BACKGROUND
most land plants engage in symbiotic associations with fungi  that colonize their roots and provide them with phosphate and other mineral nutrients  <cit> .
this association, referred to as arbuscular mycorrhiza , is found in most major taxa of land plants  <cit> , and is thought to have emerged monophyletically in an early progenitor of the vascular plants  <cit> .
the strongest argument for this assumption is the fact that mycorrhizal development requires a conserved signalling pathway that consists of approximately  <dig> genes that encode receptor components such as symrk, and signalling intermediates such as ccamk  <cit> .
the genes involved in this pathway are conserved between monocots and dicots, and occur also in lycopods and mosses  <cit> , suggesting that the origin of am dates back to the early vascular plants at the time when land became colonized  <cit> .
this assumption is consistent with the fossil record, which provides evidence for am-like associations in the sediments of the rhynie chert that is estimated to originate from the ordovician period around  <dig> my ago  <cit> .
more than  <dig> my after the evolution of am, a subsequent event in a small subset of the dicots , allowed for the emergence of a new form of symbiosis, root nodule symbiosis  with rhizobacteria  <cit> .
interestingly, rns, as well as the actinorrhizal symbiosis with cyanobacterial endosymbionts  <cit> , involve the same signalling pathway as am, which therefore is referred to as common symbiosis signalling pathway   <cit> .
a central element of the common sym pathway is calcium spiking, a rythmic change in perinuclear calcium concentration, which is perceived and transmitted by calcium and calmodulin-dependent protein kinase  to induce symbiotic gene expression  <cit> .
am is formed by more than 80% of the vascular plants  <cit> , indicating that this association provides a significant selective advantage over non-mycorrhizal plants.
however, some plant taxa do not form am, among them the brassicaceae with the best-characterized model plant species arabidopsis thaliana, and the chenopodiaceae with the economically important crop species beta vulgaris .
am-related genes are often conserved among am-competent plant species, while they are less conserved or even missing in non-mycorrhizal species.
this phenomenon has been described for vapyrin , which is essential for infection and development of the fungal feeding structures, the arbuscules, in petunia hybrida as well as in medicago truncatula  <cit> .
vpy is entirely missing from non-mycorrhizal plant species  <cit> , and the same is true for numerous genes that are expressed specifically in am  <cit> .
such a pattern of conservation was also observed in am-related genes that are members of large ubiquitous gene families such as the abc transporters stunted arbuscule  and str <dig>  or the gras-type transcription factor required for arbuscular mycorrhiza <dig> , which both belong to subfamilies which are restricted to am-competent plant species  <cit> .
similarly, several components of the common sym pathway are missing from the non-mycorrhizal model species arabidopsis thaliana  <cit> , whereas they are conserved among am-competent dicots and monocots.
traditionally, am-related genes have been identified either by mutant screenings followed by characterization of the mutated gene , or by transcript profiling, followed by mutational analysis of am-induced genes .
considering the increasing number of sequenced plant genomes, the loss of am-related genes from the genomes of non-mycorrhizal species could serve as a criterion to detect new am-related genes by comparative genomics.
this third way of gene discovery could potentially identify am-related genes that have escaped characterization via traditional genetic approaches because of functional redundancy, lethal phenotypes, or constitutive gene expression.
conceptually, such an approach represents a substractive procedure where the proteomes of non-mycorrhizal plants such as a. thaliana are substracted from a panel of proteomes of am-competent species to result in a set of proteins that are consistently conserved among am-competent plants and absent from non-mycorrhizal reference species.
here, we describe a novel approach to identify new am-related genes based on genome substraction.
the approach consists of a multistep procedure that uses protein sequence conservation and gene expression as criteria for the enrichment of potential symbiosis-related genes.
we have compared a set of six am-competent angiosperm species , and three non-am species  with an initial clustering approach that resulted in a set of potential candidate proteins comprising approximately 10% of the entire proteome.
at a second step, a clustering of these genes based on gene expression patterns in medicago truncatula provided a set of conserved genes that are induced in am.
finally, to focus on conserved constitutively expressed genes , a proteome blast of the conserved genes to an extended panel of proteomes  allowed to perform quantitative statistics on the e-values, hence providing a set of proteins that are significantly more conserved among am-competent plant species than towards the non-mycorrhizal brassicaceae.
this strategy was validated with a number of known am-related genes that passed our selection scheme.
the resulting list of predicted am-related proteins will be functionally tested by reverse genetic approaches.
RESULTS
patterns of sequence conservation in am-related genes
in order to explore the potential for differential conservation of am-related genes, we established the phylogeny of two central am-related proteins, the gras-type transcription factor required for arbuscular mycorrhiza <dig> , which is an essential regulator of am symbiosis  <cit> , and phosphate transporter <dig> , which is required for symbiotic phosphate transfer and arbuscule functioning  <cit>  .
a phylogenetic tree of ram <dig> shows a clear bisection between mycorrhizal plants , and non-mycorrhizal plants  .
notably, the sequences of am-competent dicots and monocots  grouped significantly closer together than the dicots among each other .
a similar pattern was observed in a phylogenetic tree of pt <dig> and its closest homologues in various monocot and dicot species .
as with ram <dig>  the homologues from am-competent dicots  and monocots  grouped more closely together than the homologues of the phylogenetically related groups of the am-competent dicots  and the non-mycorrhizal dicots .figure  <dig>
phylogenetic analysis of am-related genes relative to house-keeping genes.
phylograms of am-induced ram <dig>  and pt <dig> , in relation to the housekeeping genes cyclin d <dig>  and rpl <dig> .
a contrasting pattern was observed when two housekeeping genes were analysed, that encode cyclin d <dig> and chloroplast ribosomal protein l <dig> .
these proteins showed a pattern of conservation that reflects the closer relationship among the dicots, relative to the monocots, which clustered separately from all the dicot species .
taken together, this evidence suggests that pt <dig> and ram <dig>  and perhaps other am-related genes, are under diversifying selection in am-competent species.
hence, am-related genes could potentially be identified based on the relative conservation pattern of their encoded proteins.
hierarchical clustering to identify protein phylogeny
in order to identify am-related genes in a systematic way, we first applied the clustering software hieranoid  <cit>  to the proteome sequences of six am-competent species, namely medicago truncatula , glycine max , vitis vinifera , solanum lycopersicum , solanum tuberosum  and populus trichocarpa , and three non-mycorrhizal species, namely arabidopsis thaliana , arabidopsis lyrata , and brassica rapa .
pairwise clustering proceeded based on a conceptual phylogenetic tree of the involved plant species .
figure  <dig> describes the work-flow of our strategy.
briefly, the proteomes of  <dig> species were used for clustering.
the resulting trees of orthologous/paralogous proteins were filtered to yield lists of protein clusters that satisfied certain defined criteria referred to as task <dig>  task <dig>  task <dig> .
these gene lists were further processed to isolate am-related genes based on gene expression, on conservation of the protein-coding region, and on the promoter sequence, as discussed in detail in the following sections.
scripts involved in the different processes  are provided in additional file 3: file s <dig> figure  <dig>
strategy used to identify am-related genes based on sequence conservation.
the flow chart reflects the stepwise identification of potential am-related proteins based on their pattern of sequence conservation at the protein level, the pattern of gene expression, and predicted regulatory elements in their promoters.
sp: plant species.
p0-p <dig> correspond to protocols, files, or scripts provided in supplementary materials .
blue boxes: databases; green boxes: tools and processes; pink boxes: intermediate outputs; red boxes: final outputs.
after an initial round of clustering, we noticed that, unexpectedly, the known am-related vapyrin protein was not found among the trees generated by hieranoid, although it is believed to generally occur in all am-competent plants  <cit> , whereas it is absent from the brassicaceae  <cit> .
closer inspection of the mtr proteome from ensembl revealed that vapyrin, as well as the two am-related genes castor and pt <dig> were missing from the m. truncatula proteome .
however, they could be identified in the uniprotkb database  <cit>  and were added to the mtr proteome , and hieranoid was restarted.
final clustering from the proteomes of the  <dig> plant species gave rise to a total of 28’ <dig> clusters of orthologous and/or paralogous proteins .
selecting clusters with potential am-related proteins
in order to select among all the clusters generated by hieranoid those that showed the conservation pattern known for am-related proteins , we performed in silico substraction by selecting protein clusters that contained at least one protein of each of the 6 am-competent species, but none of the non-mycorrhizal species ath, aly, and bra .
in order to account for cases where an individual protein may be missing because of an incomplete proteome , we also performed a more permissive search for clusters that contained proteins of at least  <dig> of the 6 am-competent species, but none from the three non-mycorrhizal species , and we also carried out the corresponding subtractions with task <dig> and task <dig> .
the numbers of clusters passing through these filters are listed in table  <dig>  in all filtering trials, m. truncatula showed considerably lower numbers than the other species, indicating that its proteome is less complete than those of the other am-competent species .
hence, to account for the incompleteness of the m. truncatula proteome, and for other potentially missing proteins, we selected the clusters identified by task <dig> for further analysis.table  <dig>
number of protein clusters from hierarchical clustering after filtering
task3
task4
task5
task6
among the total of 28’ <dig> clusters obtained from hieranoid, those were selected that did not have a member from the brassicaceae , but had at least one hit from at least  <dig>   <dig>   <dig>  or  <dig> of the am-competent species, respectively.
these are referred to as task <dig>  task <dig>  task <dig> and task <dig>  respectively.
in addition, the occurrence of the 6 am-competent species in the respective clusters is indicated.
information of the relative representation of genes from m. truncatula  in the medicago gene atlas is provided as well.
in order to assess the efficiency of hieranoid clustering and subsequent filtering, we tested whether known am-related genes passed the selection process.
we assembled a list of  <dig> proteins with known function and/or expression pattern in am .
they are known either as components of the common sym signalling pathway , as receptor of nod factor and potentially myc factor , as genes required specifically in am development , or as genes specifically induced in mycorrhizal roots .
an additional set of proteins served as negative controls that were not expected to pass the filter, either because they function as housekeeping genes and are therefore ubiquitous , or because they are primarily involved in nodulation but not am .
as expected, most  of the components of the sym signalling pathway passed the filter applied by task <dig> , with the exception of dmi1/pollux and the nucleoporins nup <dig>  and nena, which were known before to share close homologues with a. thaliana  <cit> .
nup <dig> was lost during the hieranoid clustering and therefore cannot be used in this context.
furthermore, the lysm-type receptor kinase, as well as ram <dig> and ram <dig> were retained, whereas the abc transporter str was excluded.
genes that are induced during am, or expressed exclusively in mycorrhizal roots, were also retained through filtering, with the exception of subtilase and pr <dig>  with the latter being represented by a slightly more distant relative .
importantly, the negative controls  were eliminated by filtering .
these results show that our clustering and filtering procedure has the potential to identify am-related genes based on conservation of the protein sequence.
the fact that str was removed despite its conservation pattern that would be expected to allow it to pass through our filtering approach  <cit> , can be explained with the fact that it is part of a large gene family .
identifying am-related proteins by gene expression pattern
am-related genes can potentially be identified based on their induction during am development.
genome-wide analysis of am-related gene expression has been performed in a number of plant species including m. truncatula, l. japonicus, s. lycopersicum , oryza sativa , and petunia hybrida  <cit> .
a large set of transcriptomic data is available online for the model legume m. truncatula .
we used this resource to identify among the protein clusters resulting from task <dig> those that are induced at the transcriptional level during am.
we first extracted for all the trees retained by task <dig>  those that had a member from m. truncatula .
these clusters represented a total of  <dig> genes of m. truncatula, of which  <dig> genes had at least one affymetrix probe set in the m. truncatula gene atlas .
after removal of unreliable probesets , the expression data for 1526 m. truncatula genes representing  <dig> protein clusters were used for further analysis.
expression data are available from various conditions including mycorrhizal roots with rhizophagus irregularis and with glomus mosseae, and laser-microdissected root cortex cells with arbuscules from r. irregularis.
in addition, expression data of inoculated roots of the doesn’t make infection <dig>  mutant  <cit> , roots treated with myc factor  for 6 h and 24 h, and roots treated with low phosphate levels are available.
the values of gene expression under these treatments and of the corresponding control treatments, were extracted from the medicago gene atlas to calculate induction ratios according to  <dig> criteria .
in order to focus on proteins that are induced robustly in mycorrhizal roots, an induction threshold of 3-fold was applied for further filtering of candidate genes.
a complete list of genes identified by task <dig> with the corresponding expression ratios according to criteria 1- <dig> is provided in additional file 7: table s <dig> table  <dig>
criteria used for gene expression filtering of
m. truncatula
genes identified in task4
name
name of treatment in mtgea
these  <dig> criteria were applied to the  <dig> genes of task <dig>  for which a reliabel probe set was available in the m. truncatula gene atlas .
*expression values were averaged for the two samples inoculated by g. intraradices and g. mosseae, respectively.
combinatorial analysis revealed that  <dig> genes were commonly induced in mycorrhizal roots and in microdissected arbusculated cells, whereas  <dig> genes were induced only in mycorrhizal roots, and  <dig> were induced only in microdissected arbusculated cells, respectively .
only  <dig> genes were induced by myclco after 6 h of treatment, while none of them remained induced after 24 h of treatment .
interestingly,  <dig> genes were induced in the dmi <dig> mutant which is defective for ccamk, indicating that their expression is regulated independently of the common sym pathway.
three of these genes were also induced in am roots, in microdissected arbusculated cells, and in p-starved roots  .
it will be interesting to explore how these genes are induced in the absence of dmi <dig> figure  <dig>
venn diagram of
m. truncatula
genes up-regulated under various am-related conditions.
genes listed in additional file 7: table s <dig>  were subjected to combinatorial analysis according to the criteria listed in table  <dig>  red domain, genes induced in mycorrhizal roots; green domain, genes induced by myc-lco after 6 h; dark blue domain, genes induced in laser-microdissected cortex cells with arbuscules; light blue domain, genes induced in the dmi <dig> mutant .
criterion  <dig>  did not yield any result.
genes identified according to criterion  <dig> are marked in red.
list a, b, and c are separately shown in additional file 8: table s <dig>
while gene expression patterns can be identified based on defined conditions , global expression analysis by clustering can identify groups of genes with similar expression patterns over a large set of expression data like the  <dig> different treatments and conditions covered by the medicago gene atlas .
this approach can identify groups of genes that are co-regulated and therefore might be functionally related.
on the other hand, this approach can lead to the discovery of common regulatory elements in promoters, which are the reason for co-regulation .
hence, we decided to use pairwise average linkage and pearson correlation in order to identify genes with shared expression pattern .
all proteins identified by task <dig> were correlated based on their standardized gene expression score, i.e.
the ratios between the individual expression levels divided by the average of all expression levels as a relative indicator of expression .
particular attention was paid to groups of genes that comprised am-induced genes.
one conspicuous cluster of  <dig> genes with a significantly correlated expression pattern turned out to be highly specific for am, resulting in apparent vertical red stripes in the visual representation of the cluster .
a further relevant group comprised genes that are induced commonly in am and in rns .
these genes may encode proteins that play a general role in symbiotic interactions.
interestingly, this cluster consisted primarily of chitinases, cysteine proteases, a glucanase and several ripening-related proteins .figure  <dig>
details of the am-related gene cluster shown in additional file
9
: figure s <dig>  the hierarchical cluster contains genes induced in mycorrhizal roots.
numbers in the column “criteria” indicate under which conditions the gene was induced at least 3-fold .
numbers in the column “task” indicate in which task the gene was still retained .
ranks are assigned according to gene induction in mycorrhizal roots .
cluster of genes induced in both, mycorrhizal roots, and nodule symbiosis.
details from cluster shown in additional file 8: table s <dig>  numbers in the column “criteria” indicate under which conditions the gene was induced at least 3-fold .
numbers in the column “task” indicate in which task the gene was still retained .
ranks are assigned according to gene induction in mycorrhizal roots .
analysis of relative conservation of proteins among angiosperms
many genes with a role in am are induced during the interaction.
this includes nutrient transporters such as am-related phosphate transporters  <cit>  , and the ammonium transporter amt <dig>  <cit> , as well as regulatory components such as the transcription factor ram <dig>  <cit> .
in contrast, the genes involved in the early steps of the interaction are constitutively expressed.
for example, the expression of the nod factor receptors  and of the common sym genes is not significantly altered during am development in petunia  <cit> , consistent with their early function in symbiont recognition and signalling.
in order to identify constitutively expressed genes with a potential role in am development, we decided to compare the relative sequence conservation of proteins in the context of the three am-relevant plant groups: am-competent dicots , am-competent monocots , and non-mycorrhizal dicots  .
in order to avoid to miss relevant genes due to proteome incompleteness, we chose the relatively permissive task <dig> for this approach .
firstly, multiple sequence alignment  from the sequences represented in the individual clusters retained by task <dig> were calculated using mafft  <cit> , and secondly, these msa consensus sequences were used as queries to search by psi-blast the proteomes used for hieranoid clustering and in addition a number of monocot species, namely o. sativa , zea mays , triticum urartu , sorghum bicolor , hordeum vulgare , brachypodium distachyon , and aegilops tauschii   .
the e-values were then compared between the groups by wilcoxon test  to identify genes for which the populations of e-values between groups c and a, or between c and b were significantly different .
for proteins that exhibited significant differences, the e-values were averaged among the three groups, and the ratios between the log of these values for c/a and c/b were calculated as a relative measure for am-related sequence conservation .
the higher the conservation ratio, the farther the non-mycorrhizal homologues are from the consensus sequences relative to the homologues from am-competent species, indicative for am-related conservation.
establishing the frequency distribution of the conservation ratios revealed that several of our test genes, such as symrk, vapyrin, ram <dig>  ram <dig>  and pt <dig> passed this filter , hence their pattern of sequence conservation was significantly related to the competence to engage in am symbiosis.
surprisingly, none of the test genes passed the comparison between non-mycorrhizal dicots and mycorrhizal monocots , although at least vapyrin, ram <dig>  and pt <dig> are more closely related between the am-competent dicots and the monocots, than between the am-competent and the non-mycorrhizal dicots  <cit>  .figure  <dig>
conservation ratios of potentially am-related proteins averaged for relevant plant groups with significant difference between non-am and am species.
histograms represent the frequency distributions of the ratios of log <dig> of the e-values from psi-blast.
the query sequences for psi-blast were generated by calculating msa consensus sequences based on the results of task <dig>  these query sequences were blasted against am-competent dicot species , monocot species , and non-mycorrhizal dicot species  .
to derive conservation ratios, the e-values were averaged group-wise for groups a, b, and c, respectively, and the following ratios were generated: c/a and c/b.
conservation ratios were included only if the difference between the groups were significant .
ratios for log10/log <dig>   ratios for log10/log <dig>  note that  <dig> control genes from additional file 5: table s <dig> passed the filter in , whereas none passed in .
since the significance threshold of the wilcoxon test eliminated many genes, we sought for an alternative way to evaluate the degree of am-related conservation.
instead of a fixed threshold level, we defined the conservation ratios for a set of household proteins that do not exhibit an am-related bias in conservation.
to this end, we extracted from the hieranoid clusters those that have representative homologues from all plants, including am-competent and non-mycorrhizal species .
thus, this set contains conserved house-keeping proteins that can serve as reference for the conservation patterns of proteins identified by task <dig>  we proceeded in the same way as with the proteins identified by task <dig>  i.e.
an msa consensus sequence was calculated based on the 6 am-competent species from each protein cluster, and these msa sequences were used as queries for psi-blast against all proteomes.
as expected, most of the genes selected in this way showed a conservation pattern consistent with the closer phylogenetic relatedness among all the dicots vs. the monocots .
this fact is reflected in the phylogenetic trees of the house-keeping genes cyclin d <dig> and rpl <dig> , which are also represented in the list resulting from task <dig> .
we selected a set of  <dig> proteins from the results of task <dig> with intermediate e-values  and with genes represented in most monocots .
for these  <dig> genes , the ratios were calculated as for the genes obtained with task <dig> .
these values tended to be more to the negative, reflecting the closer position of the brassicaceae homologues from the msa consensus sequences relative to the proteins obtained with task <dig>  hence, the  <dig> reference genes resulting from task <dig> define the range of conservation ratios for housekeeping proteins and therefore allows to define the range that contains proteins with an am-related bias of conservation .
global comparison of the conservation ratios of proteins identified by task <dig> and task <dig>  revealed considerably higher values for the former group , reflecting the different conservation patterns among proteins selected by task <dig> and task <dig>  in the comparison between group a  and group c , the am-related genes ram <dig> and pt <dig> were clearly seprated from the housekeeping controls cyclin d <dig> and rpl <dig> , while this distinction was much less clear in the comparison between group b  and group c .
these results show that the conservation ratio can be used as a comparative proxy to evaluate the relative degree of conservation of a given protein among am-competent species relative to the non-mycorrhizal species.figure  <dig>
conservation ratios of potentially am-related proteins in comparison with housekeeping genes averaged for relevant groups of plant species.
conservation ratios were generated as for figure  <dig> .
however, no statistics were performed and all ratios are shown.
conservation ratios are compared for potential am-related proteins extracted by task <dig> , and for potential house-keeping genes identified by task <dig> .
for comparison, the position of the proteins represented in figure  <dig> is indicated .
ratios for log10/log <dig>   ratios for log10/log <dig>
outcome of the in silico substraction approach
the goal of this study was to identify am-related genes based on the conservation pattern of their orthologues between am-competent and non-mycorrhizal plant species.
this approach is particularly targeted to identify am-related genes that are not induced during symbiosis, hence, we focused on the proteins identified by task <dig> that are not induced at the gene level .
to evaluate the efficiency of the approach, this list was ordered according to the ratios of e-values between the averaged brassicaceae and am-competent dicot plants, respectively .
the list was sorted in descending order, since the highest values for the conservation ratios indicate the proteins that are conserved to a higher degree among am-competent species than between am-competent and non-mycorrhizal species.
in this list, the first protein, a predicted α-glucosidase/ xylosidase, was chosen to evaluate its conservation pattern in detail.
indeed, a phylogenetic tree prepared as in figure  <dig> shows an extremely skewed pattern of conservation with a clearly resolved common branch of the am-competent monocots and dicots, including the basal lineage amborella trichopoda, whereas all the non-mycorrhizal species, the brassicaceae, and b. vulgaris as a representative of the chenopodiaceae, form an outlier group .figure  <dig>
phylogenetic analysis of an α-glucosidase with high conservation ratio.
the first hit in the list of predicted am-related proteins was an α-glucosidase .
the s. lycopersicum sequence was used to retrieve the closest homologues in a wide range of species  for phylogenetic analysis.
tree as in figure  <dig> with the first hit per species identified at ncbi by blastp against non-redundant protein database using the tomato α-glucosidase  as a query.
tree with all homologues of solyc03g <dig> .
<dig> from tomato , grape vine , poplar , m. truncatula, and a. thaliana.
note the am-related branch  that has no homologue from a. thaliana.
the closest arabidopsis homologue  clusters far away.
hydrolases are often encoded by gene families, and this is also the case for this α-glucosidase/xylosidase.
in order to test whether the member with the am-related conservation pattern forms a dedicated group in am-competent species, we isolated all the homologues available from the protein database at ncbi for the species v. vinifera, p. trichocarpa, s. lycopersicum, m. truncatula and a. thaliana.
they are numbered in each species based on their similarity to the am-related homologue in s. lycopersicum.
a phylogenetic tree with all these sequences revealed that all am-competent species have a single am-related homologue, resulting in a clearly separated am-related branch .
the closest homologue of a.thaliana falls into the large containing the remaining sequences.
hence, a. thaliana misses only the am-related form of the α-glucosidase/ xylosidase gene family.
search for potential cis-regulatory elements in promoters of am-related genes
besides the conservation of the orf, we investigated the non-coding upstream sequences of am-related genes by searching for potential cis-regulatory promoter elements that may control gene activity during symbiosis.
we selected the m. truncatula proteins identified by task <dig> that were at least 3-fold induced in mycorrhizal roots relative to non-mycorrhizal control roots .
their promoter sequences , were downloaded from ensembl plants biomart , and analyzed with the pattern recognition software meme  for overrepresented sequences.
a first search revealed a series of conserved predicted promoter elements .
a highly conserved element comprised the core sequence ggggttcgaacccc  with the bold letters being almost invariant .
the palindromic nature of this element, and the fact that many cis-elements in plant promoters are palindromes  <cit>  prompted us to search specifically for palindromic sequences.
interestingly,  <dig> of the  <dig> elements predicted by the first search were confirmed by the second search for palindromes .
besides the myc <dig> element, a second element with the well-defined core sequence tgagcttagctca  emerged .
an element with completely invariant sequence was the palindrome gccggc that tended to be located within the 500 bp immediately upstream of the atg , indicating that it represents a relevant promoter element .
besides the gccggc element, none of the predicted elements were described previously.
an element that could have been expected to result from our meme search is the am-related cttc element , also known as mycs element  <cit> .
this element is present in the promoters of am-related phosphate transporter genes, and of the am-related snare ljvti <dig>  <cit> , however, it was not found by meme in our sample of 190 am-induced promoter sequences.
this surprising result prompted us to search for it in the vapyrin promoter, which is induced in mycorrhizal roots of both, m. truncatula and petunia hybrida  <cit> , indicating that this promoter may contain conserved am-related promoter elements.
indeed, we found an extended version of the cttc element  in all promoters of  <dig> vapyrin genes from various monocots and dicots, indicating that it is of a wider significance for am-related gene regulation .
the elements myc <dig> and myc <dig>  the gccggc element, and the cttc element were selected for further statistical analysis in the genome of m. truncatula.
in order to estimate their relative frequency in the promoters of am-related genes, we selected three samples: the promoters of the genes identified with task <dig> , those of the am-induced genes whithin task <dig> , and all promoters of m. truncatula .
these promoter sequences were searched by blast for the presence of the four elements of interest.
for the non-palindromic cttc element, both orientations were considered separately, and the two respective longer variants  were included as well .
indeed, element myc <dig> and myc <dig>  as well as the gccggc element were significantly overrepresented among the genes identified by task <dig> compared to the statistical expectations, and relative to the observed frequency in all promoters .
overrepresentation was even more evident when the am-induced genes were considered .
significance of these results was confirmed by binomial test.
in contrast to the myc <dig> and myc <dig> elements, and to the gccggc element, the cttc element was not overrepresented in promoters identified by task <dig>  and only moderately in the am-induced genes, when its original sequence was considered  .
however, the longer form  was significantly overrepresented in both orientations in the promoters of the genes identified by task <dig> and even more of the am-induced genes .
this indicates that the longer version is the relevant form of the cttc element.
discussion
during the past decade, many screens for am- or rns-defective mutants have been carried out and a significant number of genes involved in signalling and symbiotic functioning have been identified  <cit> .
in addition, transcript profiling has identified many am- and rns-related transcripts, however, only few of them have been functionally characterized by reverse genetic approaches.
a central finding of the majority of these genetic studies was, that the genes involved in symbiosis are well conserved among am-competent species, whereas they are conserved to a lesser extent, or entirely missing, in non-mycorrhizal species such as a. thaliana.
based on this finding, we designed a strategy for a third avenue to identify yet unexplored am-related genes using the conservation of their coding region as the main criterion.
we compared the proteomes of am-competent and non-mycorrhizal plant species, thereby isolating the proteins that are conserved only, or to a higher degree, in am-competent species.
essentially, this approach represents an in silico substraction procedure, where the proteomes of the non-mycorrhizal species are substracted from the proteomes of the am-competent species to yield a fraction of the proteome that is expected to be enriched in am-related proteins.
since function is selected for at the level of the protein, and since sequence conservation at the dna level is affected by the degeneracy of the genetic code, we compared the species at the proteome level.
ideally, an in silico substractive approach would involve an equal number of proteomes of the am-competent and the non-mycorrhizal group.
however, due to the wide success of am in nature, non-mycorrhizal species are the exception , and only few of them have been sequenced to a degree that they could be used for such an approach.
as non-mycorrhizal species, only a. thaliana, a. lyrata, and b. rapa were available as proteomes in the ensembl database.
in contrast, a wide range of am-competent species is available, among them various dicots  and cereals.
hence, in a first approach, we selected two am-competent monocots , four am-competent dicots , and the two non-mycorrhizal species a. thaliana and b. rapa.
the first step in this approach was to establish the orthologous relationships between the proteomes of the eight selected species.
this task was performed with hieranoid, which establishes clusters of orthologous/paralogous proteins in a hierarchical step-wise procedure that proceeds according to a previously defined phylogenetic tree of the involved species.
the subsequent step is to identify protein clusters that have members in all or most of the am-competent species, but none in the non-mycorrhizal species.
however, after a first run with hieranoid, we noticed that in many cases, the orthologous trees of known single proteins fell into a monocot and a dicot tree, thus preventing our strategy.
therefore, we decided to perform a clustering just using dicot species, namely g. max, m. truncatula, p. trichocarpa, v. vinifera, s. lycopersicum, and s. tuberosum, together with the three non-mycorrhizal species a. thaliana, a. lyrata, and b. rapa .
in order to assess the efficiency of the procedure, we used a number of known am-related genes and a few housekeeping genes for reference .
clustering of all nine proteomes resulted in a total of 28’ <dig> protein clusters.
subsequent selection of clusters that contain orthologues only in am-competent species established a first set of potential am-related proteins .
in order to account for cases were individual orthologues may be missing from one or two proteomes, we relaxed the stringency of the filtering by allowing also for clusters were  <dig> or  <dig> members of am-competent species were missing  .
an even more permissive filter  was applied to obtain a large population of proteins that were subsequently subjected to quantitative assessment of amino acid sequence conservation based on the comparison of e-values .
the fact that task <dig> and task <dig> yielded rather long lists of candidate proteins  was not considered a problem, since subsequent filtering and sorting narrowed the list further down .
a next step was to use gene expression as a criterion for selection, hence, we defined expression ratios relevant for am .
based on these criteria we sorted the list of proteins identified by task <dig>  by applying a stringent threshold induction ratio of 3-fold, we restricted the analysis to genes with a robust transcriptional induction in mycorrhizal roots .
besides known am-related proteins such as pt <dig>  am <dig>  vapyrin, ram <dig>  and ipd <dig>  this list included several interesting proteins like the lysm receptor kinase lyr <dig>  a close homologue of the lotus japonicus nod factor receptor nfr <dig>  <cit> , and several abc transporters and peptide transporters, that may play a role in am development.
highly induced candidates with yet unknown functions in am include a triacylglycerol lipase , neutral ceramidase , an enzyme with a role in sphingolipid metabolism  <cit> , and reticuline oxidase  an enzyme involved in the biosynthesis of the alkaloid intermediate reticuline  <cit> .
the fact that many known am-induced genes were recovered by our selection procedure  documents the potential of gene discovery by comparative phylogenetics.
the main goal of this study was to identify new am-related proteins based on conservation of the coding sequence, i.e.
independently of am-related gene induction.
in order to obtain a quantitative measure for conservation of the open reading frame  in am-competent species, we performed a secondary filtering step by blasting the conserved consensus sequences derived from the 6 am-competent species  against a wider panel of plant proteomes including dicots, monocots, the liverwort selaginella moellendorfii, the moss physcomitrella patens, and the unicellular alga chlamydomonas reinhardtii.
the interest to include these three lower plants is based on the finding that several of the am-related genes were found in liverworths and/or mosses  <cit> , potentially revealing the evolutionary origin of the common sym pathway in lower land plants  <cit> .
the psiblast was performed with the results of the relatively permissive task <dig> .
with this approach  <dig> proteins were identified for which the e-values between the three relevant groups of plant species were significantly different  between the non-mycorrhizal species, and the am-competent dicots and monocots, respectively , indicating that they are conserved particularly well in am-competent species.
interestingly, the  <dig> proteins with the lowest p-values  included  <dig> proteins with annotations as receptors, protein kinases, phosphatases, disease resistant proteins, or g-protein regulating protein, implying roles in signalling.
in view of the essential role of am in plant nutrition, a highly conserved ammonium transporter , and the oligomineral nutrient transporter nramp <dig>  are of particular interest.
ammonium is the form in which nitrogen is thought to be transferred from the am fungus to the plant  <cit> , and therefore, ammonium transporters are of particular importance in am  <cit> .
transporters for other mineral nutrients such as zinc and copper remain to be identified.
many of the conserved proteins were also found to be well conserved in the liverwort s. moelledorfii, and some of them even in the moss p. patens, whereas c. reinhardtii proved to be phylogenetically much more distant .
in order to obtain a direct quantitative measure for the degree of am-related conservation, we defined the conservation ratio, which represents the ratio of the averaged e-values from psiblast  between the non-mycorrhizal species, and the am-competent species  .
for comparison, the results from task <dig>  were included as representatives of generally well conserved housekeeping proteins.
the larger the conservation ratio, the more a protein sequence is divergent between the non-am plants and the am-competent species.
therefore, the proteins identified by task <dig> have mostly positive values, whereas the housekeeping proteins  have neutral or negative values .
of particular interest were proteins with a conservation ratio > <dig>  which comprised for example pt <dig> and ram <dig> , which are known to show a highly am-related conservation pattern .
a general overview over the conservation ratios  revealed that for  <dig> proteins, the conservation ratio could not be calculated because they were entirely missing from the psiblast results in the non-mycorrhizal species .
in addition, a further  <dig> proteins exhibited conservation ratios of > <dig>  indicative of a high degree of am-related conservation.
in order to define a range above which the conservation ratios can be considered as being skewed towards the am-competent species,  <dig> proteins identified by task <dig>  were included for reference in the results from task <dig> .
among the  <dig> proteins that were missing from the non-mycorrhizal species ,  <dig> have unknown functions.
interestingly, three proteins each are predicted chitinases , and glucanases , indicating potential roles in cell wall modification or signaling.
three predicted cle peptides  might be involved in am-related signaling as it was shown for rns  <cit> .
a known component in symbiotic signaling comprised interacting protein of dmi <dig> / cyclops , an interactor of the central symbiosis regulator ccamk encoded by dmi <dig>  <cit> .
high conservation ratios were also found for vapyrin , which is required both for am and rns  <cit>  and vapyrin-like .
<dig> proteins exhibited a value > <dig> for both conservation ratios , i.e.
they are highly conserved among both, am-competent dicots and monocots, relative to the non-mycorrhizal species.
of particular interest is task3_ <dig> which encodes a predicted α-glucosidase  which is in some species classified as α-xylosidase .
while the conserved protein is missing from a. thaliana , there are related glycosidases that occur in all examined plant species including a. thaliana, indicating that the non-mycorrhizal model species misses only the am-related isoform .
the potential role for α-glucosidase or α-xylosidase in am is unknown, but given the important contribution of xylose in the hemicelluloses of the cell wall  <cit> , it may be involved in the modifications of cell walls during am infection and functioning  <cit> .
interestingly, a predicted α-xylosidase of petunia is among the most strongly repressed genes under conditions of high phosphate, which inhibits am  <cit> .
five of the  <dig> proteins with high conservation ratios  represent receptors and protein kinases, of which three  are closely related receptor-like protein kinases  .
they are similar to s-locus receptor kinase   <cit> , which mediates sporophytic self-incompatibility in the brassicaceae  <cit> , and to the maize receptor kinase zmpk <dig>  <cit> .
phylogenetic analysis showed that they indeed fall into a large clade of proteins found in all am-competent species, but with considerably lower conservation in the non-mycorrhizal species .
a conserved c-rich stretch in the amino acid sequence is shared among the three am-related rlks, as well as with srk, and zmpk <dig> , indicating that they may have similar tertiary structures mediated by cysteine bridges.
interestingly, zmpk <dig> is expressed in roots and young seedlings, rather then floral tissues  <cit>  suggesting that this family of receptor kinases can have other functions than self-incompatibility.
taken together, the examples of α-glucosidae/xylosidase and the rlks show that our approach can identify proteins with am-related conservation patterns even if they are members of large families.
furthermore, the case of the three related rlks highlights the potential of our approach to identify proteins that could potentially escape genetic mutant screens because of functional redundancy.
transcription is controlled primarily by cis-acting regulatory sequences in the promoters of the genes.
hence, co-regulated genes can be expected to share common regulatory elements in their promoters.
based on this logic, we screened the promoters of 190 am-induced genes for overrepresented sequences that may represent cis-acting regulatory promoter elements.
the prediction program meme identified two new palindromic elements  and found the gccggc element to be overrepresented in am-induced promoters as well as in the promoters of genes identified by task <dig>  statistical analysis confirmed the overrepresentation of these elements in am-related promoters .
the statistical significance of element myc <dig> and myc <dig> is emphasized by their rather unusual length  compared to other cis-acting elements that are typically less then 10 nt in length  <cit> .
in addition, element myc <dig> and myc <dig> are often coupled , underscoring their relevance for am-related gene induction, and suggesting that they may function in conjunction.
the predominant position of the gccggc element in a range of 500 bp upstream of the atg start codon is indicative of functional relevance as well.
the gccggc element can be bound by the transcription factor rap.
<dig>  in ethylene-responsive promoters involved in the potassium starvation response of a.thaliana  <cit> , and it was classified as wound- and pathogen-inducible  <cit> .
gccggc represents a palindromic variant of the common gcc element  that is widely found in the promoters of pathogenesis-related genes and in ethylene-responsive promoters of various plants  <cit> .
in fact, gcc elements occur in different variants, which are recognized by specific members of the ethylene response factor family of transcription factors  <cit> .
the overrepresentation of the gccggc element in am-related promoters may explain the frequency of predicted stress- and pathogen-related transcripts in the transcriptome of mycorrhizal roots in plant species such as rice, potato, petunia, parsley, m. truncatula, and l. japonicus  <cit> .
surprisingly, the am-related cttc/mycs element   <cit>  was not found by meme in the 190 am-induced promoters.
furthermore, it was not overrepresented in the genes resulting from task <dig>  and only moderately in the am-induced genes .
however, an extended form , was found to be significantly overrepresented, indicating that this may represent the complete am-inducible element in m. truncatula.
an overview over the occurrence of predicted promoter elements in the analyzed promoters is provided in additional file 19: table s <dig>
taken together, we have identified two new potentially am-associated cis-regulatory promoter elements.
in addition, we identified the gccggc element as a potential am-related element that may indicate an involvement of ethylene in am development, and we present a modified  form of the cttc element.
future work will address the functional relevance of these elements for am development and functioning,
CONCLUSIONS
we describe a non-targeted approach to identify new am-related genes by genome-wide comparative sequence analysis of am-competent and non-mycorrhizal plant species.
the validity of this approach has been confirmed by the finding that it identified many known am-related genes from the genomes of am-competent plant species.
following this strategy, we identified many new potentially am-related genes based on the conservation pattern of the coding sequence, on gene expression pattern, and on predicted regulatory promoter elements.
these genes will be further evaluated for their predicted function , the degree of their conservation, and the copy number, or the number of similar genes in the case of gene families.
this process will result in a short list of genes that will be further functionally analyzed by insertional mutagenesis in p. hybrida, m. truncatula or l. japonicus using established transposon-related protocols  <cit> .

 most land plants engage in symbiotic associations with fungi that colonize their roots and provide them with phosphate and other mineral nutrients. this association, referred to as arbuscular mycorrhiza, is found in most major taxa of land plants, and is thought to have emerged monophyletically in an early progenitor of the vascular plants. the strongest argument for this assumption is the fact that mycorrhizal development requires a conserved signalling pathway that consists of approximately genes that encode receptor components such as symrk, and signalling intermediates such as ccamk. the genes involved in this pathway are conserved between monocots and dicots, and occur also in lycopods and mosses, suggesting that the origin of am dates back to the early vascular plants at the time when land became colonized. this assumption is consistent with the fossil record, which provides evidence for am-like associations in the sediments of the rhynie chert that is estimated to originate from the ordovician period around my ago. more than my after the evolution of am, a subsequent event in a small subset of the dicots, allowed for the emergence of a new form of symbiosis, root nodule symbiosis with rhizobacteria. interestingly, rns, as well as the actinorrhizal symbiosis with cyanobacterial endosymbionts, involve the same signalling pathway as am, which therefore is referred to as common symbiosis signalling pathway. a central element of the common sym pathway is calcium spiking, a rythmic change in perinuclear calcium concentration, which is perceived and transmitted by calcium and calmodulin-dependent protein kinase to induce symbiotic gene expression. am is formed by more than 80% of the vascular plants, indicating that this association provides a significant selective advantage over non-mycorrhizal plants. however, some plant taxa do not form am, among them the brassicaceae with the best-characterized model plant species arabidopsis thaliana, and the chenopodiaceae with the economically important crop species beta vulgaris. am-related genes are often conserved among am-competent plant species, while they are less conserved or even missing in non-mycorrhizal species. this phenomenon has been described for vapyrin, which is essential for infection and development of the fungal feeding structures, the arbuscules, in petunia hybrida as well as in medicago truncatula. vpy is entirely missing from non-mycorrhizal plant species, and the same is true for numerous genes that are expressed specifically in am. such a pattern of conservation was also observed in am-related genes that are members of large ubiquitous gene families such as the abc transporters stunted arbuscule and str or the gras-type transcription factor required for arbuscular mycorrhiza, which both belong to subfamilies which are restricted to am-competent plant species. similarly, several components of the common sym pathway are missing from the non-mycorrhizal model species arabidopsis thaliana, whereas they are conserved among am-competent dicots and monocots. traditionally, am-related genes have been identified either by mutant screenings followed by characterization of the mutated gene, or by transcript profiling, followed by mutational analysis of am-induced genes. considering the increasing number of sequenced plant genomes, the loss of am-related genes from the genomes of non-mycorrhizal species could serve as a criterion to detect new am-related genes by comparative genomics. this third way of gene discovery could potentially identify am-related genes that have escaped characterization via traditional genetic approaches because of functional redundancy, lethal phenotypes, or constitutive gene expression. conceptually, such an approach represents a substractive procedure where the proteomes of non-mycorrhizal plants such as a. thaliana are substracted from a panel of proteomes of am-competent species to result in a set of proteins that are consistently conserved among am-competent plants and absent from non-mycorrhizal reference species. here, we describe a novel approach to identify new am-related genes based on genome substraction. the approach consists of a multistep procedure that uses protein sequence conservation and gene expression as criteria for the enrichment of potential symbiosis-related genes. we have compared a set of six am-competent angiosperm species, and three non-am species with an initial clustering approach that resulted in a set of potential candidate proteins comprising approximately 10% of the entire proteome. at a second step, a clustering of these genes based on gene expression patterns in medicago truncatula provided a set of conserved genes that are induced in am. finally, to focus on conserved constitutively expressed genes, a proteome blast of the conserved genes to an extended panel of proteomes allowed to perform quantitative statistics on the e-values, hence providing a set of proteins that are significantly more conserved among am-competent plant species than towards the non-mycorrhizal brassicaceae. this strategy was validated with a number of known am-related genes that passed our selection scheme. the resulting list of predicted am-related proteins will be functionally tested by reverse genetic approaches.  patterns of sequence conservation in am-related genes in order to explore the potential for differential conservation of am-related genes, we established the phylogeny of two central am-related proteins, the gras-type transcription factor required for arbuscular mycorrhiza, which is an essential regulator of am symbiosis, and phosphate transporter, which is required for symbiotic phosphate transfer and arbuscule functioning. a phylogenetic tree of ram shows a clear bisection between mycorrhizal plants, and non-mycorrhizal plants. notably, the sequences of am-competent dicots and monocots grouped significantly closer together than the dicots among each other. a similar pattern was observed in a phylogenetic tree of pt and its closest homologues in various monocot and dicot species. as with ram the homologues from am-competent dicots and monocots grouped more closely together than the homologues of the phylogenetically related groups of the am-competent dicots and the non-mycorrhizal dicots.figure phylogenetic analysis of am-related genes relative to house-keeping genes. phylograms of am-induced ram and pt, in relation to the housekeeping genes cyclin d and rpl. a contrasting pattern was observed when two housekeeping genes were analysed, that encode cyclin d and chloroplast ribosomal protein l. these proteins showed a pattern of conservation that reflects the closer relationship among the dicots, relative to the monocots, which clustered separately from all the dicot species. taken together, this evidence suggests that pt and ram and perhaps other am-related genes, are under diversifying selection in am-competent species. hence, am-related genes could potentially be identified based on the relative conservation pattern of their encoded proteins. hierarchical clustering to identify protein phylogeny in order to identify am-related genes in a systematic way, we first applied the clustering software hieranoid to the proteome sequences of six am-competent species, namely medicago truncatula, glycine max, vitis vinifera, solanum lycopersicum, solanum tuberosum and populus trichocarpa, and three non-mycorrhizal species, namely arabidopsis thaliana, arabidopsis lyrata, and brassica rapa. pairwise clustering proceeded based on a conceptual phylogenetic tree of the involved plant species. figure  describes the work-flow of our strategy. briefly, the proteomes of species were used for clustering. the resulting trees of orthologous/paralogous proteins were filtered to yield lists of protein clusters that satisfied certain defined criteria referred to as task task task. these gene lists were further processed to isolate am-related genes based on gene expression, on conservation of the protein-coding region, and on the promoter sequence, as discussed in detail in the following sections. scripts involved in the different processes are provided in additional file 3: file s figure strategy used to identify am-related genes based on sequence conservation. the flow chart reflects the stepwise identification of potential am-related proteins based on their pattern of sequence conservation at the protein level, the pattern of gene expression, and predicted regulatory elements in their promoters. sp: plant species. p0-p correspond to protocols, files, or scripts provided in supplementary materials. blue boxes: databases; green boxes: tools and processes; pink boxes: intermediate outputs; red boxes: final outputs. after an initial round of clustering, we noticed that, unexpectedly, the known am-related vapyrin protein was not found among the trees generated by hieranoid, although it is believed to generally occur in all am-competent plants, whereas it is absent from the brassicaceae. closer inspection of the mtr proteome from ensembl revealed that vapyrin, as well as the two am-related genes castor and pt were missing from the m. truncatula proteome. however, they could be identified in the uniprotkb database and were added to the mtr proteome, and hieranoid was restarted. final clustering from the proteomes of the plant species gave rise to a total of 28’ clusters of orthologous and/or paralogous proteins. selecting clusters with potential am-related proteins in order to select among all the clusters generated by hieranoid those that showed the conservation pattern known for am-related proteins, we performed in silico substraction by selecting protein clusters that contained at least one protein of each of the 6 am-competent species, but none of the non-mycorrhizal species ath, aly, and bra. in order to account for cases where an individual protein may be missing because of an incomplete proteome, we also performed a more permissive search for clusters that contained proteins of at least of the 6 am-competent species, but none from the three non-mycorrhizal species, and we also carried out the corresponding subtractions with task and task. the numbers of clusters passing through these filters are listed in table  in all filtering trials, m. truncatula showed considerably lower numbers than the other species, indicating that its proteome is less complete than those of the other am-competent species. hence, to account for the incompleteness of the m. truncatula proteome, and for other potentially missing proteins, we selected the clusters identified by task for further analysis.table number of protein clusters from hierarchical clustering after filtering task3 task4 task5 task6 among the total of 28’ clusters obtained from hieranoid, those were selected that did not have a member from the brassicaceae, but had at least one hit from at least or of the am-competent species, respectively. these are referred to as task task task and task respectively. in addition, the occurrence of the 6 am-competent species in the respective clusters is indicated. information of the relative representation of genes from m. truncatula in the medicago gene atlas is provided as well. in order to assess the efficiency of hieranoid clustering and subsequent filtering, we tested whether known am-related genes passed the selection process. we assembled a list of proteins with known function and/or expression pattern in am. they are known either as components of the common sym signalling pathway, as receptor of nod factor and potentially myc factor, as genes required specifically in am development, or as genes specifically induced in mycorrhizal roots. an additional set of proteins served as negative controls that were not expected to pass the filter, either because they function as housekeeping genes and are therefore ubiquitous, or because they are primarily involved in nodulation but not am. as expected, most of the components of the sym signalling pathway passed the filter applied by task, with the exception of dmi1/pollux and the nucleoporins nup and nena, which were known before to share close homologues with a. thaliana. nup was lost during the hieranoid clustering and therefore cannot be used in this context. furthermore, the lysm-type receptor kinase, as well as ram and ram were retained, whereas the abc transporter str was excluded. genes that are induced during am, or expressed exclusively in mycorrhizal roots, were also retained through filtering, with the exception of subtilase and pr with the latter being represented by a slightly more distant relative. importantly, the negative controls were eliminated by filtering. these results show that our clustering and filtering procedure has the potential to identify am-related genes based on conservation of the protein sequence. the fact that str was removed despite its conservation pattern that would be expected to allow it to pass through our filtering approach, can be explained with the fact that it is part of a large gene family. identifying am-related proteins by gene expression pattern am-related genes can potentially be identified based on their induction during am development. genome-wide analysis of am-related gene expression has been performed in a number of plant species including m. truncatula, l. japonicus, s. lycopersicum, oryza sativa, and petunia hybrida. a large set of transcriptomic data is available online for the model legume m. truncatula. we used this resource to identify among the protein clusters resulting from task those that are induced at the transcriptional level during am. we first extracted for all the trees retained by task those that had a member from m. truncatula. these clusters represented a total of genes of m. truncatula, of which genes had at least one affymetrix probe set in the m. truncatula gene atlas. after removal of unreliable probesets, the expression data for 1526 m. truncatula genes representing protein clusters were used for further analysis. expression data are available from various conditions including mycorrhizal roots with rhizophagus irregularis and with glomus mosseae, and laser-microdissected root cortex cells with arbuscules from r. irregularis. in addition, expression data of inoculated roots of the doesn’t make infection mutant, roots treated with myc factor for 6 h and 24 h, and roots treated with low phosphate levels are available. the values of gene expression under these treatments and of the corresponding control treatments, were extracted from the medicago gene atlas to calculate induction ratios according to criteria. in order to focus on proteins that are induced robustly in mycorrhizal roots, an induction threshold of 3-fold was applied for further filtering of candidate genes. a complete list of genes identified by task with the corresponding expression ratios according to criteria 1- is provided in additional file 7: table s table criteria used for gene expression filtering of m. truncatula genes identified in task4 name name of treatment in mtgea these criteria were applied to the genes of task for which a reliabel probe set was available in the m. truncatula gene atlas. *expression values were averaged for the two samples inoculated by g. intraradices and g. mosseae, respectively. combinatorial analysis revealed that genes were commonly induced in mycorrhizal roots and in microdissected arbusculated cells, whereas genes were induced only in mycorrhizal roots, and were induced only in microdissected arbusculated cells, respectively. only genes were induced by myclco after 6 h of treatment, while none of them remained induced after 24 h of treatment. interestingly, genes were induced in the dmi mutant which is defective for ccamk, indicating that their expression is regulated independently of the common sym pathway. three of these genes were also induced in am roots, in microdissected arbusculated cells, and in p-starved roots. it will be interesting to explore how these genes are induced in the absence of dmi figure venn diagram of m. truncatula genes up-regulated under various am-related conditions. genes listed in additional file 7: table s were subjected to combinatorial analysis according to the criteria listed in table  red domain, genes induced in mycorrhizal roots; green domain, genes induced by myc-lco after 6 h; dark blue domain, genes induced in laser-microdissected cortex cells with arbuscules; light blue domain, genes induced in the dmi mutant. criterion did not yield any result. genes identified according to criterion are marked in red. list a, b, and c are separately shown in additional file 8: table s while gene expression patterns can be identified based on defined conditions, global expression analysis by clustering can identify groups of genes with similar expression patterns over a large set of expression data like the different treatments and conditions covered by the medicago gene atlas. this approach can identify groups of genes that are co-regulated and therefore might be functionally related. on the other hand, this approach can lead to the discovery of common regulatory elements in promoters, which are the reason for co-regulation. hence, we decided to use pairwise average linkage and pearson correlation in order to identify genes with shared expression pattern. all proteins identified by task were correlated based on their standardized gene expression score, i.e. the ratios between the individual expression levels divided by the average of all expression levels as a relative indicator of expression. particular attention was paid to groups of genes that comprised am-induced genes. one conspicuous cluster of genes with a significantly correlated expression pattern turned out to be highly specific for am, resulting in apparent vertical red stripes in the visual representation of the cluster. a further relevant group comprised genes that are induced commonly in am and in rns. these genes may encode proteins that play a general role in symbiotic interactions. interestingly, this cluster consisted primarily of chitinases, cysteine proteases, a glucanase and several ripening-related proteins.figure details of the am-related gene cluster shown in additional file 9: figure s the hierarchical cluster contains genes induced in mycorrhizal roots. numbers in the column “criteria” indicate under which conditions the gene was induced at least 3-fold. numbers in the column “task” indicate in which task the gene was still retained. ranks are assigned according to gene induction in mycorrhizal roots. cluster of genes induced in both, mycorrhizal roots, and nodule symbiosis. details from cluster shown in additional file 8: table s numbers in the column “criteria” indicate under which conditions the gene was induced at least 3-fold. numbers in the column “task” indicate in which task the gene was still retained. ranks are assigned according to gene induction in mycorrhizal roots. analysis of relative conservation of proteins among angiosperms many genes with a role in am are induced during the interaction. this includes nutrient transporters such as am-related phosphate transporters, and the ammonium transporter amt, as well as regulatory components such as the transcription factor ram. in contrast, the genes involved in the early steps of the interaction are constitutively expressed. for example, the expression of the nod factor receptors and of the common sym genes is not significantly altered during am development in petunia, consistent with their early function in symbiont recognition and signalling. in order to identify constitutively expressed genes with a potential role in am development, we decided to compare the relative sequence conservation of proteins in the context of the three am-relevant plant groups: am-competent dicots, am-competent monocots, and non-mycorrhizal dicots. in order to avoid to miss relevant genes due to proteome incompleteness, we chose the relatively permissive task for this approach. firstly, multiple sequence alignment from the sequences represented in the individual clusters retained by task were calculated using mafft, and secondly, these msa consensus sequences were used as queries to search by psi-blast the proteomes used for hieranoid clustering and in addition a number of monocot species, namely o. sativa, zea mays, triticum urartu, sorghum bicolor, hordeum vulgare, brachypodium distachyon, and aegilops tauschii. the e-values were then compared between the groups by wilcoxon test to identify genes for which the populations of e-values between groups c and a, or between c and b were significantly different. for proteins that exhibited significant differences, the e-values were averaged among the three groups, and the ratios between the log of these values for c/a and c/b were calculated as a relative measure for am-related sequence conservation. the higher the conservation ratio, the farther the non-mycorrhizal homologues are from the consensus sequences relative to the homologues from am-competent species, indicative for am-related conservation. establishing the frequency distribution of the conservation ratios revealed that several of our test genes, such as symrk, vapyrin, ram ram and pt passed this filter, hence their pattern of sequence conservation was significantly related to the competence to engage in am symbiosis. surprisingly, none of the test genes passed the comparison between non-mycorrhizal dicots and mycorrhizal monocots, although at least vapyrin, ram and pt are more closely related between the am-competent dicots and the monocots, than between the am-competent and the non-mycorrhizal dicots.figure conservation ratios of potentially am-related proteins averaged for relevant plant groups with significant difference between non-am and am species. histograms represent the frequency distributions of the ratios of log of the e-values from psi-blast. the query sequences for psi-blast were generated by calculating msa consensus sequences based on the results of task these query sequences were blasted against am-competent dicot species, monocot species, and non-mycorrhizal dicot species. to derive conservation ratios, the e-values were averaged group-wise for groups a, b, and c, respectively, and the following ratios were generated: c/a and c/b. conservation ratios were included only if the difference between the groups were significant. ratios for log10/log ratios for log10/log note that control genes from additional file 5: table s passed the filter in, whereas none passed in. since the significance threshold of the wilcoxon test eliminated many genes, we sought for an alternative way to evaluate the degree of am-related conservation. instead of a fixed threshold level, we defined the conservation ratios for a set of household proteins that do not exhibit an am-related bias in conservation. to this end, we extracted from the hieranoid clusters those that have representative homologues from all plants, including am-competent and non-mycorrhizal species. thus, this set contains conserved house-keeping proteins that can serve as reference for the conservation patterns of proteins identified by task we proceeded in the same way as with the proteins identified by task i.e. an msa consensus sequence was calculated based on the 6 am-competent species from each protein cluster, and these msa sequences were used as queries for psi-blast against all proteomes. as expected, most of the genes selected in this way showed a conservation pattern consistent with the closer phylogenetic relatedness among all the dicots vs. the monocots. this fact is reflected in the phylogenetic trees of the house-keeping genes cyclin d and rpl, which are also represented in the list resulting from task. we selected a set of proteins from the results of task with intermediate e-values and with genes represented in most monocots. for these genes, the ratios were calculated as for the genes obtained with task. these values tended to be more to the negative, reflecting the closer position of the brassicaceae homologues from the msa consensus sequences relative to the proteins obtained with task hence, the reference genes resulting from task define the range of conservation ratios for housekeeping proteins and therefore allows to define the range that contains proteins with an am-related bias of conservation. global comparison of the conservation ratios of proteins identified by task and task revealed considerably higher values for the former group, reflecting the different conservation patterns among proteins selected by task and task in the comparison between group a and group c, the am-related genes ram and pt were clearly seprated from the housekeeping controls cyclin d and rpl, while this distinction was much less clear in the comparison between group b and group c. these results show that the conservation ratio can be used as a comparative proxy to evaluate the relative degree of conservation of a given protein among am-competent species relative to the non-mycorrhizal species.figure conservation ratios of potentially am-related proteins in comparison with housekeeping genes averaged for relevant groups of plant species. conservation ratios were generated as for figure . however, no statistics were performed and all ratios are shown. conservation ratios are compared for potential am-related proteins extracted by task, and for potential house-keeping genes identified by task. for comparison, the position of the proteins represented in figure  is indicated. ratios for log10/log ratios for log10/log outcome of the in silico substraction approach the goal of this study was to identify am-related genes based on the conservation pattern of their orthologues between am-competent and non-mycorrhizal plant species. this approach is particularly targeted to identify am-related genes that are not induced during symbiosis, hence, we focused on the proteins identified by task that are not induced at the gene level. to evaluate the efficiency of the approach, this list was ordered according to the ratios of e-values between the averaged brassicaceae and am-competent dicot plants, respectively. the list was sorted in descending order, since the highest values for the conservation ratios indicate the proteins that are conserved to a higher degree among am-competent species than between am-competent and non-mycorrhizal species. in this list, the first protein, a predicted α-glucosidase/ xylosidase, was chosen to evaluate its conservation pattern in detail. indeed, a phylogenetic tree prepared as in figure  shows an extremely skewed pattern of conservation with a clearly resolved common branch of the am-competent monocots and dicots, including the basal lineage amborella trichopoda, whereas all the non-mycorrhizal species, the brassicaceae, and b. vulgaris as a representative of the chenopodiaceae, form an outlier group.figure phylogenetic analysis of an α-glucosidase with high conservation ratio. the first hit in the list of predicted am-related proteins was an α-glucosidase. the s. lycopersicum sequence was used to retrieve the closest homologues in a wide range of species for phylogenetic analysis. tree as in figure  with the first hit per species identified at ncbi by blastp against non-redundant protein database using the tomato α-glucosidase as a query. tree with all homologues of solyc03g. from tomato, grape vine, poplar, m. truncatula, and a. thaliana. note the am-related branch that has no homologue from a. thaliana. the closest arabidopsis homologue clusters far away. hydrolases are often encoded by gene families, and this is also the case for this α-glucosidase/xylosidase. in order to test whether the member with the am-related conservation pattern forms a dedicated group in am-competent species, we isolated all the homologues available from the protein database at ncbi for the species v. vinifera, p. trichocarpa, s. lycopersicum, m. truncatula and a. thaliana. they are numbered in each species based on their similarity to the am-related homologue in s. lycopersicum. a phylogenetic tree with all these sequences revealed that all am-competent species have a single am-related homologue, resulting in a clearly separated am-related branch. the closest homologue of a.thaliana falls into the large containing the remaining sequences. hence, a. thaliana misses only the am-related form of the α-glucosidase/ xylosidase gene family. search for potential cis-regulatory elements in promoters of am-related genes besides the conservation of the orf, we investigated the non-coding upstream sequences of am-related genes by searching for potential cis-regulatory promoter elements that may control gene activity during symbiosis. we selected the m. truncatula proteins identified by task that were at least 3-fold induced in mycorrhizal roots relative to non-mycorrhizal control roots. their promoter sequences, were downloaded from ensembl plants biomart, and analyzed with the pattern recognition software meme for overrepresented sequences. a first search revealed a series of conserved predicted promoter elements. a highly conserved element comprised the core sequence ggggttcgaacccc with the bold letters being almost invariant. the palindromic nature of this element, and the fact that many cis-elements in plant promoters are palindromes prompted us to search specifically for palindromic sequences. interestingly, of the elements predicted by the first search were confirmed by the second search for palindromes. besides the myc element, a second element with the well-defined core sequence tgagcttagctca emerged. an element with completely invariant sequence was the palindrome gccggc that tended to be located within the 500 bp immediately upstream of the atg, indicating that it represents a relevant promoter element. besides the gccggc element, none of the predicted elements were described previously. an element that could have been expected to result from our meme search is the am-related cttc element, also known as mycs element. this element is present in the promoters of am-related phosphate transporter genes, and of the am-related snare ljvti, however, it was not found by meme in our sample of 190 am-induced promoter sequences. this surprising result prompted us to search for it in the vapyrin promoter, which is induced in mycorrhizal roots of both, m. truncatula and petunia hybrida, indicating that this promoter may contain conserved am-related promoter elements. indeed, we found an extended version of the cttc element in all promoters of vapyrin genes from various monocots and dicots, indicating that it is of a wider significance for am-related gene regulation. the elements myc and myc the gccggc element, and the cttc element were selected for further statistical analysis in the genome of m. truncatula. in order to estimate their relative frequency in the promoters of am-related genes, we selected three samples: the promoters of the genes identified with task, those of the am-induced genes whithin task, and all promoters of m. truncatula. these promoter sequences were searched by blast for the presence of the four elements of interest. for the non-palindromic cttc element, both orientations were considered separately, and the two respective longer variants were included as well. indeed, element myc and myc as well as the gccggc element were significantly overrepresented among the genes identified by task compared to the statistical expectations, and relative to the observed frequency in all promoters. overrepresentation was even more evident when the am-induced genes were considered. significance of these results was confirmed by binomial test. in contrast to the myc and myc elements, and to the gccggc element, the cttc element was not overrepresented in promoters identified by task and only moderately in the am-induced genes, when its original sequence was considered. however, the longer form was significantly overrepresented in both orientations in the promoters of the genes identified by task and even more of the am-induced genes. this indicates that the longer version is the relevant form of the cttc element. discussion during the past decade, many screens for am- or rns-defective mutants have been carried out and a significant number of genes involved in signalling and symbiotic functioning have been identified. in addition, transcript profiling has identified many am- and rns-related transcripts, however, only few of them have been functionally characterized by reverse genetic approaches. a central finding of the majority of these genetic studies was, that the genes involved in symbiosis are well conserved among am-competent species, whereas they are conserved to a lesser extent, or entirely missing, in non-mycorrhizal species such as a. thaliana. based on this finding, we designed a strategy for a third avenue to identify yet unexplored am-related genes using the conservation of their coding region as the main criterion. we compared the proteomes of am-competent and non-mycorrhizal plant species, thereby isolating the proteins that are conserved only, or to a higher degree, in am-competent species. essentially, this approach represents an in silico substraction procedure, where the proteomes of the non-mycorrhizal species are substracted from the proteomes of the am-competent species to yield a fraction of the proteome that is expected to be enriched in am-related proteins. since function is selected for at the level of the protein, and since sequence conservation at the dna level is affected by the degeneracy of the genetic code, we compared the species at the proteome level. ideally, an in silico substractive approach would involve an equal number of proteomes of the am-competent and the non-mycorrhizal group. however, due to the wide success of am in nature, non-mycorrhizal species are the exception, and only few of them have been sequenced to a degree that they could be used for such an approach. as non-mycorrhizal species, only a. thaliana, a. lyrata, and b. rapa were available as proteomes in the ensembl database. in contrast, a wide range of am-competent species is available, among them various dicots and cereals. hence, in a first approach, we selected two am-competent monocots, four am-competent dicots, and the two non-mycorrhizal species a. thaliana and b. rapa. the first step in this approach was to establish the orthologous relationships between the proteomes of the eight selected species. this task was performed with hieranoid, which establishes clusters of orthologous/paralogous proteins in a hierarchical step-wise procedure that proceeds according to a previously defined phylogenetic tree of the involved species. the subsequent step is to identify protein clusters that have members in all or most of the am-competent species, but none in the non-mycorrhizal species. however, after a first run with hieranoid, we noticed that in many cases, the orthologous trees of known single proteins fell into a monocot and a dicot tree, thus preventing our strategy. therefore, we decided to perform a clustering just using dicot species, namely g. max, m. truncatula, p. trichocarpa, v. vinifera, s. lycopersicum, and s. tuberosum, together with the three non-mycorrhizal species a. thaliana, a. lyrata, and b. rapa. in order to assess the efficiency of the procedure, we used a number of known am-related genes and a few housekeeping genes for reference. clustering of all nine proteomes resulted in a total of 28’ protein clusters. subsequent selection of clusters that contain orthologues only in am-competent species established a first set of potential am-related proteins. in order to account for cases were individual orthologues may be missing from one or two proteomes, we relaxed the stringency of the filtering by allowing also for clusters were or members of am-competent species were missing. an even more permissive filter was applied to obtain a large population of proteins that were subsequently subjected to quantitative assessment of amino acid sequence conservation based on the comparison of e-values. the fact that task and task yielded rather long lists of candidate proteins was not considered a problem, since subsequent filtering and sorting narrowed the list further down. a next step was to use gene expression as a criterion for selection, hence, we defined expression ratios relevant for am. based on these criteria we sorted the list of proteins identified by task by applying a stringent threshold induction ratio of 3-fold, we restricted the analysis to genes with a robust transcriptional induction in mycorrhizal roots. besides known am-related proteins such as pt am vapyrin, ram and ipd this list included several interesting proteins like the lysm receptor kinase lyr a close homologue of the lotus japonicus nod factor receptor nfr, and several abc transporters and peptide transporters, that may play a role in am development. highly induced candidates with yet unknown functions in am include a triacylglycerol lipase, neutral ceramidase, an enzyme with a role in sphingolipid metabolism, and reticuline oxidase an enzyme involved in the biosynthesis of the alkaloid intermediate reticuline. the fact that many known am-induced genes were recovered by our selection procedure documents the potential of gene discovery by comparative phylogenetics. the main goal of this study was to identify new am-related proteins based on conservation of the coding sequence, i.e. independently of am-related gene induction. in order to obtain a quantitative measure for conservation of the open reading frame in am-competent species, we performed a secondary filtering step by blasting the conserved consensus sequences derived from the 6 am-competent species against a wider panel of plant proteomes including dicots, monocots, the liverwort selaginella moellendorfii, the moss physcomitrella patens, and the unicellular alga chlamydomonas reinhardtii. the interest to include these three lower plants is based on the finding that several of the am-related genes were found in liverworths and/or mosses, potentially revealing the evolutionary origin of the common sym pathway in lower land plants. the psiblast was performed with the results of the relatively permissive task. with this approach proteins were identified for which the e-values between the three relevant groups of plant species were significantly different between the non-mycorrhizal species, and the am-competent dicots and monocots, respectively, indicating that they are conserved particularly well in am-competent species. interestingly, the proteins with the lowest p-values included proteins with annotations as receptors, protein kinases, phosphatases, disease resistant proteins, or g-protein regulating protein, implying roles in signalling. in view of the essential role of am in plant nutrition, a highly conserved ammonium transporter, and the oligomineral nutrient transporter nramp are of particular interest. ammonium is the form in which nitrogen is thought to be transferred from the am fungus to the plant, and therefore, ammonium transporters are of particular importance in am. transporters for other mineral nutrients such as zinc and copper remain to be identified. many of the conserved proteins were also found to be well conserved in the liverwort s. moelledorfii, and some of them even in the moss p. patens, whereas c. reinhardtii proved to be phylogenetically much more distant. in order to obtain a direct quantitative measure for the degree of am-related conservation, we defined the conservation ratio, which represents the ratio of the averaged e-values from psiblast between the non-mycorrhizal species, and the am-competent species. for comparison, the results from task were included as representatives of generally well conserved housekeeping proteins. the larger the conservation ratio, the more a protein sequence is divergent between the non-am plants and the am-competent species. therefore, the proteins identified by task have mostly positive values, whereas the housekeeping proteins have neutral or negative values. of particular interest were proteins with a conservation ratio > which comprised for example pt and ram, which are known to show a highly am-related conservation pattern. a general overview over the conservation ratios revealed that for proteins, the conservation ratio could not be calculated because they were entirely missing from the psiblast results in the non-mycorrhizal species. in addition, a further proteins exhibited conservation ratios of > indicative of a high degree of am-related conservation. in order to define a range above which the conservation ratios can be considered as being skewed towards the am-competent species, proteins identified by task were included for reference in the results from task. among the proteins that were missing from the non-mycorrhizal species, have unknown functions. interestingly, three proteins each are predicted chitinases, and glucanases, indicating potential roles in cell wall modification or signaling. three predicted cle peptides might be involved in am-related signaling as it was shown for rns. a known component in symbiotic signaling comprised interacting protein of dmi / cyclops, an interactor of the central symbiosis regulator ccamk encoded by dmi. high conservation ratios were also found for vapyrin, which is required both for am and rns and vapyrin-like. proteins exhibited a value > for both conservation ratios, i.e. they are highly conserved among both, am-competent dicots and monocots, relative to the non-mycorrhizal species. of particular interest is task3_ which encodes a predicted α-glucosidase which is in some species classified as α-xylosidase. while the conserved protein is missing from a. thaliana, there are related glycosidases that occur in all examined plant species including a. thaliana, indicating that the non-mycorrhizal model species misses only the am-related isoform. the potential role for α-glucosidase or α-xylosidase in am is unknown, but given the important contribution of xylose in the hemicelluloses of the cell wall, it may be involved in the modifications of cell walls during am infection and functioning. interestingly, a predicted α-xylosidase of petunia is among the most strongly repressed genes under conditions of high phosphate, which inhibits am. five of the proteins with high conservation ratios represent receptors and protein kinases, of which three are closely related receptor-like protein kinases. they are similar to s-locus receptor kinase, which mediates sporophytic self-incompatibility in the brassicaceae, and to the maize receptor kinase zmpk. phylogenetic analysis showed that they indeed fall into a large clade of proteins found in all am-competent species, but with considerably lower conservation in the non-mycorrhizal species. a conserved c-rich stretch in the amino acid sequence is shared among the three am-related rlks, as well as with srk, and zmpk, indicating that they may have similar tertiary structures mediated by cysteine bridges. interestingly, zmpk is expressed in roots and young seedlings, rather then floral tissues suggesting that this family of receptor kinases can have other functions than self-incompatibility. taken together, the examples of α-glucosidae/xylosidase and the rlks show that our approach can identify proteins with am-related conservation patterns even if they are members of large families. furthermore, the case of the three related rlks highlights the potential of our approach to identify proteins that could potentially escape genetic mutant screens because of functional redundancy. transcription is controlled primarily by cis-acting regulatory sequences in the promoters of the genes. hence, co-regulated genes can be expected to share common regulatory elements in their promoters. based on this logic, we screened the promoters of 190 am-induced genes for overrepresented sequences that may represent cis-acting regulatory promoter elements. the prediction program meme identified two new palindromic elements and found the gccggc element to be overrepresented in am-induced promoters as well as in the promoters of genes identified by task statistical analysis confirmed the overrepresentation of these elements in am-related promoters. the statistical significance of element myc and myc is emphasized by their rather unusual length compared to other cis-acting elements that are typically less then 10 nt in length. in addition, element myc and myc are often coupled, underscoring their relevance for am-related gene induction, and suggesting that they may function in conjunction. the predominant position of the gccggc element in a range of 500 bp upstream of the atg start codon is indicative of functional relevance as well. the gccggc element can be bound by the transcription factor rap. in ethylene-responsive promoters involved in the potassium starvation response of a.thaliana, and it was classified as wound- and pathogen-inducible. gccggc represents a palindromic variant of the common gcc element that is widely found in the promoters of pathogenesis-related genes and in ethylene-responsive promoters of various plants. in fact, gcc elements occur in different variants, which are recognized by specific members of the ethylene response factor family of transcription factors. the overrepresentation of the gccggc element in am-related promoters may explain the frequency of predicted stress- and pathogen-related transcripts in the transcriptome of mycorrhizal roots in plant species such as rice, potato, petunia, parsley, m. truncatula, and l. japonicus. surprisingly, the am-related cttc/mycs element was not found by meme in the 190 am-induced promoters. furthermore, it was not overrepresented in the genes resulting from task and only moderately in the am-induced genes. however, an extended form, was found to be significantly overrepresented, indicating that this may represent the complete am-inducible element in m. truncatula. an overview over the occurrence of predicted promoter elements in the analyzed promoters is provided in additional file 19: table s taken together, we have identified two new potentially am-associated cis-regulatory promoter elements. in addition, we identified the gccggc element as a potential am-related element that may indicate an involvement of ethylene in am development, and we present a modified form of the cttc element. future work will address the functional relevance of these elements for am development and functioning,  we describe a non-targeted approach to identify new am-related genes by genome-wide comparative sequence analysis of am-competent and non-mycorrhizal plant species. the validity of this approach has been confirmed by the finding that it identified many known am-related genes from the genomes of am-competent plant species. following this strategy, we identified many new potentially am-related genes based on the conservation pattern of the coding sequence, on gene expression pattern, and on predicted regulatory promoter elements. these genes will be further evaluated for their predicted function, the degree of their conservation, and the copy number, or the number of similar genes in the case of gene families. this process will result in a short list of genes that will be further functionally analyzed by insertional mutagenesis in p. hybrida, m. truncatula or l. japonicus using established transposon-related protocols. 
the central element of our approach is based on the fact that many am-related genes are conserved only among am-competent species. our approach involves genome-wide comparisons at the proteome level of am-competent host species with non-mycorrhizal species. proteins of these clusters were then analyzed in an extended set of plant species and ranked based on their relatedness among am-competent monocot and dicot species, relative to non-mycorrhizal species. as a result we present a list of yet uncharacterized proteins that show a strongly am-related pattern of sequence conservation, indicating that the respective genes may have been under selection for a function in am. using a clustering method we first established orthologous/paralogous relationships and subsequently identified protein clusters that contain members only of the am-competent species. we present a new systematic strategy of gene discovery based on conservation of the protein-coding sequence that complements classical forward and reverse genetics.
 genes involved in arbuscular mycorrhizal symbiosis have been identified primarily by mutant screens, followed by identification of the mutated genes. in addition, a number of am-related genes has been identified by their am-related expression patterns, and their function has subsequently been elucidated by knock-down or knock-out approaches. however, genes that are members of functionally redundant gene families, or genes that have a vital function and therefore result in lethal mutant phenotypes, are difficult to identify. if such genes are constitutively expressed and therefore escape differential expression analyses, they remain elusive. the goal of this study was to systematically search for am-related genes with a bioinformatics strategy that is insensitive to these problems. the central element of our approach is based on the fact that many am-related genes are conserved only among am-competent species.  our approach involves genome-wide comparisons at the proteome level of am-competent host species with non-mycorrhizal species. using a clustering method we first established orthologous/paralogous relationships and subsequently identified protein clusters that contain members only of the am-competent species. proteins of these clusters were then analyzed in an extended set of plant species and ranked based on their relatedness among am-competent monocot and dicot species, relative to non-mycorrhizal species. in addition, we combined the information on the protein-coding sequence with gene expression data and with promoter analysis. as a result we present a list of yet uncharacterized proteins that show a strongly am-related pattern of sequence conservation, indicating that the respective genes may have been under selection for a function in am. among the top candidates are three genes that encode a small family of similar receptor-like kinases that are related to the s-locus receptor kinases involved in sporophytic self-incompatibility.  we present a new systematic strategy of gene discovery based on conservation of the protein-coding sequence that complements classical forward and reverse genetics. this strategy can be applied to diverse other biological phenomena if species with established genome sequences fall into distinguished groups that differ in a defined functional trait of interest. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords arbuscular mycorrhizasymbiosissymbiosis signalingcommon sym geneconservationgene clusteringproteome analysisbioinformaticsissue-copyright-statement© the author 2014 