Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A.
Méndez-Vilas (Ed.)
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Nitrogen metabolism in ectomycorrhizal fungi: fHANT-AC gene
regulation in Laccaria bicolor
Minna J. Kemppainen and Alejandro G. Pardo
Laboratorio de Micología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes and
CONICET. Roque Sáenz Peña 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina. E-mail:
[email protected]. Phone: 54-11-4365-7100
The mycorrhizal symbiosis is an ancient mutualistic association between fungi and the roots of the vast majority of
terrestrial plant species. In natural ecosystems the plant nutrient uptake (N, P and several micronutrients) from soil
happens via the extraradical mycelia of these symbiotic fungi. This association also improves plant water acquisition,
heavy metal tolerance and resistance to pathogens. While most herbaceous plants and tropical trees are engaged in
endomycorrhiza-type interactions, forest trees of boreal and temperate zones are typically ectomycorrhizal (ECM) plants.
These species include the majority of economically important trees and the fungal symbionts are predominantly
filamentous basidiomycetes.
In order to understand how an ECM fungus exploits soil N resources the expression profile of Laccaria fHANT-AC, a
gene cluster responsible for growth on nitrate and containing Lbnrt, Lbnr and Lbnir, was analyzed on variable N regimens
and using the RNA silencing technology. As a result a novel regulatory mechanism, not previously described for fungal
nitrate acquisition, was discovered. The repression of Laccaria fHANT-AC on ammonium seems not to be mediated via
L-glutamine as in ascomycete fungi. Growth on different organic N sources, these including also L-glutamine, results in
high transcript levels of Laccaria fHANT-AC and suggest a direct ammonium repression effect. Also uptake of nitrate
could be observed when growing on organic N. This finding indicates that the symbiotic fungus, differing from its
saprotrophic competitors in soil, has the capacity for efficient simultaneous utilization of both inorganic and organic soil N
resources. This also suggests that Laccaria can maintain active nitrate uptake from soil despite the known high hyphal free
L-glutamine concentration in ECM fungi destined for N translocation towards the host plant. This novel fHANT-AC
regulation pattern reveals an elegant adaptive response of an ECM fungus for maximizing its N acquisition especially in
soil-horizons rich in organic N and with spatial and temporal fluctuation of nitrate.
1. Introduction
Mycorrhiza is the most prevalent symbiosis on earth and it can be found in all terrestrial ecosystems. While most
herbaceous plants and tropical trees are engaged in endomycorrhiza-type interactions, forest trees of boreal and
temperate zones are typically ectomycorrhizal (ECM) plants.
The ECM symbiosis involves a large number of fungal taxa, mostly filamentous baidiomycetes, and these fungi play
an important role in seedling establishment and tree growth in different habitats across the globe [1]. It is accepted that
in ECM a mutual benefit exist for both partners due to nutrient exchange in symbiotic organs. The fungus receives C as
hexoses derived from host photosynthesis and the plant receives mainly N and P from the mycosymbiont. ECM also
improves plant access to soil water resources and increases uptake of other marco- and micronutrients.
In ECM forests nitrification rate is generally low and the amount of mineral N in soil is plant growth limiting [2, 3].
The involvement of ECM in N uptake of host plants was originally proposed by Frank [4] and several studies
demonstrated that in fact ECM improves plant N nutrition [5, 6]. While soil inorganic N can be taken up by plants, the
ammonium absorption capacity of fungal mycelium is also demonstrated to be higher than the one of plant roots. It is
this higher affinity of fungal nutrient uptake systems which further contributes to improved host N nutrition when
engaged in ECM symbioses [7]. Moreover, mycorrhization has been shown to cause down-regulation of host plant N
uptake systems. As a result ECM roots may become completely dependent on the fungal N acquisition machinery [810].
ECM fungi have evolved in N limiting ecosystems and they are clearly well adapted to low free N concentrations.
Ammonium is the dominant inorganic N form in ECM forest soils. It is also the preferred N source for many ECM
fungi even though the role of nitrate as N source may be more important also in these ecosystems than traditionally
believed [11-13]. ECM fungi can take up a range of organic N compounds such as amino acids, peptides, proteins and
N containing secondary metabolites. The main core of molecular information on basic N metabolism of ammonium,
nitrate, amino acids and peptide uptake of ECM fungi comes from studies in the basidiomycetes Hebeloma spp., [14],
Amanita muscaria [15] and the ascomycete Tuber borchii [16-21]. These studies have led to cloning and
characterization of several high and low affinity ammonium transporters and genes responsible for nitrate utilization
and growth on organic N sources.
Even though the relevance of ECM in native and managed forests the comprehension of host-fungus recognition,
establishment of symbiotic organs and nutrient exchange in ECM is limited. During the last decade the exploitation of
molecular methods such as EST-libraries and cDNA micro– and macro arrays has dramatically increased the knowledge
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on the genetics underlying the ECM interaction. Moreover, sequencing the genomes of mycobionts of the model tree
Populus trichocarpa, by the Department of Energy Joint Genome Institute (JGI), made mycorrhizal research to enter
the genomic era, and the genomic sequence of the first ECM fungus, the basidiomycete Laccaria bicolor, was
unraveled [22].
Another breakthrough in ECM research was the ability to obtain gene knock-down mutants of ECM fungi. In fact,
Kemppainen et al. [23] were able to obtain targeted gene expression modification in dikaryotic mycelium of L. bicolor.
Nitrate reductase as a test gene (Lbnr) was successfully knocked-down by hpRNA expression reducing the target
transcript level below 10 % of the control. Silencing of Lbnr resulted also in non-mycorrhizal phenotype with poplar on
nitrate, while ammonium or an organic N source restored the symbiotic capacity of the fungus. These results were the
first direct genetic proof showing that the efficient functioning of fungal N metabolism is fundamental for the
establishment of the ECM symbiosis. They also strongly suggest that the plant host controls the interaction not allowing
the fungal symbiont to behave as a parasite consuming photosynthates without delivering significant amount of N to the
host.
2. fHANT-AC gene regulation
The RNA silencing of Laccaria nitrate reductase encoding gene and the profound effect of it on the ectomycorrhizal
interaction [23] led us to study the expression profile and control of Laccaria nitrate utilization genes. Nitrogen
regulation and genetic control of secondary N sources utilization, such as nitrate, has been extensively studied in yeast
and saprotrophic filamentous ascomycetes [24, 25]. However, the knowledge on regulation of nitrate utilization or N
metabolism in basidiomycetes at the molecular level in general is poor. Nor has the expression and control of Laccaria
genes responsible for nitrate utilization been previously studied. Such studies have been conducted before only in two
ectomycorrhizal fungi, the basidiomycete Hebeloma cylindrosporum and the ascomycete Tuber borchii [18-21, 26-28].
However, previous data from these species have revealed some fundamental and highly interesting regulatory
differences with respect to nitrate utilization between them and model saprotrophic fungi. In both studied ECM fungi
the initiation of nitrate utilization does not depend on nitrate induction and also N starvation can activate nitrate
metabolic pathways. As these special regulatory features are shared by two phylogenetically distant ectomycorrhizal
species a case of convergent evolution to exploitation of the symbiotic niche has been proposed.
Besides the similarities however also clear regulatory differences exist in nitrate utilization between Hebeloma and
Tuber. It is not known whether these differences reflect different evolutionary history of basidio- and ascomycetes as
fungal taxa per se or whether they are species specific adaptations of ECM fungi to their prevalent growth environments
in nature such as different soil types. As the N metabolism has been traditionally proposed to be crucial for the
ectomycorrhizal lifestyle and this is supported further by our results on inhibition of mycorrhization via RNAi of Lbnr
[23] the expression control of Laccaria nitrate utilization genes is thus of special interest. Studying the regulation of
Laccaria nitrate usage genes would offer a comparison point for the expression pattern described in Hebeloma and give
an insight whether the special regulation pattern observed in this fungus is a conserved regulatory adaptation among
ECM homobasidiomycetes. Moreover, nitrate uptake of ectomycorrhizal roots is not only demonstrated to be carried
out by the fungal nitrate acquisition system but symbiosis leads to down-regulation of plant nitrate utilization genes.
This proposes a high host dependency on the nitrate uptake and metabolism of the fungal partner [8-10, 19]. How ECM
fungi regulate their N acquisition and how they respond to variable N conditions would therefore contribute to better
understanding of ECM symbiosis and tree nutrition in general. Besides characterizing the gene transcriptional status on
variable N regimens our access to Lbnr-silenced strains allowed testing for possible regulatory effects of nitrate
reductase activity on this metabolic pathway in Laccaria.
The utilization of nitrate is generally highly regulated in all organisms and it requires de novo synthesis of pathway
specific enzymes and transporters. In most organisms nitrate assimilation is carried out by the uptake and sequential
reduction of nitrate to nitrite and further to ammonium by nitrate transporter, nitrate reductase and nitrite reductase,
respectively. In fungi nitrate assimilation genes include a high affinity nitrate transporter (nrt2), nitrate reductase
(euknr) and ferredoxin-independent nitrite reductase (NAD(P)H-nir). These genes are present in many asco- and
basidiomycetes as a cluster abbreviated as fHANT-AC (fungal high affinity nitrate assimilation cluster). A gene cluster
organization is proposed to facilitate an efficient and coordinated genetic control and thus provide a selective advantage.
Phylogenetic analyses of this gene cluster propose its assembly in a lineage leading to oomycetes and its later transfer to
Dikarya, a fungal clade which includes asco- and basidiomycetes [30]. More interestingly Slot and Hibbett [30]
proposed that this horizontal transfer of fHANT-AC could have played a key role in the success of fungi to diversify as
free-living saprotrophs or mycorrhizal symbionts in terrestrial ecosystems.
Laccaria genome encodes one homologue of each gene needed for nitrate assimilation (Lbnrt, protein ID 254042;
Lbnr, ID 254066; and Lbnir, ID 291348; Fig. 1A). Their predicted amino acid sequences are highly similar to fHANTAC proteins of H. cylindrosporum and less related to their orthologues in filamentous ascomycetes indicating strong
sequence conservation among different phylogenetic fungal groups (Fig. 1B, C).
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A
ID 254066
Lbnr (3182 bp)
ID 254042
ID 291348
Lbnrt (1902 bp)
Lbnir (4254 bp)
13852 bp
593 bp
NR
B
NRT
100% 90% 80% 70% 60% 50% 40% 30%
100% 90% 80% 70% 60% 50% 40%
C
Aspergillus nidulans
Coprinopsis cinerea
59%
Magnaporthe grisea
Laccaria bicolor
B
Hebeloma cylindrosporum
52%
63%
Podospora anserina
46%
Phanerochaete chrysosporium
A
S
Neurospora crassa
A
Podospora anserina
59%
48%
Penicillium marneffei
42%
56%
37%
Tuber borchii
64%
56%
Laccaria bicolor
Hebeloma cylindrosporum
55%
B
Tuber borchii
Aspergillus nidulans
54%
Fusarium oxysporum
Fusarium oxysporum
Magnaporthe grisea
41%
62%
Neurospora crassa
Ustilago maydis
S
68%
68% 59%
Coprinopsis cinerea
72%
69%
53%
Phanerochaete chrysosporium
42%
Penicillium marneffei
Ustilago maydis
Fig. 1. (A) Laccaria nitrate utilization genes Lbnr, Lbnrt and Lbnir. The length of the genomic coding sequence of the genes and the
sequences separating the ORFs are indicated in the figure. The hexagon between Lbnrt and Lbnir refers to ORFs separating these two
genes in Laccaria fHANT-AC. (B) Homology tree of predicted full length amino acid sequences of fungal nitrate reductase (NR)
proteins. (C) Homology tree of fungal nitrate transporter (NRT) proteins. A: ascomycetes; B: basidiomycetes; S: sordariomycetes.
Sequence information and analysis are detailed in Kemppainen et al. [31].
Laccaria nitrate utilization genes are also arranged in a fHANT-AC-type gene cluster but this Laccaria cluster is not
as compact as in several other fungi (Fig. 2). Four other putative protein encoding sequences are present between Lbnrt
and Lbnir. One of these is a class I Copia-like retrotransposon sequence indicating that a transposomal insertion event
has occurred in Laccaria fHANT-AC.
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Fig. 2. Representation of intra-cluster organization of L. bicolor fHANT-AC and comparison of the cluster gene organization
between a set of basidiomycete and ascomycete fungi. Numbers 1-4 in Laccaria fHANT-AC refer to putative protein coding
sequences within the cluster. 1: hypothetical protein (ID 332461); 2: Class I Copia-like retrotransposon; putative gag-pol polyprotein
(ID 399468); 3: conserved hypothetical protein (ID 332464); 4: putative manganese and iron superoxide dismutase (ID 291347).
fHANT-ACs are drawn respecting their relative lengths (except for L. bicolor cluster intergenic region). nr: nitrate reductase; nrt:
nitrate transporter; nir: nitrite reductase. Figure adapted from Kemppainene et al. [31] where sequence sources of fHANT-AC are
detailed.
While protein sequences of fHANT-AC show conservation within fungal taxa, the cluster gene organization has not
been conserved during evolution. Nevertheless, the integrity of the cluster as such has been maintained in many species
(Fig. 2). This supports the idea that cluster organization of nitrate utilization genes has provided a selective advantage
for fungi in their natural habitats. These fHANT-AC species include both asco- and basidiomycete ECM fungi
suggesting that nitrate utilization has played an important role in the survival of these species as well.
Both dikaryotic Laccaria wild type and Lbnr-silenced strain (pHg/NITRSPL st. 43) [23] were analyzed for their
fHANT-AC transcriptional profiles. The expression pattern of Laccaria fHANT-AC genes was investigated on different
N source conditions (N starvation, ammonium, nitrate, ammonium/nitrate, urea, L-glutamine and L- asparagine feeding)
by both northern blot (Figs. 3 and 4B) and semi-quantitative RT-PCR (Fig. 4A) [31].
A
NH4
NO3
NH4/
NO3
-N
urea
Gln
B
NH4
NO3
NH4/
NO3
-N
urea
Gln
Lbnr
Lbnrt
Lbnir
α-tubulin
28S rRNA
Fig. 3. (A) Northern blot detection of L. bicolor wild type and (B) Lbnr-silenced strain (pHg/NITRSPL st.43) fHANT-AC transcript
levels on variable N regimens after 48 h of induction. The N conditions were adjusted to 4 mM of total N (except for NH4/NO3 for
which total N was 8 mM). NH4: ammonium (as ammonium sulphate); NO3: nitrate (as potassium nitrate); NH4/NO3: ammonium +
nitrate; N-: no nitrogen; Gln: L-glutamine. Loading controls of 28S ribosomal RNA before membrane blotting and control
hybridization with α-tubulin probe are presented. Figure adapted from Kemppainen et al. [31].
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A
4 mM NH4
8 mM Gln
8 mM urea
8 mM Asn
Relative expression levels
6
5
4
3
8 mM Gln
8 mM urea
8 mM Asn
2
Lbnr (α-tub/Gγ)
5.1x/4.7x
2.6x/2.3x
1.8x/1.6x
1
Lbnrt (α-tub/Gγ)
3.1x/2.9x
2.6x/2.4x
2x/1.7x
Lbnir (α-tub/Gγ)
2.9x/2.7x
1.8x/1.6x
2.4x/2x
0
Lbnr
B
Lbnr
α-tub
Lbnrt
4 mM
NH4
2 mM
Gln
Lbnir
8 mM
Gln
Lbnr/α-tub
2 mM Gln
8 mM Gln
4,744x
4,684x
Fig. 4. (A) Semi-quantitative RT-PCR detection of relative expression levels of fHANT-AC genes (Lbnr: nitrate reductase; Lbnrt:
nitrate transporter; Lbnir: nitrite reductase) in dikaryotic wild type mycelia grown for 48 h on 4 mM ammonium (NH4), 8 mM Lglutamine (Gln), urea and L-asparagine (Asn). The target gene amplifications were normalized separately with two control genes (αtubulin and Gγ). The results are presented as a mean of up-regulation values originating from normalization against the two control
genes with respect to the ammonium treatment (considered as 1x). The error bars represent standard deviation between the two
normalized values which are also shown in the table next to the graphic. (B) Northern blot detection of Lbnr mRNA in wild type
mycelia induced for 48 h on 4 mM ammonium (NH4) and 2 mM or 8 mM L-glutamine (Gln). Lbnr transcript levels were normalized
against α-tubulin signal. Relative gene expression up-regulation values with respect to the ammonium treatment (1x) for the two
glutamine concentration are presented in a table next to the blot. The Northern blot results confirm the degree of up-regulation
detected by semi-quantitative RT-PCR. Figure adapted from Kemppainen et al. [31] where the methodology used in these
experiments is detailed.
Based on these mRNA detection experiments in Laccaria wild type strain we could conclude that L. bicolor fHANTAC is under a concert regulation and each of the three genes respond to N availability in a similar manner. This
regulation is characterized by:
(1) A dominant repression in presence of ammonium
(2) A detectable basal transcript level under repressive conditions
(3) Independence from nitrate induction
(4) A strong and fast N starvation linked activation
(5) A strong expression in the presence of variable organic N sources, these including also L-glutamine.
Nitrate reductase has been proposed to be involved in transcriptional regulation of other nitrate assimilation genes in
fungi and algae [32-37]. Moreover, an A. nidulans nr-mutant strain shows increased transcription and elevated nitrate
transporter protein level [38]. However, silencing of Lbnr did not affect the expression levels of other cluster genes
under any of the N conditions tested (Fig. 3B). This indicates that high cellular activity of nitrate reductase is not the
key controller of Laccaria fHANT-AC cluster expression. This conclusion is further supported by recent RNA silencing
studies of Lbnrt which are pointing towards the nitrate transporter protein as a key regulator of transcription of the other
cluster genes (Kemppainen & Pardo, unpublished results).
The high transcript levels of fHANT-AC genes on variable organic N compounds tested (urea, L-glutamine, Lasparagine) revealed the high functional flexibility of this N utilization pathway in Laccaria. They also suggested
simultaneous organic and inorganic N source utilization by the fungus. However, the elevated cellular transcript levels
on organic N cannot be taken as a direct proof that the fungus would indeed take up and utilize nitrate in presence of
organic N. Other mechanisms, such as posttranslational modifications, could result in lack of transporter activity despite
the high mRNA levels, in the presence of preferred N sources. Whether Laccaria simultaneously incorporates nitrate
when growing on L-glutamine is especially intriguing. Glutamine is clearly a preferred N source by Laccaria as the
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biomass production on this compound is far superior to nitrate. Is the fungus indeed "wasting" energy for nitrate uptake
in the presence of such a good organic N source?
Due to coupled proton uptake the growth of fungi on nitrate causes an increase in the growth medium pH. This
alkalization effect can be camouflaged by production of organic acids by some fungal species but culturing of L. bicolor
on nitrate results in clear increase of medium pH [39, 40]. We used this growth medium alkalization effect as an
indirect indicator of active nitrate metabolism of the fungus. Moreover, in order to discriminate for the nitrate growth
specific pH effect we included the Lbnr-silenced strain pHg/NITRSPL43 [23] as a control in this study. This strain
shows minimum growth on nitrate but has unaffected growth capacity on other N sources, such as L-glutamine. Studies
with nitrate reductase knock-out mutants of ascomycetes have demonstrated that the function of nitrate uptake in fungi
depends on unaffected nitrate reductase activity, while in plants nitrate uptake is independent of nitrate reductase. This
fundamental difference between these two kingdoms has been suggested to be linked to the weak fungal capacity to
storage nitrate in vacuoles and hence maintain a low cytosolic nitrate concentration [41]. Also a nitrate reductase mutant
of H. cylindrosporum has been reported not to absorb nitrate [8, 42] suggesting that nitrate reductase activity could be
essential for the function of nitrate uptake system in basidiomycetes like in ascomycetes. Therefore, the Laccaria Lbnrsilenced strain was, despite its high Lbnrt-transcript levels, expected not to show significant nitrate uptake and
consequently not to cause pH alkalization when this nutrient was available. As a control this strain was expected to
allow linking the possible medium pH effects of the wild type Laccaria more firmly to the N utilization pathway of the
fungus, especially when fed with nitrate and organic N simultaneously.
The simultaneous exposure of Laccaria wild type strain to nitrate and L-glutamine caused a clear elevation of the
growth medium pH (Fig. 5).
5.96
6
5.75
5
3.74
pH
4
3
3.59 3.62
3.64
2.68 2.63
2.66 2.65
2
wild type
Lbnr-silenced strain
1
0
NH4
NO3
NH4/NO3
Gln
Gln /NO3
Fig. 5. Effect of fungal growth on different N sources on medium pH. Ammonium pre-grown fungal colonies of dikaryotic wild type
(dark bars) and Lbnr-silenced strain pHg/NITRSPL43 (white bars) were incubated on variable N sources for 5 days: 4 mM
ammonium (NH4); 4 mM nitrate (NO3); 4 mM ammonium + 4 mM nitrate (NH4/NO3); 8 mM L-glutamine (Gln); and 8 mM Lglutamine + 4 mM nitrate (Gln/NO3). Ammonium was added as ammonium sulphate and nitrate as potassium nitrate. Mean values of
triplicate cultures are presented with the standard deviation. The initial pH of the growth medium was adjusted to 5. The graphic is
adapted from Kemppainen et al. [31] where the pH growth assay is explained in more detail.
This result strongly suggests, as already proposed by the high fHANT-AC transcript levels, that the fungal nitrate
uptake system is not only transcribed but also functioning under nitrate/glutamine conditions. Besides the organic N
also the inorganic N source is utilized. Similar pH elevation was detected also during simultaneous feeding of Laccaria
with nitrate and urea or L-asparagine (data not shown). Also these results are in concordance with high fHANT-AC
transcript levels on the given organic N sources. No medium pH increase was however detected on nitrate or on
nitrate/L-glutamine media in the case of the control the Lbnr-silenced strain. (Fig. 5). This links the detected medium
pH effect of the wild type on nitrate to active nitrate utilization in the presence of L-glutamine. These results also
indicate that in basidiomycetes such as Laccaria, equally to ascomycete fungi, high cytosolic NR activity is required for
massive nitrate uptake to take place.
The control of N metabolism in ascomycete fungi is a highly complex regulatory network of simultaneously acting
global positive and negative transcription factors and pathway specific inducers. Even though some general N
metabolism control mechanisms seem to be conserved among the studied ascomycetes, significant regulatory
differences exist between Saccharomyces and filamentous model ascomycetes [25]. The control of the transcription
factors, which further control transcription of genes responsible for different N utilization pathways, is demonstrated to
be complex and can occur at different cellular levels depending on species. These proteins can be controlled
transcriptionally, via mRNA stabilization/destabilization effects and at the level of their cellular localization. Also the N
pathway specific structural genes, such as fHANT-AC, can be regulated both transcriptioanally and at mRNA stability
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level. Figure 6 summarizes the current knowledge of the main regulatory pathways responsible for transcriptional
repression and activation of nitrate utilization genes in the saprotrophic filamentous ascomycetes Asperguillus nidulans
and Neurospora. crassa. This control is mediated via global AreA/Nit2-type N regulator GATA-factors and it also
strictly depends on the activity NirA/Nit4-type pathway specific inducers. Only the positive acting transcriptional
factors are illustrated for simplifying the figure.
A
B
high NH4
high
NH4
high Gln
I
high Gln
No/low
cytosolic
NO3
*
Effect on
AreA/Nit2- type
global GATAfactor
Reduced transcription
and/or Gln-mediated
mRNA destabilization
II
*
Effect on NirA/Nit4type pathway
specific
inducers
Cytosolic
localization
Possible destabilizing
effects of Gln on cellular
fHANT-AC transcripts
Low protein level
and predominantly
cytosolic
localization
fHANT-AC
expression
not activated
nucleus
low
NH4
N starvation
low
NH4
Possible weak
NO3 uptake
by fHANT-AC
basal activity
low Gln
III
high/low
NO3
NO3 uptake
not active
Effect on
AreA/Nit2- type
global GATAfactor
low Gln
Cytosolic
NO3
accumulation
NO3
IV
Effect on NirA/Nit4type pathway
specific
inducers
high
Nuclear
NO3
Increased transcription localization
and/or increased
Possible
mRNA stability
stabilizing
effects of
NO3 on
High protein level
fHANT-AC
and
transcripts
nuclear localization
Weak AreAdependent
N starvation
uptake
activation
high NO3
Strong
induced
NO3 uptake
V
Promoter binding of
GATA-factor
promoter binding pathway specific inducer
synergistic
action
Strong NO3-induced fHANT-AC
activation
Fig. 6. Efficient nitrate utilization by saprotrophic ascomycetes depends on nuclear localization and promoter binding of a positively
acting global N regulator GATA-factor and a pathway specific inducer. (A) Repression of fHANT-AC. (I) The presence of preferred
N sources, ammonium (NH4) and L-glutamine (Gln), is believed to be sensed in both cases via high cytosolic Gln concentration.
This leads to reduced nuclear localization of the global positive regulator GATA-factor and transcription of nitrate utilization genes is
not activated. Glutamine can also have direct mRNA destabilization effects on fHANT-AC gene mRNAs. (II) The transcription of
fHANT-AC genes depends also on the nuclear localization and promoter binding of the NirA/Nit4-type pathway specific inducer.
Lack/low cytosolic concentration of nitrate (NO3) results in lack of activity of this pathway specific inducer due to its reduced
nuclear localization. *A direct destabilizing role of ammonium on AreA and/or NirA has also recently been proposed.(B) Nitrate
induction of fHANT-AC. (III) A low concentration or complete absence of repressing N sources and presence of nitrate are needed
for fHANT-AC activation. Low cytosolic Gln level is believed to cause nuclear accumulation and promoter binding of the global
regulator GATA-factor. (IV) This starvation response allows a weak activation of nitrate utilization genes which initiates cytosolic
NO3 accumulation by nitrate transporter activity. This cytosolic NO3 further activates the pathway specific inducer causing its
nuclear localization. (V) Promoter binding of the GATA-factor and the inducer (efficient promoter binding of the inducer requires
presence of the GATA-factor) fully activates fHANT-AC resulting in active nitrate growth. Besides controlling the cellular protein
localization L-glutamine has also direct effects on GATA-factor and fHANT-AC transcripts in A. nidulans (but not in N. crassa)
which lead to mRNA destabilization via deadenylation of their poly-A tail. Similarly, NO3 directly stabilizes nr and nir (but not nrt)
transcripts in A. nidulans. This effect is dominant and counteracts the glutamine effect assuring that cytosolic nitrate becomes fully
metabolized when the nutritional status of the fungus is changing from nitrate growth to the use of a repressing N source, or when the
cellular N status is high and nitrate uptake needs to be reduced. Also several other regulatory proteins participate in control of
ascomycete nitrate utilization, especially regulating the activity of the positive GATA-factor. These proteins are not included into the
illustration for simplification and also because basidiomycetes seem to lack their orthologs.
The molecular regulatory circuits of N source utilization are not yet studied in basidiomycetes. Therefore, detected
similarities and differences in regulation of Laccaria nitrate genes with respect to ascomycetes can lead at this point
only to rather speculative models. The study of the genomic sequence allows some deduction of possible molecular
mechanisms behind the observed regulation. However, the lack of homologous regulatory proteins in basidiomycete
genomes cannot be taken as a proof for lack of certain regulatory traits as these can be carried out by basidiomycete
specific proteins.
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The dominant ammonium repression of Laccaria fHANT-AC genes indicates that ascomycete AreA/NIT2-like positive
global N regulator should be active in the fungus. Laccaria genome encodes for one putative ortholog of these positive
acting GATA-factors (protein ID 313622) and this protein could be behind the observed fHANT-AC repression on
ammonium. No putative protein orthologs for negative acting GATA-factors of filamentous ascomycetes (AreB:
AF320976_3 or Asd4: AF319953_1) are present in Laccaria genome. Neither orthologs for AreA co-repressors (NmrA:
AAC39442.1 or Nmr1: CAC28734.1) could be detected in Laccaria genome. The same absence of negative acting N
regulatory GATA-factors and AreA co-repressors have been reported also for the basidiomycetes Ustilago maydis and
Cryptococcus neoformans [25] proposing that these proteins, and N regulatory circuits, could be ascomycete specific.
Hence, it is possible that N repression of Laccaria fHANT-AC is mediated solely via a positive acting GATA-factor
regulation. Interestingly A. nidulans and N. crassa nmrA and nmr-1 knock-out strains have partially lost N metabolite
repression of nitrate utilization genes [43, 44]. These mutants show elevated basal nitrate reductase transcript levels in
the presence of repressive N sources (ammonium or glutamine). This situation resembles the detected high basal
transcript level of Laccaria fHANT-AC on ammonium and further supports the absence of nmrA-type co-repressor
regulation in this fungus. Also the very recent data from A. nidulans negative acting GATA-factor, AreB, demonstrate
that the role of this protein as a nuclear counteractor of AreA promoter binding is relevant only during N limitation.
Under such conditions areB-mutants show increased N starvation activation of AreA-responsive genes [45]. This
situation resembles the strong N starvation activation of Laccaria fHANT-AC when growing in total absence of N or on
a weak N source such as urea and supports the lack of negative GATA-factor activity in the fungus. However, even
though these data are strongly pointing towards sole positively acting GATA-factor regulation of fHANT-AC in
Laccaria the existence of other basidiomycete-specific regulators of N metabolism can not be excluded.
The high transcript levels of fHANT-AC genes on nitrate but also under N starvation and especially during growth
on preferred organic N sources demonstrate that no nitrate induction is needed for activation of Laccaria nitrate
utilization pathway. Nitrogen starvation activation of some AreA-dependent pathways has been reported also in
Aspergillus and these are generally not dependent on specific induction either [46] supporting further the independence
of Laccaria fHANT-AC from nitrate induction. It is however intriguing that Laccaria genome encodes two putative
transcription factor proteins (ID 312485 and ID 317377) with certain homology (4e-15 and 7e-29) to the ascomycetelike nitrate pathway-specific inductor NIT4 of N. crassa (AAA33602.1). If these are active genes or pseudogenes is not
known. On the other hand, it is also possible that the proposed absence of both AreB- and NmrA-type regulators of
positive acting GATA-factor in Laccaria could create conditions where the activity of such a pathway specific inductor
of fHANT-AC is no longer relevant. Even though this inductor could participate in gene activation during nitrate
growth equivalent gene activation could also be reached under N limitation merely by the activity of a positive acting
GATA-factor. The possible role of these NIT4-like proteins in control of Laccaria fHANT-AC would be interesting to
study in future by RNA silencing techniques.
The same independence from nitrate induction combined with strong starvation activation is reported for Hebeloma
fHANT-AC [26-28]. Also physiological studies in the homobasidiomycete Rhizopogon roseolus indicate that no nitrate
induction is needed for maximum nitrate uptake under starvation [9] proposing that a starvation/lack of induction
response could be a conserved feature of basidiomycete fungi. However, the fact that the nitrate transporter of the
ectomycorrhizal ascomycete T. borchii, but interestingly not the nitrate reductase gene, responds to starvation and needs
no induction, shows that this mode of control is not restricted to basidiomycetes but could be more linked to the
symbiotic ECM lifestyle. The nitrate utilization activation without induction may however not be linked only to the
symbiotic fungus-plant interaction. Nitrogen starvation is reported to activate the nitrate utilization pathway in the
phytopathogen U. maydis as well [47] and this type of regulation may thus be characteristic also of the pathogenic
fungal lifestyle. Interestingly N starvation is known to promote both ectomycorrhization and pathogenic fungal-plant
interactions and several N starvation activated genes are shown to be relevant for pathogenesis and contribute to
virulence in Magnaporthe grisea [48]. Nitrate utilization is strongly repressed by ammonium as well as by L-glutamine
in saprotrophic model filamentous ascomycetes and in T. borchii. In fact, it has been demonstrated that intracellular Lglutamine is the true effector compound responsible for transcriptional repression of nitrate utilization genes observed
on ammonium in saprotrophic fungi [49]. L-glutamine acts via transcriptional and sub-cellular localization effects on
global N regulatory proteins, positively acting GATA-factors, as well as via direct transcript destabilization effects on
nitrate utilization genes in some species [25]. Therefore, the strong expression of Laccaria fHANT-AC genes in the
presence of organic N sources, and especially when growing on L-glutamine, was unexpected. This indicates that the
nitrate metabolic pathway could be repressed directly by ammonium and not via L-glutamine in Laccaria. Such a direct
repression of nitrate utilization by ammonium has not been described in fungi before. The transcript levels of Hebeloma
fHANT-AC genes on L-glutamine are unknown but these genes are relative active on other amino acid, L-glutamate
[27] suggesting that the lack of repression by L-glutamine could be a conserved feature among ECM
homobasidiomycetes. A similar direct ammonium repression, independent of L-glutamine, has also been shown for
some other GATA-factor regulated fungal genes [50, 51]. However, it has never before been linked to utilization of
nitrate and this regulatory pathway may be a crucial adaptation of ECM basidiomycetes to forest soils where the main
soil N pool consists of organic N. Figure 7 summarizes the Laccaria fHANT-AC regulation observed under repressing
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and de-repressing conditions. Our data support the global GATA-factor type regulation N repression, mediated directly
by ammonium, and nitrate independent activation of the whole cluster.
A
B
Repressing conditions: ammonium, ammonium/nitrate growth
(cytosolic NH4 concentration high)
De-repressing conditions: starvation, urea, nitrate, glutamine
growth
(cytosolic NH4 concentration low)
high Gln
Urea
high NH4
high
NH4
Transcriptional
repression
low
NH4
high Gln
Effects on LbGATA
and/or
Direct effect
on cellular
localization
Reduced
LbGATA
protein level
high Gln
low
NH4
NO3
Total N
starvation
No regulatory
effects
basal
uptake
NO3
Effects on LbGATA
and/or
Transcriptional
activation
NO3
Direct effect
on cellular
localization
No
regulatory
effects
maximum
NO3 uptake
capacity
NO3
Increased
LbGATA
protein level
Reduced nuclear
localization
Increased nuclear
localization
Basal fHANT-AC
expression level
fHANT-AC
expression activated
Fig. 7. Laccaria bicolor fHANT-AC gene regulation working model under (A) repressing and (B) de-repressing N conditions. The
nitrate utilization gene regulation seems to depend purely on activity of the positive acting global N regulator GATA-factor
(LbGATA). The repressing condition seems to be directly linked to high cytosolic concentration of ammonium (and not L-glutamine)
which leads to reduced GATA-factor activity in nucleus. This effect can be reached via variable and not yet confirmed regulatory
pathways such as transcriptional repression and/or cellular protein localization effects. Under repressing conditions the relative high
basal fHANT-AC transcription level would allow low uptake of nitrate. Different de-repressing conditions (organic N, nitrate,
starvation), would result in low cytosolic ammonium concentration producing high nuclear activity of LbGATA. This fully activates
fHANT-AC expression. Apparently no nitrate pathway-specific inductor is needed for maximum Laccaria fHANT-AC activity. This
allows simultaneous high nitrate uptake in the presence of organic N (including also L-glutamine) or at low concentration of
repressive N source.
The Laccaria fHANT-AC regulation resembles the one described in H. cylindrosporum indicating that it could be
evolutionary conserved. The same independence from nitrate induction, strong and fast N starvation response and an
important basal expression level of the whole cluster can be interpreted as an adaptation of a C sufficient mycorrhizal
fungus to optimize its maximum N uptake from soil. In boreal and temperate forests soils preferred N sources are
suggested to show patchy availability and to be under hard microbial competition. This condition is accompanied with
low and/or highly fluctuating concentrations of less preferred N sources such as nitrate. High basal fHANT-AC
expression can contribute to simultaneous nitrate and ammonium utilization. This extraordinary capacity has been
demonstrated for H. cylindrosporum and R. roseolus [10, 42] and due to high basal fHANT-AC transcription it may be
present in Laccaria as well. Also N starvation and lack of nitrate induction makes these mycorrhizal fungi faster than
their saprotrophic competitors in capturing secondary N sources, such as nitrate, when present in forest soil. More
importantly, tree roots show nitrate induction-dependent nitrate uptake [9, 10, 52-54]. This plant response is rather slow
and trees cannot respond to rapidly changing nitrate conditions in soil. However, when mycorrhized, tree roots show the
induction-independent profile of the fungal nitrate uptake system, indicating its dominance in symbiotic organs [9, 10].
Therefore, the ECM association clearly offers the host tree an increased access to nitrate in soil especially if present at
low concentrations and with temporal and spatial fluctuations.
The induction-independent fHANT-AC regulatory pattern of Laccaria is clearly of high C costs. However, such a
regulation could have evolved in fungi whose main concern is not the C acquisition from soil but instead have
especially high N demand. Both of these features are consistent with the ECM lifestyle where the fungus fulfills its C
need from host trees and faces, not only its own, but also the N demand of its symbiotic partner. Moreover, even though
possible to cultivate like saprotrophs under laboratory conditions the true capacity of symbiotic ECM fungi to compete
314
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A. Méndez-Vilas (Ed.)
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for soil C compounds with soil saprotrophs in nature has been questioned. The ECM fungi also typically inhabit soil
cores low in C compounds of positive energetic value. This further supports the idea of their high dependency on
symbiosis derived C [55]. Recently, the weakness of ligno- and cellulytic enzyme repertoire of ECM fungi and
consequently the low potential to liberate C from soil plant-derived organic resources has received confirmation from
analysis of full genome sequences of these organisms [22, 56, 57]. Laccaria is hence not primary competing for soil C
but for N (and other macro- and micronutrients) with soil saprotrophs. Furthermore, establishment of ECM structures
seems to be controlled by the host according to the fungal capacity to offer the plant N compounds [23]. Therefore,
regulatory adaptations maximizing N acquisition from soil could be expected to be competitively highly beneficial for
ECM fungi. As the high N acquisition efficiency is directly linked to establishment of symbiosis, which on the other
hand ensures a steady C supply from the host, also adaptations of high energy costs which maximize N sequestration
can be proposed to be evolutionary sustainable.
The nitrate transporter of the ectomycorrhizal ascomycete T. borchii shows lack of nitrate induction and its transcript
level increases during starvation while the nitrate reductase of the fungus depends on induction [18, 29]. Even though
the whole Tuber fHANT-AC does not have induction-free activation, the reactivity of the transporter to starvation and
to low nitrate concentrations in combination with the basal transcript levels of nitrate reductase optimize nitrate
utilization by Tuber in a similar manner than the simultaneous whole cluster activation in Laccaria and Hebeloma. This
different regulatory solution leading to the same functional outcome to optimize N sequestration from soil could reflect
different evolutionary histories of asco- and basidiomycete ECM fungi. The detected similarities may however also
represent an ancient adaptation linked to the appearance of the ectomycorrhizal symbiosis. Even though today
considered of minor importance it is possible that when the ECM interaction appeared nitrate could have had a more
predominant role as N source in early forests soils as it has today in agricultural systems [58]. The optimization of use
of this N source might have given an important competitive advantage to mycorrhizal fungi and hence influenced
evolution of this type of symbiotic interaction.
The lack of L-glutamine repression of fHANT-AC and the simultaneous uptake of organic N and nitrate in Laccaria
are highly interesting. This extraordinary regulation among the fungi studied this far seems like a perfect adaptation of
an ECM fungus to boreal and temperate soils rich in organic N sources and with fluctuating concentrations of nitrate.
Free amino acids are important constituents of the N pool in boreal forest soils. As the total amount of amino acids is
demonstrated to vary between different forests types and succession stages the composition of this pool is generally
constant and dominated by L-glutamine [59]. Free L-glutamine can therefore be considered one of the essential organic
N sources available for ECM fungi in boreal soils. On the other hand, such regulation is not present in Tuber where both
ammonium and L-glutamine repress the fHANT-AC. This difference may reflect the adaptation of ECM fungi to
different soil types during evolution. At the present ECM basidiomycetes inhabit predominantly higher soil horizons
rich in organic N while ECM ascomycetes are more abundant in mineral horizons or are pioneer species during early
forest succession with preference for higher soil pH and lower organic N conditions [60, 61]. The maximum uptake of
inorganic N may thus be the most relevant competitive feature of ectomycorrhizal ascomycetes in their natural soil
niche while strong simultaneous capacity for the use of organic and inorganic N has been of minor importance.
However, competition for both organic and inorganic N compounds must exist among micro-organisms in soils rich in
organic N. Simultaneous capacity to utilize both of these sources could have given an important adaptive benefit to
organisms showing such a metabolic flexibility, these including ECM basidiomycetes.
The lack of fHANT-AC repression by L-glutamine, besides allowing optimization of the use of inorganic and
organic N from soil, might also represent another important adaptation of Laccaria to other fundamental aspect of the
ectomycorrhizal lifestyle. Free cellular glutamine has been identified as a major sink for absorbed N in extraradical
mycelia of basidiomycete ECM fungi. This amino acid is most likely one of the dominant forms in which N is
translocated in the mycelium towards the symbiotic organs and probably also transferred across the symbiotic interface
to the plant [6]. Taking into account this proposed fundamental role of L-glutamine in functions of ECM symbiosis the
identified L-glutamine independent fHANT-AC regulation seems quite logical. It allows Laccaria to maintain nitrate
uptake optimizing thus N capture from soil independently of the high intracellular concentration of L-glutamine (or
other amino acids and organic N forms such as urea) used for translocation of N towards the plant host. As Tuber
fHANT-AC is repressed by L-glutamine this may also indicate that other amino acids such as L-asparagine, known to
be a neutral N source for fHANTAC in saprotrophic ascomycetes as well, could have evolved to play more dominant
role as N translocation compounds in ascomycete ECM fungi circumventing the intracellular fHANT-AC repression by
high cytosolic concentration of N compounds destined to the host plant. Whether this L-glutamine free regulation of
fHANT-AC is a homobasidiomycete specific adaptation to the ectomycorrhizal lifestyle or a specific feature of L.
bicolor metabolism needs to be further investigated. Interestingly, also Laccaria sulfur uptake lacks the typical fungal
feedback inhibition response [62]. In future the L-glutamine response of nitrate utilization in Hebeloma spp. and
saprotrophic basidiomycete species such as Agaricus bisporus and Coprinus cinereus would allow an interesting
comparison between saprotrophic and symbiotic lifestyles in respect to nitrate acquisition. Figure 8 shows the working
hypothesis of Laccaria fHANT-AC regulation in comparison with nitrate utilization of saprotrophic soil ascomycetes.
The figure highlights the possible ecological significance of the herein described Laccaria regulation pattern for the
ECM lifestyle.
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These N sources
do not repress
fHANT-AC
Simultaneous
uptake
urea
Efficient NO3
uptake
NO3
NO3
All three fHANT-AC
genes are activated
without NO3 induction in
absence of NH4
NO3
NO3
Efficient NO3
uptake
NO3
High expression
of fHANT-AC
Gln
Gln
NH4 represses fHANT-AC
and the basal expression level is very low
No significant simultaneous NO3 uptake
NH4
NH4
fHANT-AC repression on
ammonium is mediated by Gln
Gln
Gln
No simultaneous
uptake
Gln
Gln
Growth on Gln causes
repression of fHANT-AC
NO3
Gln
fHANT-AC repression is
mediated by Gln
Growth on urea does not
repress fHANT-AC but does
not induce the cluster either
(neutral N sources)
Potential
simultaneous
weak NO3 uptake* urea
NO3
Presence of neutral
N source and NO3
can lead to partial activation
of fHANT-AC
Efficient NO3 NO3
uptake
High expression
of fHANT-AC
NO3
NO3
NO3
N-starvation
dependent
weak fHANT-AC
activation **
NO3
Potential
weak NO3 uptake
Limited capture of
sparse or temporal
NO3 sources
independently
of NO3 induction
Gln Gln
Gln does not repress
fHANT-AC
Gln
NO3
NO3
High expression
of fHANT-AC
N starvation
Maximum expression
of fHANT-AC genes
depends strictly on NO3
induction and absence
of repressing N source
fHANT-AC repression
directly by ammonium
Gln
Simultaneous
Gln
uptake
NO3
NO3
High expression of fHANT-AC High expression of fHANT-AC
in absence of NO3
when growing on Gln
(abundant ON in boreal
soils)
High cytosolic concentration of aa, especially Gln destined for N translocation toward symbiotic organs
NH4
potential simultaneous use
of soil NH4 and NO3
Growth on Gln
(one of the dominant
free aa in boreal soils)
(NO3 rich nutrient
patches or increased
temporal concentration
as result of rainfall or
snow melts)
Fast capture and
maximized use of
NO3 in temporal or
spatial NO3
gradients
NH4
NO3
NH4 NH4
NO3
saprotrophic
ascomycete
Laccaria
Ammonium represses fHANT-AC
but the basal expression level is
relative high
(dominant ION in boreal soils)
Growth on NH4
Growth on organic
N such as urea
Growth on NO3
Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A.
Méndez-Vilas (Ed.)
_______________________________________________________________________________________
Fig. 8. Laccaria fHANT-AC N source regulation and the possible ecological significance of it for the ECM lifestyle. The detected
regulatory flexibility of fHANT-AC confers the fungus a high potential to obtain nitrate from forest soil under variable N regimens
when compared to saprotrophic ascomycetes. This regulation pattern proposes a clear superiority of the Laccaria nitrate acquisition
system especially in forest soils with elevated concentrations of organic N and low or fluctuating nitrate availability. ON: organic
nitrogen; ION: inorganic nitrogen. *10 % of the fully induced nitrate growth expression level of niiA (encodes nitrite reductase) has
been reported for urea+NO3 grown A. nidulans [49]. This potential low level simultaneous nitrate assimilation requires however
prolonged presence of nitrate. Low and temporal NO3 concentrations are therefore probably out of reach for saprotrophic
Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________
ascomycetes in the presence of urea. **Induction-independent N starvation activation of A. nidulans fHANT-AC represents less than
10 % of NO3-induced expression level [49]. Nevertheless, it can contribute to increased NO3 uptake under N starvation.
Acknowledgements. The Laccaria genome sequence data were produced by the US Department of Energy Joint
Genome Institute http://www.jgi.doe.gov/. The support by UNQ, CONICET and ANPCyT is gratefully acknowledged.
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