BACKGROUND
the association of rhizobial strains and legumes represents one of the most perfect symbiotic interactions, in which a sophisticated machinery has been developed in both partners for millions of years, now contributing the highest inputs of nitrogen on earth .
a fascinating step in the symbiosis is represented by the molecular signal dialogue established between the compatible partners, starting with the message sent with the exudation of molecules—mainly flavonoids—from the host legume, and replied with the synthesis of lipochitooligosaccharides —also known as nod factors—by the rhizobium .
the “maestro” that orchestrates this symphony in the bacterium is the regulatory nodd gene, constitutively expressed and responsible for initiating the transcriptions of the remaining nodulation genes .
rhizobium tropici is abundantly found in tropical acid soils of south america; its main characteristics are high tolerance of environmental stresses and ability to nodulate a broad range of legumes, the most economically important being common bean  .
probably the most intriguing feature of the common bean-r. tropici symbiosis is the abundance of flavonoid nod-gene inducers released by the host legume  <cit> , and the synthesis of the largest known variety of nod factors by r. tropici ciat  <dig> .
also unique are the observations that r. tropici ciat  <dig> is capable of producing lcos under abiotic stresses—such as acidic and saline conditions—in the absence of plant-molecular signals , and interestingly, some lcos are produced even in the absence of saline stress and flavonoids  <cit> .
rhizobial species described so far have one to five regulatory nodd genes.
r. tropici ciat  <dig> and closely related species carrying the symbiovar tropici  possess the highest numbers, with five copies of nodd genes  <cit> .
elucidating the roles of the five nodd genes of ciat  <dig> may help to understand their protagonism in host-range characteristics and in the strategies that the strain uses to circumvent abiotic stresses.
it may also contribute to gaining a better understanding of the evolution of symbiotic interactions, since r. tropici has a strong resemblance to the pathogen agrobacterium in terms of genes and proteins  <cit> .
as a first study, we shed light on the roles of nodd <dig> and nodd <dig> of r. tropici ciat  <dig>  with data related to the activation/repression of nodulation genes, their role in host range and showing that these two genes have functions beyond nodulation  <cit> .
here we achieve improved understanding of the mechanisms controlling regulation of the nodd genes of ciat  <dig>  with studies of nodd <dig>  nodd <dig> and nodd <dig> mutants.
RESULTS
gene localization and phenotypes in vitro of the wild type and mutant strains
nodd gene nomenclature was used as defined for the genome of r. tropici strain ciat  <dig>  <cit> .
noda <dig> precedes nodd <dig> and is close to an aquaporin; nodd <dig> precedes the operon nifhdk and nodd <dig> is downstream of an operon of hypothetical proteins .
nodd <dig>  nodd <dig> and nodd <dig> genes correspond to the cd <dig>  cd <dig> and cd <dig> nodd-hybridizing regions of ciat  <dig> described by van rhijn et al.
<cit> , respectively.
mutations in the nodd <dig>  nodd <dig> and nodd <dig> genes were achieved as described in the methods section.
in general, evaluations were performed in the absence of any inducer   <cit> , in the presence of the nod-gene-inducing molecule apigenin , or under salinity stress  that also induce the synthesis of nod factors.
the same treatments were used in our previous study with nodd <dig> and nodd <dig> mutants  <cit> .fig.
<dig> gene neighborhood of nodd <dig>  nodd <dig> and nodd <dig> genes and representation of the mutations.
a gene localizationin the symbiotic plasmid  of r. tropici strain ciat  <dig> and location of primers used to perform rtqpcr experiments ; b schematic representation of the nodd <dig>  nodd <dig> and nodd <dig> mutation
some bacterial properties may be regulated via nodd proteins, and in our previous study we showed that both nodd <dig> and nodd <dig> have a constitutive suppression role on swarming motility and an activation effect on indole acetic acid  synthesis  <cit> .
here, we found no differences between the three mutants and the wt strain in exopolysaccharide  and lipopolysaccharide  profiles, swimming and swarming motilities, biofilm formation, or iaa synthesis .
symbiotic phenotypes
symbiotic phenotypes of the wt ciat  <dig> and the mutant strains were verified in pots containing sterile substrate .
previously, we reported that common bean requires both nodd <dig> and nodd <dig>  whereas nodd <dig> was the main nodulation regulator of both leucaena  and siratro   <cit> .
for comparison, these results are shown again in table  <dig>  together with the results obtained with nodd <dig>  nodd <dig> and nodd <dig> mutants; in addition, the symbiotic properties of all five nodd mutants were assayed in two other host legumes, lotus burtii borsos and lotus japonicus  k. larsen.table  <dig> plant responses  and shoot dry weight  to inoculation of common bean, leucaena, siratro, lotus japonicus and l. burtii with r. tropici strain ciat  <dig> and derivatives.
plants evaluated after  <dig> , 42 days , or 50 days  of growth under controlled conditions
p. vulgaris
l. leucocephala
m. atropurpureum
l. japonicus
l. burtii
aafter  <cit>
*data represent means ± sd  of  <dig> jars, each with two plants.
nodd <dig>  nodd <dig> and nodd <dig> mutant parameters were individually compared with the parental strain ciat  <dig> parameters by using the mann-whitney non-parametric test.
values tagged by * and ** are significantly different at the level α =  <dig> and 5 %, respectively
for the common bean, there were no statistical differences between the wt and the three mutants in terms of the nodulation or shoot dry weight  parameters, but we should mention that there was a decrease in nodulation, which might indicate effects of minor magnitude due to the mutations, with an emphasis on nodd <dig>  where it also affected sdw.
leucaena and siratro also did not show differences in nodulation between the wt and the three mutants, but sdw of leucaena inoculated with nodd <dig> and nodd <dig> mutants was slightly, but significantly, lower .
in relation to the nodulation of lotus species, nodd <dig> was the main regulator in l. japonicus and very important for l. burtii, whereas nodd <dig> did not affect nodulation but resulted in a significative reduction in sdw of l. japonicus, indicating that it interferes with the efficiency of nitrogen fixation.
in addition, for l. japonicus, a mutation in nodd <dig> and nodd <dig> also affected nodulation and sdw, whereas no effects were observed for l. burtii .
from these data, we may conclude that full nodulation of common bean requires both nodd <dig> and nodd <dig> genes, but it is possible that nodd <dig> and nodd <dig> could make minor contributions that were not statistically detected in our experiments.
therefore, as in several other rhizobial species  <cit>  including the broadly nodulating s. fredii ngr  <dig>  <cit> , nodd <dig> is the main gene regulating nodulation of leucaena, siratro, l. burtii and l. japonicus.
siratro does not require any other nodd gene for nodulation, but a non-statistically significant decrease observed with the nodd <dig> mutant should be more fully investigated.
lotus burtii does not require any of the nodd genes except for nodd <dig>  in contrast, l. japonicus receives contributions from all the nod genes except for nodd <dig> for nodulation, because with the other nodd mutants a reduced sdw was observed .
the most intricate pattern of responses in nodulation described so far is that for sinorhizobium meliloti, which utilizes the three copies of nodd to optimize nodulation of each of its legume hosts  <cit> .
however, now we present a regulatory pattern that involves from one nodd gene  to four .
there were also indications that nodd genes influence nodule effectiveness, as shown for nodd <dig>  nodd <dig> and nodd <dig> for leucaena, and nodd <dig> for l. japonicus.
another particularity for r. tropici ciat  <dig> was that, in general, nodd <dig> was not a repressor of any of the legumes evaluated, contrary to what happens with the broadly nodulating strain ngr  <dig>  <cit> .
nod-factor patterns
the interesting roles of nod factors—which apparently can go further than nodulation—have been broadly investigated over a long period of time  <cit> .
r. tropici ciat  <dig> synthesises a large variety of nod factors when induced by flavonoids , or under abiotic stress conditions in the absence of flavonoids  <cit> ; surprisingly, nod factors are also synthesized in the absence of any known inducer  <cit> .
a variety of nod-factor structures was synthesized by the nodd <dig>  nodd <dig> and nodd <dig> mutants .
nodd <dig> mutant reduced the number of nod factors under all three conditions, control, when induced with apigenin and uneder saline conditions, while nodd <dig> mutant had a decrease in the number of nod factors under control and saline conditions .
it is noteworthy that nodd <dig> increased the number of nod factors in under salinity, without the induction of apigenin.
the larger number of nod factors was observed under saline conditions for the wt and nodd <dig> mutants, whereas similar numbers were observed with apigenin .
these results add more evidence to the hypothesis that the large production of nod factors is related to the well known properties of broad host infectivity and the high tolerance of abiotic stresses of r. tropici, being able to establish symbioses even under harsh environmental conditions .table  <dig> number of nod factors produced by the wild type r. tropici strain ciat  <dig> and the nodd <dig>  nodd <dig> and nodd <dig> mutants when grown in control b− medium  <cit> , with  <dig>  μm of apigenin or salt .
the structures of nod factors under each condition are shown in additional file 1: table s <dig>  additional file 2: table s <dig> and additional file 3: table s3
nodd3
nodd4
nodd5
in relation to the nod-factor structures, the mutations in nodd <dig> in general did not result in changes in the molecules produced both in the presence of apigenin and salt.
however, in the negative control b−, the number of molecules was drastically reduced, which indicates that nodd <dig> might be an activator of other regulatory genes such as nodd <dig> .
moreover, ciat  <dig> produced only two nod factors deacetyled in the presence of apigenin .
however, the nodd <dig> mutant produced five deacetyled nod factors that were not detected in the lcos biosynthesized by the nodd <dig> and nodd <dig> mutants.
these results suggest that the nodd <dig> gene is important for the deacetylation of nod factors produced by ciat  <dig> in the presence of apigenin.
non-deacetylated nod factors were detected in control and saline conditions .
interestingly, it has been suggested that hsnt  has a role in nod-factor decoration in neorhizobium galegae  <cit> .
under saline stress, in addition to an increase in the lcos synthesized by the nodd <dig> mutant, fatty acids of c14: <dig> were not found, nor were sulphated molecules of four units of n-acetyl-glucosamine.
not least important, the strong increase in the number of molecules in the nodd <dig> mutant might indicate that the gene is a repressor of other regulatory genes, such as nodd <dig> .
for the nodd <dig> mutant, no quantitative or qualitative differences in lcos were observed, whereas important differences were detected under saline stress.
the mutation not only resulted in a decrease in the number of lcos, but also the molecules included neither fatty acids c14: <dig>  nor sulphated molecules of  <dig> units of n-acetylglucosamine.
in addition, we did not detect the fatty acids c20: <dig> or c20: <dig> - v; v and v, found in the wt, nodd <dig> and nodd <dig> strains in the presence of salt.
it is worth mentioning that nodd <dig> and nodd <dig> mutants also do not produce these factors in the presence of salt  <cit> .
fatty acids c20: <dig> or c20: <dig> under salt stress and/or c14: <dig> o c14: <dig> with apigenin might have the participation of genes detected in the genome of ciat  <dig>  <cit> , such as node and nodf genes  <cit> .
consequently, the absence of these fatty acids in nodd <dig> mutants under saline stress indicates that other regulatory nodd genes were not activated under salt, and nodd <dig> and nodd <dig> are strong candidates, as their mutants inhibited the synthesis of these fatty acids.
we suggest that nodd <dig> could be an activator of nodd <dig> or nodd <dig> in the presence of salt to allow the expression of genes node-nodf.
this intricate regulatory mechanism for the production of lcos involving five nodd genes—demonstrated in our study—seems to have no parallel in other rhizobia, especially under saline stress.
as the tropical conditions where r. tropici is abundantly found are often extreme  <cit> , this might indicate a high degree of evolution to allow the symbioses to form and function under harmful conditions.
it is also worth mentioning that several other roles have been attributed to lcos besides being involved in early steps of nodulation.
for example, there are reports that lco effects resemble those of cytokinins  <cit> , that they have a role in mechanisms related to defense against disease  <cit>  and seed germination  <cit> ; therefore, the role of lco structure in phenotype determination deserves further investigation.
gene expression
we performed gene-expression studies with the wild type and nodd <dig>  nodd <dig> and nodd <dig> mutants.
in these studies we evaluated the expression of nodc—which controls the elongation of the oligosaccharide chain of nod factors and is transcribed with the activation of nod genes  <cit> —of the nodd <dig> gene and of each of the three nodd genes, to improve our understanding of the roles of these three genes.
as expected, endogenous expression of all nodd genes was consistently low, as these genes are constitutively expressed.
in addition, the expression of each nodd gene corresponding to each nodd mutation was confirmed as null .fig.
<dig> rt-qpcr analysis of the expression of nodd genes from r. tropici ciat  <dig> and derivatives grown under control , in the absence and in the presence of apigenin  or nacl .
expression data shown are the mean  of three biological replicates.
data were normalized in relation to the endogenous control .
the asterisks indicate a statistically significant expression at the level α = 5 %, determined by rest <dig> software.
light gray bars: wild type strain, black bars: mutant.
a, b - expression induced in the nodd <dig> mutant; c, d - expression induced in the nodd <dig> mutant; e, f - expression induced in the nodd <dig> mutant
significant expression of nodc for both the wt and the nodd <dig> mutant was verified, of 42- and 58-fold, respectively, when induced by apigenin, and, although at basal low levels, nodd <dig> expression was significantly increased in the wt strain .
under saline stress, a mutation in nodd <dig> decreased both nodc and nodd <dig> expression, confirming the results obtained with nod factors, that nodd <dig> is an activator of nodd <dig>  especially under saline conditions .
the picture obtained with nodd <dig> was somewhat different.
a mutation in nodd <dig> resulted in a 3-fold decrease, but not in total inhibition of expression of nodc with apigenin; therefore, the results indicate an activation role in the presence of apigenin .
however, in saline conditions, nodd <dig> expression in the wt strain was down-regulated, indicating a repressor role, that was confirmed by an increase of  <dig> -fold on nodc expression when the gene was mutated fig.
2d.
therefore, under apigenin nodd <dig> acted as an activator and under salt stress as a repressor of other nod genes .
in relation to nodd <dig>  the gene proved to be an inducer, increasing the expression of both nodc and nodd <dig> in the presence of both the inducer molecule apigenin  and salt stress .
these results are consistent with the results based on the synthesis of nod factors.
phylogeny of nodd genes
a phylogenetic tree was built to verify the similarities between the five copies of nodd genes of ciat  <dig>  helping to add information about these genes.
figure  <dig> shows that nodd <dig> gene of ciat  <dig> is positioned in a different cluster from the other nodd genes, showing full similarity with the nodd <dig> of r. freirei prf  <dig>  that belongs to the “r.
tropici group”  <cit> .
within the same great cluster of the phylogenetic tree, but positioned in another subgroup was the nodd <dig> genes of r. etli sv phaseoli.
the other four copies of nodd genes of ciat  <dig> were positioned in another great cluster, each one showing full identity with the correspondent nodd gene of r. freirei.
therefore, apparently nodd <dig> had a different evolutionary history from the other nodd genes, and as we pointed out before, one important host legume, leucaena does not need nodd <dig> for full nodulation, while common bean does.
one hypothesis is that nodd <dig> could have been acquired in the evolutionary process of getting the ability to nodulate common bean.fig.
<dig> phylogenetic tree of representatives nodd genes of some relevant rizobia.
the branchs length represents the evolutionary lineages changing over time.
the length of the brach represents the amount of changes and it is proportional to the number of nucleotide substitutions per site.
the bar at the bottom of the figure provides a scale for the evolution.
the numbers next to each node represent a measure of support for the node .
a high value means that there is strong evidence that the sequences to the right of the node cluster together to the exclusion of any other.
phylogenetic tree was built as described in the methods section.
in this phylogenetic tree, nodd <dig> to nodd <dig> ciat  <dig> correspond to rhizobium tropici ciat  <dig> nodd genes; nodd <dig> to nodd <dig> prf  <dig> correspond to rhizobium freirei prf  <dig> nodd genes; nodd <dig> to nodd <dig> ie  <dig> correspond to rhizobium etli sv.
phaseoli ie  <dig> nodd genes; nodd <dig> to nodd <dig>  <dig> correspond to sinorhizobium meliloti  <dig> nodd genes; and nodd <dig> and nodd <dig> usda  <dig> correspond to bradyrhizobium elkanii usda  <dig> nodd genes.
why five nodd genes?
in contrast to what we have previously reported for nodd <dig> and nodd <dig>  <cit> , nodd <dig>  nodd <dig> and nodd <dig> of r. tropici ciat  <dig> did not affect phenotypic traits such as motility or of iaa synthesis.
however, they contributed to the intricate regulatory mechanism of nodulation of different host plants that, overall, may require from one to four genes.
it remains to be determined if all five genes contribute to the nodulation of common bean, since minor, but not statistically significant, decreases in nodulation were observed with nodd <dig>  nodd <dig> and nodd <dig> mutants.
apparently the increased number of nod factors and the activation of nodd <dig> and nodc genes under abiotic stress may be critical for nodulation under abiotic stresses or for enlargement of host range, guaranteeing the survival of both symbiotic partners under harsh environmental conditions.
it is surprising that one major host, common bean, releases so many nod-gene-inducing molecules  <cit>  and that a main rhizobial symbiont synthesizes so many nod factors by the action of five nodd genes.
the results previously obtained with hosts such as leucaena and siratro—and now confirmed with lotus spp.—where nodd <dig> played the major role adds weight to the hypothesis that common bean is not the main host for r. tropici  <cit> .
one important observation was that the phylogenetic comparisons of nodd genes positioned nodd <dig> in a different cluster from the other nodd genes, what might indicate an evolution in the process of getting the ability to nodulate common bean, the requires both nodd <dig> and nodd <dig> genes, contrary to hosts as leucaena.
sharing the nodulation responsibility with more than one nodd gene, as is the case with common bean, suggests the ultimate evolution of the symbiosis, guaranteeing that nodulation occurs under abiotic stresses.
CONCLUSIONS
r. tropici ciat  <dig> is an intriguing rhizobia with high tolerance of environmental stresses, the ability to nodulate a broad range of legume hosts and carrying five copies of the regulatory nodd gene  <cit> .
the role of three nodd genes of ciat  <dig> was investigated for the first time in this study.
nodd <dig> and nodd <dig> were identified as activators of the nodd <dig> gene, whereas nodd <dig> might act as repressor or inducer, depending on the inducing environment.
a large variety of nod factors was produced by the three mutants when induced by apigenin or salt stress, and also in the absence of any inducer.
we may hypothesize that the high number of nodd copies and the synthesis of many nod factors might help r. tropici both in enhancing the host range and in the ability to nodulate the hosts under harsh environmental conditions.

 the association of rhizobial strains and legumes represents one of the most perfect symbiotic interactions, in which a sophisticated machinery has been developed in both partners for millions of years, now contributing the highest inputs of nitrogen on earth. a fascinating step in the symbiosis is represented by the molecular signal dialogue established between the compatible partners, starting with the message sent with the exudation of molecules—mainly flavonoids—from the host legume, and replied with the synthesis of lipochitooligosaccharides —also known as nod factors—by the rhizobium. the “maestro” that orchestrates this symphony in the bacterium is the regulatory nodd gene, constitutively expressed and responsible for initiating the transcriptions of the remaining nodulation genes. rhizobium tropici is abundantly found in tropical acid soils of south america; its main characteristics are high tolerance of environmental stresses and ability to nodulate a broad range of legumes, the most economically important being common bean. probably the most intriguing feature of the common bean-r. tropici symbiosis is the abundance of flavonoid nod-gene inducers released by the host legume, and the synthesis of the largest known variety of nod factors by r. tropici ciat. also unique are the observations that r. tropici ciat is capable of producing lcos under abiotic stresses—such as acidic and saline conditions—in the absence of plant-molecular signals, and interestingly, some lcos are produced even in the absence of saline stress and flavonoids. rhizobial species described so far have one to five regulatory nodd genes. r. tropici ciat and closely related species carrying the symbiovar tropici possess the highest numbers, with five copies of nodd genes. elucidating the roles of the five nodd genes of ciat may help to understand their protagonism in host-range characteristics and in the strategies that the strain uses to circumvent abiotic stresses. it may also contribute to gaining a better understanding of the evolution of symbiotic interactions, since r. tropici has a strong resemblance to the pathogen agrobacterium in terms of genes and proteins. as a first study, we shed light on the roles of nodd and nodd of r. tropici ciat with data related to the activation/repression of nodulation genes, their role in host range and showing that these two genes have functions beyond nodulation. here we achieve improved understanding of the mechanisms controlling regulation of the nodd genes of ciat with studies of nodd nodd and nodd mutants.  gene localization and phenotypes in vitro of the wild type and mutant strains nodd gene nomenclature was used as defined for the genome of r. tropici strain ciat. noda precedes nodd and is close to an aquaporin; nodd precedes the operon nifhdk and nodd is downstream of an operon of hypothetical proteins. nodd nodd and nodd genes correspond to the cd cd and cd nodd-hybridizing regions of ciat described by van rhijn et al., respectively. mutations in the nodd nodd and nodd genes were achieved as described in the methods section. in general, evaluations were performed in the absence of any inducer, in the presence of the nod-gene-inducing molecule apigenin, or under salinity stress that also induce the synthesis of nod factors. the same treatments were used in our previous study with nodd and nodd mutants.fig. gene neighborhood of nodd nodd and nodd genes and representation of the mutations. a gene localizationin the symbiotic plasmid of r. tropici strain ciat and location of primers used to perform rtqpcr experiments; b schematic representation of the nodd nodd and nodd mutation some bacterial properties may be regulated via nodd proteins, and in our previous study we showed that both nodd and nodd have a constitutive suppression role on swarming motility and an activation effect on indole acetic acid synthesis. here, we found no differences between the three mutants and the wt strain in exopolysaccharide and lipopolysaccharide profiles, swimming and swarming motilities, biofilm formation, or iaa synthesis. symbiotic phenotypes symbiotic phenotypes of the wt ciat and the mutant strains were verified in pots containing sterile substrate. previously, we reported that common bean requires both nodd and nodd whereas nodd was the main nodulation regulator of both leucaena and siratro. for comparison, these results are shown again in table  together with the results obtained with nodd nodd and nodd mutants; in addition, the symbiotic properties of all five nodd mutants were assayed in two other host legumes, lotus burtii borsos and lotus japonicus k. larsen.table plant responses and shoot dry weight to inoculation of common bean, leucaena, siratro, lotus japonicus and l. burtii with r. tropici strain ciat and derivatives. plants evaluated after, 42 days, or 50 days of growth under controlled conditions p. vulgaris l. leucocephala m. atropurpureum l. japonicus l. burtii aafter *data represent means ± sd of jars, each with two plants. nodd nodd and nodd mutant parameters were individually compared with the parental strain ciat parameters by using the mann-whitney non-parametric test. values tagged by * and ** are significantly different at the level α =  and 5 %, respectively for the common bean, there were no statistical differences between the wt and the three mutants in terms of the nodulation or shoot dry weight parameters, but we should mention that there was a decrease in nodulation, which might indicate effects of minor magnitude due to the mutations, with an emphasis on nodd where it also affected sdw. leucaena and siratro also did not show differences in nodulation between the wt and the three mutants, but sdw of leucaena inoculated with nodd and nodd mutants was slightly, but significantly, lower. in relation to the nodulation of lotus species, nodd was the main regulator in l. japonicus and very important for l. burtii, whereas nodd did not affect nodulation but resulted in a significative reduction in sdw of l. japonicus, indicating that it interferes with the efficiency of nitrogen fixation. in addition, for l. japonicus, a mutation in nodd and nodd also affected nodulation and sdw, whereas no effects were observed for l. burtii. from these data, we may conclude that full nodulation of common bean requires both nodd and nodd genes, but it is possible that nodd and nodd could make minor contributions that were not statistically detected in our experiments. therefore, as in several other rhizobial species including the broadly nodulating s. fredii ngr, nodd is the main gene regulating nodulation of leucaena, siratro, l. burtii and l. japonicus. siratro does not require any other nodd gene for nodulation, but a non-statistically significant decrease observed with the nodd mutant should be more fully investigated. lotus burtii does not require any of the nodd genes except for nodd in contrast, l. japonicus receives contributions from all the nod genes except for nodd for nodulation, because with the other nodd mutants a reduced sdw was observed. the most intricate pattern of responses in nodulation described so far is that for sinorhizobium meliloti, which utilizes the three copies of nodd to optimize nodulation of each of its legume hosts. however, now we present a regulatory pattern that involves from one nodd gene to four. there were also indications that nodd genes influence nodule effectiveness, as shown for nodd nodd and nodd for leucaena, and nodd for l. japonicus. another particularity for r. tropici ciat was that, in general, nodd was not a repressor of any of the legumes evaluated, contrary to what happens with the broadly nodulating strain ngr. nod-factor patterns the interesting roles of nod factors—which apparently can go further than nodulation—have been broadly investigated over a long period of time. r. tropici ciat synthesises a large variety of nod factors when induced by flavonoids, or under abiotic stress conditions in the absence of flavonoids; surprisingly, nod factors are also synthesized in the absence of any known inducer. a variety of nod-factor structures was synthesized by the nodd nodd and nodd mutants. nodd mutant reduced the number of nod factors under all three conditions, control, when induced with apigenin and uneder saline conditions, while nodd mutant had a decrease in the number of nod factors under control and saline conditions. it is noteworthy that nodd increased the number of nod factors in under salinity, without the induction of apigenin. the larger number of nod factors was observed under saline conditions for the wt and nodd mutants, whereas similar numbers were observed with apigenin. these results add more evidence to the hypothesis that the large production of nod factors is related to the well known properties of broad host infectivity and the high tolerance of abiotic stresses of r. tropici, being able to establish symbioses even under harsh environmental conditions.table number of nod factors produced by the wild type r. tropici strain ciat and the nodd nodd and nodd mutants when grown in control b− medium, with  μm of apigenin or salt. the structures of nod factors under each condition are shown in additional file 1: table s additional file 2: table s and additional file 3: table s3 nodd3 nodd4 nodd5 in relation to the nod-factor structures, the mutations in nodd in general did not result in changes in the molecules produced both in the presence of apigenin and salt. however, in the negative control b−, the number of molecules was drastically reduced, which indicates that nodd might be an activator of other regulatory genes such as nodd. moreover, ciat produced only two nod factors deacetyled in the presence of apigenin. however, the nodd mutant produced five deacetyled nod factors that were not detected in the lcos biosynthesized by the nodd and nodd mutants. these results suggest that the nodd gene is important for the deacetylation of nod factors produced by ciat in the presence of apigenin. non-deacetylated nod factors were detected in control and saline conditions. interestingly, it has been suggested that hsnt has a role in nod-factor decoration in neorhizobium galegae. under saline stress, in addition to an increase in the lcos synthesized by the nodd mutant, fatty acids of c14: were not found, nor were sulphated molecules of four units of n-acetyl-glucosamine. not least important, the strong increase in the number of molecules in the nodd mutant might indicate that the gene is a repressor of other regulatory genes, such as nodd. for the nodd mutant, no quantitative or qualitative differences in lcos were observed, whereas important differences were detected under saline stress. the mutation not only resulted in a decrease in the number of lcos, but also the molecules included neither fatty acids c14: nor sulphated molecules of units of n-acetylglucosamine. in addition, we did not detect the fatty acids c20: or c20: - v; v and v, found in the wt, nodd and nodd strains in the presence of salt. it is worth mentioning that nodd and nodd mutants also do not produce these factors in the presence of salt. fatty acids c20: or c20: under salt stress and/or c14: o c14: with apigenin might have the participation of genes detected in the genome of ciat, such as node and nodf genes. consequently, the absence of these fatty acids in nodd mutants under saline stress indicates that other regulatory nodd genes were not activated under salt, and nodd and nodd are strong candidates, as their mutants inhibited the synthesis of these fatty acids. we suggest that nodd could be an activator of nodd or nodd in the presence of salt to allow the expression of genes node-nodf. this intricate regulatory mechanism for the production of lcos involving five nodd genes—demonstrated in our study—seems to have no parallel in other rhizobia, especially under saline stress. as the tropical conditions where r. tropici is abundantly found are often extreme, this might indicate a high degree of evolution to allow the symbioses to form and function under harmful conditions. it is also worth mentioning that several other roles have been attributed to lcos besides being involved in early steps of nodulation. for example, there are reports that lco effects resemble those of cytokinins, that they have a role in mechanisms related to defense against disease and seed germination; therefore, the role of lco structure in phenotype determination deserves further investigation. gene expression we performed gene-expression studies with the wild type and nodd nodd and nodd mutants. in these studies we evaluated the expression of nodc—which controls the elongation of the oligosaccharide chain of nod factors and is transcribed with the activation of nod genes —of the nodd gene and of each of the three nodd genes, to improve our understanding of the roles of these three genes. as expected, endogenous expression of all nodd genes was consistently low, as these genes are constitutively expressed. in addition, the expression of each nodd gene corresponding to each nodd mutation was confirmed as null.fig. rt-qpcr analysis of the expression of nodd genes from r. tropici ciat and derivatives grown under control, in the absence and in the presence of apigenin or nacl. expression data shown are the mean of three biological replicates. data were normalized in relation to the endogenous control. the asterisks indicate a statistically significant expression at the level α = 5 %, determined by rest software. light gray bars: wild type strain, black bars: mutant. a, b - expression induced in the nodd mutant; c, d - expression induced in the nodd mutant; e, f - expression induced in the nodd mutant significant expression of nodc for both the wt and the nodd mutant was verified, of 42- and 58-fold, respectively, when induced by apigenin, and, although at basal low levels, nodd expression was significantly increased in the wt strain. under saline stress, a mutation in nodd decreased both nodc and nodd expression, confirming the results obtained with nod factors, that nodd is an activator of nodd especially under saline conditions. the picture obtained with nodd was somewhat different. a mutation in nodd resulted in a 3-fold decrease, but not in total inhibition of expression of nodc with apigenin; therefore, the results indicate an activation role in the presence of apigenin. however, in saline conditions, nodd expression in the wt strain was down-regulated, indicating a repressor role, that was confirmed by an increase of -fold on nodc expression when the gene was mutated fig. 2d. therefore, under apigenin nodd acted as an activator and under salt stress as a repressor of other nod genes. in relation to nodd the gene proved to be an inducer, increasing the expression of both nodc and nodd in the presence of both the inducer molecule apigenin and salt stress. these results are consistent with the results based on the synthesis of nod factors. phylogeny of nodd genes a phylogenetic tree was built to verify the similarities between the five copies of nodd genes of ciat helping to add information about these genes. figure  shows that nodd gene of ciat is positioned in a different cluster from the other nodd genes, showing full similarity with the nodd of r. freirei prf that belongs to the “r. tropici group”. within the same great cluster of the phylogenetic tree, but positioned in another subgroup was the nodd genes of r. etli sv phaseoli. the other four copies of nodd genes of ciat were positioned in another great cluster, each one showing full identity with the correspondent nodd gene of r. freirei. therefore, apparently nodd had a different evolutionary history from the other nodd genes, and as we pointed out before, one important host legume, leucaena does not need nodd for full nodulation, while common bean does. one hypothesis is that nodd could have been acquired in the evolutionary process of getting the ability to nodulate common bean.fig. phylogenetic tree of representatives nodd genes of some relevant rizobia. the branchs length represents the evolutionary lineages changing over time. the length of the brach represents the amount of changes and it is proportional to the number of nucleotide substitutions per site. the bar at the bottom of the figure provides a scale for the evolution. the numbers next to each node represent a measure of support for the node. a high value means that there is strong evidence that the sequences to the right of the node cluster together to the exclusion of any other. phylogenetic tree was built as described in the methods section. in this phylogenetic tree, nodd to nodd ciat correspond to rhizobium tropici ciat nodd genes; nodd to nodd prf correspond to rhizobium freirei prf nodd genes; nodd to nodd ie correspond to rhizobium etli sv. phaseoli ie nodd genes; nodd to nodd correspond to sinorhizobium meliloti nodd genes; and nodd and nodd usda correspond to bradyrhizobium elkanii usda nodd genes. why five nodd genes? in contrast to what we have previously reported for nodd and nodd, nodd nodd and nodd of r. tropici ciat did not affect phenotypic traits such as motility or of iaa synthesis. however, they contributed to the intricate regulatory mechanism of nodulation of different host plants that, overall, may require from one to four genes. it remains to be determined if all five genes contribute to the nodulation of common bean, since minor, but not statistically significant, decreases in nodulation were observed with nodd nodd and nodd mutants. apparently the increased number of nod factors and the activation of nodd and nodc genes under abiotic stress may be critical for nodulation under abiotic stresses or for enlargement of host range, guaranteeing the survival of both symbiotic partners under harsh environmental conditions. it is surprising that one major host, common bean, releases so many nod-gene-inducing molecules and that a main rhizobial symbiont synthesizes so many nod factors by the action of five nodd genes. the results previously obtained with hosts such as leucaena and siratro—and now confirmed with lotus spp.—where nodd played the major role adds weight to the hypothesis that common bean is not the main host for r. tropici. one important observation was that the phylogenetic comparisons of nodd genes positioned nodd in a different cluster from the other nodd genes, what might indicate an evolution in the process of getting the ability to nodulate common bean, the requires both nodd and nodd genes, contrary to hosts as leucaena. sharing the nodulation responsibility with more than one nodd gene, as is the case with common bean, suggests the ultimate evolution of the symbiosis, guaranteeing that nodulation occurs under abiotic stresses.  r. tropici ciat is an intriguing rhizobia with high tolerance of environmental stresses, the ability to nodulate a broad range of legume hosts and carrying five copies of the regulatory nodd gene. the role of three nodd genes of ciat was investigated for the first time in this study. nodd and nodd were identified as activators of the nodd gene, whereas nodd might act as repressor or inducer, depending on the inducing environment. a large variety of nod factors was produced by the three mutants when induced by apigenin or salt stress, and also in the absence of any inducer. we may hypothesize that the high number of nodd copies and the synthesis of many nod factors might help r. tropici both in enhancing the host range and in the ability to nodulate the hosts under harsh environmental conditions. 
phenotypic traits such as exopolysaccharide, lipopolysaccharide, swimming and swarming motilities, biofilm formation and indole acetid acid production were also perfomed. rhizobium tropici strain ciat is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodd genes—five—and the greatest diversity of nod factors. nodd and nodd were identified as activators of the nodd gene, whereas, for the first time, it was shown that a regulatory nodd gene—nodd4—might act as repressor or inducer, depending on the inducing environment, giving support to the hypothesis that nodd roles go beyond nodulation, in terms of responses to abiotic stresses. up to different structures of nod factors were detected, being higher under salt stress, except for the nodd mutant; in addition, a high number of structures was synthesized by the nodd mutant in the absence of any inducer. expression of nodc, nodd and each nodd studied by rt-qpcr confirmed that nodd is an activator of nodd both in the presence of apigenin and salt stress. here we shed light on the roles of the multiple nodd genes of ciat by reporting, for the first time, results obtained with nodd nodd and nodd mutants. transcription of nodulation genes in rhizobial species is orchestrated by the regulatory nodd gene. phenotypic and symbiotic properties, nod factors and gene expression of nodd nodd and nodd mutants were compared with those of the wild-type ciat both in the presence and in the absence of the nod-gene-inducing molecule apigenin and of saline stress. in contrast, nodd might be an inducer with apigenin and a repressor under saline stress, whereas nodd was an inducer under both conditions. finally, nodulation assays were evaluated in up to six different legumes, including common bean. nodulation required from one to four nodd genes, depending on the host legume. in addition, nodd and nodc relative gene expressions were measured by quantitative rt-pcr in the wt and derivative mutant strains. we report for r. tropici ciat the most complex model of regulation of nodulation genes described so far. nod factors were purified and identified by lc-ms/ms analyses. biological nitrogen fixationlconodd genenod factorssymbiosisissue-copyright-statement© the author 2015 the three nodd mutants were built by insertion of Ω interposon. nodulation studies demonstrated the most complex regulatory system described so far, requiring from one to four nodd genes. probable activator or repressor roles, possibly via nodd and/or nodd were attributed to the three nodd genes. five nodd genes performed different roles depending on the host plant and the inducing environment.
 transcription of nodulation genes in rhizobial species is orchestrated by the regulatory nodd gene. rhizobium tropici strain ciat is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodd genes—five—and the greatest diversity of nod factors. here we shed light on the roles of the multiple nodd genes of ciat by reporting, for the first time, results obtained with nodd nodd and nodd mutants. methods the three nodd mutants were built by insertion of Ω interposon. nod factors were purified and identified by lc-ms/ms analyses. in addition, nodd and nodc relative gene expressions were measured by quantitative rt-pcr in the wt and derivative mutant strains. phenotypic traits such as exopolysaccharide, lipopolysaccharide, swimming and swarming motilities, biofilm formation and indole acetid acid production were also perfomed. all these experiments were carried out in presence of both inducers of ciat apigenin and salt. finally, nodulation assays were evaluated in up to six different legumes, including common bean.  phenotypic and symbiotic properties, nod factors and gene expression of nodd nodd and nodd mutants were compared with those of the wild-type ciat both in the presence and in the absence of the nod-gene-inducing molecule apigenin and of saline stress. no differences between the mutants and the wt were observed in exopolysaccharide and lipopolysaccharide profiles, motility, indole acetic acid synthesis or biofilm production, either in the presence, or in the absence of inducers. nodulation studies demonstrated the most complex regulatory system described so far, requiring from one to four nodd genes. up to different structures of nod factors were detected, being higher under salt stress, except for the nodd mutant; in addition, a high number of structures was synthesized by the nodd mutant in the absence of any inducer. probable activator or repressor roles, possibly via nodd and/or nodd were attributed to the three nodd genes. expression of nodc, nodd and each nodd studied by rt-qpcr confirmed that nodd is an activator of nodd both in the presence of apigenin and salt stress. in contrast, nodd might be an inducer with apigenin and a repressor under saline stress, whereas nodd was an inducer under both conditions.  we report for r. tropici ciat the most complex model of regulation of nodulation genes described so far. five nodd genes performed different roles depending on the host plant and the inducing environment. nodulation required from one to four nodd genes, depending on the host legume. nodd and nodd were identified as activators of the nodd gene, whereas, for the first time, it was shown that a regulatory nodd gene—nodd4—might act as repressor or inducer, depending on the inducing environment, giving support to the hypothesis that nodd roles go beyond nodulation, in terms of responses to abiotic stresses. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords biological nitrogen fixationlconodd genenod factorssymbiosisissue-copyright-statement© the author 2015 