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'Leaf'cells'do'not'take'up'extracellular'AvrMBAB'and'AVR3aBmCherry' fusions'! (A)' Live3cell! imaging! of! PR1 1330 3AvrM3A 1063156 3mCherry! in! Nico%ana( benthamiana! leaves.! Images!show!a!single!op(cal!sec(on.!Cells!marked!c1!to!c5!are!pavement!cells!differen(ally! accumula(ng!the!fusion!proteins!(c1:!no!accumula(on;!c2!to!c5:!high!accumula(on).!White! arrowhead:! mCherry3labelled! puncta;! black! arrowhead:! mCherry3labelled! cytosol.! n:! nucleus.!Asterisks:!guard!cells.!Note!the!absence!of!mCherry!signal!in!c1!and!in!guard!cells.! (B)'Live3cell!imaging!of!PR1 1330 3AVR3a 22359 3mCherry!in!N.(benthamiana!leaves.!Images!show!a! maximal! projec(on! of! three! op(cal! sec(ons! (z3stack:! 2.4! µm).! Cells! marked! c1! to! c4! are! pavement!cells!differen(ally!accumula(ng!the!fusion!proteins!(c1!and!c2:!no!accumula(on;! c3! and! c4:! high! accumula(on).! White! arrowhead:! mCherry3labelled! stuctures! resembling! the!endoplasmic!re(culum;!black!arrowhead:!mCherry3labelled!cytosol.!n:!nucleus.!Note!the! absence!of!mCherry!signal!in!c1,!in!c2,!and!in!guard!cells.!!
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... that the PR1 signal peptide is highly effective in targeting proteins to the apoplast via the Endoplasmic Reticulum (ER)/Golgi secretory pathway (Hammond-Kosack et al., 1994), we expected the fusion proteins to accumulate in the apoplast. We then hypothesized that extracellular fusion proteins will be taken up by leaf cells as previously proposed (Supplementary Table 1 In addition to the apoplast, we observed accumulation of the AvrM-A and AVR3a mCherry fusion proteins inside transformed cells (Figure 2 and 3). We aimed at better determining the intracellular accumulation pattern of these proteins, but the fluorescent signal from the apoplast interfered with -and impaired optimal imaging of -the intracellular signal. ...
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... This could indicate that Foa3 accumulates at an intermediate step of the secretory path, even though it eventually reaches the apoplast, since it is present in the apoplastic fluid ( Figure 1A). Interestingly, several host-translocated effectors showed similar concentrated accumulation when expressed in planta with a signal peptide (Kloppholz et al., 2011;Petre et al., 2016). In contrast, other intracellular effectors showed a more uniform distribution, indicating that this localization pattern is effector-specific (Rafiqi et al., 2010). ...
Plant pathogens employ secreted proteins, among which are effectors, to manipulate and colonize their hosts. A large fraction of effectors is translocated into host cells, where they can suppress defense signaling. Bacterial pathogens directly inject effectors into host cells via the type three secretion system, but it is little understood how eukaryotic pathogens, such as fungi, accomplish this critical process and how their secreted effectors enter host cells. The root-infecting fungus Fusarium oxysporum (Fo) secrets numerous effectors into the extracellular space. Some of these, such as Foa3, function inside the plant cell to suppress host defenses. Here, we show that Foa3 suppresses pattern-triggered defense responses to the same extent when it is produced in planta irrespective of whether the protein carries the PR1 secretory signal peptide or not. When a GFP-tagged Foa3 was targeted for secretion it localized, among other locations, to mobile subcellular structures of unknown identity. Furthermore, like the well-known cell penetrating peptide Arginine 9, Foa3 was found to deliver an orthotospovirus avirulence protein-derived peptide into the cytosol, resulting in the activation of the matching resistance protein. Finally, we show that infiltrating Foa3 into the apoplast results in strong suppression of the pattern-triggered immune responses, potentially indicating its uptake by the host cells in absence of a pathogen.
... Other chloroplast-targeting effectors have been shown to lose their targeting ability upon addition of a reporter gene to the N-terminus (Carrie et al. 2009), but interestingly, the targeting of PstCTE1 remained functional despite an N-terminal fluorescent protein tag; this effect was also evidenced by the chloroplast targeting of the PstCTE1 with its signal peptide intact. Indeed, this observation may suggest the re-entry of an effector (Petre et al. 2016a), which is expected for a Pst effector that targets host organelles via translocation during natural disease progress. The RXLR motif in oomycetes and RXLR-like motifs in some fungi have been reported to mediate effector translocation into host cells (Ve et al. 2013). ...
... Alternatively, it was pointed out by Wawra et al. 2017 that pathogen effectors re-enter the cell in a non-specific manner (Tyler et al. 2013;Wawra et al. 2017). Nevertheless, we note that the candidate effector that we study is neither an oomycete effector, nor it has an RxLR-like motif, possible explanations for the accumulation in the cytoplasm may be that either the effector protein may escape from the secretory pathway by retrograde transport, or translation starts at alternative sites, resulting in a truncated or non-functional signal peptide at the N-terminus; as it was pointed out that it is possible in a non-host model organism (Petre et al. 2016a). If the candidate effector protein escapes from the secretory pathway, it no longer needs to cross the plasma membrane; fugitive effectors remain in the host cytoplasm and enter the chloroplast (Fig. 8). ...
... If the candidate effector protein escapes from the secretory pathway, it no longer needs to cross the plasma membrane; fugitive effectors remain in the host cytoplasm and enter the chloroplast (Fig. 8). It is reported that the RXLR effectors do not enter plant cells in the absence of pathogen (Petre et al. 2016a), in another word, the cell re-entry assays do not work in protein expressed for secretion by agro-infiltrated N. benthamiana model system (Fig. 8). The status of fungal effectors before secretion, secretion routes, and plant cell entry mechanisms are yet to be elucidated (Petre and Kamoun 2014). ...
Fungal pathogens secrete effector molecules into host plant cells to suppress host immunity to colonize plants. Ongoing efforts are being made to identify and characterize effector proteins in many fungal plant pathogens. Nevertheless, the precise biological and biochemical functions of many effectors, such as their trafficking from the pathogen to the host, have yet to be fully understood. In this study, we show that an effector candidate, matured PstCTE1 of Puccinia striiformis f. sp. tritici, localizes to chloroplasts when expressed in planta. It has no conserved transit signal region that can be detected by widely accepted prediction tools including TargetP and ChloroP, it must be carrying a unique localization signal. We have shown that N-terminal tagged red fluorescent protein has no effect on the chloroplast localization of PstCTE1, suggesting a new chloroplast translocation mechanism. We also observed the entrance of the candidate effector to the chloroplast even with the construct having the intact signal peptide on the N-terminus of the transit peptide region. Possibly due to overexpression of the protein in N. benthamina, accumulation in the ER (cytoplasm) was obvious. As previously reported, PstCTE1, similar to effector proteins, may either escape from the secretory pathway by retrograde transport, or translation may occur at alternative sites. This would result in a truncated and/or non-functional signal peptide at the N-terminus in a non-host model system (Nicotiana benthamiana), if it is not re-entering the cell from the apoplast. Our study adds PstCTE1 to the pool of few candidate effectors, experimentally shown to target the chloroplast.
... RxLR effectors are named based on a conserved RxLR amino acid motif (Arg-any amino acid-Leu-Arg) downstream of a secretory signal peptide. The RxLR motif is thought to be required for effector translocation into host cells (Whisson et al., 2007), but the mechanism of translocation is under debate (Kale et al., 2010;Tyler et al., 2013;Petre et al., 2016;Wawra et al., 2017). Recent evidence has suggested that the RxLR motif may function as an internal sorting signal that is cleaved before secretion (Wawra et al., 2017). ...
Pathogen effectors act as disease promoting factors that target specific host proteins with roles in plant immunity. Here, we investigated the function of the RxLR3 effector of the plant‐pathogen Phytophthora brassicae.
Arabidopsis plants expressing a FLAG‐RxLR3 fusion protein were used for co‐immunoprecipitation followed by liquid chromatography‐tandem mass spectrometry to identify host targets of RxLR3. Fluorescently labelled fusion proteins were used for analysis of subcellular localisation and function of RxLR3.
Three closely related members of the callose synthase family, CalS1, CalS2 and CalS3, were identified as targets of RxLR3. RxLR3 co‐localised with the plasmodesmal marker protein PDLP5 (PLASMODESMATA‐LOCALISED PROTEIN 5) and with plasmodesmata‐associated deposits of the β‐1,3‐glucan polymer callose. In line with a function as an inhibitor of plasmodesmal callose synthases (CalS) enzymes, callose depositions were reduced and cell‐to‐cell trafficking was promoted in the presence of RxLR3.
Plasmodesmal callose deposition in response to infection was compared with wild‐type suppressed in RxLR3‐expressing Arabidopsis lines. Our results implied a virulence function of the RxLR3 effector as a positive regulator of plasmodesmata transport and provided evidence for competition between P. brassicae and Arabidopsis for control of cell‐to‐cell trafficking.
... The RXLR motif may guide the translocation of Phytophthora sojae effectors into plant cells (Kale et al., 2010). Other studies suggested that re-entry signals of certain RXLR effectors (AvrM and AVR3a) might be triggered by traffic congestion of the secretion pathway, and the cleavage of the RXLR motif of AVR3a occurred before secretion (Petre et al., 2016a;Wawra et al., 2017). Another investigation of animal-pathogenic oomycete Saprolegnia parasitica found that the uptake process of a hosttargeting protein SpHtp3 is guided by a gp96-like receptor via its C-terminal YKARK region, but not by the N-terminal RXLR motif (Trusch et al., 2018). ...
Rust fungi secrete various specialized effectors into host cells to manipulate the plant defense response. Conserved motifs, including RXLR, LFLAK-HVLVxxP (CRN), Y/F/WxC, CFEM, LysM, EAR, [SG]-P-C-[KR]-P, DPBB_1 (PNPi), and ToxA, have been identified in various oomycete and fungal effectors and are reported to be crucial for effector translocation or function. However, little is known about potential effectors containing any of these conserved motifs in the wheat leaf rust fungus (Puccinia triticina, Pt). In this study, sequencing was performed on RNA samples collected from the germ tubes (GT) of uredospores of an epidemic Pt pathotype PHTT(P) and Pt-infected leaves of a susceptible wheat cultivar “Chinese Spring” at 4, 6, and 8 days post-inoculation (dpi). The assembled transcriptome data were compared to the reference genome of “Pt 1-1 BBBD Race 1”. A total of 17,976 genes, including 2,284 “novel” transcripts, were annotated. Among all these genes, we identified 3,149 upregulated genes upon Pt infection at all time points compared to GT, whereas 1,613 genes were more highly expressed in GT. A total of 464 secreted proteins were encoded by those upregulated genes, with 79 of them also predicted as possible effectors by EffectorP. Using hmmsearch and Regex, we identified 719 RXLR-like, 19 PNPi-like, 19 CRN-like, 138 Y/F/WxC, and 9 CFEM effector candidates from the deduced protein database including data based on the “Pt 1-1 BBBD Race 1” genome and the transcriptome data collected here. Four of the PNPi-like effector candidates with DPBB_1 conserved domain showed physical interactions with wheat NPR1 protein in yeast two-hybrid assay. Nine Y/F/WxC and seven CFEM effector candidates were transiently expressed in Nicotiana benthamiana. None of these effector candidates showed induction or suppression of cell death triggered by BAX protein, but the expression of one CFEM effector candidate, PTTG_08198, accelerated the progress of cell death and promoted the accumulation of reactive oxygen species (ROS). In conclusion, we profiled genes associated with the infection process of the Pt pathotype PHTT(P). The identified effector candidates with conserved motifs will help guide the investigation of virulent mechanisms of leaf rust fungus.
... A different mechanism of translocation that is mediated by external PI(3)P binding of the RXLR motif of effectors has been suggested (Kale et al. 2010). This model of effector translocation has also been under heavy debate, with more recent studies indicating that the RXLR motif is not sufficient for PI(3)P binding and that the uptake is pathogen-dependent (Yaeno and Shirasu 2013;Petre and Kamoun 2014;Petre et al. 2016). ...
p>Late blight caused by the oomycete pathogen Phytophthora infestans is one of the most destructive diseases in potato cultivation. To successfully colonize its host, P. infestans secretes a suite of effector proteins that undermine plant immunity, many of which contain a conserved N-terminal RXLR motif that strongly resembles the host targeting motif in effectors of the malaria parasite Plasmodium falciparum. In this study, we focus on three P. infestans clade 5 aspartic proteases (PiAPs) that are homologous to Plasmepsin V (PMV), a Pl. falciparum AP responsible for cleaving effectors prior to translocation into red blood cells. Malaria parasites expressing mutated PMV are impaired in effector translocation and are less virulent. To determine whether clade 5 PiAPs play similar roles in virulence, we characterized P. infestans transformants with either reduced or enhanced PiAP expression levels. Phytophthora infestans transformants with altered PiAP10 or PiAP12 expression were found to be impaired in mycelial growth and sporangia production, and are hampered in their virulence on potato leaves. This was not observed in PiAP11 transformants. Activity assays showed that PiAP10 and PiAP12 possess moderate protease activity, and can potentially cleave the RXLR effector PiAVR4, but not a PiAVR4 version with a mutated RXLR motif. These findings imply that P. infestans APs function in the proteolytic cleavage of RXLR effectors, and warrant further investigation to verify and confirm the role of clade 5 PiAPs in effector processing.</p
... In 94 addition, oomycete and fungal RxLR and RxLR-like effectors have been reported to 95 translocate into host cells in the absence of the pathogen (Plett et al., 2011;Gu et al., Yaeno et al., 2011;Wawra et al., 2012b). Furthermore, a recent study 101 indicates that cell-based re-entry assays do not support models of pathogen- proteins (Petre at al., 2016;Na et al., 2013). 105 The oomycete RxLR motif might have a function similar to the PEXEL-motif as a 106 potential internal sorting signal. ...
When plant-pathogenic oomycetes infect their hosts, they employ a large arsenal of effector proteins to establish a successful infection. Some effector proteins are secreted and are destined to be translocated and function inside host cells. The largest group of translocated proteins from oomycetes are the RxLR effectors, defined by their conserved N-terminal Arg-Xaa-Leu-Arg (RxLR) motif. However, the precise role of this motif in the host cell translocation process is unclear. Here detailed biochemical studies of the RxLR effector AVR3a from the potato pathogen Phytophthora infestans are presented. Mass spectrometric analysis revealed that the RxLR sequence of native AVR3a is cleaved off prior to secretion by the pathogen and the N-terminus of the mature effector was found likely to be acetylated. High-resolution NMR structure analysis of AVR3a indicates that the RxLR motif is well accessible to potential processing enzymes. Processing and modification of AVR3a is to some extent similar to events occurring with the export element (PEXEL) found in malaria effector proteins from Plasmodium falciparum. These findings imply a role for the RxLR motif in the secretion of AVR3a by the pathogen, rather than a direct role in the host cell entry process itself.
... In Phytophthora sojae effectors, the RxLR-dEER motif has been proposed to be sufficient for reentry into plant cells, even in the absence of the pathogen (Dou et al. 2008;Wang et al. 2011). However, a recent study in N. benthamiana failed to show re-entry into plant cells of effectors from Melampsora lini and Phytophthora infestans fused to a signal peptide and fluorescent proteins (Petre et al. 2016). Therefore, other methods may be required to test the role of the PNPi RSLL-DEEP region in plant cell entry. ...
In Arabidopsis, NPR1 is a key transcriptional co-regulator of systemic acquired resistance. Upon pathogen challenge, NPR1 translocates from the cytoplasm to the nucleus, where it interacts with TGA-bZIP transcription factors to activate the expression of several Pathogenesis-Related (PR) genes. In a screen of a yeast two-hybrid library from wheat leaves infected with Puccinia striiformis f. sp. tritici, we identified a conserved rust protein that interacts with wheat NPR1 and named it Puccinia NPR1 interactor (PNPi). PNPi interacts with the NPR1/NIM1-like domain of NPR1 via its C-terminal DPBB_1 domain. Using bimolecular fluorescence complementation assays, we detected the interaction between PNPi and wheat NPR1 in the nucleus of Nicotiana benthamiana protoplasts. A yeast three-hybrid assay showed that PNPi interaction with NPR1 competes with the interaction between wheat NPR1 and TGA2.2. In barley transgenic lines over expressing PNPi, we observed reduced induction of multiple PR genes in the region adjacent to Pseudomonas syringae pv. tomato DC3000 infection. Based on these results, we hypothesize that PNPi has a role in manipulating wheat defense response via its interactions with NPR1.
Both pathogenic and symbiotic microorganisms modulate the immune response and physiology of their host to establish a suitable niche. Key players in mediating colonization outcome are microbial effector proteins that act either inside (cytoplasmic) or outside (apoplastic) the plant cells and modify the abundance or activity of host macromolecules. We compile novel insights into the much-disputed processes of effector secretion and translocation of filamentous organisms, namely fungi and oomycetes. We report how recent studies that focus on unconventional secretion and effector structure challenge the long-standing image of effectors as conventionally secreted proteins that are translocated with the aid of primary amino acid sequence motifs. Furthermore, we emphasize the potential of diverse, unbiased, state-of-the-art proteomics approaches in the holistic characterization of fungal and oomycete effectomes.
