BACKGROUND
more than 80 % of all land plant species enter an arbuscular mycorrhiza  symbiosis with glomeromycota fungi.
this plant-microbe interaction primarily improves phosphate uptake, but also contributes to the acquisition of nitrogen, minerals, and water  <cit> .
am host plants secrete strigolactones that stimulate fungal metabolism and promote pre-symbiotic hyphal branching  <cit> .
in return, am fungal recognition by the plant is thought to be mediated by myc-factors that signal the presence of symbiotic microbes.
the chemical structure of one class of diffusible factors from am fungi was revealed by maillet et al.
<cit> , who identified a mixture of sulfated  and non-sulfated  lipochitooligosaccharide  molecules in fungal exudates.
although the n-linked acyl chain is different  <cit> , myc-lcos are structurally related to rhizobial nodulation -factor lcos.
after perception by rhizodermal lysm receptor kinase heterodimers , these lcos mediate bacterial infection and the induction of nodule primordia  <cit> .
the general similarity between rhizobial and am fungal lcos indiates that rhizobia adopted a pre-existing, much older mycorrhizal signaling program to initiate host responses  <cit> .
in addition to being involved in mycorrhization and nodulation, microbial lcos stimulate the formation of lateral roots  <cit> .
since the activation of this basic developmental process  <cit>  and the transcriptional responses of roots towards myc-lcos require nfp  <cit> , recognition of am fungal lco signals is probably mediated by this lysm receptor.
however, since nfp-type receptors are missing a functional kinase domain  <cit> , another kinase is additionally required for signal transduction.
one such candidate was proposed to be lyk <dig>  the second component of the heterodimeric nod-lco receptor and a m. truncatula orthologue of the rice chitin receptor oscerk <dig>  <cit> .
perception of myc-lco signals leads to characteristic ca2+-oscillations in rhizodermal cells  <cit> .
in m. truncatula, this ca2+ spiking depends on the lrr  receptor kinase dmi <dig>  and on dmi <dig>  a potassium channel of the nuclear membrane.
ultimately, the ca2+/calmodulin-dependent protein kinase dmi <dig>  together with its interacting transcription factor cyclops, receives the spiking pattern  <cit> .
since mycorrhizal as well as rhizobial infection is controled by the same set of dmi proteins, this signal cascade is referred to as the common symbiotic signaling pathway .
apart from myc-lcos, short chain chitooligosaccharides  were identified as am fungal signals that induce ca2+ spiking  <cit> .
in root organ cultures  <cit> , the level of co <dig> and co <dig> was enhanced by plant-derived strigolactones, and these co versions induced a spiking pattern similar to exudates from germinating spores, being more persistent than that observed for myc-lcos.
this response required dmi <dig> and dmi <dig>  but, in contrast to myc-lcos, was nfp-independent, suggesting a role for other lysm kinases  <cit> .
similar to myc-lco perception, homologues of the cerk <dig> chitin receptor might be involved here  <cit> .
in addition to diffusible myc-factors acting at a distance , several lines of evidence point to signals that require physical contact between fungal hyphae and host roots .
such contact signals coordinate a specific re-differentiation of rhizodermal and cortical cells, once hyphopodia attach to the root surface.
the most remarkable cytological response of the infected host cells is the development of a cytoplasmatic pre-penetration apparatus .
upon hyphopodium formation, this structure is established by each successively infected cell and guides fungal hyphae towards the inner cortex  <cit> .
here, they proliferate in the apoplast and ultimately form intracellular, highly branched arbuscules that serve as the major interface for nutrient exchanges  <cit> .
global transcriptional changes in m. truncatula am roots were studied extensively, resulting in a comprehensive overview of symbiotic gene expression on the tissue and cellular level .
in contrast to these studies that investigated colonized roots, genome-wide analyses of pre-symbiotic gene expression responses to diffusible myc-signals are still limited  <cit> .
based on medicago genechip hybridizations, we previously reported on the lco-related transcriptional changes in 5 days old primary roots of the m. truncatula wild type  <cit> , challenging roots for  <dig> or 24 h  with 10− <dig>  or 10- <dig> m  lcos.
a sufficient activation of symbiotic signaling at these concentrations was validated by the histological detection of epidermal mtenod <dig>  <cit>  expression.
the complex transcriptional responses towards myc-lcos were almost absolutely dependent on nfp and largely on dmi <dig>  indicating that during the pre-symbiotic stage, am fungal lco signals are predominantly transduced via the cssp  <cit> .
in a comparable experimental setup, camps et al.
<cit>  recently studied the response of 4 days old primary m. truncatula roots in the wild type and in dmi3- <dig> mutants.
the most notable differences between the two studies are the shorter incubation time of 2–4 h and the usage of 10– <dig> fold higher  concentrations of myc-lcos by camps et al.
<cit> , together leading to an identification of more differentially regulated genes.
in additon, rnaseq was applied, which allowed to measure the expression of genes not represented on medicago genechips.
similar to 10-7/- <dig> m myc-lco concentrations  <cit> , camps et al.
<cit>  demonstrated that lco-signaling is predominantly mediated by dmi <dig> also at 10− <dig> m, although a dmi3-independent signaling pathway was defined as well.
in addition to studying cssp-dependency, camps et al.
<cit>  also looked into myc-lco activated gene expression in nsp1- <dig> mutants.
interestingly, a high proportion of the myc-lco induced, dmi3-dependent genes appeared to require the gras transcription factor  nsp <dig>  as already reported for a limited number of genes  <cit> .
nsp <dig> acts immediately downstream of the cssp and was originally thought to be exclusively involved in nodulation .
delaux et al.
<cit>  and takeda et al.
<cit>  had nevertheless reported that nsp1-deficient legume mutants displayed a reduced am fungal colonization.
a comparable, non-essential function for mycorrhization was also demonstrated for the nsp1-interactor nsp <dig>  <cit> , another gras tf initially thought to be exclusively required for nodulation  <cit> .
it has to be noted that both nsp <dig> and nsp <dig> play a role in strigolactone biosynthesis  <cit> , providing further evidence for a link between nsp gras tfs and am formation.
recently, the gras tf ram <dig> was identified in a screen for am-deficient mutants  <cit> .
similar to nsp <dig>  ram <dig> interacts with nsp <dig>  <cit> , illustrating that nodulation and mycorrhization are controled by overlapping sets of regulators  <cit> .
since in contrast to nsp <dig>  ram <dig> was not described to be required for the transduction of nod-signals  <cit> , ram <dig> is currently regarded both as an essential and a specific component of myc-signaling.
in later stages of am, ram <dig> plays a role in the development of functional arbuscules both in m. truncatula and petunia  <cit> , a function consistent with the mtram <dig> promoter activity in cortical cells  <cit> .
while putative receptors  and components of the cssp mediating the pre-symbiotic transduction of lco signals have clearly been identified , the interdependency of the key regulators that activate lco-related transcriptional networks immediately downstream of the cssp remains to be adressed.
to shed light on the relative importance of the gras tfs nsp <dig> and ram <dig> for lco-activated gene expression particularly during the pre-symbiotic stage, we obtained global transcription patterns of 5 days old nsp1- <dig> and ram1- <dig> plantlet roots after a 6 h challenge with myc- and nod-lcos.
as in a previous study  <cit> , we used 10-7/- <dig> m lco concentrations, in order to be as close as possible to the lower concentrations of myc-lcos that likely exist in soils and to avoid over-activation of gene expression by higher signal concentrations.
together with the transcriptional responses of nfp- <dig> and dmi <dig> mutants, the expression fingerprints obtained served as molecular phenotypes to specify the requirement for nodulation- and mycorrhization-related gras tfs during the initial transduction of lco signals, prior to a contact between the symbionts.
our findings provide evidence that in contrast to nod-lco signaling that predominantly relies on nsp <dig>  the presence of both nsp <dig> and ram <dig> is essential for almost all myc-lco related gene expression activation in the pre-symbiotic stage.
moreover, our results link the pre-symbiotic responses with the transcriptional reprogramming during am fungal contact and early root colonization, a process largely blocked in ram1- <dig> mutants.
we propose a model, where downstream of the cssp, the ram <dig> and nsp <dig> tfs act synergistically in the transduction of diffusible myc-lco signals.
RESULTS
we here present an integrated view of the global transcriptional responses in primary roots of the m. truncatula wild type a <dig> as well as the nfp- <dig>  dmi <dig>  nsp1- <dig>  and ram1- <dig> mutants towards am fungal  and rhizobial  lcos, using concentrations of 10− <dig>  or 10− <dig> m , respectively.
for all lco treatments, we used 5 days old seedlings, being in a stage of development where the first trifoliate was just initiated.
using nsp1- <dig> and ram1- <dig> mutants that lack functional nsp <dig> and ram <dig> gras tfs, our experiments build on a study of myc-lco induced gene expression in the wild type as well as nfp- <dig> and dmi <dig> mutants  <cit> .
acting downstream of the cssp, the nsp <dig> and ram <dig> regulators can be considered as key jigsaw pieces of early myc-lco signaling.
to identify myc-lco specific effects, the transcriptional responses towards nod-lcos in the wild type  <cit>  were now complemented with results for nfp- <dig>  dmi <dig>  nsp1- <dig>  and ram1- <dig> mutants as well.
in our experimental conditions that rely on the terminal  <dig> to  <dig>  cm of 5 days old primary roots excluding the root meristem, these concentrations led to an easily visible epidermal induction of the mtenod <dig> promoter by all lcos tested, including the least efficient nsmyclcos  <cit> .
together with the marked dmi3-dependency of most gene expression responses to 10-7/- <dig> m myc-lcos  <cit> , this indicates that symbiotic ca2+ spiking was activated to a sufficient extent in our conditions.
due to the fact that gene expression responses towards all myc-lcos occurred much stronger after 6 h as compared to 24 h  <cit> , we here focus on this early time point.
a compilation of all gene expression data is available as additional file  <dig>  , and will be discussed in detail in the subsequent chapters.
activation of symbiotic marker genes by myc- and nod-lcos displays a differential requirement for the gras transcription factors nsp <dig> and ram1
to deepen our understanding of the transcriptional activation by symbiotic lcos, we first had a look at the expression properties of  <dig> genes related to symbiotic signaling .
this analysis revealed that mtnfp, mtlyk <dig>  and all components of the cssp  as well as mtcyclops were not activated in the wild type by any symbiotic lco tested.
whereas mtnsp <dig> was slightly activated only after a 24 h treatment with nod-lcos, mtnsp <dig> was  <dig> -fold up-regulated in the wild type by this factor already after 6 h .
this up-regulation depends on nfp and dmi <dig>  but was independent of both nsp <dig> itself  and ram <dig> .
mtnsp <dig> in addition displayed a tendency for activation by myc-lcos , which was absent in nsp1- <dig>  but still detectable in ram1- <dig> mutants .
the induction of mtnsp <dig> by myc-lcos thus appears to be independent of ram <dig>  an observation in line with results delaux et al.
<cit>  obtained for a 10− <dig> m s/nsmyc-lco mixture.fig.
<dig> expression of symbiotic marker genes in response to myc- and nod-lcos.
expression of mtnsp <dig> , mtenod <dig> , and mtvapyrin  in m. truncatula wild type a <dig> , nfp- <dig> , dmi <dig> , nsp1- <dig> , and ram1- <dig>  plantlet roots challenged for 6 h with 10− <dig> m smyc-lcos , 10− <dig> m nsmyc-lcos , a mixture of 10− <dig> m smyc- and 10− <dig> m nsmyc-lcos , and 10− <dig> m nod-lcos .
ratios of up-regulation in comparison to non-treated controls  is shown.
an at least  <dig> -fold  up-regulation is denoted by asterisks
a well-characterized marker gene responding to lcos is mtenod <dig>  <cit> .
this gene was found activated most strongly by nod-  and only slightly by smyc- and s/ns-myclcos  in wild type plantlet roots .
whereas mtenod11-activation by nod-lcos was not detected in nfp- <dig>  dmi <dig>  and nsp1- <dig> mutants, this gene was still up-regulated in the ram1- <dig> line, although somewhat lower .
activation of mtenod <dig> by smyc-lcos follows a similar tendency .
the ram <dig> independency of mtenod <dig> expression in response to smyc- and nod-lcos conforms to the histological results of sun et al.
<cit> .
as reported previously  <cit> , no mtenod <dig> induction by nsmyc-lcos was detected in the pooled root samples used here, probably due to a quenching of the epidermal expression observed by reporter gene studies  <cit> .
a prominent example for a symbiosis-specific gene relevant both for am fungal and rhizobial infection is mtvapyrin, an ankyrin repeat protein involved in membrane trafficking  <cit> .
in line with a report by sun et al.
<cit> , who used different concentrations of lcos applied to only 2 days old seedlings, this gene was also found to be up-regulated between  <dig> - and  <dig> -fold by all symbiotic lcos in the wild type in our conditions .
similar to many other lco-induced genes  <cit> , mtvapyrin activation by fungal lcos strictly depends on nfp .
whereas dmi <dig> and nsp <dig> were similarly required for an expression activation by nsmyc- and s/nsmyc-lcos, smyc-lcos still induced mtvapyrin in dmi <dig> and nsp1- <dig> mutant roots, although at a strongly reduced level .
in contrast, mtvapyrin up-regulation was independent of ram <dig> for all fungal lcos tested, reminiscent of the detection of vapyrin expression in mycorrhizal roots of petunia ram1- <dig> mutants  <cit> .
with respect to nod-lcos, mtvapyrin induction also required nfp and dmi <dig>  but was independent of both nsp <dig> and ram <dig> .
the differential mtvapyrin expression observed in wild type, nfp- <dig>  dmi <dig>  and nsp1- <dig> roots was comparable to results obtained for 10- <dig> m nod-lcos  <cit> .
it thus appears that while the up-regulation of mtvapyrin is largely nsp1-dependent in response to myc-, it is nsp1-independent in response to nod-lcos.
together, the activation of symbiotic marker genes by myc- and nod-lcos points to a differential requirement for the symbiotic gras tfs nsp <dig> and ram <dig>  indicating that these regulators might be of different relative importance for myc- and nod-lco signaling in general.
gene expression activation by myc-lcos strongly depends on the two gras transcription factors nsp <dig> and ram1
we previously reported on the transcriptional responses of m. truncatula wild type a <dig>  nfp- <dig>  and dmi <dig> plantlet roots towards myc-lcos, leading to the conclusion that the lysm receptor kinase nfp is almost absolutely essential for the perception of myc-lco signals and that the majority of myc-lco specific gene expression is activated via the cssp  <cit> .
in order to investigate processes occuring downstream of the cssp, we studied the transcriptional responses towards smyc-, nsmyc-, and s/nsmyc-lcos in nsp1- <dig> and ram1- <dig> plantlet roots.
to allow a direct comparison, the resulting gene expression patterns were analyzed together with previous data for wild type, nfp- <dig>  and dmi <dig> roots  <cit> .
these analyses revealed that out of  <dig> genes activated at least  <dig> -fold  by myc-lcos and not by nod-lcos after 6 h in the wild type , only  <dig> were still up-regulated at these cutoffs by any myc-lco or myc-lco combination in ram1- <dig>  equivalent to an appr.
94 % reduction .
this observation fits well to the strongly impaired am fungal root colonization observed in this mutant  <cit> .
strikingly, a comparable reduction of gene expression responses  was also observed in the nsp1- <dig> mutant, where only  <dig> genes remained activated by any myc-lco or myc-lco combination .
although nsp <dig> was recently described to be relevant for mycorrhization , the extent of reduction of gene expression responses towards myc-lcos was unexpected.fig.
<dig> myc-lco related gene expression in the m. truncatula wild type and in four symbiotic mutants.
a number of genes at least  <dig> -fold  up-regulated in m. truncatula wild type a <dig> , nfp- <dig> , dmi <dig> , nsp1- <dig> , and ram1- <dig>  plantlet roots challenged for 6 h with 10− <dig> m smyc-lcos , 10− <dig> m nsmyc-lcos , or a mixture of 10− <dig> m smyc- and 10− <dig> m nsmyc-lcos .
none of these genes is activated by nod-lcos at this cutoff .
on the left, the three myc-lco specific data columns are summed up, regardless of individual myc-lcos.
values do not add up due to an overlapping activation of transcription by different lcos.
the number of genes induced by myc-lcos in the wild type still activated in four symbiotic mutants are shown to the right of the wild type columns.
expression of myc-lco related genes in nfp- <dig> and dmi <dig> plantlet roots reported by  <cit>  were included to facilitate comparisons.
b genes still activated in dmi <dig> mutants were subtracted  to highlight cssp-dependent effects
the concomitant importance of nsp <dig> and ram <dig> for signaling downstream of the cssp prompted us to focus on  <dig> myc-lco related genes encoding transcriptional regulators or components of signal transduction reported previously  <cit> .
after removing duplicate probes identified from the most recent m. truncatula genome annotation , transcription of the remaining  <dig> genes in wild type and mutant roots was visualized in fig.
<dig>  in addition to a strong reduction of expression in nfp- <dig> and dmi <dig> mutant roots, the  <dig> myc-lco related genes in general were not significantly up-regulated in the nsp1- <dig> and ram1- <dig> background.
in all but two cases, a significant activation in mutant roots only occurred in response to another lco or lco combination as in the wild type, probably indicating some level of deregulation in the symbiotic mutants .
the general absence of an expression activation comparable to the wild type indicates that the signaling-related genes mentioned above encode components of myc-lco signaling activated not only via the cssp, but subsequently via the gras transcription factors nsp <dig> and ram <dig> fig.
<dig> expression of myc-lco related genes encoding signaling components in the m. truncatula wild type and in four symbiotic mutants.
subset of  <dig> genes encoding transcription factors or signaling-related proteins and being rapidly activated in m. truncatula wild-type roots after a 6 h treatment with 10− <dig> m smyc-lcos , 10− <dig> m nsmyc-lcos , or a mixture of both .
none of these genes is significantly activated by 10− <dig> m nod-lcos  after 6 h  <cit> .
expression responses after 6 h in the wild type  as well as the nfp- <dig> , dmi <dig> , nsp1- <dig> , and ram1- <dig>  mutants is visualized as a heat map, with shades of red representing up- and shades of green down-regulation.
an at least  <dig> -fold  up-regulation is indicated by a white dot.
expression data were scaled to the maximum of a  <dig> -fold regulation using the genesis software  <cit>
in total, only  <dig> myc-lco related genes were still activated both in nsp1- <dig> and ram1- <dig> mutants.
whereas activation of all four genes required nfp, three were dmi3-independent , indicating that some myc-lco related genes can be activated independent of the cssp by an alternative pathway that also doesn't require nsp <dig> and ram <dig>
to exclude a bias from cssp-independent effects, we subtracted all genes still activated in dmi <dig> mutants, arriving at  <dig> out of  <dig> myc-lco related genes whose expression relies on the cssp .
apart from a requirement for nfp , the strong dependency on both nsp <dig> and ram <dig> was again evident, with gene expression activation by myc-lcos being reduced by 96 % in each case .
this strong nsp1-/ram1-codependency is also evident, if the effects are monitored separately for smyc-, nsmyc-, or s/nsmyc-lco induced genes .
interestingly, the co-dependency on nsp <dig> and ram <dig> observed for genes up-regulated by myc-lcos is also a characteristic of genes down-regulated by a treatment with these symbiotic signals .
in conclusion, our results demonstrate that the two symbiotic gras tfs nsp <dig> and ram <dig> are both essential for almost all gene expression responses towards a 6 h application of 10-7/- <dig> m myc-lcos.
genes co-activated by myc- and nod-lcos display a preferential requirement for the gras transcription factor nsp1
to further investigate the differential nsp1- and ram1-dependency of myc-lco-related transcription, we studied the expression in nfp- <dig>  dmi <dig>  nsp1- <dig>  and ram1- <dig> mutants of those  <dig> genes activated at least  <dig> -fold  not only by myc-, but also by nod-lcos after 6 h in the wild type .
this analysis revealed that gene expression responses  were reduced to comparable levels in nfp- <dig> and dmi3-mutants  as observed for myc-lco related genes , confirming that these genes are primarily activated via the cssp.
interestingly, the nsp1- <dig> and ram1- <dig> mutants now displayed a lower and clearly differential level of reduction , allowing the conclusion that genes co-activated by nod-lcos  show a higher dependency on nsp <dig> as compared to ram <dig>  this was most apparent for those genes activated by smyc-lcos, sharing the highest structural similarity to nod-lcos.
here, from  <dig> genes co-activated by smyc- and nod-lcos,  <dig> remained induced by smyc-lcos in ram1- <dig> mutants, while only  <dig> displayed activation by these lcos in the nsp1- <dig> background.
in contrast, the difference in reduction of responses to nsmyc-lcos was less pronounced in the nsp1- <dig> and ram1- <dig> mutants .
similar to the situation for myc-lco related genes, exclusion of those sym-related genes activated in dmi <dig> mutants led to comparable results .fig.
<dig> sym-lco related gene expression in the m. truncatula wild type and in four symbiotic mutants.
a number of genes at least  <dig> -fold  up-regulated in m. truncatula wild type a <dig> , nfp- <dig> , dmi <dig> , nsp1- <dig> , and ram1- <dig>  plantlet roots challenged for 6 h with 10− <dig> m smyc-lcos , 10− <dig> m nsmyc-lcos , or a mixture of 10− <dig> m smyc- and 10− <dig> m nsmyc-lcos .
these genes were also activated by nod-lcos at this cutoff .
on the left, the three myc-lco specific data columns are summed up, regardless of individual myc-lcos.
values do not add up due to an overlapping activation of transcription by different lcos.
the number of genes induced by myc-lcos in the wild type still activated in four symbiotic mutants are shown to the right of the wild type columns.
b genes still activated in dmi <dig> mutants were subtracted  to highlight cssp-dependent effects
based on these results we infer that transcriptional activation is less ram1-dependent, if it is triggered not only by myc-, but also by nod-lcos.
this observation is corroborated by an analysis of the  <dig> genes identified by camps et al.
<cit>  being commonly activated by myc- and/or nod-lcos both at 10-7/- <dig>  <cit>  and 10− <dig> m  <cit> .
from the  <dig> genes of this set co-induced by myc- and nod-lcos after 6 h in our conditions, only  <dig> are activated in the nsp1- <dig>  while  <dig> remain up-regulated in the ram1- <dig> mutant .
the strong nsp1-dependency of these sym-lco related genes indicates that many of them primarily respond to nod-lcos and are only cross-activated by myc-lcos, since their structure is similar.
this is particularly relevant for the smyc-lcos, where an absence of ram <dig> appears least important, while nsp1-dependency remains high .
most sym-lco related genes thus seem to be activated by a route primarily controled by nsp <dig>
gene expression activation by nod-lcos displays a preferential requirement for the gras transcription factor nsp1
since our results provided evidence that genes co-activated by myc- and nod-lcos show a higher requirement for nsp <dig> in comparison to ram <dig>  we assessed the nsp1- and ram1-dependency of those  <dig> genes being activated at least  <dig> -fold  by nod-lcos, but not by myc-lcos after 6 h in m. truncatula a <dig> .
to achieve comparability with the results reported above, we used the same concentration  of nod-lcos as reported previously for the wild type  <cit> , and also challenged plantlet roots for a duration of 6 h. to compare nod-lco activation of gene expression in the wild type to that in nfp- <dig>  dmi <dig>  nsp1- <dig>  and ram1- <dig> mutants, expression data had to be re-normalized across wild type and mutant conditions.
this led to a slight change in the number of genes activated in the wild type at least  <dig> -fold  by nod-, but not by myc-lcos .fig.
<dig> nod-lco related gene expression in the m. truncatula wild type and in four symbiotic mutants.
a number of genes at least  <dig> -fold  up-regulated in m. truncatula wild type a <dig> , nfp- <dig> , dmi <dig> , nsp1- <dig> , and ram1- <dig>  plantlet roots challenged for 6 h with 10− <dig> m nod-lcos .
these genes were not activated by myc-lcos at this cutoff .
the number of genes induced by nod-lcos in the wild type still activated in four symbiotic mutants are shown to the right of the wild type column.
b genes still activated in dmi <dig> mutants were subtracted  to highlight cssp-dependent effects
figure  <dig> shows that gene expression responses towards nod-lcos were reduced to similar levels as observed for sym-lco related genes in the nfp- <dig>  dmi <dig>  nsp1- <dig>  and ram1- <dig> mutants , using the same cutoffs as mentioned above.
as expected  <cit>  and similar to myc-lco perception  <cit> , these results revealed an almost complete dependency of the perception of nod-lco signals on nfp, and a predominant transduction of these nodulation-related signals via the cssp.
with respect to processes occuring downstream of dmi <dig>  the requirement for nsp <dig> was much more pronounced than the requirement for ram <dig> .
similar to the situation observed for sym-lcos , this effect was still evident after subtracting dmi3-independent genes and hence cssp-independent responses .
in conclusion, the ram1-dependency of gene expression is comparably low not only for sym-lco, but also for nod-lco related transcription, indicating that nod-lco related genes are activated by a signaling pathway primarily controled by nsp <dig>  however, a fraction of the nod-lco related genes identified in our conditions also depended on ram <dig>  a gras tf previously reported to be only required for myc-signaling and mycorrhizal infection  <cit> .
ram <dig> controls infection-related genes activated by nsmyc-lcos during the pre-symbiotic stage
to address the question, if myc-lcos supplied at 10-7/- <dig> m primarily act as signals that pre-announce the presence of a mycorrhizal fungus, or if they are also able to activate genes connected to early fungal infection, we related our results to gene expression responses recorded from laser-microdissected am tissues  <cit> .
although a comparison of microdissected cell types to the tissue samples studied here might include a bias, it allows us to obtain initial insights into overlapping expression patterns of genes responding to lcos in the pre-symbiotic and the infection stage.
in the laser-microdissection study used  <cit> , gene expression in regions of mycorrhizal roots containing early infection stages as well as hyphopodia  and in corresponding non-colonized areas  was analysed.
whereas in group a,  <dig> genes with a comparable expression in app and nap were identified that were also induced by myc-lcos, group b contained  <dig> genes up-regulated in app  <cit> .
group a genes were thus thought to be root-expressed and activated by external myc-signals before fungal contact, while group b genes were related to the initial intracellular infection  <cit> .
it has to be noted that for both groups, a substantial portion of genes continued to be expressed in mature mycorrhizal roots, primarily in the arbuscule-containing cells  <cit> .
amongst the  <dig> genes from group a,  <dig> are activated not only by myc-, but also by nod-lcos , indicating that these app and nap co-induced genes respond to lcos of different microbial origin  <cit> .
in line with the results for sym-lco related genes presented above,  <dig> of these are still up-regulated in response to myc-lcos independently of ram <dig>  while the nsp1-dependency remains higher .
looking at the  <dig> genes up-regulated in regions of mycorrhizal roots containing early infection stages and hyphopodia  <cit> ,  <dig>   <dig>  and  <dig> were already activated by smyc-, nsmyc-, and s/nsmyc-lcos in wild type plantlet roots, respectively .
due to redundancies, a total of  <dig> app-induced genes were up-regulated by any myc-lco or myc-lco combination, providing evidence that a limited number of these infection-related genes responds to diffusible myc-signals already at the pre-symbiotic stage.
interestingly,  <dig> app-induced genes were activated by nod-lcos , including mtvapyrin  <cit>  and  <dig> of the  <dig> myc-lco induced genes mentioned above.
this indicates a substantial cross-activation of infection-related genes by lcos of different microbial origin, as reported by camps et al.
<cit> .
the co-activation by nod-lcos might also explain why nine of the  <dig> app-induced genes up-regulated by myc-lcos are expressed independently of ram <dig>  furthermore, the highest ram1-dependency was found for the  <dig> app-induced genes already activated by nsmyc-lcos at the pre-symbiotic stage.
of these, only  <dig> were still up-regulated by nsmyc-lcos in ram1- <dig> mutants .
we now decided to focus on nsmyc-lcos, since these lack the sulfate group that leads to a strong structural similarity between smyc- and nod-lcos.
thus, gene expression responses to this most basic fungal lco should be most instructive to identify genuine mycorrhization responses.
to shed light on this, we had a look at all genes being activated by 10− <dig> m nsmyc-lcos in the wild type, but not in ram1- <dig> mutants.
a total of  <dig> myc-related  and  <dig> sym-related  genes fulfilled this criterion .
whereas the  <dig> myc-related genes activated by nsmyc-lcos displayed an almost absolute ram1-dependency also towards smyc-, s/nsmyc-, and nod-lcos , the  <dig> sym-related genes were completely ram1-dependent only for their activation by nsmyc-lcos.
in contrast, a total of  <dig>   <dig>  and  <dig> genes remained activated by smyc, s/nsmyc-, and nod-lcos in ram1- <dig> roots .
on the one hand, this allows to conclude that the  <dig> myc-related and  <dig> sym-related genes up-regulated by nsmyc-lcos are controled by a signaling pathway that strictly requires ram <dig>  on the other hand, in response to smyc- or nod-lcos, a substantial fraction of these can be induced by an alternative pathway that that does not require ram <dig> and is apparently not triggered by nsmyc-lcos.
this indicates that downstream of the cssp, fine-tuning of gene expression activation travels along alternative roads, depending on the lco variant initially perceived and possibly also on the epidermal cell type  <cit> , where the cssp is activated.
with respect to am fungal infection, it is interesting to note that two of the  <dig> myc-related and  <dig> of the  <dig> sym-related genes activated by nsmyc-lcos are up-regulated in regions containing early infection structures and hyphopodia  or in arbuscule-containing cells , while  <dig> and  <dig> were activated in whole am roots, respectively .
considering the ram1-dependency of their activation by nsmyc-lcos in the pre-symbiotic stage, it is thus possible that those genes are activated by nsmyc-lco signals also during am fungal contact and root infection.
discussion
gene expression responses towards 10-7/- <dig> m of am fungal and rhizobial lcos in primary roots of the m. truncatula wild type as well as nfp- <dig>  dmi <dig>  nsp1- <dig>  and ram1- <dig> mutants demonstrated that during the pre-symbiotic stage, lco-induced transcription strictly relies on the nfp receptor and largely on the calcium-calmodulin dependent kinase dmi <dig> .
this indicates that lcos of whatever origin are perceived by the same lysm domain receptor kinase and that the majority of responses towards externally applied lcos is controled by the cssp.
due to the existence not only of a common receptor but also a common signal transduction pathway, specificity for myc- or nod-signals has to be generated downstream of it, where it is widely accepted that the gras transcription factors nsp <dig>  nsp <dig>  and ram <dig> play an important role  <cit> .
these symbiotic regulators were shown to form nsp1/nsp <dig> and ram1/nsp <dig> heterodimers  <cit> , respectively, and were thought to control either nodulation-  or mycorrhization-related  responses via parallel signaling pathways  <cit> .
in line with this model, we have shown here that the activation of gene expression by myc-lcos during pre-symbiotic signaling within 6 h of application almost absolutely depends on ram <dig>  a gras transcription factor essential for root colonization and arbuscule formation  <cit> .
nevertheless, the nsp <dig> gras transcription factor was equally important for an up-regulation of gene expression by myc-lcos during the pre-symbiotic stage.
it thus appears that the two symbiotic gras tfs are not only both essential for transcriptional reprogramming by myc-lcos at the lower concentrations used here, they also cannot complement each other.
while the extent of nsp1-dependency was unexpected, our observation is supported by the re-assessment of nsp <dig> function by delaux et al.
<cit>  and takeda et al.
<cit>  as well as the strong nsp1-dependency observed for up to 100-fold higher myc-lco concentrations and shorter periods of application in an otherwise similar experimental setup  <cit> .
interestingly, from the  <dig> genes that camps et al.
<cit>  identified as co-activated by 10-7/- <dig> and 10− <dig> m myc-lcos, only  <dig>   <dig>   <dig>  and  <dig> were still activated in the nfp, dmi <dig>  nsp1- <dig> and ram1- <dig> background in our conditions, respectively , principally confirming a strict nfp-dependency, a predominant cssp involvement, and a high ram <dig> as well as nsp1-requirement for myc-lco induced transcription.
with respect to nsp <dig>  it might surprise that the strict dependency of myc-lco related gene expression on this gras tf contrasts with the comparably mild am infection phenotype of nsp1- <dig> mutants  <cit> .
this discrepancy can probably be explained by the fact that, reminiscent of the situation observed for nfp mutants, most pre-symbiotic responses to a low concentration of signal molecules mainly pre-announce, but not necessarily determine am fungal contact and subsequent entry.
during the pre-symbiotic stage, and despite the structural similarities between myc- and nod-lcos that inevitably lead to a certain amount of cross-activiation of gene expression  <cit> , in particular if sufficently high concentrations of less active lco versions were used, host roots display characteristic gene expression patterns in response to microbial lco signals .
our comparative analysis of gene expression responses during pre-symbiotic signaling presented here indicates that those myc-lco induced genes being either specifically or co-activated by nod-lcos still require nsp <dig>  but are much less ram1-dependent.
it appears to us that the more nod-lco -induced a gene is, the more nsp1- and the less ram1-dependent it is.
obviously, nod-lco signals are thus primarily transduced via a signaling pathway controled by nsp <dig>  which is in line with the initial nodulation-related function proposed for this tf .
however, a substantial portion of the nod-lco related genes identified here depends on the mycorrhiza-related  <cit>  gras tf ram <dig>  it is possible that this fraction represents genuine myc-lco induced genes that were not identified as activated in our study, since the myc-lco concentrations used were too low or since alternative myc-lco molecules exist.
in this case, these ram1-dependent genes might have been cross-activated by the more active nod-lcos.
on the other hand, it cannot be excluded that ram <dig> plays a so far unknown role in nod-lco signaling and that, similar to the minor am phenotype of nsp1- <dig> mutants  <cit> , ram1- <dig> mutants exhibit a yet undetected, mild nodulation-related phenotype.
while our results indicate that in the pre-symbiotic stage, signal transduction primarily requires different sets of gras tfs, it appears that a model where nsp <dig> solely controls the nodulation- and ram <dig> the mycorrhization-related branch downstream of the cssp is too simple.
this is at least true for the pre-symbiotic responses to lcos supplied for 6 h at 10-7/- <dig> m that we studied here.
as shown by other reports  <cit> , there might well be additional signals that complete the picture of fungal recognition.
in fig.
<dig>  a model for the pre-symbiotic activation of host genes by 10-7/- <dig> m microbial lcos is proposed, focussing on the cssp-dependent responses identified here.
this model rests on the observation that downstream of the cssp, nsp <dig> and ram <dig> are both essential for the up-regulation of myc-lco related genes being not concomitantly activated by nod-lcos , while nsp <dig> is primarily required for the induction of genes by nod-lcos .
from our results it is plausible that during the pre-symbiotic stage, transcriptional activation by nod-lcos predominantly requires nsp <dig>  but at the same time, yet unidentified regulators, e.g.
gras tfs, might still come into play.
it also has be kept in mind that a substantial fraction of lco-induced gene expression changes occurs independent of dmi <dig> , suggesting the existence of parallel pre-symbiotic signaling pathways  <cit> .fig.
<dig> model for the pre-symbiotic activation of host genes by microbial lcos.
this model integrates gene expression responses towards nod- and myc-lcos in the wild type and in four symbiotic mutants at concentrations of 10-7/- <dig> m .
the two major pathways for a pre-symbiotic transduction of nod- and myc-lco signals are shown.
on top of the figure, lco structures are depicted with four n-acetylamine residues.
sulfate groups of nod- and smyc-lcos are shown in red, the acetate group typically present on nod-lcos in black, and differences in fatty acids by a different width.
all components of signal perception and transduction studied in this work are shown in green.
other components of signal transduction reported in the literature are indicated in black.
the partial dependency of myc-lco perception on lyk <dig> at high concentrations  is indicated by a question mark, since it is unknown if lyk <dig> is relevant at 10-7/- <dig> m as well.
whereas nsp <dig> and ram <dig> are both essential for an up-regulation of myc-lco related genes not responding to nod-lcos, nsp <dig> is primarily required for the induction of genes by nod-lcos.
the possible presence of additional gras tfs involved in activating nod-lco related gene expression is indicated by a dashed line.
abbreviations: cssp, common symbiotic signaling pathway; ccamk, ca2+/calmodulin-dependent protein kinase; lrr, leucine-rich receptor; tf, transcription factor
apart from their involvement in myc- and nod-signaling, lcos have a more basic function: the stimulation of root branching , thus leading to the formation of lateral roots  <cit> .
it is likely that an initial mycorrhization-related, and subsequently a rather sophisticated nodulation-related signaling evolved from this basic developmental response.
whereas ram <dig> was not essential for rbs mediated by nod-lcos, nsp <dig> was shown to be required both for rbs triggered by nod- and smyc-lcos  <cit> .
this indicates that sulfated lcos activate rbs primarily via nsp <dig>  which is in line with our observation of a primarily nsp1-dependent pre-symbiotic activation of gene expression by smyc and nod-lcos.
for nsmyc-lcos though, the nsp1- and ram1-codependency of transcriptional activation observed here is in contrast to the rbs phenotype described in other studies  <cit> .
this process was ram1-dependent  <cit> , but nsp1-independent  <cit> , indicating that nsmyc-lco induced gene expression patterns are not primarily related to rbs.
this conclusion is further supported by overlaps between nsmyc-lco activation of transcription and gene expression during fungal infection.
despite overlaps in gene expression reported here and on a broader scale by camps et al.
<cit> , obtained using  <dig> d younger seedling roots, it remains an open question whether myc-lco activated genes play an essential role during the colonization of roots by am fungi or if they are mainly necessary to facilitate it.
while nfp on the one hand is required for myc-lco dependent activation of ca2+ spiking  <cit>  and for almost all pre-symbiotic transcriptional responses towards myc-lcos supplied at 10-7/- <dig> m  <cit> , nfp- <dig> mutants on the other hand are not impaired in mycorrhization  <cit> , indicating that at low concentrations, lco signals are probably not essential for am fungal entry and the infection of root cortical cells.
however, our transcriptional analysis of the nsp1- <dig> and ram1- <dig> mutants demonstrated that downstream of the cssp, the gras tfs ram <dig> and nsp <dig> are required for the activation of gene expression by externally applied myc-lcos .
as far as processes downstream of the cssp are concerned, these findings thus correlate pre-symbiotic responses towards myc-lcos with root infection, a process impaired in nsp1- <dig>  <cit>  and largely blocked in ram1- <dig>  <cit>  mutants.
in this context, the ram1-dependency in particular of the nsmyc-lco induced gene expression is intriguing.
lower levels of and delayed fungal colonization were observed in the ram1- <dig> background both in m. truncatula and in petunia.
moreover, degenerated arbuscules developed in the more infection-permissive m. truncatula ram1- <dig> and the petunia ram1- <dig> mutants upon high inoculum pressure, indicating that the degree of ram <dig> knockout is important  <cit> .
it remains to be demonstrated, if transcriptional responses towards myc-lcos are different in m. truncatula ram1- <dig> and ram1- <dig> mutants as well.
it is an attractive hypothesis that apart from enhancing the formation of lateral roots to increase the chance of infection, myc-lcos pre-announce the presence of a beneficial fungus at a distance via the activation of the symbiotic gras tfs ram <dig> and nsp <dig>  that way priming host roots for a subsequent colonization by am fungi.
in addition, an interplay of myc-lcos and cos in the activation of ca2+ spiking and downstream signal transduction is likely and can further mediate a multi-step recognition process from lco-triggered pre-announcement via an initial physical contact to the actual infection  <cit> .
although a priming of root tissues for infection would certainly be helpful, it is probably not essential for colonization  <cit> , which would explain the nfp-independency of am formation.
lyk <dig>  the second component of the heterodimeric nod-lco receptor, has recently been identified to be involved in myc-lco perception  <cit> .
following pre-symbiotic signaling that is likely mediated by a lower concentration of diffusible signals present in the area surrounding plant roots, am fungal entry might be controled by elevated concentrations of myc-lcos, e.g.
by activating the lyk <dig> receptor after accumulating in the local area underneath hyphopodia.
this scenario is supported by the results of zhang et al.
<cit>  who reported that at 10− <dig> m, fungal lcos induced only a reduced ca2+ spiking in roots lacking lyk <dig>  apart from lcos, short chain cos that do not require nfp to induce ca2+ spiking  <cit> , secreted fungal effectors  <cit> , and plant-derived cutin monomers  <cit>  probably play a role.
CONCLUSIONS
our integrated view of the transcriptional responses of the m. truncatula wild type and of four symbiotic mutants towards am fungal and rhizobial lcos sheds light on the pre-symbiotic reprogramming of host tissues by diffusible microbial signals.
well before a physical contact between the symbiotic partners, this pre-announcement probably informs the host about the presence of an appropriate symbiont in close vicinity of the root and in additon might prepare host roots for a subsequent colonization.
while the initial perception and transduction of diffusible microbial signals occurs via the well-characterized lysm receptor kinase nfp and the cssp, the interplay of transcriptional regulators acting immediately downstream of this signaling cascade is not yet fully understood.
with respect to myc-lcos, we could clearly show that both the nsp <dig> and the ram <dig> gras tfs are essential for almost all transcriptional responses towards myc-lcos at the pre-symbiotic stage, while nod-lcos predominantly rely on nsp <dig>  this differential requirement for gras transcriptional regulators acting downstream of ca2+ spiking and the cssp can serve as a starting point to further unravel connections of the key jigsaw pieces that mediate the transduction of symbiotic signals in m. truncatula roots.
these signals might even act in a sequential manner, as suggested by nadal and paszkowski  <cit> .
future experiments have to address the functional interplay and coordination of lco and co signals, prior to an opening of the gate for symbiotic guests.
in this context, a dissection of the relevance of both am fungal and plant signals for mycorrhizal infection remains a major challenge.

 more than 80 % of all land plant species enter an arbuscular mycorrhiza symbiosis with glomeromycota fungi. this plant-microbe interaction primarily improves phosphate uptake, but also contributes to the acquisition of nitrogen, minerals, and water. am host plants secrete strigolactones that stimulate fungal metabolism and promote pre-symbiotic hyphal branching. in return, am fungal recognition by the plant is thought to be mediated by myc-factors that signal the presence of symbiotic microbes. the chemical structure of one class of diffusible factors from am fungi was revealed by maillet et al., who identified a mixture of sulfated and non-sulfated lipochitooligosaccharide molecules in fungal exudates. although the n-linked acyl chain is different, myc-lcos are structurally related to rhizobial nodulation -factor lcos. after perception by rhizodermal lysm receptor kinase heterodimers, these lcos mediate bacterial infection and the induction of nodule primordia. the general similarity between rhizobial and am fungal lcos indiates that rhizobia adopted a pre-existing, much older mycorrhizal signaling program to initiate host responses. in addition to being involved in mycorrhization and nodulation, microbial lcos stimulate the formation of lateral roots. since the activation of this basic developmental process and the transcriptional responses of roots towards myc-lcos require nfp, recognition of am fungal lco signals is probably mediated by this lysm receptor. however, since nfp-type receptors are missing a functional kinase domain, another kinase is additionally required for signal transduction. one such candidate was proposed to be lyk the second component of the heterodimeric nod-lco receptor and a m. truncatula orthologue of the rice chitin receptor oscerk. perception of myc-lco signals leads to characteristic ca2+-oscillations in rhizodermal cells. in m. truncatula, this ca2+ spiking depends on the lrr receptor kinase dmi and on dmi a potassium channel of the nuclear membrane. ultimately, the ca2+/calmodulin-dependent protein kinase dmi together with its interacting transcription factor cyclops, receives the spiking pattern. since mycorrhizal as well as rhizobial infection is controled by the same set of dmi proteins, this signal cascade is referred to as the common symbiotic signaling pathway. apart from myc-lcos, short chain chitooligosaccharides were identified as am fungal signals that induce ca2+ spiking. in root organ cultures, the level of co and co was enhanced by plant-derived strigolactones, and these co versions induced a spiking pattern similar to exudates from germinating spores, being more persistent than that observed for myc-lcos. this response required dmi and dmi but, in contrast to myc-lcos, was nfp-independent, suggesting a role for other lysm kinases. similar to myc-lco perception, homologues of the cerk chitin receptor might be involved here. in addition to diffusible myc-factors acting at a distance, several lines of evidence point to signals that require physical contact between fungal hyphae and host roots. such contact signals coordinate a specific re-differentiation of rhizodermal and cortical cells, once hyphopodia attach to the root surface. the most remarkable cytological response of the infected host cells is the development of a cytoplasmatic pre-penetration apparatus. upon hyphopodium formation, this structure is established by each successively infected cell and guides fungal hyphae towards the inner cortex. here, they proliferate in the apoplast and ultimately form intracellular, highly branched arbuscules that serve as the major interface for nutrient exchanges. global transcriptional changes in m. truncatula am roots were studied extensively, resulting in a comprehensive overview of symbiotic gene expression on the tissue and cellular level. in contrast to these studies that investigated colonized roots, genome-wide analyses of pre-symbiotic gene expression responses to diffusible myc-signals are still limited. based on medicago genechip hybridizations, we previously reported on the lco-related transcriptional changes in 5 days old primary roots of the m. truncatula wild type, challenging roots for or 24 h with 10− or 10- m lcos. a sufficient activation of symbiotic signaling at these concentrations was validated by the histological detection of epidermal mtenod expression. the complex transcriptional responses towards myc-lcos were almost absolutely dependent on nfp and largely on dmi indicating that during the pre-symbiotic stage, am fungal lco signals are predominantly transduced via the cssp. in a comparable experimental setup, camps et al. recently studied the response of 4 days old primary m. truncatula roots in the wild type and in dmi3- mutants. the most notable differences between the two studies are the shorter incubation time of 2–4 h and the usage of 10– fold higher concentrations of myc-lcos by camps et al., together leading to an identification of more differentially regulated genes. in additon, rnaseq was applied, which allowed to measure the expression of genes not represented on medicago genechips. similar to 10-7/- m myc-lco concentrations, camps et al. demonstrated that lco-signaling is predominantly mediated by dmi also at 10− m, although a dmi3-independent signaling pathway was defined as well. in addition to studying cssp-dependency, camps et al. also looked into myc-lco activated gene expression in nsp1- mutants. interestingly, a high proportion of the myc-lco induced, dmi3-dependent genes appeared to require the gras transcription factor nsp as already reported for a limited number of genes. nsp acts immediately downstream of the cssp and was originally thought to be exclusively involved in nodulation. delaux et al. and takeda et al. had nevertheless reported that nsp1-deficient legume mutants displayed a reduced am fungal colonization. a comparable, non-essential function for mycorrhization was also demonstrated for the nsp1-interactor nsp, another gras tf initially thought to be exclusively required for nodulation. it has to be noted that both nsp and nsp play a role in strigolactone biosynthesis, providing further evidence for a link between nsp gras tfs and am formation. recently, the gras tf ram was identified in a screen for am-deficient mutants. similar to nsp ram interacts with nsp, illustrating that nodulation and mycorrhization are controled by overlapping sets of regulators. since in contrast to nsp ram was not described to be required for the transduction of nod-signals, ram is currently regarded both as an essential and a specific component of myc-signaling. in later stages of am, ram plays a role in the development of functional arbuscules both in m. truncatula and petunia, a function consistent with the mtram promoter activity in cortical cells. while putative receptors and components of the cssp mediating the pre-symbiotic transduction of lco signals have clearly been identified, the interdependency of the key regulators that activate lco-related transcriptional networks immediately downstream of the cssp remains to be adressed. to shed light on the relative importance of the gras tfs nsp and ram for lco-activated gene expression particularly during the pre-symbiotic stage, we obtained global transcription patterns of 5 days old nsp1- and ram1- plantlet roots after a 6 h challenge with myc- and nod-lcos. as in a previous study, we used 10-7/- m lco concentrations, in order to be as close as possible to the lower concentrations of myc-lcos that likely exist in soils and to avoid over-activation of gene expression by higher signal concentrations. together with the transcriptional responses of nfp- and dmi mutants, the expression fingerprints obtained served as molecular phenotypes to specify the requirement for nodulation- and mycorrhization-related gras tfs during the initial transduction of lco signals, prior to a contact between the symbionts. our findings provide evidence that in contrast to nod-lco signaling that predominantly relies on nsp the presence of both nsp and ram is essential for almost all myc-lco related gene expression activation in the pre-symbiotic stage. moreover, our results link the pre-symbiotic responses with the transcriptional reprogramming during am fungal contact and early root colonization, a process largely blocked in ram1- mutants. we propose a model, where downstream of the cssp, the ram and nsp tfs act synergistically in the transduction of diffusible myc-lco signals.  we here present an integrated view of the global transcriptional responses in primary roots of the m. truncatula wild type a as well as the nfp- dmi nsp1- and ram1- mutants towards am fungal and rhizobial lcos, using concentrations of 10− or 10− m, respectively. for all lco treatments, we used 5 days old seedlings, being in a stage of development where the first trifoliate was just initiated. using nsp1- and ram1- mutants that lack functional nsp and ram gras tfs, our experiments build on a study of myc-lco induced gene expression in the wild type as well as nfp- and dmi mutants. acting downstream of the cssp, the nsp and ram regulators can be considered as key jigsaw pieces of early myc-lco signaling. to identify myc-lco specific effects, the transcriptional responses towards nod-lcos in the wild type were now complemented with results for nfp- dmi nsp1- and ram1- mutants as well. in our experimental conditions that rely on the terminal to  cm of 5 days old primary roots excluding the root meristem, these concentrations led to an easily visible epidermal induction of the mtenod promoter by all lcos tested, including the least efficient nsmyclcos. together with the marked dmi3-dependency of most gene expression responses to 10-7/- m myc-lcos, this indicates that symbiotic ca2+ spiking was activated to a sufficient extent in our conditions. due to the fact that gene expression responses towards all myc-lcos occurred much stronger after 6 h as compared to 24 h, we here focus on this early time point. a compilation of all gene expression data is available as additional file, and will be discussed in detail in the subsequent chapters. activation of symbiotic marker genes by myc- and nod-lcos displays a differential requirement for the gras transcription factors nsp and ram1 to deepen our understanding of the transcriptional activation by symbiotic lcos, we first had a look at the expression properties of genes related to symbiotic signaling. this analysis revealed that mtnfp, mtlyk and all components of the cssp as well as mtcyclops were not activated in the wild type by any symbiotic lco tested. whereas mtnsp was slightly activated only after a 24 h treatment with nod-lcos, mtnsp was -fold up-regulated in the wild type by this factor already after 6 h. this up-regulation depends on nfp and dmi but was independent of both nsp itself and ram. mtnsp in addition displayed a tendency for activation by myc-lcos, which was absent in nsp1- but still detectable in ram1- mutants. the induction of mtnsp by myc-lcos thus appears to be independent of ram an observation in line with results delaux et al. obtained for a 10− m s/nsmyc-lco mixture.fig. expression of symbiotic marker genes in response to myc- and nod-lcos. expression of mtnsp, mtenod, and mtvapyrin in m. truncatula wild type a, nfp-, dmi, nsp1-, and ram1- plantlet roots challenged for 6 h with 10− m smyc-lcos, 10− m nsmyc-lcos, a mixture of 10− m smyc- and 10− m nsmyc-lcos, and 10− m nod-lcos. ratios of up-regulation in comparison to non-treated controls is shown. an at least -fold up-regulation is denoted by asterisks a well-characterized marker gene responding to lcos is mtenod. this gene was found activated most strongly by nod- and only slightly by smyc- and s/ns-myclcos in wild type plantlet roots. whereas mtenod11-activation by nod-lcos was not detected in nfp- dmi and nsp1- mutants, this gene was still up-regulated in the ram1- line, although somewhat lower. activation of mtenod by smyc-lcos follows a similar tendency. the ram independency of mtenod expression in response to smyc- and nod-lcos conforms to the histological results of sun et al.. as reported previously, no mtenod induction by nsmyc-lcos was detected in the pooled root samples used here, probably due to a quenching of the epidermal expression observed by reporter gene studies. a prominent example for a symbiosis-specific gene relevant both for am fungal and rhizobial infection is mtvapyrin, an ankyrin repeat protein involved in membrane trafficking. in line with a report by sun et al., who used different concentrations of lcos applied to only 2 days old seedlings, this gene was also found to be up-regulated between - and -fold by all symbiotic lcos in the wild type in our conditions. similar to many other lco-induced genes, mtvapyrin activation by fungal lcos strictly depends on nfp. whereas dmi and nsp were similarly required for an expression activation by nsmyc- and s/nsmyc-lcos, smyc-lcos still induced mtvapyrin in dmi and nsp1- mutant roots, although at a strongly reduced level. in contrast, mtvapyrin up-regulation was independent of ram for all fungal lcos tested, reminiscent of the detection of vapyrin expression in mycorrhizal roots of petunia ram1- mutants. with respect to nod-lcos, mtvapyrin induction also required nfp and dmi but was independent of both nsp and ram. the differential mtvapyrin expression observed in wild type, nfp- dmi and nsp1- roots was comparable to results obtained for 10- m nod-lcos. it thus appears that while the up-regulation of mtvapyrin is largely nsp1-dependent in response to myc-, it is nsp1-independent in response to nod-lcos. together, the activation of symbiotic marker genes by myc- and nod-lcos points to a differential requirement for the symbiotic gras tfs nsp and ram indicating that these regulators might be of different relative importance for myc- and nod-lco signaling in general. gene expression activation by myc-lcos strongly depends on the two gras transcription factors nsp and ram1 we previously reported on the transcriptional responses of m. truncatula wild type a nfp- and dmi plantlet roots towards myc-lcos, leading to the conclusion that the lysm receptor kinase nfp is almost absolutely essential for the perception of myc-lco signals and that the majority of myc-lco specific gene expression is activated via the cssp. in order to investigate processes occuring downstream of the cssp, we studied the transcriptional responses towards smyc-, nsmyc-, and s/nsmyc-lcos in nsp1- and ram1- plantlet roots. to allow a direct comparison, the resulting gene expression patterns were analyzed together with previous data for wild type, nfp- and dmi roots. these analyses revealed that out of genes activated at least -fold by myc-lcos and not by nod-lcos after 6 h in the wild type, only were still up-regulated at these cutoffs by any myc-lco or myc-lco combination in ram1- equivalent to an appr. 94 % reduction. this observation fits well to the strongly impaired am fungal root colonization observed in this mutant. strikingly, a comparable reduction of gene expression responses was also observed in the nsp1- mutant, where only genes remained activated by any myc-lco or myc-lco combination. although nsp was recently described to be relevant for mycorrhization, the extent of reduction of gene expression responses towards myc-lcos was unexpected.fig. myc-lco related gene expression in the m. truncatula wild type and in four symbiotic mutants. a number of genes at least -fold up-regulated in m. truncatula wild type a, nfp-, dmi, nsp1-, and ram1- plantlet roots challenged for 6 h with 10− m smyc-lcos, 10− m nsmyc-lcos, or a mixture of 10− m smyc- and 10− m nsmyc-lcos. none of these genes is activated by nod-lcos at this cutoff. on the left, the three myc-lco specific data columns are summed up, regardless of individual myc-lcos. values do not add up due to an overlapping activation of transcription by different lcos. the number of genes induced by myc-lcos in the wild type still activated in four symbiotic mutants are shown to the right of the wild type columns. expression of myc-lco related genes in nfp- and dmi plantlet roots reported by were included to facilitate comparisons. b genes still activated in dmi mutants were subtracted to highlight cssp-dependent effects the concomitant importance of nsp and ram for signaling downstream of the cssp prompted us to focus on myc-lco related genes encoding transcriptional regulators or components of signal transduction reported previously. after removing duplicate probes identified from the most recent m. truncatula genome annotation, transcription of the remaining genes in wild type and mutant roots was visualized in fig.  in addition to a strong reduction of expression in nfp- and dmi mutant roots, the myc-lco related genes in general were not significantly up-regulated in the nsp1- and ram1- background. in all but two cases, a significant activation in mutant roots only occurred in response to another lco or lco combination as in the wild type, probably indicating some level of deregulation in the symbiotic mutants. the general absence of an expression activation comparable to the wild type indicates that the signaling-related genes mentioned above encode components of myc-lco signaling activated not only via the cssp, but subsequently via the gras transcription factors nsp and ram fig. expression of myc-lco related genes encoding signaling components in the m. truncatula wild type and in four symbiotic mutants. subset of genes encoding transcription factors or signaling-related proteins and being rapidly activated in m. truncatula wild-type roots after a 6 h treatment with 10− m smyc-lcos, 10− m nsmyc-lcos, or a mixture of both. none of these genes is significantly activated by 10− m nod-lcos after 6 h. expression responses after 6 h in the wild type as well as the nfp-, dmi, nsp1-, and ram1- mutants is visualized as a heat map, with shades of red representing up- and shades of green down-regulation. an at least -fold up-regulation is indicated by a white dot. expression data were scaled to the maximum of a -fold regulation using the genesis software in total, only myc-lco related genes were still activated both in nsp1- and ram1- mutants. whereas activation of all four genes required nfp, three were dmi3-independent, indicating that some myc-lco related genes can be activated independent of the cssp by an alternative pathway that also doesn't require nsp and ram to exclude a bias from cssp-independent effects, we subtracted all genes still activated in dmi mutants, arriving at out of myc-lco related genes whose expression relies on the cssp. apart from a requirement for nfp, the strong dependency on both nsp and ram was again evident, with gene expression activation by myc-lcos being reduced by 96 % in each case. this strong nsp1-/ram1-codependency is also evident, if the effects are monitored separately for smyc-, nsmyc-, or s/nsmyc-lco induced genes. interestingly, the co-dependency on nsp and ram observed for genes up-regulated by myc-lcos is also a characteristic of genes down-regulated by a treatment with these symbiotic signals. in conclusion, our results demonstrate that the two symbiotic gras tfs nsp and ram are both essential for almost all gene expression responses towards a 6 h application of 10-7/- m myc-lcos. genes co-activated by myc- and nod-lcos display a preferential requirement for the gras transcription factor nsp1 to further investigate the differential nsp1- and ram1-dependency of myc-lco-related transcription, we studied the expression in nfp- dmi nsp1- and ram1- mutants of those genes activated at least -fold not only by myc-, but also by nod-lcos after 6 h in the wild type. this analysis revealed that gene expression responses were reduced to comparable levels in nfp- and dmi3-mutants as observed for myc-lco related genes, confirming that these genes are primarily activated via the cssp. interestingly, the nsp1- and ram1- mutants now displayed a lower and clearly differential level of reduction, allowing the conclusion that genes co-activated by nod-lcos show a higher dependency on nsp as compared to ram this was most apparent for those genes activated by smyc-lcos, sharing the highest structural similarity to nod-lcos. here, from genes co-activated by smyc- and nod-lcos, remained induced by smyc-lcos in ram1- mutants, while only displayed activation by these lcos in the nsp1- background. in contrast, the difference in reduction of responses to nsmyc-lcos was less pronounced in the nsp1- and ram1- mutants. similar to the situation for myc-lco related genes, exclusion of those sym-related genes activated in dmi mutants led to comparable results.fig. sym-lco related gene expression in the m. truncatula wild type and in four symbiotic mutants. a number of genes at least -fold up-regulated in m. truncatula wild type a, nfp-, dmi, nsp1-, and ram1- plantlet roots challenged for 6 h with 10− m smyc-lcos, 10− m nsmyc-lcos, or a mixture of 10− m smyc- and 10− m nsmyc-lcos. these genes were also activated by nod-lcos at this cutoff. on the left, the three myc-lco specific data columns are summed up, regardless of individual myc-lcos. values do not add up due to an overlapping activation of transcription by different lcos. the number of genes induced by myc-lcos in the wild type still activated in four symbiotic mutants are shown to the right of the wild type columns. b genes still activated in dmi mutants were subtracted to highlight cssp-dependent effects based on these results we infer that transcriptional activation is less ram1-dependent, if it is triggered not only by myc-, but also by nod-lcos. this observation is corroborated by an analysis of the genes identified by camps et al. being commonly activated by myc- and/or nod-lcos both at 10-7/- and 10− m. from the genes of this set co-induced by myc- and nod-lcos after 6 h in our conditions, only are activated in the nsp1- while remain up-regulated in the ram1- mutant. the strong nsp1-dependency of these sym-lco related genes indicates that many of them primarily respond to nod-lcos and are only cross-activated by myc-lcos, since their structure is similar. this is particularly relevant for the smyc-lcos, where an absence of ram appears least important, while nsp1-dependency remains high. most sym-lco related genes thus seem to be activated by a route primarily controled by nsp gene expression activation by nod-lcos displays a preferential requirement for the gras transcription factor nsp1 since our results provided evidence that genes co-activated by myc- and nod-lcos show a higher requirement for nsp in comparison to ram we assessed the nsp1- and ram1-dependency of those genes being activated at least -fold by nod-lcos, but not by myc-lcos after 6 h in m. truncatula a. to achieve comparability with the results reported above, we used the same concentration of nod-lcos as reported previously for the wild type, and also challenged plantlet roots for a duration of 6 h. to compare nod-lco activation of gene expression in the wild type to that in nfp- dmi nsp1- and ram1- mutants, expression data had to be re-normalized across wild type and mutant conditions. this led to a slight change in the number of genes activated in the wild type at least -fold by nod-, but not by myc-lcos.fig. nod-lco related gene expression in the m. truncatula wild type and in four symbiotic mutants. a number of genes at least -fold up-regulated in m. truncatula wild type a, nfp-, dmi, nsp1-, and ram1- plantlet roots challenged for 6 h with 10− m nod-lcos. these genes were not activated by myc-lcos at this cutoff. the number of genes induced by nod-lcos in the wild type still activated in four symbiotic mutants are shown to the right of the wild type column. b genes still activated in dmi mutants were subtracted to highlight cssp-dependent effects figure  shows that gene expression responses towards nod-lcos were reduced to similar levels as observed for sym-lco related genes in the nfp- dmi nsp1- and ram1- mutants, using the same cutoffs as mentioned above. as expected and similar to myc-lco perception, these results revealed an almost complete dependency of the perception of nod-lco signals on nfp, and a predominant transduction of these nodulation-related signals via the cssp. with respect to processes occuring downstream of dmi the requirement for nsp was much more pronounced than the requirement for ram. similar to the situation observed for sym-lcos, this effect was still evident after subtracting dmi3-independent genes and hence cssp-independent responses. in conclusion, the ram1-dependency of gene expression is comparably low not only for sym-lco, but also for nod-lco related transcription, indicating that nod-lco related genes are activated by a signaling pathway primarily controled by nsp however, a fraction of the nod-lco related genes identified in our conditions also depended on ram a gras tf previously reported to be only required for myc-signaling and mycorrhizal infection. ram controls infection-related genes activated by nsmyc-lcos during the pre-symbiotic stage to address the question, if myc-lcos supplied at 10-7/- m primarily act as signals that pre-announce the presence of a mycorrhizal fungus, or if they are also able to activate genes connected to early fungal infection, we related our results to gene expression responses recorded from laser-microdissected am tissues. although a comparison of microdissected cell types to the tissue samples studied here might include a bias, it allows us to obtain initial insights into overlapping expression patterns of genes responding to lcos in the pre-symbiotic and the infection stage. in the laser-microdissection study used, gene expression in regions of mycorrhizal roots containing early infection stages as well as hyphopodia and in corresponding non-colonized areas was analysed. whereas in group a, genes with a comparable expression in app and nap were identified that were also induced by myc-lcos, group b contained genes up-regulated in app. group a genes were thus thought to be root-expressed and activated by external myc-signals before fungal contact, while group b genes were related to the initial intracellular infection. it has to be noted that for both groups, a substantial portion of genes continued to be expressed in mature mycorrhizal roots, primarily in the arbuscule-containing cells. amongst the genes from group a, are activated not only by myc-, but also by nod-lcos, indicating that these app and nap co-induced genes respond to lcos of different microbial origin. in line with the results for sym-lco related genes presented above, of these are still up-regulated in response to myc-lcos independently of ram while the nsp1-dependency remains higher. looking at the genes up-regulated in regions of mycorrhizal roots containing early infection stages and hyphopodia, and were already activated by smyc-, nsmyc-, and s/nsmyc-lcos in wild type plantlet roots, respectively. due to redundancies, a total of app-induced genes were up-regulated by any myc-lco or myc-lco combination, providing evidence that a limited number of these infection-related genes responds to diffusible myc-signals already at the pre-symbiotic stage. interestingly, app-induced genes were activated by nod-lcos, including mtvapyrin and of the myc-lco induced genes mentioned above. this indicates a substantial cross-activation of infection-related genes by lcos of different microbial origin, as reported by camps et al.. the co-activation by nod-lcos might also explain why nine of the app-induced genes up-regulated by myc-lcos are expressed independently of ram furthermore, the highest ram1-dependency was found for the app-induced genes already activated by nsmyc-lcos at the pre-symbiotic stage. of these, only were still up-regulated by nsmyc-lcos in ram1- mutants. we now decided to focus on nsmyc-lcos, since these lack the sulfate group that leads to a strong structural similarity between smyc- and nod-lcos. thus, gene expression responses to this most basic fungal lco should be most instructive to identify genuine mycorrhization responses. to shed light on this, we had a look at all genes being activated by 10− m nsmyc-lcos in the wild type, but not in ram1- mutants. a total of myc-related and sym-related genes fulfilled this criterion. whereas the myc-related genes activated by nsmyc-lcos displayed an almost absolute ram1-dependency also towards smyc-, s/nsmyc-, and nod-lcos, the sym-related genes were completely ram1-dependent only for their activation by nsmyc-lcos. in contrast, a total of and genes remained activated by smyc, s/nsmyc-, and nod-lcos in ram1- roots. on the one hand, this allows to conclude that the myc-related and sym-related genes up-regulated by nsmyc-lcos are controled by a signaling pathway that strictly requires ram on the other hand, in response to smyc- or nod-lcos, a substantial fraction of these can be induced by an alternative pathway that that does not require ram and is apparently not triggered by nsmyc-lcos. this indicates that downstream of the cssp, fine-tuning of gene expression activation travels along alternative roads, depending on the lco variant initially perceived and possibly also on the epidermal cell type, where the cssp is activated. with respect to am fungal infection, it is interesting to note that two of the myc-related and of the sym-related genes activated by nsmyc-lcos are up-regulated in regions containing early infection structures and hyphopodia or in arbuscule-containing cells, while and were activated in whole am roots, respectively. considering the ram1-dependency of their activation by nsmyc-lcos in the pre-symbiotic stage, it is thus possible that those genes are activated by nsmyc-lco signals also during am fungal contact and root infection. discussion gene expression responses towards 10-7/- m of am fungal and rhizobial lcos in primary roots of the m. truncatula wild type as well as nfp- dmi nsp1- and ram1- mutants demonstrated that during the pre-symbiotic stage, lco-induced transcription strictly relies on the nfp receptor and largely on the calcium-calmodulin dependent kinase dmi. this indicates that lcos of whatever origin are perceived by the same lysm domain receptor kinase and that the majority of responses towards externally applied lcos is controled by the cssp. due to the existence not only of a common receptor but also a common signal transduction pathway, specificity for myc- or nod-signals has to be generated downstream of it, where it is widely accepted that the gras transcription factors nsp nsp and ram play an important role. these symbiotic regulators were shown to form nsp1/nsp and ram1/nsp heterodimers, respectively, and were thought to control either nodulation- or mycorrhization-related responses via parallel signaling pathways. in line with this model, we have shown here that the activation of gene expression by myc-lcos during pre-symbiotic signaling within 6 h of application almost absolutely depends on ram a gras transcription factor essential for root colonization and arbuscule formation. nevertheless, the nsp gras transcription factor was equally important for an up-regulation of gene expression by myc-lcos during the pre-symbiotic stage. it thus appears that the two symbiotic gras tfs are not only both essential for transcriptional reprogramming by myc-lcos at the lower concentrations used here, they also cannot complement each other. while the extent of nsp1-dependency was unexpected, our observation is supported by the re-assessment of nsp function by delaux et al. and takeda et al. as well as the strong nsp1-dependency observed for up to 100-fold higher myc-lco concentrations and shorter periods of application in an otherwise similar experimental setup. interestingly, from the genes that camps et al. identified as co-activated by 10-7/- and 10− m myc-lcos, only and were still activated in the nfp, dmi nsp1- and ram1- background in our conditions, respectively, principally confirming a strict nfp-dependency, a predominant cssp involvement, and a high ram as well as nsp1-requirement for myc-lco induced transcription. with respect to nsp it might surprise that the strict dependency of myc-lco related gene expression on this gras tf contrasts with the comparably mild am infection phenotype of nsp1- mutants. this discrepancy can probably be explained by the fact that, reminiscent of the situation observed for nfp mutants, most pre-symbiotic responses to a low concentration of signal molecules mainly pre-announce, but not necessarily determine am fungal contact and subsequent entry. during the pre-symbiotic stage, and despite the structural similarities between myc- and nod-lcos that inevitably lead to a certain amount of cross-activiation of gene expression, in particular if sufficently high concentrations of less active lco versions were used, host roots display characteristic gene expression patterns in response to microbial lco signals. our comparative analysis of gene expression responses during pre-symbiotic signaling presented here indicates that those myc-lco induced genes being either specifically or co-activated by nod-lcos still require nsp but are much less ram1-dependent. it appears to us that the more nod-lco -induced a gene is, the more nsp1- and the less ram1-dependent it is. obviously, nod-lco signals are thus primarily transduced via a signaling pathway controled by nsp which is in line with the initial nodulation-related function proposed for this tf. however, a substantial portion of the nod-lco related genes identified here depends on the mycorrhiza-related gras tf ram it is possible that this fraction represents genuine myc-lco induced genes that were not identified as activated in our study, since the myc-lco concentrations used were too low or since alternative myc-lco molecules exist. in this case, these ram1-dependent genes might have been cross-activated by the more active nod-lcos. on the other hand, it cannot be excluded that ram plays a so far unknown role in nod-lco signaling and that, similar to the minor am phenotype of nsp1- mutants, ram1- mutants exhibit a yet undetected, mild nodulation-related phenotype. while our results indicate that in the pre-symbiotic stage, signal transduction primarily requires different sets of gras tfs, it appears that a model where nsp solely controls the nodulation- and ram the mycorrhization-related branch downstream of the cssp is too simple. this is at least true for the pre-symbiotic responses to lcos supplied for 6 h at 10-7/- m that we studied here. as shown by other reports, there might well be additional signals that complete the picture of fungal recognition. in fig.  a model for the pre-symbiotic activation of host genes by 10-7/- m microbial lcos is proposed, focussing on the cssp-dependent responses identified here. this model rests on the observation that downstream of the cssp, nsp and ram are both essential for the up-regulation of myc-lco related genes being not concomitantly activated by nod-lcos, while nsp is primarily required for the induction of genes by nod-lcos. from our results it is plausible that during the pre-symbiotic stage, transcriptional activation by nod-lcos predominantly requires nsp but at the same time, yet unidentified regulators, e.g. gras tfs, might still come into play. it also has be kept in mind that a substantial fraction of lco-induced gene expression changes occurs independent of dmi, suggesting the existence of parallel pre-symbiotic signaling pathways.fig. model for the pre-symbiotic activation of host genes by microbial lcos. this model integrates gene expression responses towards nod- and myc-lcos in the wild type and in four symbiotic mutants at concentrations of 10-7/- m. the two major pathways for a pre-symbiotic transduction of nod- and myc-lco signals are shown. on top of the figure, lco structures are depicted with four n-acetylamine residues. sulfate groups of nod- and smyc-lcos are shown in red, the acetate group typically present on nod-lcos in black, and differences in fatty acids by a different width. all components of signal perception and transduction studied in this work are shown in green. other components of signal transduction reported in the literature are indicated in black. the partial dependency of myc-lco perception on lyk at high concentrations is indicated by a question mark, since it is unknown if lyk is relevant at 10-7/- m as well. whereas nsp and ram are both essential for an up-regulation of myc-lco related genes not responding to nod-lcos, nsp is primarily required for the induction of genes by nod-lcos. the possible presence of additional gras tfs involved in activating nod-lco related gene expression is indicated by a dashed line. abbreviations: cssp, common symbiotic signaling pathway; ccamk, ca2+/calmodulin-dependent protein kinase; lrr, leucine-rich receptor; tf, transcription factor apart from their involvement in myc- and nod-signaling, lcos have a more basic function: the stimulation of root branching, thus leading to the formation of lateral roots. it is likely that an initial mycorrhization-related, and subsequently a rather sophisticated nodulation-related signaling evolved from this basic developmental response. whereas ram was not essential for rbs mediated by nod-lcos, nsp was shown to be required both for rbs triggered by nod- and smyc-lcos. this indicates that sulfated lcos activate rbs primarily via nsp which is in line with our observation of a primarily nsp1-dependent pre-symbiotic activation of gene expression by smyc and nod-lcos. for nsmyc-lcos though, the nsp1- and ram1-codependency of transcriptional activation observed here is in contrast to the rbs phenotype described in other studies. this process was ram1-dependent, but nsp1-independent, indicating that nsmyc-lco induced gene expression patterns are not primarily related to rbs. this conclusion is further supported by overlaps between nsmyc-lco activation of transcription and gene expression during fungal infection. despite overlaps in gene expression reported here and on a broader scale by camps et al., obtained using d younger seedling roots, it remains an open question whether myc-lco activated genes play an essential role during the colonization of roots by am fungi or if they are mainly necessary to facilitate it. while nfp on the one hand is required for myc-lco dependent activation of ca2+ spiking and for almost all pre-symbiotic transcriptional responses towards myc-lcos supplied at 10-7/- m, nfp- mutants on the other hand are not impaired in mycorrhization, indicating that at low concentrations, lco signals are probably not essential for am fungal entry and the infection of root cortical cells. however, our transcriptional analysis of the nsp1- and ram1- mutants demonstrated that downstream of the cssp, the gras tfs ram and nsp are required for the activation of gene expression by externally applied myc-lcos. as far as processes downstream of the cssp are concerned, these findings thus correlate pre-symbiotic responses towards myc-lcos with root infection, a process impaired in nsp1- and largely blocked in ram1- mutants. in this context, the ram1-dependency in particular of the nsmyc-lco induced gene expression is intriguing. lower levels of and delayed fungal colonization were observed in the ram1- background both in m. truncatula and in petunia. moreover, degenerated arbuscules developed in the more infection-permissive m. truncatula ram1- and the petunia ram1- mutants upon high inoculum pressure, indicating that the degree of ram knockout is important. it remains to be demonstrated, if transcriptional responses towards myc-lcos are different in m. truncatula ram1- and ram1- mutants as well. it is an attractive hypothesis that apart from enhancing the formation of lateral roots to increase the chance of infection, myc-lcos pre-announce the presence of a beneficial fungus at a distance via the activation of the symbiotic gras tfs ram and nsp that way priming host roots for a subsequent colonization by am fungi. in addition, an interplay of myc-lcos and cos in the activation of ca2+ spiking and downstream signal transduction is likely and can further mediate a multi-step recognition process from lco-triggered pre-announcement via an initial physical contact to the actual infection. although a priming of root tissues for infection would certainly be helpful, it is probably not essential for colonization, which would explain the nfp-independency of am formation. lyk the second component of the heterodimeric nod-lco receptor, has recently been identified to be involved in myc-lco perception. following pre-symbiotic signaling that is likely mediated by a lower concentration of diffusible signals present in the area surrounding plant roots, am fungal entry might be controled by elevated concentrations of myc-lcos, e.g. by activating the lyk receptor after accumulating in the local area underneath hyphopodia. this scenario is supported by the results of zhang et al. who reported that at 10− m, fungal lcos induced only a reduced ca2+ spiking in roots lacking lyk apart from lcos, short chain cos that do not require nfp to induce ca2+ spiking, secreted fungal effectors, and plant-derived cutin monomers probably play a role.  our integrated view of the transcriptional responses of the m. truncatula wild type and of four symbiotic mutants towards am fungal and rhizobial lcos sheds light on the pre-symbiotic reprogramming of host tissues by diffusible microbial signals. well before a physical contact between the symbiotic partners, this pre-announcement probably informs the host about the presence of an appropriate symbiont in close vicinity of the root and in additon might prepare host roots for a subsequent colonization. while the initial perception and transduction of diffusible microbial signals occurs via the well-characterized lysm receptor kinase nfp and the cssp, the interplay of transcriptional regulators acting immediately downstream of this signaling cascade is not yet fully understood. with respect to myc-lcos, we could clearly show that both the nsp and the ram gras tfs are essential for almost all transcriptional responses towards myc-lcos at the pre-symbiotic stage, while nod-lcos predominantly rely on nsp this differential requirement for gras transcriptional regulators acting downstream of ca2+ spiking and the cssp can serve as a starting point to further unravel connections of the key jigsaw pieces that mediate the transduction of symbiotic signals in m. truncatula roots. these signals might even act in a sequential manner, as suggested by nadal and paszkowski. future experiments have to address the functional interplay and coordination of lco and co signals, prior to an opening of the gate for symbiotic guests. in this context, a dissection of the relevance of both am fungal and plant signals for mycorrhizal infection remains a major challenge. 
we investigated the genome-wide transcriptional responses in 5 days old primary roots of the medicago truncatula wild type and four symbiotic mutants to a 6 h challenge with lco signals supplied at 10-7/- m. we were able to show that during the pre-symbiotic stage, sulfated myc-, non-sulfated myc-, and nod-lco-activated gene expression almost exclusively depends on the lysm receptor kinase nfp and is largely controled by the cssp, although responses independent of this pathway exist. more than 80 % of all terrestrial plant species establish an arbuscular mycorrhiza symbiosis with glomeromycota fungi. amongst others, fungal signals were identified as a mix of sulfated and non-sulfated lipochitooligosaccharides, being structurally related to rhizobial nodulation -factor lcos that in legumes induce the formation of nitrogen-fixing root nodules. our results show that downstream of the cssp, gene expression activation by myc-lcos supplied at 10-7/- m strictly required both the gras transcription factors ram and nsp whereas those genes either co- or specifically activated by nod-lcos displayed a preferential nsp1-dependency. this plant-microbe interaction primarily improves phosphate uptake, but also supports nitrogen, mineral, and water aquisition. since both ram and nsp were essential for the pre-symbiotic transcriptional reprogramming by myc-lcos, we propose that downstream of the cssp, these gras transcription factors act synergistically in the transduction of those diffusible signals that pre-announce the presence of symbiotic fungi. ram a central regulator of root colonization by am fungi, controled genes activated by non-sulfated myc-lcos during the pre-symbiotic stage that are also up-regulated in areas with early physical contact, e.g. hyphopodia and infecting hyphae; linking responses to externally applied lcos with early root colonization. using complex gene expression fingerprints as molecular phenotypes, this study primarily intended to shed light on the importance of the gras tfs nsp and ram for lco-activated gene expression during pre-symbiotic signaling. lco signals are transduced via a common symbiotic signaling pathway that activates a group of gras transcription factors. during the pre-contact stage, the am symbiosis is controled by an exchange of diffusible factors from either partner.
 more than 80 % of all terrestrial plant species establish an arbuscular mycorrhiza symbiosis with glomeromycota fungi. this plant-microbe interaction primarily improves phosphate uptake, but also supports nitrogen, mineral, and water aquisition. during the pre-contact stage, the am symbiosis is controled by an exchange of diffusible factors from either partner. amongst others, fungal signals were identified as a mix of sulfated and non-sulfated lipochitooligosaccharides, being structurally related to rhizobial nodulation -factor lcos that in legumes induce the formation of nitrogen-fixing root nodules. lco signals are transduced via a common symbiotic signaling pathway that activates a group of gras transcription factors. using complex gene expression fingerprints as molecular phenotypes, this study primarily intended to shed light on the importance of the gras tfs nsp and ram for lco-activated gene expression during pre-symbiotic signaling.  we investigated the genome-wide transcriptional responses in 5 days old primary roots of the medicago truncatula wild type and four symbiotic mutants to a 6 h challenge with lco signals supplied at 10-7/- m. we were able to show that during the pre-symbiotic stage, sulfated myc-, non-sulfated myc-, and nod-lco-activated gene expression almost exclusively depends on the lysm receptor kinase nfp and is largely controled by the cssp, although responses independent of this pathway exist. our results show that downstream of the cssp, gene expression activation by myc-lcos supplied at 10-7/- m strictly required both the gras transcription factors ram and nsp whereas those genes either co- or specifically activated by nod-lcos displayed a preferential nsp1-dependency. ram a central regulator of root colonization by am fungi, controled genes activated by non-sulfated myc-lcos during the pre-symbiotic stage that are also up-regulated in areas with early physical contact, e.g. hyphopodia and infecting hyphae; linking responses to externally applied lcos with early root colonization.  since both ram and nsp were essential for the pre-symbiotic transcriptional reprogramming by myc-lcos, we propose that downstream of the cssp, these gras transcription factors act synergistically in the transduction of those diffusible signals that pre-announce the presence of symbiotic fungi. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords arbuscular mycorrhizagras transcription factorlipochitooligosaccharidemedicago genechipnsp1pre-symbiotic signalingram1transcriptional reprogrammingissue-copyright-statement© the author 2015 