Cell Host & Microbe

Previews
Laguette, N., Bregnard, C., Hue, P., Basbous, J.,
Yatim, A., Larroque, M., Kirchhoff, F., Constantinou, A., Sobhian, B., and Benkirane, M. (2014).
Cell 156, 134145.

December 19, 2013. http://dx.doi.org/10.1016/j.

tcb.2013.11.007.

Cybulski, K.E., and Howlett, N.G. (2011). Cell Cycle

10, 17571763.

Malim, M.H., and Emerman, M. (2008). Cell Host

Microbe 3, 388398.

Svendsen, J.M., and Harper, J.W. (2010). Genes

Dev. 24, 521536.

Dehart, J.L., and Planelles, V. (2008). J. Virol. 82,

10661072.

Minocherhomji, S., and Hickson, I.D. (2013).

Trends Cell Biol., in press. Published online

Yan, N., and Chen, Z.J. (2012). Nat. Immunol. 13,

214222.

REFERENCES
Blanco-Melo, D., Venkatesh, S., and Bieniasz, P.D.
(2012). Immunity 37, 399411.

Romani, B., and Cohen, E.A. (2012). Curr Opin Virol

2, 755763.

CYCLOPS: A New Vision

on Rhizobium-Induced
Nodule Organogenesis
Erik Limpens1 and Ton Bisseling1,2,*
1Laboratory

of Molecular Biology, Wageningen University, 6708 PB Wageningen, The Netherlands

of Science, King Saud University, Riyadh 11451, Saudi Arabia
*Correspondence: ton.bisseling@wur.nl
http://dx.doi.org/10.1016/j.chom.2014.01.015
2College

The accommodation of nitrogen-fixing rhizobium bacteria inside plant cells requires reprogramming of root
cortex cells by rhizobial signals. In this issue of Cell Host & Microbe, Singh et al. (2014) reveal that CYCLOPS,
representing a novel class of transcription factors, links rhizobium-induced calcium signaling to reprogramming of root cortex cells.
In plants, intracellularly hosted bacteria
are exceptional, and in contrast to animals, intracellularly hosted pathogenic
bacteria are not known. Exceptions are
symbiotic bacteria that reduce atmospheric nitrogen within organelle-like
structures inside plant cells. However,
this intracellular accommodation requires
the formation of a complete new organ,
the root nodule. The best-studied N2fixing root nodules are those of legumes
that are formed during an interaction
with rhizobium bacteria. In such legume
nodules, specialized cells are present
that can contain thousands of bacteria,
and these are always surrounded by a
plant membrane that forms a symbiotic
interface (Figure 1).
Legume root nodules are formed from
fully differentiated root cortical cells.
These are mitotically reactivated to form
a nodule primordium that develops into
a nodule. The interaction of rhizobium
and legume plants starts at the root
epidermis, where an infection process is
initiated. In legumes like Lotus and

Medicago, this involves the formation of

a tube-like structure (infection thread)
that is bound by a plant membrane and
cell wall. These infection threads are initiated in curled root hairs, and they grow to
the nodule primordia where rhizobia are
released into the cytoplasm of nodule
cells. Lotus plants with the CYCLOPS
gene mutated are arrested after root
hairs are curled and the rhizobia have
formed a colony in the curl (Figure 1).
These structures resemble a one-eyed
Cyclops, and hence the name (Yano
et al., 2008).
Nodule organogenesis as well as
the infection process is triggered by
specific lipochito-oligosaccharides (Nod
factors) that are secreted by rhizobia.
These Nod factors activate a symbiotic
signaling pathway that includes a calcium- and calmodulin-dependent kinase
(CCaMK) as an essential and central
component. CCaMK is activated by
Nod factor-induced calcium oscillations
in the nucleus (Oldroyd, 2013). Through
the use of gain-of-function mutants, it

has been shown that the activation

of CCaMK in Lotus is sufficient to
trigger nodule organogenesis and even
allows Nod factor-independent intracellular accommodation of rhizobia (Madsen
et al., 2010).
How CCaMK triggers transcriptional responses that control these processes has
so far remained unclear. Several transcription factors have been identified
that function immediately downstream of
CCaMK. These include two GRAS-type
transcription factors, NSP1 and NSP2,
that are essential for nodule organogenesis and are considered to be primary
regulators of Nod factor-induced gene
expression. However, the mechanism by
which NSPs are activated has remained
unclear, as they are not a direct target of
CCaMK. Another transcription factor that
is essential for nodule organogenesis is
NIN, which is transcriptionally regulated
by Nod factor signaling.
The work of Singh et al. (2014) published in this issue of Cell Host & Microbe
helps to close this gap between activation

Cell Host & Microbe 15, February 12, 2014 2014 Elsevier Inc. 127

Cell Host & Microbe

Previews

Figure 1. Model Summarizing the Central Role of the CCaMK-CYCLOPS-NIN Transcriptional Cascade
See main text for details. CYCLOPS is important for: (1) Infection thread formation in the root hair curl; images represent a tubular (WT) infection thread and an
infection event arrested at the microcolony stage inside a curled root hair of the Medicago cyclops/ipd3 mutant. Rhizobia expressing GFP are visible in green.
(2) Nodule organogenesis; image depicting a wild-type Medicago nodule. (3) Intracellular accommodation of rhizobia as N2-fixing organelles called symbiosomes. Confocal image showing cells in the central tissue of the nodule, almost completely filled with (GFP-expressing) rhizobium bacteria.

of CCaMK and transcriptional activity. It is

shown that CYCLOPS, a nuclear coiledcoil protein, is a target of CCaMK and
represents a novel class of transcription
factors that induces NIN.
It is shown that CYCLOPS is phosphorylated by CCaMK at five serines, of
which two are essential for symbiosis.
When phosphorylated, CYCLOPS is
turned into an active transcription factor
that binds to the NIN promoter in a
sequence-specific manner, and phosphorylated CYCLOPS is sufficient to
induce NIN expression. A phosphomimetic CYCLOPS mutant where the two
essential serine residues are replaced by
aspartate results in an autoactive transcription factor whose expression in
Lotus is sufficient to trigger nodule for-

mation in the absence of bacteria. The

ability of autoactive CYCLOPS to induce
NIN and to trigger nodule formation is
consistent with the formation of nodulelike structures upon overexpression of
NIN in Lotus and its essential role
in nodule organogenesis. NIN in turn
activates CCAAT-box binding transcription factors that control organogenesis
(Soyano et al., 2013).
An intriguing finding of Singh et al.
(2014) is that the NSPs are not required
for CYCLOPS-induced NIN expression,
whereas they are indispensable for
CYCLOPS-induced nodule organogenesis. This finding raises questions concerning the role of NSPs and NIN in Nod
factor-induced signaling. Singh et al.
(2014) conclude that the NSPs are active

128 Cell Host & Microbe 15, February 12, 2014 2014 Elsevier Inc.

downstream of NIN or in an alternative

pathway.
However, a role of NSPs downstream
of NIN is difficult to reconcile with the
more severe phenotype of nsp mutants
compared to nin. The idea that NSPs are
involved in an alternative pathway would
have major implications for their involvement in the Nod factor signaling cascade
of which they are considered to be an integral part (Oldroyd, 2013). This putative
alternative pathway is not regulated by
the autoactive CYCLOPS, which implies
that it is active in the absence of symbiotic
signaling. In other words, this hypothesis
implicates that NSPs are not activated
by Nod factor signaling. Consistent with
this idea, the NSPs are constitutively
expressed. Further, they are active

Cell Host & Microbe

Previews
transcription factors in nonsymbiotic conditions in Medicago, as well as in
nonlegume plants, where NSPs are key
regulators of strigolactone biosynthesis
(Liu et al., 2011). The putative alternative
pathway could be involved in creating
permissive conditions in the root for
nodule organogenesis. Although strigolactones have a role in nodule initiation,
their block of synthesis in pea has only a
relatively mild (50% reduction) effect
on nodule formation, and this cannot
explain the severe effect of the NSP KO
mutations on nodulation. Therefore, in
case the NSPs are essential to create
nodule organogenesis permissive conditions, they must be involved in additional
nonsymbiotic processes. A more direct
role of the NSPs in nodulation is still
probable, since previous studies have
shown that NSPs are required for NIN induction during nodulation and NSP1 has
been found to bind to the NIN promoter
in an NSP2-dependent sequence-specific manner in Medicago (Hirsch et al.,
2009). However. if they act in concert
with other transcriptional regulators, Nod
factor signaling need not necessarily
directly regulate NSP activity. The recent
identification of a MYB coiled-coil transcription factor interacting with Lotus
NSP2 supports this idea (Kang et al.,
2014). So the study of Singh et al. (2014)
creates an interesting conundrum concerning the role of NSPs in nodulation.
This complexity might be caused by
multifunctionality of the NSPs and the
involvement of multiple steps that occur
at different location and time points in
the nodulation/infection process.
The study of Singh et al. (2014) is especially focused on the role of CYCLOPS in
inducing nodule organogenesis. Former
studies on Lotus and the Medicago ortholog of CYCLOPS (IPD3) show that
CYCLOPS/IPD3 is also a key regulator in
two other major steps of rhizobial infection (Ovchinnikova et al., 2011), namely
the start of infection thread formation in
curled root hairs and the release of
rhizobia in nodule cells to form N2-fixing
organelles. Therefore, transcriptional cas-

cades similar to those identified by Singh

et al. (2014) might play a role in these processes as well. This is supported by the
involvement of a primary target of NIN, a
pectate lyase, in infection thread formation (Xie et al., 2012). Whether NIN plays
a role in release of rhizobia in nodule cells
is not known. This leads us to propose
the model summarized in Figure 1 and
detailed below.
Ectopic expression of autoactive
CYCLOPS is sufficient to trigger nodule
organogenesis, indicating that CYCLOPS
is a key regulator of this process.
CYCLOPS shares this property with
the calcium-spiking-regulated kinase,
CCaMK, which phosphorylates CYCLOPS
and promotes induction of NIN expression. However, mutants deficient in these
genes show a major difference, as no
responses can be induced by Nod factors in ccamk (CCaMK-deficient) plants,
whereas in cyclops (CYCLOPS-deficient)
plants, Nod factor induces phenotypes
that include root hair curling as well as
cortical cell divisions. This implies that
CCaMK must have other, yet unknown,
targets (X) that contribute to the different
steps in nodule formation. NSPs are
essential for most of these steps. However, it is unlikely that NSPs are directly
regulated by Nod factor signaling, as they
are already active under nonsymbiotic
conditions. They can be involved in nodulation by creating nodulation permissive
conditions and by regulating genes
required for nodulation in concert with
other transcription factors. NIN has a
dual function. It is involved in infection
thread formation via the induction of a
pectate lyase. Further, it triggers organogenesis, which involves the activation of
CCAAT-box binding transcription factors.
Within nodules, CYCLOPS has a third
important function, which is the controlled
release of rhizobia from infection threads
to form N2-fixing organelles, called symbiosomes. Whether this process depends
on the same transcriptional cascade involving NIN remains to be examined.
It has become clear that Nod factor
signaling controls multiple steps in the

nodulation process, involving different

cell types and integrating various cues.
Such multifaceted control requires a
complex interplay between various transcription factors, with a central role for
the CCaMK-CYCLOPS-NIN transcriptional module identified by Singh et al.
(2014). Unraveling the gene regulatory
networks active in different cell types
and at various developmental stages of
nodulation will be a major challenge for
the future.

ACKNOWLEDGMENTS
E.L. and T.B. are supported by the European
Research Council (ERC-2011-AdG294790).

REFERENCES
Hirsch, S., Kim, J., Munoz, A., Heckmann, A.B.,
Downie, J.A., and Oldroyd, G.E. (2009). Plant Cell
21, 545557.
Kang, H., Chu, X., Wang, C., Xiao, A., Zhu, H.,
Yuan, S., Yang, Z., Ke, D., Xiao, S., Hong, Z., and
Zhang, Z. (2014). New Phytol. 201, 837849.
Liu, W., Kohlen, W., Lillo, A., Op den Camp, R., Ivanov, S., Hartog, M., Limpens, E., Jamil, M., Smaczniak, C., Kaufmann, K., et al. (2011). Plant Cell 23,
38533865.
Madsen, L.H., Tirichine, L., Jurkiewicz, A., Sullivan,
J.T., Heckmann, A.B., Bek, A.S., Ronson, C.W.,
James, E.K., and Stougaard, J. (2010). Nat
Commun 1, 10.
Oldroyd, G.E. (2013). Nat. Rev. Microbiol. 11,
252263.
Ovchinnikova, E., Journet, E.P., Chabaud, M.,
Cosson, V., Ratet, P., Duc, G., Fedorova, E., Liu,
W., den Camp, R.O., Zhukov, V., et al. (2011).
Mol. Plant Microbe Interact. 24, 13331344.
Singh, S., Katzer, K., Lambert, J., Cerri, M., and
Parniske, M. (2014). Cell Host Microbe 15, this
issue, 139152.
Soyano, T., Kouchi, H., Hirota, A., and Hayashi, M.
(2013). PLoS Genet. 9, e1003352, http://dx.doi.
org/10.1371/journal.pgen.1003352.
Xie, F., Murray, J.D., Kim, J., Heckmann, A.B.,
Edwards, A., Oldroyd, G.E., and Downie, J.A.
(2012). Proc. Natl. Acad. Sci. USA 109, 633638.
Yano, K., Yoshida, S., Muller, J., Singh, S., Banba,
M., Vickers, K., Markmann, K., White, C., Schuller,
B., Sato, S., et al. (2008). Proc. Natl. Acad. Sci.
USA 105, 2054020545.

Cell Host & Microbe 15, February 12, 2014 2014 Elsevier Inc. 129