band, shorter than the expected size, was not investigated further, although it may represent a different isoform of the PR1 gene in tomato. The sequences of LePR5 clones were almost identical (98–99 %) to that of PR5x (Rep et al., 2002) used for primer design (data not shown). The primers used for the amplification of P69 genes were specific for the constitutively expressed P69A and the inducible, by pathogen and SA, P69C gene (Jorda et al., 1999). In order to identify transcripts of the P69 subfamily only inducible by the compost, the primers were designed to avoid the amplification of the P69E gene, which was reported to be constitutively expressed in the root tissues (Jorda et al., 1999, 2000). Cloning and sequencing of independent amplicons, which accumulated in root tissues of plants grown on compost as compared with plants grown on peat, led to the identification of a novel clone (designated as LeP69G, GenBank accession no. DQ157774). This P69 cDNA clone shows relatively low sequence identity to the already known P69 genes in tomato (Jorda et al., 1999), indicating that LeP69G either encodes a new tomato P69 isoform or is a cultivar-specific variant of one of the known P69 genes (Fig. 2). The highest amino acid sequence homology (90–91 %) of the putative polypeptide encoded by P69G is found in comparison with the polypeptides encoded by the pathogen-induced genes P69B and P69C (Table 2).

Fig. 2.

Fig. 2.

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Comparison of the partial sequence of the P69G cDNA clone with other PR69 isoforms from tomato. The alignment shows nucleotide sequences of P69A, P69B and P69C (GenBank accession numbers Y17275, Y17276 and Y17278, respectively). Grey shading indicates the sites of the primers used in order to isolate P69G cDNA.

Table 2.

Percentage identity between the deduced amino acid sequence of P69G and P69A, P69B, P69C, P69D, P69E and P69F proteins (accession nos CAA76724, CAA76725, CAA76726, CAA76727, CAB67119 and CAB67120, respectively) from tomato plants

P69A P69B P69C P69D P69E P69F
P69B 86
P69C 82 85
P69D 98 88 83
P69E 83 84 92 84
P69F 81 80 79 82 80
P69G 84 90 91 82 89 83
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Localization of PR1b, PR5 and P69G gene expression in tomato roots

The histological localization of the LePR1 transcripts in tomato roots, grown on compost for 4 weeks, was studied with DIG-labelled riboprobes and in situ hybridization. High levels of PR1b transcript accumulation were observed in cell layers located in the periphery of the vascular stele of the main root (Fig. 3A). These cells comprise the pericycle tissue as is demonstrated in Fig. 3B, where a section of a young root is shown. A similar spatial hybridization pattern was found in lateral roots, but a lower signal intensity was observed (data not shown). Low transcript levels or no transcripts at all of the PR1b gene were found in other peripheral tissues, while no signal was detected in the root stele or in the root meristem. The expression of the LePR1 gene was barely or not detectable in tomato roots grown on peat (Fig. 3E). As a negative control, sections were hybridized to the sense RNA probe. In this case, no significant hybridization signal was detected (data not shown).

Fig. 3.

Fig. 3.

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In situ localization of PR1 and PR5 mRNA transcripts on tomato roots grown on the suppressive compost (A–D) and peat (E, F). Sections of 4 weeks post-seeding tomato roots were hybridized to digoxigenin-11-rUTP-labelled antisense RNA, and in vitro transcribed from the LePR1 cDNA clone (A and B) and LePR5 cDNA clone (C and D). The signal is detected as a blue-purple coloured precipitate. (A) Transcription of the PR1 gene is detected, almost exclusively, in the pericycle cells of the tomato roots grown on the compost. (B) The same hybridization pattern is observed in younger parts of the root. (C) A high level of accumulation of PR5 transcripts is observed in the phloem of the tomato roots grown on the compost. (D) PR5 transcripts are also detected in the phloem of crown tissues. PR1 or PR5 mRNA transcripts are not detected in sections of roots grown on peat (E and F, respectively). p, pericycle cells; c, cortex; s, root stele; ph, phloem; x, xylem. Bars represent 50 μm.

The sites of PR5x gene expression in tomato roots grown on compost, as determined by in situ hybridization, were exclusively the cells of the phloem tissue of the main root (Fig. 3C). No expression was detectable in the other tissues of the vascular system. Similarly, significant levels of PR5 gene expression were found in the phloem tissues of the crown (Fig. 3D). The expression of the LePR5 gene was not detectable in tomato roots grown on peat (Fig. 3F). No signal was observed in sections hybridized with sense probe (data not shown).

Both sense and antisense P69 RNA probes were in vitro reverse transcribed from a LeP69G clone. The pattern obtained was very characteristic and suggests that this gene is not expressed in a tissue-specific manner in the roots of plants grown on the compost. Thus, although a faint hybridization signal was detectable in almost all parenchyma cells of the main root, the accumulation of P69 transcripts was significantly enhanced in certain, usually neighbouring, cells of the root parenchyma (Fig. 4A). These groups of cells appeared sporadically at unpredictable locations. Furthermore, in some tissue sections, high levels of hybridization signal were observed in the vascular system of the main root, particularly in the phloem (Fig. 4B). The sporadic pattern of the gene's expression was observed in serial sections taken from different parts of the root and could not be attributed to the root age. Expression of the LeP69G gene was also detectable in the lateral roots (data not shown). In contrast, the absence of or considerably lower transcript levels of the LeP69G gene was observed in the parenchyma of the root tissues of tomato plants grown on peat (Fig. 4C) and the hybridization signal was equally intense among the cells. As expected, no signal was detected in tissues hybridized with the sense probe (Fig. 4D).

Fig. 4.

Fig. 4.

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In situ localization of P69 mRNA transcripts on tomato roots grown on the suppressive compost (A, B and D) and peat (C). Sections of 4 weeks post-seeding tomato roots were hybridized to digoxigenin-11-rUTP-labelled antisense (A, B and C) or sense (D) RNA, and in vitro transcribed from the P69G cDNA clone. The signal is detected as a blue-purple coloured precipitate. (A) LeP69 mRNA transcripts are observed in almost all parenchyma cells of the cortex; a more intense hybridization signal is detected in certain groups (arrows) and (B) in the root stele (arrows) of some sections. (C) Lower transcript levels of the LeP69G gene are detected in the parenchyma of the root tissues of tomato plants grown on peat. (D) No hybridization signal is detected in sections of tomato roots grown on compost hybridized to sense RNA transcribed from the LeP69G cDNA clone. c, cortex; s, root stele. Bars represent 50 μm.

Localization of PR1b and PR5 gene expression in tomato stems

To investigate the temporal accumulation of PR1 and PR5 gene transcripts, stems of tomato plants grown on the compost were collected, fixed and sliced. Afterwards, sections were hybridized with PR1 and PR5 RNA riboprobes labelled with DIG. High transcript levels of the PR1 gene were detected in the parenchyma cells of the stem (Fig. 5A). No visible hybridization signal was detected in the vascular system of the stem. Expression of the PR5 gene was mainly detected in the phloem tissues of the vascular bundles (Fig. 5B). No hybridization signal was detected in tissues of tomato plants grown on peat (Fig. 5C and D).

Fig. 5.

Fig. 5.

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In situ localization of PR1 and PR5 transcripts on stems of tomato plants grown on the suppressive compost for 4 weeks. The signal is detected as a blue-purple coloured precipitate. (A) PR1 mRNAs are detected in the parenchyma cell. No hybridization signal is detected in the stele of the stem. (B) Expression of the PR5 gene is observed in the phloem tissues of the vascular bundles (arrows). (C and D) Sections of stems from tomato plants grown on peat, hybridized to PR1 and PR5 RNA probes, respectively. No visible hybridization signal is detected. c, cortex; s, root stele; ph, phloem. Bars represent 50 μm.

Histological observation of tissues from tomato plants grown on compost

In order to examine whether the uneven distribution of P69 transcripts in the root cells was associated with alterations in the cell structures, comparative cytological studies were undertaken on root samples grown on compost and peat. Examination of root sections (25 independent observations) from tomato plants, grown on compost for 4 weeks, did not disclose alterations in the cell wall or any other appositions detectable by this approach. Interestingly, however, the colonization of the root system by fungal mycelia was observed, which is in accordance with previous reports (Kavroulakis et al., 2005) on the recovery of a non-pathogenic Fusarium-like isolate from the root tissue of tomato plants grown on this particular compost. The colonization was relatively intense in the younger parts of the main root, which was not restricted to the outer tissues of the roots but extended to the vascular system (Fig. 6A). Penetration of the fungus was not correlated with cytological changes (e.g. appositions) or extensive cell destruction. In the sections taken from the upper (older) parts of the main root, hyphae were mainly restricted to the vascular system, whereas the outer tissues were seldom colonized (Fig. 6B). In root samples from plants grown on peat, no fungal colonization was observed (data not shown). No fungus penetration was found in tomato stems grown on compost (data not shown).

Fig. 6.

Fig. 6.

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Light micrographs of tomato roots grown on the suppressive compost after toluidine blue staining. No association of fungal ingress with marked host wall alteration or tissue damage is observed. (A) A section taken from the younger parts of the main roots. Abundant multiplication of hyphae (arrows) in the cortex and vascular tissues is seen. (B) Sections taken from the upper (older) parts of the main root. Most of the hyphae (arrows) are located in the xylem tissues of the vascular tissues. Bars represent 50 μm.

DISCUSSION

The presented work reports, for the first time, the induction of PR gene expression in the root tissues of tomato plants grown on a suppressive compost. In a previous study, it was proposed that induced resistance played a key role in the suppressiveness against root and foliar fungal pathogens exhibited by the GM-EPC compost and it was shown that only a slight increase in LePR1 expression was detected systemically in the leaves of tomato plants growing in GM-EPC (Kavroulakis et al., 2005). In contrast, evidence is now presented that in the roots, the GM-EPC compost is able to elicit consistent and increased expression of certain PR genes. Thus, the accumulation of PR1, PR5 and P69 transcripts is observed. The lack of substantial changes in gene expression in the leaves of plants growing on compost resembles the induced systemic resistance mediated by rhizobacteria (i.e. Pseudomonas fluorescens WCS417r) in Arabidopsis plants, as reported by Verhagen et al. (2004). On the other hand though, in the latter case, only downregulated transcripts of PR1 were identified, locally in the roots, among 97 genes that showed a consistent change. This suggests that compost-mediated induced resistance is conferred to the plant by different signalling pathways, at least as far as concerns the expression of PR genes in root tissues. Whether this induction of PR genes plays an essential and indispensable role in the protection against root pathogen conferred by the particular compost (Kavroulakis et al., 2005) is not clear, and further investigation is needed.

Chitinases and β-1,3-glucanases are considered key plant defence enzymes acting on fungal cell wall degradation and constitute several of the PR gene families (PR2, PR3, PR4, PR8 and PR11). A prominent induction of chitinase and β-1,3-glucanase gene expression in the leaves of plants challenged by a pathogen has been reported for plants growing on compost-amended mixtures, with suppressive activity (Zhang et al., 1998). Using GM-EPC as peat amendment, no change in the expression pattern of basic and acidic chitinases and glucanases in tomato plants could be detected as compared with plants grown on peat, either in the leaves (Kavroulakis et al., 2005) or in the root (this study). It is not clear if these genes are regulated differentially in the root tissues or different signalling pathways are triggered due to the absence of a pathogen in the rhizosphere.

Tomato plants, grown on compost, showed elevated levels of expression of the PR1 gene in their roots relative to plants grown on peat. Induction of an SA- and pathogen-induced PR1 gene (Uknes et al., 1992) has also been reported in the leaves of Arabidopsis plants, grown on soil amended with paper mill residue-derived compost in the absence of a pathogen (Vallad et al., 2003). Studies concerning the spatial distribution of PR1 expression were mostly conducted following expression of reporter genes driven by PR1 promoters in heterologous systems. Thus, the promoter of the tomato PR1b1 gene drives the expression of the GUS reporter gene in the leaves of transgenic tobacco exhibiting a hypersensitive response (HR) after infection with tobacco mosaic virus (TMV) (but not in non-inoculated leaves) and is induced by both SA and ethylene (Tornero et al., 1997). The promoter of the tobacco basic PR1-like gene directs the expression of the GUS reporter gene to phloem tissues of the vascular bundle of the stem (Eyal et al., 1993). In this work, the cellular localization of PR1 gene expression in root and stem tissues was determined. The clone LePR1, which is expressed at elevated levels in the roots of tomato plants grown on the compost, was found to be expressed in a highly tissue-specific manner, limited only to the pericycle cells. The expression of the PR1 gene in these cells could be related to its putative direct antimicrobial action. For example, a basic PR1 protein has been shown to exhibit strong inhibitory activity against the late blight fungal pathogen of potato, Phytophthora infestans, in both in vitro and in vivo assays (Niderman et al., 1995). Thus, in tomato roots, the presence of PR1 in the cell layers surrounding the stele suggests a role in protection of the root from the entry of pathogens. This is further supported by the immunogold detection of the basic tomato PR1b in secondary thickenings of xylem vessels of tomato root tissues infected with F. oxysporum f.sp. radicis-lycopersici (Benhamou et al., 1991). On the other hand, high levels of accumulation of PR1 transcripts were detected in the cortex cells of the stem with only occasional and not consistent detection in the vascular bundle. The possibility that cross-hybridization occurred between genes encoding different isoforms of PR1 (expressed in the different tissues) cannot be ruled out, despite the high stringency conditions used in the experiments conducted. It should be noted, however, that a defined expression pattern was observed in a series of sections from different plants in the two experiments conducted. The plants, grown either on compost or on peat, were of similar developmental stage, and no abnormality or deficiency of plant growth that could account for the induced and highly specific expression of the gene has ever been detected. Even if different PR1-encoding genes hybridized in the different tissues, the induction of these genes by the compost occurred consistently.

The family of PR5 proteins comprises either extracellular acidic or basic vacuolar proteins with diverse functions and roles in development, protection against osmotic stress, freezing tolerance and protection against fungal and oomycete infection (Campos et al., 2002; Piggott et al., 2004). The PR5x gene studied in this work encodes a secreted PR5x isoform of PR5 proteins, which is highly similar to the basic vascular PR5 proteins, also referred to as osmotins, but not to the acidic, extracellular PR5 proteins of other plant species. This isoform accumulates early in the xylem sap of tomato plants in both compatible and incompatible interactions with the root-infecting fungal pathogen F. oxysporum f.sp. lycopersici (Rep et al., 2002). Thus, the site of gene expression in the adjacent phloem tissues in both the root system and stem of tomato plants, as revealed in the present study, suggests that the protein is exported in the xylem vessels and this is in accordance with the secreted nature of the protein and its presence in the xylem sap. To our knowledge, however, there are no further data concerning the way in which the PR5x gene is induced by other biotic or abiotic factors. Evidence is presented here that the same gene is induced in root and stem tissue of tomato plants growing on a compost in the absence of any pathogen. Nevertheless, it cannot be excluded that the presence of the fungal colonizer, revealed by toluidine blue staining of root sections of plants growing on compost, could contribute to some extent to the local and systemic induction of the PR5 gene. In preliminary experiments, in which tomato plants grown on peat were inoculated with conidia of the isolated Fusarium-like fungal strain, no increase in the expression of PR5x was detected by RT–PCR analysis. Nevertheless, further investigation is needed since the induction may be localized. In this case, further cytological data would be needed to elucidate the putative role of the fungal colonizer in the tissue-specific expression of the gene.

RT–PCR reveals that the plants grown on the compost accumulate higher levels of P69 transcripts compared with the control plants grown on peat. P69 genes belong to the gene family of subtilisin-like serine proteases (SBTs), which in tomato fall into five distinct subfamilies. Six different isoforms, encoded by different orthologues of P69 subtilisin-like protein in tomato, have been reported (Meichtry et al., 1999). These genes are tightly regulated by developmental and environmental cues in tomato plants (Jorda et al., 1999; Jorda and Vera, 2000). Only two genes, P69B and P69C, were induced by both virulent and avirulent Pseudomonas syringae strains in Arabidopsis and tomato plants as well as by exogenously applied SA.

Expression of the P69G gene could be detected in discrete groups of cells (islands) that appeared sporadically in both the parenchyma and the vascular system of the root. Heterologous expression of the gus gene driven by the P69C promoter in transgenic Arabidopsis plants and histochemical staining showed a similar pattern of scattered spots all around the leaf lamina when plants were infected with P. syringae (Jorda and Vera, 2000). P69C gene expression in the root was not reported. It may be suggested that the P69G gene (isolated in the present study) represents an inducible, root-specific orthologue of this family, playing a role in the protection against root pathogens.

CONCLUSIONS

The in situ localization studies of PR gene transcripts, induced by the suppressive compost, reveal expression patterns which are in accordance with the presence (e.g. PR5) or with the putative roles of the respective encoded proteins (e.g. PR1). Nonetheless, based on the present results and data reported previously (Zhang et al., 1998; Vallad et al., 2003; Kavroulakis et al., 2005), the compost-mediated mechanism of PR gene induction could not be specified with certainty. Previous data indicated that chemical factors may play a significant role in the induced resistance mediated by the compost used in the present study (Kavroulakis et al., 2005). Such factors may be sugars or phenolics, both of which are contained in high concentration in the compost mix used. Thus, the elevated levels of PR gene expression could be associated with such abiotic factors of the compost. Alternatively, since the experimental conditions do not involve the presence of any pathogen, the expression of the PR gene may be triggered by the microflora of the compost. For example, PR genes could be activated in the root tissues undergoing cell death due to the presence of incompatible or weak pathogenic interactions since occasionally necrotic lesions have been observed on the root tissues of plants growing on compost. The Fusarium-like fungal isolate (Kavroulakis et al., 2005) represents a good candidate for such a type of interaction.

Acknowledgments

This work was partially funded by the RECOVEG project (E.U., QLRT-2000-01458). We thank Anne Osbourn for constructive comments on the manuscript.

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