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DNA Research 2007 14(3):117-133; doi:10.1093/dnares/dsm014
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© The Author 2007. Kazusa DNA Research Institute
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Transcriptome Profiling of Lotus japonicus Roots During Arbuscular Mycorrhiza Development and Comparison with that of Nodulation

Yuichi Deguchi1, {dagger}, Mari Banba1, {ddagger}, Yoshikazu Shimoda2, , Svetlana A. Chechetka1, Ryota Suzuri1, Yasuhiro Okusako1, Yasuhiro Ooki1, Koichi Toyokura1, Akihiro Suzuki3, §, Toshiki Uchiumi3, Shiro Higashi3, Mikiko Abe3, Hiroshi Kouchi4, Katsura Izui1, || and Shingo Hata1,5,*

1 Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
2 Graduate School of Science and Technology, Kagoshima University, Kagoshima 890-0065, Japan
3 Department of Chemistry and BioScience, Kagoshima University, Kagoshima 890-0065, Japan
4 National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
5 Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan

Received 17 January 2007; accepted 22 June 2007.


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results and discussion
 Supplementary Data
 Acknowledgements
 References
 
To better understand the molecular responses of plants to arbuscular mycorrhizal (AM) fungi, we analyzed the differential gene expression patterns of Lotus japonicus, a model legume, with the aid of a large-scale cDNA macroarray. Experiments were carried out considering the effects of contaminating microorganisms in the soil inoculants. When the colonization by AM fungi, i.e. Glomus mosseae and Gigaspora margarita, was well established, four cysteine protease genes were induced. In situ hybridization revealed that these cysteine protease genes were specifically expressed in arbuscule-containing inner cortical cells of AM roots. On the other hand, phenylpropanoid biosynthesis-related genes for phenylalanine ammonia-lyase (PAL), chalcone synthase, etc. were repressed in the later stage, although they were moderately up-regulated on the initial association with the AM fungus. Real-time RT–PCR experiments supported the array experiments. To further confirm the characteristic expression, a PAL promoter was fused with a reporter gene and introduced into L. japonicus, and then the transformants were grown with a commercial inoculum of G. mosseae. The reporter activity was augmented throughout the roots due to the presence of contaminating microorganisms in the inoculum. Interestingly, G. mosseae only colonized where the reporter activity was low. Comparison of the transcriptome profiles of AM roots and nitrogen-fixing root nodules formed with Mesorhizobium loti indicated that the PAL genes and other phenylpropanoid biosynthesis-related genes were similarly repressed in the two organs.

Key words: cysteine proteinase; defense response; phenylalanine ammonia-lyase; symbiosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results and discussion
 Supplementary Data
 Acknowledgements
 References
 
Arbuscular mycorrhizal (AM) fungi of the phylum Glomeromycota1Go establish ecologically important symbiotic associations with the majority of land-plant species, allowing improved uptake of phosphate and other nutrients from the soil in exchange for plant-assimilated carbohydrates.2Go,3Go Additionally, AM fungi endow plants with tolerance to pathogens and abiotic stress.4Go,5Go In the process of colonization by AM fungi, the hyphae of extraradical mycelia branch near the host roots and form appressoria on the root surface, from which hyphae penetrate the epidermis and grow inter- and intracellularly in the root cortex. In the case of Arum-type AM (as formed in Lotus japonicus by Glomus mosseae or Gigaspora margarita), the hyphae of intraradical mycelia form arbuscules, which are highly branched structures thought to be the main site of nutrient exchange between the two symbiotic partners.2Go,3Go,6Go Early land-plant fossils contain structures that appear similar to arbuscules, suggesting the important role of AM fungi in the colonization of land by plants.7Go–9Go

In addition to AM symbiosis, leguminous plants establish a better-characterized symbiotic association with rhizobia, forming nitrogen-fixing root nodules. Recent molecular and genetic data suggest that the mechanism governing nodule formation evolved from that of AM symbiosis over time.10Go,11Go

The development of AM symbiosis is generally thought to accompany complex signal perception and transduction, but the understanding of the latter at the molecular level is very limited, mainly because AM fungi are obligate symbionts and the leading model plant Arabidopsis thaliana does not form AM roots. For a better understanding, in silico data mining,12Go the subtractive hybridization approach,13Go–15Go and cDNA and oligonucleotide array analyses16Go–20Go have been performed for Medicago truncatula, a model legume.21Go,22Go Medicago truncatula was also used to investigate the differential expression of chitinase genes in AM colonization, nodulation, and plant–pathogen interactions.20Go,23Go Lotus japonicus is another valuable model legume.24Go For example, L. japonicus has been used for elucidation of the molecular mechanisms of plant–AM fungi interactions.25Go Gene expression profiling with the aid of cDNA-amplified fragment length polymorphism has also been carried out.10Go

For transcriptome analyses of host responses to AM fungi, we here made use of a large-scale cDNA array of L. japonicus,26Go,27Go carefully eliminating the effects of contaminating microorganisms in the soil inoculants. We compared the results with a gene expression profile of root-nodule formation with Mesorhizobium loti, finding a number of genes commonly regulated during AM symbiosis and nodule formation.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results and discussion
 Supplementary Data
 Acknowledgements
 References
 
2.1. Plant material and microorganisms
Lotus japonicus Gifu B-129 seeds were scarified, surface-sterilized with 1% NaClO, rinsed eight times with sterile water, and then spread on 0.7% water agar plates for germination. The plates were placed for a week in a controlled-environment growth chamber (Sanyo, Tokyo, Japan) with a 16-h-day and 8-h-night cycle at 25°C, and a light intensity of 260 µEs–1m–2 with 60% humidity.

For AM colonization, glass tubes (30 mm diameter x 120 mm length) containing 55 mL of vermiculite supplemented with 45 mL of modified Hornum nutrient solution were autoclaved before transferring the seedlings. The concentration of phosphate was reduced from 640 µM24 to 250 µM to facilitate the colonization. The soil inoculant of G. mosseae (2 g/tube; a gift from K. Nagashima, Idemitsu Kosan, Tokyo, Japan) was suspended in sterilized water and then added to the tubes. For the control plants, the G. mosseae inoculant suspension was filtered through a 38 µm stainless mesh and the filtrate was added to the tubes. The resulting sieved carrier was free of G. mosseae spores but contaminated by microorganisms equivalent to those in the whole inoculum suspension. The seedlings were grown for up to 8 weeks in a growth chamber with occasional irrigation with the modified Hornum solution. For inoculation of G. margarita (Central Glass Co., Tokyo, Japan), large spores were picked up with forceps under a stereomicroscope, surface-treated with 0.1% NaClO for 7 min, and then rinsed five times with sterilized water. The seedlings were inoculated with the spores and grown as above. The control plants were mock-inoculated with the final rinse and then allowed to grow further. Assessment of AM colonization was carried out by the gridline intersect method28Go after staining with trypan blue.29Go

When the initial stage of AM symbiosis was examined, we modified the ‘nurse pot’ method,30Go as follows. Giant spores of G. margarita were picked up with forceps from a commercial inoculum (Central Glass Co., Tokyo, Japan), surface-treated with 0.1% NaClO, and then rinsed with sterile water. Three sterile L. japonicus seedlings (1-week-old) were inoculated with 500 spores in an autoclaved plastic container (11 cm diameter x 16 cm height; Takeya Chemical Co., Osaka, Japan) with a lid and then allowed to grow further. As a non-inoculated control, the final rinse of the sterilized spores was applied to sterile seedlings in another container, followed by further growth. After 2 months, freshly prepared sterile seedlings (2-week-old) were transplanted into the containers and then allowed to grow for a week. Then, roots of the younger plants were harvested from the container inoculated with G. margarita or the mock-inoculated container.

For root-nodule formation, L. japonicus seedlings were inoculated with M. loti Tono and then grown for 2 weeks on vermiculite supplied with nitrogen-free Broughton and Dilworth medium as described previously.31Go,32Go The resulting young nodules were harvested.

2.2. cDNA array analysis
Total RNA was extracted from AM roots, root nodules, or control roots using an RNeasy Plant Mini-Kit (Qiagen, Hilden, Germany). Labeling of target cDNA, hybridization of a large-scale nylon filter array with the target, washing of membranes under high-stringency conditions, detection of radioactive images, and data mining were all carried out as described previously.26Go

2.3. Real-time RT–PCR analysis
After treating the total RNA preparation with DNase, reverse transcription was performed with oligo(dT) and Superscript II (Invitrogen, Carlsbad, CA). Real-time PCR with a real-time RT–PCR Core Kit (Takara Bio, Otsu, Japan) and a Smart Cycler system (Cepheid, Sunnyvale, CA) was carried out as described previously.32Go,33Go The forward and reverse primer sets and annealing temperatures (in parentheses) were as follows: 5'-CAGTGACAAAAGGTTTGGACCTAC-3' and 5'-ATGCAGAGAGATGTTGCTGCTG-3' (68°C) for LjCyp2; 5'-AACTTTATTAGTAACTTTTAG-3' and 5'-CTTTCACATCCGAGGAAATTG-3' (55°C) for LjPAL1; 5'-GCTCAGGTGGCTGCCATCGCC-3' and 5'-GGCAGTGTGTGGTTTGTCTCG-3' (55°C) for LjPAL2; 5'-AACTTTACTAG TTTCTTCAGG-3' and 5'-TAATTCCATATTCCGCAAATT-3' (55°C) for LjPAL3; 5'-GAATGCAGATCTTACCCGCTA-3' and 5'-TTTGCTTAAATACAAAGAATG-3' (50°C) for LjPAL4; 5'-GAATGCAGATCTTACCCGCTG-3' and 5'-ATTGCATTTGCATAAATACAG-3' (50°C) for LjPAL5; 5'-AACTTAACCATTTATTTTTTT-3' and 5'-TTGTAATGTAATGTGAGATGG-3' (55°C) for LjPAL6; 5'-TTGGCTAGCATCGATTCAGGA-3' and 5'-GTCCAGGGTGGTGCTTAAGCC-3' (50°C) for LjPAL7; 5'-GCTCAGGTGGCTGCCATCGCA-3' and 5'-GGCAGGGTGTGAGTTGATTCA-3' (55°C) for LjPAL8; 5'-AACTTGCCTGCCAGTTATGTT-3' and 5'-CTCTTGTGTTTTTCTGTAGTG-3' (55°C) for LjPAL9; and 5'-AGAACAGTTTGTTTGTTTGAG-3' and 5'-CATAAAGGAGAACTTAAAGGA-3' (55°C) for LjPAL10. Amplification of the ß-actin gene was carried out as described previously.33Go A single amplicon of expected size, 100–300 bp, with each primer set was observed on agarose gel electrophoresis, irrespective of whether the reverse-transcribed template was from AM roots or control roots. In order to calculate the transcript level ratios, it was assumed that each PCR cycle results in exact doubling of the amounts of amplicons.

2.4. In situ hybridization
In situ hybridization of paraffin-embedded sections was carried out as described previously.31Go,33Go,34Go

2.5. Promoter-ß-glucuronidase construction, hairy root transformation and histochemical analysis of L. japonicus
The 2 kb 5' flanking region of LjPAL1 contains a BamHI site. Therefore, to amplify the region derived from genomic DNA of L. japonicus, forward primer 5'-ATGCGGCCGCTGACCGACAATGGTTTATGAAC TAGCC-3' and reverse primer 5'-ATTGATCACTTAGTATATATGATCTCTCACTTACA-3', containing NotI and BclI sites, respectively, were used for PCR. The BclI end of the promoter was ligated to the BamHI site 24 bp upstream of the coding sequence of the uidA gene for the ß-glucuronidase (GUS) reporter with a nopaline synthase terminator. Then, making use of the SalI sites at the ends of the intermediate construct, the promoter-GUS unit was ligated into the SalI site of pHKN29,35Go which is a derivative of pCAMBIA 1300 (CAMBIA, Canberra, Australia).

Hairy root transformation with Agrobacterium rhizogenes LBA 1334 was performed following the protocol of Diaz and Schlaman, Leiden University, as described previously.33Go,35Go Transformants with green fluorescent protein (GFP)-positive hairy roots were transferred to vermiculite containing the modified Hornum solution, inoculated with the entire G. mosseae inoculum or sieved carrier, and then grown as described above. When nodule formation was examined, the transformants were transferred to nitrogen-free Broughton and Dilworth medium and then inoculated with M. loti Tono.

Detached roots were stained with 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide, and then the reaction was stopped with 75% ethanol as described previously.35Go When AM fungi were re-stained, the roots were immersed in 0.02% safranin and then observed under a stereomicroscope. Quantitative assaying of GUS activity in hairy roots was performed as described previously,35Go based on the method of Jefferson et al.36Go GUS-stained roots were also fixed in 4% paraformaldehyde and 0.25% glutaraldehyde in 50 mM Na-phosphate buffer (pH 7.2), washed with Na-phosphate buffer, dehydrated in an ascending ethanol series (10, 30, 50, 60, 70, 90, and 100%), immersed in 50% Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) in ethanol, and then left to stand overnight. Then, they were embedded in Technovit 7100 at room temperature by adding the polymerization agent provided in the kit. Six-micrometer sections were prepared and re-stained with 0.02% safranin when necessary.

2.6. Accession numbers
The entire nucleotide sequences of cDNAs for cysteine proteinases and PALs were determined. The accession numbers for the sequences mentioned in this paper are as follows: AB300459 (LjCyp1), AB300460 (LjCyp2), AB300461 (LjCyp3), AB300462 (LjCyp4), AB283031 (LjPAL1), AB283032 (LjPAL2), AB283033 (LjPAL3), AB283034 (LjPAL4), AB283035 (LjPAL5), AB283036 (LjPAL6), AB283037 (LjPAL7), AB283038 (LjPAL8), AB283039 (LjPAL9), and AB283040 (LjPAL10).


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  		Table 1. Up-regulated genes in L. japonicus roots after establishment of symbiosis with G. mosseae and G. margarita

 


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  		Table 2. Down-regulated genes in L. japonicus roots after establishment of symbiosis with G. mosseae and G. margarita

 

   3. Results and discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results and discussion
Supplementary Data
 Acknowledgements
 References
 
3.1. Setting up cDNA array experiments with AM roots
In this work, we made use of a large-scale array of cDNAs from entire seedlings, pods, roots, and root nodules of L. japonicus.26Go,27Go We grew L. japonicus plants with or without AM fungi in the presence of 250 µM phosphate. At that phosphate concentration, AM fungi colonized well and the effects of phosphate depletion did not need to be taken into account. Under our growth conditions, the root length colonization by G. mosseae was 20 and 60% at 3 and 6 weeks after inoculation, respectively. The colonization by G. margarita was 10 and 30–40% at 5 and 8 weeks after inoculation, respectively. It has been pointed out that fungal transcripts account for up to 12% of the entire transcripts of AM roots,37Go which sometimes complicates analysis.10Go,12Go–15Go On the other hand, since the plant materials used for our array were grown avoiding microorganisms other than M. loti, a nitrogen-fixing symbiont of L. japonicus, our array did not contain fungal genes, making the analysis of global plant gene-expression easier.

Extraction of RNA, preparation of radioactive targets, and hybridization were principally performed for two biological replicates, although in the experiments involving G. margarita, the procedures were carried out in duplicate for a single biological replicate. The signal intensities of array filters in each experiment were normalized as described previously.26Go When the normalized signal intensities were compared after the two independent series of experiments, the variation was found to be basically within the twofold expression ratio (Supplementary Fig. S1A), indicating the sufficient reproducibility of our experiments.

In the initial experiments, we compared the gene-expression patterns of AM roots formed with a commercial inoculant and sterile non-infected roots, as in most previous studies.12Go–15Go,17Go–19Go Then, we picked up statistically significantly different genes expressed in roots 6 weeks after inoculation of the whole G. mosseae inoculum compared with those in control roots 3 weeks after inoculation of the sieved carrier, by means of the Significance Analysis of Microarrays Program.38Go Supplementary Table S1 shows a list of the apparently up-regulated genes in AM roots. Closely related genes annotated as caffeic acid O-methyltransferase were most differently expressed. Lectin genes were also differentially expressed, as previously reported.13Go,15Go,19Go The up-regulation of a gene for subtilisin-like serine protease was similar to the finding of Liu et al.,16Go although serine carboxypeptidase genes were not listed in our experiment. The differential expression of chitinase genes was in accordance with a previous study.23Go Glutathione S-transferase genes were reported to be up-regulated in AM roots.12Go–14Go,20Go In our experiment, a gene for glutathione S-transferase (GNf044a01) was also up-regulated by 1.61- and 2.51-fold 3 and 6 weeks, respectively, after inoculantion (not included in the supplementary table). The expression levels of blue copper protein genes12Go,13Go,18Go,20Go varied from experiment to experiment under our conditions (data not shown). Overall, the data in Supplementary Table S1 are consistent with those in previous papers.12Go–20Go

Notably, when the G. mosseae inoculum suspension and sieved carrier were diluted and streaked on yeast extract/peptone/glucose plates, many colonies of contaminating microorganisms appeared, their numbers and appearances being similar to each other (data not shown). Thus, the above cDNA array analysis was performed in the constant presence of background microorganisms in the AM root material. We next filtered the G. mosseae inoculant suspension through a 38 µm stainless mesh, L. japonicus seedlings were grown in the presence of the filtrate, and then the gene expression in the resulting roots was compared with that in non-infected ones. Supplementary Table S2 shows the effects of contaminating microorganisms. Genes encoding PAL, chalcone synthase and chalcone reductase, which are involved in important steps of flavonoid phytoalexin synthesis,39Go and WRKY transcription factors, which are mainly involved in tolerance to pathogen-related stress,40Go were remarkably induced. The genes annotated as caffeic acid O-methyltransferase, and those for chitinase and glutathione S-transferase were also induced. Therefore, the results in Supplementary Table S1 represent super-positioning of the effects of the AM fungus and contaminating microorganisms in the inoculant. It is noteworthy that commercial AM fungus inoculants have been used easily in a number of investigations on plant gene expression in AM roots.12Go–15Go,17Go–19Go Care must be taken regarding contamination in nurse plants used for inoculation of the AM fungus.10Go On the other hand, in the works of Liu et al.16Go,20Go and Salzer et al.23Go on M. truncatula, Guimil et al.41Go on rice, and ours on L. japonicus and G. margarita (see below), aseptic spores of AM fungi were inoculated into plants, making the populations of contaminating microorganisms, if any, similar between AM roots and control roots.

3.2. Expression profiling of up- and down-regulated plant genes after colonization by AM fungi
In order to subtract the above-described effects of contaminating microorganisms, we compared the gene expression patterns of AM roots inoculated with the whole G. mosseae inoculum and control roots inoculated with the sieved carrier only. When the average intensities on duplicate determination of gene expression were compared, the patterns indicated a significant difference in gene expression (Supplementary Fig. S1B). AM-enhanced genes were first identified after colonization by G. mosseae and G. margarita (Table 1) because they have attracted more interest than repressed ones.12Go–15Go,17Go,19Go Genes for aquaporins, also annotated as plasma-membrane intrinsic protein, tonoplast intrinsic protein, and nodulin 26-like protein, were up-regulated in AM roots, confirming the results in several reports.14Go,17Go,19Go Annexin genes were also induced in AM roots, in accordance with Manthey et al.17Go

Four cysteine proteinase genes, designated as LjCyp1-4, were most obviously up-regulated among the AM-enhanced genes (Table 1), confirming previous reports.10Go,16Go–18Go Although there were around 20 cysteine proteinase genes on our array membrane, the expression of other genes did not change or was rather repressed in AM roots. Real-time RT–PCR showed that LjCyp2, a representative of the four genes, was induced only at the late stage of G. mosseae colonization (Fig. 1A). The expression of LjCyp2 was also high in G. margarita-colonized roots at the late stage (data not shown). Our in situ localization revealed that the induced LjCyp2 gene was specifically expressed in arbuscule-containing inner cortical cells of G. mosseae-colonized roots (Fig. 1C). The LjCyp1 transcript showed a very similar localization (not shown) to that of LjCyp2. The spatial expression patterns of AM-induced genes fall into two groups. The glutathione S-transferase,13Go serine carboxypeptidase,16Go annexin,17Go and calcium-binding protein10Go genes were reported to be expressed not only in arbuscule-containing cells but also in the cells around them. In contrast, the endoglucanase (MtCel1),16Go cysteine-rich antifungal protein,19Go and AM-induced phosphate transporter33Go genes were specifically expressed in cells that contained fungal arbuscules. The present study revealed that the Lotus cysteine proteinase genes are members of the latter group. It is noteworthy that the cysteine proteinase genes are expressed early in cells containing arbuscules just after maturation, whereas their levels are quite low in cells with very young arbuscules (Fig. 1E). The induced cysteine proteinases may be involved in the degradation of arbuscules, short-lived fungal organs,2Go since the PSORT program (http://psort.nibb.ac.jp/) predicted that they are secreted proteins. Alternatively, these proteases may stay within the cells, e.g. in vacuoles42Go,43Go and play important roles in remodeling of intracellular structures, cell cycle progression, protein turnover etc. It is also interesting that the four cysteine proteinase genes are exactly the same genes as those that are highly induced in early-senescent root nodules of ineffective nitrogen fixation.27Go


Figure 1
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  		Figure 1. Expression levels and in situ localization of the LjCyp2 gene. The expression levels of the LjCyp2 (A) and ß-actin (B) genes relative to those in control roots 3 weeks after inoculation of the sieved carrier (SC3, level = 1) were determined by real-time RT–PCR. The means and variation of two independent experiments are shown. ni3, sterile non-infected roots grown for 3 weeks; Gm6, roots 6 weeks after inoculation of the whole G. mosseae inoculum; gni1, roots 1 week after mock-inoculation; and Gi1, roots 1 week after inoculation of G. margarita (see Materials and Methods and the legend to Table 3 for details). Longitudinal AM root sections were probed with digoxygenine-labeled antisense RNA prepared from the entire LjCyp2 cDNA (C and E). Hybridization signals are visible as a dark blue color. When sense RNA was used as a negative control probe, much lower hybridization signals were detected except in central cylinders (D). Bars, 50 µm.

 
A promoter region of a calcium-binding protein gene of L. japonicus was reported to be activated during AM development.10Go We found that a cDNA for the calcium-binding protein (MWM036h04_r) is present on our array membrane. Unexpectedly, however, the mRNA level did not show significant variation under our experimental conditions. The mRNA level of the gene did not vary on root-nodule formation, either (http://est.kazusa.or.jp/en/plant/lotus/EST/cDNA.html). The promoter activity of the gene may not coincide with its transcript level.

In the present study, AM-repressed genes were also identified after colonization by G. mosseae or G. margarita (Table 2). Five PAL genes were repressed most drastically after colonization by G. mosseae. In addition, four and two genes for chalcone synthase and chalcone reductase, respectively, were found to be repressed. These three enzymes catalyze key reactions in the biosynthesis of phenylpropanoid compounds. Another series of duplicate experiments involving G. margarita supported this finding. Thus, the reproducibility of the repression of phenylpropanoid biosynthesis-related genes was confirmed unequivocally. Liu et al. presented a small list of AM-repressed genes.16Go Our finding that particular forms of phosphoenolpyruvate carboxylase and glutathione S-transferase are repressed is in accordance with their results. Hohnjec et al.,18Go Kistner et al.,10Go and Guimil et al.41Go presented larger lists of AM-repressed genes of M. truncatula, L. japonicus, and rice, respectively, but neither PAL genes nor chalcone synthase ones were included in the lists. In the work of Hohnjec et al.,18Go for example, many stress-related genes were listed as AM-repressed genes, because they were highly up-regulated in the phosphate-starved control roots. Very recently, Liu et al. presented the largest list of AM-repressed genes in M. truncatula roots as well as those in other portions.20Go Again, however, PAL genes were not included in their list of repressed genes. We will confirm our current results by promoter analysis and discuss the discrepancy (see below). Besides phenylpropanoid biosynthesis-related genes, a phosphate transporter gene (LjPT1) was also repressed (Table 2). This finding is in accord with the general tendency that the expression of common phosphate transporters is suppressed in AM roots.44Go–46Go A recently found AM root-enhanced phosphate transporter gene of L. japonicus33Go was not found on the present nylon filter.

3.3. Differential expression of plant genes caused by G. margarita infection in the initial stage of symbiosis
In contrast to the later stage of symbiosis (Table 1), a number of genes were found to be up-regulated or down-regulated on the initial association with the AM fungus (Table 3). In accordance with previous reports,16Go,47Go–52Go the genes for enzymes involved in defense-related secondary metabolism and the pathogen response, such as PALs, chalcone synthases, and peroxidases, were moderately up-regulated at this stage. A number of genes for transcription or translation were also induced, suggesting that a dynamic cellular change in plant roots occurs at the initial stage of the AM association. In addition, several genes involved in signal transduction were up-regulated (Table 3). For example, the gene for a pathogen-induced receptor protein kinase with a characteristic extracellular domain was induced.53Go,54Go Transcripts for a heterotrimeric G protein-coupled receptor, small GTP-binding proteins, protein serine/threonine kinases, and a mitogen-activated protein kinase were also accumulated. These gene products may represent signal transduction pathways for AM colonization.


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  		Table 3. Transcriptional changes caused by G. margarita infection in the initial stage of symbiosis

 
3.4. Expression patterns of PAL genes in L. japonicus
PALs connect primary and secondary metabolism in plants, catalyzing common rate-limiting steps of flavonoid phytoalexin synthesis, lignin synthesis, salicylic acid synthesis, etc. The expression patterns of PAL genes in our experiments are very characteristic compared with those in previous studies.10Go,12Go–20Go,41Go Since PAL genes are known to form a family in a number of plant species,55Go we first checked how many PAL genes were present on the array membrane and found nine non-redundant ones. In addition, we found a TAC clone (Accession no. AP004502) containing a unique PAL gene, LjPAL5, in the databases. As shown in Table 4, most PAL genes were induced in the initial stage of AM infection and then repressed in the later stage. However, LjPAL10 did not seem to be expressed differentially. In addition, other genes, LjPAL7 and LjPAL9, might be of the intermediate type. Thus, as pointed out previously,55Go care must be taken that PAL genes do not show similar expression patterns. Although the array membrane was washed under high-stringency conditions after hybridization, cross hybridization among the gene family members could not be excluded since the members are more than 80% identical to each other at the nucleotide level in their coding regions. Therefore, we performed real-time RT–PCR experiments with gene-specific primer sets to validate the differential expression of the PAL genes. The results of RT–PCR for all PAL genes were more or less the same as those of array analysis (Table 4). In addition, we found that the LjPAL5 gene, which was not found on the array membrane, was severely down-regulated in AM roots (Table 4).


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  		Table 4. L. japonicus genes for PALs and their expression patterns

 
To further confirm the repression of some PAL genes after AM colonization, we searched for genomic sequences of the PAL genes in databases, finding that LjPAL1, LjPAL4, and LjPAL5 lie in tandem on a single TAC clone, AP004502. We chose the LjPAL1 promoter, which shows typical differential expression, for further analysis. This promoter, 2 kb in size, was amplified by PCR, fused with the uidA reporter for GUS, and then introduced into L. japonicus by the hairy root method with A. rhizogenes. The transformants showed basal activity, especially in central cylinders, in the absence of any microorganisms (Fig. 2A). The GUS activity was augmented throughout the roots in the presence of contaminating microorganisms in the sieved carrier (Fig. 2B). When the transformants were inoculated with the whole G. mosseae inoculum, the area of expression decreased (Fig. 2C). The specific GUS activity levels in the entire hairy roots of the above transformants were 1.2 ± 0.2, 7.1 ± 2.9, and 4.1 ± 1.5 pmol/min/µg protein, respectively. Unexpectedly, when GUS-stained AM roots were re-stained with safranin, a red dye that stains fungal cells better than plant cells, it turned out that G. mosseae only colonized where GUS activity was low (Fig. 2C). To confirm this observation, sections of GUS-stained AM roots were prepared and then re-stained with safranin. As shown in Fig. 2D and E, the root portions exhibiting high LjPAL1 promoter activity did not contain G. mosseae. In contrast, the AM fungus colonized well where the GUS level was low (Fig. 2F and G). In some cases, G. mosseae was observed where GUS activity was also significant, but the level of GUS was not very high either (Fig. 2H and I). As described above, the whole G. mosseae inoculum and the sieved carrier contained equivalent amounts of contaminating microorganisms. Therefore, host plants repress PAL gene expression where AM fungi colonize, preventing infection by pathogenic microorganisms. This repression pattern is similar to that of isoflavone reductase of M. truncatula previously reported,50Go but different from those of PAL and chalcone synthase observed in that study. Comprehensive expression analysis of every family member for the latter enzymes of M. truncatula would be necessary to resolve this discrepancy.


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  		Figure 2. Spatial patterns of LjPAL1 promoter activity in L. japonicus hairy roots. GUS activity is visible as a blue color in intact roots (A–C) or longitudinal sections of AM roots (D–I). A, a non-infected root. (B), 4 weeks after inoculation of the sieved carrier containing contaminating microorganisms. (C) An AM root, 4 weeks after addition of the whole G. mosseae inoculum. The arrow indicates the region heavily colonized by the AM fungus. After regular GUS staining, the root was re-stained with safranin. Sections of GUS-stained roots were prepared (D, F, and H) and then stained on slide glasses with safranin (E, G, and I). The small arrows in G and I indicate arbuscules of the AM fungus. (J and K) LjPAL1 promoter activity in nodules on L. japonicus hairy roots. Two weeks after inoculation of M. loti, GUS activity in the nodulated roots was examined under a stereomicroscope. (J) A nodule primordium. (K) A more mature nodule. Bars, 100 µm.

 
3.5. Commonly repressed genes of L. japonicus in AM roots and nitrogen-fixing nodules
When the results of cDNA array experiments on AM roots with G. mosseae, and ones on G. margarita and mature root nodules with M. loti were compared with each other, the overlapping of induced genes or repressed genes was found to be limited (Supplementary Fig. S2), in accord with previous reports.17Go,18Go However, when the commonly regulated genes in G. mosseae-colonized roots and mature root nodules were listed up, it was obvious that many defense-related and stress-induced genes were included in the commonly repressed list (Table 3). They include genes for WRKY transcription factors, which are up-regulated in response to biotic or abiotic stress,40Go,56Go and those for BURP domain proteins, one of which is a stress-induced transcription factor,57Go besides PAL genes. These results suggest that host plants accept AM fungi and compatible rhizobia in similar manners, their defense mechanisms being suppressed.

Because LjPAL1 is one of the commonly repressed genes in AM roots and nodules (Table 5), we inoculated M. loti into hairy roots transformed with the LjPAL1 promoter-GUS construct. As shown in Fig. 2J and K, strong GUS activity was detected at the top of a nodule primordium, but it had soon disappeared in a slightly more mature nodule, in accordance with the results of the array experiments (Table 5).


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  		Table 5. Co-regulated genes of L. japonicus in AM roots and nitrogen-fixing nodules

 
3.6. Concluding remarks
We performed comprehensive transcriptome analysis and spatial examination of gene expression in AM roots and root nodules of L. japonicus, taking into account the effects of contaminating microorganisms. We found that several cysteine protease genes were specifically induced in arbuscule-containing cells of AM roots. Moreover, we also found that PAL and other phenylpropanoid biosynthesis-related genes were moderately induced on the initial infection of the symbionts and then repressed concomitant with the establishment of the two symbioses. Characteristic expression patterns were observed both in the absence of contaminating microorganisms (Table 4, experiments with G. margarita; Fig. 2J and K) and more drastically in their presence (Table 4, experiments with G. mosseae; Fig. 2A–I). So far, it has been suggested that defense genes for AM fungi or rhizobia are initially up-regulated and then down-regulated.16Go,26Go,47Go–52Go,58Go Nevertheless, the current study is unexpectedly the first demonstration that this prediction is correct especially for AM root formation with G. mosseae and G. margarita using a large scale cDNA array. Then, why did previous works on AM roots not reveal the unique expression patterns of PAL and other phenylpropanoid biosynthesis-related genes? When the expression levels of these genes in roots with commercial inoculants of AM fungi applied were examined,12Go–15Go,17Go–19Go it is possible that their induction by contaminating microorganisms and their repression by AM fungus colonization were super-imposed, resulting in comparable levels to those in sterile non-infected roots. Actually, when we did a similar experiment,12Go–15Go,17Go–19Go we did not detect the differential expression of most PAL genes except LjPAL10, which was moderately up-regulated (Supplementary Table S1). Other previous works in which aseptic spores of AM fungi were inoculated did not show significant down-regulation of these phenylpropanoid biosynthesis-related genes, either.16Go,20Go,41Go On the other hand, our experiments involving NaClO-treated G. margarita spores revealed repression of the genes. It is difficult at present to fully explain this discrepancy. As revealed in this work, however, the varying microbial population around AM roots significantly affects gene expression and hence the reproducibility of the experiments. If our surface-sterilization of the spores was not complete, for example, the differential expression of plant genes on G. margarita colonization might be similar to that on application of a commercial G. mosseae inoculant.

The presence of contaminating microorganisms is, in a sense, closer to natural field conditions than the inoculation of aceptical spores of AM fungi into sterile plants. The spatial investigation in this study revealed that a PAL gene, LjPAL1, is repressed where AM fungi colonized. Although PALs are multi-functional enzymes, we consider that the defense response including de novo synthesis of flavonoid phytoalexins against other microorganisms than AM fungi is suppressed. In nature, host plants may accept microsymbionts by suppressing their defense reactions to a minimum level at which they may still prevent infection by pathogens.


   Supplementary Data
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results and discussion
 Supplementary Data
Acknowledgements
 References
 
Supplementary data are available at www.dnaresearch.oxfordjournals.org


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results and discussion
 Supplementary Data
 Acknowledgements
References
 
We wish to thank Drs. K. Akiyama, Osaka Prefecture University, and Y. Tamada, Kyoto University, for the valuable discussions. We also thank Drs. C. L. Diaz-Argueta (Leiden University), K. Nagashima (Idemitsu Kosan Co.), and K. Nakamori (Kyoto University) for providing the protocol for hairy root transformation with A. rhizogenes LBA1334, the kind gift of the G. mosseae inoculum, and the instructions for embedding AM roots in plastic resin, respectively. This work was supported in part by an AIST Research Grant and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government. The array experiments were carried out at the Radioisotope Research Center, Kyoto University.


   Footnotes
 
* To whom correspondence should be addressed. Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Tel. +81 75-753-6141. Fax. +81 75-753-6470. E-mail: shing{at}kais.kyoto-u.ac.jp

Edited by Satoshi Tabata

{dagger} Current address: Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. Back

{ddagger} Current address: National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. Back

Current address: Kazusa DNA Research Institute, Chiba 292-0812, Japan. Back

§ Current address: Faculty of Agriculture, Saga University, Saga 840-8502, Japan. Back

|| Current address: Department of Biotechnological Science, Kinki University, Wakayama 649-6493, Japan. Back


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 Abstract
 1. Introduction
 2. Materials and methods
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 Acknowledgements
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