Research

Rapid report
NSP1 is a component of the Myc signaling pathway

Authors for correspondence: Pierre-Marc Delaux1, Guillaume B
ecard2,3 and Jean-Philippe Combier2,3
Jean-Philippe Combier 1
Department of Agronomy, University of Wisconsin Madison, 219 Moore Hall, 1575 Linden Drive, Madison, WI 53706, USA;
Tel: +33 (0)5 34 32 38 11
2
Email: combier@lrsv.ups-tlse.fr Laboratoire de Recherche en Sciences V
eg
etales, UMR5546, Universit
e de Toulouse, 31326, Castanet-Tolosan CEDEX, France;
3
CNRS, UMR5546, 31326, Castanet-Tolosan CEDEX, France
Guillaume B
ecard
Tel: +33 (0)5 34 32 38 20
Email: becard@lrsv.ups-tlse.fr
Received: 3 April 2013
Accepted: 23 April 2013

New Phytologist (2013) 199: 59–65
doi: 10.1111/nph.12340
Key words: AM symbiosis, GRAS
transcription factor, Medicago truncatula,
NSP1, SYM pathway.
Summary
 Nodulation and arbuscular mycorrhization require the activation of plant host symbiotic
programs by Nod factors, and Myc-LCOs and COs, respectively. The pathways involved in the
perception and downstream signaling of these signals include common and distinct components.
Among the distinct components, NSP1, a GRAS transcription factor, has been considered for
years to be specifically involved in nodulation.
 Here, we analyzed the degree of conservation of the NSP1 sequence in arbuscular mycorrhizal
(AM) host and non-AM host plants and carefully examined the ability of Medicago truncatula
nsp1 mutants to respond to Myc-LCOs and to be colonized by an arbuscular mycorrhizal fungus.
 In AM-host plants, the selection pressure on NSP1 is stronger than in non-AM host ones. The
response to Myc-LCOs and the frequency of mycorrhizal colonization are significantly reduced
in the nsp1 mutants.
 Our results reveal that NSP1, previously described for its involvement in the Nod factor
signaling pathway, is also involved in the Myc-LCO signaling pathway. They bring additional
evidence on the evolutionary relatedness between nodulation and mycorrhization.

legume nodulation by Nod factors (Oldroyd et al., 2009).

Introduction
The arbuscular mycorrhizal (AM) symbiosis plays a key role in the
nutrient uptake of a majority of land plants and could potentially be
an important component of low input sustainable agriculture. The
establishment of this symbiosis involves signal molecules released
by both partners. The plant produces strigolactones, which are an
ancient class of plant hormones that are also required for AM
symbiosis (Gomez-Roldan et al., 2008; Umehara et al., 2008;
Delaux et al., 2012). At extremely low concentrations strigolac-
tones stimulate AM fungal metabolism, germination and pre-
symbiotic growth (Akiyama et al., 2005; Besserer et al., 2006,
2008). The fungus releases lipochitooligosaccharides (Myc-LCOs)
and short oligosaccharides (COs), which prepare the plant for
symbiosis by activating a symbiotic (Sym) signaling pathway and
promoting lateral root formation (Maillet et al., 2011; Genre et al.,
2013). The Sym pathway is also involved in the activation of
Following these early signaling events, the fungus contacts the
plant root epidermis, develops hyphopodia and colonizes the root
inter- and intra-cellularly. Finally, it forms highly branched
structures, called arbuscules, in cortical cells where the nutrient
exchanges between the two partners take place.
To date, only two transcription factors of the Sym pathway,
RAM1 and NSP2, have been identified as being involved in the
Myc signaling pathway (Maillet et al., 2011; Gobbato et al., 2012;
Lauressergues et al., 2012). NSP2 is a GRAS transcription factor
also involved in the Nod signaling pathway. It interacts with NSP1
and promotes the activation of symbiotic marker genes, such as
ENOD11 (Hirsch et al., 2009). NSP1 is another GRAS transcrip-
tion factor which has been described as a specific component of the
Nod signaling pathway (Catoira et al., 2000; Smit et al., 2005). It
has been proposed that RAM1 could play a role in mycorrhization
similar to that of NSP1 during nodulation by interacting with

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NSP2 and activating symbiotic genes (Gobbato et al., 2012).
However, the fact that nsp1 mutant is affected in the synthesis of
strigolactones (Liu et al., 2011) strongly suggests that NSP1 could
also be involved in the early steps of the establishment of the AM
symbiosis.
To test this hypothesis, we performed a phylogenetic study of
NSP1 in plants and carefully examined the mycorrhizal phenotype
of the Medicago truncatula nsp1 mutant. We found that NSP1 is
well-conserved in angiosperms across the AM host plants and is
more divergent in the non-AM host plants of the Brassicaceae
family. Also, NSP1 is required for optimum mycorrhization and is
involved in the Myc signaling pathway. Taken together, our results
show that NSP1 is not specific to the Nod signaling pathway. They
bring additional evidence on the evolutionary relatedness between
nodulation and mycorrhization.
Materials and Methods
Phylogenetic analyses
The NSP1 sequences of angiosperms, of the moss Physcomitrella
patens and of the lycophyte Selaginella moellendorffii were collected
by BLASTp against the GenBank nr database (Supporting
Information Table S1). The gymnosperm sequences were obtained
by tBLASTn on the available ESTs. The Marchantia polymorpha
sequence was identified using the http://www.one-KP.com dataset.
In addition, the charophyte databases available on GenBank were
also screened. The collected sequences were aligned using MAFFT
(http://mafft.cbrc.jp/alignment/server/). The phylogenetic tree
was generated by maximum-likelihood using MEGA5 as previ-
ously described (Delaux et al., 2012). AtSHR and AtSCL32 and
their orthologs in Gymnosperms, S. moellendorffii and P. patens
were used as outgroups (Engstrom, 2011). The average pairwise
distances of the LHR domains were calculated between the
Brassicaceae, AM host dicots and Carica papaya using MEGA5
after gap removal (Tamura et al., 2011).
Estimation of x parameter
The codon sequences were aligned using MUSCLE (codon) via
MEGA5. The gaps were automatically removed. The ratio of non-
synonymous substitutions per non-synonymous site (dN) to
synonymous substitutions per synonymous site (dS), the x
parameter, was calculated using MEGA5. To determine the x
parameter in each branch of the NSP1 gene tree, a branch-specific
approach was conducted using codeml (Yang, 1998). The free-ratio
and one-ratio models were generated as proposed by Yang (1998).
To determine the most likely model, twice the difference between
the log likelihood of each model was compared by Chi-square. For
the Chi-square test, df = number of branches in the free-ratio
model – 1.
Biological materials
Rhizophagus irregularis sterile spores were purchased from Agro-
nutrition (Carbonne, France). Medicago truncatula A17 seeds were
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surface-sterilized and germinated on agar plates in the dark for 5 d
at 4°C. The nsp1 B85 and C54 (Smit et al., 2005), nsp2-1 (Kal
o
et al., 2005), ram1-1 (Gobbato et al., 2012) and ipd3-1 (Horv
ath
et al., 2011) alleles were used. The plants were then cultivated in
200 ml pots on Oil-Dri US-Special Substrate (Damolin, Den-
mark) for 6–8 wk in a growth chamber and watered every 2 d with
Long Ashton medium containing 7.5 lM phosphate (Lauresser-
gues et al., 2012). For inoculation with R. irregularis, we used either
400 or 1200 spores per liter of substrate.
Bioactive chemicals
Myc-LCOs used in this study are an equimolar mix of the four
(Syn)Myc-LCOs: LCO-IV(C16 : 0), LCO-IV(C16 : 0,S), LCO-
IV(C18 : 1D9Z) and LCO-IV(C18 : 1D9Z,S) described in Maillet
et al. (2011) at a final concentration of 10 8 M. The plants treated
with Myc-LCOs (12 h) were cultivated on Fahraeus medium as
described in Combier et al. (2008), containing a low concentration
of phosphate (7.5 lM) and a high concentration of nitrogen
(10 mM NH4NO3). Some plants were grown in the same
conditions but treated with Nod factors of Sinorhizobium meliloti
(Lerouge et al., 1990) at a final concentration of 10 8 M. COs with
four residues (CO4) (Genre et al., 2013) were used at a final
concentration of 10 8 M in the same conditions as Myc-LCOs.
Quantitative reverse transcription polymerase chain reaction
(qRT-PCR) analyses
RNAs of Medicago truncatula roots were extracted using the
RNeasy Plant Mini Kit (Qiagen). The reverse transcription (RT)
was performed using the SuperScript II Reverse Transcriptase
(Invitrogen) on 500 ng of total RNA. For the experiment
comparing the mycorrhizal and non-mycorrhizal roots, six inde-
pendent plants were analyzed per condition. For the experiments
with molecule treatments, ten independent plants were pooled per
replicate. Three replicates (n = 3) were performed with two
technical replicates each. Each experiment has been repeated two
to three times. The quantitative polymerase chain reaction (qPCR)
amplifications were conducted on a Roche LightCycler 480 System
(Roche Diagnostics) (see Lauressergues et al., 2012).
Statistical analyses
The mean values of relative gene expression or mycorrhization rates
were compared by using the Kruskal–Wallis test and, when
significant, a pairwise comparison was made using the non-
parametric Mann–Whitney test. The error bars represent the
standard error of the mean (SEM). The stars indicate significant
differences (P < 0.05).
Results and Discussion
NSP1 is divergent in non-AM host species
To address the question of the involvement of NSP1 in AM
symbiosis, we first looked at its conservation in the green lineage by
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in silico analysis. Indeed, most of the AM symbiosis-related genes
are well conserved in land plants and poorly conserved or absent in
the Brassicaceae, a non-AM host family (Delaux et al., 2013). We
screened the available genomic data of land plants and chlorophyte
green algae, the transcriptomic data of Gymnosperms and the
recently released transcriptomic sequences of charophyte green
algae (Wodniok et al., 2011; Timme et al., 2012) for the presence
of NSP1. We identified GRAS domain-containing proteins in land
plants and charophyte green algae datasets but not in the
chlorophyte sequences. To assess their identity, the collected
sequences were aligned and a maximum-likelihood tree was
resolved. As previously determined by Engstrom (2011), we found
clear orthologs of NSP1 in all the available angiosperm genomes
and in the genomes of the Lycophyte Selaginella moellendorffii and
of the moss Physcomitrella patens (Fig. 1a). In addition, we found
orthologs of NSP1 in the liverwort Marchantia polymorpha and in
the Gymnosperm Pinus taeda (Fig. 1a). By contrast, the GRAS
domain-containing proteins found in the charophyte green algae
belonged to other clades of the GRAS family (data not shown).
To determine whether NSP1 is under different evolutionary
constraints in AM host and non-AM host species, we calculated
several x values. The x value reflects the selection constraints
(Yang, 1998). We applied the pairwise comparison approach and
calculated the x values within the symbiotic dicots and the
Brassicaceae for NSP1 and four other GRAS transcription factors:
SCARECROW (Di Laurenzio et al., 1996), SHORTROOT (Benfey
et al., 1993), PAT1 (Bolle et al., 2000) and NSP2 (Kal
o et al.,
2005). We found that, for NSP1 and NSP2, the x value of the
Brassicaceae is significantly (P < 0.001) higher than that of the AM
host dicots (Fig. 1b). By contrast, the x parameters of
SCARECROW and PAT1 are higher (P < 0.001) in the symbiotic
dicots than in the Brassicaceae (Fig. 1b). For SHORTROOT, the x
parameters are similar in both groups (Fig. 1b). Then, to take into
account the evolutionary rate of the most common ancestor, we
performed a branch-specific analysis on NSP1. In this approach,
two models are compared: one which assumes the same x for all the
branches (one-ratio) and the other which assumes a different x
parameter for each branch in the tree (free-ratio). We found that the
free-ratio model fits the data significantly better than the one-ratio
model (P < 0.001). In this model, the branch of the tree at the base
of the Brassicaceae lineage displays a x value (x = 0.5165) higher
than the x value of the symbiotic dicot branch (x = 0.1750). These
results obtained with the pairwise and the branch-specific
approaches suggest that a relaxed selection constraint occurred on
NSP1 in the non-AM host Brassicaceae family.
Proteins of the GRAS family contain five domains (LHRI,
VHIID, LHRII, PFYRE and SAW) which are well-conserved in
each sub-clade of the family (Engstrom, 2011). The two LHR
domains are required for the binding of NSP1 to the promoter of
MtENOD11, a gene upregulated during the early steps of AM and
root nodule symbioses (Boisson-Dernier et al., 2005; Hirsch et al.,
2009). To determine the impact of the relaxed selection constraint
on NSP1 in the Brassicaceae, the LHR domains of angiosperms
were aligned. Then, we calculated the pairwise distance of the LHR
domains of the Brassicaceae and of the symbiotic dicots to the LHR
domains of Carica papaya. We used C. papaya as reference because
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it is a non-Brassicaceae member of the Brassicales order and can
form AM symbioses (Khade et al., 2010; Franzke et al., 2011).
While no significant differences were found for the LHRII domain,
the LHRI domain seems to be highly divergent in the Brassicaceae
compared to the other, AM host, dicots (Fig. 1c).
Most of the genes involved in the AM symbiosis have been lost in
the Brassicaceae (Delaux et al., 2013). However, some of them are
still present (DMI1 or NSP2 for instance) suggesting the occurrence
of alternative functions that led to their conservation. The purifying
selection acting on NSP1 and NSP2 (x values < 1) argue for this
hypothesis. The involvement of these two genes in the regulation of
the biosynthesis of the plant hormones strigolactones has been
recently demonstrated (Liu et al., 2011) and could explain their
conservation. By contrast, some domains, LHRI in NSP1 or the
mir171 h-binding domain in NSP2 (Lauressergues et al., 2012),
which are important for the symbiotic function, are more
divergent, likely due to a locally relaxed selection pressure.
Taken together, these analyses suggest that NSP1 appeared early
in the Embryophytes lineage and diverged in the Brassicaceae, an
evolutionary pattern shared with NSP2, another GRAS transcrip-
tion factor of the Sym pathway.
Mycorrhization and Myc-LCOs induce NSP1 expression
NSP1 is known to play a role during nodulation (Smit et al., 2005)
and the Medicago gene atlas (http://mtgea.noble.org/v2/) reveals
that NSP1 expression is induced during this symbiosis. Interest-
ingly, the Medicago gene atlas also reveals that NSP1 expression is
slightly induced in mycorrhizal roots. We first confirmed by qRT-
PCR that the expression of NSP1 is slightly (two-fold compared to
the control) but significantly induced during mycorrhization
(Fig. 2a). In parallel, by analogy with the role of NSP1 in the Nod
signaling pathway, we investigated whether the expression of NSP1
could be induced by a mixture of sulfated and non-sulfated Myc-
LCOs. We found, under conditions of high nitrogen fertilization
(10 mM NH4NO3), which inhibits the Nod signaling pathway
(Fig. S1), that NSP1 expression is induced by the Myc-LCO
treatment (Fig. 2b). Moreover, this induction is still present in the
ram1 and nsp2 mutants but not in the ipd3 mutant (Fig. 2b),
suggesting that the induction of NSP1 expression by Myc-LCOs
requires IPD3, which acts upstream of NSP1 (Horv
ath et al., 2011)
but is independent of RAM1 and NSP2. The high expression of
NSP1 in the non-treated ipd3 mutant was not expected but may
suggest the occurrence in the wild-type plant of some negative
feedback regulation by IPD3 of the expression of downstream
genes. Interestingly, we found no induction of NSP1 expression by
treatment with CO4 (Fig. S2), confirming that the symbiotic
signaling pathways induced by Myc-LCOs and COs are distinct
(Genre et al., 2013).
Mycorrhization is affected in the Mtnsp1 mutant
The screening for mutants affected in the AM symbiosis was
initially performed with strong fungal inoculants: a high number of
spores or fragments of mycorrhized roots. By using a smaller
inoculum (fewer spores), NSP2, which was initially identified as
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(a)
(b)
(c)
exclusively involved in nodulation (Oldroyd & Long, 2003), was
recently found to be also involved in the AM symbiosis and to be a
member of the common Sym pathway (Maillet et al., 2011). We
first inoculated the Mtnsp1 B85 allele and wild-type plants with a
high number of spores (1200 spores per liter). In this condition, the
mutant and wild-type plants were similarly colonized (Fig. 3a).
However, with a smaller fungal inoculum (400 spores per liter), the
mycorrhization of Mtnsp1 mutant plants was significantly reduced
compared to the wild-type after either 6, 8 or 12 wk post-
inoculation (Fig. 3a). The colonization rate in the mutant plants
was two- to three-fold lower than in the wild-type plants (Fig. 3a–
c), while in the colonized root sections the frequency and the
structure of the arbuscules were the same (Fig. 3b). In addition to
the B85 Mtnsp1 allele we tested the Mtnsp1 C54 allele (Smit et al.,
2005) for its ability to get colonized. As for B85 we found a reduced
colonization rate in Mtnsp1 C54 (Fig. 3c), confirming the
requirement of NSP1 for a proper AM symbiosis. These results
suggest that NSP1 has a role in mycorrhization, but the protein is
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Fig. 1 The evolution of NSP1 in land plants.
(a) Maximum-likelihood tree of NSP1. (b)
Pairwise estimation of x values in the
Brassicaceae family (Bra) and in the arbuscular
mycorrhizal (AM) host dicots (AM host) for
NSP1, NSP2, SHORTROOT (SHR), PAT1 and
SCARECROW (SCR). Asterisks indicate a
significant difference according to the Mann–
Whitney test (P < 0.001). (c) Pairwise distance
comparison of the LHR domains (I and II) of
Carica papaya to the same LHR domains of the
Brassicaceae family (Bra) and of the AM host
dicots (AM host). ***, Significant difference
according to Student’s t-test (P < 0.001); ns,
not significant. Accession numbers and species
names are available in Supporting Information
Table S1.
probably non-essential, suggesting an overlapping function,
perhaps with other GRAS proteins.
Nsp1 participates in the Myc-LCO signaling pathway
To further investigate the mycorrhizal phenotype of the Mtnsp1
mutant, we looked at whether NSP1 is involved in the Myc
signaling pathway. For this purpose we tested the effect of Myc-
LCOs on the expression of four symbiotic marker genes: ENOD11
(Boisson-Dernier et al., 2005) and three genes known to be up-
regulated in Medicago truncatula roots treated with Myc-LCOs
(Mtr.52092.1.S1_s_at, Mtr.37912.1.S1_at and Mtr.35524.1.S1_at,
which encode for a putative pectinesterase, a NADP-dependent
oxidoreductase, and a member of the subtilase family, respectively;
Maillet et al., 2011; Lauressergues et al., 2012). To make sure that
the Myc rather than the Nod signaling pathway was activated,
Mtnsp1 and wild-type plants were treated for 24 h with
non-sulfated Myc-LCOs. These LCOs are the most remote to
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(a) (a)

(b)

(b)

Fig. 2 NSP1 expression during arbuscular mycorrhizal (AM) symbiosis. (a)

Quantification of NSP1 expression by qRT-PCR in non-inoculated (MYC–)
and inoculated roots (MYC+) of Medicago truncatula with Rhizophagus
irregularis and cultivated for 9 wk (n = 6 independent plants). (b)
Quantification of NSP1 expression by qRT-PCR in wild-type (A17) and
mutant lines treated or not with 10 8 M of a mixture of sulfated and non-
sulfated Myc-LCOs (n = 3 independent pools of 10 plants). Error bars (c)
represent standard error of mean (SEM); *, significant difference between
the two treatments according to the Mann–Whitney test (P < 0.05).

the cognitive Nod factors of Medicago whose sulfate group is

essential to activate the nodulation process (Lerouge et al., 1990).
In wild-type plants expression of these genes was up-regulated by
non-sulfated Myc-LCOs, except for ENOD11, which was previ-
ously known to be non-inducible by these molecules (Maillet et al.,
2011) (Fig. 4). By contrast, treatment with non-sulfated Myc-
LCOs did not affect the expression level of these genes in Mtnsp1
mutant plants (Fig. 4). To strengthen this result, wild-type and
Mtnsp1 plants were grown on a medium supplemented with
10 mM NH4NO3. This concentration is sufficient to inhibit plant
response to Nod factors (Fig. S1). The plants were then treated for
12 h with a mixture of sulfated and non-sulfated Myc-LCOs. The
four genes were up-regulated in wild type plants after treatment
with Myc-LCOs (Fig. S3). By contrast, their induction was
abolished in the Mtnsp1 mutant plants, confirming that NSP1 is
required for the normal response of the plant to Myc-LCOs. Once
gene expression and plant responses activated by tetrameric and
pentameric chitin oligomers (CO4/CO5, Genre et al., 2013) will
be described, similar experiments can be performed to investigate
the role of NSP1 in CO-induced signaling pathway.
While RAM1 seems to be required for the penetration of the
fungus (Gobbato et al., 2012), NSP1, like NSP2 (Lauressergues
et al., 2012), seems to control the fungal colonization in the root. As
Fig. 3 Mycorrhizal phenotype of the Mtnsp1 mutant. (a) Percentage of
colonization in the roots of wild-type Medicago truncatula A17 and nsp1 B85
allele inoculated with 400 or 1200 spores per liter of Rhizophagus irregularis
and cultivated for 6, 8 and 12 wk (wpi, week post-inoculation). (b)
Quantification of mycorrhization in wild-type A17 and nsp1 B85 allele
12 wk after inoculation according to Trouvelot et al. (1986). ‘F’, the
frequency of colonization in the root system; ‘a’, the arbuscule abundance (in
percentage) in the colonized root sections. (c) Percentage of colonization in
the roots of wild type (A17) and two alleles of nsp1 mutants (B85 and C54),
8 wk after inoculation. Error bars represent standard error of mean (SEM).
*, Significant difference when compared with control according to the
Kruskal–Wallis test (n = 6, P < 0.05).
NSP1 and NSP2 activate genes involved in the biosynthesis of
strigolactones (Liu et al., 2011), this control might require local
adjustments of strigolactone content.

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(a) (b)
(c) (d)
When examining the lateral root formation of Medicago truncatula
in response to Myc-LCOs, Maillet et al. (2011) found that this
response was not dependent on NSP1. This result suggested that
NSP1 was not involved in the Myc signaling pathway. In light of
our results here, showing that the nsp1 mutants are affected in
mycorrhizal colonization and in the Myc-LCO induction of
mycorrhization marker genes, we hypothesize that the positive
induction by Myc-LCOs observed by Maillet et al. (2011) of both
mycorrhization and lateral root formation actually involves distinct
signaling pathways. Together, these results suggest that NSP1
would be necessary to transduce the Myc-LCO promotion of
fungal colonization but not the Myc-LCO stimulation of root
development. Finally, as we know that both RAM1 and NSP1
interact with NSP2 (Hirsch et al., 2009; Gobbato et al., 2012),
future work will have to further investigate the respective role of
each interaction during the fungal penetration of the root and, later,
during the fungal colonization and autoregulation of this coloni-
zation.
Acknowledgements
This work was carried out in the Laboratoire de Recherche en Sciences
V
e g
e tales in Toulouse, which belongs to the Laboratoire d’Excellence
entitled TULIP (ANR -10-LABX-41). The authors thank Fabi-
enne Maillet (LIPM, Toulouse, France) for providing mutant seeds
and Nod factors, Eric Samain, S
ebastien Fort and Sylvain Cottaz
(CERMAV, Grenoble, France) for providing the Myc-LCOs and
Kari L. Forshey for critical reading of the manuscript. The authors
would like to thank the anonymous reviewers for their valuable
comments and suggestions to improve the quality of the paper.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 The response of Medicago truncatula roots to Sinorhizobium
meliloti Nod factors on a low or high nitrogen medium.
Fig. S2 The response of Medicago truncatula roots to COs.
Fig. S3 The response of Medicago truncatula roots to Myc-LCOs
on a high nitrogen medium.
Table S1 Accession numbers and species names of genes used in
Fig. 1
Please note: Wiley-Blackwell are not responsible for the content or
functionality of any supporting information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
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