The retromer CSC subcomplex is recruited by MoYpt7 and sequentially sorted by MoVps17 for effective conidiation and pathogenicity of the rice blast fungus

Abstract In eukaryotic cells, Rab GTPases and the retromer complex are important regulators of intracellular protein transport. However, the mechanistic relationship between Rab GTPases and the retromer complex in relation to filamentous fungal development and pathogenesis is unknown. In this study, we used Magnaporthe oryzae, an important pathogen of rice and other cereals, as a model filamentous fungus to dissect this knowledge gap. Our data demonstrate that the core retromer subunit MoVps35 interacts with the Rab GTPase MoYpt7 and they colocalize to the endosome. Without MoYpt7, MoVps35 is mislocalized in the cytoplasm, indicating that MoYpt7 plays an important role in the recruitment of MoVps35. We further demonstrate that the expression of an inactive MoYpt7‐DN (GDP‐bound form) mutant in M. oryzae mimicks the phenotype defects of retromer cargo‐sorting complex (CSC) null mutants and blocks the proper localization of MoVps35. In addition, our data establish that MoVps17, a member of the sorting nexin family, is situated at the endosome independent of retromer CSC but regulates the sorting function of MoVps35 after its recruitment to the endosomal membrane by MoYpt7. Taken together, these results provide insight into the precise mechanism of retromer CSC recruitment to the endosome by MoYpt7 and subsequent sorting by MoVps17 for efficient conidiation and pathogenicity of M. oryzae.


| INTRODUC TI ON
Vesicle transport exists in all eukaryotic cells to mediate material and signal communication, and it involves defined and dynamic space-time coupled processes regulated by many different proteins (Faini et al., 2013;Jahn & Scheller, 2006;Seaman, 2008). Rab GTPases are among the essential components and regulators of vesicle transport that mediate various steps of membrane trafficking (Hutagalung & Novick, 2011;Li & Marlin, 2015;Pfeffer, 2013;Wandinger-Ness & Zerial, 2014). They are evolutionarily conserved in many organisms ranging from yeast to humans, and function as molecular switches by alternating between inactive (guanosine diphosphate [GDP]-bound) and active (guanosine triphosphate [GTP]-bound) states to promote vesicle formation, scission, movement, and fusion at the target membrane (Hutagalung & Novick, 2011;Li & Marlin, 2015;Pfeffer, 2013;Wandinger-Ness & Zerial, 2014). In particular, Rab7 is a conserved protein that plays an important role in the late endocytic pathway and in lysosome biogenesis in mammalian cells (Chavrier et al., 1990;Modica et al., 2017). In budding yeast (Saccharomyces cerevisiae) or fission yeast (Schizosaccharomyces pombe), the homolog of Rab7, Ypt7, is localized primarily to the vacuolar membrane and mediates docking and fusion of late endosomes with vacuoles; it is also necessary for vacuole-vacuole fusion (Balderhaar et al., 2010;Kashiwazaki et al., 2009). In Arabidopsis, RABG3f, a plant Rab7 homolog, localizes to prevacuolar compartments and the tonoplast, and it is involved in vacuolar trafficking, vacuole biogenesis, and plant growth (Cui et al., 2014;Ebine et al., 2014). Although the localization and functions of Rab7 have been investigated in various organisms, the molecular details of Ypt7-mediated protein trafficking pathways in the endosomal system are still not well understood.
Retromer is a multisubunit protein transport complex that mediates transportation of various transmembrane proteins/receptors, including immune protein receptors, mannose-6-phosphate receptors, Shiga toxin, and Wnt receptors from early endosomes to the trans-Golgi network (TGN) (Bonifacino & Rojas, 2006;Brown et al., 2001;Eaton, 2008;Seaman, 2005;Verges, 2007). Dysfunction of retromer abrogates various physiological processes such as secretion, storage, transport, and immune responses (Abubakar et al., 2017). Retromer is composed of two subcomplexes: a sorting nexin (SNX) dimer and a cargo-sorting complex (CSC). The CSC consists of three vacuolar protein-sorting (Vps) proteins, namely Vps35, Vps26, and Vps29. The C-terminus of Vps35 binds to the phosphodiesterase-folded region of Vps29 and its N-terminus binds to the C-terminal domain of Vps26, making it the central protein of the retromer complex. The SNX dimer consists of Vps5 and Vps17 proteins, which both contain PX (phox homology) and BAR (Bin-Amphyphysin-Rvs) domains. The PX domain binds to phosphatidylinositol on the endosomal membrane surface for effective anchorage of the retromer, whereas the BAR domain induces membrane curvature for vesicle formation (Seaman, 2005).
Studies in mammals and S. cerevisiae have shown that Rab7/ Ypt7 coordinates retromer-mediated cargo transport during vesicle-vacuole fusion (Balderhaar et al., 2010;Rojas et al., 2008). In mammals, GTP-binding Rab7 protein recruits the retromer CSC to the endosomal membrane (Rojas et al., 2008). Silencing the RAB7 gene prevents the association of the retromer CSC with the endosomal membrane, which in turn results in mis-sorting of acid hydrolase D and inhibition of retromer-mediated retrograde transport of cation-independent mannose-6-phosphate receptor (CI-MPR) (Rojas et al., 2008). Further study revealed that the retromer complex and Rab7 colocalize on endosomes and that GTP-Rab7 protein interacts directly with the retromer CSC (Rojas et al., 2008). In addition, Seaman et al. showed that TBC1D5 acts as a GTPase activating protein of Rab7, which interacts with the retromer complex and negatively regulates the recruitment of retromer CSC to the endosomal membrane (Seaman et al., 2009). In yeast, the retromer CSC acts as a downstream effector of Ypt7 and overexpression of YPT7 causes the degradation of CSC in the vacuoles, indicating that Ypt7 is essential for proper localization of the CSC (Balderhaar et al., 2010).
Magnaporthe oryzae is a heterozygous filamentous fungus that causes the devastating rice blast disease, which hampers rice production worldwide and threatens world food security. The organism has emerged as an important model organism for studying the development and mechanism of pathogenesis in filamentous fungi (Ebbole, 2007;Valent & Chumley, 1991). M. oryzae infection begins when the fungal spores land on the host surface and begin to germinate and differentiate to produce appressoria, which are required for physical penetration of the host cuticles (Mentlak et al., 2012;Veneault-Fourrey et al., 2006;Wilson & Talbot, 2009). In our previous studies, we demonstrated that ΔMoypt7 and ΔMovps35 deletion mutants exhibit similar phenotypes, including defective conidiogenesis and pathogenicity (Liu et al., 2015;Zheng et al., 2015b).
MoYpt7 is localized on the late endosome/vacuolar membrane and is important for the fungal growth, conidiation, vacuolization, autophagy, stress responses, and pathogenicity (Liu et al., 2015). The ΔMovps35 mutant has delayed lipid and glycogen degradation, and is significantly reduced in conidiation and virulence on rice and barley. Further study showed that appressorial turgor pressure in the ΔMovps35 mutant is significantly lower than that of the wild type, and autophagy is blocked during conidial germination (Zheng et al., 2015b). Both MoYpt7 and MoVps35 are important for autophagy and may work in the same trafficking pathway to regulate vegetative growth, conidiation, and pathogenesis of M. oryzae.
However, our understanding of the relationship between Rab GTPases and the retromer complex in filamentous fungi is completely unknown. In this study, we dissect the molecular interactions and functional mechanisms of these two intracellular trafficking machineries in M. oryzae. Using affinity purification/ mass spectrometry assay, coimmunoprecipitation (Co-IP), and colocalization studies, we show that MoYpt7 interacts with MoVps35, the core subunit of the retromer complex. Loss of MoYpt7 results in mislocalization of MoVps35 to the cytoplasm, which reduces M. oryzae conidiation and pathogenesis. Furthermore, MoVps35 is recruited to the late endosomes by MoYpt7 in a nucleotide-dependent manner. Finally, we show that the retromer-sorting nexin protein MoVps17 replaces MoYpt7 for sequential sorting of retromer CSC in pathogenicity of the rice blast fungus. Our data address the key question of how a plant pathogen balances vesicular recruitment and subsequent sorting events to govern its development and pathogenesis.

| Punctate MoVps35 associates with MoYpt7 on late endosomal and vacuolar membranes
MoYpt7 is mainly localized on late endosomal and vacuolar membranes, while MoVps35, a core subunit of the retromer complex, appears as fast-moving punctate structures close to FM4-64marked vacuolar membranes in M. oryzae (Liu et al., 2015;Zheng et al., 2015b). We therefore investigated the possible colocalization of the two proteins. mCherry-MoYpt7 and MoVps35-GFP were coexpressed in the Guy11 strain via transformation of the respective plasmid constructs, and their intracellular localization was examined by laser scanning confocal microscopy. We found that MoYpt7 appeared on ring-shaped vacuoles and dotted structures in both mycelial and conidial cells, while MoVps35 exhibited its usual punctate appearance inside the cells (Figure 1a,b). Strikingly, the majority of MoVps35 punctate structures were aligned at the surface of the MoYpt7 vacuoles and dotted endosomes, and this was further verified by line scan assays (Figure 1a,b). These data clearly demonstrate that MoVps35 closely associates with MoYpt7 on the late endosomal and vacuolar membranes.

| MoVps35 interacts with MoYpt7 in M. oryzae
The partial colocalization of MoVps35 and MoYpt7 prompted us to check whether there is direct interaction between the two proteins.
We first expressed MoVps35-GFP in the ΔMovps35 strain and then used GFP-Trap beads to extract MoVps35-GFP and its associated proteins in pull-down assays. Considering the fact that MoYpt7 and retromer complex have both been found to be involved in autophagy, which is essential for M. oryzae conidiogenesis and pathogenicity (Liu et al., 2015;Zheng et al., 2015b), we analysed the MoVps35 interactomes obtained from nutrient-sufficient and nitrogen-deficient conditions for uninduced and induced autophagy, respectively. Two negative control strains, Guy11 (wild type) and Guy11-GFP (wild type expressing green fluorescent protein, GFP), were introduced for parallel analysis. Proteins bound to the GFP-Trap beads were eluted and fractionated by 10% sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie brilliant blue R-250. As shown in Figure 1c, many protein bands were observed in the MoVps35-GFP strains as opposed to only a few in the two negative controls (Guy11 and Guy11-GFP). Digestion with trypsin and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that the retromer CSC subcomplex, MoVps35-MoVps26-MoVps29, was repeatedly captured as one of the interacting proteins in all the replicates of MoVps35-GFP pulldown (Table 1), and this was considered as high-confidence positive control. Remarkably, MoYpt7 was also captured repeatedly in the MoVps35-GFP pull-down assays, suggesting a high possibility of interaction between MoYpt7 and MoVps35 (Table 1).

Next, we tested for in vivo interaction between MoYpt7 and
MoVps35 by Co-IP. To this end, we generated a Guy11 transformant coexpressing GFP-MoYpt7 and MoVps35-FLAG and subjected it to immunoprecipitation (using GFP-Trap_A beads) and subsequent immunoblot analysis (using an anti-FLAG antibody). A 51 kDa band corresponding to GFP-MoYpt7 and a 102 kDa band corresponding to MoVps35-FLAG were identified, suggesting an in vivo interaction between the retromer subunit, MoVps35, and MoYpt7 ( Figure 1d).    ΔMoypt7 to the wild-type phenotype. We also performed a reciprocal experiment by overexpressing MoYpt7 in the ΔMovps35 mutant.

| Ectopic expression of dominant negative
The results were the same ( Figure S3), suggesting that the regulatory relationship between MoYpt7 and MoVps35 is not decided by transcriptional control but depends on their accurate spatial positioning in the cells.

| MoVps17 releases MoVps35 after its recruitment to the endosomal membrane by MoYpt7
The precise sorting event of the retromer CSC after recruitment to the endosomal membrane and any potential cross-regulation among the retromer CSC, SNX, and Ypt7 have not been investigated in depth, even in mammals and yeast. To our knowledge, this process is also com- and ΔMovps17 mutants appear less pathogenic than the mutants of the retromer CSC subunits.

| D ISCUSS I ON
In this study, we investigated the molecular mechanism of the indispensable vesicular transport machinery, the retromer CSC, in the phytopathogenic fungus M. oryzae. To summarize our findings, the model in Figure 8a is proposed, in which MoYpt7 initially recruits the retromer CSC from the cytosol to the endosomal membrane.
After this recruitment, MoVps17 subsequently replaces MoYpt7 and assembles with the retromer CSC to mediate protein sorting/ trafficking. In M. oryzae, protein sorting is essential for inducing autophagy, which is required for effective conidiation and pathogenicity (Abubakar et al., 2017;Zheng et al., 2015b). In addition, the sorting nexin proteins MoVps17/MoVps5 play distinct roles in the regulation of endosome dynamics during fungal development and plant infection, which was documented in our previous study .  (Liu et al., 2015;Zheng et al., 2015bZheng et al., , 2017. The present study confirmed that the pathogenicity of ΔMovps35, ΔMovps26, and ΔMovps29 mutants were significantly reduced compared to the wild-type strain, while ΔMoypt7 and ΔMovps17 mutants almost lost their pathogenicity in the host (Figure 7), indicating that MoYpt7 and MoVps17 play stronger regulatory roles in pathogenicity than the retromer CSC complex in M. oryzae. Our previous study showed that the SNX protein MoVps17 localizes to endosomes and is essential for regulating endosome dynamics such as endosome budding, fusion, and endocytosis, which are independent of the retromer CSC , meaning that the tethering of MoVps17 to the endosomal membrane further strengthens the ability of the pathogen to infect the host plant. MoYpt7 acts as a molecular switch for vesicle transport where deletion of the MoYpt7 gene prevents the transition from early to late endosomes or vacuole/lysosomes, which leads to defects in multiple regulatory pathways and autophagy (Liu et al., 2015). This explains why the ΔMoypt7 mutant in this study almost lost the ability to infect the host.
Endosomes are important protein-sorting centres during endocytosis in eukaryotes (Huotari & Helenius, 2011). The cargoes of the endocytic system could be retained in mature endosomes and delivered to lysosomes for degradation, or could be transported to other organelles (Seaman et al., 1998). The retromer complex prevents unnecessary degradation of cargoes by sorting them from endosomes and channelling them to the Golgi network via the retrograde trafficking pathway (Attar & Cullen, 2010;Rojas et al., 2008;Seaman et al., 1998). In mammals, Rab7 interacts with the retromer CSC complex Vps26-Vps29-Vps35. A pull-down assay indicated that Rab7 colocalizes with retromer complex on the endosomes, and the retromer CSC complex Vps26-Vps29-Vps35 is recruited to the endosomal membrane by GTP-bound Rab7 protein (Priya et al., 2015;Rojas et al., 2008). In Arabidopsis, the Rab7 homolog RABG3f plays a role in recruiting the retromer CSC to the endosomal membrane through interaction with Vps35 (Zelazny et al., 2013). This recruitment requires Ypt7, a protein that coordinates retromer-mediated retrograde trafficking, membrane recycling, and fusion of late endosomes with the vacuole in S. cerevisiae (Balderhaar et al., 2010;Harrison et al., 2014;Liu et al., 2012;Purushothaman et al., 2017).
These results suggest that the interaction of Ypt7 with Vps35 is conserved among different species.
Rab proteins regulate various pathways by interacting with some effector proteins. In yeast, previous studies on the retromer complexes showed that the retromer CSC interacts with Ypt7-GTP (Balderhaar et al., 2010;Liu et al., 2012;Purushothaman et al., 2017).
Our results showed that both the GTP-bound (MoYpt7-CA) and GDPbound (MoYpt7-DN) forms of MoYpt7 interacted with MoVps35 ( Figure 3c), which means that this interaction is independent of the F I G U R E 7 The Rab GTPase MoYpt7 and the retromer complex are important for pathogenicity in Magnaporthe oryzae. (a) Disease symptoms due to mycelial blocks from wild type (Guy11), ΔMoypt7, ΔMoypt7-C, ΔMovps17, ΔMovps17-C, ΔMovps35, ΔMovps35-C, ΔMovps26, ΔMovps26-C, ΔMovps29, and ΔMovps29-C strains, inoculated on intact and wounded barley leaves for 7 days. Compared to the wild type and each complemented (-C) strain, ΔMovps35, ΔMovps26, and ΔMovps29 are significantly reduced in virulence while ΔMoypt7 and ΔMovps17 almost lost their pathogenicity on the host. (b) Quantification of lesion area from the inoculated barley leaves. The level of significance was measured using an unpaired t test (**p < .01, n = 9 lesions, error bars indicate standard deviation).(c) Disease symptoms due to mycelial blocks from wild type, ΔMoypt7, ΔMoypt7-C, ΔMovps17, ΔMovps17-C, ΔMovps35, ΔMovps35-C, ΔMovps26, ΔMovps26-C, ΔMovps29, and ΔMovps29-C strains, inoculated on intact and wounded rice leaves for 7 days. Compared to the wild type and each complemented strain, ΔMovps35, ΔMovps26, and ΔMovps29 are significantly reduced in virulence while ΔMoypt7 and ΔMovps17 almost lost their pathogenicity on the host. (d) Quantification of lesion area from the inoculated rice leaves. The level of significance was measured using an unpaired t test (**p < .01, n = 9 lesions, error bars indicate standard deviation).(e) Microscopic observations for penetration of wild type, ΔMoypt7, ΔMoypt7-C, ΔMovps17, ΔMovps17-C, ΔMovps35, ΔMovps35-C, ΔMovps26, ΔMovps26-C, ΔMovps29, and ΔMovps29-C mycelia inoculated on barley leaves for 2 and 3 days. Extensive hyphae are found in the barley epidermal cells when inoculated with the wild type and each of the complemented strains. ΔMovps35, ΔMovps26, and ΔMovps29 can also penetrate the barley epidermal cells and produce invasive hyphae, while the hyphal growth of ΔMoypt7 and ΔMovps17 ceased completely. Bar = 50 µm activate or inactivate state of MoYpt7. Our finding is consistent with a study from mammalian cells that showed that the dissociation constants for the interactions of Vps35 with GppNHp-and GDP-bound Rab7 were similar, indicating Vps35 does not show any preference for the active form of Rab7 (Priya et al., 2015). Most effector proteins interact with Rab proteins in their activate states, but there are a few effector proteins that interact with inactivate Rab proteins (Shirane & Nakayama, 2006). Therefore, the MoVps35 may not act as a typical effector of MoYpt7 in M. oryzae. This clearly demonstrates a difference in the regulation of retromer function between yeast and filamentous fungi.
We also revealed that the M. oryzae retromer exhibited unexpected features compared to its yeast counterparts. For example, we found that MoVps35-GFP fluorescence was aggregated into plaquelike structures and the discrete fluorescent spots were significantly reduced in the ΔMovps17 mutant compared to the wild type (Guy11) ( Figure 5). In yeast, although the localization of retromer CSC to endosomes requires the Vps5 and Vps17 sorting nexins, it does not show aggregated structures in the Δvps5vps17 double mutant (Liu et al., 2012). In addition, loss of Ypt7 does not lead to mislocalization of Vps35-GFP to the cytoplasm in the yeast (Liu et al., 2012). A further study found that yeast Vps35-GFP and Vps26-GFP localize to the cytosol in a Δvps5vps17ypt7 triple mutant (Liu et al., 2012). These data indicate that the M. oryzae sorting nexin MoVps17 itself plays a vital role in retromer CSC sorting. We also investigated the connection between MoYpt7 and MoVps17 and found that the punctate localization of MoVps17 was partially affected in a MoYpt7 mutant, and MoVps17 was not required for endosomal/vacuolar membrane localization of MoYpt7 (Figure 6a,b). The punctate localization of MoVps17 plays a leading role in vesicular sorting, which is clearly different from its role in yeast.
Overexpression of MoYpt7 in ΔMovps35 did not restore the phenotypic defects of ΔMovps35 ( Figure S3). Likewise, overexpression of MoVps35 in ΔMoypt7 did not restore the phenotypic defects observed in ΔMoypt7 (Figure 4). These results indicate that the biological function in conidogenesis and pathogenicity depends on correct recognition of the retromer by MoYpt7 at the right time and in the right space. Therefore, our results fill the gap in knowledge between retromer complex recruitment and its sorting function in filamentous fungi, which could lay a foundation for effective prevention and control of rice blast disease.

| Construction of fusion vectors and point mutations
MoYpt7 was tagged at the N-terminus with mCherry, with the gene under the control of its native promoter, to generate the native-mCherry-MoYpt7 vectors. This insertion was verified by PCR using the specific primer pairs presented in Table S1. The mCherry fragment was amplified from the FgSpa2-mCherry plasmid (Zheng et al., 2015a) and then cloned into the pCB1532 plasmid digested with SalI and BamHI. The MoYpt7 protein was also tagged with GFP at its N-terminus, with the gene under the control of native and RP27 promoters. The GFP fragment was isolated from the GFP-FgRab fusion vector (Zheng et al., 2015a), whereas the RP27 fragment was isolated from the pTE11 vector and cloned into plasmid pKNT following digestion with XhoI and HindIII restriction enzymes. For generation of MoYpt7-CA and MoYpt7-DN constructs, we first amplified MoYpt7-CA1, -CA2, -DN1, and -DN2 genes from the cDNA of Guy11 using the pairs of primers presented in Table S1. We then introduced the point mutations in MoYpt7-CA and MoYpt7-DN fragments using splicing-byoverlap extension PCR and cloned them (with an RP27 promoter) into pKNT vector and verified the constructs by sequencing. MoVps35 was also tagged with GFP at its C-terminus, with the gene under the control of its native or RP27 promoter, to form the native-MoVps35-GFP or RP27-MoVps35-GFP vectors. The constructed vectors were transformed into Guy11, ΔMoypt7, or ΔMovps35 mutant protoplasts.

| Co-IP assay
We coexpressed MoYpt7 fused with GFP and MoVps35 tagged with FLAG in Guy11 protoplasts. The total proteins were extracted from the transformants and GFP-Trap beads (ChromoTek Inc.) were used for a pull-down assay. The interacting proteins were separated by SDS-PAGE. The GFP-MoYpt7 and the MoVps35-FLAG proteins were detected by α-GFP (MBL) and α-FLAG (Abmart) antibodies, respectively.

| Quantitative reverse transcription PCR
Total RNA was extracted from the wild type and mutants' mycelia harvested from liquid CM that was inoculated and incubated at 28 °C with constant shaking at 110 rpm for 3 days, using an Eastep Super Total RNA Extraction Kit (Promega). Reverse transcription was used to generate cDNA using a reverse transcription kit (Takara).
Quantitative PCR was finally conducted using a SYBR kit (Takara) with specific primers (Table S1). M. oryzae β-tubulin (Table S1) was used as the endogenous reference gene, and the expression levels were calculated using the 2 −ΔΔCt method as previously reported (Livak & Schmittgen, 2001). All RT-qPCR assays were repeated three times for each sample.

| Subcellular localization assay
For cellular localization assay, all the strains were cultured in liquid/ solid CM at 28 °C. The mycelia and conidia of the strains involved were observed using a Nikon A1R laser scanning confocal microscope. Unless noted otherwise, all microscope images were taken within the same focal plane section.

| Phenotypic analysis
For growth rate assays, mycelial blocks 3 mm in diameter (the peripheral edge of the colony) were inoculated at the centre of CM, SYM, and RBM plates. We measured the diameters of the colonies after 10 days of incubation at 28 °C. For conidiation assays, the strains were cultured on RBM plates and incubated at 28 °C and the first aerial hyphae were stripped out after they had filled up the plates, and reincubated. After 4 days of the second incubation, the cultures were washed with double-distilled water and conidia were collected and counted using a haemocytometer. For the pathogenicity assay, fresh mycelial blocks were inoculated on intact and wounded barley (cv. Golden Promise) as well as rice (CO39) leaves. The set-up was kept humid and incubated in the dark for 24 hr at 28 °C, then transferred to a 12 hr photoperiod at 28 °C. The lesions were observed and compared after 7 days. The lesion area was analysed using ImageJ software.