Oomycete and fungal effector entry, a microbial Trojan horse

Authors


  • Shiv D. Kale was a finalist for the 2011 New Phytologist Tansley Medal for excellence in plant science, which recognises an outstanding contribution to research in plant science by an individual in the early stages of their career; see the Editorial by Dolan, 193: 821–822.

Author for correspondence:
Shiv D. Kale
Tel: +1 540 231 3784
Email: sdkale@vt.edu

Summary

Oomycete and fungal symbionts have significant impacts on most commercially important crop and forest species, and on natural ecosystems, both negatively as pathogens and positively as mutualists. Symbiosis may be facilitated through the secretion of effector proteins, some of which modulate a variety of host defense mechanisms. A subset of these secreted proteins are able to translocate into host cells. In the oomycete pathogens, two conserved N-terminal motifs, RXLR and dEER, mediate translocation of effector proteins into host cells independent of any pathogen-encoded machinery. An expanded ‘RXLR-like’ motif [R/K/H]X[L/M/I/F/Y/W]X has been used to identify functional translocation motifs in host-cell-entering fungal effector proteins from pathogens and a mutualist. The RXLR-like translocation motifs were required for the fungal effectors to enter host cells in the absence of any pathogen-encoded machinery. Oomycete and fungal effectors with RXLR and RXLR-like motifs can bind phospholipids, specifically phosphatidylinositol-3-phosphate (PtdIns-3-P). Effector-PtdIns-3-P binding appears to mediate cell entry via lipid raft-mediated endocytosis, and could be blocked by sequestering cell surface PtdIns-3-P or by utilizing inositides that competitively inhibit effector binding to PtdIns-3-P. These findings suggest that effector blocking technologies could be developed and utilized in a variety of important crop species against a broad spectrum of plant pathogens.

Oomycete and fungal symbiosis

Oomycetes and fungi affect the productivity of many economically important plant species. Important diseases include wheat head blight, rice blast, soybean rust and root rot, late blight on potatoes (Solanum tuberosum) and tomatoes (Solanum lycopersicum), and cereal rusts. Conversely, beneficial mycorrhizal fungi improve disease and drought resistance and nutrient uptake by plants. Fungi and oomycetes can also cause disease in animals and humans, and in certain cases mortality (Mendoza et al., 1993; Heitman, 2011). Through convergent evolution, oomycetes and fungi have acquired striking similarities in their mechanisms of host colonization, including physiological adaptations, mechanisms of adhesion, modulation of host defenses, and strategies of nutrient acquisition (Fig. 1) (Meng et al., 2009).

Figure 1.

Role of effectors in fungal and oomycete colonization. Oomycete and fungal pathogens and mutualists secrete effectors to facilitate host colonization. A subset of effectors localize and function in the apoplast. Other effectors are able to translocate into host cells and localize in diverse host compartments. These effectors have a variety of cellular targets and many contribute to colonization by modulating host defense machinery. Some effectors are delivered through the haustorium, a site of intimate interaction between pathogen and host formed by certain pathogens, while other effectors enter directly from the apoplast.

Many symbiotic microbes manipulate their hosts’ physiology through the use of secreted effector proteins. Some effectors act in the apoplast, while others translocate into host cells. Fungal and oomycete effectors can suppress a variety of host defense mechanisms. These defense responses fall under two broad overlapping categories, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) (Jones & Dangl, 2006). PTI, a relatively weak yet broad-spectrum defense response, is triggered by the recognition of nonself components, such as chitin and flagellin, by pattern recognition receptors. ETI, a more robust yet generally pathogen-specific defense response, is triggered by the recognition of effectors by resistance (R) proteins, most commonly of the nucleotide-binding, leucine rich repeat (NB-LRR) variety.

From the microbe to the host

Microbes have evolved a variety of mechanisms to deliver effector proteins into host cells. Bacterial effector translocation occurs via specialized type III, IV, and VI secretion systems (Tseng et al., 2009). Toxoplasma and many other apicomplexan parasites deliver effectors via a secretory organelle, known as a rhoptry, that directly injects effectors into host cells (Boothroyd & Dubremetz, 2008). Plasmodium species utilize a translocon to deliver cleaved Pexel-motif-containing effectors into red blood cells (de Koning-Ward et al., 2009). The rice blast fungus, Magnaporthe oryzae, forms a novel structure, known as the biotroph interface complex, which is believed to mediate effector entry into host cells through an unknown mechanism (Khang et al., 2010). Several oomycetes and fungi deliver effector proteins through the extrahaustorial matrix, which is formed at the interface between the pathogen and the host. In the oomycetes, a large number of these effectors share a conserved N-terminus RXLR-dEER motif (Jiang et al., 2008).

Oomycete RXLR effector translocation

The amino acid sequence motifs, RXLR and dEER, defined using hidden Markov models (HMMs), are the hallmark of a very large superfamily of predicted effectors in oomycete genomes (Jiang et al., 2008). HMMs for RXLR effectors revealed that the oomycete genomes of Phytophthora sojae, Phytophthora ramorum, Phytophthora infestans, and Hyaloperonospora arabidopsidis contain large reservoirs of potential effectors (396, 374, 550, and 134, respectively) (Tyler et al., 2006; Jiang et al., 2008; Haas et al., 2009; Baxter et al., 2010). These reservoirs facilitated the identification of five new avirulence genes (Avr) in P. sojae and five in P. infestans (Fig. 2a) (reviewed in Tyler, 2011). The mechanisms by which these effectors suppress host immunity and promote virulence are just beginning to be unraveled (e.g. Dou et al., 2008a; Oh et al., 2009; Bos et al., 2010; Mukhtar et al., 2011; Wang et al., 2011). In this review, however, I focus on how studies of the RXLR motif and, to a lesser extent, the dEER motif have illuminated the mechanisms of host cell entry by these effectors and by some fungal effectors.

Figure 2.

Oomycete and fungal effector sequences. (a) Nearly all oomycete avirulence proteins and their close homologs share a highly conserved RXLR motif (bold; functional motifs highlighted in gray) followed by a dEER motif (underlined italics). These two motifs mediate cell entry in the absence of any pathogen-encoded machinery. In certain cases, such as Avr1b, a noncanonical and nonfunctional RXLR motif (not highlighted) may be present. (b) Several intracellular fungal avirulence proteins and effectors contain RXLR-like motifs capable of mediating cell entry without the requirement of pathogen-encoded machinery. For a number of effectors with multiple RXLR-like motifs, one is functional (bold; highlighted in gray) and the others are not (bold). In the case of AvrM, translocation may be mediated by several RXLR-like motifs, but this has not yet been proved. Ps, Phytophthora sojae; Pi, Phytophthora infestans; Ha, Hyaloperonospora arabidopsidis; Fol, Fusarium oxysporum f. sp. lycopersici; Lm, Leptosphaeria maculans; Ml, Melampsora lini; Lb, Laccaria bicolor.

The N-terminal domains of oomycete effectors, containing RXLR and dEER motifs, were early on thought to facilitate entry into host cells because the motifs were conserved in avirulence gene products. The role of RXLR in effector translocation was validated using P. infestans and P. sojae stable transformants expressing, respectively, Avr3a and Avr1b RXLR and dEER mutant avirulence proteins; these mutant proteins lost their ability to provoke a defense response in plants carrying their corresponding R genes, R3a (in potato) and Rps1b (in soybean (Glycine max)) (Whisson et al., 2007; Dou et al., 2008b). Expression of the mutant avirulence proteins inside the plant cells restored their ability to provoke a response, suggesting that the mutations caused only a loss of translocation into the host cells. Furthermore, in P. infestans stable transformants, the N-terminal RXLR-EER domain of Avr3a could translocate β-glucuronidase into potato cells during infection (Whisson et al., 2007). Similarly, the N-terminal RXLR-dEER domains of P. sojae Avr4/6 and H. arabidopsidis Ha341 could translocate the C-terminus of Avr1b into soybean cells during infection (Dou et al., 2008b), and the RXLR-dEER domains of H. arabidopsidis ATR1 (A. thaliana Recognized 1) and ATR13 could translocate the C-terminus of Avr3a into potato cells during infection (Grouffaud et al., 2008).

To more directly test the hypothesis that RXLR domains mediate translocation, and to test if translocation can occur in the absence of the pathogen, two separate experimental strategies were utilized. Using a novel double-barrel particle bombardment system, soybean leaf cells were transformed with plasmids encoding the secreted form of Avr1b, together with DNA encoding β-glucuronidase (Dou et al., 2008b). Upon re-entry into soybean cells after secretion, Avr1b could interact with its corresponding resistance gene product, Rps1b, inducing a cell death response visualized through an ablation of blue-staining, β-glucuronidase-positive tissue patches (Dou et al., 2008b). RXLR and dEER mutants of Avr1b abolished this cell re-entry activity. Similar experiments with mutations in the RXLR or dEER motifs of P. sojae Avh5 (Avirulence homolog 5) and Avh331 also resulted in a loss of translocation (Kale et al., 2010). In a separate assay to directly detect cell entry, Avr1b, Avh5 and Avh331 proteins were fused to GFP and expressed in Escherichia coli, and then the purified fusion proteins were applied to soybean cells (Dou et al., 2008b; Kale et al., 2010). Cell entry was observed when each protein was applied to soybean root cells and soybean root suspension culture cells. Mutations in the RXLR or dEER motifs of each effector resulted in a loss of translocation. In similar experiments in soybean leaves, full-length Avr1b and Avh331 proteins were purified from E. coli and then infiltrated into leaves containing the Rps1b or Rps1k resistance genes (Avh331 is recognized by Rps1k) (Shan et al., 2004; Kale et al., 2010). Leaves with Rps1b or Rps1k exhibited a strong cell death response to Avr1b or Avh331 proteins, respectively, and the responses required intact RXLR and dEER motifs in each case (Kale et al., 2010). These experiments support the hypothesis that the RXLR domains of oomycete effectors mediate pathogen-independent entry into plant cells. Recently, the Saprolegnia parasitica putative RXLR effector SpHtp1 (S. parasitica host targeting protein 1) was also shown to translocate into fish cells with and without the pathogen present, suggesting that the same mechanism may be operative in oomycete–animal interactions (van West et al., 2010). Concordant with that observation, RXLR effectors from P. sojae could also enter human airway epithelial cells (Kale et al., 2010).

Fungal RXLR-like effector translocation

Several well-studied fungal effectors, including Melampsora lini AvrL567 and AvrM (Rafiqi et al., 2010), Magnaporthe oryzae Avr-Pita (Khang et al., 2010), Fusarium oxysporum Avr1, Avr2, and Avr3 (Houterman et al., 2008), and Leptosphaeria maculans AvrLm6 (Fudal et al., 2007), have been inferred to enter plant cells as a consequence of matching intracellular R gene products. Through systematic mutagenesis of the Avr1b RXLR motif, Kale et al. (2010) defined a broadened ‘RXLR-like’ motif, [R/K/H]X[L/M/I/F/Y/W]X, that could be found in many intracellular fungal effectors such as those listed above (Fig. 2b). Kale et al. (2010) demonstrated that several of these fungal effectors could enter plant cells in a pathogen-independent manner, and that entry depended on at least one RXLR-like motif (Kale et al., 2010). For example, both F. oxysporum Avr2 and L. maculans AvrLm6 each contained a functional motif, RIYER and RYWT, respectively. Both also contained a second nonfunctional motif, suggesting that certain flanking sequences are also required for the function of the motif, a situation that also occurs in Avr1b. In the case of M. lini AvrL567, two independent groups (Kale et al., 2010; Rafiqi et al., 2010) showed that a single N-terminal RXLR-like motif, RFYR, mediated translocation in the absence of the pathogen. Rafiqi et al. (2010) also showed that an N-terminal region of M. lini AvrM, containing three RXLR-like motifs, mediated translocation into plant cells (Rafiqi et al., 2010). Mutation of each of the three RXLR-like motifs individually did not abolish translocation, while the triple mutant did, suggesting that there may be cooperation among the motifs to facilitate translocation of this effector. Recently a secreted effector, MiSSP7 (mycorrhizal induced small secreted protein 7), has been identified from Laccaria bicolor, a mutualistic ectomycorrhizal fungus of Populus trichocarpa (poplar) (Plett et al., 2011). MiSSP7 was shown to enter poplar and soybean root cells in the absence of any pathogen-encoded machinery, and entry was dependent on the RXLR-like motif, RALG (Plett et al., 2011). Silencing of MiSSP7 strongly reduced the ability of L. bicolor to initiate colonization of poplar roots; exogenous MiSSP7 protein, but not RALG mutant protein, could complement the MiSSP7 deficiency (Plett et al., 2011). This is the first case of an RXLR-like effector promoting a beneficial interaction with a host. It is not yet known if fungal RXLR-like domains and oomycete RXLR domains share similar folds.

Other translocation motifs

In addition to RXLR and RXLR-like motifs, a number of other N-terminal motifs have been identified based on the analysis of putative secreted proteins predicted from fungal and oomycete genomes. Schornack et al. (2010) showed that the conserved KFLAK domain from several oomycete crinkling- and necrosis-inducing (CRN) proteins could replace the RXLR domain of Avr3a in delivering the C-terminus of Avr3a into host cells during infection of Nicotiana benthamiana by P. capsici (Schornack et al., 2010). Similarly, a new CHXC motif found in putative effectors of the biotrophic oomycete Albugo laibachii could also translocate the C-terminus of P. infestans Avr3a into host cells (Kemen et al., 2011). In both cases the mutation of the CHXC or KFLAK resulted in a loss of translocation. The mechanisms of CRN and CHXC translocation remain to be investigated. Several other cell entry motifs have been postulated (Ridout et al., 2006; Yoshida et al., 2009; Godfrey et al., 2010; Levesque et al., 2010), but they have not yet been experimentally tested.

Mechanism of entry

In addition to pathogen translocation machines, proteins can enter cells through a variety of pathways, including clathrin-mediated endocytosis, caveolae-mediated endocytosis, noncaveolar lipid raft-mediated endocytosis, dynamin-independent endocytosis, and macropinocytosis (reviewed in Doherty & McMahon, 2009). Some pathways such as clathrin-mediated and lipid raft-mediated endocytosis exhibit a strong requirement for binding of the internalized protein to a membrane receptor, while other pathways such as dynamin-independent endocytosis and macropinocytosis can endocytose liquid-phase molecules (reviewed in Doherty & McMahon, 2009). Kale et al. (2010) observed small vesicle-like structures when both oomycete and fungal effector-GFP fusion proteins entered soybean suspension culture cells or human airway epithelial cells (Kale et al., 2010). Entry of both Avr1b and AvrL567 into soybean suspension cultures and human airway epithelial cells was inhibited when lipid rafts were disrupted by filipin and nystatin (Kale et al., 2010). However, entry was not impeded by inhibitors of clathrin-mediated endocytosis or of macropinocytosis (Kale et al., 2010). Thus, the mechanism of entry was inferred to be a form of lipid raft-mediated endocytosis for both the oomycete and the fungal effectors. MiSSP7 entry was also blocked by a number of general endocytosis inhibitors, but the role of lipid rafts was not tested (Plett et al., 2011).

Binding of phospholipids by RXLR(-like) effectors

The similarities among the entry mechanisms of RXLR and RXLR-like effectors from oomycetes and fungi, respectively, suggested that they might bind a common host cell surface molecule to facilitate cell entry via receptor-mediated endocytosis. The receptor should be essential to cellular function, as negative selection pressure from the pathogens would select for a loss of the receptor. By binding an essential surface receptor, oomycete and fungal symbionts could hijack the trafficking machinery of the host cell. A key clue to the identification of the cell surface receptor was a plant phosphatidylinositol-4-kinase which contains 11 tandem RXLR-dEER motifs that could bind phosphatidylinositol (PtdIns), PtdIns-4-phosphate (PtdIns-4-P) (Lou et al., 2006). Several examples exist of lipid receptor-mediated entry mechanisms for pathogen-secreted proteins (Fig. 3). Most notably, bacterial toxins such as tetanus, botulinum and Shiga toxins bind glycosphingolipids in order to enter human cells via lipid raft-mediated endocytosis (Sandvig & van Deurs, 2005). Utilizing two assays, lipid filter binding and liposome binding, oomycete RXLR effectors and fungal RXLR-like effectors were confirmed to bind phosphoinositides with a preference for PtdIns-3-P (Kale et al., 2010; Plett et al., 2011). For all seven effectors tested, mutation of the RXLR or RXLR-like motifs required for cell entry also resulted in a loss of binding to PtdIns-3-P; mutation of motifs not required for cell entry did not result in loss of PtdIns-3-P binding, providing a strong correlation between loss of PtdIns-3-P binding and cell entry (Kale et al., 2010; Plett et al., 2011).

Figure 3.

Pathogen-independent cell entry. A variety of bacterial and eukaryotic effectors and toxins are capable of entering cells by endocytosis after binding cell surface receptors. RXLR and RXLR-like effectors bind phosphatidylinositol-3-phosphate (PtdIns-3-P), probably in lipid rafts, to enter by endocytosis. It is unknown if these effectors escape vesicles as they navigate to the Golgi or are released via retrograde translocation. CagA (cytotoxin associated protein A) binds Ptd-Serine and is able to enter cells via a non-endocytotic mechanism. Several toxins use glycosphingolipids as receptors. Toxins escape endosomes either by retrograde translocation (ricin, shiga, cholera, and pertussis toxins) or by directly crossing the endosomal membrane in transit (diphtheria, tetanus, and anthrax toxins). Several RGD motif-containing proteins bind integrins and enter cells by caveolae-mediated endocytosis. Integrin-mediated cell entry also occurs for whole bacteria and viruses. CRN and CHXC proteins have been shown to enter cells during pathogenesis, but it is unknown if they can enter in the absence of the pathogen, or what pathway they enter by.

Phosphoinositides have been best characterized as intracellular lipids with roles in three broad arenas (reviewed in Falasca & Maffucci, 2009). They function as signaling molecules, as regulators of integral membrane proteins, and as membrane localization sites. For example, in plants and animals, PtdIns-4,5-P is hydrolyzed by PLC (phospholipase C) into d-myo inositol (IP3) during abiotic and biotic stress responses. IP3 leads to activation of intracellular calcium release, which cascades into a multitude of cell responses. PtdIns-3-P and PtdIns-4-P localize to the endosomal vesicles and endocytotic recycling compartments, respectively. Their localization leads to the recruitment of a diverse array of proteins, which assist in the shuttling and processing of these compartments. PtdIns-Ps have also been shown to regulate ion channels through proximal contact and concentration. More recently, PtdIns-3-P has been demonstrated to play an active role at the plasma membrane of animal cells, particularly with respect to secretion of insulin and neurotransmitters, and during the response of adipocytes to insulin (Falasca & Maffucci, 2009; Krag et al., 2010; Dominguez et al., 2011). Plasma membrane PtdIns-3-P appears to be controlled by a novel wortmannin-resistant PtdIns-3-kinase that is recruited to the plasma membrane through the action of small GTPases TC10 and Rab5 (Falasca & Maffucci, 2009).

Although PtdIns-3-P had been identified at the plasma membrane under certain conditions, it had not previously been identified on the outer leaflet, where it would be needed if it were to mediate effector entry. Kale et al. (2010) used three well-characterized and structurally different PtdIns-3-P binding proteins (Hrs-2xFYVE, Vam7p-PX, and PEPP1-PH domains), each fused to GFP or mCherry, as biosensors to detect PtdIns-3-P on the outer leaflet of soybean root cells and human lung epithelial cells (Kale et al., 2010; Plett et al., 2011). In the case of human red blood cells, though, neither the PtdIns-3-P biosensors nor any effectors bound to the outside of these cells, which appears to rule out any of the lipids, glycolipids, carbohydrates, glycoproteins etc. found on these cells as alternative targets for the PtdIns-3-P biosensors or for the effectors. The strongest evidence for the hypothesis that PtdIns-3-P directly mediates cell entry was obtained by showing that the three PtdIns-3-P-binding proteins could all block entry into soybean root and leaf cells and into human epithelial cells (Kale et al., 2010; Plett et al., 2011), whereas control proteins that bound only PtdIns-4-P did not. Plett et al. (2011) subsequently showed independently that a PtdIns-3-P-binding protein could block entry of MiSSP7 into poplar root cells. Further support was obtained from the observation that the PtdIns-3-kinase inhibitor wortmannin causes a depletion of cell surface PtdIns-3-P in human cells and loss of entry into human, soybean, and poplar cells (Kale et al., 2010; Plett et al., 2011).

Recently, Gan et al. (2010) reported that they could not detect RXLR-dependent binding of AvrL567 or AvrM to PtdIns-3-P and Yaeno et al. (2011) reported that they could not detect RXLR-dependent binding of Avr3a or Avr1b to PtdIns-3-P (Gan et al., 2010; Yaeno et al., 2011). It is currently difficult to reconcile these reports with those of Kale et al. (2010) and Plett et al. (2011) as the different studies used different binding conditions in their lipid blot assays. Furthermore, Gan et al. (2010) and Yaeno et al. (2011) did not validate their results using liposome binding. Under the conditions of their lipid blot assays, Gan et al. (2010) could detect binding of the C-terminus of AvrM to anionic lipids (PtdIns, PtdSer, PtdIns-3-P, PtdIns-4-P and Ptd-5-P) while Yaeno et al. (2011) detected binding of the C-terminus of Avr1b and Avr3a to all PtdIns monophosphates. It is not yet clear if or how these C-terminal binding sites may contribute to cell entry, as none of the studies so far have carefully quantified entry activity by the relevant mutant effectors. It is interesting to speculate that some of the most essential pathogen effectors may have evolved additional high-affinity PtdIns-3-P binding sites to aid in their entry into host cells in competition with other effectors. Alternatively, if membrane PtdIns-3-P levels are low before infection, or conversely are down-regulated by the plant during infection as a defense measure, then effectors with high PtdIns-3-P affinity could still gain entry.

The hypothesis that PtdIns-3-P acts as the entry receptor for oomycete and fungal RXLR and RXLR-like effectors opens up many important questions regarding extracellular phosphoinositides. For example, does PtdIns-3-P reach the outside of cells by passive diffusion or via an active process? Is there an extracellular kinase or are the phosphoinositides exported by exocytosis or via a floppase? Why is the distribution different in different cell types? Does the symbiont play a role in stimulating or depositing more PtdIns-3-P during infection? How are PtdIns-3-P and other phospholipids modulated during infection? Are there essential host proteins that utilize PtdIns-3-P-mediated endocytosis?

Blocking entry of effectors into host cells: biotechnology applications

The discovery of PtdIns-3-P-mediated cell entry by RXLR and RXLR-like effectors included the development of two different methods to block effector cell entry into plant and animal cells. Cell entry of all seven oomycete RXLR and fungal RXLR-like effectors could be blocked by sequestering PtdIns-3-P through the use of PtdIns-3-P binding proteins, but not by a PtdIns-4-P binding protein (Kale et al., 2010; Plett et al., 2011). Effector entry could also be blocked by inositol-1,3-bisphosphate (1,3IP2) or inositol-1,4-bisphosphate (1,4IP2), which are competitive inhibitors of PtdIns-3-P binding (Kale et al., 2010; Plett et al., 2011). Blockage of effector entry by either sequestering PtdIns-3-P or competitive inhibition of the effectors’ binding sites could potentially be used to inhibit infection by pathogens that depend on such effectors (Fig. 4).

Figure 4.

Blocking effector entry for disease resistance. Interfering with phosphatidylinositol-3-phosphate (PtdIns-3-P)-mediated effector entry may provide protection against pathogens that depend on such effectors. (A) Secreted RXLR and RXLR-like effectors are capable of translocating into host cells through receptor-mediated endocytosis after binding PtdIns-3-P. (B) Secretion of PtdIns-3-P-binding proteins could sequester surface PtdIns-3-P, thereby blocking effector binding. (C) Secretion of small molecules or peptides that mimic the structure of inositol-1,3-bisphosphate (1,3IP2), a competitive inhibitor of PtdIns-3-P binding, could also block binding. (D) Secretion of enzymes capable of modifying or degrading PtdIns-3-P also might prevent effector entry. Controlled or targeted expression of the blocking molecules (B–D) may be required as the intrinsic role of external PtdIns-3-P is still unknown.

Conceptually, a reduction in translocated effectors should result in decreased virulence by pathogens and improved disease resistance. Because many effectors are targeted, in principle it would be difficult for multiple effectors to simultaneously hijack one of the several other pathways for cell entry. On the other hand, constitutive expression of a protein to block cell surface PtdIns-3-P may alter the development and physiology of the plant and/or interfere with effector proteins required for beneficial plant microbe interactions. Controlling the timing and site of deployment of effector blocking molecules may be key to successfully implementing the technology in field settings to maximize yield and protection.

Perspective

The deployment of effectors is essential for successful infection by many pathogenic and mutualistic symbionts. Bacterial and apicomplexans pathogens utilize a variety of specialized secretion systems for effector delivery. By contrast, several reports (Dou et al., 2008b; Kale et al., 2010; Rafiqi et al., 2010; van West et al., 2010; Plett et al., 2011) now support the hypothesis that oomycete and fungal effectors enter host cells via a pathogen-independent mechanism, probably a form of endocytosis, thus resembling the endocytotic entry of bacterial toxins into animal cells via binding to glycolipid receptors. Oomycete RXLR effectors and a number of fungal effectors containing RXLR-like motifs appear to utilize PtdIns-3-P binding to initiate receptor-mediated endocytosis, although the literature is not yet fully consistent on this point and further work is needed. It is not yet known why evolutionarily diverse effectors should target PtdIns-3-P to mediate entry when many other surface molecules are available. Nevertheless, these other molecules may be the target of emerging conserved protein translocation motifs such as the oomycete crinkler motif or the Albugo CHXC motif. It will be of interest to determine if other classes of host-associated symbionts, such as parasitic nematodes and insects, produce cell-entering effectors that utilize PtdIns-3-P or other lipids to enter host cells. By identifying and characterizing effector translocation mechanisms, novel strategies for broad-spectrum disease resistance can potentially be developed.

Acknowledgements

I would like to thank Brett Tyler, Chris Lawrence, John McDowell, Daniel Capelluto, Weixing Shan, Daolong Dou, Biao Gu, Vincenzo Antigiani, Amanda Rumore and Ryan Anderson for valuable discussions. This work was supported by the Agriculture and Food Research Initiative of the USDA National Institute of Food and Agriculture, grant number #2007-35319-18100, and by the US National Science Foundation, grant number IOS-0924861. S.D.K. was supported in part by a U.S. National Science Foundation predoctoral fellowship.

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