Perspective

Innate Immune Defense Through RNA Interference

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Science's STKE  13 Jun 2006:
Vol. 2006, Issue 339, pp. pe27
DOI: 10.1126/stke.3392006pe27

Abstract

RNA interference (RNAi, also known as RNA silencing) has recently emerged as a fundamental and widespread regulator of gene expression. New developments in this field implicate RNAi in the innate immune response to infection in plants and animals. Evidence from plants, tissue culture cells, and Caenorhabditis elegans–based systems previously suggested that RNAi plays a role in the defense against viral infection, but definitive evidence using viruses and whole animals has been lacking. Two recent reports now show that both Drosophila embryos and adult flies mount a substantial innate immune response to insect viruses that requires the RNAi machinery. This innate response is distinct from known bacterial and fungal defense systems provided by the Toll and immune deficiency (Imd) pathways, thus defining a previously unrecognized strategy to fight viral infection. Whether RNAi, aside from its function in counteracting viruses, is also used to fight bacterial infection remained enigmatic. New evidence, however, now shows that in Arabidopsis, the bacterial component, flagellin, induces the expression of a specific microRNA, which in turn leads to the down-regulation of the signaling pathways that are implicated in disease susceptibility. This down-regulation then increases the plant's resistance to infection. Whether RNAi mechanisms also exist for combating bacterial diseases in animals remains an intriguing question for future studies.

RNA interference (RNAi) is a process by which eukaryotes modulate gene expression, mainly through the degradation of specific mRNAs or through translational repression. Two major classes of small RNAs, siRNAs (small interfering RNAs) and miRNAs (microRNAs), are responsible for RNAi, and despite their similar size (21 to 26 nucleotides), are generated by distinct cellular machineries (1, 2). siRNAs are formed from long, double-stranded RNAs (dsRNAs) that are processed by Dicer [an endonuclease of the ribonuclease (RNase) III type]; miRNAs, in contrast, are produced from transcripts that fold into stem-loop structures and are processed sequentially, first in the nucleus by the RNase III enzyme Drosha and then in the cytosol by Dicer. In a second step, siRNAs or miRNAs guide the cleavage of cognate RNA through RNA-induced silencing complexes (RISCs).

RNA-based silencing was first identified in plants, but the critical importance of this phenomenon took center stage with the discovery that RNAi could be used in an animal, Caenorhabditis elegans, to specifically inhibit gene expression (3). Seminal discoveries then highlighted the regulatory role played by RNAi in animals in biological processes as diverse as ontogenesis and maintenance of genome integrity. More recently, RNAi has been suggested to function in innate immune defense of animals against viral infection. Indeed, using an infection model of Drosophila S2 cells, Li et al. put forward the concept that viral infection could lead to an RNAi-mediated antiviral response in animals (4). Similarly, ex vivo studies have confirmed that RNAi-mediated antiviral immunity also occurs in C. elegans (57). Despite these advances, it remained unknown, however, whether RNAi could confer a protective innate antiviral immunity in vivo. Two reports provide the missing evidence by showing that the RNAi machinery protects adult Drosophila from viral infections (8, 9).

In these studies, both groups infect adult flies with Flock house virus (FHV), a RNA virus of the Nodaviridae family that can replicate in a wide range of hosts, including yeast, plant, insect, and mammalian cells. Moreover, to demonstrate that their findings are not restricted to this virus, both groups substantiate their data by infections with distinct RNA viruses. To show that the RNAi machinery of Drosophila is involved in the defense against viral pathogens, both groups demonstrate that flies mutant for Dicer-2 display enhanced susceptibility to viral infections. The defective innate immunity in these mutant flies is specific to viruses, because responses to bacterial (8, 9) and fungal (9) pathogens remain normal (Fig. 1).

Fig. 1.

Drosophila innate immune pathways. Bacterial and fungal infections are recognized by pattern recognition receptors that trigger the activation of either the Toll or immune deficiency (Imd) pathway. The Toll pathway is triggered during infection by fungi and Gram-positive bacteria. Components of these microbes are sensed by Gram-negative binding proteins (GNBP), peptidoglycan recognition proteins (PGRPs), or both. Detection then triggers the cleavage and activation of the cytokine Spätzle, which then binds and activates the Toll pathway. In contrast, Gram-negative bacteria are sensed by a different set of PGRPs that lead to the activation of the IMD pathway. Conserved molecules, such as members of the nuclear factor κB family and c-Jun N-terminal kinase–related proteins, mediate the expression of antimicrobial peptides, which are essential for the insect to fight bacterial and fungal infection. For viral infections, the Jak-STAT pathway activated by the receptor Domeless triggers host defense. New evidence shows that a second form of antiviral immunity is represented by RNA interference (RNAi). The sensors or receptors involved in this RNAi pathway are still unknown.

The FHV viral genome inhibits the host RNAi machinery by expressing B2, a protein that binds to the long dsRNA molecule, thereby inhibiting the formation of siRNAs by the Dicer endonuclease. When Drosophila embryos were injected with the plus-strand RNA molecule (R1) encoding the B2 protein, FHV RNAs accumulated; in contrast, injection of a mutant RNA molecule did not lead to FHV RNA accumulation (8). The importance of B2 in FHV RNA accumulation was also addressed by Galiana-Arnoux et al. (9), who used transgenic flies expressing the R1 or R1 mutant RNAs. In adult flies, the accumulation of FHV RNAs was dependent on B2, suggesting that under normal circumstances, the host RNAi machinery efficiently controls accumulation of viral RNA.

The "take-home message" from these studies is that RNAi serves a protective role in innate antiviral immunity in vivo in Drosophila; however, the same mechanisms apparently leave antibacterial and antifungal immunity unaffected (Fig. 1). The question remains, however, whether innate immune defense through RNAi is solely used to fight off viral infection, and, more generally, whether RNAi is a crucial regulator of immunity in animals, as it appears to be in plants.

In the RNAi field, studies in plants appear to be one step ahead of those in animals. Indeed, Navarro et al. reported that plant miRNAs induced by a bacterial antigen contribute to antibacterial immunity by down-regulating host proteins implicated in the susceptibility to the pathogen (10).

Plants, like animals, can recognize potentially pathogenic microorganisms and mount efficient defenses. Nonhost- and host-specific responses are the two general types of plant immunity (11). Much recent research has focused on host-specific responses, which constitute a form of disease resistance relying on the presence of specific resistance genes (R genes) that confer immunity to particular pathogens (12). Plant proteins encoded by R genes mediate recognition of factors specified by particular avirulence (Avr) genes in the pathogens. Many Avr proteins are pathogenicity factors that exert host-modifying activity either extracellularly or by gaining entry into the host cell through a type III secretion apparatus.

In addition to R gene–related disease resistance, plants have a broader, more basal perception system for patterns characteristic for entire groups or classes of microorganisms. These so-called general elicitors are conceptually equivalent to the pathogen-associated molecular patterns (PAMPs) sensed by pattern recognition molecules (PRMs), such as peptidoglycan recognition proteins (PGRPs), Toll-like receptors (TLRs), and NACHT-LRR proteins (NLRs) in insects and vertebrates. Examples of microbial constituents that act as general elicitors in plants include chitin and ergosterol from fungi, and flagellin and lipopolysaccharides from bacteria (13).

However, these two different perception processes are not mutually exclusive, but indeed, overlap and have multiple levels of cross-talk (Fig. 2). Evidence for bridging the conceptual gap between nonhost- and host-specific responses comes mostly from studies of flagellin perception (13). Sensing of this bacterial PAMP in plants occurs through recognition of the most conserved domain in the flagellin N terminus, represented by the peptide flg22. Perception of this domain is dependent on the leucine-rich repeat (LRR)–type receptor-like kinase (LRR-RLK) FLS2 (flagellin sensing 2), which activates a downstream mitogen-activated protein kinase pathway conferring bacterial disease resistance. Analysis of the molecular mechanisms revealed that leaf infiltration with flg22 induces the up-regulation of genes involved in the perception and transmission of the flg22 signal. Moreover, the same stimulus induces massive expression of R genes, leading to enhanced sensitivity of the plant and enabling it to sense the presence of other microbial constituents (14). Furthermore, the transcriptional response to flg22 in Arabidopsis overlaps with the one induced by Avr genes (15). FLS2-mediated flagellin perception also triggers a rapid down-regulation of a certain subset of genes (15). Navarro et al. show that specific RNAi induced by flg22 perception leads to the suppression of a specific gene subset, contributing to antibacterial resistance (10).

Fig. 2.

Plant innate immune pathways. Nonhost-specific perception of the general elicitor, flagellin (flg22), by the LRR-RLK (leucine-rich repeat–receptor-like kinase) protein FLS2 (flagellin sensing 2) mediates expression of R genes (A), leading to enhanced host-specific immunity and transcription of defense mediators, and regulators implicated in perception and transmission of general elicitor sensing (B), thus providing an amplification loop and collectively leading to enhanced nonhost-specific immunity. A third regulatory loop induced by flagellin sensing up-regulates RNAi, blocking the action of the phytohormone auxin and leading to enhanced disease resistance.

Transgenic expression of viral proteins from different plant viruses that suppress miRNA- and siRNA-guided functions was used to demonstrate that flg22-stimulated transcript repression is reversed for a subset of mRNAs. These include the mRNAs of three F-box proteins, which have recently been described to function as receptors for the plant hormone auxin: TIR1 (transport inhibitor response 1) and AFB2 and AFB3 (auxin signaling F-box proteins 2 and 3) (16). Perception of flg22 results in enhanced transcription of a conserved miRNA (miR393), which targets all three F-box mRNAs (TIR1, AFB2, and AFB3). The observed repression by the specific miRNA was further shown to be compromised in seedlings deficient for DCL1, which is a miRNA-producing enzyme, providing genetic evidence that a miRNA-directed pathway contributes to the auxin-receptor down-regulation.

Auxin signaling acts by promoting the degradation of Aux/IAA (auxin or indole-3-acetic acid) proteins that function as transcriptional repressors of ARF proteins that bind auxin-responsive element (AuxRE) in DNA, thus controlling transcription of auxin-responsive genes. Auxin binding to TIR or AFB forms a complex with SCF-type E3 ubiquitin ligases and the COP9 signalosome, catalyzing the covalent addition of a ubiquitin molecule to Aux/IAA proteins, thereby targeting them for destruction (17). RNAi regulation of TIR1 mRNA transcripts in response to flg22 perception leads to a rapid reduction in TIR1 protein levels, conversely resulting in enhanced Aux/IAA protein stability. But does this regulatory loop affect the bacterial disease resistance? Upon infection with virulent Pseudomonas syringae, Arabidopsis lines constitutively expressing high levels of miR393 display 5- to 10-fold lower bacterial titers than do control plants. Therefore, these findings indicate that repression of auxin signaling by RNAi contributes to antibacterial disease resistance.

Although Navarro et al. provide indications that exogenous application of auxin enhances disease symptoms, they leave open the question of how suppression of auxin signaling leads to enhanced immunity. Auxin, an essential plant hormone regulating primarily development and growth, has only recently been implicated in infection (18). Preventing the action of phytohormones like ethylene and salicylic acid has also been shown to reduce disease symptoms (18). Whether this is also the case for auxin still needs to be tested. However, it is clear that flagellin, which is a classical PAMP that induces nonhost-specific responses, is implicated in regulating plant immunity by RNAi (Fig. 2). Application of crude bacterial extracts enhances disease resistance independently of flagellin and FLS2 (14). Whether this is due to the activity of other PAMPs acting through RNAi remains to be tested.

An intriguing question for the future is whether RNAi mechanisms also exist for combating bacterial and fungal diseases in animals, and whether RNAi plays a more general role in innate immune defense of higher animals, including mammals. Although at present it is unclear whether RNA silencing naturally restricts viral infection in vertebrates, there are indications that this is indeed the case. Many mammalian viruses encode suppressors of the RNAi pathway, and host-encoded miRNAs have been shown to both repress and enhance intracellular levels of viral RNA (19). Given the exciting progress in this field, one can only anticipate that future research will be able to reveal if RNA silencing plays an important role in host defense against microbial infections in general.

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