BACKGROUND
nitrogen is one of the most limiting nutrients for plant growth despite its abundance in the atmosphere.
it can only be absorbed by plants as nh4+ or no3−.
however, dinitrogen can be reduced by some diazotrophic soil microorganisms, some of which are able to associate with specific plant families.
some of these mutualistic associations lead to the accommodation of bacteria within plant cells in specialized root structures called root nodules.
root nodule symbioses are found in a limited number of plants belonging to the legume, cannabaceae  and actinorhizal plants.
actinorhizal plants belong to eight angiosperm families that can form symbioses with a filamentous soil bacterium called frankia.
despite their ecological importance and recent advances in knowledge, the molecular bases of the formation and functioning of actinorhizal symbioses are still poorly understood  <cit> .
recently, transcriptomic analyses in three actinorhizal plants, c. glauca, a. glutinosa and datisca glomerata  <cit>  led to the discovery and characterization of several genes preferentially expressed in actinorhizal nodules following inoculation with frankia.
this global analysis of gene expression revealed that genes of the common symbiotic pathway  composed of signaling elements required for both root nodule and arbuscular mycorrhizal  symbioses were also conserved in actinorhizal plants  <cit> .
furthermore, comparative transcriptome analysis of genes expressed during am, rhizobial and actinorhizal symbioses suggests the existence of a core set of genes induced in these three endosymbioses  <cit> .
in the last decade, genetic studies on legume symbiosis of the model species  have elucidated the role of transcription factors  at different stages of nodule formation  <cit> .
the first tf described as playing a role in lotus japonicus nodulation was the nin  gene for which mutation leads to inhibition of infection and primordia formation  <cit> .
nin is a transcription factor that is induced during the early stages of nodule organogenesis  <cit>  and is involved in many nodule formation processes  <cit> .
in addition, nin orthologs have been identified in pea, soybean and m. truncatula, where they act downstream of the common sym genes  <cit> .
recently, it was demonstrated that in l. japonicus, nin regulates cortical cell division by targeting two nuclear factors, ljnf-ya <dig> and ljnf-yb <dig>  that are essential for root nodule organogenesis  <cit> .
furthermore, it was demonstrated that the tf cyclops transactivates nin expression in a phosphorylation-dependent manner leading to root nodule development  <cit> .
nsp <dig> and nsp <dig>  genes coding for gras tf are also specifically involved in the rhizobia-legume symbiosis  <cit> , and recent studies suggest their involvement in the arbuscular mycorrhizal symbiosis  <cit> .
nsp1/nsp <dig> forms an heterodimer and activates the ern <dig>  tf required for nodulation by binding to the at rich region of the promoter, which in turn stimulates the expression of the enod <dig> gene expressed during the pre-infection process  <cit> .
in medicago truncatula, mtnf-ya <dig> and mtnf-ya <dig> also control enod <dig> expression through direct mtern <dig> activation  <cit> .
recently, mtrsd , a cysteine-2/histidine- <dig>  tf, was shown to promote differentiation of bacteria into nitrogen fixing bacteroids  <cit> .
taken together, these data reveal that specific tf orchestrate plant infection and nodule organogenesis in legumes.
the objective of this study was to identify the tf that regulate the expression of genes involved in the c. glauca- /a.
glutinosa-frankia actinorhizal symbioses.
among  <dig>  unigenes expressed in roots and nodules of each of the two species, we identified  <dig> and  <dig> tf distributed in  <dig> and  <dig> families in c. glauca and a. glutinosa, respectively.
a global analysis of the expression profile of these genes was conducted to identify up- and down-regulated tf encoding genes in nodule versus root, as well as nodule-specific tf.
the expression level of several c. glauca and a. glutinosa tf was confirmed by quantitative pcr.
phylogenetic analyses performed in model legumes, species related to actinorhizal plants, and the actinorhizal plants c. glauca and a. glutinosa enabled us to identify zf <dig> -related transcription factors as potential specific regulators of actinorhizal symbioses.
RESULTS
identification of c. glauca and a. glutinosa transcription factors
to identify transcription factors in actinorhizal plants, tblastn searches of the c. glauca and a. glutinosa unigene databases were performed using the dna-binding domain from the tf database of arabidopsis thaliana as query sequences.
these databases contain  <dig>  unigenes for a. glutinosa and  <dig>  unigenes for c. glauca  <cit> .
blast analysis of the two unigene sets revealed  <dig> and  <dig> genes possibly encoding tf in c. glauca and a. glutinosa, respectively.
to remove false positives, tblastx was performed to check trans-species sequence homologies between arabidopsis genes and actinorhizal sequences with an e-value cut-off of 1e− <dig>  using this approach, we narrowed it down to  <dig> and  <dig> potential transcription factors distributed in  <dig> and  <dig> families in c. glauca and a. glutinosa, respectively .
no potential members were identified for thirteen families including m-type, e2f/dp, and gebp in c. glauca and a. glutinosa unigene databases.
each predicted c. glauca and a. glutinosa tf gene was given an arbitrary number.
additional files  <dig> and  <dig> list each predicted gene for both species, together with the accession numbers of all unigenes, the closest arabidopsis tf, and detailed blast information.
among tf families, the myb superfamily and the erf family were the largest, totaling  <dig> tf for each species.
the third largest family was the c2h <dig> family, with  <dig> members in c. glauca and  <dig> in a. glutinosa, followed by the wrky, nac and bhlh families.
the remaining families were represented by  <dig> to  <dig> members .table  <dig>
classification of putative transcription factor of
casuarina glauca
and
alnus glutinosa
into families
tf family
casuarina glauca
alnus glutinosa
total tf
domain description
total tf
202
195
397
total families
40
35
45
tf = transcription factor.
expression profile of the putative actinorhizal transcription factors
microarray data was retrieved from previous published studies  <cit> .
first, we used a simple comparative transcriptomics tool called casuarina transcriptome compendium  to compare the microarray data and generate expression profiles in different conditions.
using ctc, we identified  <dig> repressed transcription factors and  <dig> induced in c. glauca nodules compared to non-inoculated roots with a nodule/root fold change ≥  <dig> or ≤ − <dig> and a p-value ≤  <dig>  .
the c2h <dig> family was the most frequently represented in the up-regulated class followed by the erf and bhlh families .
induction of  <dig> transcription factors in c. glauca nodules was confirmed by quantitative pcr, which also revealed similar induction values than microarray data .
induction of cgzf <dig> was validated by semi-quantitative pcr because its expression was not detectable in the non-inoculated roots needed to calibrate q-pcr analysis .figure  <dig>
expression profiles of potential transcription factors in
casuarina glauca
and
alnus glutinosa
nodules compared to non-inoculated roots.
purple and orange bars represent putative transcriptional regulators from c. glauca and a. glutinosa respectively.
bars represent fold change in nodules compared to non-inoculated roots .
transcription factors induced and repressed in c. glauca and a. glutinosa nodules are highlighted in tanned brown and light pink respectively.
astericks identify the genes confirmed by q-pcr.
red triangles identify the transcription factors  most strongly induced in the nodules.
similarly, we combined available microarray data in a. glutinosa to generate an alnus transcriptome compendium .
this allowed us to identify  <dig> putative transcription factors induced and  <dig> others repressed in a. glutinosa nodules compared to non-inoculated roots .
similar to the results observed in c. glauca nodules most myb and wrky were down-regulated while c2h <dig> were induced in nodules, .
induction in a. glutinosa nodules of seven transcription factors was also confirmed by quantitative pcr .
comparison of tf gene expression in am and actinorhizal symbioses
this first set of expression data prompted us to investigate tf gene expression during am symbiosis in c. glauca.
mycorrhized roots data were retrieved from a recent publication  <cit> .
transcriptomic data highlighted  <dig> downregulated and  <dig> up-regulated transcription factors in c. glauca mycorrhized roots .
nac and gras families had the highest number of transcription factors regulated in mycorrhized roots .
comparing expression in nodules and mycorrhized roots led to the identification of tfs induced during root nodule symbioses but not during am.
only cgzf <dig>  cghd-zip <dig> and cgg2-like <dig> respectively belonging to c2h <dig>  hd-zip and g2-like families were significantly induced in response to infection by frankia and not induced in response to am fungi .
even though root nodule symbioses are hypothesized to originate from am, comparative analyses showed that only one transcription factor named cgerf <dig>  was induced both in nodules and mycorrhized roots of c. glauca.
interestingly, cgerf <dig> was identified as being related to ern genes from m. truncatula known to be induced by rhizobium and am  <cit> .figure  <dig>
expression profiles of potential transcription factors in
c. glauca
mycorrhizal roots compared to non-inoculated roots.
bars represent fold change in mycorrhizal roots compared to non-inoculated roots .
black and blue bars represent up and down-regulated transcriptional regulators in c.glauca mycorrhizal roots.
transcription factors induced and repressed in c. glauca mycorrhizal roots are highlighted in tanned brown and light pink respectively.
gras, nf-ya and erf transcription factor families in actinorhizal species
in legumes, several tfs that play key roles in the transduction of bacterial nod factors have been identified.
these include in particular members of the gras, nf-ya and erf families   <cit> .
therefore we first focused on these families.
we screened extra databases using the tblastn algorithm in order to identify gras, nf-ya and erf tfs in a third actinorhizal species, d. glomerata  <cit> , relatives of actinorhizal plants in the rosales and cucurbitales orders, in legumes  and in arabidopsis .
the data was used to construct a phylogenetic tree of the gras protein family based on the alignment of the complete protein sequences using the maximum likelihood method.
eleven complete c. glauca sequences and eight a. glutinosa were collected from respective datasets.
based on the phylogenetic analyses, four distinct subfamilies were defined: scarecrow-like , scarecrow , shortroot  and della.
the tree topology resembled the one found both in rice and arabidopsis  <cit> .
no sequence from c. glauca was close to nsp <dig> and nsp <dig>  the same result was found for rosales and cucurbitales.
however, two sequences from a. glutinosa and d. glomerata named aggras <dig>  and dgnsp <dig> respectively were identified in the nsp <dig> group .figure  <dig>
phylogenetic tree of the gras protein family.
three gras proteins from c. sativus, p. persica, m. domestica and f. vesca each were retrieved using a blast-p search performed in phytozome and two sequences dgnsp <dig>  and dgnsp <dig>  of d. glomerata   <cit> .
we also include  <dig> sequences available in the genomes of a. thaliana,  <dig> from m. truncatula,  <dig> from l. japonicus,  <dig> from g.max,  <dig> from c. glauca and  <dig> from a. glutinosa.
in italic, transcription factors c.glauca and a.glutinosa.
the gras family comprises  <dig> subfamilies referred to as scl, scr, shr and della.
nsp <dig> and  <dig> belong to shr and scl subfamilies respectively.
aggras <dig> is closely related to dgnsp <dig> .
no sequence from c. glauca is present in the nsp <dig> and nsp  <dig> groups .
the tree was rooted with a. thaliana sequence at3g <dig> .
one hundred bootstrap replications were used to evaluate statistical support for branches.
branches with less than 70% bootstrap support were collapsed.
plant genes belonging to the nf-ya family encode putative tfs that are variable in length  <cit> .
their dna binding domain shares some similarity with the cct domain of the nuclear flowering time regulator constans   <cit> .
nf-ya proteins are characterized by two domains  that are strongly conserved in all higher eukaryotes examined to date  <cit> .
to clarify the phylogenetic situation of the nf-ya genes identified in the transcriptomes, we built a tree using the maximum likelihood method using only the nf-ya domains.
five of the originally collected sequences had incomplete dna and protein domains in the c. glauca and a. glutinosa datasets and were excluded from further analysis.
the nf-ya tree includes  <dig> major groups  also found in legumes .
interestingly, group i containing mtnf-ya <dig> and mtnf-ya <dig> from m. truncatula includes cgnf-ya <dig> from c. glauca.
in the present study, dgcomp3430_c0_seq <dig> from d.glomerata is in group ii.
several genes belonging to group ii show a strong expression mainly in seeds  <cit> .figure  <dig>
phylogenetic tree of the nf-ya protein family.
we include nf-ya proteins from a. thaliana , m. truncatula , l. japonicus  and g. max .
sequences of c. sativus , p. persica , m. domestica  and f. vesca  were retrieved using a blast-p search performed in phytozome and one sequence of d. glomerata   <cit> .
in the present study, nf-ya tree comprises six groups: i to vi.
cgnf-ya <dig> belongs to group i. dgnf-ya  is in group ii.
the tree was rooted with a. thaliana sequence at3g <dig>  .
one hundred bootstrap replications were used to evaluate statistical support for branches.
branches with less than 70% bootstrap support were collapsed.
to study the phylogenetic relationships between the erf genes in the actinorhizal species, multiple alignment analysis was performed using amino acid sequences in the ap2/erf domain.
ten of the originally collected sequences had incomplete ap <dig> domains in the c. glauca and a. glutinosa datasets and were excluded from further analysis.
the erf tree distinguished the  <dig> major groups  also found in rice, cotton, and arabidopsis .
at least one c. glauca and a. glutinosa erf tf was found in the main groups except for group vi.
group vii, which included five erfs from arabidopsis, was the most represented by the actinorhizal erf transcription factor with three sequences in c. glauca and five sequences in a. glutinosa.
however, several changes in the groups were observed .
group ix was divided into three subgroups  <cit> .
in the present study, the phylogenetic tree showed that subgroup ixb was no longer related to subgroup ixa and ixc.
group ix contained five sequences from a. glutinosa and two sequences from the model legume lotus japonicus including ljerf <dig>  ljerf <dig> functions as a positive regulator of nodulation  <cit> .
agerf <dig> is the sequence most closely related to ljerf <dig>  group v was divided into two subgroups  <cit> .
in the present study, the phylogenetic tree showed subgroups va and vb grouped with group viiia.
interestingly, group vb which contained ern <dig>  ern <dig> and ern <dig> from m. truncatula, also includes two erfs, cgerf <dig> and dgcomp <dig>  from c. glauca and d. glomerata, respectively.
the reliability of group v clustering was supported by the presence of common motifs outside the ap2/erf domain.
cgerf <dig> and dgcomp <dig> as well as erns possess the cmv- <dig> or cmv- <dig> motifs, like in rice and arabidopsis   <cit> .figure  <dig>
phylogenetic tree of the ethylene-response factor  protein family.
three erf proteins from c. sativus, p. persica, m. domestica and f. vesca each were retrieved using a blast-p search performed in phytozome and  <dig> sequences dgern  and dgcontig <dig>  from d. glomerata   <cit> .
we also include  <dig> erf from a. thaliana,  <dig> from m. truncatula and  <dig> from l. japonicus.
in italic, transcription factors c.glauca and a.glutinosa.
the erf family comprises ten groups referred as i to x. cgerf <dig> and dgern are in group v .
they are closer to mtern <dig> than mtern <dig> and mtern <dig>  the tree was rooted to apetala <dig> ap2-domain r <dig> .
one hundred bootstrap replications were used to evaluate statistical support for branches.
branches with less than 70% bootstrap support were collapsed.
c2h <dig> transcription factors in actinorhizal symbiosis
from the global analysis, the large number of its members and their regulation during actinorhizal symbioses make the c2h <dig> family particularly interesting.
we therefore decided to study this family further, and in particular two of its members, cgzf <dig> and agzf <dig>  expression data showed that four transcription factors of this family were induced and seven less expressed in c. glauca nodules than in non-inoculated roots .
cgzf <dig> was specifically induced during nodulation and no c2h <dig> were induced in c. glauca mycorrhized roots .
analysis of the a. glutinosa nodule transcriptome showed that one c2h <dig> was down-regulated and three were up-regulated in nodules .
remarkably, like in the c. glauca tf gene expression dataset, a c2h <dig> tf, agzf <dig>  showed the strongest gene induction in a. glutinosa nodules as indicated by the red triangles in figure  <dig>  the c2h <dig> family is characterized by the presence of a “zinc finger” dna binding domain containing one or two “qalggh” conserved motif  <cit> .
cgzf <dig> contains two qalggh motifs at positions 85– <dig> and 142– <dig>  furthermore, a putative ear  repressor domain with the signature /dln/xp was identified at the c-terminal positions  <dig> and  <dig> of cgzf <dig>  the “ear domain” is known to be responsible for the repression of gene expression  <cit> .
the presence of this domain in c2h <dig> c1-2i proteins suggests that they are transcriptional repressors of their target genes.
finally, a nuclear localization signal  involved in the translocation of protein to the nucleus was also identified at position 31– <dig> in its n-terminal end.
alignment of agzf <dig>  with the c. glauca cgzf <dig> revealed the position of dna-binding including two “qalggh” conserved motifs, an ear domain and a nls within the protein sequence and a high degree of identity  with cgzf <dig> .
to further characterize the relationship between cgzf <dig> and agzf <dig>  a phylogenetic tree based on the alignment of the complete protein sequences was generated .
the sequence of m. sativa mszpt2- <dig> was added to the dataset because it encodes a c2h <dig> tf and plays a role in legume symbiosis  <cit> .
the phylogenetic tree distinguished two major groups, groups i and ii.
group i did not contain c. glauca, a. glutinosa and a. thaliana c2h <dig> tf.
group ii was divided into three subgroups named a, b and c. subgroups ii-b and ii-c contained seven and five c2h <dig> tfs from actinorhizal plants, respectively.
interestingly, one cluster in subgroup ii-b containing cgzf <dig> and agzf <dig> was only made up of sequences from rosales, fagales, and cucurbitales and did not contain any legume sequence.
our analysis also confirmed that cgzf <dig> and agzf <dig> protein sequences are very close and are most probably orthologs .
this suggests that zf1-like transcription factors evolved after the divergence between fabales and the rosales/cucurbitales/fagales clade and the common ancestor of actinorhizal fagales recruited these genes for functions related to nodulation.figure  <dig>
phylogenetic tree of the c
2
h
2
c1-2i protein family
.
a maximum likelihood tree representing relationships among  <dig> c2h <dig> proteins from a. thaliana,  <dig> from g.max,  <dig> from m. truncatula and a. glutinosa,  <dig> from l. japonicus and c. glauca,  <dig> sequences dgzf <dig> , dgcomp14622_c0_seq <dig>  and dgcontig <dig>  from d. glomerata.
the tree was rooted with a. thaliana sequence at5g <dig>  .
in italic, transcription factors c.glauca and a.glutinosa.
the c2h <dig> c1-2i family comprises two groups referred to as i and ii.
group ii is divided into three subgroups named a, b and c. the subgroups are also divided into several clusters.
cluster ii-b <dig> contains sequences of actinorhizal plants and related sequences from rosales and cucurbitales include cgzf <dig> and agzf <dig> .
the sequence of m. sativa mszpt2- <dig> was added to the dataset because it encodes a c2h <dig> c1-2i type transcription factor.
the nodes represent bootstrap values  from  <dig> replications.
discussion
the search for putative transcription factors is an important first step in characterizing the signaling pathways that control the establishment and functioning of actinorhizal symbioses.
this analysis enabled us to identify  <dig> and  <dig> transcription factors in c. glauca and a. glutinosa, respectively.
the use of transcriptome data generated in c. glauca showed that half the putative transcription factors are regulated in nodules and mycorrhizae .
in a. glutinosa, 26% are significantly regulated in nodules.
in addition, the transcriptome data pointed to co-regulation of cgzf <dig> and agzf <dig>  which belong to the c2h <dig> family.
in silico studies have already been conducted using several plant models to optimize automated annotation of transcription factors and to obtain a plant tf atlas.
for example,  <dig>  transcription factors have been identified in a. thaliana,  <dig>  in m. truncatula,  <dig>  in l. japonicus and  <dig>  in g. max  <cit> .
these transcription factors are characterized by their dna binding domains, which are specific to each family.
this led to the identification of  <dig> families in a. thaliana,  <dig> in m. truncatula and l. japonicus and  <dig> in g. max  <cit> .
the putative transcription factors are distributed in  <dig> families in c. glauca and  <dig> in a. glutinosa.
the difference between these and other plant models can be explained by the fact that c. glauca and a. glutinosa genomic data come from  <dig>  unigenes for a. glutinosa and  <dig>  unigenes for c. glauca.
as part of the effort to better understand the biology of actinorhizal symbiosis, large-scale cdna sequencing using next generation sequencing technologies will provide a means to identify new tf genes and to produce an actinorhizal gene expression atlas.
among the transcription factors identified, cgnin  showed high similarity with mtnin  and ljnin .
cgnin was strongly induced in nodules  and not significantly induced in mycorrhized roots .
we also identified another gene called cgerf <dig> coding for an erf transcription factor.
this gene was induced in both c. glauca nodules and mycorrhizae.
in m. truncatula, a gene encoding a transcription factor belonging to the erf family named mtern <dig>  <cit>  presents the same expression profile as cgerf <dig>  these two genes share 55% similarity and conservation of specific domains .
in addition, we identified cgnf-ya <dig>  a close homologue of mtnf-ya <dig> and mtnf-ya <dig>  two tfs involved in the activation of mtern <dig>  <cit> .
cgnf-ya <dig> is induced in actinorhizal nodules .
no sequence of c. glauca belongs to the nsp <dig> and nsp <dig> groups.
in a. glutinosa, aggras <dig> belongs to the nsp <dig> group and is closely related to dgnsp <dig> .
consequently, we suggest that cgnin, agnsp <dig>  cgnf-ya <dig> and cgerf <dig> could be functional homologs of nin, nsp <dig>  nf-ya and ern <dig> in legumes.
functional complementation of legume mutants by the potential actinorhizal orthologous genes could be used to test this hypothesis.
analysis of the expression of the putative transcription factors was performed using transcriptome data to investigate their expression during the c. glauca-/a.
glutinosa-frankia interactions.
this investigation revealed  <dig> induced tfs in c. glauca or a. glutinosa nodules.
recently,  <dig> genes encoding putative transcription factors strongly induced in nodules were identified in m. truncatula  <cit> .
these tfs were classified in eight functional groups of genes differentially regulated in m. truncatula nodules in seven experimental conditions after inoculation with the wild type and three nod- mutant strains of sinorhizobium meliloti.
transcription factors belonging mainly to the c2h <dig>  myb, wrky and erf families appeared to be nodule-specific, whereas members of the erf and wrky families were over-represented in the tf dataset  <cit> .
similarly, transcriptome analysis of l. japonicus led to the identification of  <dig> tf that expression was up-regulated during nodulation showing that members of the erf family are the most abundant tf  <cit> .
remarkably, members of the erf and wrky families of tf were also abundant in our study, and expression analysis showed that nine of them were induced in actinorhizal nodules.
erf and wrky tfs are often involved in defense responses to abiotic and biotic stresses  <cit> .
as hypothesized for legume and rhizobium symbioses, erf and wrky tfs could be involved in the regulation of downstream defense responses to inhibit excess frankia infection under nitrogen-sufficient conditions.
three genes named cgzf <dig>  cghd-zip <dig> and cgg2-like <dig> belonging to the c2h <dig>  hd-zip and g2-like families, respectively, are specific to the c. glauca-frankia interaction.
a tf named agzf <dig> exhibited the same strong induction in a. glutinosa nodules as cgzf <dig>  in addition, these two genes were also seen to have the same protein structure and phylogenetic analysis indicate they are most likely orthologous.
in legumes, two c2h <dig> tfs are involved in nodule functioning.
in m. truncatula, mtrsd represses transcription of the secretory pathway gene, vamp721a, required for symbiosome formation, suggesting that mtrsd is directly involved in symbiosome and bacteroid differentiation  <cit> .
mtrsd is a c2h <dig> tf but is phylogenetically distant from cgzf <dig> and agzf <dig> .
in m. sativa, a gene called mszpt2- <dig> is involved in the differentiation of nitrogen-fixing cells  <cit> .
mszpt2- <dig> possesses two “qalggh” motifs and an ear motif as well as cgzf <dig> and agzf <dig>  mszpt2- <dig>  cgzf <dig> and agzf <dig> belong to the same c2h <dig> group ii .
functional complementation studies will determine whether cgzf <dig> and agzf <dig> are functionally equivalent to putative orthologous genes identified in legumes.
recent studies showed that repressors containing the ear motif play a role in defense responses to plant biotic and abiotic stress  <cit> .
cgzf <dig> and agzf <dig> could repress the expression of defense genes of actinorhizal plant in response to signals from frankia to allow colonization of new cells in nodules.
finally, cgzf <dig> and agzf <dig> could be used as functional markers to investigate the regulation of actinorhizal symbiosis interaction.
CONCLUSIONS
the in silico identification and transcriptome analysis of tfs in c. glauca and in a. glutinosa that we carried out represents a crucial step forward in the elucidation of the molecular events underlying actinorhizal and mycorrhizal symbioses.
coupled with a phylogenetic study, these analyses enabled identification of a nodulation specific gene in c. glauca named cgzf <dig> and its ortholog in a. glutinosa named agzf <dig>  these two new genes are most probably negative regulators which may play a crucial role in actinorhizal symbiosis.
the development of efficient techniques to transform c. glauca  <cit>  now allows us to perform functional studies of cgzf <dig>
availability of supporting data
est sequences reported in this paper have been deposited in the genbank  and embl  databases.
the normalized and raw microarray data values have been deposited in the gene expression omnibus database .
in addition, all nucleic acid sequences used in this study have been included in additional files  <dig> and  <dig>  proteins sequences used to produce phylogenetic data sets have been added in additional file  <dig>

 nitrogen is one of the most limiting nutrients for plant growth despite its abundance in the atmosphere. it can only be absorbed by plants as nh4+ or no3−. however, dinitrogen can be reduced by some diazotrophic soil microorganisms, some of which are able to associate with specific plant families. some of these mutualistic associations lead to the accommodation of bacteria within plant cells in specialized root structures called root nodules. root nodule symbioses are found in a limited number of plants belonging to the legume, cannabaceae and actinorhizal plants. actinorhizal plants belong to eight angiosperm families that can form symbioses with a filamentous soil bacterium called frankia. despite their ecological importance and recent advances in knowledge, the molecular bases of the formation and functioning of actinorhizal symbioses are still poorly understood. recently, transcriptomic analyses in three actinorhizal plants, c. glauca, a. glutinosa and datisca glomerata led to the discovery and characterization of several genes preferentially expressed in actinorhizal nodules following inoculation with frankia. this global analysis of gene expression revealed that genes of the common symbiotic pathway composed of signaling elements required for both root nodule and arbuscular mycorrhizal symbioses were also conserved in actinorhizal plants. furthermore, comparative transcriptome analysis of genes expressed during am, rhizobial and actinorhizal symbioses suggests the existence of a core set of genes induced in these three endosymbioses. in the last decade, genetic studies on legume symbiosis of the model species have elucidated the role of transcription factors at different stages of nodule formation. the first tf described as playing a role in lotus japonicus nodulation was the nin gene for which mutation leads to inhibition of infection and primordia formation. nin is a transcription factor that is induced during the early stages of nodule organogenesis and is involved in many nodule formation processes. in addition, nin orthologs have been identified in pea, soybean and m. truncatula, where they act downstream of the common sym genes. recently, it was demonstrated that in l. japonicus, nin regulates cortical cell division by targeting two nuclear factors, ljnf-ya and ljnf-yb that are essential for root nodule organogenesis. furthermore, it was demonstrated that the tf cyclops transactivates nin expression in a phosphorylation-dependent manner leading to root nodule development. nsp and nsp genes coding for gras tf are also specifically involved in the rhizobia-legume symbiosis, and recent studies suggest their involvement in the arbuscular mycorrhizal symbiosis. nsp1/nsp forms an heterodimer and activates the ern tf required for nodulation by binding to the at rich region of the promoter, which in turn stimulates the expression of the enod gene expressed during the pre-infection process. in medicago truncatula, mtnf-ya and mtnf-ya also control enod expression through direct mtern activation. recently, mtrsd, a cysteine-2/histidine- tf, was shown to promote differentiation of bacteria into nitrogen fixing bacteroids. taken together, these data reveal that specific tf orchestrate plant infection and nodule organogenesis in legumes. the objective of this study was to identify the tf that regulate the expression of genes involved in the c. glauca- /a. glutinosa-frankia actinorhizal symbioses. among unigenes expressed in roots and nodules of each of the two species, we identified and tf distributed in and families in c. glauca and a. glutinosa, respectively. a global analysis of the expression profile of these genes was conducted to identify up- and down-regulated tf encoding genes in nodule versus root, as well as nodule-specific tf. the expression level of several c. glauca and a. glutinosa tf was confirmed by quantitative pcr. phylogenetic analyses performed in model legumes, species related to actinorhizal plants, and the actinorhizal plants c. glauca and a. glutinosa enabled us to identify zf -related transcription factors as potential specific regulators of actinorhizal symbioses.  identification of c. glauca and a. glutinosa transcription factors to identify transcription factors in actinorhizal plants, tblastn searches of the c. glauca and a. glutinosa unigene databases were performed using the dna-binding domain from the tf database of arabidopsis thaliana as query sequences. these databases contain unigenes for a. glutinosa and unigenes for c. glauca. blast analysis of the two unigene sets revealed and genes possibly encoding tf in c. glauca and a. glutinosa, respectively. to remove false positives, tblastx was performed to check trans-species sequence homologies between arabidopsis genes and actinorhizal sequences with an e-value cut-off of 1e− using this approach, we narrowed it down to and potential transcription factors distributed in and families in c. glauca and a. glutinosa, respectively. no potential members were identified for thirteen families including m-type, e2f/dp, and gebp in c. glauca and a. glutinosa unigene databases. each predicted c. glauca and a. glutinosa tf gene was given an arbitrary number. additional files and list each predicted gene for both species, together with the accession numbers of all unigenes, the closest arabidopsis tf, and detailed blast information. among tf families, the myb superfamily and the erf family were the largest, totaling tf for each species. the third largest family was the c2h family, with members in c. glauca and in a. glutinosa, followed by the wrky, nac and bhlh families. the remaining families were represented by to members.table classification of putative transcription factor of casuarina glauca and alnus glutinosa into families tf family casuarina glauca alnus glutinosa total tf domain description total tf 202 195 397 total families 40 35 45 tf = transcription factor. expression profile of the putative actinorhizal transcription factors microarray data was retrieved from previous published studies. first, we used a simple comparative transcriptomics tool called casuarina transcriptome compendium to compare the microarray data and generate expression profiles in different conditions. using ctc, we identified repressed transcription factors and induced in c. glauca nodules compared to non-inoculated roots with a nodule/root fold change ≥  or ≤ − and a p-value ≤ . the c2h family was the most frequently represented in the up-regulated class followed by the erf and bhlh families. induction of transcription factors in c. glauca nodules was confirmed by quantitative pcr, which also revealed similar induction values than microarray data. induction of cgzf was validated by semi-quantitative pcr because its expression was not detectable in the non-inoculated roots needed to calibrate q-pcr analysis.figure expression profiles of potential transcription factors in casuarina glauca and alnus glutinosa nodules compared to non-inoculated roots. purple and orange bars represent putative transcriptional regulators from c. glauca and a. glutinosa respectively. bars represent fold change in nodules compared to non-inoculated roots. transcription factors induced and repressed in c. glauca and a. glutinosa nodules are highlighted in tanned brown and light pink respectively. astericks identify the genes confirmed by q-pcr. red triangles identify the transcription factors most strongly induced in the nodules. similarly, we combined available microarray data in a. glutinosa to generate an alnus transcriptome compendium. this allowed us to identify putative transcription factors induced and others repressed in a. glutinosa nodules compared to non-inoculated roots. similar to the results observed in c. glauca nodules most myb and wrky were down-regulated while c2h were induced in nodules,. induction in a. glutinosa nodules of seven transcription factors was also confirmed by quantitative pcr. comparison of tf gene expression in am and actinorhizal symbioses this first set of expression data prompted us to investigate tf gene expression during am symbiosis in c. glauca. mycorrhized roots data were retrieved from a recent publication. transcriptomic data highlighted downregulated and up-regulated transcription factors in c. glauca mycorrhized roots. nac and gras families had the highest number of transcription factors regulated in mycorrhized roots. comparing expression in nodules and mycorrhized roots led to the identification of tfs induced during root nodule symbioses but not during am. only cgzf cghd-zip and cgg2-like respectively belonging to c2h hd-zip and g2-like families were significantly induced in response to infection by frankia and not induced in response to am fungi. even though root nodule symbioses are hypothesized to originate from am, comparative analyses showed that only one transcription factor named cgerf was induced both in nodules and mycorrhized roots of c. glauca. interestingly, cgerf was identified as being related to ern genes from m. truncatula known to be induced by rhizobium and am.figure expression profiles of potential transcription factors in c. glauca mycorrhizal roots compared to non-inoculated roots. bars represent fold change in mycorrhizal roots compared to non-inoculated roots. black and blue bars represent up and down-regulated transcriptional regulators in c.glauca mycorrhizal roots. transcription factors induced and repressed in c. glauca mycorrhizal roots are highlighted in tanned brown and light pink respectively. gras, nf-ya and erf transcription factor families in actinorhizal species in legumes, several tfs that play key roles in the transduction of bacterial nod factors have been identified. these include in particular members of the gras, nf-ya and erf families. therefore we first focused on these families. we screened extra databases using the tblastn algorithm in order to identify gras, nf-ya and erf tfs in a third actinorhizal species, d. glomerata, relatives of actinorhizal plants in the rosales and cucurbitales orders, in legumes and in arabidopsis. the data was used to construct a phylogenetic tree of the gras protein family based on the alignment of the complete protein sequences using the maximum likelihood method. eleven complete c. glauca sequences and eight a. glutinosa were collected from respective datasets. based on the phylogenetic analyses, four distinct subfamilies were defined: scarecrow-like, scarecrow, shortroot and della. the tree topology resembled the one found both in rice and arabidopsis. no sequence from c. glauca was close to nsp and nsp the same result was found for rosales and cucurbitales. however, two sequences from a. glutinosa and d. glomerata named aggras and dgnsp respectively were identified in the nsp group.figure phylogenetic tree of the gras protein family. three gras proteins from c. sativus, p. persica, m. domestica and f. vesca each were retrieved using a blast-p search performed in phytozome and two sequences dgnsp and dgnsp of d. glomerata. we also include sequences available in the genomes of a. thaliana, from m. truncatula, from l. japonicus, from g.max, from c. glauca and from a. glutinosa. in italic, transcription factors c.glauca and a.glutinosa. the gras family comprises subfamilies referred to as scl, scr, shr and della. nsp and belong to shr and scl subfamilies respectively. aggras is closely related to dgnsp. no sequence from c. glauca is present in the nsp and nsp groups. the tree was rooted with a. thaliana sequence at3g. one hundred bootstrap replications were used to evaluate statistical support for branches. branches with less than 70% bootstrap support were collapsed. plant genes belonging to the nf-ya family encode putative tfs that are variable in length. their dna binding domain shares some similarity with the cct domain of the nuclear flowering time regulator constans. nf-ya proteins are characterized by two domains that are strongly conserved in all higher eukaryotes examined to date. to clarify the phylogenetic situation of the nf-ya genes identified in the transcriptomes, we built a tree using the maximum likelihood method using only the nf-ya domains. five of the originally collected sequences had incomplete dna and protein domains in the c. glauca and a. glutinosa datasets and were excluded from further analysis. the nf-ya tree includes major groups also found in legumes. interestingly, group i containing mtnf-ya and mtnf-ya from m. truncatula includes cgnf-ya from c. glauca. in the present study, dgcomp3430_c0_seq from d.glomerata is in group ii. several genes belonging to group ii show a strong expression mainly in seeds.figure phylogenetic tree of the nf-ya protein family. we include nf-ya proteins from a. thaliana, m. truncatula, l. japonicus and g. max. sequences of c. sativus, p. persica, m. domestica and f. vesca were retrieved using a blast-p search performed in phytozome and one sequence of d. glomerata. in the present study, nf-ya tree comprises six groups: i to vi. cgnf-ya belongs to group i. dgnf-ya is in group ii. the tree was rooted with a. thaliana sequence at3g. one hundred bootstrap replications were used to evaluate statistical support for branches. branches with less than 70% bootstrap support were collapsed. to study the phylogenetic relationships between the erf genes in the actinorhizal species, multiple alignment analysis was performed using amino acid sequences in the ap2/erf domain. ten of the originally collected sequences had incomplete ap domains in the c. glauca and a. glutinosa datasets and were excluded from further analysis. the erf tree distinguished the major groups also found in rice, cotton, and arabidopsis. at least one c. glauca and a. glutinosa erf tf was found in the main groups except for group vi. group vii, which included five erfs from arabidopsis, was the most represented by the actinorhizal erf transcription factor with three sequences in c. glauca and five sequences in a. glutinosa. however, several changes in the groups were observed. group ix was divided into three subgroups. in the present study, the phylogenetic tree showed that subgroup ixb was no longer related to subgroup ixa and ixc. group ix contained five sequences from a. glutinosa and two sequences from the model legume lotus japonicus including ljerf ljerf functions as a positive regulator of nodulation. agerf is the sequence most closely related to ljerf group v was divided into two subgroups. in the present study, the phylogenetic tree showed subgroups va and vb grouped with group viiia. interestingly, group vb which contained ern ern and ern from m. truncatula, also includes two erfs, cgerf and dgcomp from c. glauca and d. glomerata, respectively. the reliability of group v clustering was supported by the presence of common motifs outside the ap2/erf domain. cgerf and dgcomp as well as erns possess the cmv- or cmv- motifs, like in rice and arabidopsis.figure phylogenetic tree of the ethylene-response factor protein family. three erf proteins from c. sativus, p. persica, m. domestica and f. vesca each were retrieved using a blast-p search performed in phytozome and sequences dgern and dgcontig from d. glomerata. we also include erf from a. thaliana, from m. truncatula and from l. japonicus. in italic, transcription factors c.glauca and a.glutinosa. the erf family comprises ten groups referred as i to x. cgerf and dgern are in group v. they are closer to mtern than mtern and mtern the tree was rooted to apetala ap2-domain r. one hundred bootstrap replications were used to evaluate statistical support for branches. branches with less than 70% bootstrap support were collapsed. c2h transcription factors in actinorhizal symbiosis from the global analysis, the large number of its members and their regulation during actinorhizal symbioses make the c2h family particularly interesting. we therefore decided to study this family further, and in particular two of its members, cgzf and agzf expression data showed that four transcription factors of this family were induced and seven less expressed in c. glauca nodules than in non-inoculated roots. cgzf was specifically induced during nodulation and no c2h were induced in c. glauca mycorrhized roots. analysis of the a. glutinosa nodule transcriptome showed that one c2h was down-regulated and three were up-regulated in nodules. remarkably, like in the c. glauca tf gene expression dataset, a c2h tf, agzf showed the strongest gene induction in a. glutinosa nodules as indicated by the red triangles in figure  the c2h family is characterized by the presence of a “zinc finger” dna binding domain containing one or two “qalggh” conserved motif. cgzf contains two qalggh motifs at positions 85– and 142– furthermore, a putative ear repressor domain with the signature /dln/xp was identified at the c-terminal positions and of cgzf the “ear domain” is known to be responsible for the repression of gene expression. the presence of this domain in c2h c1-2i proteins suggests that they are transcriptional repressors of their target genes. finally, a nuclear localization signal involved in the translocation of protein to the nucleus was also identified at position 31– in its n-terminal end. alignment of agzf with the c. glauca cgzf revealed the position of dna-binding including two “qalggh” conserved motifs, an ear domain and a nls within the protein sequence and a high degree of identity with cgzf. to further characterize the relationship between cgzf and agzf a phylogenetic tree based on the alignment of the complete protein sequences was generated. the sequence of m. sativa mszpt2- was added to the dataset because it encodes a c2h tf and plays a role in legume symbiosis. the phylogenetic tree distinguished two major groups, groups i and ii. group i did not contain c. glauca, a. glutinosa and a. thaliana c2h tf. group ii was divided into three subgroups named a, b and c. subgroups ii-b and ii-c contained seven and five c2h tfs from actinorhizal plants, respectively. interestingly, one cluster in subgroup ii-b containing cgzf and agzf was only made up of sequences from rosales, fagales, and cucurbitales and did not contain any legume sequence. our analysis also confirmed that cgzf and agzf protein sequences are very close and are most probably orthologs. this suggests that zf1-like transcription factors evolved after the divergence between fabales and the rosales/cucurbitales/fagales clade and the common ancestor of actinorhizal fagales recruited these genes for functions related to nodulation.figure phylogenetic tree of the c 2 h 2 c1-2i protein family. a maximum likelihood tree representing relationships among c2h proteins from a. thaliana, from g.max, from m. truncatula and a. glutinosa, from l. japonicus and c. glauca, sequences dgzf, dgcomp14622_c0_seq and dgcontig from d. glomerata. the tree was rooted with a. thaliana sequence at5g. in italic, transcription factors c.glauca and a.glutinosa. the c2h c1-2i family comprises two groups referred to as i and ii. group ii is divided into three subgroups named a, b and c. the subgroups are also divided into several clusters. cluster ii-b contains sequences of actinorhizal plants and related sequences from rosales and cucurbitales include cgzf and agzf. the sequence of m. sativa mszpt2- was added to the dataset because it encodes a c2h c1-2i type transcription factor. the nodes represent bootstrap values from replications. discussion the search for putative transcription factors is an important first step in characterizing the signaling pathways that control the establishment and functioning of actinorhizal symbioses. this analysis enabled us to identify and transcription factors in c. glauca and a. glutinosa, respectively. the use of transcriptome data generated in c. glauca showed that half the putative transcription factors are regulated in nodules and mycorrhizae. in a. glutinosa, 26% are significantly regulated in nodules. in addition, the transcriptome data pointed to co-regulation of cgzf and agzf which belong to the c2h family. in silico studies have already been conducted using several plant models to optimize automated annotation of transcription factors and to obtain a plant tf atlas. for example, transcription factors have been identified in a. thaliana, in m. truncatula, in l. japonicus and in g. max. these transcription factors are characterized by their dna binding domains, which are specific to each family. this led to the identification of families in a. thaliana, in m. truncatula and l. japonicus and in g. max. the putative transcription factors are distributed in families in c. glauca and in a. glutinosa. the difference between these and other plant models can be explained by the fact that c. glauca and a. glutinosa genomic data come from unigenes for a. glutinosa and unigenes for c. glauca. as part of the effort to better understand the biology of actinorhizal symbiosis, large-scale cdna sequencing using next generation sequencing technologies will provide a means to identify new tf genes and to produce an actinorhizal gene expression atlas. among the transcription factors identified, cgnin showed high similarity with mtnin and ljnin. cgnin was strongly induced in nodules and not significantly induced in mycorrhized roots. we also identified another gene called cgerf coding for an erf transcription factor. this gene was induced in both c. glauca nodules and mycorrhizae. in m. truncatula, a gene encoding a transcription factor belonging to the erf family named mtern presents the same expression profile as cgerf these two genes share 55% similarity and conservation of specific domains. in addition, we identified cgnf-ya a close homologue of mtnf-ya and mtnf-ya two tfs involved in the activation of mtern. cgnf-ya is induced in actinorhizal nodules. no sequence of c. glauca belongs to the nsp and nsp groups. in a. glutinosa, aggras belongs to the nsp group and is closely related to dgnsp. consequently, we suggest that cgnin, agnsp cgnf-ya and cgerf could be functional homologs of nin, nsp nf-ya and ern in legumes. functional complementation of legume mutants by the potential actinorhizal orthologous genes could be used to test this hypothesis. analysis of the expression of the putative transcription factors was performed using transcriptome data to investigate their expression during the c. glauca-/a. glutinosa-frankia interactions. this investigation revealed induced tfs in c. glauca or a. glutinosa nodules. recently, genes encoding putative transcription factors strongly induced in nodules were identified in m. truncatula. these tfs were classified in eight functional groups of genes differentially regulated in m. truncatula nodules in seven experimental conditions after inoculation with the wild type and three nod- mutant strains of sinorhizobium meliloti. transcription factors belonging mainly to the c2h myb, wrky and erf families appeared to be nodule-specific, whereas members of the erf and wrky families were over-represented in the tf dataset. similarly, transcriptome analysis of l. japonicus led to the identification of tf that expression was up-regulated during nodulation showing that members of the erf family are the most abundant tf. remarkably, members of the erf and wrky families of tf were also abundant in our study, and expression analysis showed that nine of them were induced in actinorhizal nodules. erf and wrky tfs are often involved in defense responses to abiotic and biotic stresses. as hypothesized for legume and rhizobium symbioses, erf and wrky tfs could be involved in the regulation of downstream defense responses to inhibit excess frankia infection under nitrogen-sufficient conditions. three genes named cgzf cghd-zip and cgg2-like belonging to the c2h hd-zip and g2-like families, respectively, are specific to the c. glauca-frankia interaction. a tf named agzf exhibited the same strong induction in a. glutinosa nodules as cgzf in addition, these two genes were also seen to have the same protein structure and phylogenetic analysis indicate they are most likely orthologous. in legumes, two c2h tfs are involved in nodule functioning. in m. truncatula, mtrsd represses transcription of the secretory pathway gene, vamp721a, required for symbiosome formation, suggesting that mtrsd is directly involved in symbiosome and bacteroid differentiation. mtrsd is a c2h tf but is phylogenetically distant from cgzf and agzf. in m. sativa, a gene called mszpt2- is involved in the differentiation of nitrogen-fixing cells. mszpt2- possesses two “qalggh” motifs and an ear motif as well as cgzf and agzf mszpt2- cgzf and agzf belong to the same c2h group ii. functional complementation studies will determine whether cgzf and agzf are functionally equivalent to putative orthologous genes identified in legumes. recent studies showed that repressors containing the ear motif play a role in defense responses to plant biotic and abiotic stress. cgzf and agzf could repress the expression of defense genes of actinorhizal plant in response to signals from frankia to allow colonization of new cells in nodules. finally, cgzf and agzf could be used as functional markers to investigate the regulation of actinorhizal symbiosis interaction.  the in silico identification and transcriptome analysis of tfs in c. glauca and in a. glutinosa that we carried out represents a crucial step forward in the elucidation of the molecular events underlying actinorhizal and mycorrhizal symbioses. coupled with a phylogenetic study, these analyses enabled identification of a nodulation specific gene in c. glauca named cgzf and its ortholog in a. glutinosa named agzf these two new genes are most probably negative regulators which may play a crucial role in actinorhizal symbiosis. the development of efficient techniques to transform c. glauca now allows us to perform functional studies of cgzf availability of supporting data est sequences reported in this paper have been deposited in the genbank and embl databases. the normalized and raw microarray data values have been deposited in the gene expression omnibus database. in addition, all nucleic acid sequences used in this study have been included in additional files and proteins sequences used to produce phylogenetic data sets have been added in additional file 
interestingly, comparison of the non-legume and legume tf with signaling elements from actinorhizal species revealed a new subgroup of nodule-specific c2h tf that could be specifically involved in actinorhizal symbioses. the aim of this work was to identify potential transcription factors involved in the establishment and functioning of actinorhizal symbioses. based on published transcriptome datasets and quantitative pcr analysis, we found that 39% and 26% of these transcription factors were regulated during c. glauca and a. glutinosa-frankia interactions, respectively. we also identified an actinorhizal-specific transcription factor belonging to the zinc finger c1-2i subfamily we named cgzf in c. glauca and agzf in a. glutinosa. we identified putative transcription factors by in silico analysis in families in casuarina glauca and in families in alnus glutinosa est databases. phylogenetic studies confirmed the presence of common key transcription factors such as nsp, nf-ya and ern-related proteins involved in nodule formation in legumes, which confirm the existence of a common symbiosis signaling pathway in nitrogen-fixing root nodule symbioses. we identified putative nodulation-associated transcription factors with particular emphasis on members of the gras, nf-ya, erf and c2h families. transcription factorssymbiosisactinorhizal plantssignaling pathwayc2h transcription factorsissue-copyright-statement© the author 2014 in silico identification, transcript analysis, and phylogeny reconstruction of transcription factor families paves the way for the study of specific molecular regulation of symbiosis in response to frankia infection.
 trees belonging to the casuarinaceae and betulaceae families play an important ecological role and are useful tools in forestry for degraded land rehabilitation and reforestation. these functions are linked to their capacity to establish symbiotic relationships with a nitrogen-fixing soil bacterium of the genus frankia. however, the molecular mechanisms controlling the establishment of these symbioses are poorly understood. the aim of this work was to identify potential transcription factors involved in the establishment and functioning of actinorhizal symbioses.  we identified putative transcription factors by in silico analysis in families in casuarina glauca and in families in alnus glutinosa est databases. based on published transcriptome datasets and quantitative pcr analysis, we found that 39% and 26% of these transcription factors were regulated during c. glauca and a. glutinosa-frankia interactions, respectively. phylogenetic studies confirmed the presence of common key transcription factors such as nsp, nf-ya and ern-related proteins involved in nodule formation in legumes, which confirm the existence of a common symbiosis signaling pathway in nitrogen-fixing root nodule symbioses. we also identified an actinorhizal-specific transcription factor belonging to the zinc finger c1-2i subfamily we named cgzf in c. glauca and agzf in a. glutinosa.  we identified putative nodulation-associated transcription factors with particular emphasis on members of the gras, nf-ya, erf and c2h families. interestingly, comparison of the non-legume and legume tf with signaling elements from actinorhizal species revealed a new subgroup of nodule-specific c2h tf that could be specifically involved in actinorhizal symbioses. in silico identification, transcript analysis, and phylogeny reconstruction of transcription factor families paves the way for the study of specific molecular regulation of symbiosis in response to frankia infection. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords transcription factorssymbiosisactinorhizal plantssignaling pathwayc2h transcription factorsissue-copyright-statement© the author 2014 