Pathogen small RNAs: a new class of effectors for pathogen attacks

Introduction

Over recent decades, profound findings in plant pathology research have made tremendous contributions to our understanding of how pathogens are able to colonize the biological niche of a living plant. Genetic approaches have determined pathogenicity or virulence factors, and the exploration of these factors has broadened our understanding of host–pathogen interactions. A group of virulence genes that code for secreted proteins are called effectors, and have received much attention, because effectors interfere with and manipulate host defence pathways for infection. To counter against pathogen effectors, host plants evolve resistance (R) gene‐encoding proteins to interact directly or indirectly with pathogen effectors which mount a strong immune reaction, a process called effector‐triggered immunity (ETI). An evolutionary arms race occurs between hosts and pathogens, which drives the pathogens to reinvent their effector molecules to undermine host plant immunity, and drives the hosts to update their molecular immune fence line to recognize effectors and to defeat pathogens by intensifying its immune response.

RNA interference (RNAi) or gene silencing is a mechanism in which small RNAs (sRNAs) guide the transcriptional and post‐transcriptional silencing of gene expression. It is an ancient and conserved mechanism present in almost all eukaryotic life forms, including plants, animals, fungi and oomycetes. Typically, sRNAs are generated by Dicer‐like proteins (DCLs), which process double‐stranded RNAs or single‐stranded RNAs with partial double‐stranded regions into mature sRNAs. The mature sRNAs are loaded into Argonaute (AGO) proteins, and form the RNA‐induced silencing complex (RISC). The RISC silences genes with complementary sequences to sRNAs. RNAi and sRNAs are important players in defence against viruses and other invading DNA elements, such as transposons (TE) and transgenes. Moreover, sRNAs also play an important role in the regulation of the expression of endogenous genes. Gene silencing occurs in diverse cellular processes, including plant defence pathways against various pathogen attacks.

The regulatory role of plant endogenous sRNAs in plant innate immunity has been studied intensively. Recent evidence has also demonstrated the important roles of pathogen‐derived sRNAs in host–microbe interaction. Here, we mainly discuss the roles of sRNAs from eukaryotic plant pathogens, which regulate gene expression within pathogens or hosts during infection. In this regard, pathogen‐produced sRNAs can be categorized into two groups: (i) pathogen endogenous sRNAs which regulate important virulence genes (effectors) during infection within pathogen cells; and (ii) sRNAs which translocate from the pathogens into the host plant cells during infection to silence host immunity genes. These pathogen‐produced sRNAs, which direct the silencing of host immunity genes, are termed sRNA effectors. Host gene silencing by pathogen sRNA effectors describes a new chapter of cross‐kingdom RNAi events during host–pathogen interaction. Indeed, there is much more to be discovered about RNA and RNAi‐based communication between pathogens and host plants.

Pathogen sRNAs Regulate Effector Genes Within Pathogen Cells to Achieve Virulence

Many pathogens produce effectors to suppress host plant immunity as part of their virulence strategy. Two of the best‐characterized eukaryotic effector classes are RxLR motif‐containing effectors and Crinkler (CRN)‐type effectors. Both classes of effectors are commonly known from the oomycete plant‐pathogenic Phytophthora spp. Host plants of Phytophthora evolve R gene‐based resistance which recognizes such RxLR and CRN effectors, and triggers ETI.

Phytophthora infestans is the causal agent of late blight and of the disastrous potato famines in the 18th century. Phytophthora infestans is expected to produce hundreds of protein effectors during infection. In total, more than 500 RxLR and over 300 CRN effector genes have been predicted in the P. infestans genome; however, only a few have been proven to be essential for pathogenicity, which is probably a result of combinatorial effects, host‐specific activity and redundant functionality. Remarkably, tight spatial–temporal regulation of effector expression occurs.

Recently, genome‐wide transcriptomic studies have revealed that Phytophthora produces masses of sRNAs that map to genomic regions of RxLR and CRN genes. This observation suggests that the expression of effector genes is controlled by regulatory sRNAs. Indeed, the accumulation of sRNAs has been shown to correlate with the silencing of these effector genes. Remarkably, sRNA populations are distinct among phytopathogenic Phytophthora spp. (Fahlgren et al., 2013). Moreover, significant differences in sRNAs which map to effector gene sites have been revealed between two P. infestans isolates that show different virulence levels on the host potato (Vetukuri et al., 2012). We are awaiting a more detailed study on the relationship between sRNA accumulation intensities at effector gene sites and the virulence performance of different Phytophthora strains.

The soybean pathogen Phytophthora sojae is a close relative of P. infestans. Qutob et al. (2013) observed sRNA‐mediated silencing of another effector gene, Ps‐Avr3a. Interestingly, silencing was observed in the P. sojae virulent strain ACR10, but not in the avirulent strain P7076, when infecting Rps3a‐carrying soybean plants. In support of this, the level of sRNAs derived from the Avr3a locus was much higher in the ACR10 strain than in the avirulent P7076. Here, unlike the usual positive role of effectors in host plant infection, silencing of an effector gene seems to be of advantage to the pathogen. Under the described circumstances, keeping an effector gene silenced might help to avoid it being detected by the corresponding host R protein to escape host immune reaction to achieve compatibility. This shows that a P. sojae strain has evolved such an adaptive strategy to bypass R gene‐mediated resistance in host plants. By silencing of an effector, the host ETI trigger, the ACR10 strain is able to infect its host plant without triggering a fatal resistance. The reversible silencing of an effector gene by sRNAs is assumed to be more advantageous than the irreversible loss of effector function by a gene mutation, because the re‐activation of a silenced effector might strengthen virulence when its producer infects a new host plant that lacks the corresponding R gene to this effector.

Phytophthora effectors often reside in TE‐rich regions, which give rise to many sRNAs. The fine‐tuned expression patterns of these effectors during infection are possibly regulated by sRNAs in order to adapt to various host plants. However, we are still at the beginning of our understanding of how effector gene expression is controlled and what are the underlying mechanisms. sRNAs act through RNAi machinery and guide gene silencing, and the RNAi pathway components are indeed functional in Phytophthora. Fungal RNAi pathways are very diverse and complex, with only a subgroup of sRNAs being DCL dependent (Jin and Zhu, 2010; Lee et al., 2010). Similarly, only a subgroup of sRNAs from Phytophthora are dependent on DCLs. The sRNAs that map to effector gene loci are mostly DCL1 dependent and probably regulate the expression of effector genes.

Transcriptional control via sRNA‐guided DNA methylation has been observed in animal and plant species, predominantly in TE‐rich regions. Local spreading of DNA methylation patterns from TEs to nearby protein‐coding genes has been described. Although sRNA‐directed DNA methylation has not been observed in fungal or oomycete systems, epigenetic control, such as histone modification, has been proposed to regulate the gene silencing pathway in P. infestans (Vetukuri et al., 2011), as silencing of a sporulation‐associated gene was found to require a histone deacetylase.

Similar genomic organization of effector genes in TE‐rich regions has been found in other notorious fungal plant pathogens, such as Fusarium, Blumeria and Leptosphaeria. In Leptosphaeria maculans, epigenetic control of effector genes is linked to heterochromatin formation via the methylation of histone H3 lysine 9. Many effector genes are activated during infection, some possibly through epigenetic activation (Soyer et al., 2014).

Future research is needed to clarify the extent and conservation of the regulation of expression of effectors or other virulence factors by sRNAs among diverse pathogens. Silencing of effectors to avoid ETI might be a special virulence strategy that has evolved in Phytophthora. The activation of effectors, which are host immunity suppressors and infection facilitators, is expected to be more common during infection. Indeed, several sRNAs have been found in the rice blast pathogen Magnaporthe oryzae which have been predicted to target virulence‐related genes, among them the avirulence gene ACE1. The expression of ACE1 was de‐repressed in RNAi mutants of M. oryzae, probably as a result of blocking of the production of regulatory sRNAs (Raman et al., 2013). The expression of ACE1 is strictly controlled and is induced only during appressoria formation, a specialized cell formation for initial penetration into plant tissue. It is likely that sRNAs silence ACE1 under non‐infectious situations, whereas sRNAs are switched off at local sites of host infection in order to activate ACE1 expression. We speculate that pathogen sRNAs that suppress virulence genes under non‐infectious situations and during saprophytic growth are very common. For infection, the expression of such sRNAs might be switched off, leading to the activation of virulence genes.

Pathogen sRNAs Are Delivered into Host Cells and Act as Effectors to Suppress Host Immunity

Pathogen effectors are molecules that are delivered into host cells to suppress host immunity. Most effectors that have been studied so far are proteins. A recent study has assigned a similar behavior to Botrytis cinerea sRNAs (Bc‐sRNAs), which are non‐proteinous effectors in its virulence arsenal. Botrytis cinerea is an aggressive pathogen with a broad host range, which can infect more than 200 different plant species. Bc‐sRNAs are transported into host cells during infection and silence important plant immunity genes, as shown in two hosts: Arabidopsis thaliana and tomato (Solanum lycopersicum). In total, more than 70 Bc‐sRNAs have been identified to be potential effectors based on in planta expression and target gene prediction in both Arabidopsis and tomato hosts, for which three sRNA effectors have been demonstrated experimentally to silence host plant immunity genes by hijacking host RNAi machinery. The silencing of host immune genes ensures successful infection of B. cinerea in host plants (Weiberg et al., 2013). These Bc‐sRNA effectors share common features with host sRNAs that are favourably sorted into Arabidopsis AGO1 (AtAGO1) protein, and thus utilize the host RNAi machinery by loading into host AGO1 to silence host immunity genes. In support of this, the Arabidopsis mutant ago1‐27 was less susceptible to B. cinerea, because the Bc‐sRNA effectors were no longer functional in guiding the host gene silencing without the appropriate AGO protein (Weiberg et al., 2013).

This is the first report of pathogen sRNAs acting as effectors to inhibit host immunity. Future research will unveil whether this novel sRNA‐based virulence pathway also exists in other plant eukaryotic pathogens. Indeed, another aggressive fungal pathogen, Verticillium dahilae, may have evolved a similar strategy of hijacking the host plant RNAi machinery to suppress host immunity. Similar to that observed during B. cinerea infection, the Arabidopsis ago1‐27 mutant was more resistant against Verticillium spp., whereas several other Arabidopsis RNAi mutants exhibited enhanced susceptibility (Ellendorff et al., 2009). Thus, Arabidopsis AGO1 is also required for Verticillium pathogenicity.

Long‐Terminal Repeat (LTR) Retrotransposons Produce Masses of sRNAs That Provide a Large Selective Pool of sRNA Regulators for Pathogenicity

TEs are mobile genomic elements that drive genome evolution. TE replication and transposition are associated with genomic DNA rearrangements and mutations. Although temporal transposition activity has beneficial effects in terms of adaptive evolution, it is obvious that such elements can be detrimental. The class of LTR retrotransposons is widespread among eukaryotes. LTRs proliferate by transcription of an RNA intermediate that is reversely transcribed into complementary DNA and subsequently re‐integrates into the host genome by random insertion. LTR regions are hot spots of sRNA production. LTR RNA intermediates probably serve as templates for RNA‐dependent RNA polymerases that synthesize a complementary RNA strand. Double‐stranded RNAs are processed by DCLs to produce masses of sRNA molecules. The primary function of these sRNAs within fungal pathogens is to silence LTRs to maintain genome integrity.

Protein effector genes are often clustered and located in TE‐enriched chromosomal regions, where housekeeping genes are largely depleted. For instance, RxLR and CRN effectors of Phytophthora spp. are often located in close vicinity to LTRs. The spread of transcriptional silencing from LTR loci onto nearby coding genes has been found in other eukaryotes. Indeed, RxLR and CRN genes are often found to be within a distance of 2 kb of LTRs in P. infestans, which represents an evolutionary advantage for the fast turnover of effectors (Vetukuri et al., 2012; Whisson et al., 2012). The majority of Bc‐sRNAs predicted to silence host plant genes are also derived from a class of LTRs in B. cinerea, the so‐called Boty‐like elements. Such gene arrangement suggests that Boty LTRs possibly play a positive role in driving the fast evolution of Bc‐sRNA effectors in Botrytis. This might lead to the rapid adaptation of Botrytis to a wide range of host plants, rendering this fungal pathogen into a highly aggressive, broad‐spectrum pathogen. The temporal activation of TEs under stress has been observed in different organisms. Likewise, transcriptional expression of LTRs is strongly induced in various eukaryotic pathogens, such as P. infestans, during sporulation, germination and appressoria formation. Apparently, the induction of LTRs results in greater accumulation of LTR‐associated sRNAs, which not only control LTR expression, but also provide a large pool of sRNAs for selection of effectors towards different hosts. In certain cases, LTR‐derived sRNAs can silence neighbour protein effector genes, which may also be an adaptive strategy during infection to escape ETI, as discussed above.

Interestingly, Boty elements genetically associate with virulence and host preference in B. cinerea. Population genetics studies have revealed that B. cinerea field isolates collected from geographically diverse and independent locations show a domination of Boty‐carrying isolates (called transposa) in areas of massive crop (host plant) production. Transposa isolates are significantly more virulent than others. Bc‐sRNA effectors physically link to Boty elements and may facilitate the fast turnover of Bc‐sRNAs, which would be of evolutionary advantage for the pathogen during the molecular arms race against host plants (Weiberg et al., 2014).

Cross‐Kingdom RNAi in Host Plant–Pathogen Interaction

Cross‐kingdom RNAi describes the phenomenon in which a donor organism produces an RNAi trigger that moves into a recipient organism and causes gene silencing. Cross‐kingdom RNAi occurs during host plant–pathogen interaction, and can take place in both directions: (i) sRNAs produced by a pathogen to be delivered into host cells to silence host genes; and (ii) a host‐produced gene silencing trigger to suppress pathogen gene(s). The sRNA effectors that are produced by B. cinerea translocate into host cells to silence plant immunity genes. Host‐induced gene silencing (HIGS) studies have shown that a transgenic silencing trigger is expressed in plants, which then translocates into infecting pathogen cells to turn down virulence gene expression. HIGS is a well‐established molecular tool to achieve plant resistance against various pathogens and pests.

HIGS is based on an artificially designed RNAi trigger against pathogen virulence genes. We speculate that the export ‘channel’ for the RNAi trigger is not only prepared for artificial transgenic sRNAs, but that some host endogenous RNAi triggers or sRNAs are also transported into certain pathogen cells for gene regulation. This is quite likely because cross‐kingdom RNAi has been described in diverse biological systems. For instance, sRNAs from plants consumed as food have been detected in human and animal serum (Zhang et al., 2012). HIGS is effective in diverse plant species and against different pathogens and pests, indicating that the basic cellular inventory required for cross‐kingdom RNAi seems to exist ubiquitously in plants, animals and filamentous microbes. Thus, the identification of a natural plant‐produced gene silencing trigger has great potential as a novel molecular marker in host resistance against pathogens and pests.

Cross‐kingdom RNAi events demonstrate that gene silencing signals can travel extracellularly over long distances and, in terms of plant–microbe interaction, across plant and pathogen cell walls, membranes, cuticular layers and other cellular boundaries. However, the underlying mechanisms of trafficking of RNAi signals still remain enigmatic. For example, the application of HIGS is successful in Phytophthora capsici, but does not seem to work efficiently in a related species Phytophthora parasitica. Although more experiments on other HIGS‐targeting genes are needed to confirm this observation, an understanding of how the RNAi signals travel between hosts and pathogens/pests is a major task in the field, and will help to address this question. In addition, another open question concerns what form and nature of mobile gene silencing signals exist in cross‐kingdom RNAi: single‐stranded sRNAs, double‐stranded sRNAs or long double‐stranded sRNA precursors? Systemic RNA gene silencing has been shown in plants and animals. In plants, mature sRNAs can spread from cell to cell at approximately 10–15 adjacent cells from the origin of production, most probably via plasmodesmata. RNAi signals can also move systemically over long distances via the phloem to mediate gene silencing. In contrast, systemic RNAi in Caenorhabditis elegans is associated with longer RNA molecules, the precursors of mature sRNAs. Systemic RNAi‐deficient (SID) genes have been identified to be required for the cellular uptake of environmental RNA and cell‐to‐cell RNA transport. Interestingly, SID genes have been exclusively found in invertebrates, but not in plants, oomycetes or fungi, indicating a unique pathway of environmental and systemic RNAi in invertebrates.

The characterized Bc‐sRNA effectors possibly translocate as sRNA duplexes or mature sRNAs, rather than longer RNA precursors, and load directly into the plant AGO protein to silence host immunity genes. Infection assays on Arabidopsis dcl1 (Atdcl1) mutants with B. cinerea revealed an enhanced susceptibility phenotype, which indicates that Bc‐sRNA‐induced host gene silencing was not disturbed in the Atdcl1 mutant, and the host RNAi pathway may contribute to plant natural defence against B. cinerea. Moreover, a B. cinerea dcl1/dcl2 mutant was unable to produce Bc‐sRNA effectors, and consequently failed to suppress host immunity genes during infection, thus exhibiting a weakened virulence phenotype compared with the B. cinerea wild‐type (Weiberg et al., 2013). It would be worthwhile to determine whether other eukaryotic pathogens could also utilize similar strategies to deliver sRNA effectors into host cells to trigger the silencing of host plant immunity genes.

Future research is needed to elucidate what are the underlying molecular mechanisms of RNA export from an infecting pathogen cell and uptake into the host plant cell. How do sRNAs move across diverse cellular boundaries? Is this process based on an active specific transport ‘channel'? It seems that there is a selective process for choosing Bc‐sRNAs to be delivered into host cells, because not all Bc‐sRNAs are found in host cells. What is the selection mechanism? Softening of the plant cell wall and membrane by pathogen‐secreted degrading enzymes might ease the entrance of sRNA effectors into host cells during the infection process. Another fundamental yet basic question is what protect cross‐kingdom sRNAs from degradation in the extracellular matrix. In mammals, extracellular sRNAs are often associated with RNA‐protective protein complexes and/or encapsulated into extracellular vesicles (Mittelbrunn and Sanchez‐Madrid, 2012). Do such protective proteins and vesicles also exist for the transport of sRNAs between plants and microbes?

Can Pathogen RNA Species Trigger a Host Immune Response?

The discovery of pathogen RNA effectors that suppress host immunity has increased our understanding of the molecular arms race between pathogens and host plants. sRNA‐triggered interspecies gene silencing seems to be an additional regulatory layer for host–pathogen interaction. From the evolutionary point of view, the physical contact of pathogen RNA effectors with host cellular components must enforce the evolution of a counter‐defence strategy to defeat RNA attack. Normally, host plant receptor proteins recognize conserved pathogen‐associated molecular patterns (PAMPs) or pathogen protein effectors, and induce a host immune reaction. Are microbial RNA molecules recognized by receptor molecules directly or indirectly to stimulate defence responses? The receptor proteins that recognize PAMPs or effectors and initiate immune responses in animals usually belong to the class of Toll‐like receptors (TLRs). TLRs are described as resistance factors, which can also recognize conserved pathogen DNA elements to stimulate immunity. Interestingly, a recent report has claimed that a bacterial pathogen‐derived ribosomal RNA molecule activated TLR signalling and induced an immune response (Oldenburg et al., 2012). In addition, it has been demonstrated that endogenous extracellular sRNAs, such as microRNAs (miRNAs), activate membrane‐associated TLR receptors for immune reaction in human natural killer cells (Fehniger, 2013). It would be worthwhile to determine whether plants have evolved similar receptors that recognize microbial RNA molecules to trigger innate immune responses against microbial attackers. In this context, we speculate that extracellular RNA molecules might be multifunctional in host–pathogen interaction. In particular, cell‐non‐autonomous sRNAs might be a lingua franca in interspecies RNAi communication affairs.