BACKGROUND
the arbuscular mycorrhiza is the name given to a symbiotic association of plants and arbuscular mycorrhizal  fungi.
the am symbiosis occurs widely throughout the plant kingdom and involves angiosperms, gymnosperms, pteridophytes and some bryophytes.
the fungi that participate in this symbiosis are all members of the glomeromycota,  <cit> , a likely sister group to the ascomycota and basidiomycota.
the symbiosis develops in the plant roots, where am fungi form extensively branched hyphae, called arbuscules, in the cortical cells.
in addition to growth within the root, the fungus develops a network of extra-radical hyphae that extends into the rhizosphere.
inorganic phosphate  and nitrogen , acquired by the extra-radical hyphae, are translocated to the arbuscules and released to the plant.
in return the plant provides the fungus with carbon.
p and n are the two mineral nutrients that plants require in the greatest quantities and therefore the symbiosis has an important influence on plant health and consequently on ecosystem function  <cit> .
there has been significant progress in understanding the signaling associated with the early stages of development of am symbiosis  <cit> .
some of the signal molecules have been identified, including strigolactones, which act as pre-contact signals to stimulate am fungal growth and metabolism  <cit> .
in addition, several components of a plant 'symbiosis signaling' pathway required for fungal entry into the root have been cloned  <cit> .
these genes were identified initially in legumes, where the symbiosis signaling pathway is required also for the interaction with rhizobia.
however, orthologs of these genes have been identified in tomato and rice, where they are required for am symbiosis  <cit> .
this further supports the hypothesis that the symbiosis signaling pathway arose initially for interactions with am fungi, and in legumes it was co-opted to enable interactions with rhizobia  <cit> .
currently, for am symbiosis, the downstream genes controlled by this pathway are not known.
in general, events associated with fungal development in the cortex are less well understood, and genes controlling arbuscule formation, have yet to be identified.
arbuscule development is an active process for both symbionts.
the fungal hypha penetrates the cortical cell wall and undergoes repeated dichotomous branching, while the plant cell undergoes significant cellular changes, including reorganization of the cytoskeleton, endoplasmic reticulum and development of the peri-arbuscular membrane, which envelops the arbuscule  <cit> .
reorganization of the cytoskeleton and endoplasmic reticulum is initiated prior to arbuscule formation suggesting that a mobile signal may trigger these events  <cit> .
currently, information about the lipid and protein content of the peri-arbuscular membrane is limited, but it is clear that the membrane contains some unique proteins, including symbiosis-specific pi transporters that function to import pi into the cortical cell  <cit> .
the transporters involved in n acquisition are as yet unknown, but a series of nuclear magnetic resonance  spectroscopy and gene expression studies indicate that the fungus assimilates n in the external hyphae, translocates this to the intra-radical hyphae as arginine, and then disassembles the arginine to retain the carbon molecules, while providing n to the plant, probably as ammonium   <cit> .
although not shown directly, it is likely that nh4+ transfer to the plant occurs at the arbuscule-cortical cell interface also.
arbuscules are terminally differentiated hyphae with a finite life and following development, they undergo a degeneration phase in which the arbuscule branches collapse, and the arbuscule gradually disappears from the cell  <cit> .
this process is not well understood, but it is clear that arbuscule development and degeneration occur cell autonomously and arbuscules in neighboring cells may be in different phases of the life cycle.
although the arbuscule-cortical cell interface is central to the am symbiosis, currently, we understand little of the processes that enable its development or that control its degeneration.
to gain insight into the molecular changes that accompany development of am symbiosis, a variety of transcript profiling approaches have been undertaken.
in rice, surveys using a whole genome chip identified mycorrhiza-responsive genes and revealed a partial overlap in the transcriptional responses to mutualistic and pathogenic biotrophic fungi  <cit> .
in medicago truncatula, suppressive subtractive hybridization, cdna arrays and long oligonucleotide arrays have been used to document transcript changes associated with am symbiosis  <cit> .
the inclusion of m. truncatula symbiosis mutants enabled gene expression to be associated with different phases of the symbiosis, for example appressoria formation  <cit> .
a mycorrhizal root includes a heterogeneous mix of colonized and non-colonized cells, and therefore spatial gene expression information is particularly important.
in situ hybridization and/or analysis of promoter-reporter fusions demonstrated that genes whose expression was induced in colonized roots could be further separated into two groups; those expressed exclusively in cells containing arbuscules and those expressed in cells containing arbuscules and in neighboring non-colonized cells  <cit> .
these data indicate that the different cortical cells of the mycorrhizal root system have unique transcript profiles, and point to at least two signaling pathways active in regulating mycorrhiza-induced gene expression.
while these approaches have advanced information about gene expression in colonized cortical cells, in situ hybridization or promoter-reporter gene studies are labor intensive and this precludes surveys of a large number of genes.
our current knowledge of genes expressed in cells containing arbuscules is limited to less than  <dig> genes.
in addition, the genes that have been analyzed are those that have the highest transcript levels and relatively few receptors or transcriptional regulators have been investigated.
beyond plant gene expression in the colonized cortical cell, almost nothing is known about gene expression in the arbuscule.
laser microdissection  offers a new way to obtain rna from a subset of cells, and coupled with transcript profiling approaches, it provides an opportunity to monitor gene expression in an individual cell type more easily than promoter-reporter gene fusions.
lm was originally developed to isolate specific animal cells from complex tissues; a breakthrough for cancer and neuronal research  <cit> .
in plants, lm was used initially to isolate phloem cells from rice leaves  <cit> .
subsequently, epidermal and vascular tissues were isolated from maize coleoptiles  <cit>  and then various cell or tissue types, including bundle sheath, shoot tip protoderm, shoot apical meristem, leaf primordium, seedling procambium, hypocotyls and root meristems have been harvested from maize, rice, radish and tomato  <cit> .
so far, most studies have focused on shoot tissues.
in a few cases, lm has been used to isolate plant cells during interactions with other organisms including cyst or root-knot nematode feeding sites in soybean roots  <cit> .
in addition, fungus-plant interactions were assessed in maize by dissecting parenchyma cells infected with colletotrichum graminicola and the rna samples were used for analysis of c. graminicola gene expression  <cit> .
recently, this technology was used to isolate cells from arbuscular mycorrhizal or non-mycorrhizal tomato roots  <cit>  where it enabled the analysis of phosphate transporter gene expression in the plant and the fungus.
toward the ultimate goal of understanding how arbuscules develop, we initiated experiments to identify genes expressed in cortical cells containing arbuscules.
we first undertook a profiling experiment with a new m. truncatula genechip®, and then developed lm of m. truncatula root cortical cells to enable cell-type specific analyses of gene expression by rt-pcr.
this approach led to the identification of novel plant and fungal genes expressed in colonized cortical cells and in arbuscules, including members of the lysm-receptor like kinase family, gras transcription factor family, a novel ammonium transporter that is a strong candidate for mediating symbiotic ammonium transport, and am fungal genes of the urea cycle.
RESULTS
use of the medicago genechip® to monitor transcript profiles in m. truncatula colonized by glomus intraradices
previously in m. truncatula, a long oligonucleotide-based array containing  <dig>  features, was used to profile gene expression in a number of different am symbioses  <cit> .
while these studies provided important insights into gene expression during the am symbiosis, they surveyed only a small fraction of the m. truncatula transcriptome.
in order to expand these gene expression studies, we used the affymetrix medicago genechip®.
it contains  <dig>  probe sets of which  <dig>  were designed based on sequences from the m. truncatula est database and  <dig>  were based on gene predictions from the international medicago genome annotation group  .
the genechip® was hybridized with three biological replicate rna samples from m. truncatula and m. truncatula/g.
intraradices mycorrhizal roots and using significance criteria established previously  <cit> ,  <dig> genes that showed a 2-fold or greater change in gene expression in mycorrhizal roots relative to the mock-inoculated controls were identified.
of these,  <dig> genes showed increased transcript levels in g. intraradices-colonized roots, and  <dig> genes showed decreased transcript levels .
our previous experiments with the m. truncatula 16k array had identified  <dig> genes that showed a 2-fold or greater change in gene expression  <cit> .
<dig> induced genes were present in both data sets  <cit> .
thus, the genechip dataset has extended the transcriptional information available for m. truncatula/g.
intraradices mycorrhizal roots substantially.
new am-induced genes with potentially important roles in the symbiosis include an ammonium transporter , which shows a  <dig>  fold induction in mycorrhizal roots .
this transporter is a candidate for mediating symbiotic n transport.
other transporters including a zinc transporter , organic-cation transporter , and copper transport protein  were induced in mycorrhizal roots, along with genes encoding transporters of the peptide transporter family  and abc transporter family .
agenes tested by rt-pcr have different id
1expression ratio for genes from the medicago genechip® experiment that showed differential expression in m. truncatula roots colonized by g. intraradices relative to mock-inoculated control roots.
for complete data set see additional file  <dig>
carbon metabolism and carbon transport in the am symbiosis are also areas of great importance that are currently poorly understood.
a putative alpha-amylase gene  was up-regulated  <dig>  fold suggesting a potential increase in starch breakdown.
also, a putative snf1-related protein kinase gene  was up-regulated  <dig>  fold .
the snf <dig> and snf1-related kinases are widely conserved in eukaryotes and play a central role in sugar and energy signaling  <cit> .
two genes, one predicted to encode a protein involved in lipid biosynthesis, palmitoyl-acp thioesterase, and one encoding an enzyme of lipid breakdown, a triacylglycerol/steryl ester lipase  were highly up-regulated.
the latter showed a 102-fold increase in transcript levels .
this increase in both biosynthetic and degradative activities may be associated with the ongoing development and breakdown of the peri-arbuscular membrane associated with the different phases of the arbuscule lifespan.
currently, transcription factors that regulate am-induced gene expression are unknown.
here,  <dig> probesets representing members of the gras family of transcription factors  indicate that several members of this gene family are up-regulated more than 7-fold.
two gras family transcription factors are essential for nodulation  <cit> , and given the conserved symbiosis signaling pathway, there is a strong possibility that members of this family will be involved in regulating gene expression in am symbiosis also.
other genes related to signaling or perception of signal, include a putative inositol polyphosphate phosphatase  that was up-regulated  <dig> -fold .
inositol polyphosphate 5-phosphatases are involved in the modulation of phosphoinositide signaling.
in plants, inositol polyphosphate 5-phosphatases are members of large gene families and their individual roles are mostly unknown.
while this signaling pathway has not been implicated in the am symbiosis, lysophosphatidyl choline, was recently shown to induce expression of symbiotic pi transporters in potato and tomato  <cit> .
finally, one of the most intriguing findings was a gene encoding a lysine motif-receptor-like kinase  called mtlyr <dig>  <cit>  which showed a  <dig> -fold increase in mycorrhizal roots .
mtlyr <dig> is a close relative of nfp, a lysm-rlk gene that is likely a nod factor receptor  <cit> .
glomus intraradices genes are represented on the medicago genechip®
the medicago genechip® includes  <dig>  probes designed to sequences from the m. truncatula gene index .
the gene index includes ests from mycorrhizal root cdna libraries, consequently, there is the potential that some probes were designed, unintentionally, to ests that represent genes from the am fungal symbionts.
the discovery that some probes represent genes with blast hits exclusively to fungal sequences suggested that this may be the case.
therefore, we systematically mined the array data to identify probes that might represent am fungal genes.
probes that showed an average expression value on the mock-inoculated arrays near background levels  and a higher average expression value on g. intraradices-inoculated arrays, and corresponded to ests present exclusively in mycorrhizal cdna libraries, were considered strong candidates.
subsequent tblastx analysis indicated that  <dig> probesets with the criteria outlined above represented genes that showed best blast hits to fungal sequences .
there were additional probes designed to ests that showed similarity to fungal sequences but the ests were short and the blast scores were unreliable, so they were excluded from further analysis.
of the  <dig> genes identified as putative fungal genes,  <dig> are represented by a single est, and all are present exclusively in m. truncatula/g.
intraradices mycorrhizal root cdna libraries and are therefore likely to represent g. intraradices genes.
experimental analysis of ten genes confirmed that they are g. intraradices genes.
they could be amplified by pcr from g. intraradices genomic dna and not from m. truncatula genomic dna .
except for the putative g. intraradices a-tubulin a <dig> gene, which is highly conserved, the primers designed to the g. intraradices genes did not amplify transcripts from g. versiforme.
it has been shown that despite a common genus name, these two fungi are only distantly related  <cit> .
eight of these genes  were represented on the 16k array although their origin was not demonstrated  <cit> .
previous estimates suggest that total rna extracted from a highly colonized, whole root system contains not more than 12% am fungal rna  <cit> .
consequently, it might be predicted that the fungal transcripts represented in the est libraries and consequently represented on the genechip, are likely to represent the most highly expressed fungal genes.
interestingly, the fungal genes represented on the array include genes predicted to encode enzymes of n metabolism, specifically, the urea cycle .
development of a laser microdissection procedure suitable for the capture of cortical cells from m. truncatula mycorrhizal roots
to enable lm of cortical cells containing arbuscules, the first issue was to identify suitable regions of the root system to fix and embed.
in an am symbiosis, the fungus proliferates in the inner cortex of the root and in m. truncatula mycorrhizal roots, the colonized regions are not readily visible without fixing and staining.
in particular, it is difficult to identify regions of the root with infections that contain large numbers of arbuscules.
to facilitate this, our goal was to first develop a m. truncatula transgenic plant line in which the colonized regions of the root would be marked by expression of green fluorescent protein .
previously, we had identified mtscp <dig>  a gene that is expressed only in mycorrhizal roots, specifically in colonized regions of the roots  <cit> .
analysis of an mtscp <dig> promoter-gfp fusion construct in m. truncatula transgenic roots indicated that the construct reported the presence of the am fungus in the cortex  <cit> .
the mtscp <dig> promoter-gfp fusion construct was transferred to agrobacterium tumefaciens and m. truncatula transgenic plants were generated.
the transgenic plants showed the same expression patterns as seen previously and strong gfp fluorescence reported the presence of the fungus in the cortex .
using the transgenic plants, we were able to identify and dissect out highly colonized regions of the root.
these were then used for lm experiments.
in addition, rna extracted from these intact colonized root pieces is highly enriched for plant and fungal transcripts associated with colonized regions of the root, and are useful rna resource.
a review of lm and its use in plants, revealed that a wide variety of protocols have been used for the preparation of plant tissue for lm  <cit> .
each different plant tissue has unique characteristics and therefore it is necessary to determine empirically, the most appropriate fixation and embedding regime to retain satisfactory cellular morphology, while still enabling extraction of high rna quality.
we based our approach on a protocol developed by kerk et al.
<cit>  and then evaluated aspects of fixation, embedding, rna extraction and amplification to find conditions optimal for m. truncatula mycorrhizal roots.
rna quality was evaluated using a bioanalyzer and/or agarose gel electrophoresis.
tissues were fixed in farmer's fixative and fixation times of  <dig> or  <dig> h yielded rna with an rna integrity number  of  <dig>   as assessed by a bioanalyzer.
in addition, fixation at  <dig> and  <dig> hours yielded larger size amplified, antisense rna  molecules, than a  <dig> h fixation .
we experienced considerable difficulties with rna degradation during the paraffin embedding and xylene de-waxing stages, so as an alternative to paraffin wax, we tested steedman's wax, a polyester wax used for immunolocalization  <cit> .
steedman's wax has the advantages that it can be mixed under rnase-free conditions, has a lower melting temperature , and sections can be de-waxed easily with ethanol.
the rna quality extracted from root samples embedded in steedman's was good  and the morphology of the sections were comparable to sections made from tissues embedded in paraffin wax .
lm and laser pressure catapulting was carried out with the p.a.l.m.
microbeam system.
between  <dig> and  <dig> cortical cells were harvested directly into lysis buffer and rna was isolated within  <dig> hours of harvest.
two rounds of linear rna amplification yielded over  <dig> μg of arna .
to validate the lm rna, we examined the expression of mtpt <dig>  a gene expressed exclusively in cells containing arbuscules.
in addition, we assessed expression of mtscp <dig>  and mtbcp <dig>  two genes expressed in this cell type that are suitable as additional markers.
the latter two genes are not unique to colonized cortical cells  <cit> .
mtpt <dig>  mtscp <dig>  and mtbcp <dig> transcripts were detected in two biological replicate samples of rna from cells with arbuscules and not from cortical cells captured from mock-inoculated roots.
elongation factor 1-alpha  transcripts were present in rna samples from both cell types .
the length of individual molecules present in the arna sample was evaluated by examining mtpt <dig> transcripts  <cit> .
cdna was synthesized using random priming and the length of molecules in the samples was evaluated by rt-pcr using primers distributed across the mtpt <dig> gene.
although we were unable to amplify the intact  <dig>  kb mtpt <dig> transcript, primers sets that amplified less than  <dig> bp fragments at the 5' end, middle, or 3' end of the coding sequence showed that the arna represents complete transcripts but not necessarily as single intact molecules .
m. truncatula gene expression in cortical cells containing g. versiforme and g. intraradices arbuscules
following development and validation of the lm method, we generated cell-type specific rna samples from cortical cells of m. truncatula mock-inoculated roots, and cortical cells containing either g. versiforme or g. intraradices arbuscules.
in addition, we prepared rna from the highly-colonized 'mycorrhiza-enriched' root pieces from g. versiforme-colonized roots.
genes selected for expression analysis in these samples included  <dig> genes that were previously reported as mycorrhiza-induced, but whose spatial expression patterns were not known, and  <dig> novel genes that showed increased transcript levels in the genechip® experiment .
initially, transcript levels were monitored in the rna from the intact, enriched root piece samples.
all  <dig> new genes showed an increase in transcript levels in the rna from enriched mycorrhizal root samples relative to that of the mock-inoculated controls confirming the expression patterns observed on the array .
this was observed for both m. truncatula roots colonized with g. versiforme and g. intraradices.
transcripts for  <dig> of these genes were extremely low or not detectable in mock-inoculated roots.
in the rna samples from laser microdissected cells,  <dig> of  <dig> genes assayed showed expression in cortical cells containing arbuscules and not in the cortical cells from mock-inoculated control roots .
for  <dig> genes, transcripts were detected in only one of the g. versiforme arbuscule-cortical cell samples indicating some variability in the samples.
however, analysis in g. intraradices arbuscule-cortical cell samples confirmed the expression in this cell-type .
it is possible that sample variability arises from variation in the developmental stages of the arbuscules in the sample.
this could lead to differences in the samples and would likely have the greatest effects on low abundance transcripts or on genes whose expression is associated with a specific phase of arbuscule development.
using this approach, transcripts of two putative transcription factors of the gras family, a peptide transporter, an ammonium transporter, three genes associated with lipid biosynthesis or degradation and a lysm-receptor like kinase were detected in cortical cells containing arbuscules and were not detected in cortical cells from the mock-inoculated controls.
four mycorrhiza-induced genes, the putative copper transport protein, dihydrodipicolinate synthase, inositol polyphosphate phosphatase and an snf1-like kinase were not detected in cortical cells containing arbuscules.
as transcripts of the latter  <dig> genes were detected in the colonized root pieces , it can be concluded that they are expressed in mycorrhizal roots but in a cell type other than the cortical cells containing arbuscules.
analysis of g. intraradices gene expression in arbuscules
the expression of  <dig> g. intraradices genes identified on the arrays was evaluated in rna from both the colonized root pieces and the lm cell-type specific samples .
the genes selected for analysis included those predicted to encode enzymes of n metabolism, in particular the urea cycle including a putative arginase gene.
consistent with the genechip results, transcripts for all  <dig> genes were present in m. truncatula/g.
intraradices mycorrhizal roots samples.
in addition, all genes tested showed expression in arbuscules.
this suggests that the urea cycle is likely to be active in this tissue.
previous nmr studies had predicted this but a direct demonstration of the presence of transcripts in arbuscules had not been made.
in addition to genes associated with n and amino acid metabolism, we also detected a putative g. intraradices aut <dig> homolog and a putative phospholipids-transporting atpase in arbuscules.
aut <dig> is part of the autophagic machinery and is considered a marker of cells undergoing an autophagic response.
the presence of this transcript in arbuscules is the first molecular hint that arbuscule turnover might involve autophagy.
the phospholipid-transporting atpases are p-type atpases whose activity is required to maintain phospholipid asymmetry in the membrane bilayers  <cit> .
in magnaporthe grisea, the activity of a phospholipid-transporting atpase was required for development of the penetration hypha and further growth of the fungus within the plant cells  <cit> .
discussion
here, we used the medicago genechip® to extend the transcript dataset for mycorrhizal roots and coupled this with lm and rt-pcr to assess gene expression specifically at the cortical cell-arbuscule interface.
at the time that this work was initiated, there were only a few reports of lm of root cells or roots associated with other organisms, consequently, it was necessary to test and modify existing protocols to develop a protocol suitable for m. truncatula mycorrhizal roots.
the protocol developed was based largely on kerk et al.
<cit>  and the main modification was the nature of the embedding material.
following initial difficulties with the quality of the rna extracted from paraffin-embedded tissues, we tested steedman's wax with the idea that rna degradation would be minimized by exposing roots to lower temperatures during embedding, and the use of ethanol for de-waxing would be less detrimental than xylene.
this proved to be the case and this low-melting-temperature, ethanol-soluble wax enabled us to extract rna from m. truncatula roots of superior quality.
lm of approximately  <dig> m. truncatula root cortical cells, followed by two rounds of linear rna amplification provided microgram quantities of rna for rt-pcr analyses.
these cell-type specific rna samples were validated by monitoring the expression of three genes, mtpt <dig>  mtscp <dig> and mtbcp <dig>  whose expression patterns have been described previously  <cit> .
recently, balestrini et al.
<cit>  reported the use of methacarn fixation and paraffin embedding for lm of cells from tomato roots colonized by the am fungus g. mosseae.
however, they did not couple this with linear rna amplification and consequently, it was necessary to harvest a large quantity of cells, between  <dig>  and  <dig>  cells to obtain nanogram quantities of rna.
although the linear rna amplification used in our approach may lead to a 3' end bias in the transcript molecules, comparative analyses are possible with careful primer design.
the genechip analysis provided a greatly expanded view of genes induced in the am symbiosis, including several transcription factors and receptors.
as reported previously, development of the am symbiosis is accompanied by significant alterations in gene expression including the expression of novel 'mycorrhiza-specific' genes.
surprisingly, transcription factors responsible for mycorrhiza-regulated gene expression are entirely unknown.
here we identified  <dig> members of the gras transcription factor family induced in mycorrhizal roots .
the gras family of proteins is unique to plants and its name derives from three members that were initially identified gai, rga, and scr  <cit> .
the arabidopsis gras family contains at least  <dig> members including scarecrow and short root, two proteins that play a central role in root development  transcripts increased in g. versiforme or g. intraradices-colonized roots.
moreover, the mtlyr <dig> gene was detected in cortical cells with arbuscules.
like nfp, mtlyr <dig> has three lysm domains and a ser-thr kinase domain that lacks the activation and p-loops and consequently, it probably lacks kinase activity  <cit> .
it is possible that these 'inactive' kinases dimerize with an active receptor kinase to form a receptor with kinase activity and consistent with this, heterodimerization of nfr <dig> and nfr <dig> has been demonstrated.
lysm domains recognize n-acetyl glucosamine or structurally similar molecules such as nod factors  <cit> .
it is possible that the lysm domain of mtlyr <dig> recognizes the n-acetyl glucosamine residues in chitin in the am fungal cell walls and given its expression pattern, it could play a role in signaling during arbuscule development.
cell biological analyses of the arbuscule-cortical cell interface suggest that the peri-arbuscular membrane increases the membrane area within the cortical cell at least 10-fold.
consistent with this, metabolite profiles of mycorrhizal roots indicate a significant increase in fatty acids  <cit> .
the genechip analyses identified two new mycorrhiza-induced genes predicted to be involved in fatty acid metabolism; a putative palmitoyl-acp thioesterase gene  and a triacylglycerol/steryl ester lipase-like protein gene  that show a 49- and 102-fold increase in transcript levels respectively.
rt-pcr analysis indicated that tc <dig> and tc <dig> transcript levels increase in cortical cells with arbuscules.
fatty acid synthesis in plants is a cyclic process and the extension cycles involves the binding of acyl chains to a soluble acyl carrier protein , and specific thioesterases hydrolyze the newly formed acyl-acp into fatty acids and acp  <cit> .
an increase in palmitoyl-acp thioesterase gene expression suggests an increase in the biosynthesis of palmitic acid.
consistent with this, a recent metabolite profile of g. intraradices/m.
truncatula roots showed that mycorrhizal roots contained high levels of palmitic and oleic acids during active phases of am symbiosis  <cit> .
oleic acid  is created by a desaturation reaction catalyzed by a delta- <dig> desaturase.
surprisingly, a g. intraradices delta- <dig> desaturase gene was expressed highly in mycorrhizal roots and specifically in cells with arbuscules.
previous studies had speculated that the fungus might obtain palmitic acid from the plant, and even suggested that am fungi might lack fatty acid synthase activity and thus be dependent on host lipid  <cit> .
in the absence of a full genome sequence, it is not possible to determine whether this is correct, however, our findings of complementary gene expression patterns in the two symbionts is intriguing.
other metabolic changes documented in mycorrhizal roots include an increase in amino acids  <cit> .
in plants, there are at least three protein families involved in transport of small peptides, the abc transporters, the di/tripeptide transporters  and the oligopeptide  family.
two genes  of the ptr family were expressed in mycorrhizal roots and tc <dig> was detected in cells containing arbuscules.
the genechip experiment also led to the identification of other new genes predicted to encode transport proteins including a copper transport protein  and an ammonium transporter  and the latter was expressed in cells with arbuscules.
the identification of a mycorrhiza-induced ammonium transporter is particularly interesting.
it is clear from many studies that in addition to pi, n is also transferred from the fungus to the plant and recent analyses indicate that n is likely transferred to the plant as ammonium.
this ammonium transporter is a strong candidate for a role in the acquisition of ammonium delivered via the fungus.
metabolic labeling studies and gene expression analyses led to a model in which inorganic n taken up by the fungal extra-radical mycelium is converted into arginine which is transported into the intra-radical mycelium, where it is broken down into amino acids.
ammonium, resulting from arginine breakdown is proposed to be exported to the plant  <cit> .
in further support of the labeling studies, govindarajulu et al.
<cit>  showed elevated transcripts of g. intraradices ornithine aminotransferase, a urease accessory protein and an ammonium transporter in the intra-radical mycelium relative to the extra-radical mycelium.
interestingly, the g. intraradices genes present on the m. truncatula array include genes predicted to encode enzymes of the urea cycle as well as amino acid metabolism.
rt-pcr analysis showed that these genes were expressed in g. intraradices-mycorrhizal roots, and analysis of the cell-type specific samples, indicates that they are expressed in the arbuscules.
these data extend the previous findings, showing that the genes of urea and amino acid metabolism are expressed in the arbuscules.
in addition, our data set includes a gene predicted to encode arginase that breaks down arginine to release ornithine and urea.
this is a key step for the release of ammonia and further supports the model proposed by govindarajulu et al.
<cit> .
in addition to the urea cycle, we detected g. intraradices asparagine synthetase and glutamine synthetase gene expression in arbuscules.
this suggests that ornithine, released via arginase activity, may be cycled into asparagine and glutamine through the action of asparagine synthetase and glutamine synthetase.
interestingly, the metabolite profiling studies found higher levels of specific amino acids, including glu, asp, and asn in mycorrhizal roots  <cit> .
from these studies, it is not possible to determine whether the amino acids are in the plant or am fungus, however, coupled with the gene expression studies, there is evidence to suggest that at least some of this amino acid accumulation may result from activities in the fungus.
CONCLUSIONS
using a combination of arrays, lm and rt-pcr, we have extended the m. truncatula/g.
intraradices mycorrhizal root transcriptome and identified novel m. truncatula and g. intraradices genes that are expressed in cortical cells harboring arbuscules.
by virtue of their expression patterns, the genes expressed in this cell type are candidates for mediating aspects of development or function of the symbiotic arbuscule-cortical cell interface.
novel genes potentially involved in signaling and in n transport and metabolism are of particular interest.
this dataset will be valuable for future studies aimed at determining the molecular events that control arbuscule development, and n transport and metabolism in the am symbiosis.

 the arbuscular mycorrhiza is the name given to a symbiotic association of plants and arbuscular mycorrhizal fungi. the am symbiosis occurs widely throughout the plant kingdom and involves angiosperms, gymnosperms, pteridophytes and some bryophytes. the fungi that participate in this symbiosis are all members of the glomeromycota,, a likely sister group to the ascomycota and basidiomycota. the symbiosis develops in the plant roots, where am fungi form extensively branched hyphae, called arbuscules, in the cortical cells. in addition to growth within the root, the fungus develops a network of extra-radical hyphae that extends into the rhizosphere. inorganic phosphate and nitrogen, acquired by the extra-radical hyphae, are translocated to the arbuscules and released to the plant. in return the plant provides the fungus with carbon. p and n are the two mineral nutrients that plants require in the greatest quantities and therefore the symbiosis has an important influence on plant health and consequently on ecosystem function. there has been significant progress in understanding the signaling associated with the early stages of development of am symbiosis. some of the signal molecules have been identified, including strigolactones, which act as pre-contact signals to stimulate am fungal growth and metabolism. in addition, several components of a plant'symbiosis signaling' pathway required for fungal entry into the root have been cloned. these genes were identified initially in legumes, where the symbiosis signaling pathway is required also for the interaction with rhizobia. however, orthologs of these genes have been identified in tomato and rice, where they are required for am symbiosis. this further supports the hypothesis that the symbiosis signaling pathway arose initially for interactions with am fungi, and in legumes it was co-opted to enable interactions with rhizobia. currently, for am symbiosis, the downstream genes controlled by this pathway are not known. in general, events associated with fungal development in the cortex are less well understood, and genes controlling arbuscule formation, have yet to be identified. arbuscule development is an active process for both symbionts. the fungal hypha penetrates the cortical cell wall and undergoes repeated dichotomous branching, while the plant cell undergoes significant cellular changes, including reorganization of the cytoskeleton, endoplasmic reticulum and development of the peri-arbuscular membrane, which envelops the arbuscule. reorganization of the cytoskeleton and endoplasmic reticulum is initiated prior to arbuscule formation suggesting that a mobile signal may trigger these events. currently, information about the lipid and protein content of the peri-arbuscular membrane is limited, but it is clear that the membrane contains some unique proteins, including symbiosis-specific pi transporters that function to import pi into the cortical cell. the transporters involved in n acquisition are as yet unknown, but a series of nuclear magnetic resonance spectroscopy and gene expression studies indicate that the fungus assimilates n in the external hyphae, translocates this to the intra-radical hyphae as arginine, and then disassembles the arginine to retain the carbon molecules, while providing n to the plant, probably as ammonium. although not shown directly, it is likely that nh4+ transfer to the plant occurs at the arbuscule-cortical cell interface also. arbuscules are terminally differentiated hyphae with a finite life and following development, they undergo a degeneration phase in which the arbuscule branches collapse, and the arbuscule gradually disappears from the cell. this process is not well understood, but it is clear that arbuscule development and degeneration occur cell autonomously and arbuscules in neighboring cells may be in different phases of the life cycle. although the arbuscule-cortical cell interface is central to the am symbiosis, currently, we understand little of the processes that enable its development or that control its degeneration. to gain insight into the molecular changes that accompany development of am symbiosis, a variety of transcript profiling approaches have been undertaken. in rice, surveys using a whole genome chip identified mycorrhiza-responsive genes and revealed a partial overlap in the transcriptional responses to mutualistic and pathogenic biotrophic fungi. in medicago truncatula, suppressive subtractive hybridization, cdna arrays and long oligonucleotide arrays have been used to document transcript changes associated with am symbiosis. the inclusion of m. truncatula symbiosis mutants enabled gene expression to be associated with different phases of the symbiosis, for example appressoria formation. a mycorrhizal root includes a heterogeneous mix of colonized and non-colonized cells, and therefore spatial gene expression information is particularly important. in situ hybridization and/or analysis of promoter-reporter fusions demonstrated that genes whose expression was induced in colonized roots could be further separated into two groups; those expressed exclusively in cells containing arbuscules and those expressed in cells containing arbuscules and in neighboring non-colonized cells. these data indicate that the different cortical cells of the mycorrhizal root system have unique transcript profiles, and point to at least two signaling pathways active in regulating mycorrhiza-induced gene expression. while these approaches have advanced information about gene expression in colonized cortical cells, in situ hybridization or promoter-reporter gene studies are labor intensive and this precludes surveys of a large number of genes. our current knowledge of genes expressed in cells containing arbuscules is limited to less than genes. in addition, the genes that have been analyzed are those that have the highest transcript levels and relatively few receptors or transcriptional regulators have been investigated. beyond plant gene expression in the colonized cortical cell, almost nothing is known about gene expression in the arbuscule. laser microdissection offers a new way to obtain rna from a subset of cells, and coupled with transcript profiling approaches, it provides an opportunity to monitor gene expression in an individual cell type more easily than promoter-reporter gene fusions. lm was originally developed to isolate specific animal cells from complex tissues; a breakthrough for cancer and neuronal research. in plants, lm was used initially to isolate phloem cells from rice leaves. subsequently, epidermal and vascular tissues were isolated from maize coleoptiles and then various cell or tissue types, including bundle sheath, shoot tip protoderm, shoot apical meristem, leaf primordium, seedling procambium, hypocotyls and root meristems have been harvested from maize, rice, radish and tomato. so far, most studies have focused on shoot tissues. in a few cases, lm has been used to isolate plant cells during interactions with other organisms including cyst or root-knot nematode feeding sites in soybean roots. in addition, fungus-plant interactions were assessed in maize by dissecting parenchyma cells infected with colletotrichum graminicola and the rna samples were used for analysis of c. graminicola gene expression. recently, this technology was used to isolate cells from arbuscular mycorrhizal or non-mycorrhizal tomato roots where it enabled the analysis of phosphate transporter gene expression in the plant and the fungus. toward the ultimate goal of understanding how arbuscules develop, we initiated experiments to identify genes expressed in cortical cells containing arbuscules. we first undertook a profiling experiment with a new m. truncatula genechip®, and then developed lm of m. truncatula root cortical cells to enable cell-type specific analyses of gene expression by rt-pcr. this approach led to the identification of novel plant and fungal genes expressed in colonized cortical cells and in arbuscules, including members of the lysm-receptor like kinase family, gras transcription factor family, a novel ammonium transporter that is a strong candidate for mediating symbiotic ammonium transport, and am fungal genes of the urea cycle.  use of the medicago genechip® to monitor transcript profiles in m. truncatula colonized by glomus intraradices previously in m. truncatula, a long oligonucleotide-based array containing features, was used to profile gene expression in a number of different am symbioses. while these studies provided important insights into gene expression during the am symbiosis, they surveyed only a small fraction of the m. truncatula transcriptome. in order to expand these gene expression studies, we used the affymetrix medicago genechip®. it contains probe sets of which were designed based on sequences from the m. truncatula est database and were based on gene predictions from the international medicago genome annotation group. the genechip® was hybridized with three biological replicate rna samples from m. truncatula and m. truncatula/g. intraradices mycorrhizal roots and using significance criteria established previously, genes that showed a 2-fold or greater change in gene expression in mycorrhizal roots relative to the mock-inoculated controls were identified. of these, genes showed increased transcript levels in g. intraradices-colonized roots, and genes showed decreased transcript levels. our previous experiments with the m. truncatula 16k array had identified genes that showed a 2-fold or greater change in gene expression. induced genes were present in both data sets. thus, the genechip dataset has extended the transcriptional information available for m. truncatula/g. intraradices mycorrhizal roots substantially. new am-induced genes with potentially important roles in the symbiosis include an ammonium transporter, which shows a fold induction in mycorrhizal roots. this transporter is a candidate for mediating symbiotic n transport. other transporters including a zinc transporter, organic-cation transporter, and copper transport protein were induced in mycorrhizal roots, along with genes encoding transporters of the peptide transporter family and abc transporter family. agenes tested by rt-pcr have different id 1expression ratio for genes from the medicago genechip® experiment that showed differential expression in m. truncatula roots colonized by g. intraradices relative to mock-inoculated control roots. for complete data set see additional file carbon metabolism and carbon transport in the am symbiosis are also areas of great importance that are currently poorly understood. a putative alpha-amylase gene was up-regulated fold suggesting a potential increase in starch breakdown. also, a putative snf1-related protein kinase gene was up-regulated fold. the snf and snf1-related kinases are widely conserved in eukaryotes and play a central role in sugar and energy signaling. two genes, one predicted to encode a protein involved in lipid biosynthesis, palmitoyl-acp thioesterase, and one encoding an enzyme of lipid breakdown, a triacylglycerol/steryl ester lipase were highly up-regulated. the latter showed a 102-fold increase in transcript levels. this increase in both biosynthetic and degradative activities may be associated with the ongoing development and breakdown of the peri-arbuscular membrane associated with the different phases of the arbuscule lifespan. currently, transcription factors that regulate am-induced gene expression are unknown. here, probesets representing members of the gras family of transcription factors indicate that several members of this gene family are up-regulated more than 7-fold. two gras family transcription factors are essential for nodulation, and given the conserved symbiosis signaling pathway, there is a strong possibility that members of this family will be involved in regulating gene expression in am symbiosis also. other genes related to signaling or perception of signal, include a putative inositol polyphosphate phosphatase that was up-regulated -fold. inositol polyphosphate 5-phosphatases are involved in the modulation of phosphoinositide signaling. in plants, inositol polyphosphate 5-phosphatases are members of large gene families and their individual roles are mostly unknown. while this signaling pathway has not been implicated in the am symbiosis, lysophosphatidyl choline, was recently shown to induce expression of symbiotic pi transporters in potato and tomato. finally, one of the most intriguing findings was a gene encoding a lysine motif-receptor-like kinase called mtlyr which showed a -fold increase in mycorrhizal roots. mtlyr is a close relative of nfp, a lysm-rlk gene that is likely a nod factor receptor. glomus intraradices genes are represented on the medicago genechip® the medicago genechip® includes probes designed to sequences from the m. truncatula gene index. the gene index includes ests from mycorrhizal root cdna libraries, consequently, there is the potential that some probes were designed, unintentionally, to ests that represent genes from the am fungal symbionts. the discovery that some probes represent genes with blast hits exclusively to fungal sequences suggested that this may be the case. therefore, we systematically mined the array data to identify probes that might represent am fungal genes. probes that showed an average expression value on the mock-inoculated arrays near background levels and a higher average expression value on g. intraradices-inoculated arrays, and corresponded to ests present exclusively in mycorrhizal cdna libraries, were considered strong candidates. subsequent tblastx analysis indicated that probesets with the criteria outlined above represented genes that showed best blast hits to fungal sequences. there were additional probes designed to ests that showed similarity to fungal sequences but the ests were short and the blast scores were unreliable, so they were excluded from further analysis. of the genes identified as putative fungal genes, are represented by a single est, and all are present exclusively in m. truncatula/g. intraradices mycorrhizal root cdna libraries and are therefore likely to represent g. intraradices genes. experimental analysis of ten genes confirmed that they are g. intraradices genes. they could be amplified by pcr from g. intraradices genomic dna and not from m. truncatula genomic dna. except for the putative g. intraradices a-tubulin a gene, which is highly conserved, the primers designed to the g. intraradices genes did not amplify transcripts from g. versiforme. it has been shown that despite a common genus name, these two fungi are only distantly related. eight of these genes were represented on the 16k array although their origin was not demonstrated. previous estimates suggest that total rna extracted from a highly colonized, whole root system contains not more than 12% am fungal rna. consequently, it might be predicted that the fungal transcripts represented in the est libraries and consequently represented on the genechip, are likely to represent the most highly expressed fungal genes. interestingly, the fungal genes represented on the array include genes predicted to encode enzymes of n metabolism, specifically, the urea cycle. development of a laser microdissection procedure suitable for the capture of cortical cells from m. truncatula mycorrhizal roots to enable lm of cortical cells containing arbuscules, the first issue was to identify suitable regions of the root system to fix and embed. in an am symbiosis, the fungus proliferates in the inner cortex of the root and in m. truncatula mycorrhizal roots, the colonized regions are not readily visible without fixing and staining. in particular, it is difficult to identify regions of the root with infections that contain large numbers of arbuscules. to facilitate this, our goal was to first develop a m. truncatula transgenic plant line in which the colonized regions of the root would be marked by expression of green fluorescent protein. previously, we had identified mtscp a gene that is expressed only in mycorrhizal roots, specifically in colonized regions of the roots. analysis of an mtscp promoter-gfp fusion construct in m. truncatula transgenic roots indicated that the construct reported the presence of the am fungus in the cortex. the mtscp promoter-gfp fusion construct was transferred to agrobacterium tumefaciens and m. truncatula transgenic plants were generated. the transgenic plants showed the same expression patterns as seen previously and strong gfp fluorescence reported the presence of the fungus in the cortex. using the transgenic plants, we were able to identify and dissect out highly colonized regions of the root. these were then used for lm experiments. in addition, rna extracted from these intact colonized root pieces is highly enriched for plant and fungal transcripts associated with colonized regions of the root, and are useful rna resource. a review of lm and its use in plants, revealed that a wide variety of protocols have been used for the preparation of plant tissue for lm. each different plant tissue has unique characteristics and therefore it is necessary to determine empirically, the most appropriate fixation and embedding regime to retain satisfactory cellular morphology, while still enabling extraction of high rna quality. we based our approach on a protocol developed by kerk et al. and then evaluated aspects of fixation, embedding, rna extraction and amplification to find conditions optimal for m. truncatula mycorrhizal roots. rna quality was evaluated using a bioanalyzer and/or agarose gel electrophoresis. tissues were fixed in farmer's fixative and fixation times of or h yielded rna with an rna integrity number of as assessed by a bioanalyzer. in addition, fixation at and hours yielded larger size amplified, antisense rna molecules, than a h fixation. we experienced considerable difficulties with rna degradation during the paraffin embedding and xylene de-waxing stages, so as an alternative to paraffin wax, we tested steedman's wax, a polyester wax used for immunolocalization. steedman's wax has the advantages that it can be mixed under rnase-free conditions, has a lower melting temperature, and sections can be de-waxed easily with ethanol. the rna quality extracted from root samples embedded in steedman's was good and the morphology of the sections were comparable to sections made from tissues embedded in paraffin wax. lm and laser pressure catapulting was carried out with the p.a.l.m. microbeam system. between and cortical cells were harvested directly into lysis buffer and rna was isolated within hours of harvest. two rounds of linear rna amplification yielded over μg of arna. to validate the lm rna, we examined the expression of mtpt a gene expressed exclusively in cells containing arbuscules. in addition, we assessed expression of mtscp and mtbcp two genes expressed in this cell type that are suitable as additional markers. the latter two genes are not unique to colonized cortical cells. mtpt mtscp and mtbcp transcripts were detected in two biological replicate samples of rna from cells with arbuscules and not from cortical cells captured from mock-inoculated roots. elongation factor 1-alpha transcripts were present in rna samples from both cell types. the length of individual molecules present in the arna sample was evaluated by examining mtpt transcripts. cdna was synthesized using random priming and the length of molecules in the samples was evaluated by rt-pcr using primers distributed across the mtpt gene. although we were unable to amplify the intact kb mtpt transcript, primers sets that amplified less than bp fragments at the 5' end, middle, or 3' end of the coding sequence showed that the arna represents complete transcripts but not necessarily as single intact molecules. m. truncatula gene expression in cortical cells containing g. versiforme and g. intraradices arbuscules following development and validation of the lm method, we generated cell-type specific rna samples from cortical cells of m. truncatula mock-inoculated roots, and cortical cells containing either g. versiforme or g. intraradices arbuscules. in addition, we prepared rna from the highly-colonized'mycorrhiza-enriched' root pieces from g. versiforme-colonized roots. genes selected for expression analysis in these samples included genes that were previously reported as mycorrhiza-induced, but whose spatial expression patterns were not known, and novel genes that showed increased transcript levels in the genechip® experiment. initially, transcript levels were monitored in the rna from the intact, enriched root piece samples. all new genes showed an increase in transcript levels in the rna from enriched mycorrhizal root samples relative to that of the mock-inoculated controls confirming the expression patterns observed on the array. this was observed for both m. truncatula roots colonized with g. versiforme and g. intraradices. transcripts for of these genes were extremely low or not detectable in mock-inoculated roots. in the rna samples from laser microdissected cells, of genes assayed showed expression in cortical cells containing arbuscules and not in the cortical cells from mock-inoculated control roots. for genes, transcripts were detected in only one of the g. versiforme arbuscule-cortical cell samples indicating some variability in the samples. however, analysis in g. intraradices arbuscule-cortical cell samples confirmed the expression in this cell-type. it is possible that sample variability arises from variation in the developmental stages of the arbuscules in the sample. this could lead to differences in the samples and would likely have the greatest effects on low abundance transcripts or on genes whose expression is associated with a specific phase of arbuscule development. using this approach, transcripts of two putative transcription factors of the gras family, a peptide transporter, an ammonium transporter, three genes associated with lipid biosynthesis or degradation and a lysm-receptor like kinase were detected in cortical cells containing arbuscules and were not detected in cortical cells from the mock-inoculated controls. four mycorrhiza-induced genes, the putative copper transport protein, dihydrodipicolinate synthase, inositol polyphosphate phosphatase and an snf1-like kinase were not detected in cortical cells containing arbuscules. as transcripts of the latter genes were detected in the colonized root pieces, it can be concluded that they are expressed in mycorrhizal roots but in a cell type other than the cortical cells containing arbuscules. analysis of g. intraradices gene expression in arbuscules the expression of g. intraradices genes identified on the arrays was evaluated in rna from both the colonized root pieces and the lm cell-type specific samples. the genes selected for analysis included those predicted to encode enzymes of n metabolism, in particular the urea cycle including a putative arginase gene. consistent with the genechip results, transcripts for all genes were present in m. truncatula/g. intraradices mycorrhizal roots samples. in addition, all genes tested showed expression in arbuscules. this suggests that the urea cycle is likely to be active in this tissue. previous nmr studies had predicted this but a direct demonstration of the presence of transcripts in arbuscules had not been made. in addition to genes associated with n and amino acid metabolism, we also detected a putative g. intraradices aut homolog and a putative phospholipids-transporting atpase in arbuscules. aut is part of the autophagic machinery and is considered a marker of cells undergoing an autophagic response. the presence of this transcript in arbuscules is the first molecular hint that arbuscule turnover might involve autophagy. the phospholipid-transporting atpases are p-type atpases whose activity is required to maintain phospholipid asymmetry in the membrane bilayers. in magnaporthe grisea, the activity of a phospholipid-transporting atpase was required for development of the penetration hypha and further growth of the fungus within the plant cells. discussion here, we used the medicago genechip® to extend the transcript dataset for mycorrhizal roots and coupled this with lm and rt-pcr to assess gene expression specifically at the cortical cell-arbuscule interface. at the time that this work was initiated, there were only a few reports of lm of root cells or roots associated with other organisms, consequently, it was necessary to test and modify existing protocols to develop a protocol suitable for m. truncatula mycorrhizal roots. the protocol developed was based largely on kerk et al. and the main modification was the nature of the embedding material. following initial difficulties with the quality of the rna extracted from paraffin-embedded tissues, we tested steedman's wax with the idea that rna degradation would be minimized by exposing roots to lower temperatures during embedding, and the use of ethanol for de-waxing would be less detrimental than xylene. this proved to be the case and this low-melting-temperature, ethanol-soluble wax enabled us to extract rna from m. truncatula roots of superior quality. lm of approximately m. truncatula root cortical cells, followed by two rounds of linear rna amplification provided microgram quantities of rna for rt-pcr analyses. these cell-type specific rna samples were validated by monitoring the expression of three genes, mtpt mtscp and mtbcp whose expression patterns have been described previously. recently, balestrini et al. reported the use of methacarn fixation and paraffin embedding for lm of cells from tomato roots colonized by the am fungus g. mosseae. however, they did not couple this with linear rna amplification and consequently, it was necessary to harvest a large quantity of cells, between and cells to obtain nanogram quantities of rna. although the linear rna amplification used in our approach may lead to a 3' end bias in the transcript molecules, comparative analyses are possible with careful primer design. the genechip analysis provided a greatly expanded view of genes induced in the am symbiosis, including several transcription factors and receptors. as reported previously, development of the am symbiosis is accompanied by significant alterations in gene expression including the expression of novel'mycorrhiza-specific' genes. surprisingly, transcription factors responsible for mycorrhiza-regulated gene expression are entirely unknown. here we identified members of the gras transcription factor family induced in mycorrhizal roots. the gras family of proteins is unique to plants and its name derives from three members that were initially identified gai, rga, and scr. the arabidopsis gras family contains at least members including scarecrow and short root, two proteins that play a central role in root development transcripts increased in g. versiforme or g. intraradices-colonized roots. moreover, the mtlyr gene was detected in cortical cells with arbuscules. like nfp, mtlyr has three lysm domains and a ser-thr kinase domain that lacks the activation and p-loops and consequently, it probably lacks kinase activity. it is possible that these'inactive' kinases dimerize with an active receptor kinase to form a receptor with kinase activity and consistent with this, heterodimerization of nfr and nfr has been demonstrated. lysm domains recognize n-acetyl glucosamine or structurally similar molecules such as nod factors. it is possible that the lysm domain of mtlyr recognizes the n-acetyl glucosamine residues in chitin in the am fungal cell walls and given its expression pattern, it could play a role in signaling during arbuscule development. cell biological analyses of the arbuscule-cortical cell interface suggest that the peri-arbuscular membrane increases the membrane area within the cortical cell at least 10-fold. consistent with this, metabolite profiles of mycorrhizal roots indicate a significant increase in fatty acids. the genechip analyses identified two new mycorrhiza-induced genes predicted to be involved in fatty acid metabolism; a putative palmitoyl-acp thioesterase gene and a triacylglycerol/steryl ester lipase-like protein gene that show a 49- and 102-fold increase in transcript levels respectively. rt-pcr analysis indicated that tc and tc transcript levels increase in cortical cells with arbuscules. fatty acid synthesis in plants is a cyclic process and the extension cycles involves the binding of acyl chains to a soluble acyl carrier protein, and specific thioesterases hydrolyze the newly formed acyl-acp into fatty acids and acp. an increase in palmitoyl-acp thioesterase gene expression suggests an increase in the biosynthesis of palmitic acid. consistent with this, a recent metabolite profile of g. intraradices/m. truncatula roots showed that mycorrhizal roots contained high levels of palmitic and oleic acids during active phases of am symbiosis. oleic acid is created by a desaturation reaction catalyzed by a delta- desaturase. surprisingly, a g. intraradices delta- desaturase gene was expressed highly in mycorrhizal roots and specifically in cells with arbuscules. previous studies had speculated that the fungus might obtain palmitic acid from the plant, and even suggested that am fungi might lack fatty acid synthase activity and thus be dependent on host lipid. in the absence of a full genome sequence, it is not possible to determine whether this is correct, however, our findings of complementary gene expression patterns in the two symbionts is intriguing. other metabolic changes documented in mycorrhizal roots include an increase in amino acids. in plants, there are at least three protein families involved in transport of small peptides, the abc transporters, the di/tripeptide transporters and the oligopeptide family. two genes of the ptr family were expressed in mycorrhizal roots and tc was detected in cells containing arbuscules. the genechip experiment also led to the identification of other new genes predicted to encode transport proteins including a copper transport protein and an ammonium transporter and the latter was expressed in cells with arbuscules. the identification of a mycorrhiza-induced ammonium transporter is particularly interesting. it is clear from many studies that in addition to pi, n is also transferred from the fungus to the plant and recent analyses indicate that n is likely transferred to the plant as ammonium. this ammonium transporter is a strong candidate for a role in the acquisition of ammonium delivered via the fungus. metabolic labeling studies and gene expression analyses led to a model in which inorganic n taken up by the fungal extra-radical mycelium is converted into arginine which is transported into the intra-radical mycelium, where it is broken down into amino acids. ammonium, resulting from arginine breakdown is proposed to be exported to the plant. in further support of the labeling studies, govindarajulu et al. showed elevated transcripts of g. intraradices ornithine aminotransferase, a urease accessory protein and an ammonium transporter in the intra-radical mycelium relative to the extra-radical mycelium. interestingly, the g. intraradices genes present on the m. truncatula array include genes predicted to encode enzymes of the urea cycle as well as amino acid metabolism. rt-pcr analysis showed that these genes were expressed in g. intraradices-mycorrhizal roots, and analysis of the cell-type specific samples, indicates that they are expressed in the arbuscules. these data extend the previous findings, showing that the genes of urea and amino acid metabolism are expressed in the arbuscules. in addition, our data set includes a gene predicted to encode arginase that breaks down arginine to release ornithine and urea. this is a key step for the release of ammonia and further supports the model proposed by govindarajulu et al.. in addition to the urea cycle, we detected g. intraradices asparagine synthetase and glutamine synthetase gene expression in arbuscules. this suggests that ornithine, released via arginase activity, may be cycled into asparagine and glutamine through the action of asparagine synthetase and glutamine synthetase. interestingly, the metabolite profiling studies found higher levels of specific amino acids, including glu, asp, and asn in mycorrhizal roots. from these studies, it is not possible to determine whether the amino acids are in the plant or am fungus, however, coupled with the gene expression studies, there is evidence to suggest that at least some of this amino acid accumulation may result from activities in the fungus.  using a combination of arrays, lm and rt-pcr, we have extended the m. truncatula/g. intraradices mycorrhizal root transcriptome and identified novel m. truncatula and g. intraradices genes that are expressed in cortical cells harboring arbuscules. by virtue of their expression patterns, the genes expressed in this cell type are candidates for mediating aspects of development or function of the symbiotic arbuscule-cortical cell interface. novel genes potentially involved in signaling and in n transport and metabolism are of particular interest. this dataset will be valuable for future studies aimed at determining the molecular events that control arbuscule development, and n transport and metabolism in the am symbiosis. 
the arbuscule-cortical cell interface is the primary functional interface of the symbiosis and is of central importance in nutrient exchange. to determine the molecular events the underlie arbuscule development and function, it is first necessary to identify genes that may play a role in this process. lm coupled with rt-pcr provided spatial gene expression information for both symbionts and expanded current information available for gene expression in cortical cells containing arbuscules. toward this goal we used the affymetrix genechip® medicago genome array to document the m. truncatula transcript profiles associated with am symbiosis, and then developed laser microdissection of m. truncatula root cortical cells to enable analyses of gene expression in individual cell types by rt-pcr. a laser microdissection protocol that incorporates low-melting temperature steedman's wax, was developed to enable laser microdissection of m. truncatula root cortical cells. the symbiosis develops in the roots where am fungi colonize the root cortex and form arbuscules within the cortical cells. this approach led to the identification of novel m. truncatula and g. intraradices genes expressed in colonized cortical cells and in arbuscules. within the arbuscule, expression of genes associated with the urea cycle, amino acid biosynthesis and cellular autophagy was detected. analysis of gene expression in the colonized cortical cell revealed up-regulation of a lysine motif -receptor like kinase, members of the gras transcription factor family and a symbiosis-specific ammonium transporter that is a likely candidate for mediating ammonium transport in the am symbiosis. transcript profiling using the affymetrix genechip® medicago genome array provided new insights into gene expression in m. truncatula roots during am symbiosis and revealed the existence of several g. intraradices genes on the m. truncatula genechip®.
 most vascular flowering plants have the capacity to form symbiotic associations with arbuscular mycorrhizal fungi. the symbiosis develops in the roots where am fungi colonize the root cortex and form arbuscules within the cortical cells. arbuscules are enveloped in a novel plant membrane and their establishment requires the coordinated cellular activities of both symbiotic partners. the arbuscule-cortical cell interface is the primary functional interface of the symbiosis and is of central importance in nutrient exchange. to determine the molecular events the underlie arbuscule development and function, it is first necessary to identify genes that may play a role in this process. toward this goal we used the affymetrix genechip® medicago genome array to document the m. truncatula transcript profiles associated with am symbiosis, and then developed laser microdissection of m. truncatula root cortical cells to enable analyses of gene expression in individual cell types by rt-pcr.  this approach led to the identification of novel m. truncatula and g. intraradices genes expressed in colonized cortical cells and in arbuscules. within the arbuscule, expression of genes associated with the urea cycle, amino acid biosynthesis and cellular autophagy was detected. analysis of gene expression in the colonized cortical cell revealed up-regulation of a lysine motif -receptor like kinase, members of the gras transcription factor family and a symbiosis-specific ammonium transporter that is a likely candidate for mediating ammonium transport in the am symbiosis.  transcript profiling using the affymetrix genechip® medicago genome array provided new insights into gene expression in m. truncatula roots during am symbiosis and revealed the existence of several g. intraradices genes on the m. truncatula genechip®. a laser microdissection protocol that incorporates low-melting temperature steedman's wax, was developed to enable laser microdissection of m. truncatula root cortical cells. lm coupled with rt-pcr provided spatial gene expression information for both symbionts and expanded current information available for gene expression in cortical cells containing arbuscules. 