Review

Research review
Small regulatory RNAs and the fine-tuning of plantbacteria
interactions
Author for correspondence:
Wafa Achouak
Tel: +33 (0)442254961
Email: wafa.achouak@cea.fr
Received: 25 August 2014
Accepted: 22 October 2014

Lamia Harfouche1,2,3, Feth el Zahar Haichar4 and Wafa Achouak1,2,3

1

CEA, DSV, IBEB, Lab of Microbial Ecology of the Rhizosphere & Extreme Environment (LEMiRE), 13108 Saint Paul-Lez-Durance,

France; 2CNRS, BVME UMR 7265, ECCOREV FR 3098 13108 Saint Paul-Lez-Durance, France; 3Aix Marseille Universit
e, 13284
Marseille Cedex 07, France; 4Universit
e Lyon1, CNRS, UMR5557, INRA, USC1364 Ecologie Microbienne, Groupes Fonctionnels
Microbiens et Cycle de lAzote, 69622 Villeurbanne, France

Summary
New Phytologist (2014)
doi: 10.1111/nph.13195

Key words: beneficial bacteria, pathogenic

bacteria, plantbacteria interactions,
regulation, small regulatory RNAs (sRNAs).

Small regulatory RNAs (sRNAs) play a key role in many physiological and adaptive responses in
bacteria. Faced with rapidly changing environments, it is more advantageous for bacteria to use
sRNA-mediated responses than regulation by protein transcriptional factors, as sRNAs act at the
post-transcriptional level and require less energy and time for their synthesis and turnover. The
use of RNA deep sequencing has provided hundreds of sRNA candidates in different bacterial
species that interact with plants. Here, we review the most recent results for the involvement of
bacterial sRNAs in beneficial as well as deleterious plantbacteria interactions. We describe the
current view for the role of sRNAs, which are suggested to improve competition for both niches
and resources in plant-interacting bacteria. These sRNAs also help plant-associated bacteria
individually adapt to the rapidly changing conditions to which they are exposed, during different
stages of this interaction.

Introduction
The successful interaction of bacteria with their eukaryotic hosts
requires intricate regulatory networks to control host tissue
colonization and utilize host resources. These features are common
(to some extent) to both pathogenic and beneficial plant-associated
bacteria. Even though the same strategies and structures that
promote host colonization might be used, interactions with
pathogenic bacteria will cause damage to the host, whereas
phytobeneficial bacteria interactions are harmless or at least
promote plant growth and/or pathogen protection. While phytopathogenic microbes employ a variety of strategies to overcome
host defenses, phytobeneficial bacteria must compete with other
microorganisms for niches and resources (Montesinos, 2010; Goh
et al., 2013).
Phytobeneficial bacteria promote plant growth either by
producing phytohormones or antifungal compounds, or by
facilitating nutrient uptake from the environment, including
sulfur and phosphate (Soto et al., 2009). Pathogenic bacteria
restrict plant development by utilizing a broad array of virulence
factors, including hydrolases and effectors delivered mainly by
the type III secretion system (T3SS) (Block et al., 2008). Highly
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complex biological processes occur in the plant rhizosphere and

phylosphere, where bacteria constantly face environmental
changes and must respond quickly to survive by adjusting their
physiology. Small regulatory RNAs (sRNAs) are appropriate for
such responses as they play critical roles in a variety of cellular
processes such as virulence, quorum sensing (QS), iron
deficiency response, oxidative stress response, and carbon
starvation response (Hoe et al., 2013). The best characterized
classes of sRNAs act via antisense base-pairing with their target
mRNAs, or by interacting with proteins to form a ribonucleoprotein complex (Waters & Storz, 2009). Based on their
genomic location, sRNAs can be subdivided into cis-encoded
sRNAs, which map to the strand of DNA complementary to
their mRNA targets, and trans-encoded sRNAs, which are
located remote from their mRNA target and often exhibit only
partial complementarity to them (Waters & Storz, 2009). Transencoded RNAs usually require the RNA chaperone protein Hfq,
which facilitates the formation of sRNAmRNA duplexes and
their subsequent degradation by the RNA degradosome (Waters
& Storz, 2009). Hfq generally binds A/U-rich regions present in
the sRNA and the target mRNA in order to promote and
stabilize sRNAmRNA interaction (Gottesman & Storz, 2011).
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Research review

Small regulatory RNAs may influence target mRNA translation

by sequestering RNA-binding regulatory proteins. In
Escherichia coli, the sRNAs CsrB and CsrC (part of the wellstudied Csr ribonucleoprotein regulatory system) function as
antagonists of the RNA-binding protein CsrA, by sequestering it
through multiple CsrA-binding sites (Seyll & Van Melderen,
2013).
Recently, a large number of potential sRNAs have been
discovered through the use of high-throughput RNA-sequencing
approaches in different phytopathogenic bacteria as well as in the
plant-beneficial bacteria. This review is focused on the involvement
of sRNAs in the control of a variety of biological functions required
for both beneficial and deleterious plantbacteria interactions. A
number of them induced during plant interaction in model
microorganisms are mentioned in Table 1.

Beneficial and deleterious shared sRNA-mediating

interactions between Gammaproteobacteria and
plants
The bacterial Gac/Rsm system: advantages and
disadvantages for plants
The two-component system comprising the GacS sensor kinase
and its response regulator, GacA (homologous to E. coli BarA/
UvrY), is present in a wide variety of Gammaproteobacteria,
including many plant-beneficial and plant-pathogenic bacteria.
This system helps the bacteria sense and respond to external
stimuli, in order to adapt to changing environmental conditions (Heeb & Haas, 2001). However, little is known about
the genes and environmental factors that activate the GacS/
GacA system.
Small regulatory RNAs, including CsrB, CsrC (in certain
enterobacteria), RsmB (in Pectobacterium and Dickeya), and RsmX,
RsmY and RsmZ (in Pseudomonas and related bacteria), have been
well established as central regulatory elements in the GacS/GacA
signal transduction pathway (Lapouge et al., 2008). These sRNAs
prevent the translational repression of target mRNAs that typically
encode virulence factors, antifungal metabolites or carbon storage
compounds, by sequestering dimeric RNA-binding proteins of the
RsmA family (functionally similar to the carbon storage regulator
CsrA in E. coli), which can promote the decay of these target
mRNAs (Sonnleitner & Haas, 2011; Seyll & Van Melderen,
2013).
RsmA was initially discovered in the phytopathogen
Pectobacterium carotovorum (formerly Erwinia carotovora) as a
negative regulator of extracellular enzyme production, QS, and
pathogenicity (Ma et al., 2001). RsmA, RsmB and RsmC are the
major components of the Gac/Rsm global regulatory system in
P. carotovorum, in which rsmB expression is negatively controlled
by the transcriptional repressors RsmC, KdgR, and HexA within a
complex regulatory network. On the other hand, RsmC positively
regulates the expression of rsmA, whose activity is antagonized by
rsmB (Chatterjee et al., 2010).
The Gac/Rsm system controls many different traits involved in
the pathogenicity of Pectobacterium and Dickeya, including
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production of extracellular enzymes, phytohormones, antibiotics,
pigments, polysaccharides, flagella, and levels of the QS signal
N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). This system
also affects virulence and the production of P. carotovorum harpin
(harpinEcc), the elicitor of the hypersensitive reaction (Chatterjee
et al., 2010). In P. carotovorum, RsmA negatively regulates the
expression of flhDC, the master regulator of flagellar genes, and fliA,
a flagellum-specific r factor. These observations contrast with
positive regulation of flhDC and motility by CsrA (= RsmA) in
E. coli (Chatterjee et al., 2010). Furthermore, the QS signal OHHL
and (p)ppGpp (an alarmone involved in the stringent response) in
Pectobacterium atrocepticum independently contribute to regulate
the level of free RsmA. Whereas QS acts to reduce the synthesis of
RsmA, (p)ppGpp is required to stimulate the synthesis of the RsmA
antagonist, rsmB (Fig. 1). Indeed, rsmB-mediated antagonism of
RsmA is sufficient to restore production of plant cell wall-degrading
exoenzymes to DrelADspoT (pppGpp synthase) mutants (Bowden
et al., 2013). The overlap and coordination between the different
pppGpp/QS/FlhD/Rsm signals result in a high degree of complexity in the regulatory networks that fine-tune bacterial gene
regulation. This favors a less costly metabolic pathway, ensuring a
quick response in a hostile environment.
The Gac/Rsm system in Dickeya dadantii 3937 (formerly
Erwinia chrysanthemi) is involved in the regulation of T3SS
genes (Yang et al., 2008). Under T3SS-inducing conditions,
GacA positively regulates the transcription of hrpA (a structural
protein of the T3SS pilus), hrpN (a T3SS harpin), and dspE (a
T3SS effector) through the Rsm post-transcriptional regulatory
pathway, by increasing rsmB expression; this thereby inhibits
the degradation effect of RsmA on hrpL mRNA (Yang et al.,
2008). Moreover, the Gac/Rsm pathway is involved in the
expression of various virulence factors in the plant-tumorigenic
bacteria Pantoea agglomerans pv. gypsophilae, including the
T3SS. Mutants of gacS, gacA or rsmB significantly reduce the
size and colonization of host plants by P. agglomerans pv.
gypsophilae (Panijel et al., 2013).
The expression of five rsmX sRNAs has been confirmed in
Pseudomonas syringae pv. tomato DC3000, along with rsmY and
rsmZ (Moll et al., 2010). Furthermore, rsmA overexpression
diminishes disease and colonization of plant tissue in all tested
strains of P. syringae (Kong et al., 2012). These results appear to
reliably indicate that rsmA is important in the main phenotypes of
disease and endophytic colonization, suggesting that the Gac/Rsm
pathway is required for full virulence and pathogenicity. Nevertheless, the mechanism by which RNA-binding proteins function
within the Gac/Rsm signal transduction pathway to regulate
disease and other lifestyles in P. syringae remains unknown, and
clearly deserves to be investigated.
More recently, a role for RsmA was reported in the virulence of
the phytopathogenic bacteria Xanthomonas citri ssp. citri (XCC), as
well as its contribution to the hypersensitive response (HR) in
nonhost plants (Andrade et al., 2014). Unlike the RsmA signaling
pathway in Erwinia, which destabilizes hrp transcripts (Fig. 1),
Xanthomonas RsmA activates the expression of T3SS-encoding hrp/
hrc genes by directly binding to the 50 untranslated region (UTR) of
hrpG, the master regulator of the hrp/hrc genes in XCC. However,
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Virulence

Metabolite/nutrition

AbcR1/AbcR2

RprA and RyhA

Agrobacterium tumefaciens

X. campestris pv. campestris

(Xcc) strain 8004
Erwinia amylovora vs
Nicotiana tabacum

X. oryzae pv. oryzae (Xoo)

sRNA-Xoo1
to sRNA-Xoo4

sRNA-Xcc1

Pseudomonas syringae
pv tomato DC3000

CrcX, Y and Z

Xanthomonas campestris
pv. vesicatoria
(Xcv strain 85-10) vs
pepper plants

Erwinia carotovora ssp.

carotovora strain
71 vs Apple

RsmB (AepH)

sX12 and sX13

Pseudomonas fluorescens
CHA0 vs
Pythium ultimum

Bacteria vs
Plant

RsmX, Y
and Z

sRNA

Trans-encoded sRNA. Hfq is not

requiredActs upstream of HrpGEmploys
C-rich loops to repress mRNAs harboring
G-rich motifs in the proximity of
translation start sites
Integron-encoded transposon and plasmidtransferred trans-acting sRNAs
Trans-encoded sRNA Hfq-dependentRegulate
translation of the stationary-phase sigma factor
RpoS in E. coliRegulatory mechanism in E.
amylovora under investigation
Trans-encoded sRNA, Hfq-dependentInhibition
of atu2422 (periplasmic components of ABC
transporters) initiation translation by masking
its ShineDalgarno sequence

Protein-binding sRNAGacAdependent small

RNAsAntagonize the activities
of RsmA/RsmE
proteins by exposing several
GGA recognition motifs,
resulting in sequestration of
the RNA-binding proteins
Protein-binding sRNAGacAdependent small
RNAsAntagonize the activities
of RsmA (RNA-binding protein)
by exposing several GGA recognition
motifs, resulting in sequestration
of the RNA-binding proteins
Protein-binding sRNACatabolism
repression controlIncrease in the
presence of less preferred carbon
sourcesBind to and sequester Crc
(repressor of mRNAs, encoding
functions involved in uptake of
different carbon sources)
Trans-encoded sRNAHfq-dependent

sRNA characteristics/
regulatory mechanism

Table 1 Selected small regulatory RNAs (sRNAs) induced during plant interactions in model microorganisms

GABA and proline uptake regulation

Contribution to virulence in E. amylovora

May play a role in the virulence of Xcc

Wilms et al. (2012)

Zeng et al. (2013)

Chen et al. (2011)

Schmidtke et al. (2013)

Liang et al. (2011)

Filiatrault et al. (2013)

Competition for nutrientsInfluence

susceptibility to antibiotics, T3SS and
QS expression

Xoo1: genes involved in amino acid

metabolism and protein transportXoo3:
oxidation reduction; response to stress
and metabolismXoo4: DNA replication,
rRNA processing protein transport and
metabolic processes
Promote the synthesis of the type III
secretion systemContribute to Xcv
virulence

Ma et al. (2001)

Lapouge et al. (2008)

Production of antibiotic
compoundsBiological
control of root-pathogenic
fungiRegulation of
biocontrol factor synthesisStress
thermic resistance

Production of extracellular
polysaccharide (pectate lyase,
polygalacturonase, cellulase,
protease)Control of pathogenicity
factors (harpin, motility, flagellum
formation, antibiotic, pigment)

References

Bacterial response

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Review 3

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4 Review

Vercruysse et al. (2010)

Rhizobium etli
Rec43 and
Rec49

Trans-encoded regulatory sRNAs

Sinorhizobium meliloti
AbcR1/AbcR2

Trans-encoded sRNAHfq-dependent AbcR1:

Post-transcriptional silencing of the periplasmic
SBP LivK mRNA

Torres-Quesada et al.
(2013)

Madhugiri et al. (2012)

Different expressions between free-living

bacteria and bacteroids in root
nodulesBjrC1505
expression decreased in nodulesBjrC2a,
BjrC68, BjrC80, and BjrC174 expressions
increased in nodulesBjrC2a is an antisense
RNA regulating the expression of inositolmonophosphatase
AbcR1: transcribed in actively dividing
bacteriaAbcR2: transcribed upon entry
into stationary phase and under abiotic
stressAbcR1 and AbcR2: dispensable for
symbiosis
Highly expressed during the stationary
growth phase and during symbiosis
Bradyrhizobium/
Rhodopseudomonas
Symbiosis

BjrC

Antisense RNAProbably regulate gene expression

at the post-transcriptional levelBjrC1505 target:
blr4023 encoding acetolactate synthasePotential
base-pairing of BjrC2 with the 50 -untranslated
region of bll7804 (encoding 3-hydroxyacyl-CoA
dehydrogenase type II)

References
Bacterial response
sRNA characteristics/
regulatory mechanism
Bacteria vs
Plant
sRNA
Table 1 (Continued)

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the mechanism of RsmA regulation by sRNAs in Xanthomonas is

still unknown. No RsmB homologs have been identified in the
XCC genome, although sRNAs whose expression levels are
controlled by HrpG have been found, suggesting a potential
involvement of the RsmA signaling pathway that controls hrp
expression (Andrade et al., 2014).
The role of the Gac/Rsm signaling pathway has also been
demonstrated in the regulation of phytobeneficial traits. Indeed,
the Gac/Rsm pathway is essential, in Pseudomonas fluorescens, for
the expression of different antimicrobial compounds and lytic
enzymes, including DAPG, HCN, pyoluteorin, pyrrolnitrin,
orfamide A, and the protease AprA, which are all beneficial to
plants (Lapouge et al., 2008).
Moreover, the Gac/Rsm signal transduction pathway appears
to be required for the induction of systemic resistance in
plants, which contributes to plant protection from diseases
caused by pathogenic fungi, nematodes, and bacteria (Sonnleitner & Haas, 2011). The P. fluorescens GacS/GacA pathway
is required for the expression of RsmX, RsmY, and RsmZ,
which exhibit high affinities for RsmA, as well as the RsmA
paralog RsmE (Lapouge et al., 2008). Eight copies of Rsm
sRNA (rsmZ1 through rsmZ7 and rsmY) have been identified
from the genome of the free-living diazotroph bacteria
Azotobacter vinelandii; these control expression of algD, which
encodes the key enzyme of the alginate biosynthetic pathway
(Hernandez-Eligio et al., 2012). Inactivation of any of the
rsmZ/Y genes in A. vinelandii reduces polyhydroxybutyrate
biosynthesis. The general characteristics of the Gac/Rsm signal
transduction pathway in P. carotovorum (a phytopathogenic
bacteria) and in P. fluorescens (a plant-beneficial bacteria) are
outlined in Fig. 1.
Improved use of carbon sources by the carbon catabolite
repression system
The activity and growth of the microbial biomass are limited by
substrate availability, especially carbon. In the rhizosphere, microbial diversity and function are strongly influenced by the nature of
plant root exudates. To respond to a need to optimize efficiency and
ecological fitness in a variety of different environments, bacteria
have developed a sophisticated mechanism, the carbon catabolite
repression system (CCR), which allows them to choose energetically favorable, balanced diets (Rojo, 2010). The CCR system
plays a key role in the competition between different bacterial
species sharing the same natural habitats. Initially described in
E. coli, CCR is a global control system in which the expression of
genes required for the utilization of resources is prevented by the
presence of a preferred substrate, that is, favorable carbon and
energy sources (Rojo, 2010). Interestingly, the CCR molecular
mechanism differs significantly between bacterial genera. In the
case of Pseudomonas, a group of metabolically versatile bacteria
studied for their role in plant health, the Crc regulatory protein is
the main actor in the CCR process. This protein acts as a posttranscriptional repressor of genes involved in the use of nonpreferred carbon sources and in different functions such as virulence,
QS, and biofilm formation (Rojo, 2010). For example, when
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Research review
Krebs cycle intermediates

Pectobacterium carotovorum

Signals ?

Pseudomonas fluorescens

Other signals ?

GacS

LadS

GacS

FliA
Flagella

RetS

35C

FlhDC

GacA

HexA

RsmB

GacA

RelA

Mobility
5

ppGpp

SpoT

RsmA

OHHL

Review 5

AHL

ppGpp
RelA
SpoT

Rsm X/Y/Z
RsmA

RsmE

RpoS

ExpR
PCWDE
I

II

Hrp
III

Effector proteins
Degradation of
plant cell walls
and membranes

Manipulation of
plant defense

PvdS

Pyoverdine

AHL

AlgU
AmrZ
HCN, pyocyanine, elastase,
DAPG, pyoluteorin,
phospholypase, pyrrolnitrin

FleQ

Suppression of
plant pathogen
(biocontrol)

Flagella
root colonization

Fig. 1 Role of the Gac/Rsm system in the regulation of genes inducing phytobeneficial (Pseudomonas fluorescens) vs phytopathogenic (Pectobacterium
carotovorum) effects. The GacA two-component system, which is controlled by a phosphorelay system kinase GacS (with RetS and LadS in P. fluorescens),
activates Rsm-sRNA expression. Rsm-sRNAs inhibit the function of the RNA-binding protein(s) RsmA (and RsmE in P. fluorescens) by a sequestering
mechanism. This in turn inhibits different genes whose expression has either beneficial (in P. fluorescens) or deleterious (in P. carotovorum) effects on plants.
Further explanation is provided in the text. Arrow, positive control; perpendicular lines, negative control; dashed lines, indirect control; Pvds, pyoverdine; AHL,
N-acyl homoserine lactone; AlgU, RNA polymerase sigma factor; AmrZ, alginate biosynthesis transcriptional activator; FleQ, flagellar regulator; ppGpp,
guanosine 30 ,50 -bispyrophosphate; RelA/SpoT, (p)ppGpp synthetase I/(p)ppGpp synthetase II; HexA, hexosaminidase A; FlhDC, transcriptional regulator of
flagellar and nonflagellar operons; FliA, flagellar biosynthesis sigma factor; RpoS, RNA polymerase sigma S; ExpR, AHL receptor; Hrp, hypersensitive response
and pathogenicity gene; PCWDE, plant cell wall-degrading enzymes; OHHL, oxo-hexanoyl homoserine lactone.

Pseudomonas putida is cultivated in a rich medium, crc inactivation

modifies the expression of at least 134 genes that are mostly
involved in the transport and assimilation of amino acids or sugars
(Moreno et al., 2012).
Computational analysis of Pseudomonas genomic sequences has
revealed variation in Crc-sRNA number and distribution within
Pseudomonas genomes (Filiatrault et al., 2013). P. putida contains a
second sRNA, CrcY, in addition to CrcZ (Moreno et al., 2012).
P. syringae strains appear to have an additional Crc-regulating
sRNA, CrcX, which appears to be unique to all P. syringae strains
sequenced to date (Filiatrault et al., 2013). P. syringae produces a
large array of small disease-causing effector proteins that are
transported into the cytosol of plant cells through the T3SS, and
which elicit diverse disease symptoms in infected host plants
(Moreno et al., 2012). The role of CrcX may be crucial to the
phytopathogenic P. syringae strains, which have less metabolic
versatility than other Pseudomonas species. It has also been
suggested that CrcX is involved in the control of the limited
pathways used for primary metabolism (Filiatrault et al., 2013).
As with the rsmX sRNAs, P. syringae strains contain multiple
copies of crc sRNAs (Moll et al., 2010; Filiatrault et al., 2013). The
possible advantage of having several RsmA and Crc antagonists,
rather than one of each, suggests either that each sRNA may
respond to a different signal, or that redundancy may help to
respond rapidly and efficiently under certain conditions. This
amplification of sRNA copy number within plant-interacting
bacteria represents their importance to the different lifestyles that
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these bacteria adopt in the presence (and absence) of their hosts. It is

also possible that this phenomenon is related to the selection
pressure exerted by plants.

sRNAs involved in ATP-binding cassette (ABC)

transporter regulation in Alphaproteobacteria
The natural habitat of rhizobia is the rhizosphere, a densely
populated, highly dynamic, and competitive niche. Bacteria must
monitor their surroundings and adapt their gene expression
accordingly in order to survive in this highly coveted environment
and to successfully infect plant cells.
The genomes of rhizobia and agrobacteria are predicted to
encode a large repertoire of ABC transporters and regulators of
transcription, which guarantees that bacteria can endure oligotrophic soil conditions (Wilms et al., 2012). Indeed, Sinorhizobium
meliloti and its related plant pathogen, Agrobacterium tumefaciens,
both encode more than 150 ABC transporter systems (Dean,
2011). Recent analyses of the Hfq regulon in these two species have
revealed a massive misregulation of ABC transporter genes in the
respective hfq mutants. In addition, these mutants appear to be
affected in their interaction with plants (Wilms et al., 2012).
Plants respond to A. tumefaciens by activating a complex defense
system, including the synthesis of the nonproteinogenic amino acid
c-aminobutyric acid (GABA), which stimulates degradation of
the QS signal homoserine lactone (Shelp et al., 2006). In
A. tumefaciens, plant-derived GABA induces the expression of the
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lactonase AttM, which in turn degrades the bacterial QS signal

OC8HSL, thereby attenuating virulence (Shelp et al., 2006).
GABA is transported into A. tumefaciens via the ABC transporter
Bra, in combination with the periplasmic binding protein Atu2422
that is under the control of the sRNA AbcR1. AbcR1 downregulates two periplasmic binding proteins of the ABC transporter
family (Atu2422 and Atu1879), leading to a decrease in GABA
transport. In fact, AbcR1 inhibits initiation of atu2422 translation
by masking its ShineDalgarno sequence, thereby reducing
stability of the atu2422 transcript. Consequently, AttM expression
declines and OC8HSL pools increase, leading to an augmented
Ti-plasmid copy number and a higher bacterial aggressiveness
(Wilms et al., 2012).
The sequence of the sRNA AbcR2 is similar to AbcR1, and plays
only a minor role in the control of A. tumefaciens GABA. Whether
these sRNAs are redundant, or whether they are hierarchical or
independent of each other remains to be explored. Hfq involvement in the regulation of the GABA transporter has also been
reported, suggesting that sRNAs may play important roles during
Agrobacteriumplant interactions (Wilms et al., 2012).
The plant symbiont Rhizobium leguminosarum harbors an
Atu2422 ortholog (RL3745) that is involved in GABA transport
(Prell et al., 2009). GABA is present in high amounts in
R. leguminosarum-induced nodules and plays an important role
in energy production of the symbiont (Prell et al., 2009). Sequence
alignments have revealed that AbcR homologs likely exist in
R. leguminosarum, implying the sRNA-mediated regulation of
GABA uptake in this organism as well. Interestingly, homologs of
AbcR1 and atu2422 have been predicted in Rhizobium,
Sinorhizobium, Mesorhizobium, Brucella, and Ochrobactrum (Wilms et al., 2012). The occurrence of sRNA-mediated control of
ABC transporters in hostmicrobe interactions therefore appears to
be a common theme.
Recent systems-level surveys of the noncoding RNomes of
S. meliloti have yielded large sRNA catalogs, including the
homologs of AbcR1 and AbcR2 sRNA. Recently, it was demonstrated that AbcR1 down-regulates the LivK periplasmic SBP
protein, which mediates the uptake of branched-chain amino acids
in S. meliloti (Torres-Quesada et al., 2013). However, expression of
this transcript has not been observed in mature nodules, revealing
the complexity of signaling between symbiotic partners (TorresQuesada et al., 2013).
AbcR1 and AbcR2 respond to different environmental cues to
optimize the uptake of available nutrients in free-living and
undifferentiated nodule-invading rhizobia. This most probably
occurs through the post-transcriptional regulation of largely
independent sets of ABC transporter systems in an Hfq-dependent
manner. Given that similar ABC transporters and sRNAs are
present in various Rhizobiaceae, it is likely that this sRNA-mediated
control influences a number of hostmicrobe interactions.

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into plant cells. The T3SS is an essential pathogenicity factor for
many phytopathogenic bacteria, including P. syringae pathovars,
Erwinia amylovora and Xanthomonas campestris (Dean, 2011). The
loss of T3SS severely reduces bacterial growth and symptom
causation, as well as their ability to suppress defenses in host plants
(Dean, 2011).
Among the Gammaproteobacteria, the genus Xanthomonas can
cause spots and blights on leaves, stems, and fruits in a wide variety of
plant species. Four sRNAs have been identified in X. campestris pv.
campestris (Xcc1 through Xcc4) and eight sRNAs have been
identified in Xanthomonas oryzae pv. oryzae (Xoo1 through Xoo8),
using an RNomics-based screen for sRNAs (Jiang et al., 2010; Liang
et al., 2011). Despite these reports, sRNAs within this genus are
poorly understood. The one exception is Xcc1, which is reported to
be an integron-encoded transposon and plasmid-transferred transacting sRNA, whose transcription is under the positive control of the
key virulence regulators HrpG and HrpX (Chen et al., 2011). More
recently, a genome-wide transcriptome analysis of X. campestris pv.
vesicatoria (Xcv), the causal agent of bacterial spot disease on pepper
and tomato, has identified 24 sRNAs (Schmidtke et al., 2013). A
novel class of sRNA-mediated adaptive and virulence modulating
processes has been identified through a highly detailed characterization of Xcv. This includes sX12 (Schmidtke et al., 2013), from the
first study to link virulence to sRNAs in phytopathogenic bacteria,
and sX13 (Schmidtke et al., 2013). Both sX12 and sX13 contribute
to virulence by affecting the interaction of X. campestris with its host
plants, and by contributing to environmental adaptation in Xcv.
These actions promote the synthesis of the T3SS, encoded by hrp,
which is essential to the pathogenicity of Xcv on susceptible host
plants (Schmidtke et al., 2013).
The tumor-inducing (Ti) plasmid from A. tumefaciens is
required to cause crown gall tumors in a wide variety of plants. It
is replicated via the products of the repABC genes (Li & Farrand,
2000). A novel gene encoding a small antisense RNA repE was
identified while analyzing the repABC operon on the Ti plasmid
(Chai & Winans, 2005). The 54-nucleotide sRNA is encoded in
the repB-repC intergenic region and binds repC mRNA, thereby
modulating Ti plasmid replication. Sequence analyses suggest that
this sRNA-mediated regulation of replication may be widespread
among plasmids of this family (Chai & Winans, 2005).
More recently, 384 novel sRNAs were identified in A. tumefaciens
C58 through whole transcriptome sequencing combined with an
sRNA search algorithm (Lee et al., 2013). Many of these sRNAs
were discovered on the complementary strand of important
virulence genes and operons, such as virA, virB, virC, virD, and
virE, suggesting a possible role in virulence regulation (Lee et al.,
2013). The plant-produced phenolic compound acetosyringone,
which acts as an inducer of Agrobacterium vir genes, induced 15 and
repressed seven such sRNAs (Lee et al., 2013). However, their roles
in physiological and cellular responses, as well as their role in plant
Agrobacterium interactions, have not yet been determined.

sRNA-mediated control of virulence in pathogenic

bacteria

sRNAs specific to symbiotic nitrogen-fixing bacteria

In bacteria, a successful host plant interaction often depends on a

protein secretion system for translocating bacterial effector proteins

del Val et al. (2007) detected experimentally seven sRNAs in the

nitrogen-fixing endosymbiont S. meliloti, which are differentially

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Research review

CrcZ/CrcX
Pseudomonas

HrpK

Rhizosphere
Root
exudation

Crc
Type III secretion
Quorum sensing
Virulence factors

AbcR1/AbcR2
(A. tumefaciens)
Perception
& GABA uptake

Catabolic pathway
(for less preferred
substrates)

ABC transporter
(Atu2422)

Microbial
community

Hfq

RsmX/RsmY/RsmZ
Pseudomonas

RsmE

Leucine, isoleucine & valine

uptake

Biocontrol factor synthesis

Virulence factor synthesis

ABC transporter
(LivK)

Symbiosis establishment

RsmA

AbcR1/AbcR2
(S. meliloti)

Successful tumor
formation

Carbon starvation

DspE
Type III secretion system
Motility
Biofilm formation
Amylovoran, EPS production

Fire blight disease

RprA, RyhA
(Erwinia
amylovora)

Plant signals

Review 7

Fig. 2 Small regulatory RNAs (sRNAs) involved in plantbacteria interactions. A schematic of how some bacterial sRNAs are involved in a complex regulatory
network among plants and bacteria, established in the rhizosphere (root exudation, microbial community) and phylosphere (plant signal). Selected sRNAs play a
key role in fine-tuning beneficial (Pseudomonas CrcZ/CrcX and RsmX/RsmY/RsmZ; Sinorhizobium meliloti AbcR1/AbcR2) or pathogenic (Erwinia RprA and
RyhA; Agrobacterium tumefaciens AbcR1/AbcR2) interactions. Further explanations of these sRNA regulation mechanisms are provided in the text. DspE,
T3SS effector; HrpK, T3SS helper protein; ABC, ATP binding cassette; Hfq, RNA-binding protein; GABA, gamma-aminobutyric acid; Crc, catabolite repression
control; Rsm, regulator of secondary metabolism. Arrow, positive control; perpendicular lines, negative control; dashed lines, indirect control; HCN, hydrogen
cyanide compound; DAPG, 2,4-diacetylphloroglucinol.

regulated in free-living and symbiotic bacteria. These findings

suggest novel regulatory functions for sRNAs in the a-proteobacteriaeukaryote interactions.
Several studies have been also performed in S. meliloti to assess
the impact of knocking out hfq on free-living and symbiotic
phenotypes (Barra-Bily et al., 2010; Torres-Quesada et al., 2013).
The common pattern among these studies is that the absence of Hfq
affects growth, motility, and tolerance of environmental stresses.
These types of changes associated with a lack of Hfq are reflected in
symbiotic interactions within Medicago sativa roots, revealing a
spectrum of symbiotic defects including reduced competitiveness
for infection, a delay in nodule appearance and development, a high
proportion of ineffective nodules, and reduced nitrogen fixation
and plant growth (Torres-Quesada et al., 2013).
The intergenic transcriptome of Rhizobium etli was recently
examined at various time points during free-living growth in
defined medium, as well as during nitrogen-fixing endosymbiosis
with its eukaryotic host plant P. vulgaris (Vercruysse et al., 2010).
Out of 89 sRNAs, the authors identified ReC43 and ReC49 as
potential transcriptionally independent trans-regulatory sRNAs
that are highly expressed under specific conditions during the
stationary phase and symbiosis, respectively.
The expression of seven sRNAs predicted by the genomic
comparisons of Bradyrhizobium and Rhodopseudomonas members
was recently assayed from the soybean symbiont Bradyrhizobium
japonicum USDA110 (Madhugiri et al., 2012). The sRNAs
BjrC2a, BjrC2b, and BjrC2c belong to the RNA family RF00519,
whereas the other sRNAs are orphans. The authors observed
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expression differences between free-living bacteria and bacteroids

in root nodules for several sRNAs. For example, the amount of
BjrC1505 was decreased in nodules, whereas BjrC2a, BjrC68,
BjrC80, BjrC174, and the previously described 6S RNA were
increased in nodules; the accumulation of truncated forms of these
sRNAs was also observed. Comparative genomics and deep
sequencing suggest that BjrC2a is an antisense RNA that regulates
inositol-monophosphatase expression in Rhizobiales. This observation is in agreement with the reported need for fine-tuning of the
synthesis of this protein in R. leguminosarum, to ensure successful
symbiosis with plants (Madhugiri et al., 2012).
Advances in sequencing technologies have accelerated the
identification of complex sRNAs from the genomes of nitrogenfixing a-rhizobia. Nevertheless, functions must still be assigned for
the vast majority of these newly identified sRNAs. Therefore, this
catalog of sRNAs constitutes a good starting reference for
investigating the mechanism of rhizobial physiology adjustment
during the transition from a free-living to an endosymbiotic state.
Finally, there is clear and unambiguous evidence that sRNAs
regulate the genes involved in the pathogenic and benefic
interactions with plants (Fig. 2).

Concluding remarks and future thoughts

The fitness of bacteria is principally determined by their ability to
rapidly and accurately detect and respond to environmental
change. Key components in this respect are sRNAs, which can
contribute to maintaining cell viability and competitiveness in
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8 Review

Research review

various environmental niches. Bacteria also acclimate to environmental change by altering gene expression levels. This may be
favored by increasing the copy number of very useful genes when
high levels of their expression are required.
Small regulatory RNAs appear to have been acquired in a
hereditary manner and are lineage-specific, although copy number
appears to be niche-specific. Certain sRNAs have been inherited
within the Gammaproteobacteria while others are more specific to
Alphaproteobacteria. These genes not only were maintained during
evolution, but were amplified in relation to the ecological niche of
the bacteria. This has been shown for Rsm RNAs, with more than
five rsmX copies present in P. syringae. The sRNA duplications
during evolution have thereby introduced redundancy and/or
increased activity of this gene. Paralogs retain the ancestral function
and increase survival under selective pressure, suggesting that the
function is beneficial. This same feature has been reported for CrcZ
sRNAs, in which three copies are found in P. syringae, as Crc is
essential for optimizing the carbon sources that are necessary for an
efficient metabolism. This raises the question as to whether plantassociated bacteria increase sRNA copy number because it is
extraordinarily advantageous for bacterial interactions with plants,
or because the multiple copies are required for bacterial survival in
the absence of a host plant.
Various methods to discover and characterize new Hfq-binding
RNAs have been reported as cross-linking immunoprecipitation
(CLIP). Their use should promise new discoveries and better
understanding of some specific pathways required for plant
bacteria interactions. Moreover, as more sRNAs are predicted by
bioinformatics searches and are detected in deep sequencing
experiments in diverse bacterial species, establishing their functions
will be a major challenge. Further investigation of these important
fine-tuning agents should reveal new insights into the regulation of
cellular processes within plant-interacting bacteria.

Acknowledgements
We knowledge funding from the CNRS in the frame of
Environmental Microbiology action, within the Continental and
Costal Ecospheres Program (EC2CO). We thank Ahmed Hamitouche for the design of the figures.

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