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Transcript Profiling Coupled with Spatial Expression Analyses Reveals Genes Involved in Distinct Developmental Stages of an Arbuscular Mycorrhizal Symbiosis

^a Boyce Thompson Institute for Plant Research, Ithaca, New York 14853
^b Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108
^c The Institute for Genomic Research, Rockville, Maryland 20850

¹ To whom correspondence should be addressed. E-mail mjh78{at}cornell.edu; fax 607-254-6779

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
The formation of symbiotic associations with arbuscular mycorrhizal (AM) fungi is a phenomenon common to the majority of vascular flowering plants. Here, we used cDNA arrays to examine transcript profiles in Medicago truncatula roots during the development of an AM symbiosis with Glomus versiforme and during growth under differing phosphorus nutrient regimes. Three percent of the genes examined showed significant changes in transcript levels during the development of the symbiosis. Most genes showing increased transcript levels in mycorrhizal roots showed no changes in response to high phosphorus, suggesting that alterations in transcript levels during symbiosis were a consequence of the AM fungus rather than a secondary effect of improved phosphorus nutrition. Among the mycorrhiza-induced genes, two distinct temporal expression patterns were evident. Members of one group showed an increase in transcripts during the initial period of contact between the symbionts and a subsequent decrease as the symbiosis developed. Defense- and stress-response genes were a significant component of this group. Genes in the second group showed a sustained increase in transcript levels that correlated with the colonization of the root system. The latter group contained a significant proportion of new genes similar to components of signal transduction pathways, suggesting that novel signaling pathways are activated during the development of the symbiosis. Analysis of the spatial expression patterns of two mycorrhiza-induced genes revealed distinct expression patterns consistent with the hypothesis that gene expression in mycorrhizal roots is signaled by both cell-autonomous and cell-nonautonomous signals.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
In natural ecosystems, the roots of most plants are actually symbiotic organs called mycorrhizae, a term with Greek origins that literally means "fungus root." There are many types of mycorrhizal symbioses, the most common of which is the arbuscular mycorrhizal (AM) symbiosis, formed between vascular flowering plants and fungi of the order Glomales (Smith and Read, 1997). These associations are widespread throughout the world, and both fossil evidence and molecular data indicate that the AM symbiosis existed in the earliest land plants, which underlies suggestions that the association may have enabled plants to colonize land (Pirozynski and Malloch, 1975; Remy et al., 1994; Redeker et al., 2000). The most frequently described outcomes of the AM symbiosis include a carbon supply for the fungus and an enhanced mineral nutrient supply, in particular phosphorus, for the plant. Phosphorus is an essential mineral nutrient that is frequently limiting for plant growth; consequently, the association has far-reaching effects on plant health and subsequently on ecosystem structure (Smith and Read, 1997; van der Heijden et al., 1998).

The development of the symbiosis requires significant alterations in both symbionts that are assumed to be coordinated via reciprocal signal exchange. In general, the fungus penetrates the epidermis via an appressorium, and intercellular and intracellular growth through the outer cortex ensues. In the inner cortex, the fungus differentiates within the cortical cells, forming dichotomously branched hyphae, termed arbuscules. These elaborate structures are enveloped in an extension of the plasma membrane of the cell, and the resulting arbuscule–cortical cell interface is the site of phosphorus transfer from the fungus to the plant (Bonfante-Fasolo, 1984; Gianinazzi-Pearson, 1996). The cytoskeleton of the invaded cortical cell undergoes massive transient rearrangements, presumably to enable the development of the arbuscular interface (Genre and Bonfante, 1997, 1998, 1999; Blancaflor et al., 2001), and recent analyses suggest that reorganization may be signaled before penetration of the cell (Blancaflor et al., 2001).

Arbuscule development is accompanied by increases in the expression of genes that encode enzymes of flavonoid and jasmonic acid biosynthesis (Harrison and Dixon, 1993, 1994; Hause et al., 2002). Chitinase, glutathione S-transferase, proline-rich protein, sugar transporter, arabinogalactan protein, and tubulin transcripts also are increased in these cells (Bonfante et al., 1996; Harrison, 1996; Strittmatter et al., 1996; van Buuren et al., 1999; Salzer et al., 2000; Journet et al., 2001; Wulf et al., 2003). The roles of these gene products in arbuscule development or activity are mostly unknown, but it is predicted that some of them function in the development of the symbiotic interface, either in the matrix or in the periarbuscular membrane. Phosphate transporters expressed exclusively in the symbiosis have been described in potato, rice, and Medicago truncatula (Rausch et al., 2001; Harrison et al., 2002; Paszkowski et al., 2002), and in the latter species, the phosphate transporter was shown to be located in the periarbuscular membrane, where it is assumed to play a role in the uptake of phosphate released from the arbuscule. Despite the continual invasion of cortical cells and extensive levels of fungal biomass that can accumulate within the root, plant defense responses generally are not induced significantly. Small, transient increases in defense gene transcripts have been observed in the initial stages of the symbiosis, but these decline as the symbiosis develops (Gianinazzi-Pearson et al., 1992; Harrison and Dixon, 1993; Lambais and Mehdy, 1993; Kapulnik et al., 1996).

Relatively little is known about the signal molecules involved in plant–AM fungal communication. Secondary metabolites, including flavonoids and isoflavonoids, have been implicated in plant fungal signaling but do not appear to be absolutely required for the development of the symbiosis (Siqueira et al., 1991; Poulin et al., 1993; Bécard et al., 1995; Phillips and Kapulnik, 1995; Xie et al., 1995; Poulin et al., 1997; Akiyama et al., 2002). A tomato mutant impaired in its ability to elicit spore germination may provide information on these early signaling events (David-Schwartz et al., 2001). There is evidence for a mobile fungal signal that elicits changes in plant gene expression before contact with the root (Kosuta et al., 2003) and also evidence that appressoria form in response to a plant signal endogenous to the epidermal cell wall (Nagahashi and Douds, 1997). However, the nature of the signal molecules involved remains unknown. Plant hormones may mediate some of the downstream developmental changes, but to date, causal relationships have not been established (van Rhijn et al., 1997; Barker and Tagu, 2000; Hause et al., 2002; Shaul-Keinan et al., 2002). Although the signals are unknown, in legumes, one component of a signaling pathway required for the development of both the mycorrhizal and rhizobium/legume symbioses has been identified. Mutants lacking the NORK/SYMRK Leu-rich repeat receptor kinase are unable to establish symbioses with either rhizobium or mycorrhizal fungi, and the fungi are restricted largely to the epidermis (Endre et al., 2002; Stracke et al., 2002). Other loci required for both symbioses have been identified genetically and may encode additional components of a nodule/mycorrhizal signaling cascade (Gianinazzi-Pearson et al., 1991; Sagan et al., 1995; Wegel et al., 1998).

To date, the molecular mechanisms that underlie the development of symbiosis remain poorly understood. Only a few of the genes that play roles in the development of the AM symbiosis have been identified. Genomics programs, particularly those focused on the model legumes M. truncatula and Lotus japonicus, have resulted in a tremendous increase in symbiosis-associated sequence information, and by electronic and molecular analyses, the cataloging of the transcriptomes of nodules and mycorrhizal roots is progressing (Fedorova et al., 2002; Journet et al., 2002; Wulf et al., 2003). Here, we used cDNA arrays and expression profiling to obtain insights into the transcriptional alterations associated with the development of a mycorrhizal symbiosis between M. truncatula and an AM fungus, Glomus versiforme. Sixty-seven genes whose expression is altered significantly in the AM symbiosis were identified, along with an additional 25 genes that show transcriptional changes in response to alterations in phosphate nutrition. The majority of these genes were not known previously to be regulated in response to the development of an AM symbiosis, and clusters of genes with distinct expression patterns were identified. Included in this set are genes that encode components implicated in the biosynthesis of the periarbuscular matrix as well as a significant number of proteins potentially involved in transcriptional regulation and signaling. The spatial expression patterns of two genes expressed only in the AM symbiosis were analyzed in transgenic roots. These showed unique cell type–specific expression patterns that suggest activation by distinct signals and support roles in overlapping developmental phases of the symbiosis.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
ESTs from Mycorrhizal Roots
A cDNA library was prepared from M. truncatula roots colonized with G. versiforme. Roots were sampled at 10, 17, 22, 31, and 38 days after inoculation; consequently, the library contains cDNAs representing genes expressed at all stages of development of the symbiosis and from both symbionts. A total of 7351 ESTs from this library have been deposited in GenBank and in the TIGR M. truncatula Gene Index. A comparison of these ESTs with the total M. truncatula EST collection (182,460 ESTs) revealed that the M. truncatula/G. versiforme library has 888 unique ESTs not found in any other libraries. The number of EST sequences from G. versiforme present in this library is unknown, but by extrapolating from estimates of the proportion of plant and fungal RNA in mycorrhizal RNA samples, we suggest that it is not >5%.

Arrays and Experimental Materials
To identify genes whose expression is regulated differentially in response to the development of the AM symbiosis, cDNA inserts from 2268 unique cDNA clones from the M. truncatula/G. versiforme library were arrayed at high density on nylon membranes. Each clone was spotted in duplicate, and the membranes each contained 768 cDNAs, 12 of which were controls. The controls included genes shown previously to be regulated in response to the development of the symbiosis. However, at the time we initiated these experiments, gene expression information was limited, and the genes reported as induced in the AM symbiosis actually showed only modest changes in transcript levels in mycorrhizal roots. Therefore, we included the AW587100 cDNA and a fragment of the AW587100 3' untranslated region (UTR) sequence as additional controls. A preliminary array experiment had indicated that AW587100 and a second gene, AW585598, showed increased transcripts in mycorrhizal roots (J. Liu and M.J. Harrison, unpublished data). Both were present in the 2268-unigene set.

cDNA probes were prepared from replicate sets of M. truncatula mycorrhizal roots harvested at 8, 15, 22, 31, and 36 days after inoculation with G. versiforme and the corresponding mock-inoculated controls. The mycorrhizal root samples showed between 8.7 and 68.9% of the root length colonized (Table 1). In the 8-day samples, G. versiforme hyphae and appressoria were abundant on the surface of the root. Some of the appressoria had penetrated the root and colonization of the outer cortex had been initiated, but few or no arbuscules were present. The development of the symbiosis is not a synchronous process, and after the initial colonization of the root cortex, secondary infection events commence and the invasion process is reiterated. Consequently, the 15-, 22-, 31-, and 36-day samples contained all of the structures associated with the symbiosis as G. versiforme continued to spread throughout the root system. The relative amounts of RNA from M. truncatula and G. versiforme in the RNA samples prepared from these roots were estimated by RNA gel blot analysis using short species-specific internal transcribed spacer sequence probes (Maldonado-Mendoza et al., 2002). As expected, the percentage of RNA from G. versiforme increased with increasing colonization of the root system, reaching 4.5 and 8.1% in 36-day samples in replicate experiments 1 and 2, respectively (Table 1).

Reproducibility of Array Data
After hybridization of the arrays with probes from the replicate mycorrhiza and phosphate experiments, the signals from the duplicate spots were averaged and adjusted to account for differences in the labeling and hybridization of the probes and the amount of M. truncatula RNA in the mycorrhizal root samples. For each replicate experiment, the ratio of the average adjusted signal in mycorrhizal/nonmycorrhizal roots and high/low phosphate was calculated for each gene and expressed as a log-10 expression ratio (LR). The reproducibility of the duplicate spots on the arrays and the variation observed between replicate experiments were assessed. The data obtained from duplicate spots on the same array were highly reproducible, and 99% of the values fell within ±0.2 LR of the mean (Figures 1A and 1B) . As expected, the data from replicate experiments showed greater variability (Figure 1C). For the three filters that encompass the 2268-gene set, a comparison of the average LRs for each spot in the two replicate experiments revealed a normal distribution, with 94% of the spots within ±0.33, ±0.37, and ±0.34 LR of the mean for filters A, B, and C, respectively. This finding is similar to the variation noted previously on microarrays (Wang et al., 2000; Kawasaki et al., 2001). We elected to use these values as significance thresholds, and in subsequent experiments, only data for genes whose change in expression (LR) was greater than the appropriate filter threshold in both replicate experiments were considered significant.

View larger version (36K): [in this window] [in a new window]   Figure 1. Array Quality and Variation within and between Experiments. (A) TIFF image of a cDNA array hybridized with a 33P-labeled cDNA probe from mock-inoculated M. truncatula roots harvested at 31 days after inoculation. The image shown is from filter C. (B) Scatterplot comparing the LRs of duplicate spots on the arrays. Ninety-nine percent of the spots fall within ±0.2 LR of the mean. The data shown are from filter C. Data from filters A and B fall within the same limits. (C) Scatterplot comparing the average LRs from two experiments involving independent biological samples. Ninety-four percent of the spots fall within ±0.34 LR of the mean. The data shown are from filter C. Values from filters A and B fall within ±0.33 and ±0.37 LR of the mean, respectively.

View larger version (51K): [in this window] [in a new window]   Figure 2. Cluster Analysis of 92 Transcripts during the Development of the AM Symbiosis and during Growth with 2 mM Versus 0.02 mM Phosphate. Clustered expression ratios visualized by Treeview (Eisen et al., 1998). The rows represent the individual clones, and the GenBank numbers of the ESTs are indicated. The rows labeled 53, 41, and 89 represent the AW587100, AW587100 3' UTR, and Mt4 controls, respectively. The ratios of expression in mycorrhizal/nonmycorrhizal roots at 8, 15, 22, 31, and 36 days postinoculation (dpi) and in high/low phosphate (Pi) are represented. Biological replicates are adjacent to each other. The color reflects the LR. Red represents a positive LR, green represents a negative LR, and black represents a LR of zero. In each case, the saturation reflects the magnitude of the change. Seven groups of transcripts with distinct expression patterns are indicated.

View larger version (54K): [in this window] [in a new window]   Figure 3. RNA Gel Blot Analysis of a Selection of the Genes Identified on the Arrays. (A) and (B) RNA gel blot analysis with total RNA (12 µg) from noncolonized M. truncatula roots (Mt) and M. truncatula roots colonized with G. versiforme (Mt/Gv) at 8, 15, 22, 31, and 36 days postinoculation (dpi). The blots were probed, stripped, and reprobed sequentially with the probes indicated at right. All of the genes except AW586356 are members of cluster 1. AW586356 is a member of cluster 5. (C) RNA gel blot analysis with root RNA (12 µg) from M. truncatula plants grown for 31 days (experiment 1) and 42 days (experiment 2) with differing levels of phosphate fertilization. The blots were probed, stripped, and reprobed sequentially with the probes indicated at right. The Mt4 gene was included as a control, and transcript levels reflect the degree of phosphate starvation (Burleigh and Harrison, 1998).

Genes Regulated in Response to Phosphate
The final two clusters contain transcripts that change significantly in response to growth under high- versus low-phosphate conditions. Genes in cluster 6 showed significant increases in transcript levels during growth in high-phosphate conditions (Table 7). Apart from a putative phosphoenolpyruvate carboxykinase, the 14 genes in this cluster were not known previously to be responsive to phosphate. Included in this cluster are two ESTs that are similar to kinases that interact with calcineurin B–like calcium sensor proteins and that have been implicated in signaling pathways involved in responses to stress, hormones, and environmental cues (Albrecht et al., 2001). A M. truncatula zinc transporter also is present in this cluster and shows a 10-fold increase in transcripts. Transcriptional profiling has revealed overlaps in responses to mineral nutrient stresses (Wang et al., 2001, 2002), and in barley, zinc nutrition is known to affect the expression of phosphate transporter genes (Huang et al., 2000). Our data suggest that the opposite effect also occurs and that changes in phosphorus nutrition affect the expression of a zinc transporter.

By contrast with cluster 6, genes represented in cluster 7 showed a decrease in transcript levels under high-phosphate conditions, and the expression of some of these genes was downregulated in the AM symbiosis (Table 8). A well-described phosphate starvation–induced gene, Mt4, falls into this cluster, as does a putative acid phosphatase (Baldwin et al., 2001). As seen with the genes in cluster 7, some members of this group showed marginally significant changes in mycorrhizal roots that require additional validation to be conclusive. Small, localized decreases in transcripts in mycorrhizal roots may be difficult to detect with the arrays.

View larger version (80K): [in this window] [in a new window]   Figure 4. Expression of a Selection of the Mycorrhiza-Regulated Genes in Nodulated Roots. (A) RNA gel blot analysis with total RNA (12 µg) from M. truncatula roots and M. truncatula roots colonized with G. versiforme (31 days after inoculation) (lanes 1 and 2) and M. truncatula roots and M. truncatula nodulated roots harvested at 11 (lanes 3 and 4) and 18 (lanes 5 and 6) days after inoculation. The blots were probed, stripped, and reprobed sequentially with the probes indicated at right. The leghemoglobin gene is induced in nodules and was included as a control (Barker et al., 1988). (B) RT-PCR analysis of the same RNA samples shown in (A) with primer pairs for AW585594, AW587040, and EF-1.

Cell Type–Specific Expression Patterns of Two Mycorrhiza-Induced Genes, MtSCP1 and MtCel1
The arrays enabled the identification of novel genes whose expression is regulated during the development of the symbiosis. However, because the colonization process is asynchronous, information concerning the spatial expression patterns cannot be obtained from these analyses. With the goal of understanding the role of these genes in the development of the symbiosis, we used promoter-reporter gene fusions to gain insight into the spatial expression patterns of two novel mycorrhiza-induced genes, MtSCP1 (AW587100) and MtCel1 (AW585598).

The cDNA clone from which the MtSCP1 EST sequence (AW587100) was derived was a full-length clone and was designated MtSCP1. The cDNA is predicted to encode a protein of 495 amino acids that shares 53 to 56% identity with Ser carboxypeptidase II proteins from barley, wheat, and Arabidopsis (Breddam and Ottesen, 1987; Soerensen et al., 1987; Degan et al., 1994; Li et al., 2001). The AW585598 EST was derived from a partial cDNA of 1408 bp. A combination of library screening and RT-PCR analysis enabled the identification of a genomic clone and subsequently a full-length cDNA clone designated MtCel1. The MtCel1 cDNA is predicted to encode a protein of 601 amino acids that shares 43 to 45% identity with membrane-anchored endo-1,4--D-glucanases (EGases; EC 3.2.1.4), Arabidopsis KOR1, Brassica napus Cel16, barley Cel1, and tomato Cel3.

To examine the expression patterns of the MtSCP1 and MtCel1 genes, DNA regions 5' of the respective open reading frames were fused to the uidA and green fluorescent protein (GFP) reporter genes, and M. truncatula plants containing transgenic roots carrying these constructs were created (Boisson-Dernier et al., 2001). The plants were inoculated with G. versiforme, and after the development of the symbiosis, the roots were examined for the expression of the reporter genes.

In the transgenic roots carrying the MtCel1 promoter–GFP fusion, strong green fluorescence was visible exclusively in cells containing arbuscules (Figures 5A and 5B) . This pattern of expression was confirmed in transgenic roots carrying the MtCel1 promoter–-glucuronidase (GUS) fusion, in which strong blue staining, indicating the presence of GUS activity, was visible in the cortex (Figure 5C) and was associated specifically with cells containing arbuscules (Figures 5D and 5E). Reporter gene expression was not visible in epidermal cells or in cells through which an intracellular hypha had passed, suggesting that expression is correlated tightly with arbuscule development. This pattern is the same as that reported recently for a M. truncatula mycorrhiza-specific phosphate transporter (Harrison et al., 2002) and may reflect the presence of a cell-autonomous signal that induces the expression of genes associated with arbuscule development and function.

View larger version (120K): [in this window] [in a new window]   Figure 5. M. truncatula Roots Expressing the uidA and GFP Genes under the Control of the MtCel1 Promoter. (A) and (B) Light and corresponding epifluorescence micrographs of a M. truncatula root expressing GFP under the control of the MtCel1 promoter. Roots were colonized with G. versiforme. Arrows indicate cells containing arbuscules, and GFP is present in cells containing arbuscules. Bar = 150 µm. (C) Histochemical staining for GUS activity in a M. truncatula root expressing the uidA gene under the control of the MtCel1 promoter. Arrows indicate positive staining in cortical cells containing arbuscules. Bar = 200 µm. (D) and (E) Epifluorescence and corresponding light micrographs of cortical cells from a M. truncatula root expressing the uidA gene under the control of the MtCel1 promoter. The root was stained histochemically to detect GUS activity and then counterstained with acid fuschin to reveal the fungus. Cortical cells were released by squashing. Acid fuschin fluoresces yellow when viewed via epifluorescence microscopy. Arrows indicate positive GUS staining in cells containing arbuscules. Bar = 40 µm.

View larger version (91K): [in this window] [in a new window]   Figure 6. M. truncatula Roots Expressing GFP under the Control of the MtSCP1 Promoter. Roots were colonized with G. versiforme. (A) and (B) Light and corresponding epifluorescence micrographs. Arrows indicate cells containing arbuscules, and a strong green fluorescence signal is present in these cells. Arrowheads indicate cells within the same cell file that do not contain arbuscules but show strong green fluorescence. Bar = 40 µm. (C) and (D) Light and corresponding epifluorescence micrographs. Arrowheads indicate cells in the inner cortex that do not contain arbuscules but display strong green fluorescence. Cells containing arbuscules are present but are not in the field of focus. Arrows indicate cells in the outer cortex that show weak green fluorescence. Hyphae, but not arbuscules, are present in these cells. Bar = 40 µm. (E) and (F) A laser scanning confocal microscopy image and the corresponding bright-field image of a hypha traversing the epidermis (e) and outer cortical cells. Arrows indicate outer cortical cells penetrated by the hypha, and green fluorescence is visible in these cells. Green fluorescence is not visible in the epidermal cell. (G) Merged image showing both green fluorescence and bright-field views. Bar = 75 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

The criterion selected for ascribing significance to changes in gene expression on the arrays was relatively stringent, and it is likely that some differentially regulated genes have been overlooked. However, the application of these significance thresholds resulted in a robust data set, and genes that were tested subsequently showed the expression patterns predicted from the arrays. In addition, the arrays contained a number of unanticipated internal controls that further validated the results. Some of the ESTs described initially in the TIGR M. truncatula Gene Index as singletons were shown later to represent the same gene. For example, AW584415 and AW584136 represent a single mitogen-activated protein kinase gene. They are both present on the arrays and cluster together in cluster 2. Likewise, AW584210 and AW584656 represent a putative acid phosphatase gene, and both cluster together in cluster 7.

The development of the AM symbiosis is not a synchronous process, so temporal differences in expression observed in the 15- to 36-day samples should be interpreted cautiously. In spite of this caveat, clusters of genes with distinct expression patterns were apparent. One theme that emerged from these analyses relates to defense-response genes. A significant number of genes that share sequence similarity with defense- or stress-regulated genes, including a chitinase, a pathogenesis-related (PR) protein, a putative defense-associated acid phosphatase, chalcone synthase, a stress-related mitogen-activated protein kinase, an SRC2 homolog, two 3-hydroxy 3-methyl glutaryl–CoA reductases, and a defense-related AGP, were found in cluster 2. Additional defense- and stress-related genes were present in clusters with related expression patterns, clusters 3 and 4. Previous analyses had shown that defense-response genes, largely those that encode PR proteins and enzymes of phytoalexin biosynthesis, show transient increases in expression during the early stages of the symbiosis and then the transcript levels subsequently decline (Spanu et al., 1989; Harrison and Dixon, 1993, 1994; Gianinazzi-Pearson et al., 1996; Kapulnik et al., 1996).

The expression patterns observed here are in accord with the earlier analyses and extend them to include a much broader array of defense- and stress-response genes. Furthermore, these data indicate that some defense-response genes do not show an initial transient increase in expression but simply are downregulated as the symbiosis develops, suggesting that at least two different signals regulate the expression of defense-response genes in the AM symbiosis. In addition to genes that encode defense-response proteins, there are a number of genes that encode putative defense-associated signal transduction components in cluster 2, including a protein phosphatase, a mitogen-activated protein kinase, a Leu-rich repeat kinase, and a putative RING zinc finger ankyrin protein that shares identity with a protein involved in the ubiquitination of the Ser/Thr receptor-like kinase, Xa-21. The distributions of ESTs for these genes indicate that they are expressed broadly in M. truncatula tissues; therefore, they are not representatives of mycorrhiza-specific signaling pathways. However, they may provide clues to the mechanisms by which defense responses are downregulated in the AM symbiosis.

Among the genes induced specifically in the AM symbiosis but not in response to phosphate (cluster 1) are two genes (AW586261 and AW587040) that encode unknown proteins that are similar to genes induced in grapes during fruit ripening (Davies and Robinson, 2000). At first glance, these events appear very different; however, some of the underlying physiological changes may be similar. Both processes are accompanied by significant modifications to cell wall architecture (Bonfante-Fasolo et al., 1981; Bonfante and Perotto, 1995; Davies and Robinson, 2000). The functions of the grape and M. truncatula proteins are unknown, but they are predicted to contain cleavable signal peptides and are potentially located in the cell wall.

In addition to these proteins, a third gene, MtCel1, is predicted to be involved in cell wall modifications and is induced specifically in mycorrhizal roots. The MtCel1 gene, MtCel1, product shares identity with members of the E-type EGase subfamily III (Brummell et al., 1997), including the Arabidopsis KOR1 protein (Nicol et al., 1998; Zuo et al., 2000), tomato Cel3 (Brummell et al., 1997), B. napus Cel16, and barley Cel1. These EGases are distinct in that they lack the typical eukaryotic cleavable signal peptide found in other EGases and are predicted to be type-II integral membrane proteins. Tomato Cel3, the first member of this class to be identified, is associated with expanding tissue and has been suggested to play a role in cellulose biosynthesis (Brummell et al., 1997). The Arabidopsis KOR protein is required for both cell elongation and cell plate formation and likewise is proposed to function in cell wall assembly (Nicol et al., 1998; Zuo et al., 2000). Here, we found MtCel1 expression associated specifically with cells that contain arbuscules. Arbuscule formation is accompanied by the assembly of a new extracellular matrix in the interfacial space that develops between the arbuscule and the periarbuscular membrane. Considering the membrane domain, it is possible that MtCel1 is located in the periarbuscular membrane and involved in the assembly of this cellulose/hemicellulose matrix. EGase gene expression has been reported widely in plant–microbe interactions, and plant EGases are induced during giant cell and syncytia formation in compatible nematode interactions (Goellner et al., 2001). In contrast to the AM symbiosis, they are all secreted EGases, which may reflect a greater requirement for degradative and remodeling activities than for biosynthetic activities.

A significant proportion of the mycorrhiza-induced genes are similar to signal transduction components. For instance, among the genes in cluster 1 are three putative transcription factors (AW584487, AW585594, and AW584152). One of these, AW585594, shows increased transcript levels in mycorrhizal roots, and based on RT-PCR analyses and the EST distribution within the gene index, expression is restricted to mycorrhizal roots. The encoded protein is similar to a group of myb/coiled-coil domain transcription factors that includes the PSR1 gene from Chlamydomonas reinhardtii and PHR1 from Arabidopsis (Wykoff et al., 1999; Rubio et al., 2001). PSR1 and PHR1 are required for adaptation to phosphate deprivation, and loss of function results in an inability to express a selection of phosphate starvation–response genes, including phosphate transporters, acid phosphatases, and the TPSI1/Mt4 gene family. The PSR1 and PHR1 genes are expressed constitutively, but transcript levels are increased, albeit only moderately for PHR1, during phosphate starvation. By contrast, AW585594 transcripts were not regulated by phosphate but were increased coincident with the invasion of the root system by the fungus. The development of the AM symbiosis occurs most readily in phosphate-starved roots, and colonization of the root system results in the rapid, systemic, transcriptional downregulation of high-affinity phosphate transporters, acid phosphatases, and members of the TPSI1/Mt4 gene family (Burleigh and Harrison, 1998; Liu et al., 1998). The mechanisms that underlie this regulation are unknown, but because the myb/coiled-coil domain factors act as dimers, heterodimerization between existing PSR1/PHR1-type proteins and a new mycorrhiza-specific partner, such as the AW585594 protein, might be one mechanism by which the expression of a large set of phosphate starvation–associated genes could be redirected.

Of the 20 mycorrhiza-induced genes in cluster 1, four are predicted to encode proteases. One of these, AW585765, shares 81% amino acid identity with a Cys protease from Astralagus sinicus nodules that is proposed to play a role in the recycling of nitrogenous compounds from senescing bacteroids (Naito et al., 2000). The M. truncatula Cys protease is expressed in both senescing nodules and mycorrhizal roots, suggesting that the cellular processes associated with arbuscule and nodule senescence may be common to both symbioses.

There is considerable evidence for a role for proteases in plant–microbe signaling pathways, and two putative Ser carboxypeptidases (AW586622 and MtSCP1) also were expressed highly in mycorrhizal roots. Ser carboxypeptidases were identified initially in cereal grains, where they are thought to function in the degradation of storage proteins (Soerensen et al., 1987; Degan et al., 1994); however, they also have been shown to play a role in signaling pathways. Overexpression of a Ser carboxypeptidase suppressed an extracellular domain mutant of the Leu-rich repeat receptor kinase BRI1, and it was proposed that the SCP functions in the processing of a BRI1 ligand (Li et al., 2001). Our analyses indicated that the MtSCP1 gene was expressed coordinately with the invading hyphae and displayed transient expression in the outer cortex and sustained expression in the inner cortex, with a spatial expression pattern that suggested regulation in response to a mobile signal. Given these expression patterns, a role in signaling is possible. It was shown recently that a Leu-rich repeat kinase, NORK/SYMRK, is required for both the development of the mycorrhizal symbiosis and nodulation (Endre et al., 2002; Stracke et al., 2002), and the NORK gene is expressed constitutively in mycorrhizal roots (L.A. Blaylock and M.J. Harrison, unpublished data). The other components of this signaling pathway are not known; however, by analogy with BRI1, MtSCP1 might be involved in ligand processing and could provide a mechanism by which the NORK/SYMRK kinase could transmit a mycorrhiza-specific signal.

One other possibility that should not be discounted is that the Ser carboxypeptidase–like proteins may not be proteases but rather acyltransferases (Lehfeldt et al., 2000; Li and Steffens, 2000; Steffens, 2000). The catalytic triad typical of the Ser carboxypeptidases is found in a wide range of enzymes, and Ser carboxypeptidase–like proteins were shown recently to function in the transesterification of secondary metabolites. Although the enzymatic role of these proteins requires further verification, the specific spatial and temporal expression patterns support a role in the development of the symbiosis.

In conclusion, transcriptional profiling succeeded in associating a set of previously unknown genes with the development of the AM symbiosis and provided insights into the molecular response that occurs during the development of the symbiosis and clues to the specific players involved. This approach, coupled with spatial expression information, has offered a glimpse of the complexity of transcriptional changes and the signaling that occurs during the development of the AM symbiosis and provides a blueprint for future functional analyses.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Plant Materials and Growth Conditions
Medicago truncatula cv Jemalong, line A17, was used throughout this work. Plants were grown in growth rooms under a 16-h-light/8-h-dark (25/22°C) regime. The growth rooms contained F4012/Triten 50 light bulbs (www.lightbulbs4sale.com), and the light intensity at shelf height was 260 µE·m^-2·s^-1.

The growth and mycorrhizal colonization procedures were as described previously (Harrison and Dixon, 1993; Harrison et al., 2002). Briefly, 2-week-old M. truncatula seedlings were transplanted to sterile Turface (seven plants per 11-cm pot) and inoculated with 5000 surface-sterilized Glomus versiforme spores. Control plants were mock-inoculated with the final, distilled-water wash from the sterilization procedure. The plants were fertilized twice weekly with half-strength Hoagland solution (Arnon and Hoagland, 1940) containing 0.02 mM KH₂PO_4. Plants were harvested at 8, 15, 22, 31, and 36 days after inoculation. The root systems of the seven plants from one pot were pooled, and a random sample was assessed for colonization as described previously (McGonigle et al., 1990). The remaining tissues were frozen immediately in liquid N₂ and stored at -80°C for RNA isolation and phosphate content analysis. The colonization levels of the two independent replicate experiments are listed in Table 1.

For the phosphate experiments, 10-day-old M. truncatula seedlings (nine plants per 11-cm pot) were transplanted to acid-washed, sterilized river sand and fertilized twice weekly with half-strength Hoagland solution containing 0.001, 0.02, 0.2, or 2.0 mM KH₂PO₄. Levels of potassium were adjusted by the addition of the appropriate amounts of K₂SO₄ and KNO₃. Root and shoot materials were harvested 31 or 42 days later, immediately frozen in liquid N₂, and stored at -80°C for RNA isolation and phosphate content analysis. The phosphate content of the leaves grown under low- and high-phosphate conditions were 7.3 ± 0.4 and 31.4 ± 1.5 nmol/mg fresh weight for experiment 1 and 6.6 ± 0.3 and 28.2 ± 1.0 nmol/mg fresh weight for experiment 2, respectively.

For experiments involving nodulation, 11-day-old M. truncatula seedlings were transplanted to sterile Turface (eight plants per 11-cm pot) and inoculated with Sinorhizobium meliloti, strain 2011. The plants were fertilized twice weekly with fertilizer containing 1 mM KNO₃ and harvested at 11 and 18 days after inoculation. White nodules were visible at 11 days after inoculation, and pink nodules were clearly visible at 18 days after inoculation.

RNA Isolation and cDNA Library Construction
The M. truncatula/G. versiforme mycorrhizal cDNA library was created from RNA prepared from a pooled root sample composed of 0.70, 1.50, 2.25, 3.36, and 3.50 g of root tissue from mycorrhizal roots harvested at 10, 17, 22, 31, and 38 days after inoculation, respectively. Total RNA was extracted according to Chomczynski and Sacchi (1987), and poly(A)⁺-enriched RNA was prepared using the Oligotex mRNA Midi Kit (Qiagen, Valencia, CA). cDNA was prepared using a ZAP-cDNA synthesis kit, directionally ligated to ZAP II, and packaged using Gigapack III Gold packaging extracts (Stratagene, La Jolla, CA). Plasmids containing cDNA inserts were excised using Ex-Assist helper phage and propagated in SOLR cells according to the manufacturer's instructions (Stratagene). ESTs were generated by 5' end sequencing and submitted to GenBank. The ESTs also are available at the M. truncatula Gene Index developed at TIGR (http://www.tigr.org/tdb/mtgi/). The library designation is MHAM, and the library catalog number used in the gene index is T1682.

Preparation of the cDNA Arrays
The cDNA inserts of 2344 unique clones selected from the MHAM cDNA library were amplified with the following primers (5'-TGTAATACGACTCACTATAGGGC-3' and 5'-CGCTCTAGAACTAGTGGATCCC-3') in 100-µL PCR samples (0.3 µM each primer, 125 µM deoxynucleotide triphosphates, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl₂, 1.5 units of Taq DNA polymerase, and 10 ng of plasmid template). The amplification program included a 4-min denaturation at 95°C and 35 cycles of 55 s at 94°C, 55 s at 60°C, and 3 min at 72°C followed by a final extension of 8 min. All of the PCR products were examined by agarose gel electrophoresis, and only the reactions that yielded one band with an approximate DNA concentration of 100 to 200 ng/µL were selected for spotting.

A total of 2268 cDNA inserts were spotted in duplicate on GeneScreen Plus membranes in a format containing 96 blocks of 16 inserts using the High-Density Replicating Tool and a Biomek 2000 robot (Beckman, Fullerton, CA). Approximately 20 to 40 ng of DNA was applied per spot. The set of 2268 cDNAs was encompassed on three membrane filters designated A, B, and C, and each filter also contained a set of control spots. The controls included an elongation factor-1 (EF-1) cDNA (AW585952) whose expression does not change during the development of the symbiosis or in response to high- or low-phosphate conditions and a fragment of the pBluescript SK- vector (the region between 1168 and 2588 bp) as a negative control. Both the EF-1 and pBluescript fragments were spotted twice at different locations on each filter.

Additional controls included the Mt4 cDNA, whose expression is induced in response to phosphate starvation and downregulated in response to mycorrhizal colonization (Burleigh and Harrison, 1997), isoflavone reductase, which was shown previously to be repressed during the AM symbiosis (Harrison and Dixon, 1993, 1994), a mycorrhiza-induced cDNA, AW587100 (MtSCP1), which was identified in a preliminary array experiment as significantly induced in response to mycorrhizal colonization, and a 439-bp fragment of the 3' untranslated region (UTR) of the AW587100 cDNA as a second mycorrhiza-induced control spot. After spotting, the filters were subjected to alkaline denaturation, neutralization, and UV light cross-linking according to standard procedures (Sambrook et al., 1989). One set of filters was hybridized with an -³²P-dATP–labeled 222-bp fragment of the pBluescript polylinker, which was created by PCR with M13 forward and reverse primers, to verify that the arrays were spotted evenly.

Probe Preparation and Hybridization of the Arrays
The arrays were hybridized with ³³P-labeled first-strand cDNA probes prepared from RNA from mycorrhizal roots harvested at 8, 15, 22, 31, and 36 days after inoculation, the corresponding mock-inoculated controls, and root samples from plants grown under high-phosphate (2 mM) and low-phosphate (0.02 mM) conditions (Figure 1A). The entire experiment was replicated by hybridizing a second set of identical filters with probes from a replicate set of biological materials. For the synthesis of ³³P-labeled first-strand cDNA probes, 1 µg of poly(A)⁺-enriched RNA was reverse transcribed with 200 units of SuperScript II reverse transcriptase (Gibco BRL) in the presence of 40 µCi of -³³P-dATP and 40 µCi of -³³P-dCTP at 37°C. The filters were hybridized in 0.5 M NaHPO₄, pH 7.4, 7% SDS, and 1 mM EDTA (Church and Gilbert, 1984) for 16 to 20 h at 65°C and then washed once in 2x SSPE (1x SSPE is 0.115 M NaCl, 10 mM sodium phosphate, and 1 mM EDTA, pH 7.4) and 0.2% SDS at 65°C for 20 min, once in 0.5x SSPE and 0.2% SDS at 65°C for 20 min, once in 0.2x SSPE and 0.1% SDS at 65°C for 20 min, and once in 0.1x SSPE and 0.1% SDS at 65°C for 20 min. Filters were exposed for 2 to 4 days, scanned using a Storm 820 PhosphorImager (Molecular Dynamics/Amersham Pharmacia, Sunnyvale, CA), and quantified using ArrayVision 5.1 software (Imaging Research, Brock University, St. Catharines, Ontario, Canada).

Data Analysis
To calculate the signal intensities for each spot, a grid was overlaid on the array image. The total intensity of all pixels within each grid was determined using the volume quantitation method (ImageQuant, Piscataway, NJ). Local background around each 4 x 4 subarray grid was subtracted. Signals from the duplicate spots were averaged, and adjustments for differences in the labeling and hybridization were made based on the EF-1 spots and also on the overall signal on each filter (Hardwick et al., 1999). Because the majority of the genes on the arrays are derived from M. truncatula, an additional correction for the amount of M. truncatula RNA in the samples from mycorrhizal roots (Table 1) was included. For each replicate experiment, the ratio of the average adjusted signal in mycorrhizal/nonmycorrhizal roots and high/low phosphate was calculated for each gene and expressed as a log-10 expression ratio. Data from noisy spots, in which the replicates showed a fivefold or greater difference, were discarded. Hierarchical clustering using the correlation-centered similarity metric and the complete linkage clustering option was performed using Cluster, and the data were displayed using Treeview software (http://rana.lbl.gov/EisenSoftware.htm) (Eisen et al., 1998).

RNA Gel Blot Analyses
M. truncatula total RNA was extracted according to Chomczynski and Sacchi (1987), and RNA gel blots were prepared and hybridized according to standard protocols (Church and Gilbert, 1984; Sambrook et al., 1989). The blots shown in Figures 3 and 4 were sequentially stripped and reprobed with the probes indicated in each figure. The probes consisted of the entire cDNA inserts from the corresponding clone except for the MtSCP1 3' UTR probe, which consisted of 439 bp of the 3' UTR region of the MtSCP1 gene. The blots were imaged and analyzed with a Storm 820 PhosphorImager (Molecular Dynamics/Amersham Pharmacia).

Estimations of the amounts of M. truncatula and G. versiforme RNA in the mycorrhizal root RNA samples were made using gene-specific Gv-ITS and Mt-ITS probes as described previously (Maldonado-Mendoza et al., 2002).

Reverse Transcriptase–Mediated PCR Analyses
Reverse transcriptase reactions were performed with 2 µg of RNA using SuperScript II RNase H^- reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Five percent of the reverse transcription reaction was used as a template in a 25-µL PCR sample. The primers used were EF-1 forward (5'-TCTCTCTCTGCGGCTGAGAGTTCC-3') and reverse (5'-TTAGGTCACAAGGCAGATTGCAGG-3'), AW585594 forward (5'-GGTTCCACTCGCTCAGATGTGCTC-3') and reverse (5'-TAGGCACCAAAGGTGATTGGTAGGT-3'), and AW587040 forward (5'-TTTCTGATTATCATGTTGCACTCTGC-3') and reverse (5'-GTTCTTTATCGCATCCTGCTCTC-3'). These primers generate products of 1430 bp (EF-1), 701 bp (AW585594), and 400 bp (AW587040).

Identification of MtCel1 and MtSCP1 Promoters
Genomic clones containing the MtCel1 and MtSCP1 genes and 5' proximal regions were identified from a M. truncatula FixII genomic library using the 1408-bp partial MtCel1 cDNA and the 439-bp MtSCP1 3' UTR fragment as probes. The 5' proximal sequences of the MtCel1 gene (2085 bp) and the MtSCP1 gene (1992 bp) have been deposited in GenBank. Comparisons of the promoter regions of these genes and the mycorrhiza-induced MtPT4 promoter (Harrison et al., 2002) revealed no unique sequence motifs.

Creation of the MtCel1 and MtSCP1 Promoter-Reporter Gene Fusion Constructs
PCR-generated PstI-NcoI fragments containing 2085 bp of the MtCel1 5' proximal region and 1992 bp of the MtSCP1 5' proximal region were ligated to the same sites of the pCAMBIA3301 vector (CAMBIA, Canberra, Australia) upstream of the uidA gene. The primers used for the amplification of the MtCel1 5' proximal region were 5'-AGCAAGCTTCTGCAGAATATGCAAGGTTTGAGGTTCAAAC-3' and 5'-CGACGGATCCATGGGATTGATGAAGATTTTTGTACTTCAG-3'. The primers used for the amplification of the MtSCP1 5' proximal region were 5'-AGCAAGCTTCTGCAGCAAACTGGCCCATTATAATAATAG-3' and 5'-CGACGGATCCATGGCTTGTTCTCAACTTGGTTTCAACA-3'. In both cases, this cloning strategy resulted in the insertion of 10 additional bases (CCATGGATCC) before the first ATG of the uidA gene.

For the preparation of MtCel1 and MtSCP1 promoter–sGFP fusion constructs, the same 5' regions containing HindIII-BamHI ends were ligated into the HindIII-BamHI sites of a pCAMBIA3300 vector carrying a HindIII-EcoRI fragment from the CaMV35S-sGFP(S65T)-nos plasmid (Chiu et al., 1996). Insertion of the MtCel1 or MtSCP1 5' region replaces the 35S promoter in each instance. This cloning strategy introduces two additional bases (CC) before the first ATG of the GFP gene. Binary vectors were transformed into Agrobacterium rhizogenes strain ARquaI using standard methods.

Preparation and Analysis of M. truncatula Plants Containing Transformed Roots
M. truncatula plants with transformed roots were created and grown as described previously (Boisson-Dernier et al., 2001; Harrison et al., 2002) with the following modifications. The seedlings were grown on a modified medium (1 mM NH₄NO₃, 0.9 mM CaCl₂, 0.5 mM MgSO₄, 20 µM KH₂PO₄, 10 µM Na₂HPO₄, and 20 µM ferric citrate supplemented with 100 µg/L H₃BO₃, 33 µg/L MnCl₂, 33 µg/L CuSO₄, 7 µg/L ZnCl₂, 33 µg/L Na₂MoO₄, and 200 mg/L Mes, pH 6.4, containing 5 mg/L phosphinothricin). To prevent contact of the leaves with phosphinothricin in the medium, sterilized pieces of Parafilm were placed on the agar below the cotyledons. Plants with transgenic root systems were transplanted and inoculated with G. versiforme spores as described previously (Harrison et al., 2002) and examined between 6 and 33 days after inoculation. The expression of the uidA gene was evaluated by GUS staining in 0.1 M phosphate buffer, pH 7.0, containing 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 10 mM EDTA, 1 mg/mL 5-bromo-4-chloro-3-indolyl -D-glucuronic acid, and 1% (v/v) N,N-dimethylformamide. Roots were counterstained with acid fuschin to enable the localization of G. versiforme within the roots. GFP expression was evaluated by fluorescence microscopy. Roots were hand-sectioned and examined by light, epifluorescence, and confocal microscopy as described previously (Harrison et al., 2002). The expression patterns described were observed in a minimum of 10 independent transgenic root systems.

Determination of Phosphate Content
Freshly harvested shoot samples were frozen in liquid nitrogen, and total phosphate in ashed and hydrolyzed samples was determined by phosphomolybdate colorimetric assay (Ames, 1966).

Upon request, materials integral to the findings presented in this publication will be made available in a timely manner to all investigators on similar terms for noncommercial research purposes. To obtain materials, please contact Maria J. Harrison, mjh78{at}cornell.edu.

Accession Numbers
The accession numbers for the sequences mentioned in this article are as follows: MtSCP1 cDNA, AY308957; MtSCP1 5' proximal sequence, AY308958; MtCel1 cDNA, AY308955; MtCel1 5' proximal sequence, AY308956, M. truncatula Cys protease cDNA, AY336982; KOR, AF073875; Cel16, CAB51903; Cel1, BAA94257; Cel3, AAC49704; and IFR, X58078.

	Acknowledgments

 
We thank members of our laboratory and members of the plant biology division of The Samuel Roberts Noble Foundation (Ardmore, OK) for helpful discussions and critical reading of the manuscript. Funding for this work was provided by the National Science Foundation Plant Genome Research Program (NSF DBI-9872664 and NSF DBI-0110206) and by The Samuel Roberts Noble Foundation.

	Footnotes

 
Online version contains Web-only data.

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.014183.

Received May 29, 2003; accepted July 12, 2003.

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