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An antimicrobial peptide essential for bacterial survival
in the nitrogen-fixing symbiosis
Minsoo Kim
a
, Yuhui Chen
b
, Jiejun Xi
c
, Christopher Waters
a
, Rujin Chen
b
, and Dong Wang
a,1
a
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003;
b
Plant Biology Division, The Samuel Roberts Noble
Foundation, Ardmore, OK 73401; and
c
Department of Grassland Science, College of Animal Science and Technology, Northwest A&F University, Yangling,
Shaanxi 712100, China
Edited by Frederick M. Ausubel, Harvard Medical School, Massachusetts General Hospital, Boston, MA, and approved October 26, 2015 (received for review
January 5, 2015)
In the nitrogen-fixing symbiosis between legume hosts and rhizobia,
the bacteria are engulfed by a plant cell membrane to become in-
tracellular organelles. In the model legume Medicago truncatula,
internalization and differentiation of Sinorhizobium (also known
as Ensifer)meliloti is a prerequisite for nitrogen fixation. The host
mechanisms that ensure the long-term survival of differentiating in-
tracellular bacteria (bacteroids) in this unusual association are un-
clear. The M. truncatula defective nitrogen fixation4 (dnf4)mutant
is unable to form a productive symbiosis, even though late symbiotic
marker genes are expressed in mutant nodules. We discovered that
in the dnf4 mutant, bacteroids can apparently differentiate, but they
fail to persist within host cells in the process. We found that the
DNF4 gene encodes NCR211, a member of the family of nodule-specific
cysteine-rich (NCR) peptides. The phenotype of dnf4 suggests that
NCR211 acts to promote the intracellular survival of differentiating
bacteroids. The greatest expression of DNF4 was observed in the nod-
ule interzone II-III, where bacteroids undergo differentiation. A trans-
lational fusion of DNF4 with GFP localizes to the peribacteroid space,
and synthetic NCR211 prevents free-living S. meliloti from forming
colonies, in contrast to mock controls, suggesting that DNF4 may in-
teract with bacteroids directly or indirectly for its function. Our find-
ings indicate that a successful symbiosis requires host effectors that
not only induce bacterial differentiation, but also that maintain in-
tracellular bacteroids during the host–symbiont interaction. The dis-
covery of NCR211 peptides that maintain bacterial survival inside
host cells has important implications for improving legume crops.
nitrogen-fixing symbiosis
|
Sinorhizobium meliloti
|
Medicago truncatula
|
legume
|
NCR antimicrobial peptides
Many legume plants satisfy their nitrogen needs by interacting
with nitrogen-fixing bacteria (rhizobia) to form a specialized
symbiotic organ, the root nodule. As rhizobia penetrate root hair
cells through invaginations of host membrane called infection
threads, the cortical cells underneath start dividing and eventually
build the nodule in which the invading rhizobia are internalized to
form intracellular organelles known as symbiosomes.
Among legumes, Medicago truncatula belongs to a group that
forms indeterminate nodules, where a meristem continuously pro-
duces new nodule cells. An indeterminate nodule can be divided
into four zones harboring different cell types (1). The meristem of
indeterminate nodules is located in the apical region (zone I),
which constantly supplies new cells to the nodule. These new cells
then become infected with rhizobia in zone II, where the rhizobial
cells are released from the infection thread into the host cytoplasm
to form symbiosomes. On release into the host cytoplasm, the
bacteroids are encapsulated with a host-derived membrane (the
peribacteroid membrane), blocking the rhizobia from directly con-
tacting the cytoplasm. The released bacteroids multiply and grad-
ually colonize the host cell. In interzone II-III, rhizobial nif genes
are turned on as the bacteroids expand, primarily by elongation,
occupying a majority (∼65%) of the host cell volume. The vol-
ume of vacuoles decreases dramatically, down to ∼30% of the
cell volume, which is correlated with the suppression of HOPS
(homotypic fusion and vacuole protein sorting complex) gene
expression in infected cells (2). In zone III, as the infected cells
stop expanding, bacteroids become terminally differentiated and
actively convert atmospheric nitrogen into ammonia, which can
be readily transferred to the host plant for assimilation into amino
acids. In older nodules, zone IV (also called the senescence zone)
is present, where the host cells and bacteroids degenerate.
Although in certain legumes the nitrogen-fixing bacteroids
remain morphologically similar to free-living bacteria and are
capable of reverting back to the nonsymbiotic lifestyle, bacteroids
in nodules formed on the inverted-repeat lacking clade (IRLC) of
legumes, such as M. truncatula, undergo remarkable transforma-
tions to differentiate terminally, such that they are no longer able
to survive independent of their host. Features of bacterial dif-
ferentiation include genome amplification and cell elongation,
and the host cell also expands by endoreduplicating its own ge-
nome (3, 4). Studies have shown that the differentiation of
bacteroids requires the delivery of host effectors, such as nodule-
specific cysteine-rich (NCR) peptides, through the endoplasmic
reticulum secretory system (5, 6). Present only in IRLC legumes,
NCRs are defensin-like antimicrobial peptides, some of which
drive the differentiation of bacteroids (5, 7, 8). They are now
recognized as major players in the interaction between the le-
gume host and rhizobia. The importance of this family is under-
scored by their massive expansion in the IRLC lineage, with more
than 500 members encoded in the M. truncatula genome.
The best evidence for the requirement of the NCR peptides to
date has been obtained by disrupting the nodule-specific protein
secretory pathway, where intracellular rhizobia no longer differ-
entiate (6). However, blocking protein secretion in the nodule
Significance
Legumes form a root structure, the nodule, in which nitrogen-
fixing bacteria (rhizobia) reside. In this symbiotic relationship,
the bacteria provide nitrogen to the plant and in return obtain
fixed carbon from the host. Once released into the cytoplasm of
the host cell, the rhizobia undergo a remarkable transformation,
including genome amplification and cell elongation, before reach-
ing the differentiated nitrogen-fixing state. Small plant-derived
peptides with antimicrobial activities have been known to play
critical roles in the differentiation of rhizobia in legumes that form
indeterminate nodules. By studying the Medicago truncatula dnf4
mutant, we discovered that an antimicrobial peptide, NCR211,
plays a critical role in the survival andfunctionofdifferentiated
rhizobia in host cells for successful symbiotic nitrogen fixation.
Author contributions: M.K., R.C., and D.W. designed research; M.K., Y.C., J.X., C.W., and
D.W. performed research; Y.C., J.X., and C.W. contributed new reagents/analytic tools;
M.K., Y.C., C.W., R.C., and D.W. analyzed data; and M.K., R.C., and D.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. Email: dongw@biochem.umass.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1500123112/-/DCSupplemental.
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indiscriminately is a blunt instrument that offers no insight into the
specificity of individual NCR peptides. The large size of the NCR
family and the limited sequence homology among its members
hinder efforts to generalize their role in the symbiosis. Their se-
quence diversity and distinct temporal and spatial expression
patterns have led to the suggestion that they may perform diverse
functions, with different bacterial targets and modes of action (9),
but direct proof of functional diversity is still lacking.
Our best understanding is of the subset of peptides that induce
bacterial differentiation. NCR247 has been studied in particular
detail, and has been reported to enter bacteroids and interact
with a plethora of proteins (10). In addition, NCR247 treatment
causes massive transcriptome changes in the bacteria (∼15% of
the Sinorhizobium meliloti genome), affecting critical cell cycle
regulators and cell division genes (11). Genetically, the only in
planta evidence supporting the role of NCR peptides in bacterial
differentiation comes from ectopic expression of NCR035 in
Lotus japonicus (5). Although loss-of-function genetic results
would be desirable, it is generally assumed that within such a
large group, the contribution from any single NCR peptide will
not be sufficient for its absence to cause a significant phenotype.
Here, by studying the defective in nitrogen fixation mutant,
dnf4, we report that an individual NCR peptide can be in-
dispensable for the nitrogen-fixing symbiosis. The symbiotic
mutant phenotype indicates that the M. truncatula DNF4 gene is
required for the survival and function of differentiating bacte-
roids. Map-based cloning followed by whole genome sequencing
identified the DNF4 gene as encoding NCR211. Our results in-
dicate that while some NCRs such as NCR247 have been sug-
gested to induce bacterial differentiation, DNF4/NCR211 on the
other hand acts on protecting differentiating bacteroids in sym-
biosomes from degeneration.
Results
dnf4 Is Defective in Maintaining Differentiating Bacteroids Inside the
Host Cell. The dnf4 mutant was isolated from a screen for
M. truncatula mutants defective in nitrogen fixation (12). In growth
medium lacking exogenous nitrogen, the inability of dnf4 mutant
plants to use atmospheric nitrogen resulted in growth retardation
and chlorotic leaves (Fig. 1A). The nodules of dnf4 also remained
small and white, whereas wild type nodules elongated and de-
veloped a pink color, which derives from the accumulation of
leghemoglobin (Fig. 1B). Toluidine blue-stained nodules of dnf4
at 10 d postinoculation (dpi) appeared normal in zones I and II, as
well as in interzone II-III, where the dark color suggests that the
host cells were fully occupied by bacteria (Fig. 1C); however, in the
nitrogen-fixing zone III, bacteroids appeared to disappear from
the central part of some host cells proximal to the root. Under
higher magnification, fully infected dnf4 cells exhibited a mor-
phology similar to that of WT cells, with elongated bacteroids
arranged around the central vacuole; however, cells that may have
once contained bacteroids were clearly degenerated (Fig. 1D).
To further analyze the nature of the defect in dnf4,weex-
amined the activity of the nifH bacterial gene using a promoter
fusion construct with the GUS gene that encodes β-glucuroni-
dase (12). The nifH gene encodes one of the subunits of the
nitrogenase enzyme complex, and thus is required for nitrogen
fixation. At 10 dpi, nodules of wild type and dnf4 plants showed
similar nifH expression (Fig. 2A); however, in dnf4 nodules, nifH
expression was lost by 14 dpi (Fig. 2B), whereas younger nodules
that formed later on the lateral roots (arrowhead in Fig. 2B) still
displayed nifH expression. These data indicate that the nifH
gene was expressed in young dnf4 nodules, but its expression was
abolished in older ones.
Host leghemoglobin proteins bind free oxygen to create a
microoxic environment in nitrogen-fixing cells, protecting the ni-
trogenase enzyme from inactivation by oxygen. nifH is expressed
only in low-oxygen conditions, implying that leghemoglobin is
present in dnf4 nodules. We observed that 10–20% of dnf4 nod-
ules were slightly pink, suggesting that these nodules express
leghemoglobin proteins. This proposition was confirmed by im-
munoblot analyses of proteins extracted from 10-dpi nodules. The
10-dpi nodules of dnf4 also accumulated leghemoglobin proteins
at a level near that measured in wild type, in contrast to the early
nodulation mutant dnf1, in which leghemoglobin protein was not
detected (12) (Fig. 2C). Thus, dnf4 nodules are deficient in the
symbiosis even though both partners express genes characteristic
Fig. 1. dnf4 is unable to develop functional nodules. (A)Plantswereinocu-
lated with S. medicae ABS7 and grew for 4 wk. Yellow leaves are the sign of
nitrogen deficiency. (Scale bar: 10 mm.) (B)dnf4 nodules remain small and
white, whereas the wild type nodules elongate and show pink color. (Scale bar:
1mm.)(C) Toluidine blue staining of 10-dpi nodules inoculated with ABS7.
(Scale bar: 100 μm.) (D) Magnification of boxed areas in C. Elongated bacte-
roids stain dark blue. dnf4 nodules show degradation ofsymbiosomes and host
cells at the nitrogen fixation zone proximal to the root. (Scale bar: 20 μm.)
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of mature nitrogen-fixing nodules. These results are also con-
sistent with a previous report of the dnf4 mutant being defective
in later stages of nodule development, unlike mutants affected in
earlier stages of nodule development, such as dnf1,dnf2, and
dnf5 (12).
To observe microscopic details of dnf4 defects, mutant nodules
were inoculated with GFP-expressing S. meliloti Rm1021 and
stained with propidium iodide (PI), a fluorescence dye that can
penetrate only compromised biological membranes. Wild type
nodules at 14 dpi had few PI-positive bacteroids. In contrast, dnf4
nodule cells showed widespread PI staining in bacteroids. Fur-
thermore, dnf4 bacteroids, including ones staining positive for
PI, appeared elongated and differentiated (Fig. 2D), unlike other
early-stage mutants (6, 13), although the degree of bacteroid
differentiation was unclear. Coupling PI staining with SYTO9, a
commonly used viability dye, yielded a similar pattern. In WT
nodules, most nitrogen-fixing bacteroids were green (Fig. S1A).
In dnf4, most cells containing differentiated bacteroids were red,
suggesting that the bacteria in these cells were dead; however,
bacteria in recently infected cells were still alive, and in some
nodules, these newly infected cells formed a sharp band in the
infection zone, suggesting that the infection process itself was
normal (Fig. S1B). Some bacteroids in dnf4 cells appeared to
reach the final stage of differentiation, when they are radially
aligned (Fig. S1 Cand D). In summary, the phenotypic charac-
terization of dnf4 nodules strongly suggests that this mutation
causes defects in the maintenance of differentiating bacteroids
within host cells.
Deletion of NCR211 Is Responsible for the dnf4 Phenotype. To identify
the causal mutation of the dnf4 phenotype, we crossed the dnf4
mutant (in the Jemalong background) with the A20 ecotype. Using
map-based cloning, we narrowed the region to the upper arm of
chromosome 4, between molecular markers 003F07 and h2_133k2b.
Genomic DNA pooled from phenotypically mutant plants was se-
quenced. An unrelated line in the A17 background (derived from
Jemalong) was also sequenced as a reference. Alignment of sequence
reads to the A17 reference genome identified a deletion of ∼35 kb
unique to dnf4 close to the mapping interval (Fig. 3A). This deletion,
verified by PCR amplification (Fig. S2), eliminated five predicted
genes. Two of these five genes encode NCR211 and NCR178, both
of which are expressed in nodules, whereas the other three genes
encode hypothetical proteins not expressed in nodules according to
available RNA-seq data (www.jcvi.org/medicago/index.php).
The two NCR genes were introduced into the dnf4 mutant by
hairy root transformation to test their ability to rescue the dnf4
phenotype. Only the NCR211-encoding gene (Medtr4g035705)
proved capable of complementing the dnf4 mutant, confirming
that it corresponds to DNF4 (Fig. 3B). The rescue of dnf4 phe-
notype by NCR211 was observed in all transgenic plants (n≥10)
selected by the red fluorescence of the DsRED1 marker gene.
The DNF4 gene is predicted to produce a 58-aa-long preprotein.
Cleavage of the signal peptide [the first 24 residues, as predicted by
SignalP (14)] would yield an anionic (pI =5.38) NCR211 peptide
34 aa long, with four conserved cysteines. A BLASTp search
identified NCR178 (Medtr4g035725) as the closest homolog of the
mature DNF4 (61.8% identity), suggesting that these two NCR
genes may have arisen from a recent local duplication event.
Our phenotypic analysis of the dnf4 mutant suggests that
NCR211’s function is necessary to maintain the viability of
differentiating bacteroids.
Fig. 2. dnf4 nodules express fixation genes but fail to sustain differentiated
bacteroids. (Aand B) Activity of a nifH::GUS reporter in WT and dnf4 nodules
at 10 dpi (A) and 14 dpi (B). The arrowhead in Bdenotes a young dnf4
nodule on a lateral root. (Scale bar: 1 mm.) (C) Accumulation of leghemo-
globin in 10-dpi nodules. Ponceau S-stained membrane is shown as a loading
control. (D) Live/dead staining of bacteroids in dnf4 compared with WT
nodules (14 dpi). Live bacteroids have a green signal (GFP) and dying bac-
teroids have a red signal from propidium iodide (PI). PI can only enter bac-
teroids with compromised membranes. (Scale bar: 10 μm.)
Fig. 3. Identification of the DNF4 gene. (A) Read density plot of dnf4 mutant (pool of mapping population) and an unrelated EMS mutant as a reference,
showing that the deletion is unique to dnf4. The blue bars on the bottom represent gene models. (B) Nodule morphology on hairy roots transformed with
empty vector or NCR211 or NCR178 genomic constructs. (Scale bar: 1 mm.)
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www.pnas.org/cgi/doi/10.1073/pnas.1500123112 Kim et al.
dnf4 Is Allelic to esn1.We noticed that the defects in dnf4 are similar
to two M. truncatula symbiotic mutants deficient in nitrogen fixa-
tion, FN6753 and FN9669 (named early senescent nodule1,or
esn1) (15) from a different irradiation-generated mutant collec-
tion. Under nitrogen-replete conditions, FN6753 and FN9669 are
indistinguishable from wild type (15). Inoculated with S. meliloti
Rm1021 under nitrogen depletion, FN6753 and FN9669 exhibited
symbiotic phenotypes resembling the phenotype of the dnf4 mu-
tant (Fig. S3 A–C) (15). To assess whether the symbiotic defects of
FN6753 and FN9669 result from mutations in NCR211,wefirst
examined whether NCR211 is deleted in these mutants. PCR re-
sults showed that NCR211 is absent in both mutants (Fig. S3D).
We next carried out a linkage analysis using a backcrossed F2
population of the esn1 mutant. Wild type and esn1 mutants seg-
regated at a ratio close to 3:1 (95 wild type and 44 mutant), and
the deletion in NCR211 was linked to the symbiotic phenotype
(Fig. S3E). Based on these results, we conclude that FN9669 and
FN6753 represent additional alleles of the dnf4 mutant.
DNF4 Is Highly Expressed in Nodules and Localizes to the Symbiosome.
Microarray analysis of NCR genes during nodule development
allowedustosearchfortheexpressionpatternofbothDNF4
(NCR211)andNCR178 on Rm1021 inoculation (16). As shown in
Fig. 4A, both genes were induced starting at 4 dpi, and their ex-
pression level continued to increase up to 40 dpi; however, the
expression was much higher (by ∼22-fold at 14 dpi) for DNF4
compared with NCR178. Spatial gene expression profiling of
Sm2011-inoculated nodules was recently performed with laser-
capture microdissection (LCM) followed by RNA-seq (17). Data
from this study indicate that expression of DNF4 was high in the
proximal half of zone II and reached its highest level in interzone
II-III (Fig. 4B). NCR178 exhibited a similar expression pattern
(Fig. S4A). To confirm this pattern derived from LCM, promoter-
GUS constructs of DNF4 and NCR178 were introduced into
M. truncatula with hairy root transformation. Consistent with the
LCM data, the promoter activity of DNF4 was highest in interzone
II-III (Fig. 4C). GUS activity was also present in zone III, as well
as in proximal zone II. The promoter of NCR178 showed an ex-
pression pattern similar to that of DNF4 (Fig. S4B). The promoter-
GUS expression analyses were performed with nodules from more
than five independent transgenic roots, with similar results.
Previous studies of some NCR peptides showed localization
inside bacteroids or on the bacteroid membrane (5, 10). Here the
genomic sequences of DNF4 and NCR178 were fused in frame
with a GFP sequence to create translational fusion constructs for
localization studies. The constructs were introduced into both wild
type and dnf4 plants by hairy root transformation. The DNF4-GFP
construct was able to fully rescue the defective nodule phenotype
of dnf4 in all transgenic plants recovered (n>10), indicating that
the GFP fusion does not interfere with the function of DNF4 (Fig.
S4C). DNF4-GFP was observed in the peribacteroid space be-
tween the host and the bacteroid membranes (Fig. 4D). An
NCR178-GFP fusion protein (in wild type background) also lo-
calized to the peribacteroid space (Fig. S4D). The localization of
DNF4 to the symbiosome suggests that the NCR211 peptide may
directly or indirectly interact with bacteroids.
Exogenously Supplied NCR211 Does Not Reverse Membrane Perme-
ability of the Symbiosome. The mature DNF4 peptide has certain
sequence similarities with the scorpion toxin BmKK2, which acts
as a blocker of eukaryotic potassium channels embedded in the
cell membrane (18). As the DNF4 peptide localizes to the sym-
biosome, it could be in close proximity to three membranes: the
host peribacteroid membrane, the bacterial outer membrane, and
the bacterial inner membrane. Bacteroids in dnf4 nodules readily
take up PI, suggesting that all three membranes are permeabilized.
To test whether DNF4 acts on the membranes of differentiated
symbiosomes to block membrane permeabilization, we applied a
synthetic NCR211 peptide to dnf4 symbiosomes either extracted
from nodules or in situ, and then proceeded with PI staining. In
neither case did NCR211 prevent the uptake of the PI dye (Fig.
S5). Along with later tests demonstrating that the synthetic NCR211
peptide is biologically active (next section and Fig. 5), these results
suggest that NCR211 might not block membrane permeability in
the symbiosome.
DNF4 Blocks the Proliferation of Free-Living Rhizobia. Although the
synthetic NCR211 peptide does not appear to repair compro-
mised symbiosome membranes, the localization pattern of DNF4
nonetheless suggests that it may interact with the bacteroids in
some fashion. To test this hypothesis, we applied synthetic NCR211
and a different NCR peptide, NCR247, to free-living S. meliloti
Rm1021. NCR247 treatments blocked subsequent colony formation,
as reported previously (5). NCR211 treatments also blocked colony
formation (Fig. 5). Although the kinetics of the two NCR peptides
appeared different, at the highest concentration the inhibitory effect
of NCR211 was comparable to that of NCR247 (Fig. 5).
Discussion
Approximately 600 genes encoding NCR peptides are predicted
in the M. truncatula genome (19). The lack of symbiotic mutants
in this gene family to date has been attributed to gene redundancy
(16). Here we report that removing a single NCR peptide
(NCR211) leads to defects in nitrogen fixation. Phenotypic char-
acterization suggests that dnf4 causes problems in the symbiosis at
stages later than other dnf mutants, such as dnf1,dnf2,anddnf5,
becauseanumberoflatesymbioticgenesfrombacteria(nodF,
bacA and nifH)andplants(LB1,CAM1 and Nodulin 31) are still
expressed in dnf4 (12). This classification is supported by our mi-
croscopic analyses demonstrating that differentiating bacteroids
are present in dnf4 mutant nodules, in contrast to those of dnf1
Fig. 4. DNF4 (NCR211) is highly expressed in interzone II-III, and the GFP
fusion protein localizes to the peribacteroid space. (A) Temporal expression
pattern of DNF4 and NCR178 after inoculation with Rm1021. (Inset)Ex-
pression at early time points. (B) Spatial expression pattern of DNF4. I, zone I;
IId, zone II distal; IIp, zone II proximal; IZ, interzone II-III; III, zone III. (C)DNF4
promoter GUS activity. (Scale bar: 100 μm.) (D) Localization of DNF4-GFP
translational fusion in the peribacteroid space. (Scale bar: 10 μm.)
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and dnf2, in which the bacteroids barely differentiate (6, 13).
Furthermore, PI staining of differentiated bacteroids in the dnf4
mutant suggests that NCR211 functions to maintain the viability of
differentiating bacteroids. The loss of bacterial viability in dnf4 is
further supported by the lack of signals from the bacterial GFP
reporter in PI-stained bacteroids (Fig. 1D), because doubly fluo-
rescent bacteroids are rarely observed.
The deletion in the dnf4 mutant includes two NCR genes,
NCR211 and NCR178. Although these are the closest homologs
based on mature peptide sequences, only NCR211 can rescue the
dnf4 phenotype. The promoter-GUS activity, LCM RNA-seq,
and microarray data show similar temporal and spatial expres-
sion patterns for NCR211 and NCR178; however, the expression
level of NCR211 is much higher than that of NCR178. Therefore,
the lack of complementation of dnf4 by NCR178 might be re-
lated to its lower transcript level. An alternative explanation is
that the differences in sequences of the two peptides might be
sufficient to confer different biochemical activities and distinct
biological functions. Promoter swapping analysis of the two
genes could answer this question.
Structurally similar to defensins, some NCR peptides have
bactericidal activities at high concentrations. At lower concentrations,
certain NCR peptides induce membrane permeabilization, genome
amplification, and cell elongation, which are features of terminal
bacteroid differentiation in IRLC legumes (5). One of the NCR
peptides, NCR247, was recently shown to bind many bacterial pro-
teins, including ribosomal proteins and GroEL, indicating that this
peptide may control bacteroids through multiple mechanisms (10).
Massive transcriptome changes (∼15% of the genome) by NCR247
are likely the result of numerous compromised cellular targets (10).
DNF4 is very different from NCR247 in terms of amino acid
composition, charge, and length.Furthermore,whereasNCR247
has been reported to penetrate bacteroid membranes, DNF4-GFP
is observed in the peribacteroid space (Fig. 4D), although this ob-
servation requires further verification using additional methods. The
fact that nodules without DNF4 contain differentiating bacteroids
indicates that DNF4 acts after the bacteroids have initiated the
differentiation program. Based on these observations, we propose
that DNF4 functions to promote the survival of intracellular bac-
teroids during their differentiation. The expression of DNF4 is high
in the older parts of zone II and the highest in interzone II-III,
where bacteroids undergo the final steps of differentiation before
reaching maximum size. It appears that DNF4 may be maximally
expressed in these cells to protect terminally differentiating bacteria
from otherwise lethal conditions.IntheabsenceofDNF4,these
bacteria perish either before or after reaching full differentiation.
The expression pattern of DNF4 is similar to that of NCR247 and
also overlaps with the pattern of NCR035 in interzone II-III (5, 10);
therefore, DNF4 could be in a position to balance the differentia-
tion induced by other NCRs, such as NCR247 and NCR035. Finally,
our finding that synthetic NCR211 is active on S. meliloti in culture
suggests that this effect may be direct.
At first sight, it may appear perplexing that DNF4/NCR211
supports the survival of differentiating bacteroids in planta while
also blocking free-living bacteria from forming colonies in cul-
ture. These two activities may reflect the same mode of action by
NCR211 on bacterial biology, however. Differentiating bacte-
roids and free-living bacteria have different physiologies. The
manipulation of the same bacterial target by DNF4 in these two
very different physiological contexts could manifest itself in dis-
tinct outcomes. For bacteria, differentiation can be stressful, or
even lethal if left unprotected. The induction of DNF4 could
provide a timely intervention to establish a sublethal, stable state
in bacteroids. DNF4’s action may be detrimental to free-living
and proliferating bacteria, however. Uncontrolled proliferation
of the intracellular bacterial population is clearly a risk to the
host. The dual effect of DNF4/NCR211 may reflect a mechanism
to ensure that the rhizobia stay in a properly differentiated state.
Host control of terminal bacteroid differentiation has evolved
in multiple lineages of legumes, indicating a possible fitness
benefit to the host plant (20). Furthermore, nodules with differ-
entiated bacteroids returned more benefit to the host (20). In an
accompanying report, Horváth et al. (28) identified M. truncatula
DNF7 encoding NCR169, suggesting that more than one NCR
peptide can be indispensable for the nitrogen-fixing symbiosis. In
another companion study, Price et al. (29) recovered a rhizobial
peptidase capable of degrading host NCR peptides. This collec-
tion of discoveries demonstrates the evolving nature in control-
ling bacterial differentiation in classical host–microbe mutualism.
Materials and Methods
Plant Materials and Growth Conditions. M. truncatula Jemalong ecotype and
dnf4 mutant plants were grown in vermiculite in a growth chamber (22/18 °C,
16-h/8-h day/night) under fluorescent lamps (∼100 μmol·m
−2
·s
−1
). Plants were
inoculated with Sinorhizobium medicae strain ABS7 carrying pHemA::lacZ (21),
S. meliloti Rm1021 carrying pNifH::uidA (12), or Rm1021 carrying pTrp::GFP (22).
Plasmid Constructs. For promoter-GUS fusion constructs, genomic DNA was
amplified with the primers DNF41-F1 (5′-ggatccCGGAGTGTAGGGGTGATGTT-3′)
and DNF41-R3 (5′-CAAACTTGAGAATTTCAGCCA-3′)forNCR211,aswellasDNF42-
F10 (5′-ggatccCCTC-TTAAATTGATTAAAGGC-3′) and DNF42-R5 (5′-TTTTTTTTTT-
AACTTTGCCTCT-3′)forNCR178. The products were cloned into the pCR8/GW/
TOPO vector (Life Technologies), checked for sequence errors, and transferred to
the pMDC163 binary vector (23) by LR recombination. For the translational fusions
of GFP to the C-termini of NCRs, genomic DNA was amplified with primers DNF41-
F1 and DNF41-R4 (5′-CTTGGGATAGCTCACA-CAATT-3′)forNCR211 and DNF42-
F10 and DNF42-R6 (5′-GTGGGTACGACAAT-CACAA-3′)forNCR178,andthen
cloned into the pCR8/GW/TOPO vector. The Gateway-compatible GFP vector
pK7FWG2-R (24) was cut with HindIII/SpeI, blunted with Klenow enzyme, and self-
ligated to remove the 35S promoter in front of the Gateway cassette, resulting in
pMK77. The inserts in the pCR8/GW/TOPO vector were checked for sequence
errors and then transferred to the pMK77 binary vector by LR recombination.
Microscopy. For toluidine blue staining, nodules were fixed in 2.5% (vol/vol)
glutaraldehyde in 0.05 M cacodylate buffer, washed, dehydrated, and em-
bedded in Technovit 7100 (Heraeus Kulzer), according to the manufacturer’s
instructions. Then 5-μm sections were cut on a microtome (Reichert-Jung) and
stained with 0.05% toluidine blue solution in 1×PBS buffer. Photographs were
taken with an Eclipse TE2000-S microscope (Nikon).
Transgenic nodules were selected based on DsRED1 expression using
an Olympus SZ61 binocular fitted with an ET590lp optical filter (Chroma
Fig. 5. A synthetic NCR211 peptide blocks the proliferation of free-living
S. meliloti Rm1021. Bacteria were treated with either NCR211 or NCR247 for
3 h at the indicated concentrations, and spotted on solid media. The number
of colonies was recorded 2 d later. Error bars indicate SDs (n=3). The graph
represents a typical outcome from multiple (>3) replications.
15242
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www.pnas.org/cgi/doi/10.1073/pnas.1500123112 Kim et al.
Technology). A Nikon NI-150 illuminator fitted with an ET560/40x opticalfilter
cube (Chroma Technology) was used for excitation of DsRED1.
PI staining was done wit h hand-sectioned nodules at 30 μM for 20 min.
Sections were then briefly washed with water and observed under a confocal
microscope (Olympus FluoView FV1000).
The GFP/SYTO9 (Life Technologies) signal was detected by excitation with
a 473-nm laser and emission with a 490- to 540-nm bandpass filter. The
PI/DsRED1 signal was detected by excitation with a 559-nm laser and emission
with a 575- to 675-nm bandpass filter.
SDS/PAGE and Immunoblot Analysis. Nodule samples were harvested from
21-dpi plants. Total protein extracts were prepared by grinding the samples in
a 1.5-mL microtube with SDS sample buffer [60 mM Tris·HCl pH 6.8, 60 mM
DTT, 2% (wt/vol) SDS, 15% (wt/vol) sucrose, 5 mM e-amino-N-caproic acid, and
1 mM benzamidine]. Protein concentration was measured with a Coomassie
blue binding assay (25). Here 10 μg of total nodule proteins were separated by
SDS/PAGE and blotted onto a nitrocellulose m embrane for immunoblot
analysis. The membrane was probed with an antibody against alfalfa leghe-
moglobin provided by Dr. Carroll Vance (University of Minnesota). A secondary
antibody conjugated with horseradish peroxidase was incubated with the
membrane. The signal was visualized by enhanced chemiluminescence
(Thermo Scientific).
Identification of the DNF4 Gene. The dnf4 mutant was isolated from muta-
genized M. truncatula Gaertn. cv Jemalong seeds created by fast neutron
bombardment (12). The dnf4 mutant was crossed with the A20 ecotype for
rough mapping. The location was narrowed down to the upper arm of chro-
mosome 4 between markers 003F07 and h2_133k2b. Then genomic DNA was
extracted from a pool of 40 mutant plants isolated from the mapping pop-
ulation and sent to the J. Craig Venter Institute for whole-genome sequencing
using an Illumina HiSeq. Visualization of sequence reads was done with the
Integrative Genomics Viewer (26). The primers used to amplify across the de-
letion break point in esn1 are esn1-LB (5′-GGTCCTGAAGTACCATATCTTAGT-3′)
and esn1-RB (5′-GATTAAGTGCAAGTATACAT-TGTCC-3′).
Complementation of the dnf4 Mutant. Genomic DNA sequences encoding
NCR211 (Medtr4g035705) and NCR178 (Medtr4g035725) were amplified and
cloned into pCR8/GW/TOPO with primers DNF41-F1 and DNF41-R1 (5′-ggatcc-
GGAGGGGGTCTAT-GAGAGCA-3′) for NCR211 and DNF42-F1 (5′-ggatcc-
GCAGGTTTGACATCCTCACC-3′) and DNF42-R1 (5′- CCTCACTAATCACTGACG-
GACC-3′) for NCR178. The cloned inserts were checked for errors and transferred
to a binary vector, pKGW-R (24). Hairy root transformation was performed with
Agrobacterium rhizogenes strain ARqua1 harboring the constructs according to
the method outlined by Boisson-Dernier et al. (27). More than 10 independent
transgenic roots were selected by red fluorescence of the DsRED1 marker
gene for scoring of nodule phenotype.
GUS Staining. Roots with nodules were fixed in cold 90% (vol/vol) acetone for
30 min and then rinsed with cold water. Samples were then immersed in
50 mM sodiumphosphate buffer(pH 7.2) containing2 mM 5-bromo-4-chloro-3-
indoxyl-β-D-glucuronide cyclohexylammonium salt (X-Gluc), 0.2% Triton X-100,
and 2 mM each potassium ferricyanide and ferrocyanide under vacuum for
30 min, followed by further incubation at 37 °C for 2–5 h. The nodules were
embedded in 6% (wt/vol) low-melting agarose and sectioned in 70-μm slices
with a vibratome (Vibratome 100 Plus). Nodules from more than five in-
dependently transformed roots showed the same GUS staining pattern.
Peptide Treatment of Free-Living S. meliloti. S. meliloti Rm1021 cultures were
grown to an OD
600
of 0.3–0.6 in LB media and washed with 5 mM Mes-KOH
buffer at pH 5.8. Then 200 μL of bacteria (diluted to an OD
600
of 0.1) were
treated with peptides (synthesized by GenScript) at the indicated concen-
trations and incubated for 3–4 h at 30 °C. After the peptide treatment,
bacterial suspensions were serially diluted and plated out in triplicate on
selective media. Colony-forming units per milliliter were measured after 2 d
of incubation at 30 °C.
ACKNOWLEDGMENTS. We thank Erik Limpens for the binary vectors with
a fluorescence marker, Carroll Vance for the anti-leghemoglobin antisera,
Siyeon Rhee for technical assistance with toluidine blue staining of nodules,
Alice Cheung for access to a microtome, Tobias Baskin for access to a vibratome,
and Jeanne Harris and Peter Chien for insightful discussions of the manu-
script. Funding was provided by the University of Massachusetts (D.W.) and by
the Samuel Roberts Noble Foundation and the National Science Foundation
(Grant IOS-1127155, to R.C.).
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Supporting Information
Kim et al. 10.1073/pnas.1500123112
Fig. S1. Live-dead staining of whole nodules with SYTO9 and PI. The procedures are similar to that used for Fig. 2D, except that the bacteria do not contain
fluorescent proteins. (Aand B)WT(A) and dnf4 (B) whole nodule sections. (Scale bar: 100 μm.) (Cand D) Images of individual WT (C) and dnf4 (D) cells. (Scale
bar: 10 μm.)
Fig. S2. Verification of a deletion in dnf4.(A) Sequences of the primers used for PCR verification. CW8 amplifies a distant gene near the deletion site as a
positive control. (B) DNA agarose gel picture of PCR products. Expected sizes of PCR products are shown to the left of the gels. The asterisk indicates a
nonspecific band. M, size marker.
Kim et al. www.pnas.org/cgi/content/short/1500123112 1of3
Fig. S3. FN6753 and FN9669 (esn1) are allelic to dnf4.(A–C) Symbiotic nitrogen fixation-deficient phenotypes of FN6753 (B) and FN9669 (C) compared with wild type
A17 (A). (D) PCR analysis showing deletion of DNF4 in both mutants. (E) Genetic linkage analysis showing a linkage between the dnf4/esn1 deletion as illustrated at the
top to the mutant phenotype in a backcrossed F2 population. All 44 samples are esn1 mutants. The PCR analysis was designed to amplify across the deletion interval.
Fig. S4. NCR178 is highly expressed in the interzone II-III, and the GFP fusion protein localizes to the peribacteroid space. (A) Spatial expression pattern of
NCR178. I, zone I; IId, zone II distal; IIp, zone II proximal; IZ, interzone II-III; III, zone III. (B)NCR178 promoter GUS activity. (Scale bar: 100 μm.) (C) Comple-
mentation of dnf4 by C-terminal GFP fusion constructs. NCR211-GFP transformed nodules recover the wild type-like nodule morphology, whereas NCR178-GFP
transformed nodules do not. The transgenic nodules were selected by DsRED1 fluorescence from hairy root-transformed roots. NT, nontransgenic root. (Scale
bar: 1 mm.) (D) Localization of NCR178-GFP translational fusion in the peribacteroid space. (Scale bar: 10 μm.)
Kim et al. www.pnas.org/cgi/content/short/1500123112 2of3
Fig. S5. Exogenous application of NCR211 does not prevent the uptake of PI dye. (A) Symbiosomes were extracted from the dnf4 nodules and incubated for
4 h with (Right) or without (Left)20μM NCR211 peptide. SYTO9 (green, 5 μM) and PI (red, 30 μM) staining was done afterward. (Scale bar: 5 μm.) (B) Hand-
sectioned dnf4 nodules were incubated for 4 h with (Right) or without (Left)10μM NCR211 peptide. SYTO9 (green, 5 μM) and PI (red, 30 μM) staining was done
afterward. (Scale bar: 5 μm.)
Kim et al. www.pnas.org/cgi/content/short/1500123112 3of3
