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1 Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
2 Aberdeen Oomycete Group, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK
3 University of Dundee, Division of Plant Sciences, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
Correspondence
Stephen C. Whisson
Steve.Whisson{at}scri.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Ellis et al., 2007; He et al., 2007; Birch et al., 2008). These proteins are termed effectors and may perform a variety of functions in the establishment of host infection, but are generally considered to reprogramme the host cell to the benefit of the pathogen. This may be through redirection of cellular resources, cell structural reorganization, or suppression of defence responses. The mechanisms by which effector proteins are translocated into host cells may include direct injection, as for plant-pathogenic nematodes (Davis et al., 2008), delivery via a specialized type III secretion system (T3SS), as for bacteria (Gálan & Wolf-Watz, 2006), or by as yet undetermined mechanisms mediated through host-targeting (HT) peptide motifs, as for malaria parasites (Hiller et al., 2004; Marti et al., 2004), fungi (Dodds et al., 2004; Ellis et al., 2006; Catanzariti et al., 2007) and oomycetes (Rehmany et al., 2005; Armstrong et al., 2005; Whisson et al., 2007; Birch et al., 2008).
Evolutionarily distinct from true fungi, oomycetes encompass a wide range of destructive filamentous plant pathogens that include the genera Phytophthora and Pythium, and the downy mildews (genera Hyaloperonopsora, Bremia, Plasmopara). Oomycetes belong within the stramenopiles (heterokonts) and are phylogenetically rooted with the apicomplexan genera such as Plasmodium (malaria parasites), Cryptosporidium and Toxoplasma (Baldauf et al., 2000; Burki et al., 2007). Phytophthora infestans is the best-studied oomycete, following its role in precipitating the Irish potato famines in the mid-19th century. It remains the most economically important potato pathogen worldwide.
Ph. infestans, like many other oomycete and fungal pathogens, produces specialized intracellular infection structures called haustoria through invagination of the host cell membrane, allowing the pathogen to form an intimate association with the host cell. Haustoria are thus encased in the host plasma membrane and this situation is reminiscent of the parasitophorous vacuole produced by Pl. falciparum in infected animal erythrocytes. The pathogen/parasite exports proteins into the extrahaustorial matrix/lumen of the parasitophorous vacuole by means of a conventional hydrophobic signal sequence. Transport of effector/virulence proteins through the host-derived membrane requires an additional HT signal, located near the N-terminal signal peptide of many secreted proteins, the core of which is RxLxE/D/Q (Plasmodium) or RxLR (oomycetes) (Hiller et al., 2004; Marti et al., 2004; Bhattacharjee et al., 2006, 2008; Whisson et al., 2007; Birch et al., 2008; MacKenzie et al., 2008). A subset of RxLR effectors also possess a region enriched for acidic residues following the RxLR motif, and terminating in EER. Peptide sequences surrounding the core RxLxE/D/Q or RxLR signal also have a role in translocation (Lopez-Estraño et al., 2003; Bhattacharjee et al., 2006; Whisson et al., 2007) and have additional properties such as hydrophobicity profile relevant to subcellular location (Hiss et al., 2008).
The avirulence gene Avr3a from the oomycete Ph. infestans encodes the RxLR-EER effector protein Avr3a, which is recognized by the R3a resistance protein in the host cytoplasm, triggering the hypersensitive response (HR) (Armstrong et al., 2005; Whisson et al., 2007). The HR is a form of programmed cell death in the resistant plant that limits the spread of pathogen growth, as the pathogen requires living host tissue for survival. The C-terminal region of Avr3a is sufficient for recognition (Bos et al., 2006), and both of the RxLR and EER motifs are essential for translocation of Avr3a into the host cell (Whisson et al., 2007). Furthermore, the RxLR-EER domain was capable of translocating other fused proteins, such as the β-glucosidase (gusA) gene, without the involvement of any additional peptide sequences from the C-terminal region of Avr3a (Whisson et al., 2007). The validated recognition of Avr3a only within host cells yields a useful bioassay to study the specificity and mechanisms of effector translocation in oomycetes. That is, transformation of a virulent Ph. infestans isolate (carrying the non-recognized avr3a allele) with various Avr3a constructs, in which the endogenous translocation motif has been replaced with alternative sequences, should generate avirulent transformants if the alternative sequence is functionally similar.
Previously, alanine (AAAA-EER and RxLR-AAA) and physico-chemically conservative (KMIK-DDK) substitutions of the RxLR-EER motifs in Avr3a were shown to abolish translocation of the effector following its secretion from haustoria (Whisson et al., 2007). However, some RxLR effectors from Ph. infestans, Phytophthora sojae and Hyaloperonospora parasitica exhibit potential amino acid variation in the ‘EER’ motif of candidate effectors (Rehmany et al., 2005; Win et al., 2007; Whisson et al., 2007). Examples are the Avr1b protein from Ph. sojae, which contains the variant GER sequence instead of EER (Rehmany et al., 2005), and the ATR13 protein from H. parasitica, which lacks a recognizable EER motif (Allen et al., 2004). Although the RxLR-AAA and KMIK-DDK variants of Avr3a were not translocated, investigation of the translocation potential of ATR13-like RxLR sequences lacking an EER motif was not addressed (Whisson et al., 2007). ATR13 contains heptad leucine/isoleucine repeats commencing within 10 residues of the RxLR motif (Fig. 1; Allen et al., 2004) where the EER motif is located in ATR1NdWsB and Avr3a, and it is possible that these repeats could also be involved in the translocation process.
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), implying that plant and animal eukaryotic pathogens may share some conserved mechanism to deliver effector/virulence proteins into the host cell. Here we have performed the reciprocal experiments with Ph. infestans transformants to demonstrate function of the Pl. falciparum host targeting signal in translocation. Additionally, we have demonstrated that the RxLR and RxLR-EER motifs found in avirulence proteins from a distantly related oomycete can function to deliver the Avr3a effector into host cells. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Transformation vector construction.
The National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) accession numbers for the genes used in this study are: AJ893356 (Ph. infestans Avr3a), AY842877 (H. parasitica ATR1NdWsB), AY785301 (H. parasitica ATR13) and AAD31511 (Pl. falciparum PfHRPII). ATR1NdWsB (Rehmany et al., 2005) and ATR13 (Allen et al., 2004) are cloned avirulence genes from the oomycete H. parasitica, the causal agent of downy mildew in the model plant Arabidopsis thaliana. The primary positions of the RxLR-EER motifs relative to each other and relative to the signal peptide are similar between Avr3a and ATR1NdWsB (Fig. 1
). To conserve the primary physical location between the signal peptide and the 33 aa functional core of the Pl. falciparum histidine-rich protein II (PfHRPII) HT sequence (Bhattacharjee et al., 2006), 10 residues downstream of the signal peptide of Avr3a were also retained, as this physical spacing might be required for function (Fig. 1).
For all transformation vectors, the C-terminal, elicitor-active region of the Ph. infestans Avr3a gene encoding amino acids 61–147 was amplified by PCR from an existing plasmid clone (Whisson et al., 2007) using the following conditions: each 50 µl PCR contained 0.4 U Phusion Hot Start polymerase (New England BioLabs), 10 µl of 5x reaction buffer (New England BioLabs), 15 mM deoxynucleotide triphosphates (Promega), 30 µM forward and reverse primers (Table 1), and 10 ng Avr3a plasmid DNA. All primer pairs for PCR of Avr3a sequence encoding the C-terminal 86 aa used the Avr3aStopSacR reverse primer. Forward primers were Avr3aPspF (fusion to ATR1NdWsB SP-RxLR-EER), Avr3aNotF (fusion to ATR13 SP-RxLR only), Avr3aBssF (fusion to ATR13 SP-RxLR-5xHeptad), and AVRPfHTSalF2 (fusion to PfHRPII RxLxE). Thermocycling conditions were as follows: 96 °C for 5 min, followed by 35 cycles of 96 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s. A final extension step of 72 °C for 10 min was included. Plasmid clones of H. parasitica avirulence genes ATR1NdWsB and ATR13 were obtained from J. Beynon, University of Warwick, UK. Sequences encoding the N-terminal 31 or 42 aa of Ph. infestans Avr3a, 62 aa of H. parasitica ATR1NdWsB, and 43 or 85 aa of H. parasitica ATR13 were amplified by PCR as described above using the same thermocycling conditions. Primer pairs were as follows: ATR1ClaF and ATR1PspR (ATR1NdWsB SP-RxLR-EER), ATR13claF and ATR13NotR (ATR13 SP-RxLR), ATR13claF and ATR13BssR (ATR13 SP-RxLR-5xHeptad), Avr3aClaF2 and AVRPfHTSalR2 (Avr3a 31 aa including PfHRPII RxLxE). All PCR products were purified with the Qiagen Minelute kit using the supplied protocol. The two sections of the chimeric Avr3a effector genes were digested with the following restriction endonucleases: PspOMI (ATR1NdWsB), NotI or BssHII (ATR13), and SalI (PfHRPII). Ligations of the two sections of gene used T4 DNA ligase (Promega) and were carried out at 4 °C for 16 h. Ligated fragments were separated by agarose gel electrophoresis and DNA fragments of the expected size purified from the gel (Qiagen Minelute kit) using the supplied protocol. Purified ligation products were digested with ClaI and SacII, purified again, and ligated (T4 DNA ligase, 4 °C, 16 h) into the pTOR oomycete expression vector (Blanco & Judelson, 2005; GenBank accession no. EU257520) digested with the same enzymes.
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Transformation of Ph. infestans.
Stable transformation of Ph. infestans was achieved as described by Whisson et al. (2007), using a modified PEG-CaCl2-Lipofectin protocol (Judelson et al., 1991). A version of this protocol can be found at http://138.23.152.128/protocols/protocols.html. The protocol was modified by substituting Novozym 234 with a mixture of 5 mg ml–1 lysing enzymes (from Trichoderma harzianum; Sigma L1412) and 2 mg ml–1 cellulase (from Trichoderma reesei; Sigma C8546) to digest cell walls of germinating sporangia for 30 min to release protoplasts. The protocol was scaled up to use 1 ml of prepared protoplasts per transformation. For each transformation, 50 µg of plasmid DNA, purified with the Qiagen Plasmid Maxi kit, using the supplied protocol, was mixed with 86 µl Lipofectin reagent (Invitrogen) and water to a final volume of 142 µl. Protoplasts suspended in 1 ml mannitol/Tris (MT) osmoticum were mixed with the 142 µl of DNA/Lipofectin mixture. After transformation, protoplasts were regenerated for 48–72 h in pea broth (Whisson et al., 2005) containing 20 g sucrose l–1 and 1 M mannitol, prior to selection of transformants on RyeA agar amended with 5 µg geneticin ml–1. Transformants were then transferred to, and maintained on, RyeA agar amended with 20 µg geneticin ml–1 and 10 µg pimaricin ml–1.
Inoculation of potato leaves with Ph. infestans transformants.
Sporangia (5x104 ml–1) of stable transformants were inoculated in 10 µl droplets on either side of the midvein of detached leaflets of ‘Pentland Ace’ (R3a), ‘Bintje’ or ‘Craigs Royal’ (no R genes) potato plants. Inoculated leaflets were incubated at 20 °C for 5–7 days in high relative humidity in sealed clear plastic boxes to enable disease symptoms to develop. For each transformant, six leaflets were inoculated. Samples were taken at 5 days for RNA extraction and RT-PCR analysis. Non-transformed isolate 88069 was used as a control for virulence on R3a ‘Pentland Ace’. Transformant K-7 (Whisson et al., 2007), overexpressing Avr3a, was used as an avirulent control for Avr3a recognition in R3a ‘Pentland Ace’. Transformants were classified as virulent on R3a ‘Pentland Ace’ if an expanding necrotic lesion was formed, and aerial sporulation was visible at the lesion centre at 5 days post-inoculation. Transformants were classified as avirulent on R3a ‘Pentland Ace’ if necrotic flecking or spotting, or restricted lesion with no sporulation, was formed at the inoculation sites. Infected leaves were photographed under polarized light to minimize reflection from the leaf surface.
Total RNA isolation, first-strand cDNA synthesis, and RT-PCR.
Total RNA was isolated from frozen or fresh infected leaf tissue with the Qiagen RNeasy Plant Mini kit, using the protocol for fungi supplied by the manufacturer. First-strand cDNA was synthesized by oligo dT priming with the First Strand cDNA Synthesis kit (GE Healthcare), using the supplied protocol. PCR from first-strand cDNA used the following conditions: each 20 µl PCR contained 0.5 U Taq polymerase (Molzym), 2 µl of 10x reaction buffer (New England BioLabs), 15 mM deoxynucleotide triphosphates (Promega), 30 µM forward and reverse primers (Table 1), and 10 ng first-strand cDNA. Thermocycling conditions were as follows: 96 °C for 5 min, followed by 40 cycles of 96 °C for 30 s, and 60 °C for 60 s. Amplification of the Ph. infestans ActA gene was used as a constitutively expressed positive control for pathogen presence in infected leaf tissue. PCR products were separated on 2 % agarose gels in 1x Tris/borate/EDTA (TBE) buffer (Sambrook et al., 1989) and visualized on a UV transilluminator after staining with SYBRsafe (Invitrogen).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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): constructs ATR1NdWsB : : Avr3a, ATR13(SP-RxLR-5xHeptad) : : Avr3a and ATR13(SP-RxLR) : : Avr3a, respectively. Construct ATR13(SP-RxLR) : : Avr3a encoded a protein that included only the three alanine residues normally following the ATR13 RxLR.
Inoculation of ‘Bintje’ or ‘Craigs Royal’ (no R genes) potatoes with stable Ph. infestans ATR1NdWsB : : Avr3a, ATR13(SP-RxLR-5xHeptad) : : Avr3a and ATR13(SP-RxLR) : : Avr3a transformants showed the majority to have wild-type or near wild-type levels of aggressiveness on susceptible ‘Craigs Royal’ or ‘Bintje’ potatoes. The exceptions were transformants ATR1Avr3a-16 and 22, which exhibited reduced aggressiveness but were still capable of infection. In contrast, on ‘Pentland Ace’ potatoes (expressing the R3a resistance gene), transformants ATR1Avr3a-1, 2, 8, 14, 16 and 22 exhibited an avirulent phenotype (restricted necrotic flecking or pitting), indicating recognition of Avr3a inside the host plant cells. The positive control transformant K-7, overexpressing intact Avr3a (from Whisson et al., 2007), also exhibited an avirulent phenotype (Fig. 2). Occasionally, a small, restricted lesion developed on R3a leaves for transformants (including positive control K-7) in individual virulence tests that was characteristic of a ‘trailing’ HR, in that lesion size was greatly reduced compared to the lesion on ‘Bintje’ or ‘Craigs Royal’, and no sporulation was observed (results not shown). A virulent reaction on R3a ‘Pentland Ace’ leaves was observed for transformants ATR1Avr3a-9, 15, and 17 (Table 2).
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; Fig. 2 for example). Transformants HEPT-1, 3, 4, 5 and 6 expressing the ATR13(SP-RxLR-5xHeptad) : : Avr3a fusion were also avirulent on R3a leaves, whereas transformants HEPT-2, 7 and 8 failed to trigger R3a-mediated recognition (Table 2).
RT-PCR assays using primers specific to the sequences encoding the RxLR-EER or RxLR domains of ATR1NdWsB or ATR13, respectively, for the transformants revealed expression of the transgene fusion in all cases (Table 2). Additionally, RT-PCR assays using primers specific to the endogenous avr3a sequence demonstrated that the endogenous avr3a expression had not been transcriptionally silenced in transformants exhibiting a virulent phenotype.
The host-targeting (HT) signal from the apicomplexan Pl. falciparum is functional in Ph. infestans
The region encoding RxLR and EER motifs from Avr3a was replaced with the HT signal from the Pl. falciparum PfHRPII protein, a virulence protein known to be translocated into erythrocytes during infection (Bhattacharjee et al., 2006). Inoculation of potato leaves with PfHRPII(HT) : : Avr3a transformants, as for ATR1NdWsB : : Avr3a and ATR13 : : Avr3a fusions (Figs 1 and 2), revealed the majority (PfHT-1, 6, 9, 14, 15, 17, 20, and 22) to be avirulent on potato expressing the resistance gene R3a (Table 2; Fig. 2 for example), implying that the product of the transgene had been translocated into the host cell. Transformants PfHT-5, 7, 8, 18, 19 and 23 were virulent on R3a ‘Pentland Ace’ potato leaves (Table 2). With the exceptions of transformants PfHT-6 and 14, all transformants exhibited wild-type aggressiveness. Transformants PfHT-6 and 14 exhibited reduced aggressiveness on the susceptible ‘Bintje’ and ‘Craigs Royal’ potatoes (results not shown).
RNA was extracted from the infected leaves, and the expression of the PfHRPII : : Avr3a transgene assayed by RT-PCR using primers (Table 1) specific to the PfHRPII HT sequence (Table 2). All transformants eliciting an avirulent phenotype also expressed the transgenic Avr3a sequence. Virulent transformants (on ‘Pentland Ace’) PfHT-5, 8, 18, and 19 exhibited no transgene expression, while expression was detected in PfHT-7 and 23.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Along with this previous demonstration (Bhattacharjee et al., 2006), our findings confirm that the host specificity of the originating organisms has little bearing on the generic function of these motifs in translocating effector proteins to the inside of host animal and plant cells.
The majority of, but not all, Ph. infestans transformants containing H. parasitica or Pl. falciparum translocation sequences fused to Avr3a triggered the HR on R3a ‘Pentland Ace’ potato leaves. For some of these virulent transformants, no expression of the transgenic sequences could be detected by RT-PCR. Detection by RT-PCR of expression for the endogenous avr3a allele indicated that transcriptional silencing of both transgene and endogenous avr3a had not occurred. These transformants were not considered further in this study. There remained some transformants for which transgene expression could be detected, but which were consistently virulent on R3a ‘Pentland Ace’ leaves. The proportion of virulent transformants with detectable transgene expression in the present study is similar to that observed previously (two out of nine transformants; data from Whisson et al., 2007 shown in Table 2) for Avr3a transformants. There are at least two explanations as to how this may occur. Firstly, the level of the chimeric protein may be below a threshold to either translocate the effector protein, or be detected by the R3a protein. Alternatively, disruption of the fusion transgene during genomic integration may have led to detection of expression by the primers targeted to the unique 5' region of the chimeric transgene, while the sequence encoding the recognized Avr3a elicitor may have been disrupted. Transformation of Ph. infestans uses intact plasmids, which presumably integrate into the genome through random breaks in the plasmid sequence; disruption of the promoter-transgene-terminator will therefore occur in a proportion of transformants. This possibility was not investigated further here. However, that all avirulent transformants exhibited transgene expression provides evidence of Avr3a translocation by the alternative RxLR, RxLR-EER or RxLxE/D/Q domains, since R3a recognition of Avr3a has been shown to occur inside host cells (Armstrong et al., 2005; Whisson et al., 2007).
Our results support the hypothesis that some plant and animal eukaryotic pathogens share a conserved mechanism to deliver effector/virulence proteins into the host cell, inform on the flexibility of these protein translocation motifs, and raise the question of whether RxLxE/D/Q and RxLR (including RxLR-EER) domains are evolutionarily convergent solutions to the common requirement of effector delivery to the inside of host cells (Birch et al., 2008). The motif RxL is clearly shared between Plasmodium and oomycetes, as is the position relative to the signal peptide, and the requirement of surrounding sequences (Fig. 1). These properties have been proposed as signifying involvement of secondary or tertiary peptide structures (Bhattacharjee et al., 2006). Sequencing of either genomes or expressed sequence tags (ESTs), and functional characterization of motifs from a broader phylogenetic spectrum across the stramenopile and apicomplexan lineages, may reveal if both translocation motifs arose from a common ancestor or independently. Amongst the oomycetes, ESTs encoding candidate RxLR proteins have been identified from Aphanomyces euteiches and Saprolegnia parastica, both of which are more distantly related to Ph. infestans than is H. parasitica (van West, 2006; Gaulin et al., 2008; Phillips et al., 2008).
It was intriguing that the ATR13(RxLR) : : Avr3a construct was sufficient to translocate the Avr3a elicitor. This suggests that additional downstream sequences, such as an aspartic/glutamic acid-enriched stretch ending in EER (Avr3a, ATR1NdWsB), or heptad leucine repeats (ATR13), are not always required for translocation and that the peptide sequence upstream of the RxLR motif may also function in effector delivery. The predicted cytoplasmic location of the RPP13 resistance protein (Bittner-Eddy et al., 2000), and recent experimental evidence with delivery of ATR13 from the T3SS of the bacterium Pseudomonas syringae pv. tomato DC3000, have demonstrated that translocation is required for recognition to occur (Sohn et al., 2007; Rentel et al., 2008). The implications of our results with ATR13 is that the genomic pool of potentially translocated effectors in Ph. infestans, and other oomycetes, may be far greater than existing predictions based on the dual RxLR-EER motif. A regular expression prediction from signal-peptide-encoding ORFs in the Ph. infestans genome revealed 1960 SP-RxLR-containing sequences (Whisson et al., 2007). Whilst this prediction is likely to contain many false positives, real-time RT-PCR of seven RxLR-only genes selected from ESTs demonstrated expression profiles for these genes similar to those of RxLR-EER genes in being upregulated during plant infection (Whisson et al., 2007).
The RxLR-EER domain from the Ph. sojae avirulence protein Avr1b has recently been demonstrated to translocate a fusion protein into plant cells in the absence of the oomycete pathogen (Dou et al., 2008). Various arginine-rich short peptides have been reported to translocate through the cell plasma membrane. These cell-penetrating peptides (CPPs) include Tat derived from the HIV-1 Tat protein (Frankel & Pabo, 1988; Mann & Frankel, 1991), penetratin derived from the Antennapedia homeodomain from Drosophila melanogaster (Derossi et al., 1994, 1996), and oligoarginine peptides (Futaki et al., 2001). Despite an absence of obvious sequence or structural homology, common internalization mechanisms have been suggested for these CPPs on the basis of their abundance of arginine residues (Suzuki et al., 2002). Thus, by virtue of arginine content and in function, peptide domains with core RxLR and RxLxE/D/Q motifs may also be considered as CPPs.
Two models for oomycete effector translocation have been proposed. The first involves interactions with pathogen-derived chaperones in a ‘translocon’ to mediate effector translocation (Morgan & Kamoun, 2007). The second model is based on pathogen mimicry of RxLR-EER motifs in host proteins to gain entry to host cells via interactions with the phospholipid membrane and endocytosis (Birch et al., 2008). Although both Pl. falciparum and oomycete motifs appear to be functionally analogous, the mechanisms mediating the delivery of effectors into the host cell are presently unknown and will be the focus of future research.
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ACKNOWLEDGEMENTS |
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Edited by: C. Boucher
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Received 8 July 2008;
revised 15 August 2008;
accepted 25 August 2008.
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