MoErv14 mediates the intracellular transport of cell membrane receptors to govern the appressorial formation and pathogenicity of Magnaporthe oryzae

Magnaporthe oryzae causes rice blasts posing serious threats to food security worldwide. During infection, M. oryzae utilizes several transmembrane receptor proteins that sense cell surface cues to induce highly specialized infectious structures called appressoria. However, little is known about the mechanisms of intracellular receptor tracking and their function. Here, we described that disrupting the coat protein complex II (COPII) cargo protein MoErv14 severely affects appressorium formation and pathogenicity as the ΔMoerv14 mutant is defective not only in cAMP production but also in the phosphorylation of the mitogen-activated protein kinase (MAPK) MoPmk1. Studies also showed that either externally supplementing cAMP or maintaining MoPmk1 phosphorylation suppresses the observed defects in the ΔMoerv14 strain. Importantly, MoErv14 is found to regulate the transport of MoPth11, a membrane receptor functioning upstream of G-protein/cAMP signaling, and MoWish and MoSho1 function upstream of the Pmk1-MAPK pathway. In summary, our studies elucidate the mechanism by which the COPII protein MoErv14 plays an important function in regulating the transport of receptors involved in the appressorium formation and virulence of the blast fungus.

Introduction Rice blast is one of the most devastating fungal diseases on rice, and it seriously threats world food supply security [1]. When infecting the host, the blast fungus Magnaporthe oryzae recognizes external physical and chemical signals, such as surface hardness and hydrophobicity, to produce specific infectious structures called appressoria. The robust turgor generated in appressoria leads to the penetration of the host epidermis and the production of invasive hyphae.
Previous studies indicated that several cell surface transmembrane receptor proteins, including MoPth11, play a role in sensing the host surface for appressorium production. MoPth11 is an atypical G protein-coupled receptor (GPCR) that senses surface hydrophobicity leading to endocytosis, which regulates the cAMP-signal pathway and appressorium formation [2,3]. In addition, the receptor proteins MoMsb2 and MoSho1 recognize the host surface keratinous monomer to activate the MoMst11-MoMst7-MoPmk1 MAP kinase signaling pathway that also controls appressorium formation. A recent study suggested that a GPCR protein, MoWish, is involved in appressorium formation by also recognizing host hydrophobic signals [4]. Although the above studies showed that surface transmembrane proteins are important for signal perception and appressorium induction, the molecular mechanism by which these transmembrane proteins are transported intracellularly and exert their function remains unknown.
In eukaryotic cells, protein transport and exchange of macromolecular substances between different organelles depend on vesicle trafficking. Vesicles are membrane-coated cargos containing various macromolecules that shuttle between different subcellular structures or organelles. According to their different forms of membrane coating, vesicles are divided into clathrin vesicles, COPI (cytoplasmic envelope complex I or coat protein complex I) vesicles, and COPII (cytoplasmic envelope complex II, coat protein complex II) vesicles. Clathrin vesicles mainly function in the transport of cargo from the Golgi apparatus to lysosomes and cell membranes and the endocytosis of extracellular or membrane substances into the cell. COPI vesicles mediate transport from the Golgi back to the endoplasmic reticulum (ER), and COPII vesicles mediate the material transport from the ER to the Golgi [5].
The transport of substances from the ER to the Golgi apparatus mediated by COPII is an essential component of the secretion pathway in eukaryotes that is essential for cells to perform their normal functions and maintain the dynamic balance of various organelles [6]. For example, in the budding yeast Saccharomyces cerevisiae, the Sec23/Sec24 complex forms the inner membrane, while the Sec13/Sec31 complex forms the outer membrane when coating on COPII vesicles [7]. ERV (ER-derived vesicles) proteins, divided according to their molecular weights [8,9], are a group of conserved cargo receptor proteins that is mainly responsible for recruiting specific cargo proteins and directing their ER to Golgi transport [9]. Different ERV proteins recognize specific cargo proteins [10], but the transport of the cargo containing transmembrane receptor proteins remains unclear.
We previously reported that MoErv29 specifically mediates the secretion of protein effectors during infection [11]. Here, we characterized the function of MoErv14, another member of the COPII vesicle receptor family in M. oryzae, and we revealed that MoErv14 mediates the transport of various transmembrane receptor proteins that promote the appressorium formation and pathogenicity of M. oryzae.

Identification and characterization by host infection of ERV family genes in M. oryzae
We previously reported that the COPII cargo receptor MoErv29 promotes the apoplastic effector secretion and the virulence of M. oryzae [11]. In order to systematically elucidate functions of the ERV COPII cargo receptor family proteins, we screened the available genomes of M. oryzae by BLAST using the S. cerevisiae ERV proteins' sequence as a reference and identified five MoERV genes in M. oryzae: MoERV14 (MGG_08132), MoERV25 (MGG_08210), MoERV26 (MGG_08706), MoERV41 (MGG_00949), and MoERV46 (MGG_01245) (Fig 1A and 1B).
To examine the roles of the above-identified ERV proteins in M. oryzae, we generated respective mutant strains that were verified by Southern blot analysis (S1 Fig) and carried out pathogenicity studies. Conidial suspensions of the mutants were sprayed onto 2-week-old rice seedlings (Oryza sativa cv. CO-39), along with the wild-type strain Guy11. Seven days after inoculation, ΔMoerv14 mutants failed to produce any leaf lesions, while ΔMoerv25, ΔMoerv26, ΔMoerv41, and ΔMoerv46 all produced numerous spindle-shaped lesions, comparable to Guy11. Similar results were also observed on detached barley leaves. However, in the ricesheath penetration assay, lesions produced by the ΔMoerv14 mutant showed 95% Type 1 and 5% Type 2 infectious hyphal growth, compared to 9% Type 1, 20% Type 2, 31% Type 3, and 40% Type 4 in Guy11. The severe defect in the pathogenicity of ΔMoerv14 was restored in the complemented strain (Fig 1C-1F). We concluded that MoErv14 plays a critical role in host penetration and invasive hyphae growth.

MoErv14 is involved in the vegetative growth, conidium formation, and appressorium development of M. oryzae
We selected MoErv14 for further studies based on its importance in pathogenicity. Firstly, to determine whether Erv14 is conserved among species, we used cDNA of MoErv14 to complement a yeast Δerv14 mutant. As expected, MoErv14 could partially suppress the growth defect of the yeast Δerv14 mutant upon dithiothreitol (DTT)-induced stress (S2 Fig). Despite the partial suppression, it suggested that MoErv14 could be functionally homologous to ScErv14. We then observed the vegetative growth of ΔMoerv14 mutants that showed significant growth reduction on CM, MM, SDC, and OM media in comparison to Guy11 and the complemented strain (Fig 2A-2B). The colony formed by the ΔMoerv14 mutant was much thinner on day 7 ( Fig 2C). We also found that ΔMoerv14 mutants produced significantly fewer conidia on cornmeal agar (SDC) (Fig 2D-2E). These results indicated that MoErv14 is important for the hyphae growth and asexual development of M. oryzae.
To further assess hyphae growth and asexual development as the cause of decreased virulence of the ΔMoerv14 mutant, we estimated conidia germination and appressorium formation at various time points [12][13][14]. The conidia germination of the ΔMoerv14 mutant was not significantly different from Guy11. However, its appressorium formation rate was significantly lower than Guy11, as~20% of the conidia developed appressoria in 24 hours (Fig 2F-2G).

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MoErv14 mediates transmembrane sensor transportation to control appressorium formation in M. oryzae

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MoErv14 mediates transmembrane sensor transportation to control appressorium formation in M. oryzae Meanwhile, the appressoria of the ΔMoerv14 mutant showed a higher collapse ratio than the control, indicating a decreased turgor pressure (S3 Fig). In addition, MoErv14 appeared not to be involved in a septin ring formation (S4 Fig). This results suggested that MoErv14 is important for appressorium formation and function.

MoErv14 is a vesicle traffic-associated protein
We then generated a MoErv14-GFP fusion protein and found MoErv14-GFP is located at vesicle-like spots that could be stained by the lipophilic dye FM4-64 ( Fig 3A). With reference to RFP-tagged MoLhs1 and MoSft2 proteins that are markers of ER and Golgi, respectively
Moreover, by using gradient centrifugation to fractionate subcellular organelles, we confirmed that MoErv14 was mainly accumulated in Golgi and ER (S5C Fig). We further coexpressed MoErv14-S and MoSec24-2-GFP in Guy11 and extracted the vesicular proteins. Proteins were then analyzed by Western blotting using S tag and GFP-specific antibodies. The results showed that MoErv14 and MoSec24-2 were detected in total vesicular proteins, which indicated that MoErv14 is a vesicular protein (S5D Fig). In yeast, Erv14 was reported to function as a COPII component shuttling between ER and Golgi. We then employed Brefeldin A (BFA, an inhibitor of ER to Golgi protein transport) and live-cell time-lapse imaging analysis revealed that the dynamic localization pattern of MoErv14 can be specifically inhibited by BFA ( Fig 3C) [18]. Taken together, these results suggested that MoErv14 is a COPII component that shuttles between ER and Golgi.
We also examined whether the ΔMoerv14 mutant is defective in endocytosis. By staining cells with FM4-64, we observed its internalization, but there was no apparent difference between the ΔMoerv14 mutant and Guy11 2 and 5 min after staining (S6 Fig).

MoErv14 mediates cAMP and MoPmk1-MAPK signaling pathways
Previous studies demonstrate that both cAMP and MoPmk1-MAPK signaling pathways are implicated in transducing hydrophobic surface signals to regulate appressorium formation in M. oryzae [1]. When MoPth11 senses surface signals, cAMP signaling is activated to promote appressorium formation. MoMsb2 and MoSho1, on the other hand, control the formation of appressoria by sensing host surface signals to activate the Mst11-Mst7-Pmk1 signaling pathway through protein phosphorylation. We measured cellular levels of cAMP and MoPmk1 phosphorylation, respectively, and the results showed that both cAMP levels and MoPmk1 phosphorylation were decreased significantly in the ΔMoerv14 mutant (S7A- S7B Fig).
We further analyzed the effect of externally supplementing cAMP on the ΔMoerv14 mutant. At 10 and 20 mM levels, the ΔMoerv14 mutant can germinate and form appressoria, but not at the levels of the wild-type strain (Fig 4A-4B). Exogenous cAMP also suppressed the virulence defect of ΔMoerv14 mutants on rice and barley ( Fig 4C).
We also transformed a MoMST7 S212D, T216E (MoMST7 DE ) construct into the ΔMoerv14 mutant to constitutively activate the MoPmk1-MAPK1 pathway ( Fig 5A). The results showed that MoMst7 DE effectively suppressed the defect of the ΔMoerv14 mutant in appressorium formation (Fig 5B-5C). Seven days after inoculation, the virulence of ΔMoerv14/ MoMST7 DE was partially restored when compared with the ΔMoerv14 mutant. ΔMoerv14/ MoMST7 DE also exhibited a higher penetration rate and enhanced infectious hyphal growth (Fig 5D-5F). Based on these results, we concluded that MoErv14 is involved in regulating cAMP and MoPmk1-MAPK signaling pathways required for appressorium formation and pathogenicity.

MoErv14 interacts with and mediates the transport of MoPth11, MoWish, and MoSho1
Previous studies suggested that transmembrane receptors, including MoPth11, MoWish, and MoSho1, are important for cell surface perception and appressorium formation in M. oryzae [3,4,19]. We demonstrated that MoErv14 regulates cAMP and MoPmk1-MAPK signaling pathways during appressorium formation and pathogenicity. To test whether MoErv14

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MoErv14 mediates transmembrane sensor transportation to control appressorium formation in M. oryzae interacts with these receptors, yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays were conducted that showed MoErv14 interacting with all three proteins (Fig 6A-6F). Also, bimolecular fluorescence complementation (BiFC) was used to confirm the interaction between MoErv14 and these proteins following BFA treatment (Fig 6G-6I). However, no interaction was detected among these transmembrane receptor proteins (S8A- S8C Fig).
As MoErv14 functions in COPII vesicle trafficking, we speculated that MoErv14 also interacts with these proteins to control their transport. Hence, we fused GFP with these proteins and expressed them in Guy11 and the ΔMoerv14 mutant, respectively. The results showed that MoPth11, MoWish, and MoSho1 were all located at the cell membrane surface of Guy11. However, in ΔMoerv14 mutants, they were all retained in ER (Fig 7A-7C), which was similar with the BFA treatment of Guy11 (S9 Fig). Using gradient centrifugation to fractionate subcellular organelles, we found that MoPth11-GFP, MoWish-GFP, and MoSho1-GFP were mainly distributed in ER fractions of the ΔMoerv14 mutant, along with MoLhs1 (Fig 7D-7F).
To distinguish whether these proteins were transported to the cell surface, we observed their locations with or without Latrunculin B (LatB) which inhibits endocytosis. In Guy11 and during the appressorium formation stage, these proteins were transported into the cytoplasm by endocytosis from the cell membrane following signal perception to mediate appressorial formation. In the presence of LatB, these proteins were retained in the cell membrane surface. In contrast, these proteins failed in transport to the cell surface and were trapped on the ER in

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MoErv14 mediates transmembrane sensor transportation to control appressorium formation in M. oryzae the mutants, with or without LatB treatment (Fig 8A-8C). We further compared the ΔMoerv14 mutant and Guy11 in the transport of MoPth11, MoWish, and MoSho1 by fluorescence recovery after photobleaching (FRAP). We intended to bleach the fluorescence from the regions where MoPth11-GFP, MoWish-GFP, and MoSho1-GFP were accumulated in germ tubes, and the recovery of fluorescence can reflect the rate of endocytosis. In the FRAP assay, we bleached 90% of fluorescence of a region using 488 nm light. For MoPth11-GFP, fluorescence was recovered at post-photobleach 35 s in Guy11, but not in the ΔMoerv14 mutant. In addition, the recovery level of MoWish-GFP and MoSho1-GFP in the ΔMoerv14 mutant was

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MoErv14 mediates transmembrane sensor transportation to control appressorium formation in M. oryzae also significantly impaired when compared with Guy11 at the same post-photobleach ( Fig  8D-8F). Collectively, these results suggested that MoErv14 is involved in the transport of MoPth11, MoWish, and MoSho1 to the Golgi, and MoErv14 affects the transportation of these proteins to the cell membrane.

Discussion
Understanding the mechanisms by which pathogen transmembrane receptors transduce plant surface signals promotes the development of strategies in controlling the rice blast. Previous studies revealed that MoEnd3 and MoCrn1 undergo endocytosis following the perception of external signals in M. oryzae [20,21]. This current study demonstrated that MoErv14 mediated the signal transduction of receptors MoPth11 and MoSho1 by governing their intracellular transport. MoPth11 and MoSho1 regulate cAMP and Pmk1-MAPK signaling pathways, respectively, to promote appressorium formation and pathogenicity of the blast fungus. Based on these results, we concluded that COPII coat complex proteins, such as MoErv14, played critical roles in recognizing and transmitting host surface signals (Fig 9). Previous studies also showed that COPII coat complex proteins-mediated ER to Golgi transport is an important component of eukaryotic secretion pathways [22]. In S. cerevisiae, individual ERV proteins recognize the different groups of cargo proteins and are important in ER-Golgi transport [8,[23][24][25]. In M. oryzae, this process is closely linked to its physiology and pathogenicity. Following our previous studies of MoErv29 promoting apoplastic effector secretion contributing to the virulence of M. oryzae [11], we here showed that another ERV protein, MoErv14, was involved in appressorium formation and pathogenicity by governing intracellular transport of several receptor proteins. In addition, even though we found a severe defect of appressorium formation in the ΔMoerv14 mutant, no defect was found in conidium germination. Similar results were also observed in the ΔMowish and ΔMosho1 mutants [4,19]. These observations indicated that conidium germination may be regulated by different signal pathways.
The increased knowledge of each specific cargo receptor makes it feasible to hypothesize the presence of specificities between cargo coat proteins and cargo contents. Indeed, the examination of the cargo spectrum relying on ScErv14 suggested that this large and non-homogenous group of proteins is recognized based on the length of their transmembrane domains (TMDs), rather than their sequences [26]. We have noticed that MoPth11, MoSho1, and

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MoErv14 mediates transmembrane sensor transportation to control appressorium formation in M. oryzae MoWish all contain a long TMD; however, MoErv14 is not known to regulate MoMsb2 that also contains a long TMD. These findings indicated that the mechanisms of MoErv14 in recognizing these transmembrane proteins may be more complex.
Attenuation of appressorium formation would provide a means for managing the rice blast. Despite this extraordinary potential, however, relatively few fungicides targeting appressoria have been developed. Previously, identifying chemicals inhibiting very long-chain fatty acids (VLCFA) biosynthesis, which prevents septin-mediated appressorium formation, offers an effective strategy for controlling fungal diseases [27]. Our study could provide another avenue for controlling the disease by interfering with MoErv14-mediated ER-Golgi trafficking.

Strains and cultural conditions
The M. oryzae strain Guy11 was used as the wild-type strain for transformation in this research. All strains were cultured on a complete medium (CM) at 28˚C [28,29]. For vegetative growth, small blocks were cut from the edge of 7-day-old cultures and placed onto fresh media, followed by incubation in the dark at 28˚C. The radial growth was then measured after

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MoErv14 mediates transmembrane sensor transportation to control appressorium formation in M. oryzae incubation for seven days. Other media, including oatmeal agar medium and minimal medium, were prepared as described previously [27]. Liquid CM was used to prepare the vegetative mycelia for DNA and RNA extraction. For conidiation, mycelial blocks were inoculated on straw decoction and corn agar medium (SDC) (100 g of rice straw decoction was boiled in 1 L of ddH 2 O for 20 min and filtered, and the filtrate was mixed with 40 g of cornmeal and 15 g of agar and adjusted to 1 L with ddH 2 O) at 28˚C for 7 d in the dark and followed by 3 days of continuous illumination under fluorescent light [30].

Co-immunoprecipitation (co-IP) assay
The empty GFP construct was obtained in our previous work [28]. The MoERV14 DNA fragment fused with the S tag was inserted into the pXY203 construct (MoERV14-S) containing the hygromycin resistance, and the MoPTH11, MoWISH, and MoSHO1 DNA fragment fused with GFP fluorescent protein was inserted into the pYF11 construct, respectively. Then MoPth11-GFP/MoErv14-S, MoWish-GFP/MoErv14-S, MoSho1-GFP/MoErv14-S, and MoErv14-S/GFP fusion constructs were co-transformed into wild-type strain Guy11, and transformants resistant to hygromycin and bleomycin were isolated. Total proteins were extracted from the transformants using protein lysis buffer (50 mM Tris-HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100 [Sigma-Aldrich, T8787]) and incubated with anti-GFP agarose for 4 h, followed by washing the affinity gel with Tris-buffered saline (TBS) (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) four times. The proteins bound to the affinity gel were eluted by 0.1 M glycine HCl (pH 2.5) and were detected by anti-S and anti-GFP antibodies (Abmart, Shanghai, China) [11].

Yeast two-hybrid assay
Full-length cDNA of MoPth11, MoWish, and MoSho1 (see primers in S1 Table) was cloned into the prey pGADT7 and the bait pGBKT7-MoErv14 constructs, respectively, and the plasmid pairs were co-transformed into the yeast strain AH109 according to the description (Clontech, USA). The transformants from SD-Trp-Leu plates were then isolated and spotted on SD-Trp-Leu-His and SD-Trp-Leu-His-Ade media for further growth testing.

MoERV gene family deletion and complementation
Five ΔMoerv mutants were generated by using the one-step gene replacement strategy. Two fragments with 1.0 kb of sequences flanking the targeted gene were PCR amplified with primer pairs (S1 Table). The resulting PCR products were ligated to the hygromycin resistance cassette (HPH) released from pCX62. The 3.4-kb fragment, which included the flanking sequences and the HPH cassette, was transformed into Guy11 protoplasts [31]. Putative mutants were screened by PCR and further confirmed by Southern blot analysis. The complement fragment, which contains the entire MoERV14 gene coding region and its native promoter region, was amplified by PCR with primers (S1 Table) and inserted into the pYF11 construct to complement the respective mutant strain [32].

Conidial germination and appressorium formation
Conidial germination and appressorium formation were measured on a hydrophobic surface as described before [33][34][35]. Conidial suspensions of 25 μL (5×10 4 spores/mL) were dropped onto a hydrophobic surface and placed in a humidified box at 28˚C. The appressorium formation rate was counted at 24 h post inoculation (hpi) under a microscope, and more than 200 appressoria were counted for each strain.

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MoErv14 mediates transmembrane sensor transportation to control appressorium formation in M. oryzae

Host penetration and pathogenicity assays
Conidia were collected from mutants grown on SDC agar for 10 days and re-suspended by 0.2% (w/v) gelatin solution to a concentration of 5×10 4 spores/ml. For spraying assay, twoweek-old rice seedlings (O. sativa cv. CO39) were sprayed with 4 ml of the conidial suspension of each treatment and kept in a growth chamber at 25˚C with 90% humidity in the dark for the first 24 h, followed by a 12-h-light and 12-h-dark cycle [36,37]. Lesion formation was daily checked and photographed after seven days of inoculation. The 7-day-old barley leaves were drop-inoculated with three droplets (20 μl) of conidial suspension, and photographs were taken 5 days after infection. Each experiment was repeated more than three times, and the experimental condition was kept consistent (e.g., temperature, humidity, illumination, and the age of the plants). For the 'relative fungal growth' assay, total DNA was extracted from 1.5 g disease leaves and tested by qRT-PCR (HiScript II Reverse Transcriptase, Vazyme Biotech Co., Nanjing, China) with 28S/Rubq1 primers (S1 Table) [38,39]. For rice sheath penetration and invasive hyphae expansion, conidial suspension (1×10 5 spores/ml) was inoculated into the sheaths. After incubation for 36 h at 28˚C, the sheath cuticle cells were observed under a Zeiss Axio Observer A1 inverted microscope [40].

Organelle isolation
Protoplasts were prepared and maintained in a buffer containing 100 mM Tris-HCl, 0.1 mM MgCl 2 , 10 mM DTT, and 1.1 M sorbitol plus proteinases inhibitor mix (8215, Sigma-Aldrich), as previously described [32]. Organelles were isolated according to the established protocols with minor modifications as follows. Briefly, protoplasts were lysed in a buffer containing 10 mM Tris-HCl, 0.5 mM MgCl 2 , 8% Ficoll, and a proteinase inhibitor mixture, and the lysates were placed on the top of a centrifuge tube containing a bottom layer of the lysis buffer (10 ml) and a top layer of the same buffer containing 4% Ficoll (10 ml). Intact organelles, which are viewed as an opaque layer at the top of the centrifuge tube following centrifugation (50 000 g, 45 min), were collected. The supernatants were loaded into a cushion of 18% iodixanol (Sigma-Aldrich) for density gradient self-generation. After precipitation at 100 000 g for 2 h in a swinging bucket rotor, the crude membranes were collected at the interface, adjusted to 16% in iodixanol, and spun again at 350 000 g for 3 h. Fractions of 0.5 ml were harvested and washed with 0.8 ml 160 mM Na 2 CO 3 for 30 min at 4˚C. The membrane fraction was precipitated (100 000 g), washed with water, and then precipitated again. Membranes were solubilized in a buffer (0.1 ml of 25 mM triethylammonium bicarbonate/8 M urea/2% Triton X-100/0.1% SDS), and concentrations were determined.

FRAP assay
Germinated conidia with 3 h of incubation were treated with cycloheximide and benomyl as described [19]. FRAP was performed using a fluorescence microscope Zeiss LSM710. Regions containing MoPth11-GFP, MoWish-GFP, and MoSho1-GFP in germ tubes were selected for photo-bleaching. Photobleaching was carried out using an Argon-multiline laser at a wavelength of 488 nm with 90% laser power and 150 iterations in ROI. Images were acquired with 2% laser power at a wavelength of 488 nm every 5 sec. For quantitative analyses, the fluorescence intensity was measured using the ZEISS ZEN blue software, and fluorescence recovery curves were fitted using the following formula: F(t) = Fmin + (Fmax-Fmin)(1-exp-kt), where F(t) is the intensity of fluorescence at time t, Fmin is the intensity of fluorescence immediately post-bleaching, Fmax is the intensity of fluorescence following complete recovery, and k is the rate constant of the exponential recovery. Mobile Fraction was calculated as the following formula: Mf = (Fend-F0)/(Fpre-F0), where Fend is the stable fluorescent intensity of the

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MoErv14 mediates transmembrane sensor transportation to control appressorium formation in M. oryzae punctae after sufficient recovery, F0 is the fluorescent intensity immediately after bleaching, and Fpre is the fluorescent intensity before bleaching. The germinated conidia (3 h) with cycloheximide inhibit protein biosynthesis, which may prevent Golgi resident MoPth11-GFP, MoWish-GFP, and MoSho1-GFP from entering endosomes. The germinated conidia also treated with benomyl for 10 min to inhibit endosome trafficking.

The extraction and purification of vesicles
About 5 g mycelia were ground in liquid nitrogen with a mortar and pestle, and the mycelia powder was suspended in 15 ml of 0.1 M sodium acetate (containing 0.07% β-mercaptoethanol), then stirred at 4˚C for 2 h. The mixture was centrifuged at 6500 g for 30 min and the supernatant was transferred to a new centrifuge tube. The precipitation was resuspended in 15 ml 0.1 M sodium acetate containing 0.07% β-mercaptoethanol. After being well-suspended, the mixture was centrifuged at 6500 rpm for 30min, and the supernatant was mixed with the previous one. In order to remove the debris, the mixed supernatant was centrifuged at 7500 g for 30 min at 4˚C. The supernatant was again centrifuged at 50000 g for 90 min. The supernatant was removed and the precipitate dissolved in 1 ml of TMD buffer (50 mM Tris pH 7.5, 10 mM MgCl 2 , 5 mM DTT) for 30 min. The mixture was again precipitated at 8000 g for 3 min at 4˚C, and glycerol was added to a final concentration of 15% to the supernatant. The suspension was stored at -80˚C.

The lysis and extraction of vesicle proteins
For protein extraction, 400 μl vesicle samples were dissolved with 200 μl of lysis buffer (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, 40 mM Tris, 0.07% β-mercaptoethanol, 1 mM PMSF) and mixed uniformly. Proteins were precipitated by adding two volumes of pre-cold acetone containing 10% TCA and 0.07% β-mercaptoethanol and kept at -20˚C for 30 min before precipitating at 13000 g, 4˚C for 30 min. The pellet was washed with pre-cold acetone 3 times and air dried. The protein sample was re-dissolved in 200 μl of lysis buffer and centrifuged at 13000 g to increase impurity. Further purification was carried out using the 2D Clean-Up kit (GE Healthcare).