Journal of Experimental Botany, Vol. 51, No. 353, pp. 1969-1977,
December 2000
© 2000 Oxford University Press
Original Papers |
Induction of Ltp (lipid transfer protein) and Pal (phenylalanine ammonia-lyase) gene expression in rice roots colonized by the arbuscular mycorrhizal fungus Glomus mosseae
Departamento de Microbiología del Suelo y Sistemas Simbioticos, Estación Experimental del Zaidín, CSIC Pofesor Albareda, 1, 18008 Granada, Spain
Received 31 January 2000; Accepted 26 June 2000
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Abstract |
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The expression of a lipid transfer protein (LTP) gene is regulated in Oryza sativa roots in response to colonization by the mycorrhizal fungus Glomus mosseae. Transcript levels increased when the fungus forms appressoria and penetrates the root epidermis and decreased at the onset of the intercellular colonization of the root cortex. The analysis of histochemical GUS staining in transgenic rice plants carrying the Ltp/Gus construct confirm the induction of Ltp gene associated with fungal appressoria formation and penetration area. The induction of Ltp gene expression coincided in time with a transient increase in the expression of a phenylalanine ammonia-lyase (Pal) gene and a transient accumulation of salicylic acid (SA) in the mycorrhizal roots. The expression of Ltp and Pal was induced in rice roots after treatment with SA and Pseudomonas syringae indicating that both genes could be implicated in the plant defence response. The exogenous application of SA to rice interacting with the mycorrhizal fungus did not affect appressoria formation but, instead, resulted in a transient delay of root mycorrhization. Nevertheless, although Ltp maintained a prolonged SA-induced expression level, mycorrhizal formation could still proceed.
Key words: Arbuscular mycorrhiza, plant defence, lipid transfer protein.
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Introduction |
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The endosymbiosis formed by plant roots and arbuscular mycorrhizal fungi (AM) are probably the most widespread symbiotic associations to have evolved in the plant kingdom. The establishment of this mutualistic association is a successful strategy to improve the nutritional status of both partners.
Fungal penetration and establishment in the host roots involves a complex sequence of events and intracellular modifications (Bonfante-Fasolo and Perotto, 1992
). The compatibility between plant roots and AM fungi implies a clear and selective recognition by the plant host that recognizes the beneficial feature of AM fungi.
The key to understanding the phenomenon of compatibility is to study recognition mechanisms and molecules involved in the early stages of the AM interaction. In this sense there is evidence that the phenomena of root colonization by AM fungi and root nodule induction and formation by Rhizobia share some cellular and molecular features (reviewed by Hirsch and Kapulnik, 1998).
Although AM fungi are considered as biotrophic micro-organisms and biotrophs generally exhibit a high degree of host specificity, most AM fungi that have been studied show little or no specificity, and are not thought to induce typical defence responses in host plants. Nevertheless, some plant resistance markers have been investigated in compatible symbiotic AM fungus–root interactions and the early activation of certain plant defence genes has been shown (reviewed by Gianinazzi-Pearson et al., 1996). Since the plant host can elicit a weak defence response to the invading fungus, this may be a natural mechanism to control the number and/or location of infections. Furthermore, some phenomena of suppression of defence responses have been demonstrated in mycorrhizal roots (Volpin et al., 1995; David et al., 1998). Whether this suppression is systemic or restricted to the infected area, or whether products of symbiosis-related plant genes suppress the defence genes directly or through activation of fungal-derived suppressors, remains to be elucidated. So far, it is not known how the induction/suppression of mechanisms associated with plant resistance could participate in the phenomenon of compatibility between plant roots and AM fungi. The investigation of early events and molecules involved in fungal–plant interactions are crucial for a better understanding of symbiosis.
One of the major inducible plant defence responses is the accumulation of plant defence proteins, including typical pathogenesis-related (PR) proteins and other proteins with toxic or inhibitory activity towards other organisms, including bacterial and fungal pathogens. In this sense, plant lipid transfer proteins (LTPs), previously thought to be involved in the transfer of a broad range of lipids between membranes in vitro (Kader, 1996) have also been implicated in plant defence (Molina and García-Olmedo, 1993; García-Olmedo et al., 1995). The defensive role of plant LTPs was found because of their ability to inhibit bacterial and fungal pathogens, their distribution at high concentrations over exposed surfaces, and the response of Ltp gene expression to infection with pathogens (García-Olmedo et al., 1995).
In rice there are at least two different Ltp gene families. The Ltp type 1 genes do indeed constitute a complex multigene family with at least seven members grouped into three, possibly four, differentially regulated subfamilies (Vignols et al., 1997). Northern blot experiments have demonstrated that they are differentially regulated in the different tissues, and some abiotic compounds such as salt, salicylic acid and abscisic acid were shown to modulate Ltp gene expression (Vignols et al., 1997).
In the present study, the possible co-ordination between the expression of genes implicated in plant defence and the activation of molecular mechanisms mediating gene expression during AM formation was investigated. The expression of two inducible defence rice genes, Ltp and phenylalanine ammonia-lyase (PAL), were analysed during the early stages of plant root colonization by the arbuscular mycorrhizal fungus Glomus mosseae. In addition to their role in plant defence, both genes are involved in the plant response to the environment and their expression is regulated by biotic and abiotic agents (Kader, 1996; Dixon and Paiva, 1995). The expression pattern was followed using a RT-PCR approach and a GUS chimeric gene in transgenic rice plants. The focus was on the possible induction/suppression of gene expression associated with the penetration of the epidermal tissue by the fungus. In addition, the accumulation rate of an inductor of both genes, salicylic acid, was determined during the early stages of AM formation and the effect of exogenous salicylic acid application on Ltp gene expression and AM development was analysed.
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Plant material
The seeds of rice (Oryza sativa L. cv. Thainato) plants were surface-sterilized in 10% sodium hypochlorite solution, and germinated at 28 °C. The seedlings were grown in a sand:vermiculite (50:50, v:v) seedbed until transplanting and inoculation.
Rice T3 and T1 transgenic plants (cv. Taipei 309) were constructed in the Département des Cultures Annuelles, CIRAD-CA, Montpellier, France, by electroporation of protoplasts. T3 plants carried a Ltp:Gus fusion obtained by cloning a 1177 bp BamHI–Pvu II Ltp 1 gene promoter fragment from pBBO4A (Vignols et al., 1994) into pGUS-3 digested with BamHI and SmaI. T1 control plants carried an out-of-frame fusion between the Ltp promoter fragment and Gus gene.
Fungal inoculation and determination of mycorrhizal colonization
Experiments were carried out in 80 ml pots containing a sterile mixture of sand:vermiculite:peat (50:50:15, by vol.). The seedlings were inoculated with 1 g of soil inoculum of Glomus mosseae (Nico. & Gerd.) Gerd. and Trappe per pot at the moment of transplanting. Control plants were mock inoculated with a distilled water filtrate corresponding to 1 g of inoculum (McAllister et al., 1997).
Plants grown under greenhouse conditions were harvested at 3, 4, 5, 6, 7, 9, and 11 d after inoculation and the root system was collected to make the different determinations. Mycorrhizal colonization (entry points, appressoria and internal hyphae) was measured as described previously (Ocampo et al., 1980).
Exogenous application of SA
The effect of exogenously applied SA on mycorrhization kinetics was analysed in rice plants cultivated as described earlier, except that the roots of these seedlings were treated by soaking for 2 h in solutions of 0.5, 1 and 1.5 mM salicylic acid (SA) (pH 7) before transplanting. After transplanting and fungal inoculation the plants were irrigated every 3 d with these same solutions. Only 0.5 g of soil inoculum per pot was used. Mycorrhization and numbers of entry points were measured as described previously (Ocampo et al., 1980) in 1 cm root segments (40 random segments per sample) obtained 15 d and 25 d after G. mosseae inoculation.
Extraction and quantification of free SA
Root tissue (1 g) was frozen in liquid nitrogen and the free SA content was analysed using the method described earlier (Blilou et al., 1999, and according to Rasmussen et al., 1991), but the concentrated ethanol extracts were resuspended in 5% trichloroacetic acid and extracted into 2 vols of cyclopentane/ethyl acetate/isopropanol (Malamy et al., 1992), before TLC analysis. Each data point is the average of three replicate samples (roots of three different pots) from one representative experiment. The value of each replicate is the average of 10 fluorescent readings taken over 10 s. The limit of detection for salicylic acid in a final volume of 1 ml was 1 nmol. Each experiment was performed at least three times with similar results. The data were analysed by one-way analysis of the variance. The standard error of means is given.
Bacterial inoculation and SA treatment
Rice plants were axenically grown in glass test tubes (20x200 mm) containing a strip of filter paper to support the seeds and filled with 15 ml of Long Ashton nutrient solution (Hewitt, 1952). The seeds were previously surface-sterilized in 10% sodium hypochlorite solution, and germinated at 28 °C. After germination, five seedlings per tube were grown. One week later, when the root system of the plant was well developed, each tube was inoculated with 108 cells ml-1 of Pseudomonas syringae pv. syringae. Control plants were mock inoculated with sterile distilled water. Bacteria were grown in nutrient broth medium at 28 °C. Cells were washed twice to remove the culture medium and resuspended in sterile distilled water before inoculation. The P. syringae isolate used was obtained from the Spanish National Collection of Type Cultures (CECT).
The same culture system was used to treat plant roots with SA. Each tube was inoculated with 0.5 ml of a 30 mM SA solution (adjusted to pH 7) to reach a final concentration of 1 mM. The SA solution was prepared in Long Ashton nutrient solution and sterilized by filtration. Control plants were inoculated with 0.5 ml of sterile Long Ashton nutrient solution.
Plant roots were harvested 24 h after inoculation of P. syringae or SA solution and frozen in liquid nitrogen.
Enzyme histochemical GUS assays
GUS staining was performed with 1 cm long detached root pieces and the observation of GUS staining was made by light microscopy.
Histological staining was performed according to Jefferson, with incubation in the dark for 24 h at 37 °C (Jefferson, 1987). After staining, the root pieces were fixed in 4% glutaraldehyde, 0.1 M sodium cacodylate (pH 7.2) overnight at room temperature as previously described (Couteaudier et al., 1993).
RNA isolation, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Northern analysis
Total RNA was obtained from frozen roots by phenol/chloroform extraction (Kay et al., 1987). RT was carried out in a final volume of 20 µl, and was started by mixing 1 µg total RNA and 2 µM oligo d(T)15 (final concentration) at 65 °C for 10 min. After 5 min incubation at 37 °C, 4 µl of 5x RT buffer (250 mM TRIS-HCl, pH 8.3, 250 mM KCl, 2.5 mM spermidine, 50 mM MgCl2, 50 mM DTT), 200 µM of dNTPs, 25 units of RNase inhibitor (Boehringer Mannheim), and 200 units of M-MLV reverse transcriptase (Promega) was added. Incubation was prolonged at 37 °C for 1 h and 1 µl of the single strand cDNA was used for PCR amplification. PCR reaction was performed in a volume of 20 µl using 1 unit of Taq polymerase and 2 µM of specific primers from 3' untranslated region of Lipid Transfer Protein b1, a15 and b21 cDNA clones (Vignols et al., 1997). The protocol for PCR was 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min and elongation at 72 °C for 1 min. The synthesized DNA was separated in 1.25% agarose gels and transferred to Hybond N membranes (Amersham). The membrane was hybridized with the specific digoxigenin-labelled insert of each Ltp clone according the DIG DNA labelling and detection protocol (Boehringer Mannheim).
As an internal control of the amount of RNA and its quality, the same single-strand cDNA was used to perform a PCR reaction with specific oligonucleotides to a polyubiquitin gene (Ubi-1: 5'-ATGCAGAT(C/T)TTTGTGAAGAC-3' and Ubi-2: 5'-ACGCAGACCGAGG TGGAG-3') that specific priming the 228 bp monomers fragment of the rice Rubq1 polyubiquitin gene (Huq et al., 1997).
The RT-PCR approach was also used to isolate a PAL cDNA fragment of 545 bp. This fragment was obtained by RT-PCR from RNA isolated from rice root tissue 24 h after treatment with P. syringae. The specific primers PAL1 (5'-CGAGCAGCACAACCAGGA-3') and PAL2 (5'-GAGCGGATACGACCTGCA-3') used in the PCR amplification correspond to conserved sequences present in the rice PAL gene ZB8 (Zhu et al., 1995). The 545 bp DNA fragment was cloned into pGEM-T vector (Promega) and was sequenced on both strands. DNA sequencing was performed in the ABI 373 A automatic DNA sequencer (Applied Biosystems Industries), according to the manufacturer's instructions. Sequence data and sequence analyses were carried out with EditView and DNA strider programs, respectively. Homology searches were carried out using the NCBI blast E-mail server.
Northern blot analysis was performed by total RNA obtained from root tissues. The RNA was electrophoresed in formaldehyde/agarose gels and blotted onto Hybond N membranes following standard procedures. Hybridization with 32P-labelled specific probes were carried out at 42 °C in a 50% formamide-based solution (Sambrook et al., 1989). Final washes of filters were performed twice in 0.2x SSPE, 0.1% SDS for 20 min at 65 °C.
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Transient induction of Ltp in rice roots inoculated with the AM fungus G. mosseae
Ltp mRNA accumulation was examined in a time-course experiment during the early stage of fungal root colonization. By Northern blot little or no Ltp mRNA accumulation was detected (data not shown), probably because of the dilution of the transcripts by total RNA, or the possible low level or transient expression in roots (Vignols et al., 1997
). Therefore, the expression pattern was investigated by using a RT-PCR approach. This method, which is more sensitive, allowed the examination of the expression of the three Ltp genes independently, through the use of specific oligonucleotides primers. The cDNA amplification of the constitutively expressed polyubiquitin gene was used as the internal control in the RT-PCR reactions. After quantification, cDNA was amplified with the gene-specific primers and the PCR products were hybridized to specific-gene probes.
Figure 1 shows the results of time-course RT-PCR expression studies of Ltp genes in rice root inoculated or not by G. mosseae. The transcript corresponding to the b21 gene was expressed at the same levels in roots of non-inoculated and inoculated rice plants. The level of transcript accumulation was unchanged throughout the period of fungal colonization assayed in this experiment. The a15 gene was also expressed in non-inoculated and inoculated rice roots. No differences in the level of transcript accumulation were observed between roots of non-inoculated and inoculated rice plants.
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The pattern of b1 gene expression in non-inoculated roots was similar to that observed for b21 and a15 genes and the level of transcript accumulation was unchanged throughout the period assayed. Interestingly, a transient increase of Ltp b1 transcript was observed between the third and seventh day of inoculation with G. mosseae, which was particularly evident at 6 d (Fig. 1
). This transient increase in transcript level coincided with the period of fungal appressoria formation in the surface of the root and fungal penetration. Nine and 11 d after inoculation the levels of transcript in roots of inoculated plants were similar to that shown for non-inoculated plants, coincident with the onset of the intercellular development of fungal hyphae into the root. At 9 and 11 d after inoculation the percentage of root length colonization reached 7±1% and 12±3%, respectively. In order to investigate the possible relationship between appressoria formation and fungal penetration with Ltp b1 expression, the transgenic rice line T3 was used to study the activation of the chimerical Lt:Gus construct in rice roots during the early stages of arbuscular mycorrhiza development. T3, carrying the Ltp type b1 promoter fused to the Escherichia coli uidA (Gus) gene, and T1 (promoter Ltp:Gus with frameshift) negative control rice plants were inoculated with G. mosseae. Since the method of fungal inoculation did not ensure a synchronous infection, and in order to ensure fungal appressoria formation, plant roots were harvested daily between days 6 and 10 after inoculation. Root samples were taken for estimating mycorrhizal infection and histochemical staining for GUS enzyme activity.
No GUS activity was detected in the cortex of inoculated or non-inoculated roots from T1 plants (Fig. 2A, B). In T3 plant roots, GUS activity was sometime detected associated with the vascular cylinder (Fig. 2C), but in T3 inoculated plant roots it was also clearly associated with the epidermal root regions where the fungus form the appresorium (Fig. 2D).
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Only a small portion (less than 10%) of the inoculated root pieces showed GUS staining, but always associated with an area of fungal appresorium and penetration. No GUS activity was found in external living fungal hyphae infecting roots (Fig. 2D
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Expression of Ltp b1 gene was related to a plant defence response
Whether the expression pattern of Ltp b1 gene in the root of inoculated rice plants was related to the expression of other genes implicated in plant defence responses was investigated. Specifically, the expression of a Pal gene during the early stage of G. mosseae rice interactions was studied.
Firstly, using the RT-PCR methodology a 545 bp cDNA fragment was cloned that showed 97% of similarity at nucleotide level with the ZB8 rice Pal gene (Zhu et al., 1995). To ensure that the Pal gene was implicated in the defence response, the cDNA clone was obtained from RNA extracted from root tissues inoculated with the bacterial pathogen P. syringae.
The analysis of Pal gene expression by Northern-blot or RT-PCR (Fig. 3) clearly showed that the transient induction of Pal gene expression coincided in time with the period of Ltp b1 induction in rice roots inoculated with G. mosseae. In the Northern experiments (Fig. 3A) a transient induction of Pal transcript accumulation (3–4 times) was observed from days 3–7 after inoculation. The same result was obtained by the RT-PCR approach (Fig. 3B). The RT-PCR using the specific primers for the polyubiquitin gene demonstrated that the amount of RNA in each RT reaction was approximately equal. To determine whether, biological and/or chemical inducers of plant defence response were involved in Pal and Ltp b1 gene induction, the accumulation of both transcripts in roots after inoculation with P. syringae or 1 mM salicylic acid was analysed by RT-PCR. The appearance of HR and the induction of PR proteins have previously characterized the interaction between rice and P. syringae (Smith and Métroux, 1991). As shown in Fig. 4, the expression of both genes was induced after 24 h of P. syringae inoculation or 1 mM SA treatment. This result was consistent with the proposed participation of Pal and Ltp genes in the plant defence response.
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Transient SA accumulation in roots and effect of exogenous SA application during early stages of fungal colonization
To determine whether AM fungi activate the accumulation of free SA in the rice root system the time-course of SA accumulation during the early stages of interaction with G. mosseae was studied. A transient peak of SA in roots was observed 6 d after inoculation with G. mosseae (Fig. 5). The appearance of this peak coincided with the beginning of fungal root interactions since initial entry points and appressoria formation was observed after 4 d and 6 d after inoculation, whereas longitudinal internal hyphae were observed after 6 d and 7 d. After 11 d of inoculation the AM root length colonization reached 11±2%.
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The effect of exogenously applied SA on mycorrhization kinetics was analysed in rice plants inoculated with G. mosseae and treated with different SA concentrations. It had previously been assessed that these amounts of exogenous SA did not affect plant development, measured as shoot and root fresh weights (data not shown). Exogenous SA application caused a significant delay in the mycorrhization of roots at 15 d after inoculation, which directly correlated with the SA added to the soil (Table 1
). Interestingly, SA application did not affect appressoria formation (Table 1). Thus, the levels of exogenous SA tested were not inhibitory to the capacity of fungi to attach to the root under these experimental conditions. Twenty-five days after inoculation, however, mycorrhization levels in SA-treated plants were similar to the untreated controls. These data suggested that exogenously applied SA could delay mycorrhiza formation, but only in a transient manner.
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The effect of exogenous SA application on plant gene expression was analysed in rice roots treated with 0.5 or 1 mM of SA added to the soil. RT-PCR methods were used to determine changes in the Ltp b1 gene, whose mRNA levels have been shown previously to increase in roots of seedlings grown in the presence of SA. Figure 6
shows the bands of hybridization after RT-PCR of the Ltp b1 gene and a constitutive polyubiquitin gene. Clearly the bands corresponding to 3' region of Ltp were enhanced in plants treated with both 0.5 and 1 mM SA after 15 d and 25 d of treatment. These data confirm that the SA added to the soil was effective in modulating expression of defence-related genes.
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Discussion |
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The establishment of a successful compatible AM symbiosis require clear recognition by the plant host, which recognizes the invading fungus as a beneficial symbiont instead of a pathogen. Although the mechanism of recognition is completely unknown, AM fungi do not seem to activate a host defence response or they have developed some mechanism to suppress the host defences. In this regard, the expression of host plant defence response is unco-ordinated, weak and/or very localized (Gianinazzi-Pearson et al., 1996
), although the precise role of this defensive phenomenon has not been elucidated in the compatible AM association. In this paper, it was examined whether the expression of Ltp genes, implicated in epidermal differentiation and plant response to the environment, including defence response, is co-ordinated with plant defence responses as a consequence of fungal–plant interaction. The RT-PCR approach allowed us to correlate the expression of three Ltp genes with the early stages of AM formation.
No differences in Ltp a15 and b21 gene expression were observed between plant roots inoculated or not by G. mosseae. Nevertheless, the Ltp b1 gene was induced in the rice host plant by the mycorrhizal fungus. The level of this transcript increased when the fungus formed appressoria and penetrated the root epidermis and decreased at the onset of the intercellular development of fungal hyphae into the root. A similar pattern of gene expression was reported for the early nodulin gene psENOD12A in pea plants inoculated with Glomus margarita (Albrecht et al., 1998) indicating that the process of appresorium formation and penetration is a crucial step in AM symbiosis. This process is characterized by specific morphologies and genetic alterations in both partners. In this sense, some authors suggest that plant defence reactions may occur only after appresorium formation when the fungus has changed its state from saprophytic to infective (Giovannetti et al., 1994).
The analysis of histochemical GUS staining in transgenic rice plants carrying the Gus gene under the control of rice Ltp promoter confirm the data of induction of Ltp gene expression in the root epidermis associated with fungal appressoria formation and penetration. The constitutive expression of the Ltp:Gus chimerical gene observed in the vascular tissue of inoculated or non-inoculated root seedlings was in agreement with previous results with transgenic Arabidopsis thaliana plants containing a similar Ltp:Gus fusion (Thoma et al., 1994). In inoculated rice roots this constitutive expression localized into the vascular cylinder, a tissue that AM fungi never colonize, was clearly distinguishable from the epidermal AM induced.
The putative role of LTP during mycorrhizal formation is unknown. Plant LTPs were thought to participate in membrane biogenesis and regulation of the intracellular fatty acid pools. Nevertheless, further isolation and analysis of Ltp genes has revealed novel roles for LTPs including adaptation of plants to various environmental conditions, cutin formation, embryogenesis and defence reactions against phytopathogens (Kader, 1996).
In mycorrhizal roots the expression of Ltp coincided in time with a transient increase in the expression of a Pal gene implicated in plant defence responses, and the expression of both genes was induced after treatment with SA and P. syringae. It is possible that the increase of Ltp gene expression might be part of the weak plant host defence response to the invading fungus. In the context of this early activation of a plant defence mechanism, the SA peak observed in rice plants inoculated with G. mosseae should also be considered. The SA peak coincided in time with the increase in Ltp gene expression and SA may initiate this induction. The weak rise in SA is not significant as compared with the strong (several hundred- fold) SA accumulation induced after pathogen infection in tobacco or cucumber (Malamy et al., 1990; Métroux et al., 1990). Thus, the significance of this weak SA accumulation in the mycorrhizal interaction should be further investigated. However, a weak rise in SA can have very significant effects during the development of Systemic Acquired Resistance (SAR) (Vernooij et al., 1994) and thermotolerance (Dat et al., 1998).
In these experiments, the activation of defence genes by exogenous SA only resulted in a transient delay of the mycorrhization process, and possibly the defence mechanism activated (including Ltp expression) was not effective in stopping the AM fungus. In this sense, constitutive expression of chitinase genes and other pathogenesis-related proteins in transgenic plants did not affect the time-course or the final level of root colonization by AM fungi (Vierheilig et al., 1993, 1995). If SA induces a defence response, it might be assumed that the mycorrhizal fungus would encounter more difficulties in penetrating the root system. Nevertheless, once this initial challenge is overcome, the intra-radical development of the fungus should be normal.
The phenomenon of resistance to AM fungi associated with mutation in symbiotic genes (Myc-mutant plants) (Duc et al., 1989) and the existence of cellular and molecular defence responses induced in these mutant plants challenged with AM fungi (Gollotte et al., 1993) suggest that AM fungi are capable of inducing defence-related mechanisms. Interestingly, the character Myc- is under control of specific plant symbiotic genes that are necessary to establish a compatible interaction between legumes and Rhizobium or AM fungi. One of these genes, Sym 8, is required to induce expression of some nodulin genes (Albrecht et al., 1998). The mutation in the Sym 30 gene causes an increase in SA accumulation in the roots of the symbiosis-resistant P2 pea mutant interacting with G. mosseae or R. leguminosarum (Blilou et al., 1999) suggesting that Sym 30 could be implicated in a common pathway leading to the suppression of a SA-dependent defence mechanism in legume plants against Rhizobium and AM fungi. In addition, it has been shown that some defence genes are differentially induced by a mycorrhizal fungus and a Rhizobium strain in wild-type and symbiosis-defective pea genotypes (Ruíz-Lozano et al., 1999). It is clear that symbiotic genes control the initial establishment of AM infection, and the present study demonstrates that several SA-inducible plant defence-related genes are differentially regulated during AM development. These observations suggest a link between symbiotic genes and the possible induction/suppression of a defence response in mycorrhizal interactions. The analysis of defence-related genes expressed during the compatible and incompatible interactions between AM fungi and wild and/or symbiotic mutant plants will be a valuable approach for defining genes involved in regulating AM formation and development.
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Acknowledgments |
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This work was supported in part by grants from the Spanish Instituto de Cooperación con el Mundo Arabe (ICMA) for I Blilou, and from The Ministerio de Educación y Cultura (MEC) for JM García-Garrido. The authors wish to thank Drs E Guiderdoni and F Vignols for the gift of the transgenic rice lines and LTP cDNA clones, respectively. The Comisión Interministerial De Ciencia y Tecnología of Spain (CICYT) provided financial support for this study (PB-97 1202).
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1 To whom correspondence should be addressed. Fax: +34 58 129600. E-mail: JoseManuel.Garcia{at}eez.csic.es1
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