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Originally published In Press as doi:10.1074/jbc.M500447200 on May 17, 2005 Originally published In Press as doi:10.1074/jbc.M500447200 on February 14, 2005

J. Biol. Chem., Vol. 280, Issue 28, 26241-26247, July 15, 2005
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The Endophytic Fungus Piriformospora indica Stimulates the Expression of Nitrate Reductase and the Starch-degrading Enzyme Glucan-water Dikinase in Tobacco and Arabidopsis Roots through a Homeodomain Transcription Factor That Binds to a Conserved Motif in Their Promoters*

Irena Sherameti{ddagger}, Bationa Shahollari{ddagger}, Yvonne Venus{ddagger}, Lothar Altschmied§, Ajit Varma¶**, and Ralf Oelmüller{ddagger}||

From the {ddagger}Institute of General Botany and Plant Physiology, Friedrich Schiller University Jena, 07743 Jena, Germany, §Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben, Germany, and School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India

Received for publication, January 13, 2005 , and in revised form, February 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Piriformospora indica, an endophytic fungus of the Sebacinaceae family, promotes growth of Arabidopsis and tobacco seedlings and stimulates nitrogen accumulation and the expression of the genes for nitrate reductase and the starch-degrading enzyme glucan-water dikinase (SEX1) in roots. Neither growth promotion nor stimulation of the two enzymes requires heterotrimeric G proteins. P. indica also stimulates the expression of the uidA gene under the control of the Arabidopsis nitrate reductase (Nia2) promoter in transgenic tobacco seedlings. At least two regions (–470/–439 and –103/–89) are important for Nia2 promoter activity in tobacco roots. One of the regions contains an element, ATGATAGATAAT, that binds to a homeodomain transcription factor in vitro. The message for this transcription factor is up-regulated by P. indica. The transcription factor also binds to a CTGATAGATCT segment in the SEX1 promoter in vitro. We propose that the growth-promoting effect initiated by P. indica is accompanied by a co-regulated stimulation of enzymes involved in nitrate and starch metabolisms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Often nitrogen is the limiting source for plant growth and development. It is recruited by plants either as nitrate or ammonium or for a few species by nitrogen fixation with the help of rhizobia (1, 2). Mycorrhizal fungi also play an important role in delivering either nitrate or ammonium to the root cells. It is believed that mycorrhizal fungi preferentially recruit ammonium rather than nitrate from the soil and that amino acids represent the major compounds that serve to transfer nitrogen to the host plant (cf. Refs. 3 and 4). We studied Piriformospora indica, an endophytic fungus of the Sebacinaceae family, which colonizes the roots of a wide variety of plant species and promotes their growth (5–10). The interaction of the endophytic fungus with plant roots is accompanied by an enormous requisition of nitrogen from the environment. By analyzing the interaction of P. indica with Arabidopsis and tobacco roots we found that in contrast to mycorrhizal associations, nitrate reduction in the roots is stimulated by P. indica. A homeodomain transcription factor responds to the fungus and binds to promoter regions of the P. indica-responsive Nia2, SEX1, and 2-nitropropane dioxygenase genes. These results suggest that the expression of P. indica-responsive target genes may be controlled by common regulatory elements and trans-factors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic Tobacco—Transgenic seeds of Nicotiana tabacum L., var. Samsun NN were obtained from greenhouse-grown plants (6). They were sterilized and germinated on Murashige-Skoog medium (11) supplemented with 2% (w/v) sucrose and 0.8% (w/v) agar in temperature-controlled (25 °C) growth chambers under a 16-h light/8-h dark cycle. 80 µg of ml–1 (w/v) kanamycin was added to the medium. Four-week-old plantlets were transferred to soil to obtain seeds for the physiological experiments. The antisense lines for the heterotrimeric G protein subunit {beta} were described previously (12).

Growth Conditions of Plant and Fungus—For physiological experiments in Petri dishes, transgenic or wild-type tobacco or Arabidopsis seeds were surface-sterilized and placed on Petri dishes containing Murashige and Skoog (11) nutrient medium. After cold treatment at 4 °C for 48 h, plates were incubated for 10 days (Arabidopsis thaliana) or 14 days (Nicotiana tabacum) at 22 °C under continuous illumination (100 µmol m–2 sec–1 photosynthetic active radiation). P. indica, a cultivable plant growth-promoting root endophyte (10), and Pisolithus tinctorius were cultured as described previously (6, 13).

Co-cultivation Experiments, Determination of Fresh and Dry Weight, Protein Content, and Nitrate Uptake—14-day-old tobacco (or 10-day-old Arabidopsis) seedlings were transferred to nylon disks (mesh size 70 µm) and placed on top of a modified MMN1 culture medium (MMN1/10 medium with a 1/10 ratio of nitrogen and phosphorus and no carbohydrate) (14) in 90-mm Petri dishes. After 24 h, fungal plugs of 5 mm in diameter were placed at a distance of 1 cm from the roots. Plates were incubated at 22 °C under continuous illumination from the side (maximum 80 µmol m–2 s–1 photosynthetic active radiation).

Root length was measured with a ruler. The fresh weight of the roots and aerial parts was determined directly. Proteins were extracted into a 5-ml extraction buffer (50 mM Tris-HCl, pH 8.0, 0.1% SDS), precipitated with trichloroacetic acid, and the protein concentration was determined according to Lowry. Dry weight of the roots and aerial parts was determined after incubation of the tissue at 105 °C for 16 h.

For pot experiments, 9-day-old sterile tobacco seedlings were transferred to sterile soil in pots (25-cm diameter). Aliquots of the soil were mixed with 1% (w/v) fungus and put into preformed holes surrounding the roots of the seedling. Growth was followed in temperature-controlled (25 °C) growth chambers under a 16-h light/8-h dark cycle.

Analysis of the Nia2 Promoter—Some transgenic tobacco lines harboring Nia2-promoter::uidA gene fusions were described previously (15). For the analyses performed here, the Nia2 5'-upstream region –1088/–1 (relative to the ATG start codon) of At1g37130 was fused to the uidA reporter gene. Manipulations of the –1088/–1 fragment occurred in pBSC+ (Stratagene, San Diego, CA).

Starting from the –1088/–1 Nia2 fragment in pBSC+, 5'-deletions were obtained with exonuclease III digestions. After religation, the clones were sequenced. Further analyses were performed with the following fragments: –1088/–1, –650/–1, –628/–1, –470/–1, –438/–1, –361/–1, –310/–1, –111/–1, and –89/–1. Although seeds from all plants were analyzed in parallel, only those plants that gave important information for this study are mentioned under "Results."

Site-directed mutagenesis was performed according to Mikaelian and Sergeant (16) and specified in Ref. 17. The 5'-end of the promoter segment, obtained after PCR with genomic DNA from A. thaliana, ecotype Landsberg, was located toward the 3'-end of the vector. The mutagenized DNA was obtained by three successive PCRs. An oligonucleotide with the mutagenized region and 12 authentic nucleotides on each side and the T7 primer were used for the first reaction. The second reaction was performed with the T3 primer and an oligonucleotide (5'-AAAAAACCGCTCTAGAACTAGTG-3'), which primes 20 bp 3' of the T7 primer. This oligonucleotide contains a 5'-mismatched end (cf. Ref. 16). The amplified fragments were purified on agarose gels, and 10 ng of each of them was used for the third PCR reaction with the T3 and T7 primers. The final products were ligated into pBSC+ and sequenced before transfer to pBI101 (18) as BamHI/SalI fragments. After triparental mating (19) and plant transformation, 20 independent lines per construct were generated. For physiological experiments with the seeds of the F1 generation, detailed analyses were performed only for those constructs relevant for this study. The transcription start site of the Nia2 promoter was determined with Arabidopsis root RNA and primer extension analysis.

For GUS staining, seedlings were harvested and immediately put into 5-bromo-4-chloro-3-indolyl {beta}-D-glucopyranoside (X-Glc) solution (50 mg of X-Glc, 1 ml of dimethylformamide, 4.9 ml of 50 mM sodium phosphate, pH 7.0, 250 µl of Me2SO, 500 µl of potassium hexacyanoferrate (III) (100 mM), 500 µl of potassium hexacyanoferrate (II) (100 mM)) and incubated overnight at 37 °C. After washing with water, the seedlings were incubated in 70% ethanol and stored at 4 °C. For GUS staining of root hairs, seeds were germinated and seedlings were grown in liquid Murashige and Skoog medium to avoid hair damage.

RNA Preparation and Quantitative RT-PCR—Total RNA from root material was isolated with the TRIzol reagent (Invitrogen). RT-PCR analysis was performed by reverse transcription of 5 µg of total RNA with gene-specific reverse primers (see below). First strand synthesis was performed with a kit (K1631) from MBI Fermentas (St. Leon-Roth, Germany). After 20 PCR cycles, the products were analyzed on 1.5% agarose gels and stained with ethidium bromide; visualized bands were quantified with the Image Master Video System (Amersham Biosciences). For Northern analysis, gene-specific primers were designed to amplify four DNA fragments from our cDNA library (20): Nia2 (At1g37130), SEX1 (At1g10760), and the genes for the homeodomain transcription factor (At2g35940) and for 2-nitropropane dioxygenase (At5g64250). The primers were designed such that they amplified the entire coding region including 4 nucleotides up- and downstream of the genes.

Gel Mobility Shift Assays—Gel mobility assays were performed with a fraction enriched in root nuclei proteins from A. thaliana. Approximately 10 g of Arabidopsis roots was used to isolate a fraction enriched in nuclei (21). The extracted proteins were further purified on heparin-Sepharose (Amersham Biosciences) columns. After elution with 700 mM KCl, the protein fraction was dialyzed against NEB buffer (25 mM HEPES, pH 7.8, 50 mM KCl, 0.1 mM EDTA, 14 mM {beta}-mercaptoethanol) and concentrated using spin columns (Amicon, Witten, Germany). These protein factions were used for gel mobility shift assays and the filter binding assay.

Five pairs of oligonucleotides (Nia2-a, ATGATAGATAAT, Nia2-b, ATTATCTATCAT; Nia2mu-a, ATGATGCATAAT; Nia2mu-b, ATTATGCATCAT; SEX1-a, CTGATAGATCT; SEX1-b, AGATCTATCAG; SEX1mu-a, CTGATGCATCT; SEX1mu-b, AGATGCATCAG; NpdO-a, AGGATCGATGA; NpdO-b, TCATCGATCCT) were annealed and cloned into the SmaI site of pBSC+. After restriction of the recombinant plasmid DNA with EcoRI and XbaI, the recessive ends were filled in with Klenow enzyme and radiolabeled nucleotides, and the insert was isolated by polyacrylamide gel electrophoresis. For the filter binding assay, the fragments were excised from the plasmid with XbaI and EcoRI and purified on polyacrylamide gels (5%).

Enzyme Assays—The nitrate reductase (NR) and GUS assays were described earlier (15, 17). In both instances the system of reference was an equal amount of fresh weight.

Mass Spectrometry—Proteins extracted from membrane fractions were further purified by two rounds of methanol precipitation before digestion with trypsin (6). Alternatively, silver-stained gel spots from the gels were excised and the proteins extracted into 500 µl of 50 mM ammonium bicarbonate supplemented with 60 ng/µl trypsin. After lyophilization, the pellet was resuspended in 5 µl of water/acetonitrile/formic acid (95:5:0.1) prior to liquid chromatography-MS analysis. Peptide analyses, analyte sampling, chromatography, and acquisition of data were performed on a LC (Famos-Ultimate; LC-Packings) coupled with an LCQ Deca XP ion trap mass spectrometer according to the manufacturer's instructions.

The measured MS-MS spectra were matched with the amino acid sequences of tryptic peptides from the A. thaliana data base in FASTA format. Cys modification by carbamidomethylation (+57 Da) was taken into account, and known contaminants were filtered out. Raw MS-MS data were analyzed by the Finnigan Sequest/Turbo Sequest software (revision 3.0; ThermoQuest, San Jose, CA). The parameters for the analysis by the Sequest algorithm were set according to Stauber et al. (22). The similarity between the measured MS-MS spectrum and the theoretical MS-MS spectrum, reported as the cross-correlation factor (xcorr) was above 2.95 and 3.85 for doubly or triply charged precursor ions, respectively. To identify corresponding loci, identified protein sequences were subjected to BLAST search at NCBI (www.ncbi.nlm.nih.gov/) and FASTA searches by using the AGI protein data base at The Arabidopsis Information Resource (www.arabidopsis.org/).

Macroarray Analyses—RNA was isolated from Arabidopsis roots 2 and 5 days after co-cultivation with P. indica. The macroarray filters used for the hybridization and the hybridization conditions have been described previously (23). Genes identified in these studies and those that responded to P. indica were further analyzed in separate RT-PCR studies. Only those genes that are relevant for this study are mentioned here.

Miscellaneous—DNA extraction was performed according to standard protocols (24). Western analyses with polyclonal antibodies raised against nitrate reductase (gift from Dr. K.-J. Appenroth) and the {beta}-subunit of heterotrimeric G proteins from tobacco (25) were performed according to Stöckel and Oelmüller (26). Denaturing polyacrylamide gel electrophoresis was performed with the buffer system from Laemmli (27). Determination of total nitrogen was performed with Kjeldahl equipment and protocols provided by "behr Labor-Technik" (Düsseldorf, Germany). Roots of control and inoculated plants were stained with cotton (blue) before examination under the light microscope (Zeiss Axioplan model MC 100).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Co-cultivation of Tobacco Seedlings with P. indica—14-Day-old tobacco seedlings were transferred to MMN1/10 medium and inoculated with P. indica. The fungal inoculum was placed 1 cm away from the roots. MMN1/10 medium was chosen because it contains low concentrations of phosphate and nitrate and no carbon source, conditions known to promote the interaction between plants and symbiotic fungi. The fungus grew slowly on the co-cultivation medium and produced only a few spores. A difference in root growth was not observed within the first 2 days of co-cultivation. After 5 days, a stimulatory effect of P. indica on root growth became visible. After 7 days, the inoculated seedlings were significantly larger and heavier when compared with control seedlings, and after 10 days the size of the tobacco seedlings was substantially larger (cf. Fig. 1A, a and b, cf. also B–D). We also inoculated plants with the ectomycobiont P. tinctorius because it was shown that hypaphorine, a major indolic compound from this fungus, has an impact on Arabidopsis root growth (28). However, seedlings inoculated with P. tinctorius did not differ from the uninoculated controls (Fig. 1A, c–f). The stimulatory effect of P. indica on the growth of tobacco was still detectable 6 weeks after transfer of the seedlings to soil, and inoculated plants were much bigger compared with their controls (Fig. 1E). This is not surprising considering the extensive colonization of the outer cell layers of the roots by the fungal hyphae (Fig. 1, F and G). After the initial lateral growth (Fig. 1F), the fungus produced spores, which could be detected in almost all outer root cells (Fig. 1G). It can be concluded that under the given conditions, growth of tobacco seedlings is substantially stimulated by P. indica, similar to previous observations with A. thaliana seedlings2 (6, 30, 31).



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  		FIG. 1.
Tobacco plants grown in the presence or absence of P. indica or P. tinctorius. A, 24-day-old tobacco seedlings grown either in the absence (a and f) or presence of P. indica (b) or P. tinctorius (c–e). Bars represent 1 cm. B–D, 24-day-old tobacco seedlings overexpressing an antisense block for the {beta}-subunit of heterotrimeric GTP-binding proteins. B, line 14-4; C, line 15-4; D, line 15-6, representing independent primary transformants. Top, without P. indica; bottom, with P. indica. E, tobacco plants 6 weeks after transfer of the seedlings to pots. Middle, without P. indica; left and right, with P. indica. Bar represents 10 cm. F, the outer cell layers of inoculated roots under the light microscope (Zeiss Axioplan model MC 100). Arrowhead, fast dividing fungal cells, which are in focus. Bar represents 100 µm. G, root epidermal cells stained with cotton (blue). Bar represents 100 µm.

 
P. indica Stimulates Nitrate Uptake and Nitrate Reductase Gene Expression—After 10 days of co-cultivation with P. indica the fresh weight of the aerial parts of the tobacco seedlings was enhanced by 42.2 ± 3.8%, the dry weight by 41.0 ± 4.0%, and the total protein content by 42.2 ± 3.1% (n = 480 seedlings). This was accompanied by substantial recruitment of nitrogen from the agar medium. Based on equal amounts of fresh weight, the nitrogen content in the aerial parts of the seedlings increased by 21.4 ± 4.4%. Thus, considering the larger size of the cotyledons, ~60% more nitrogen must have been transferred from the medium to the aerial parts of the seedlings. This prompted us to investigate the effect of P. indica on nitrate assimilation in more detail. We focused on seedlings grown on agar because quantitative analyses were much easier when compared with adult plants grown on soil.

Co-cultivation of tobacco seedlings with P. indica causes a 50.2 ± 4.2% increase in the plant-specific NADH-dependent NR activity in the roots. Western analysis with antibodies against plant NR confirmed that the higher NADH-dependent enzyme activity correlated with an increase in the amount of the root enzyme (Fig. 2). Equal loading of root protein extracts was confirmed with an antibody against the {beta}-subunit of heterotrimeric GTP-binding proteins (25). Thus, in contrast to mycorrhizal symbioses, the endophytic fungus P. indica stimulates the assimilatory enzyme NR. The stimulatory effect of P. indica on NR is predominantly found in roots and to a much lesser extent in the shoots (12.2 ± 1.2%). Thus, the higher nitrogen level in the aerial parts of the seedlings after cocultivation with P. indica must be caused by more efficient nitrate assimilation in the roots. In principle, the same results were obtained for Arabidopsis seedlings except that the overall stimulation of NR activity in the roots was only 29.8 ± 3.1%.



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  		FIG. 2.
Western analyses with antibodies against nitrate reductase (left) and the G{beta} (right, control). Protein extracts from 24-day-old tobacco roots were either grown in the absence (–) or presence (+) of P. indica. 25 µg of protein was loaded per lane.

 
The Arabidopsis Nia2 5'-upstream region –1088/–1 (relative to the ATG codon, the transcription start site is located 88 nucleotides upstream of the ATG codon) fused to the {beta}-glucuronidase gene (uidA) was introduced into Nicotiana. When co-cultivated with P. indica, a stimulatory effect can also be monitored at the level of transgene expression (Table I). Comparable with the results obtained for NR activity, P. indica stimulated uidA gene expression in tobacco roots by ~50%. Again, a much lower stimulatory effect was observed in shoots (~10–15%).


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  		TABLE I
GUS activity in 24-day-old transgenic tobacco seedlings, grown in either the presence (+) or absence (-) of P. indica

The uidA gene was expressed under the control of the Arabidopsis Nia2 promoter fragment –1088/–1, –470/–1, –438/–1 or Mu (–470/–1, in which AG at position –454/–453 was replaced by GC). Experimental details, cultivation of the seedlings, and the enzyme assays are described under "Materials and Methods." The enzyme activity was determined separately for shoots and roots. For each value, 490 individual plants were analyzed. The system of reference is fresh weight. The protein content in the shoots and roots increased linearly with the fresh weight. Errors are given as S.E.s.

 
Deletions of the promoter from –1088 to –470 had no significant effect on uidA expression, whereas further deletion to –438 caused an ~60% decrease in uidA expression in roots but not in shoots. Thus, the region –470/–439 appears to be important for the promoter activity in roots.

Replacement of AG by GC in the ATGATAGATAAT sequence (–459/–448) within this region causes the same decrease in GUS activity as deletion from –470/–1 to –438/–1 (Table I). Furthermore, although the –111/–1 segment is active in roots and shoots, deletion to –89 completely abolishes the promoter activity. Thus, an additional crucial cis-element(s) for Nia2 expression appears to be located in the region between –111 and the transcription start site at position –88. Random site-directed mutagenesis in the latter region in the context of the –1088/–1 fragment followed by expression analysis in transgenic tobacco revealed that the two GT nucleotides directly upstream of the transcription start site and the CA nucleotides at position –97/–96 play a crucial role in the promoter activity in vivo because replacement of GT by AA and of CA by GC completely abolished gene expression. The close vicinity of the CA and GT motifs to the transcription start site makes it unlikely that this region functions as a TATA box element. Taken together, the –470/–439 and –97/–89 regions upstream of the ATG codon are essential for Nia2 promoter activity in transgenic tobacco.

GUS staining revealed that the promoter was active in almost all of the living cells of the roots (Fig. 3). High GUS activity is found in the living cells of the vascular tissue. In the larger cells surrounding the vascular tissue, the stain is mainly detectable in the narrow cytoplasmic tubes attached to the plasma membrane. Most of the GUS staining was observed in the cytoplasm of the root hair (Fig. 3, upper panel, cf. also Ref. 15). Semiquantitative analysis revealed that the stimulatory effect of P. indica on uidA gene expression in the root hairs is at least 2-fold, i.e. significantly higher than the effect observed for the entire root (data not shown). When grown in the presence of P. indica, a significant stimulation of uidA expression in the roots was detected for all active fragments tested. However, the extent of the stimulatory effect in the roots declines dramatically when the promoter is deleted from –470 to –438 (Table I). Thus, in addition to its specific role for Nia2 promoter activity in roots, the –470/–439 region also functions as a P. indica-responsive element in the Nia2 promoter. A stimulatory effect in response to P. indica was still measurable for the –110/–1 promoter fragment (24 ± 2%), although to a lesser extent. Thus sequences within this segment in combination with or in addition to the P. indica-responsive element are involved in P. indica-mediated Nia2 expression. A fusion of the –470/–439-bp region to the –90-bp cauliflower mosaic virus minimal promoter did not respond to P. indica (data not shown).

A double-stranded nucleotide from the –459/–448 segment ATGATAGATAAT shows a retarded band in gel mobility shift assays with nuclear extracts from Arabidopsis roots (Fig. 4). No retardation is detectable with the double-stranded mutant oligonucleotide ATGATGCATAAT. Furthermore, the binding can be competed with an excess of the original but not with mutant oligonucleotide (Fig. 4). These and other results (cf. below) indicate that root nuclei from Arabidopsis contain a protein(s), which binds to the P. indica-responsive element in the Nia2 promoter.

The Nia2 Promoter Binds a Homeodomain Transcription Factor in Vitro, and the Message for the Transcription Factor Is Up-regulated in Response to P. indica—The Nia2 (and the mutant) double-stranded promoter segments were immobilized on nylon membranes. The membranes were then incubated with nuclear extract from Arabidopsis roots for ~60 h at 6 °C under continuous shaking. The filters were washed five times with 90 mM NaCl (10 min each, first at 6 °C, then at 18 °C). Finally, DNA-bound proteins were solubilized by boiling the membranes in loading buffer, and the extracted proteins were analyzed on SDS-polyacrylamide gels. Two dominant protein bands of ~75 and 160 kDa, which are not detectable in protein extracts from the control oligonucleotide (Fig. 5), can be eluted from the Nia2 oligonucleotide. The bands were extracted from the gel and analyzed by mass spectrometry after trypsin digestion. The lower band corresponds to the Arabidopsis fragments SLGEEDSVSGVGR and TSDETMMQPINADFSSNEK. Searches of the literature show that the upper band correspond to GSCGNDK and PVELGTAER. The four peptides are present in the homeodomain protein At2g35940. In the upper band, we could also identify peptides that correspond to other homeodomain transcription factors; however, the xcorr values were too low to allow conclusions. It appears that the upper band contains components of a larger, unresolved complex together with At2g35940 (cf. "Discussion").



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  		FIG. 3.
Upper panel, cross-section through a root from a 24-day-old tobacco seedling stained for GUS. Lower panel, a growing root hair; GUS staining is visible in the cytoplasm. The seedling expresses the uidA gene under the control of the –1088/–1 bp Nia2 promoter fragment from A. thaliana.

 



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  		FIG. 4.
Gel mobility shift assays with the Nia2 promoter fragment ATGATAGATAAT, the NpdO promoter fragment AGGATCGATGA, and the SEX1 fragment CTGATAGATCT with nuclear extract from Arabidopsis roots. The figure also shows cross-competition experiments between the binding activities to these fragments. –, lanes without nuclear extract. Lane 1, retardation assay with ATGATAGATAAT (Nia2); lanes 2–5, competition with increasing concentrations of cold fragment (Nia2); lanes 6–9, competition with increasing concentrations of cold ATGATGCATAAT fragment (Nia2). Lanes a and d, retardation assay with AGGATCGATGA (NpdO); lane b, competition with excess Nia2 fragment ATGATAGATAAT (NpdO); lane c, competition with excess SEX1 fragment CTGATAGATCT (NpdO). Lane 1, retardation assay with CTGATAGATCT (SEX1); lanes 2–5, competition with increasing concentrations of the Nia2 fragment ATGATAGATAAT (SEX1). For experimental details, cf. "Materials and Methods."

 



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  		FIG. 5.
Silver-stained gel with proteins extracted from filter-bound oligonucleotides. Wild-type Nia2, ATGATAGATAAT; wild-type SEX1, CTGATAGATCT; mutant Nia2, ATGATGCATAAT; mutant SEX1, CTGATGCATCT. Left, sizes in kDa.

 
Northern analyses uncovered that the message for At2g35940 is up-regulated in roots co-cultivated with P. indica (Fig. 6). Stimulation of the homeodomain protein mRNA level was observed before the Nia2 mRNA level increased.

The Expression-relevant Upstream Region ATGATAGATAAT of the Nia2 Promoter Exhibits Sequence Similarities to a SEX1 Promoter Region for the Starch-degrading Enzyme Glucan-water Dikinase and to a Region in the 2-Nitropropane Dioxygenase (At5g64250) Promoter, and the Messages of All Three Genes Are Up-regulated in Response to P. indica—Computer analyses uncovered that sequences with similarities to the expression-relevant ATGATAGATAAT element are also present in the SEX1 promoter (–1182, CTGATAGATCT, –1172) and the promoter of the 2-nitropropane dioxygenase (At5g64250) (–238, AGGATCGATGA, –228). A gel shift assay with double-stranded oligonucleotide sequences from these two promoter regions confirmed that they also bind to protein factors from root nuclei extracts (Fig. 4). Because both binding activities competed with the Nia2 promoter sequence, it is likely that they bind to the same or similar DNA-binding proteins (Fig. 4). Furthermore, filter binding assays with the SEX1 promoter segment led to the identification of the same two protein bands of 75 and 160 kDa, which also bind to the Nia2 promoter sequence, although the amounts of the two bands relative to each other differed for the two fragments (Fig. 5). Mass spectrometry identified a peptide that corresponds to PVELGTAER in the lower band and to LSNMLHEVEQR in the upper band; both are present in At2g35940. No reproducible data with this assay could be obtained for the At5g64250 promoter segment, presumably because the binding activity to this fragment was too low (data not shown). Macroarray and Northern analyses confirmed that the SEX1 and At5g64250 messages also respond to P. indica. Although the SEX1 and Nia2 messages accumulate with similar kinetics (Fig. 6), the At5g64250 message begins to accumulate earlier, although later than the message for the homeodomain transcription factor (Fig. 6). The enzyme glucan-water dikinase catalyzes the phosphorylation of starch by a dikinase-type reaction in which the {beta}-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glycosyl residue of amylopectin. As a consequence, transitory starch is more rapidly degraded (cf. Refs. 32–34). Up-regulation of the message for this enzyme in response to P. indica might have two functions. Starch breakdown products released from the root amyloplasts might be required for growth that is promoted by P. indica or for the export to the fungus, which is dependent on sucrose supply from the plant. It appears that nitrate assimilation and starch degradation is co-regulated in Arabidopsis roots via the same cis-elements in their promoters. 2-Nitropropane dioxygenase might have different functions in the interaction. The enzyme incorporates molecular oxygen into 2-nitropropane and releases acetone and nitrite. Thus, the enzyme might detoxify 2-nitropropane, which is generated under stress or as a defensive toxin (35), and generates nitrite, which might serve as an additional nitrogen source. Plants also detoxify 2-nitropropane from the environment because it is used commercially as a solvent, although it is a known mutagen (36, 37).



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  		FIG. 6.
Northern analyses for the homeodomain transcription factor (Homeo), Nia2, SEX1, and 2-nitropropane dioxygenase (NpdO) genes. RNA was extracted from Arabidopsis roots 0, 3, 6, and 10 days after co-cultivation with P. indica. An equal amount of RNA (25 µg) was loaded per lane.

 
Growth Promotion and Stimulation of Nitrate Assimilation by P. indica Does Not Require Heterotrimeric G Proteins—We tested whether P. indica can stimulate growth and NR in tobacco lines expressing an antisense construct for the {beta}-subunit of heterotrimeric GTP-binding proteins (G{beta}). Previously, we have characterized three tobacco lines in which G{beta} was severely reduced in green leaves (12). Fig. 7 demonstrates that a comparable reduction of G{beta} can also be observed in root. Severely reduced levels of G{beta} do not inhibit the stimulatory effect of the fungus on growth (Fig. 1, B–D) and NR activity (data not shown). Thus, bulk G{beta} is not required for P. indica action.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Nitrate Assimilation Is Stimulated by P. indica—We demonstrate that co-cultivation of tobacco and Arabidopsis seedlings with P. indica is accompanied by a massive transfer of nitrogen from the agar plates into the aerial part of the seedlings, an observation that is not surprising considering the growth promotion caused by the fungus. This effect is associated with a stimulation of the NADH-dependent NR, the key enzyme of nitrate assimilation in plants. Whether the stimulation of nitrate assimilation by P. indica is the reason for the growth promotion or the result of it remains to be determined. A stimulatory effect of mycorrhizal associations has also been reported for nitrate uptake into tomato root cells (38). However, recruitment of nitrogen in endophytic interactions differs from mycorrhizal interactions in which the fungus preferentially recruits ammonium rather than nitrate from the soil (cf. Refs. 3 and 4). Moreover, several studies have demonstrated that after the establishment of ectomycorrhizal symbioses, the fungal NR is increased and the plant enzyme down-regulated (4, 38, 39). Apparently in mycorrhizal symbioses, amino acids represent the major compounds that serve to transfer nitrogen to the host plant (38). We did not study NR in the fungal hyphae, and thus we cannot exclude that the fungal NR also contributes to nitrate assimilation. However, it appears that the fungal NR alone cannot account for the entire nitrate assimilation.



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  		FIG. 7.
Western blot for G{beta} in root protein extracts from three antisense plants. Lane 1, wild-type extract; lanes 2–4, extract from three antisense lines (14-4, 15-4, and 15-6, representing independent primary transformants). The protein extracts were prepared from the roots of the seedlings shown in Fig. 1, B–D. Bottom, stained gel to show equal protein loading. 25 µg of protein was loaded per lane.

 
A Homeodomain Transcription Factor Appears to Be Involved in Nitrate Assimilation—P. indica activates uptake of nitrate and induces a signaling pathway, which ultimately leads to a higher transcription of Nia2, SEX1, and the gene for 2-nitropropane dioxygenase. The ATGATAGATAAT sequence in the Nia2 promoter was shown to be crucial for the regulation of the expression, and a homeodomain transcription factor binds to this sequence in vitro. Binding activity was also observed to a related motif in the SEX1 promoter. The 2-nitropropane dioxygenase promoter also contains a conserved motif; however, we could not demonstrate conclusively that this element also binds to the same transcription factor. The homeodomain factor was identified by mass spectrometry. Most strikingly, the message for the transcription factor itself was up-regulated by the fungus in Arabidopsis roots. This was first discovered by macroarray analyses with 4000 randomly chosen cDNAs from Arabidopsis; the homeodomain transcription factor attracted attention because its message was among the few transcription factor messages, which was up-regulated by the fungus in Arabidopsis roots.3 Later, this result was confirmed by RT-PCR and Northern analyses. Because homeodomain proteins are involved in the transcriptional regulation of key eukaryotic developmental processes, we propose that this factor might play a crucial role in coupling the expression of P. indica-responsive genes to upstream signaling events. We also propose that the genes identified in this study (for enzymes involved in nitrate assimilation, starch degradation, and detoxification of nitroalkanes) are not the only ones that are regulated by this transcription factor. Homeodomain proteins bind to DNA as monomers or as homo- and/or heterodimers in a sequence-specific manner, and thus their target genes depend on their interaction partners. The upper band on the SDS gel, which contains the homeodomain transcription factor, might therefore represent an unresolved protein-DNA complex, which contains another homeodomain partner(s). The best studied homologs of the protein identified in this study are the BEL1-like transcription factors from potato (40). Seven members of the BEL1 protein family interact with KNOX transcription factors, and they control vegetative development and tuber formation. A rice homolog of the homeodomain protein is inducible by benzothiadiazole, a component that induces disease resistance in Arabidopsis and wheat by activation of the systemic acquired resistance signal transduction pathway (41, 42). Benzothiadiazole also activates resistance in sunflower to the root-parasitic weed Orobanche cumana (43). This suggests that the identified homeodomain transcription factor might also be involved in signaling pathways related to plant/microbe interaction.

Hoth et al. (44) have identified At2g35940 as one of the genes that are up-regulated in response to abscisic acid. Whether abscisic acid- and P. indica-signaling in roots is related to each other is unknown at present.

Heterotrimeric GTP-binding Proteins Are Not Involved in P. indica-induced Growth Promotion and Nitrate Assimilation—Heterotrimeric GTP-binding proteins are tested here because they are involved in many signaling events including those for plant/microbe interactions. However, antisense tobacco lines with severely reduced G{beta} protein levels exhibit a normal growth response to P. indica and contain elevated levels of NR. This indicates that bulk levels of heterotrimeric G proteins are not involved in P. indica-induced signaling events. A similar result has been described for the defense of barley against the powdery mildew fungus (45).

A Comparative Analysis of Promoter Regions Led to the Identification of New Genes with a Similar Response Pattern—We identified four genes that are up-regulated in response to P. indica, i.e. the gene for the homeodomain transcription factor itself as well as three genes that share a conserved sequence element. For two of these elements (the ATGATAGATAAT motif in the Nia2 promoter and the CTGATAGATCT motif in the SEX1 promoter) we could show binding to the homeodomain transcription factor in vitro. Comparison of these two sequences with the AGGATCGATGA element in the promoter of the 2-nitropropane dioxygenase suggests that the central GAT(A/C) GAT(C/T) sequence might be crucial for binding. TAGA is also part of the binding site of the homeodomain protein POU3F1 (29). The three enzymes have in common that they are involved in key metabolic processes in roots (nitrate assimilation, starch degradation, and detoxification of nitro compounds). It remains to be determined whether other genes contain similar DNA binding sites and thus might also be regulated by the homeodomain transcription factor.


   FOOTNOTES
 
* This work was supported by Grant SFB604 from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** Present address: Amity Institute of Herbal and Microbial Studies, Sector 125, Noida 201303, Uttar Pradesh, India. Back

|| To whom correspondence should be addressed: Inst. of General Botany and Plant Physiology, Friedrich Schiller University Jena, Dornburger Strasse 159, 07743 Jena, Germany. Tel.: 49-3641-949230; Fax: 49-3641-949232; E-mail: b7oera{at}hotmail.com.

1 The abbreviations used are: MMN, Marx-Melin-Norkrans; RT, reverse transcription; NR, nitrate reductase; MS-MS, tandem mass spectrometry; G{beta}, {beta}-subunit of heterotrimeric GTP-binding proteins; GUS, {beta}-glucuromidase. Back

2 Oelmüller, R., Peskan-Berghöfer, T., Shallohari, B., Trebicka, A., Sherameti, I., and Varma, A. (2005) Physiol. Plant. 124, 152–166. Back

3 L. Altschmied and R. Oelmüller, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. K.-J. Appenroth for carefully reading the manuscript and for providing the antibodies against NR, Günther Gessner for nitrate determination, Drs. T. Peskan-Berghöfer, Victor Kusnetsov, and Sudhir Sopory for generating the transgenic lines used in these studies, and Martin Rienth and Heidi Renz for the generation of Nia2 promoter deletions and for the initial studies with the promoter segments, which ultimately lead to the identification of the homeodomain protein.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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