Effector proteins are key elements in plant–fungal interactions. The rice blast fungus Magnaporthe oryzae secretes numerous effectors that are suspected to be translocated inside plant cells. However, their cellular targets and the mechanisms of translocation are still unknown. Here, we have identified the open reading frame (ORF3) corresponding to the M. oryzae avirulence gene AVR1–CO39 that interacts with the rice resistance gene Pi–CO39 and encodes a small secreted protein without homology to other proteins. We demonstrate that AVR1–CO39 is specifically expressed and secreted at the plant–fungal interface during the biotrophic phase of infection. Live-cell imaging with M. oryzae transformants expressing a translational fusion between AVR1–CO39 and the monomeric red fluorescent protein (mRFP) indicated that AVR1–CO39 is translocated into the cytoplasm of infected rice cells. Transient expression of an AVR1–CO39 isoform without a signal peptide in rice protoplasts triggers a Pi–CO39-specific hypersensitive response, suggesting that recognition of AVR1–CO39 by the Pi–CO39 gene product occurs in the cytoplasm of rice cells. The native AVR1–CO39 protein enters the secretory pathway of rice protoplasts as demonstrated by the ER localization of AVR1–CO39:mRFP:HDEL translational fusions, and is correctly processed as shown by Western blotting. However, this secreted AVR1–CO39 isoform triggers a Pi–CO39-specific hypersensitive response and accumulates inside rice protoplasts as shown by Western blotting and localization of AVR1–CO39:mRFP translational fusions. This indicates that AVR1–CO39 is secreted by rice protoplasts and re-enters into the cytoplasm by unknown mechanisms, suggesting that translocation of AVR1–CO39 into rice cells occurs independently of fungal factors.
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Effector proteins are key players in plant–microbe interactions, and manipulate plant immunity and physiology to promote infection (Martin and Kamoun, 2011). Some effectors, named avirulence (AVR) proteins, are recognized by so-called plant resistance (R) proteins. Most of these R proteins contain nucleotide-binding (NB) and leucine-rich repeat (LRR) domains, and are highly polymorphic (Dodds and Rathjen, 2010; Elmore et al., 2011; Jones and Dangl, 2006). Recognition of Avr proteins by R proteins triggers a strong and rapid plant defence response frequently associated with a localized programmed cell death called the hypersensitive response (HR). Effectors recognized by plant R proteins generally display signatures of strong diversifying selection (Martin and Kamoun, 2011). Genome analyses revealed that eukaryotic filamentous pathogens have large numbers of effectors (200–500; Martin and Kamoun, 2011). These effectors are secreted into the plant tissue during infection, and act either outside the host cells or are translocated into plant cells and act intracellularly (de Jonge et al., 2011; Stergiopoulos and de Wit, 2009). The mechanism underlying translocation is unknown, and may differ among fungal effectors. No molecular structure for the secretion and delivery of effectors inside plant cells has been identified in fungi and oomycetes. This strongly differs from plant pathogenic bacteria, which deliver effectors into the plant cytoplasm by a conserved type III secretion system. Conserved translocation signals such as RXLR and LXLFLAK motifs have been identified in oomycete effectors (Schornack et al., 2010; Whisson et al., 2007). The highly conserved RxLR motif was detected at the N–terminus of hundreds of oomycete candidate effectors, and is present in all known oomycete AVR proteins (Stassen and Van den Ackerveken, 2011). In fungi, translocation motifs necessary and sufficient for the internalization of effectors into host plant cells have been identified for only four effectors: ToxA from the wheat tan spot fungus Pyrenophora tritici-repentis, MISP7 from the symbiotic basidiomycete fungus Laccaria bicolor, and AvrL567 and AvrM from the biotrophic flax rust fungus Melampsora lini (Manning et al., 2008; Plett et al., 2011; Rafiqi et al., 2010). However, these domains do not display either sequence homology or shared structural properties. In ToxA, a solvent-exposed loop containing an RGD cell attachment motif acts as a translocation signal (Manning et al., 2008). In MISP7, a motif distantly related to the RxLR motif is necessary and sufficient for its translocation into poplar root cells (Plett et al., 2011). In AvrL567 and AvrM, small unrelated N–terminal motifs that are necessary and sufficient for translocation into host cells were identified (Rafiqi et al., 2010). Highly degenerate RXLR-like sequences were also identified in other effector proteins, but the relevance of RXLR-like sequences for host cell translocation is controversial (Gan et al., 2010; Kale et al., 2010; Rafiqi et al., 2010).
Magnaporthe oryzae is an ascomycete fungus that causes disease on more than 50 cultivated or wild monocotyledonous species (Couch et al., 2005). It has a hemibiotrophic lifestyle characterized by intracellular growth in living host cells at early stages of infection (biotrophy) and a switch to a necrotrophic phase with active killing of host cells at a later stage of infection (Ebbole, 2007). Isolates that are pathogenic on rice cause blast disease, the most serious fungal disease of rice (Skamnioti and Gurr, 2009). Isolates that are pathogenic on wheat have recently emerged in South America, and are responsible for a rapidly expanding disease (Urashima et al., 1993). A large number of molecular and genomic tools have been developed for both rice and M. oryzae, and allow detailed investigation of the molecular mechanisms underlying their interaction (Liu et al., 2010).
Effectors are thought to be central to the pathogenic lifestyle of M. oryzae, but little is known about their exact role during infection and their mode of action on rice (Mentlak et al., 2011; Valent and Khang, 2010). The first M. oryzae effectors were identified as cultivar- or species-specific Avr proteins (Li et al., 2009; Orbach et al., 2000; Sweigard et al., 1995). Independently, additional Avr effectors and many more putative effector-encoding genes have been identified in large-scale genome analyses (Dean et al., 2005; Mosquera et al., 2009; Yoshida et al., 2009). Experimental evidence indicates that some M. oryzae effectors are translocated into the cytoplasm of infected host plant cells (Valent and Khang, 2010). Four different M. oryzae AVR effectors, Avr-Pita, Avr-Pia, Avr-Pik/km/kp and AvrPiz–t, are recognized by presumably cytoplasmic R gene-encoded proteins, and transient expression of Avr-Pita, Avr-Pia, Avr-Pik and Avr-Pii in the cytoplasm of plants carrying the respective R genes triggers the HR, suggesting that these effectors act intracellularly (Jia et al., 2000; Yoshida et al., 2009). Three other M. oryzae effectors, Pwl1, Pwl2 and Bas1, were detected in the cytoplasm or nucleus of host plant cells during infection, when expressed as translational fusions with fluorescent proteins by transgenic M. oryzae strains (Khang et al., 2010). These results provide direct evidence for the translocation of M. oryzae effectors into rice cells. However, until now, neither host cell translocation or activity inside host cells have been demonstrated for any M. oryzae effector. Live-cell imaging of rice epidermis infected with M. oryzae strains expressing effector:fluorescent protein (effector:FP) fusions highlighted a particular structure, called the biotrophic interfacial complex (BIC), that accumulates translocated effector:FPs in infected tissues (Khang et al., 2010). The BIC is a punctate membrane-rich structure located at the biotrophic fungal–plant interface. Promoter sequences from effector-coding genes were suspected to be involved in the accumulation of effector:FPs at the BIC (Khang et al., 2010). Effector:FPs that were not translocated into rice cells displayed weak accumulation in BICs, and uniform outlining of infectious hyphae at the fungal–plant interface. The biological significance of BICs is unclear, but they have been proposed to play a role in translocation of effectors into the host cytoplasm (Khang et al., 2010; Valent and Khang, 2010).
AVR1–CO39 is an M. oryzae Avr gene that triggers the HR and resistance in rice cultivars carrying the Pi–CO39R gene. Pi–CO39 was mapped on chromosome 11 at a locus containing a cluster of NB–LRR R genes, but its molecular identity is unknown (Chauhan et al., 2002; Leong, 2008). The AVR1–CO39 locus was mapped to a 1.06 kb fragment located on chromosome 1 of M. oryzae (Farman and Leong, 1998). Transgenic M. oryzae strains carrying this 1.06 kb fragment become avirulent on a large range of indica and japonica rice cultivars that are postulated to carry Pi–CO39. Four potential open reading frames (ORFs) were identified in the 1.06 kb fragment. Among them, ORF3, which is 270 bp long, has been suggested to encode AVR1–CO39 (Farman and Leong, 1998; Peyyala and Farman, 2006). However, although preliminary data support this identity (Leong, 2008), it has not been demonstrated experimentally. AVR1–CO39 is deleted or inactivated in all rice-infecting M. oryzae isolates, but is widely distributed among M. oryzae isolates from other host plants such as wheat (Triticum aestivum), foxtail millet (Setaria italica) or finger millet (Eleusine coracana) that are non-pathogenic on rice (Farman et al., 2002; Tosa et al., 2005; Peyyala and Farman, 2006). AVR1–CO39 is therefore considered to be a non-host Avr protein in rice, and loss of AVR1–CO39 was suggested to be associated with the evolution of M. oryzae isolates adapted to Setaria spp. during their possible host switch to rice (Couch et al., 2005; Farman et al., 2002; Tosa et al., 2005).
In the present study, we confirmed that ORF3 is the avirulence gene AVR1–CO39. AVR1–CO39 was found to be expressed specifically during plant infection and to encode a small secreted protein. Live-cell imaging revealed that AVR1–CO39 accumulates at the plant–fungal interface during the biotrophic phase of infection, and is translocated into rice cells. Avirulence activity and translocation were found to be independent of AVR1–CO39 promoter sequences. Transient protoplast assays indicated that AVR1–CO39 acts inside rice cells to trigger HR-like cell death in the presence of Pi–CO39. We also present evidence suggesting that translocation of AVR1–CO39 is independent of additional fungal factors, and relies entirely on rice cell components.
The avirulence gene AVR1–CO39 encodes a small secreted protein
The ORF3 of the 1.06 kb fragment containing AVR1–CO39 (Farman and Leong, 1998; Peyyala and Farman, 2006) encodes a protein that shows the hallmarks of a fungal effector. It has a small size (89 amino acids), no homologies to other proteins, three cysteine residues and a predicted signal peptide for secretion in its N–terminus (Figure 1a). To test whether ORF3 encodes AVRCO39, it was isolated from the M. oryzae strain RW12 that is pathogenic on Eleusine coracana, and expressed under the control of the constitutive RP27 promoter from M. oryzae (Bruno et al., 2004) in the M. oryzae isolate Guy11 that is pathogenic on rice cultivars carrying Pi–CO39 (Guy11pRP27::ORF3). Inoculation experiments showed that Guy11pRP27::ORF3 strains were avirulent on rice cultivars carrying Pi–CO39, such as CO39 (Figure 1b) and Kitaake (data not shown), and virulent on rice cultivars lacking Pi–CO39, such as Nipponbare (data not shown) and Maratelli (Figure 1b). Wild-type Guy11 or transgenic Guy11 isolates carrying an empty vector were virulent on all tested rice varieties including CO39 (Figure 1b) and Kitaake (data not shown).
Antibodies were raised against the ORF3-encoded protein (Figure 1a) and used for Western blot analysis (Figure 2a). ORF3 protein antibodies detected a signal exclusively among proteins from culture filtrates of Guy11pRP27::ORF3 transformants. The size of the band (8 kDa) corresponded to the size expected for the ORF3-encoded protein after cleavage of the signal peptide (7.7 kDa). No signals were detected in mycelium protein extracts from pRP27::ORF3 transformants or in protein extracts from either mycelium or culture filtrates of Guy11 transformants carrying an empty vector (Figure 2a).
These experiments demonstrate that ORF3 is AVR1–CO39, and confers specific avirulence on Pi–CO39 rice cultivars. In addition, successful use of the RP27 promoter in the complementation of Guy11 with AVR1–CO39 indicates that the native promoter of ORF3 is not necessary for its avirulence activity.
Validation of the AVR1–CO39 signal pepide
To functionally validate the hypothetical signal peptide of AVR1–CO39, the corresponding sequence was fused to the GUS+ gene, which is an optimized reporter for secreted proteins (Broothaerts et al., 2005), and expressed under the control of the RP27 promoter in M. oryzae Guy11 (AVR1–CO391-22:GUS+). Culture filtrates of AVR1–CO391-22:GUS+ transformants displayed strong secreted GUS activity, while those from GUS+ transformants, empty vector transformants or wild-type strains did not display GUS activity (Figure 2b). As expected, protein extracts from the mycelium of GUS+ and AVR1–CO391-22:GUS+ transformants displayed strong GUS activities, while wild-type and empty vector strains lacked GUS activity. These results indicate that the AVR1–CO39 signal peptide is functional and directs secretion of this small protein.
AVR1–CO39 is expressed specifically during plant infection
Most fungal effector-coding genes are exclusively expressed during plant infection (Stergiopoulos and de Wit, 2009). AVR1–CO39 transcript levels were determined by quantitative RT–PCR using total RNA extracted from in vitro grown mycelium, conidiospores, appressoria and infected leaves. As AVR1–CO39 is deleted or inactivated in all rice-infecting M. oryzae isolates (Farman et al., 2002), for these experiments, the isolates BR32, US71 and CD156 carrying AVR1–CO39 and isolated from wheat, foxtail millet and finger millet, respectively, were inoculated onto their respective host plants and their common host barley (Hordeum vulgare). AVR1–CO39 transcripts were not detected in in vitro grown mycelium, conidia or appressoria (data not shown) and were only found in infected leaves. The expression kinetics varied among the various interactions, but maximum expression occurred in all cases during the biotrophic phase of infection, 1–3 days after inoculation (Figure 3). These results show that AVR1–CO39 is specifically expressed at an early and biotrophic stage of infection.
AVR1–CO39 is translocated into rice cells during infection
To determine whether AVR1–CO39 is translocated into rice cells, we generated transgenic Guy11 M. oryzae strains expressing a translational fusion between AVR1–CO39 and mRFP (monomeric red fluorescent protein) under the control of the RP27 promoter (AVR1–CO39:mRFP). In addition, control Guy11 strains expressing the mRFP protein alone (mRFP) or a fusion between the AVR1–CO39 signal peptide and mRFP (AVR1–CO391-22:mRFP) were constructed. In vitro grown wild-type Guy11 (data not shown) or empty vector transformants did not display any red fluorescence, while mRFP strains showed strong red fluorescence in their cytoplasm (Figure 2c). AVR1–CO391-22:mRFP and AVR1–CO39:mRFP transformants did not show fluorescence in their cytoplasm, but faint and diffuse red fluorescence outlined in vitro grown hyphae and co-localized with the blue fluorescence of calcofluor-stained fungal cell walls. Occasionally, some AVR1–CO391-22:mRFP and AVR1–CO39:mRFP hyphae showed weak red fluorescence inside their vacuoles (data not shown). These results demonstrate that AVR1–CO39:mRFP is secreted in AVR1–CO39:mRFP strains, and that the first 22 amino acids of AVR1–CO39 are sufficient to direct secretion of the heterologous mRFP protein as previously observed with GUS+.
For further characterization, the transgenic strains were inoculated on rice cultivars with or without Pi–CO39 resistance. AVR1–CO39:mRFP strains were avirulent on the rice cultivar CO39 carrying Pi–CO39, and virulent on the susceptible cultivar Maratelli (Figure S1). Empty vector, mRFP and AVR1–CO391-22:mRFP transformants behaved as the Guy11 recipient strain, and were virulent on both CO39 and Maratelli cultivars. These results show that AVR1–CO39:mRFP retains its avirulence activity towards Pi–CO39.
To investigate the localization of AVR1–CO39: mRFP during fungal infection, live-cell imaging of rice leaf epidermis infected with the transgenic Guy11 strains was performed. No red fluorescence was observed in rice leaf epidermis infected with wild-type Guy11 or empty vector transformants. Twenty-four hours post-inoculation (hpi), the fungus had started to penetrate into rice cells, and formed thin primary invasive hyphae. From 26–36 hpi, the fungus progressively filled the first invaded cell with bulbous secondary infectious hyphae (Figure 4a). During intracellular growth in the first invaded cell, the red fluorescence of AVR1–CO39:mRFP and AVR1–CO391-22:mRFP outlined invasive hyphae, suggesting that these secreted proteins accumulate at the fungus–plant interface (Figure 4b,d). The intensity of the fluorescence was heterogeneous, and bright fluorescent spots were observed. At 32 hpi, the cytoplasm of rice cells colonized by AVR1–CO39:mRFP strains displayed red fluorescence (Figure 4c, observed in >70% of 100 analysed infection sites). Red fluorescence was restricted to the host cytoplasm located at the cell periphery between the plasma membrane and the vacuole, and enlarged at the corners of the cells. It was never visible in the plant cell wall. Rice epidermal cells infected by mRFP or AVR1–CO391-22:mRFP strains never displayed red fluorescence in their cytoplasm (more than 100 infection sites analysed for each strain, Figure 4d). At 36 hpi, AVR1–CO39:mRFP strains began to invade adjacent cells. The infectious hyphae colonizing these newly infected cells displayed strong red fluorescence both at the fungus–plant interface outlining invasive hyphae and in punctuate structures located either at the hyphal tips or beside invasive hyphae (data not shown). At 40 hpi, the cytoplasm of newly invaded rice cells displayed strong red fluorescence (Figure 4e, observed in >60% of 100 analysed infection sites). Red fluorescence was restricted to the cytoplasm of cells colonized by AVR1–CO39:mRFP strains, and was never observed in non-invaded neighboring cells. To confirm the cytoplasmic localization of AVR1–CO39:mRFP, a transgenic rice line expressing cytoplasmic GFP was inoculated with Guy11 AVR1–CO39:mRFP strains. The red fluorescence from AVR1–CO39:mRFP and the green fluorescence from GFP co-localized in the cytoplasm of infected rice cells (Figure 4f). These results indicate that AVR1–CO39 is secreted during infection and accumulates at the interface between the fungal cell wall and the host plasma membrane. In addition, AVR1–CO39 is clearly translocated into the cytoplasm of infected rice cells, but appears not to migrate into non-infected adjacent host cells.
The RxLR-like motif of AVR1–CO39 is not necessary for its avirulence activity
RxLR-like motifs have been proposed to be involved in the translocation of fungal effectors (Kale et al., 2010). AVR1–CO39 carries one sequence, KVFV, corresponding to the consensus for RxLR-like motifs (Figure 1a) (Kale et al., 2010). To test whether this motif is important for AVR1–CO39 avirulence activity, an AVR1–CO39 allele was generated in which the KVFV motif was replaced by QVVV (AVR1–CO39QVVV), which does not fit the consensus for RxLR-like motifs (Kale et al., 2010). Transgenic M. oryzae Guy11 strains carrying this construct were inoculated into the rice cultivars Maratelli and Kitaake. AVR1–CO39QVVV strains were avirulent on the Pi–CO39-containing cultivar Kitaake and fully virulent on the susceptible cultivar Maratelli (Figure 5). This demonstrates that replacement of the RxLR-like motif does not impair the avirulence activity of AVR1–CO39.
AVR1–CO39 is recognized inside host cells by its cognate R protein
To test the hypothesis that the interaction between AVR1–CO39 and its cognate R protein occurs inside plant cells, secreted and non-secreted isoforms of AVR1–CO39 were expressed in rice leaf protoplasts, and their potential to trigger HR-like cell death was assayed. Leaf protoplasts of the resistant cultivar Kitaake (Pi–CO39) and the susceptible cultivar Nipponbare (pi–CO39) were co-transfected with reporter constructs for constitutive GUS expression and constructs encoding AVR1–CO39, AVR1–CO39 from which the signal peptide had been deleted (AVR1–CO3923–89) or the empty vector. The same level of GUS activity was detected in Nipponbare protoplasts transformed with AVR1–CO39, AVR1–CO3923–89 or the empty plasmid (Figure 6a). This indicates that AVR1–CO39 has no toxic activity on rice protoplasts. However, in Kitaake, AVR1–CO39 and AVR1–CO3923–89 expression resulted in a significant reduction of GUS activity indicative of R gene-mediated cell-death induction. Elicitation of HR-like cell death by transient expression of the cytoplasmic isoform AVR1–CO3923–89 indicates that AVR1–CO39 is probably recognized by the Pi–CO39 product in the rice cytoplasm.
AVR1–CO39 expressed in rice is directed to the ER, secreted and translocated back into the rice cytoplasm
Expression of AVR1–CO39 in rice protoplasts also induced Pi–CO39-dependent cell death, although the AVR1–CO39 protein produced contains its signal peptide and should therefore be secreted. This suggests that, after secretion from rice protoplasts, AVR1–CO39 may re-enter the cytoplasm to trigger R gene-dependent cell death. To confirm accumulation of AVR1–CO39 in the cytoplasm, cellular protein extracts from Nipponbare rice protoplasts expressing AVR1–CO39 were analysed by immunoblotting with anti-AVR1–CO39 antibodies. No signal was detected in protein extracts of protoplasts transformed with the empty vector, while extracts from AVR1–CO39-transformed protoplasts displayed a single band of 8 kDa (characteristic of AVR1–CO3923–89 cleaved from its signal peptide) (Figure 6b). No band was observed with extracts from AVR1–CO3923–89-transformed protoplasts (data not shown). To increase the sensitivity of detection, AVR1–CO3923–89 fused at the C–terminus to a triple HA epitope tag (AVR1–CO3923–89:HA) was expressed in Nipponbare protoplasts. Protein extracts were analysed by immunoblotting with anti-HA antibodies, and a single band of the expected size was detected (Figure S2).
To further confirm cytoplasmic accumulation of AVR1–CO39, AVR1–CO39:mRFP and AVR1–CO3923–89:mRFP fusions, as well as mRFP alone, were co-expressed in protoplasts together with cytoplasmic GFP. Both constructs, AVR1–CO39:mRFP and AVR1–CO3923–89:mRFP, produced a staining pattern similar to that observed for cytoplasmic GFP or mRFP alone, characterized by strong fluorescence in the cytoplasm and nucleus of transformed rice protoplasts (Figure 7a). Analysis of cellular protein extracts from transfected protoplasts by SDS–PAGE (Figure 7b) and immunoblotting (Figure 7c) detected recombinant proteins corresponding in size to AVR1–CO3923–89:mRFP, indicating that the fusion proteins are produced properly and that the signal peptide is cleaved from AVR1–CO39:mRFP.
To functionally validate the activity of the AVR1–CO39 signal peptide in rice protoplasts, we relied on the fact that proteins entering the secretion pathway necessarily transit through the ER and may be trapped there by addition of an ER retention signal such as the tetrapeptide HDEL (Rafiqi et al., 2010). AVR1–CO39:mRFP:HDEL and AVR1–CO3923–89:mRFP:HDEL fusions were co-expressed with cytoplasmic GFP in rice protoplasts, and the cellular localization of red fluorescence was analysed. The staining pattern observed with AVR1–CO39:mRFP:HDEL differed significantly from the cytoplasmic and nuclear fluorescence observed with AVR1–CO39:mRFP. Indeed, the localization of AVR1–CO39:mRFP:HDEL was typical of ER staining, showing tubular structures in the cytoplasm and outlining of the nucleus (Figure 7a). In contrast, expression of AVR1–CO3923–89:mRFP:HDEL produced red fluorescence in the cytoplasm and the nucleus similar to AVR1–CO3923–89:mRFP. This result indicates that AVR1–CO39 is delivered to the ER secretion pathway in rice protoplasts, and that the AVR1–CO39 signal peptide is active in plants.
Taken together, these experiments strongly suggest that, upon expression in rice protoplasts, AVR1–CO39 enters the secretion pathway in a signal peptide-dependent manner, is secreted from rice protoplasts and re-accumulates inside the cytoplasm by unknown mechanisms.
Previous genetic analysis identified a 1.06 kb genomic fragment of M. oryzae that is sufficient to confer avirulence towards rice cultivars carrying the Pi–CO39 resistance gene (Chauhan et al., 2002; Farman and Leong, 1998). Subsequent studies suggested that the third ORF of this locus (ORF3) is AVR1–CO39 (Leong, 2008; Peyyala and Farman, 2006). However, experiments confirming this identity were not reported. The present study demonstrates that AVR1–CO39 is indeed ORF3. Expression of ORF3 in transgenic M. oryzae strains under control of the constitutive M. oryzae RP27 promoter confers specific avirulence on rice cultivars carrying Pi–CO39. The pRP27::ORF3 transformants secrete a mature protein of the expected size that is detected by AVR1–CO39-specific antibodies. These results clearly establish the molecular identity of AVR1–CO39, and confirm that it displays all the hallmarks of fungal effectors, such as small size, a signal peptide for secretion, specific expression during plant infection and the absence of homology to other proteins.
We hypothesize that AVR1–CO39 contributes to M. oryzae pathogenicity as it is specifically expressed during infection, translocated into host cells and conserved among M. oryzae strains isolated on other hosts than rice (Peyyala and Farman, 2006; Tosa et al., 2006). However, no contribution of AVR1–CO39 to pathogenicity was observed when transgenic M. oryzae strains expressing Avr1–CO39 were inoculated onto susceptible rice varieties. Indeed, the virulence of Avr1–CO39-expressing strains was indistinguishable from that of control strains carrying an empty vector. Additional experiments using strains from other host plants than rice such as wheat or finger millet and carrying a functional AVR1–CO39 allele are required to investigate its possible role in pathogenicity. In addition, determining the proteins and cellular pathways targeted by AVR1–CO39 in host plants will highlight important features of the activity of AVR1–CO39.
The translocation and biological activity of fungal effectors inside plant cells has only been established recently (de Jonge et al., 2011). Indeed, a limited number of fungal effectors were localized inside plant cells either by immunocytology or by live-cell imaging, and it was shown that some fungal effectors expressed inside plant cells induce an R gene-specific HR. In M. oryzae, Avr-Pita, Avr-Pia, Avr-Pii and Avr-Pik are able to trigger an R gene-mediated HR when expressed in cells or protoplasts of rice cultivars carrying their cognate R gene (Jia et al., 2000; Yoshida et al., 2009). Translational fusions between effectors and fluorescent proteins indicate that BAS1, Pwl1 and Pwl2 are translocated into rice cells during infection (Khang et al., 2010). Five effectors from other fungal plant pathogens were shown to be translocated and to have biological activity inside plant cells: AvrM, AvrL567, MISP7, ToxA and CMU1 (Catanzariti et al., 2006; Djamei et al., 2011; Dodds et al., 2004; Manning and Ciuffetti, 2005; Rafiqi et al., 2010). Apart from ToxA, which is a susceptibility toxin, all these effectors are from basidiomycete fungi. Using translational fusions between AVR1–CO39 and mRFP, host cell translocation was documented, and the ability of cytoplasmic AVR1–CO39 to trigger R gene-mediated HR was demonstrated by protoplast transformation. Therefore, AVR1–CO39 is the first effector from a hemibiotrophic ascomycete for which translocation into host cells and HR-inducing activity inside host cells have been established. This result strengthens the view that host cell translocation is an intrinsic and important feature of fungal effectors and occurs in a large range of phylogenetically unrelated fungi.
The molecular identity of the Pi–CO39 R gene has not been determined yet, but the fact that 16 of the 17 cloned rice blast resistance genes encode NB–LRR proteins (Liu et al., 2010) and that Pi–CO39 maps to an NB–LRR gene cluster (Chauhan et al., 2002; Leong, 2008) suggest that Pi–CO39 encodes an NB–LRR protein. This type of protein is generally localized in the cytoplasm of plant cells, in agreement with the host cell translocation and cytoplasmic activity of AVR1–CO39.
Close host–fungus contacts have been suggested to be required for effector translocation (Valent and Khang, 2010). In particular, the accumulation of effector:FP fusion proteins in the BIC suggests that this structure may play a role in M. oryzae effector translocation (Khang et al., 2010). However, this does not appear to apply to AVR1–CO39 as its host cell translocation is independent of any fungal factors. In addition, promoter sequences that have been suggested to be required for the targeting of effectors to the BIC (Khang et al., 2010) are not necessary for AVR1–CO39 translocation or function. Taken together, these results suggest that the BIC is not necessary for AVR1–CO39 translocation. Therefore, we propose the following model for AVR1–CO39 translocation into host cells. AVR1–CO39 is secreted by M. oryzae into the extra-invasive hyphal matrix at the interface between the fungal cell wall and the plant plasma membrane during early stages of infection. Subsequently, AVR1–CO39 enters plant cells by a mechanism that may rely, as for other fungal effectors, on the presence of an as yet unknown translocation motif. This translocation motif is different from previously described fungal sequences such as the RGD motif of ToxA (Manning et al., 2008) or the hydrophobic motif of AvrL567 (Rafiqi et al., 2010), which are both absent from the AVR1–CO39 sequence. It also differs from the RXLR-like motif (Kale et al., 2010), which, although identified in AVR1–CO39, is not required for its avirulence activity. Identification of the AVR1–CO39 translocation motif will be of great interest for understanding of the translocation of fungal effectors into host cells.
Growth of plants and fungi and infection assays
Plants were grown as described previously (Faivre-Rampant et al., 2008). For preparation of in vitro grown mycelia or culture filtrate, the fungus was grown for 3 days in Tanaka complete medium (Villalba et al., 2008). For determination of interaction phenotypes, fungal conidiospores (2.5 × 104 spores/ml) were spray-inoculated onto the leaves of 3-week-old plants (Berruyer et al., 2003). For microscopic analysis, spore suspensions (2.5 × 104 spores/ml) were infiltrated into the hollow interior of rice leaf sheaths prepared from 6-week-old plants (Kankanala et al., 2007).
Protoplasts of Guy11 were prepared as described previously (Bohnert et al., 2004), and transformed with the HindIII-linearized plasmids listed in Table S2. At least eight independent transgenic fungal lines were isolated for each plasmid. Resistant colonies were purified by monospore isolation and characterized by PCR using a Phire Plant Direct PCR kit (Thermo Scientific, Waltham, MA, USA) and the primers listed in Table S1.
RNA extraction and quantitative RT–PCR analysis
RNA extraction and reverse transcription were performed as described previously (Delteil et al., 2012), and quantitative PCR was performed using LC 480 SYBR Green I Master Mix (Roche, Basel, Switzerland) and a LightCycler 480 instrument (Roche). For AVR1–CO39 and EF1α (MGG_03641), oligonucleotides oCS17 and oCS18 and oligonucleotides oJH27 and oJH28 were used (Table S1).
Protein extraction and Western blotting
Soluble proteins from fungal mycelia were prepared in extraction buffer [50 mm Tris/HCl pH 7.4, 150 mm NaCl, 1 mm dithiothreitol, 0.1% Triton X–100, 0.5% polyvinylpolypyrrolidone, 10 mm phenylmethanesulfonyl fluoride and 1% protease inhibitor (Sigma, St Louis, MI, USA)]. Proteins from fungal culture filtrates were prepared by filtration of the culture medium 4 days after inoculation, dialysis (cut-off 1 kDa) against water, lyophilization and resuspension in PBS buffer (10 mm NaPO4, 150 mm NaCl, pH 7.5). Protein extracts from protoplasts were prepared by resuspension of protoplasts in ice-cold extraction buffer and centrifugation for 10 min at 16 000 g at 4°C. For Western blot analysis, 50 μg of total proteins were separated by polyacrylamide gel electrophoresis using NuPAGE 4–12% gels (Invitrogen, Carlsbad, CA, USA) or Tris/Tricine gels (Schagger and von Jagow, 1987). Proteins were electro-blotted onto nitrocellulose membrane (0.2 μm, Millipore, Billerica, MA, USA), and analysed using anti-HA antibodies coupled to horseradish peroxidase (clone 3F10, Roche, diluted 2000×) or anti-AVR1–CO39 polyclonal antibodies from rabbit (diluted 1000×) and anti-rabbit antibodies coupled to horseradish peroxidase (Sigma, diluted 10 000×). The anti-AVR1–CO39 antibodies were generated against the peptides CIIQRYKDGD and CPVILPDKSVLSGDFT and affinity-purified against peptide CPVILPDKSVLSGDFT (Eurogentec, Liège, Belgium).
Rice protoplast production and transformation
Rice protoplasts were prepared as described previously (Yoshida et al., 2009) and transformed by poly(ethylene glycol) treatment with addition of 10 μg of the reporter plasmid pUGN (Nielsen et al., 1999), allowing expression of GUS under the control of the maize ubiquitin 1 promoter, 10 μg of the effector plasmid (Chen et al., 2006). After transformation, protoplasts were incubated for 24 h in the dark. For the determination of GUS activity, protoplasts were resuspended in extraction buffer and assayed for GUS activity using 4–methylumbelliferyl-glucuronide (Sigma) substrate (Hartmann et al., 1998). Plasmids and PCR primers are listed in Tables S1 and S2, and cloning procedures are described in Methods S1.
Histochemical staining and confocal microscopy
For histological determination of GUS activity, 3-day-old mycelia were incubated in GUS staining solution (1 mm 5–bromo-4–chloro-3–indoxyl-β–glucuronide (X-Gluc), 0.5 mm potassium ferrocyanide, 0.5 mm potassium ferricyanide, 0.05% Triton X–100, 50 mm sodium phosphate pH 7). Culture filtrates were filtered (0.2 μm pore size) and complemented with GUS staining solution (1:1). For staining of fungal cell walls, in vitro grown mycelia were incubated in 0.01% calcofluor white solution (10% KOH, 10% glycerol). Epidermal layers of inoculated leaf sheath were cut and analysed using a 63× oil immersion objective lens and a confocal laser-scanning microscope (LSM 700; Zeiss, Jena, Germany). Calcofluor, GFP and mRFP were excited at 405, 488 and 555 nm, respectively. Images were processed using the Zeiss confocal software ZEN 2009.
This work was supported by a grant from Genoplante Programme (Project ‘Interaction Rice Magnaporthe’) and a grant from the Agropolis Foundation (0802-023). We acknowledge the technical support of M.S. Vernerey, C. Michel and L. Fontaine. We thank X.R. Xu (Department of Botany and Plant Pathology, Purdue University, West Lafayette, USA) for providing the pDL2 plasmid. We are particularly grateful to K. Yoshida and R. Terauchi (Iwate Biotechnology Research Center, Kitakami, Japan) for help with protoplast transformation and for providing pACH17_LUC plasmid. We thank B. Tyler (Department of Plant Pathology, Physiology and Weed Science, Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, USA) for discussion of fungal RXLR-like motifs.