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Review
Tansley insight
RNA–protein interactions in plant disease:
hackers at the dinner table
Author for correspondence:
Pietro D. Spanu
Tel: +44 207 5945384
Email: [email protected]
Pietro D. Spanu
Department of Life Sciences, Room 610 SAFB, Imperial College Road, London, SW7 2AZ, UK
Received: 26 February 2015
Accepted: 31 March 2015
Contents
Summary
991
IV. Conclusion
994
I.
Introduction: effectors – from affectation to explanation
991
References
994
II.
From manipulating hardware, to interfering with software
992
III.
Delivering the message
994
Summary
New Phytologist (2015) 207: 991–995
doi: 10.1111/nph.13495
Key words: gene silencing, haustoria, plant
pathogenic microbes, powdery mildews,
RALPH (RNase-like proteins associated with
haustoria) effectors, RNA effectors, RNA
interference, RNA–protein interactions.
Plants are the source of most of our food, whether directly or as feed for the animals we eat. Our
dinner table is a trophic level we share with the microbes that also feed on the primary
photosynthetic producers. Microbes that enter into close interactions with plants need to evade
or suppress detection and host immunity to access nutrients. They do this by deploying molecular
tools – effectors – which target host processes. The mode of action of effector proteins in these
events is varied and complex. Recent data from diverse systems indicate that RNA-interacting
proteins and RNA itself are delivered by eukaryotic microbes, such as fungi and oomycetes, to
host plants and contribute to the establishment of successful interactions. This is evidence that
pathogenic microbes can interfere with the host software. We are beginning to see that
pathogenic microbes are capable of hacking into the plants’ immunity programs.
I. Introduction: effectors – from affectation to
explanation
Interactions between organisms are controlled by exchanges of
signals between partners. For nearly two decades, the molecular
signals delivered by microbial pathogens have been called effectors
(Cornelis & Wolf-Watz, 1997) – a term now extensively used for
signalling molecules in many types of interactions between
different organisms. Effector biology has become a sub-discipline
in itself, to which many workshops and conference sessions have
been devoted. Inevitably, as befits the twenty-tens, the term
effectoromics has been coined (Champouret, 2010; Vleeshouwers
et al., 2011).
In plant science, the best characterized effectors are proteins
encoded by microbial pathogens secreted into host tissues and cells,
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where they are involved in modulating metabolism and immunity
for the benefit of the microbial partner. The studies have generally
centred around the effect on immune perception and signal
transduction. This was fuelled at least in part by what was known
about avirulence determinants and their corresponding resistance
genes, and was eventually integrated conceptually in effectorperception models (Jones & Dangl, 2006; van der Hoorn &
Kamoun, 2008; Boller & Felix, 2009).
The centrality of effector proteins in the biology of plant
pathogenic microbes is demonstrated by the presence of vast arrays
of effector-like genes that are found in practically all pathogen
genomes. This is particularly striking in the genomes of the obligate
biotrophic fungi that cause powdery mildews (Spanu et al., 2010;
Wicker et al., 2013). In these fungi, many commonly large gene
families are reduced to very few members, and some genes are lost
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992 Review
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altogether. In sharp contrast, the effector-like gene superfamilies
described in cereal powdery mildews comprise over 7% of the
conventional protein-coding gene capacity of the genomes. This
situation is evidently not restricted to the powdery mildews, but is
found again and again, for example in the taxonomically unrelated
rust fungi (Hacquard et al., 2012) and many oomycetes (Kamoun,
2006).
In general, effector proteins interfere with recognition of
microbes at the surface of cells and intercellular spaces; they can
also target the intracellular immune-signalling pathways all the way
up to and including the activation of transcription of genes involved
in resistance and the defence response. Indeed, the study of protein
effectors is a useful instrument to investigate and define the
mechanisms of the immune response itself. Protein effectors have
become molecular probes for immune signalling (Bozkurt et al.,
2011, 2012). From the pathogens’ point of view, these effectors are
essentially tools to gain entry and switch off the hosts’ alarm
mechanisms.
II. From manipulating hardware, to interfering with
software
In recent years, we have glimpsed a new facet of the microbial
armoury. Several lines of independent evidence indicate that some
effectors may target gene expression by interfering with RNA-based
processes and signalling.
For instance, the barley powdery mildew candidate effectors
(candidate secreted effector proteins, CSEPs) identified in the work
cited above were initially defined as proteins predicted to be
secreted and have no evident homologues outside the powdery
mildews (as revealed by BLAST searches). They are encoded by small
to moderately sized gene families (Spanu et al., 2010); in a followon study, a very large proportion of these proteins were predicted to
have structural features that resemble microbial RNases (Pedersen
et al., 2012). Nearly all of these small RNase-like genes have one
intron in a conserved position and thus are most likely to have
originated from a single ancestral RNase. Alignment of the
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consensus sequences reveals that amino acids required for hydrolytic activities are missing, hinting that they are probably not
involved in RNA degradation, but instead may act as RNAinteracting proteins. A separate study of proteins associated with
cells penetrated by powdery mildew haustoria, found that two
powdery mildew effectors (BEC1011/CSEP0264 and BEC1054/
CSEP0064) necessary for full pathogenic development are RNaselike proteins (Fig. 1a) (Pliego et al., 2013); we therefore call them
RALPH (RNase-like proteins associated with haustoria) effectors.
Overall, ~120 candidate effectors in the barley powdery mildew
genomes may be classified as RALPH effectors. In addition to the
exceptionally large numbers of paralogs, RALPH effector genes are
also generally expressed at high levels in infected tissues and have
clear signatures of diversifying selection within the families
(Pedersen et al., 2012). It remains to be seen how these important
effectors work, and what is the functional significance of the RNaselike structure.
Other results that implicate RNA-interacting proteins come
from a study in which candidate effector genes of the soybean
pathogen Phytophthora sojae were screened to identify proteins that
interfere with RNA-mediated silencing (Qiao et al., 2013).
Transgenic Nicotiana benthamiana expressing GFP was infiltrated
with Agrobacterium tumefaciens to deliver oomycetes effectors
together with additional GFP into the plant cells. In this assay,
expression of the GFP transgene is normally silenced by the
additional transient expression of GFP T-DNA. Two P. sojae
genes, PSR1 and PSR2, encoding canonical RXLR effectors result
in reduction of gene silencing and recovery of the transgenic GFP
signal. The authors reported that the effectors act on different stages
of the silencing pathway (Fig. 1b): PSR1 inhibits RNA processing
catalysed by the dicer enzyme DCL1. Conversely, PSR2 affects the
accumulation of some small interfering RNA that may be involved
in controlling the expression of nucleotide-binding leucine-rich
repeat proteins, which include many well-known disease resistance
genes (Qiao et al., 2013). PSR2 was then shown to be part of a large
effector gene family present in several Phytophthora and stable
expression of PSR2 as a transgene induces increased susceptibility
Fig. 1 Movement of RNA and RNA-interacting proteins between plants and eukaryotic microbes. (a) The fungi that cause cereal powdery mildews encode
> 500 effector-like proteins of which c. 120 are RNase like proteins associated with haustoria (RALPH). Two functionally validated effectors in barley powdery
mildew, BEC1011 and BEC1054, are RALPHs. These were discovered by host-induced gene silencing (HIGS), a process that requires expression of dsRNA in the
host, transfer of RNA into the fungus and suppression of the target genes. The precise mechanisms of how this is achieved are not understood, but are presumed
to be under the control of conventional RNAi processes, which are conserved in these fungi (Spanu et al., 2010). We do not understand how RNA is transferred
across the host–pathogen interface. Once inside host cells, functional haustoria produce RALPH effectors to suppress host immunity. (b) A screen for protein
effectors from Phytophthora sojae identified two Phytophthora suppressors of RNA silencing (PSR1 and PSR2). These are canonical RXLR effectors that target
the RNA silencing programme which controls expression of plant immunity in the plant hosts at various different levels: PSR1 inhibits the activity of several dicerlike (DCL) proteins, whereas PSR2 inhibits the accumulation of trans-acting small interfering iRNA (ta-siRNA) (Qiao et al., 2013). The resulting reduction in
immune responses leads to increased susceptibility, and the PSR2 effector is widely conserved in the oomycetes (Xiong et al., 2014). PSR1 and PSR2 are
assumed to be secreted by the oomycetes by conventional secretion mechanisms, and presumably diffuse through the haustorial matrix (HMx). The
mechanisms for uptake of RXLR protein effectors into the host cells (Knip et al., 2014) have been the subject of intense research and debate. (c) The production
of specific small RNA (sRNA) in the fungal pathogen Botrytis cinerea is dependent on DCL proteins. sRNA then acts in several plant hosts, in an Argonaute
(AGO)-dependent manner, to silence the expression of genes encoding elements of the signal transduction pathways necessary for mounting a successful
immune response (Weiberg et al., 2013). In this case, the RNA must pass through the complex barriers posed by the plasma membranes (PM), the apoplast and
the cell walls (CW) of both fungus and plant. There is no current explanation of how this transfer occurs. (d) Direct transfer of short double-stranded RNA
(dsRNA) across the PM in the nematode Caenorhabditis elegans may be a model for RNA movement between plants and microbes. In this organism, transfer is
mediated by SID transporters (systemic RNA interference deficient) which are the basis of the well-characterized AGO-dependent RNA silencing process.
Various other, less well-understood instances of inter-kingdom RNA transfer have been observed between parasites and human cells, reviewed in Knip et al.
(2014). It will be interesting to see whether such RNA channels exist in plants and eukaryotic microbe plasma membranes.
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(a)
Blumeria graminis
Barley, wheat
?
?
?
dsRNA
Gene
expression
HIGS
BEC1011 ?
BEC1054
Effector
genes
PM
RALPH
effectors
? e.g.
BEC1011
BEC1054...
?
Immunity
HMx PM
(b)
Phytophthora spp.
Arabidopsis, Nicotiana benthamiana, soybean
?
PSR1
PSR1
DCL1
DCL2
DCL4
Pri-miRNA
Pre-miRNA
ta-siRNA
?
PSR2
PM
(c)
PSR2
Arabidopsis, tomato
?
B.c. sRNA
?
?
?
?
DCL1
DCL2
B.c. pri-RNA
PM
dsRNA
Outside
Immune
signalling
HMx PM
Botrytis cinerea
(d)
Gene
expression
B.c. sRNA
AGO
Gene
expression
Immune
signalling
CW
CW
PM
Caenorhabditis elegans
SID
dsRNA
AGO
Gene
expression
Inside
PM
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to Phytophthora capsici in soybean root cultures and Arabidopsis
thaliana (Xiong et al., 2014). In some respects, this is analogous to
the observation that the bacterium Pseudomonas syringae secretes
effector proteins to suppress host RNA silencing to promote disease
in Arabidopsis (Navarro et al., 2008).
In the examples already described, the effectors are proteins that
may interact with RNA or interfere with RNA-based information
processing. One study reports that the fungal pathogen Botrytis
cinerea actually produces and delivers small RNA molecules to the
host, where they target the expression of specific components of
signalling downstream of the pathogen perception systems
(Weiberg et al., 2013) (Fig. 1c). Suppression of host signalling is
dependent on pathogen dicer enzymes (DCL1 and DCL2),
necessary for generation of small RNAs, as well as on the host
Argonaute protein AGO1, which directs small RNAs to their
target. In this interaction, it will be interesting to see whether a
polyphagic generalist such as B. cinerea encodes small RNAs that
are specifically able to target signalling in such a diverse set of hosts.
This compelling, so far unique, case is the first indication of the
capacity of eukaryotic microbes to deliver RNA molecules to their
hosts to manipulate them for their own benefit: here, B. cinerea
appears capable of hacking into the programs encoding essential
immune response programs, and gaining control of their
expression.
In summary, we observe microbial pathogens (fungi and
oomycetes) use the universal language of life to hack into their
hosts without the need to resort to learning ‘plantish’ or other
dialects (Bonfante & Genre, 2015).
III. Delivering the message
The idea that small RNA molecules are transferred between
organisms may once have seemed extraordinary, but it is now
gaining wide acceptance (Knip et al., 2014). This is due to the
increasing number of instances where RNA is known to be taken up
by eukaryotic cells and thereby affect gene expression (Sarkies &
Miska, 2014). In host induced gene silencing (HIGS), first
described in cereal powdery mildews (Nowara et al., 2010),
expression of double-stranded (ds)RNA in plant cells leads to the
silencing of gene expression in a fungal pathogen infecting that cell.
HIGS has also been observed in various other plant–fungus
interactions (Koch et al., 2013; Panwar et al., 2013). In all these
cases, dsRNA is the starting point for the induction of gene
silencing, much like the case for RNAi silencing in Caenorhabditis
elegans, described in the original publication which earned Mello
and Fire a Nobel Prize for Physiology or Medicine in 2006 (Fire
et al., 1998). Whilst C. elegans takes up RNA directly via
transmembrane RNA carriers (SID proteins; Fig. 1d) (Sarkies &
Miska, 2014), in plant–microbe interactions we do not understand
how the RNA is transferred from one organism to another:
significant barriers have to be overcome such as host and microbial
membranes and cell walls or the haustorial matrix. Vesicular
transport may be involved: for example, in the powdery mildew–
plant systems RNA-loaded vesicles are observed at the host–
pathogen interface (Knip et al., 2014), although it is unclear
whether such vesicles could cross the haustorial matrix. In HIGS,
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RNA is transferred from the host to the microbial partners.
Movement in the opposite direction needs to be explained in the
case of B. cinerea small RNAs. In either case, it is probable that
signalling RNA associates with RNA-binding proteins; these
proteins would act essentially as RNA chaperones. This could be
one role for the abundant RALPH effectors described in the cereal
powdery mildews.
IV. Conclusion
These observations raise some important questions: how widespread is interference with host gene silencing by eukaryotic
pathogens? Are RALPH effectors found in fungi other than the
cereal powdery mildews? How are small RNAs transferred between
plants and their microbial partners? What are the programs affected
by microbial hacks?
Irrespective of the exact mechanisms that underpin the signal
exchanges between plants and microbes, a detailed understanding of these processes has the potential for delivering strategies
and tools for the control of plant pathogenic fungi, and could
ultimately contribute to ensuring safe and sustainable crops to
feed us. Maybe, we will be able to limit the presence of
unwanted guests at our dinner table by learning how to restrict
their hacks.
References
Boller T, Felix G. 2009. A renaissance of elicitors: perception of microbe-associated
molecular patterns and danger signals by pattern-recognition receptors. Annual
Review of Plant Biology 60: 379–406.
Bonfante P, Genre A. 2015. Arbuscular mycorrhizal dialogues: do you speak
‘plantish’ or ‘fungish’? Trends in Plant Science 20: 150–154.
Bozkurt TO, Schornack S, Banfield MJ, Kamoun S. 2012. Oomycetes, effectors,
and all that jazz. Current Opinion in Plant Biology 15: 483–492.
Bozkurt TO, Schornack S, Win J, Shindo T, Ilyas M, Oliva R, Cano LM, Jones
AME, Huitema E, van der Hoorn RAL et al. 2011. Phytophthora infestans effector
AVRblb2 prevents secretion of a plant immune protease at the haustorial
interface. Proceedings of the National Academy of Sciences, USA 108: 20 832–
20 837.
Champouret N. 2010. Functional genomics of Phytophthora infestans effectors and
Solanum resistance genes. Wageningen, the Netherlands: Wageningen University.
Cornelis GR, Wolf-Watz H. 1997. The Yersinia Yop virulon: a bacterial system for
subverting eukaryotic cells. Molecular Microbiology 23: 861–867.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. 1998. Potent and
specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
Nature 391: 806–811.
Hacquard S, Joly DL, Lin YC, Tisserant E, Feau N, Delaruelle C, Legue V,
Kohler A, Tanguay P, Petre B et al. 2012. A comprehensive analysis of
genes encoding small secreted proteins identifies candidate effectors in
Melampsora larici-populina (Poplar Leaf Rust). Molecular Plant–Microbe
Interactions 25: 279–293.
van der Hoorn RAL, Kamoun S. 2008. From guard to decoy: a new model for
perception of plant pathogen effectors. Plant Cell 20: 2009–2017.
Jones JDG, Dangl JL. 2006. The plant immune system. Nature 444: 323–329.
Kamoun S. 2006. A catalogue of the effector secretome of plant pathogenic
oomycetes. Annual Review of Phytopathology 44: 41–60.
Knip M, Constantin ME, Thordal-Christensen H. 2014. Trans-kingdom crosstalk: small RNAs on the move. PLoS Genetics 10: e1004602.
Koch A, Kumar N, Weber L, Keller H, Imani J, Kogel K-H. 2013. Host-induced
gene silencing of cytochrome P450 lanosterol C14a-demethylase–encoding genes
confers strong resistance to Fusarium species. Proceedings of the National Academy
of Sciences, USA 110: 19 324–19 329.
Ó 2015 The Author
New Phytologist Ó 2015 New Phytologist Trust
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Phytologist
Navarro L, Jay F, Nomura K, He SY, Voinnet O. 2008. Suppression of
the microRNA pathway by bacterial effector proteins. Science 321:
964–967.
Nowara D, Gay A, Lacomme C, Shaw J, Ridout C, Douchkov D, Hensel G,
Kumlehn J, Schweizer P. 2010. HIGS: Host-Induced Gene Silencing in the
obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22: 3130–
3141.
Panwar V, McCallum B, Bakkeren G. 2013. Host-induced gene silencing of wheat
leaf rust fungus Puccinia triticina pathogenicity genes mediated by the Barley
stripe mosaic virus. Plant Molecular Biology 81: 595–608.
Pedersen C, Ver Loren van Themaat E, McGuffin LJ, Abbott JC, Burgis TA,
Barton G, Bindschedler LV, Lu X, Maekawa T, Weßling R et al. 2012. Structure
and evolution of barley powdery mildew effector candidates. BMC Genomics 13:
694.
Pliego C, Nowara D, Bonciani G, Gheorghe D, Xu R, Surana P, Whigham E,
Nettleton D, Bogdanove AJ, Wise R et al. 2013. Host-induced gene silencing
based pathogen effector discovery in barley powdery mildew. Molecular Plant–
Microbe Interactions 26: 633–642.
Qiao Y, Liu L, Xiong Q, Flores C, Wong J, Shi J, Wang X, Liu X, Xiang Q, Jiang S
et al. 2013. Oomycete pathogens encode RNA silencing suppressors. Nature
Genetics 45: 330–333.
Tansley insight
Review 995
Sarkies P, Miska EA. 2014. Small RNAs break out: the molecular cell biology of
mobile small RNAs. Nature Reviews Molecular Cell Biology 15: 525–535.
Spanu PD, Abbott JC, Amselem J, Burgis TA, Soanes DM, St€
uber K, Ver Loren
van Themaat E, Brown JKM, Butcher SA, Gurr SJ et al. 2010. Genome
expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme
parasitism. Science 330: 1543–1546.
Vleeshouwers V, Raffaele S, Vossen JH, Champouret N, Oliva R, Segretin ME,
Rietman H, Cano LM, Lokossou A, Kessel G et al. 2011. Understanding and
exploiting late blight resistance in the age of effectors. Annual Review of
Phytopathology 49: 507–531.
Weiberg A, Wang M, Lin F-M, Zhao H, Zhang Z, Kaloshian I, Huang H-D, Jin H.
2013. Fungal small RNAs suppress plant immunity by hijacking host RNA
interference pathways. Science 342: 118–123.
Wicker T, Oberhaensli S, Parlange F, Buchmann JP, Shatalina M, Roffler S, BenDavid R, Dolezel J, Simkova H, Schulze-Lefert P et al. 2013. The wheat powdery
mildew genome shows the unique evolution of an obligate biotroph. Nature
Genetics 45: 1092–1096.
Xiong Q, Ye WW, Choi D, Wong J, Qiao YL, Tao K, Wang YC, Ma WB. 2014.
Phytophthora suppressor of RNA silencing 2 is a conserved RxLR effector that
promotes infection in soybean and Arabidopsis thaliana. Molecular Plant–Microbe
Interactions 27: 1379–1389.
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