ABSTRACT
Most fungal pathogens secrete effector proteins into host cells to modulate their immune responses, thereby promoting pathogenesis and fungal growth. One such fungal pathogen is the ascomycete Phyllachora maydis, which causes tar spot disease on leaves of maize (Zea mays). Sequencing of the P. maydis genome revealed 462 putatively secreted proteins of which 40 contain expected effector-like sequence characteristics. However, the subcellular compartments targeted by P. maydis effector candidate (PmECs) proteins remain unknown and it will be important to prioritize them for further functional characterization. To test the hypothesis that PmECs target diverse subcellular compartments, cellular locations of super Yellow Fluorescent Protein (sYFP)-tagged P. maydis effector candidate proteins were identified using a Nicotiana benthamiana-based heterologous expression system. Immunoblot analyses showed that most of the PmEC-fluorescent protein fusions accumulated protein in N. benthamiana, indicating the candidate effectors could be expressed in dicot leaf cells. Laser-scanning confocal microscopy of N. benthamiana epidermal cells revealed most of the P. maydis putative effectors localized to the nucleus and cytosol. One candidate effector, PmEC01597, localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane while an additional putative effector, PmEC03792, preferentially labelled both the nucleus and nucleolus. Intriguingly, one candidate effector, PmEC04573, consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). Collectively, these data suggest effector candidate proteins from P. maydis target diverse cellular organelles and may thus provide valuable insights into their putative functions as well as host processes potentially manipulated by this fungal pathogen.
INTRODUCTION
Plant pathogens secrete virulence proteins known as effectors to modulate host immune responses that often have a functional role in facilitating infection (Jones and Dangl, 2006; Kamoun, 2007; Wang et al., 2011; Zipfel, 2014). Secreted effectors can be either retained in the plant extracellular space (apoplastic effectors) or translocated into host cells (cytoplasmic effectors) and can localize to diverse subcellular compartments (Lorrain et al., 2018; Whisson et al., 2016). For example, an effector protein from the oomycete Phytophthora infestans, PITG_04097, localizes to the host nucleus, and such nuclear localization is required for suppression of host defense responses and pathogen virulence (Zheng et al., 2014). The Pseudomonas syringae effector HopG1, which targets the mitochondria, suppresses host defense responses and promotes cell death in Arabidopsis (Rodriguez-Puerto et al., 2022). The Magnaporthe oryzae effector, AVR-Pii, is localized to the host cytosol where it suppresses host production of reactive oxygen species through its inhibition of the rice NADP-malic enzyme2, thereby disrupting immunity to this fungal pathogen (Singh et al., 2016). Elucidating how crop pathogen effectors function in host cells is critical, in part, for understanding pathogenicity and virulence mechanisms of fungal pathogens for which control strategies are currently limited.
Phyllachora maydis is a foliar, ascomycete fungal pathogen that causes tar spot disease on maize (Zea mays subsp. mays) (Rocco da Silva et al., 2021; Ruhl et al., 2016; Valle-Torres et al., 2020). Though endemic to Central and South America, P. maydis was recently identified in the continental United States in 2015 and has since spread to most maize production regions, indicating this fungal pathogen is capable of significant global expansion (Mottaleb et al., 2019; Ruhl et al., 2016; Valle-Torres et al., 2020). Notably, P. maydis has been shown to significantly reduce maize yields especially under favorable environmental conditions, imposing severe financial constraints to growers (Mueller et al., 2020; Valle-Torres et al., 2020). Temperate-derived maize inbreds and commercial hybrids provide only partial resistance to P. maydis, and no fully resistant maize cultivar has been identified (Telenko et al., 2019). For these reasons, P. maydis is now considered one of the most economically important foliar pathogens of maize in the U.S. (Mueller et al., 2020; Rocco da Silva et al., 2021; Valle-Torres et al., 2020).
To gain initial insights into P. maydis virulence mechanisms, Telenko and colleagues provided its first draft genome sequence (Telenko et al., 2020). Analysis of the P. maydis genome revealed 462 proteins comprising the predicted secretome, of which 59 contain effector-like sequence characteristics as predicted by EffectorP (v2.0) (Telenko et al., 2020). To date, our understanding of how P. maydis utilizes its effector repertoire to promote virulence as well as the subcellular compartments targeted by these putative effectors remains limited even though this fungal pathogen represents a serious economic concern for maize growers (Helm et al., 2022; Mueller et al., 2020; Valle-Torres et al., 2020). The inability to culture or genetically manipulate P. maydis (Rocco da Silva et al., 2021; Valle-Torres et al., 2020) substantially hinders investigations aimed at characterizing its effector repertoire (Helm et al., 2022). To circumvent these limitations, the field of effector biology utilizes a surrogate plant system to express epitope-tagged candidate effectors directly inside leaf cells using Agrobacterium-mediated infiltration (agroinfiltration) (Lorrain et al., 2018). Nicotiana benthamiana is a well established and extensively used model plant for heterologous expression of crop pathogen effectors and has been used to investigate the subcellular compartments targeted by putative effector proteins produced by filamentous fungal pathogens (Alfano, 2009; Figueroa et al., 2021; Lorrain et al., 2018; Ma et al., 2012; Petre et al., 2017; Win et al., 2011; Dinne et al., 2021).
In the present study, we refined previous effector predictions performed by Telenko et al. (2020) using EffectorP (v3.0) as well as additional selection criteria including i) protein size less than 300 amino acids; ii) presence of a signal peptide (as predicted by SignalP v6.0); and iii) lack of a transmembrane domain. We discovered that among the 59 proteins originally identified by Telenko and colleagues (2020), 40 contain effector-like protein characteristics that fulfilled our more selective criteria. Intriguingly, several of the effector candidates from P. maydis encode subcellular targeting sequences including nuclear localization signals (NLS) and chloroplast and mitochondrial transit peptides, suggesting they may be targeted to specific subcellular locations. To test this hypothesis, we investigated the subcellular compartments targeted by P. maydis effector candidate proteins (PmECs) using a Nicotiana benthamiana-based heterologous expression system. Laser-scanning confocal microscopy of N. benthamiana epidermal cells revealed most of the P. maydis putative effectors localized to the nucleus and cytosol. However, one candidate effector, PmEC01597, consistently localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane while an additional putative effector, PmEC03792, preferentially labelled both the nucleus and nucleolus. Intriguingly, the candidate effector, PmEC04573, consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). These data indicate effector candidate proteins from P. maydis target diverse cellular organelles and thus lay the foundation for future studies to investigate their putative functions as well as host processes potentially manipulated by this fungal pathogen.
MATERIALS AND METHODS
Plant growth conditions
Nicotiana benthamiana seeds were sown in plastic pots containing either ProMix or Berger Seed and Propagation Mix supplemented with Osmocote slow-release fertilizer (14-14-14). Plants were maintained in a growth chamber with a 16:8 h photoperiod (light:dark) at 24°C with light and 20°C in the dark and 60% humidity with average light intensities at plant height of 120 µmols/m2/s.
In silico selection of candidate effectors from P. maydis Indiana isolate PM-01
To select candidate effector proteins, we began with the effector predictions generated previously by Telenko et al., (2020) from the predicted P. maydis secretome. We extracted all 59 candidate effector protein sequences and used EffectorP (v3.0) (Sperschneider and Dodds, 2022) (http://effectorp.csiro.au/) to further improve the effector prediction performed by Telenko et al. (2020). We also employed SignalP (v6.0) (Teufel et al., 2022) (https://services.healthtech.dtu.dk/service.php?SignalP) to predict the presence of signal peptide sequences. LOCALIZER (v1.0) (Sperschneider et al., 2017) (http://localizer.csiro.au/) was used to predict the subcellular localizations of the putative effectors. TMHMM (v2.0; https://services.healthtech.dtu.dk/service.php?TMHMM-2.0) was used to predict transmembrane helices within the candidate effector proteins. NoD (Scott et al., 2011) (http://www.compbio.dundee.ac.uk/www-nod/) was used to predict the presence of predicted Nucleolar targeting signal (NoLS). The refined catalog of candidate effector proteins as determined by our in silico pipeline (Figure 1) is shown in Table 1.
Generation of plant expression constructs
All constructs in this study were generated using a modified multisite Gateway cloning system (Invitrogen). The AtUBQ10-NLS:mCherry, AtFLS2:mCherry, RbcS-TP:mCherry, AtFIB2:mCherry, 3xHA:sYFP (free sYFP), and 3xHA:mCherry (free mCherry) constructs have been described previously (Denne et al., 2021; Gu et al., 2011; Helm et al., 2019; Nelson et al., 2007; Qi et al., 2012; Robin et al., 2018).
A commercial gene synthesis service (Azenta Life Sciences, South Plainfield, New Jersey) was used to synthesize the open reading frames (ORFs) of each P. maydis effector candidate (PmEC) (without the signal peptide) with codon optimization for plant expression. The attL1 and attL4 Gateway sequences were added to the 5’ and 3’ ends, respectively, of each P. maydis candidate effector to generate Gateway-compatible DONR clones. The resulting sequences were inserted into the plasmid vector pUC57 by the service provider. We designated the resulting constructs pDONR(L1-L4):PmEC.
To generate the PmEC-sYFP protein fusions, the pDONR(L1-L4):PmEC constructs were mixed with pBSDONR(L4r-L2):sYFP (Qi et al., 2012), and the Gateway-compatible expression vector pEG100 (Earley et al., 2006), which places the transgene under control of the 35S promoter. All plasmids were recombined by the addition of LR Clonase II (Invitrogen) and were incubated overnight at 25°C following the manufacturer’s instructions. The resulting constructs were transformed into Agrobacterium tumefaciens GV3101 (pMP90) by electroporation and subsequently used for transient expression in Nicotiana benthamiana.
Agrobacterium-mediated transient protein expression in Nicotiana benthamiana
The CaMV 35S-driven constructs described above were mobilized into Agrobacterium tumefaciens GV3101 (pMP90) and grown on LB medium plates containing 25 μg of gentamicin sulfate and 50 μg of kanamycin per milliliter for 2 days at 30°C. Cultures were prepared in liquid media (10 ml) supplemented with the appropriate antibiotics and were shaken overnight at 30°C at 225 rpm on an orbital shaker. Following overnight incubation, the cells were pelleted by either centrifuging at 3,600 rpm for 3 minutes or 3,000 rpm for 8 minutes at room temperature. The bacterial pellet was then resuspended in either 10 mM MgCl2 or induction medium (10 mM MES, pH 5.5, 3% sucrose), adjusted to an optical density at 600 nm (OD600) of 0.4 (final concentration for each strain in a mixture), and incubated with either 100 or 200 μM acetosyringone (Sigma-Aldrich) for 3-4 hours at room temperature with constant shaking. Bacterial suspensions were mixed in equal ratios (1:1) and infiltrated into the underside of 3- to 4-week-old Nicotiana benthamiana leaves with a needleless syringe. Leaf samples were collected 24 hours after agroinfiltration for immunoblot analyses or confocal microscopy.
Protein extraction and immunoblot analyses
N. benthamiana leaf samples (0.5g) were collected at 24 hrs following agroinfiltration and flash frozen in liquid nitrogen. Tissue was homogenized with protein extraction buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Nonidet P-40, 1% 2,2’- dipyridyl disulfide [DPDS] and 1% protease inhibitor cocktail) (Sigma Aldrich). Homogenates were briefly mixed and centrifuged twice at 10,000 x g for 10 min at 4°C to pellet cell debris. Total protein lysates were combined with 4X Laemmli Sample Buffer (277.8 mM Tris-HCl [pH 6.8], 4.4% LDS, 44.4% (v/v) glycerol, 0.02% bromophenol blue, and 10% β-mercaptoethanol), and the mixtures were boiled at 95°C for 10 min. Protein samples were separated on 4-20% Tris-glycine stain-free polyacrylamide gels (Bio-Rad) at 175 V for 1 hr in 1X Tris/glycine/SDS running buffer. Total proteins were transferred to nitrocellulose membranes (GE Water and Process Technologies) at 100 V for one hour. Ponceau staining was used to confirm equal loading and transfer of total protein samples. Membranes were washed with 1X Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl [pH 6.8]) solution containing 0.1% Tween20 (TBST) and incubated with 5% Difco skim milk for 1 hr at room temperature or overnight at 4°C with gentle shaking. Proteins were subsequently detected with horseradish peroxidase (HRP)-conjugated anti-GFP antibody (1:5,000) (Miltenyi Biotec) for 1 hr at room temperature with gentle shaking. Following antibody incubation, membranes were washed at least three times for 15 minutes in 1x TBST solution. Protein bands were imaged using equal parts of Clarity Western ECL substrate peroxide solution and luminol/enhancer (BioRad) solution (Thermo Scientific), with incubation at room temperature for 5 minutes. Immunoblots were developed using X-ray film.
Confocal microscopy
Live-cell imaging of N. benthamiana epidermal cells was performed 24 hours following agroinfiltration using a Zeiss LSM880 Axio Examiner upright confocal microscope as described previously (Denne et al., 2021). Briefly, N. benthamiana leaf sections were excised and mounted in sterile water between a slide and a coverslip (adaxial surface toward the objective) and subsequently imaged using a Plan Apochromat 20x/0.8 objective, pinhole 1.0 AU. For plasmolysis, leaf sections were prepared as described above, submerged in 0.8 M mannitol solution for 20 minutes, and imaged shortly thereafter. The sYFP protein fusions were excited using a 514-nm argon laser and fluorescence was detected between 517-562 nm. Fluorescence from the mCherry-tagged constructs was excited with a 561-nm helium-neon laser and detected between 565-669 nm. All confocal micrographs were captured on the Zeiss LSM880 upright confocal microscope and processed using the Zeiss Zen Blue Lite program (Carl Zeiss Microscopy, USA).
RESULTS
In silico selection and generation of super Yellow Fluorescent Protein (sYFP)- tagged P. maydis effector candidate proteins (PmECs) from Indiana isolate PM-01
To identify promising secreted effector candidates, we leveraged effector predictions previously generated by Telenko et al. (2020). These authors identified 59 P. maydis proteins that contain effector-like characteristics as determined by EffectorP (v2.0). To further refine the previous analyses by Telenko et al. (2020), we mined through the 59 P. maydis effector candidates (PmECs) and selected proteins that fulfilled more selective criteria: i) size fewer than 300 amino acids; ii) presence of a signal peptide (as predicted by SignalP v6.0); and iii) lack of a transmembrane domain (as predicted by TMHMM v2.0). We next leveraged EffectorP (v3.0) to identify putative effector proteins from this narrowed set of proteins. Among the 59 potential effectors originally identified by Telenko et al. (2020), 40 contain effector-like protein characteristics that fulfilled these more selective criteria (Figure 1A). The selected effector candidates ranged in size from 55 to 299 amino acids (Table 1). We next employed LOCALIZER (v1.0) to identify predicted nuclear localization signals (NLS), chloroplast transit peptides, or mitochondrial targeting sequences. As shown in Table 1, many of the candidates are not predicted to target specific subcellular compartments. However, several PmECs encode predicted nuclear localization signals including PmEC01597, PmEC03792, and PmEC05617 (Table 1). Two effector candidates, PmEC03153 and PmEC04573, contain predicted chloroplast transit peptides, and a mitochondrial targeting sequence was identified in PmEC03493 (Table 1). Intriguingly, PmEC00848 encoded both a chloroplast transit peptide and a mitochondrial-targeting sequence and PmEC03706 encoded both a chloroplast transit peptide and NLS, suggesting these proteins may localize to multiple subcellular compartments (Table 1).
To investigate the subcellular compartments targeted by the P. maydis effector candidates, the predicted open reading frames (ORFs) of each of the 40 putative effectors, without the predicted signal peptides, were synthesized and fused to the N terminus of super Yellow Fluorescent Protein (PmEC:sYFP) (Figure 1B). The resulting constructs were recombined into the plant expression binary vector pEarleyGate100 (pEG100) (Earley et al., 2006), which places the candidate effectors downstream of a 35S promoter. The resulting constructs were inserted into Agrobacterium tumefaciens for subsequent Nicotiana benthamiana-based heterologous expression assays (Figure 1C-E).
Candidate P. maydis effector-fluorescent protein fusions accumulate protein in planta
Prior to assessing the subcellular localization of the P. maydis effector-fluorescent protein fusions, we tested whether these effectors accumulate protein in dicot leaf cells. This was accomplished by transiently expressing each of the fusion proteins in N. benthamiana using Agrobacterium-mediated infiltration (agroinfiltration). Immunoblot analyses revealed that, of the 40 candidate effectors screened, 37 accumulated at detectable levels when transiently expressed in N. benthamiana (Figure 2). Three putative effectors (PmEC01936, PmEC02451 and PmEC06216) consistently failed to express detectable protein suggesting these fusion proteins do not accumulate when transiently expressed in N. benthamiana leaf cells (Figure 2). Though most of the fusion proteins accumulated at the expected molecular weight, several putative effectors accumulated protein at lower molecular weights than predicted, suggesting post-translational modifications (Figure 2). As most P. maydis effector-fluorescent protein fusions accumulated protein, we conclude that N. benthamiana is an appropriate surrogate plant system and can thus be used to investigate the subcellular localization patterns of P. maydis effector-fluorescent protein fusions. Based on these data, we discarded the effector candidates with insufficient protein expression (PmEC01936, PmEC02451 and PmEC06216) and retained the remaining candidate effectors for further in planta analyses.
The majority of P. maydis effector candidate-fluorescent protein fusions localize to the nucleus and cytosol
Live-cell imaging of epidermal cells using laser-scanning confocal microscopy revealed that among the 37 sYFP-tagged PmECs that accumulate protein, 29 showed subcellular distribution patterns in the nucleo-cytosol and were indistinguishable from the free sYFP control (Supplemental Figure 1; Table 1). Furthermore, fluorescence signal from five sYFP-tagged derivatives, PmEC03436, PmEC03493, PmEC03706, PmEC04014, and PmEC05617, predominantly accumulated in the cytosol (Supplemental Figure 2; Table 1). Interestingly, PmEC03436:sYFP signal labeled punctate bodies on the cell periphery, and PmEC04014:sYFP signal was observed in irregular, cytosolic aggregates (Supplemental Figure 2). In addition to localizing in the cytosol, PmEC03493:sYFP accumulated in the nucleus as well as sub-nuclear structures (Supplemental Figure 2). The remaining effector-fluorescent protein fusions preferentially localized to specific subcellular compartments within the plant cells.
The P. maydis effector candidate PmEC01597 localizes to multiple subcellular compartments
PmEC01597 encodes a predicted nuclear localization signal (NLS) at its C terminus as predicted by LOCALIZER (Sperschneider et al., 2017) (Table 1; Figure 3A) and a nucleolar targeting signal (NoLS) as predicted by NoD (http://www.compbio.dundee.ac.uk/www-nod/; Scott et al., 2011), suggesting this effector candidate localizes to both the nucleus and nucleolus. To confirm the specific localization of PmEC01597 to these subcellular compartments, we co-expressed PmEC01597:sYFP with mCherry-tagged AtUBQ10-NLS or AtFIB2, Arabidopsis proteins known to localize to the nucleus and nucleolus, respectively (Nelson et al., 2007; Robin et al., 2018). As predicted, the PmEC01597:sYFP fluorescence signal consistently co-localized with both the AtUBQ10-NLS:mCherry and AtFIB2:mCherry fluorescence signals, demonstrating that PmEC01597:sYFP accumulates in the nucleus and nucleolus (Figure 3C-D). Furthermore, PmEC01597:sYFP also overlapped fluorescence signals (Figure 3E) with mCherry-tagged FLS2 (FLS2:mCherry), an Arabidopsis protein known to localize on the plasma membrane (Helm et al., 2019). Plasmolysis of N. benthamiana epidermal cells expressing PmEC01597:sYFP and AtFLS2:mCherry revealed separation of the plasma membrane from the cell wall, further supporting plasma-membrane localization of PmEC01597:sYFP (Supplemental Figure 3). Collectively, these data demonstrate that PmEC01597 targets multiple subcellular compartments when expressed in N. benthamiana.
The P. maydis effector candidate PmEC03792 is imported into the nucleus and nucleolus
Fungal pathogens have been shown to express and translocate effectors that preferentially accumulate protein within the host nucleus (Kemen et al., 2005; Petre et al., 2015). We, therefore, investigated whether any of the P. maydis effector candidates specifically targeted the nucleus. Analysis of the PmEC03792 protein sequence revealed a NLS motif at its N terminus (aa 8-30) as well as a nucleolar-targeting sequence (aa 4-35), suggesting that it may localize to the nucleus as well as the nucleolus (Sperschneider et al., 2017; Scott et al., 2011) (Table 1). To test our hypothesis, we transiently expressed the PmEC03792:sYFP protein fusion and assessed the subcellular localization pattern in N. benthamiana epidermal cells. Live-cell imaging using laser scanning confocal microscopy showed that PmEC03792 preferentially localized to subcellular compartments resembling the nucleus and nucleolus, whereas free sYFP predominantly localized to the cytoplasm and the nucleus (Figure 4). To confirm PmEC03792 is indeed localized to the nucleus and nucleolus, we transiently co-expressed PmEC03792:sYFP with either mCherry-tagged AtUBQ10-NLS or AtFIB2. Consistent with our hypothesis, live-cell imaging revealed that the PmEC03792:sYFP fluorescence signal co-localized with both the AtUBQ10-NLS:mCherry and AtFIB2:mCherry fluorescence signals, demonstrating that PmEC03792:sYFP accumulates in the nucleus and nucleolus (Figure 4).
PmEC04573 targets the chloroplasts
Chloroplasts often have an essential role in coordinating an effective plant immune response against pathogens and, as such, are often targeted by proteinaceous effectors from filamentous fungal pathogens (Littlejohn et al., 2021). Indeed, several of the P. maydis effector candidates encode predicted chloroplast transit peptide (cTP) sequences (Table 1), suggesting these effector candidates may target host chloroplasts. We, therefore, investigated whether any of the P. maydis effector-fluorescent protein fusions localized to these subcellular compartments. Intriguingly, fluorescence from the PmEC04573:sYFP-fluorescent protein fusion was consistently detected in organelles resembling chloroplasts as well as the nucleo-cytosol (Figure 5A-D). To test whether PmEC04573:sYFP is indeed chloroplast-localized, we co-expressed PmEC04573:sYFP with RbcS-TP:mCherry, a subcellular marker for plastids (Nelson et al., 2007). Consistent with our hypothesis, PmEC04573:sYFP fluorescence signal overlapped with RbcS-TP:mCherry on chloroplasts as well as stromules (stroma-containing tubules), confirming PmEC04573:sYFP does indeed accumulate on chloroplasts (Figure 5B-C).
Interestingly, immunoblot analyses with the PmEC04573:sYFP protein fusion consistently revealed two distinct protein products; the larger protein product was near the predicted molecular weight while the smaller protein band coincided with free sYFP, suggesting cleavage of sYFP from PmEC04573 had occurred (Figure 2; Supplemental Figure 4). To exclude that the chloroplast localization observed with PmEC04573:sYFP was an artifact caused by free sYFP, we coexpressed sYFP with RbcS-TP:mCherry in N. benthamiana leaf cells. As predicted, sYFP fluorescence signal was not observed on chloroplasts, even when the sYFP signal was saturated (Supplemental Figure 5). These protein expression data, coupled with the observation that free sYFP did not localize to chloroplasts, suggests that the nucleo-cytosol localization observed with PmEC04573:sYFP (Figure 5D) may be attributed to processed sYFP diffusing throughout the nucleoplasm and cytoplasm.
DISCUSSION
Recent sequencing of the Phyllachora maydis genome revealed that this fungal pathogen encodes putatively secreted effector candidate proteins (Telenko et al., 2020). However, our general understanding of host cell compartments targeted by the P. maydis effector repertoire is limited (Helm et al., 2022). In this study, we leveraged the availability of the P. maydis genome and refined previous effector predictions to investigate the subcellular compartments targeted by candidate effector proteins using a Nicotiana benthamiana-based heterologous expression system (Figure 1). We found that 37 of the 40 putative effectors accumulated detectable protein in planta (Figure 2). Among the 37 P. maydis effector-fluorescent protein fusions tested, 29 displayed a nucleo-cytoplasmic distribution that was indistinguishable from the free sYFP control (Supplemental Figure 1) and five predominantly localized to the cytosol (Supplemental Figure 2). One effector candidate, PmEC01597, localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane (Figure 3), while PmEC03792 was specifically imported into both the nucleus and nucleolus with no observable cytoplasmic stranding (Figure 4). Another putative effector, PmEC04573, consistently localized to chloroplasts as well as stromules (Figure 5).
Collectively, our data suggest that candidate effector proteins from P. maydis localize to distinct subcellular compartments and may associate with host proteins in these locations. It should be acknowledged that our approach relied on fusing a large fluorophore to the C terminus of each candidate effector as well as overexpression in a heterologous model plant. Furthermore, the selected putative effectors are predicted to encode signal peptides and are thus likely to be secreted; however, there is no direct evidence these proteins are translocated into host cells. Nevertheless, the observation that some PmECs accumulated protein and were targeted to specific subcellular locations within leaf cells suggests that these proteins are bona fide cytoplasmic effectors. Hence, knowledge of the subcellular localization patterns of the P. maydis effector repertoire will be informative in the identification of the host proteins they target as well as the cellular pathways they alter. Determining whether any of the P. maydis effector candidates have a functional role in manipulating host immune responses as well as their potential host targets in maize is a focus for future investigations.
One effector candidate, PmEC01597, consistently localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane (Figure 3). Though it is unclear of the functional significance of PmEC01597 localization to the nucleolus, effectors from fungi and oomycetes have been shown to target this subcellular compartment (Lorrain et al., 2018). A candidate effector from the poplar leaf rust pathogen (Melamspora larici-populina), termed Mlp124478, encodes a predicted nuclear localization peptide sequence, and accumulated in the nucleus and nucleolus of N. benthamiana epidermal cells (Ahmed et al., 2018; Petre et al., 2015). Furthermore, transgenic Arabidopsis constitutively expressing Mlp124478 displayed altered leaf morphology as well as repressed defense gene expression (Ahmed et al., 2018). Intriguingly, Chip-PCR analyses revealed that this candidate effector associates with the TGA1a-binding DNA sequence, suggesting Mlp124478 binds the TGA1a promoter region and represses expression of defense genes (Ahmed et al., 2018). Though the biological significance of PmEC01597 accumulating in the nucleolus remains to be investigated, we hypothesize this putative effector may interfere with host cell transcriptional machinery of ribosomal RNA (rRNA) genes or processing of ribosomal RNA synthesis.
In addition to targeting the nucleus and nucleolus, PmEC01597 consistently localized on the plasma membrane when transiently expressed in N. benthamiana epidermal cells (Figure 3). Proteinaceous effectors from filamentous phytopathogens have indeed been shown to target the plasma membrane where they often modulate host immune responses (Fabro, 2022; Lorrain et al., 2018). For example, work by Gaouar and colleagues (2016) showed that a different putative effector from poplar leaf rust, Mlp124202, localized on the plasma membrane when transiently expressed in N. benthamiana and in stable transgenic Arabidopsis. Consistent with the subcellular localization, yeast two-hybrid assays revealed that Mlp124202 associated with plasma membrane-localized synaptotagmin A (SYTA; At2g20990), suggesting this poplar leaf rust effector may have a functional role in modulating vesicle-mediated trafficking. Furthermore, the Phytophthora sojae-secreted effector, Avh240, preferentially accumulated on the host plasma membrane where it associated with and inhibited secretion of the soybean aspartic protease, GmAP1, thereby suppressing host immune responses (Guo et al., 2019). Murphy and colleagues (2018) showed that the Phytophthora infestans effector, Pi17316, interacts directly with VASCULAR HIGHWAY1-interacting kinase from potato (StVIK). Importantly, transgenic overexpression of StVIK in potato enhanced Phytophthora infestans virulence and colonization, demonstrating StVIK functions at least in part as a susceptibility factor (Murphy et al., 2018). We, therefore, predict PmEC01597 associates with host proteins on the plasma membrane to modulate host immune responses, similar to those of other plasma membrane-localized effectors from fungal and oomycete pathogens. Hence, future functional characterization of PmEC01597 should prioritize identifying host proteins from maize that interact with this putative effector.
Numerous filamentous phytopathogens often express and translocate effectors inside host cells where they specifically localize to host nuclear compartments and manipulate host immune responses (Caillaud et al., 2012; Schornack et al., 2010; Stam et al., 2013). Here, we show one putative effector, PmEC03792, was specifically targeted to the nucleus and nucleolus (Figure 4). Consistent with the subcellular localization pattern, PmEC03792 encoded a predicted NLS motif as well as a nucleolar-targeting sequence, suggesting this putative effector may manipulate host nucleolar functions. Indeed, numerous nuclear-localized fungal and oomycete effectors have been shown to disrupt many cellular processes by reprogramming transcriptional mechanisms to suppress host immune response. For example, AVR2, an effector from Phytophthora infestans, targets the nucleus and suppresses host immune responses through its association with a brassinosteroid-responsive bHLH transcription factor (Boch and Bonas, 2010; Turnbull et al., 2017). Furthermore, the Colletotrichum graminicola effector, CgEP1, specifically targets the host nucleus where it binds to chromatin, indicating that CgEP1 may modulate host transcription (Vargas et al., 2016). Moreover, the Ustilago maydis effector, See1, is localized to the host nucleus and interacts with the host SGT1 protein to reactivate DNA synthesis and cell division in infected leaves (Redkar et al., 2015). We, therefore, speculate nuclear-localized PmEC03792 may have a functional role in manipulating host transcription by targeting host proteins associated with nuclear compartments. Future functional characterization of PmEC03792 should prioritize identifying host proteins targeted by this candidate effector.
Our finding that the P. maydis putative effector PmEC04573 labels chloroplasts suggests that this fungal pathogen may target this organelle to modulate chloroplast-mediated host immune responses (Figure 5). Indeed, filamentous fungal pathogens have evolved intracellular effectors that localize to chloroplasts wherein they subvert chloroplast-derived immune responses (Littlejohn et al., 2021). For example, Melampsora larici-populina secretes several putative effectors, termed Chloroplast Targeting Proteins (CTP1, CTP2, and CTP3), that accumulate in the stroma of chloroplasts when transiently expressed in N. benthamiana (Petre et al., 2015; Petre et al., 2016). Importantly, CTP1, CTP2, and CTP3 encode predicted chloroplast transit peptides that are cleaved upon their translocation to chloroplasts, and which are necessary and sufficient for chloroplast localization (Petre et al., 2016). The observation that PmEC04573 also encodes a predicted chloroplast transit peptide sequence and localizes to chloroplasts suggests the cTP sequence may be necessary for chloroplast localization.
The wheat stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst) has also been shown to express and translocate several effectors into host cells where they subsequently traffic to chloroplasts (Figueroa et al., 2021; Littlejohn et al., 2021). For example, Pst_12806 is a haustorium-specific effector that, when secreted, localizes to host chloroplasts, and interacts with the photosynthesis-related protein TaISP (Xu et al., 2019). Importantly, the direct association between Pst_12806 and TaISP suppresses chloroplast-derived immune responses and photosynthesis, thereby promoting pathogen growth (Xu et al., 2019). Furthermore, two additional wheat stripe rust effectors, Pst_4 and Pst_5, were recently shown to interact with TaISP and attenuate chloroplast-derived immune responses (Wang et al., 2021). However, unlike Pst_12806, Pst_4 and Pst_5 associate with TaISP in the cytoplasm and such interaction likely prevents TaISP trafficking to the chloroplast, thereby suppressing chloroplast-derived production of reactive oxygen species (Wang et al., 2021). We, therefore, predict the subcellular targeting of chloroplasts by P. maydis may be important for facilitating infection. Future work should focus on identifying host proteins from maize that associate with PmEC04573, and what effect such interactions have on facilitating P. maydis infection.
We leveraged the availability of the P. maydis genome generated using short-read sequencing technology to select the candidate effectors investigated in our study (Telenko et al., 2020). However, given the relatively low BUSCO (benchmarking sets of universal single-copy orthologs) score and the high percentage of repetitive sequences within the fungal genome, we speculate the current P. maydis genome assembly is incomplete. Hence, future work should aim to generate an improved P. maydis genome using both short- and long-read sequencing technologies as such an improved genome will likely identify additional P. maydis effector candidates.
In summary, we show the majority of putative effectors from P. maydis accumulate protein in planta, and several localize to specific plant cell compartments including the nucleus, nucleolus, plasma membrane, and chloroplasts. Our data provide valuable insights into the putative functions of the P. maydis effector candidates as well as the host processes potentially manipulated by this fungal pathogen. Lastly, our findings can be used to generate testable hypotheses for addressing the functional roles of P. maydis effectors during pathogenicity as well as identifying their host targets in maize.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon request.
CONFLICT OF INTEREST
The authors declare that they have no competing interests and that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
AUTHOR CONTRIBUTIONS
M.H. and R.S. conceived and designed the study. M.H., R.S., R.H., N.J., and A.M., performed the experiments. M.H., R.S., R.H., N.J., A.S.I-P, and S.B.G. analyzed the data. M.H. and R.S. drafted and wrote the manuscript. All authors edited the manuscript and approved the final version.
SUPPLEMENTARY FIGURE LEGENDS
ACKNOWLEDGEMENTS
The authors thank Darcy Telenko (Purdue University) for providing sequences of the candidate effectors from P. maydis Indiana isolate PM-01, and Roger Innes (Indiana University) for providing the AtFLS2:mCherry, AtFIB2:mCherry, 3xHA:sYFP (free sYFP), and 3xHA:mCherry (free mCherry) constructs. The authors would like to thank the Purdue University Imaging Facility for access to the Zeiss LSM880 Axio Examiner upright confocal microscope. We also thank Morgan Carter and Martin Darino for insightful discussions and critical reading of the manuscript. The funding bodies had no role in designing the experiments, collecting the data, or writing the manuscript. All opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of USDA, DOE, or ORAU/ORISE. USDA is an equal opportunity provider and employer.
Footnotes
Funding: This work was supported by the United States Department of Agriculture, Agriculture Research Service (USDA-ARS) research project 5020-21220-014-00D and an appointment to the Oak Ridge Institute for Science and Education (ORISE). This research was also funded by the Foundation for Food and Agriculture Research New Innovator Award and Indiana Hatch Funds (IND011293) awarded to A.I.P.