The seven transmembrane domain protein MoRgs7 functions in surface perception and undergoes coronin MoCrn1-dependent endocytosis in complex with Gα subunit MoMagA to promote cAMP signaling and appressorium formation in Magnaporthe oryzae

Regulator of G-protein signaling (RGS) proteins primarily function as GTPase-accelerating proteins (GAPs) to promote GTP hydrolysis of Gα subunits, thereby regulating G-protein mediated signal transduction. RGS proteins could also contain additional domains such as GoLoco to inhibit GDP dissociation. The rice blast fungus Magnaporthe oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8) that have shared and distinct functions in growth, appressorium formation and pathogenicity. Interestingly, MoRgs7 and MoRgs8 contain a C-terminal seven-transmembrane domain (7-TM) motif typical of G-protein coupled receptor (GPCR) proteins, in addition to the conserved RGS domain. We found that MoRgs7, but not MoRgs8, couples with Gα MoMagA to undergo endocytic transport from the plasma membrane to the endosome upon sensing of surface hydrophobicity. We also found that MoRgs7 can interact with hydrophobic surfaces via a hydrophobic interaction, leading to the perception of environmental hydrophobiccues. Moreover, we found that MoRgs7-MoMagA endocytosis is regulated by actin patch-associated protein MoCrn1, linking it to cAMP signaling. Our studies provided evidence suggesting that MoRgs7 could also function in a GPCR-like manner to sense environmental signals and it, together with additional proteins of diverse functions, promotes cAMP signaling required for developmental processes underlying appressorium function and pathogenicity.


Introduction
In the rice blast fungus Magnaporthe oryzae, the appressorium is a special infection structure produced by the fungus to penetrate the host plant. Appressorium formation and function depend on signal transduction pathways, including G protein-coupled receptors (GPCRs)/ G protein-mediated cAMP signaling [1,2]. Once extracellular surface cues are sensed by GPCRs, such as the non-canonical GPCR Pth11 at the plasma membrane (PM), the GPCR stimulates the specific G-protein Gα subunit for activating the cAMP signaling pathway [2]. M. oryzae contains three distinct Gα subunits (MoMagA, MoMagB, and MoMagC) [3,4] and other conserved pathway components, such as adenylate cyclase MoMac1, cAMP-dependent protein kinase A catalytic subunits MoCpkA, and MoCpk2 [1,[5][6][7]. Together, they regulate not only growth but also appressorium formation and pathogenesis.
In addition, M. oryzae contains at least eight RGS (regulator of G-protein signaling) and RGS-like proteins (MoRgs1 to MoRgs8). Previous studies found that all these RGS proteins have certain regulatory functions in various aspects of growth and pathogenicity with MoRgs1, MoRgs2, MoRgs3, MoRgs4, MoRgs6, and MoRgs7 being mainly involved in appressorium formation and MoRgs1, MoRgs3, MoRgs4, and MoRgs7 in full virulence [3,8]. Despite such understandings, detailed mechanisms associated with specific RGS proteins remain not fully understood. In particular, RGS-like MoRgs7 and MoRgs8 proteins that also contain a seventransmembrane domain (7-TM)‚which is a hallmark of GPCRs important in signal perception and transduction. In eukaryotes, GPCRs are well known for their role as heterotrimer ligandregulated guanine-nucleotide exchange factors (GEFs) [9]. A ligand/agonist binding to a GPCR activates GPCR and promotes GPCR to mediate the exchange of GTP on the Gα subunit of the heterotrimer, leading to Gα dissociation from the Gβ-Gγ and activation of G protein-mediated signal transduction pathway, including the cAMP signaling pathway. In plant pathogenic fungi and oomycetes, it is generally considered that GPCRs have functions in perception of the environmental cues. This function enables plant pathogens to coordinate their metabolism with environment and to develop infection structures [10][11][12], although how these GPCRs detect environmental cues remains not clear.
Previous studies have found that RGS proteins such as human Rgs14 contain a C-terminal GoLoco/G protein regulatory motif that exhibits an in vitro GDP-dissociation inhibitor for Gα(i) [13]. Since MoRgs7 or MoRgs8 contain the 7-TM domain, we were interested in revealing whether MoRgs7 or MoRgs8 has additional functions mimicking a GPCR. Here we found that MoRgs7, but not MoRgs8, is involved in a distinct regulating mechanism. MoRgs7 couples with MoMagA to undergo endocytosis that is triggered by sensing surface hydrophobicity. Interestingly, MoRgs7 can sense environmental hydrophobic cues through interacting with the hydrophobic surface. In addition, MoRgs7 endocytosis depends on the actin-binding coronin homologue protein MoCrn1. Together, they contribute to G-protein/cAMP signaling required for appressorium function and pathogenicity.

MoRgs7 function requires RGS and 7-TM domains
Despite containing a relatively conserved RGS/RGS-like domain, 8 RGS proteins of the blast fungus are structurally divergent [8]. MoRgs7 and MoRgs8, in particular, contain a long C-terminus domain that was analyzed by transmembrane domain prediction systems (http:// mendel.imp.univie.ac.at/sat/DAS/DAS and www.cbs.dtu.dk/services/TMHMM) to have a GPCR-like 7-TM motif (S1 and S2A Figs). MoRgs7 was demonstrated to have a role in appressorium function and pathogenicity, and this role is dependent on the 7-TM domain [8,14].
To dissect the roles of MoRgs7 domains, the RGS domain was deleted (S2A Fig) and the mutant allele containing the 7-TM was fused to GFP and expressed in the ΔMorgs7 mutant. The fusion proteins MoRgs7 Δ7-TM :GFP and MoRgs7:GFP [14] were also expressed in the ΔMorgs7 mutant as a control. Analytical results showed that the ΔMorgs7 mutant expressing 7-TM:GFP still remained a relatively high cAMP concentration, similar to the ΔMorgs7 mutant [8] but not the wild-type strain (S2B Fig). In hydrophobic surfaces, about 8% of ΔMorgs7 conidia improperly generated two appressoria (S2C Fig), which could also be observed in the ΔMorgs7/7-TM strain (S2C Fig). The ΔMorgs7/7-TM strain was also attenuated in virulence, similar to the ΔMorgs7 mutant (S2D and S2E Fig). In contrast, the expression of MoRgs7-GFP was able to suppress most of the defects in the ΔMorgs7 strain. These tests showed that the 7-TM and RGS domains are important for MoRgs7 function in cAMP and virulence. However, the test failed to establish an independent role of the 7-TM.

MoRgs7 and Gα protein MoMagA undergo internalization in response to exposure of hydrophobic surfaces
MoMagA plays a major role in cAMP signaling, appressorium formation and pathogenesis in M. oryzae and it is also one of the three Gα subunits demonstrated to interact with MoRgs7 [8]. To investigate functional mechanisms of MoRgs7-MoMagA interaction, we first validated the interaction through co-immunoprecipitation (co-IP). In addition to MoMagA, the constitutively active form of MoMagA, MoMagA G187S [3] was also included in the test. The result showed that MoRgs7 can interact with both MoMagA and MoMagA G187S and that the 7-TM and the RGS domain both can interact with MoMagA (Fig 1A and 1B).
Since GPCRs undergo endocytosis for receptor recycling [15], and both of MoRgs7 and MoMagA were localized to late endosomes that are the main components of the endocytic pathway, we hypothesized that MoRgs7 and MoMagA may also undergo actin-dependent endocytosis. To test this, we employed actin polymerization inhibitor latrunculin B (LatB) to disrupt endocytosis as previously described [16,17]. At 3 h post-inoculation, MoRgs7:RFP and MoMagA:RFP signals remained very strong at the PM of the germ tube, in contrast to DMSO control (Fig 1C and 1D). Given that 4-bromobenzaldehyde N-2,6-dimethylphenyl (EGA) inhibits early to late endosome transport [18], it was applied and that led to an appearance of MoRgs7 and MoMagARFP signals in MoRab5:GFP-labeled early endosomes in germ tubes, in contrast to DMSO control (Fig 1E and 1F). Without EGA treatment, MoRgs7:RFP was  G187S . T represents total proteins, which were extracted from the mycelium of the strain expressing MoRgs7:GFP or GFP and MoMagA:S or MoMagA G187S :S. Total proteins were used for incubation with anti-GFP beads. E represents elution, which contains the proteins precipitated with GFP fusion proteins. GFP was used as the negative control. These samples were probed using anti-GFP and anti-S antibodies. (B) Co-IP assay for predominantly localized to Rab7:GFP-labeled late endosomes (Fig 1E). These co-localizations of proteins with endosomes were corroborated by Pearson correlation coefficient statistical analysis. Taken together, MoRgs7 and MoMagA movement follows the common endocytic pathway.
To further validate MoRgs7 and MoMagA endocytosis, we photobleached MoRgs7 and MoMagA fluorescence in late endosomes of the germ tubes on hydrophobic surfaces and examined the fluorescence recovery dynamic using Fluorescence Recovery After Photobleaching (FRAP) at 3 h post-inoculation. In addition, we applied the microtubule-destabilizing benomyl to inhibit endosome trafficking via microtubuleand cycloheximide to inhibit newly synthesized fluorescent proteins moving into endosomes [19]. We found that endocytosis promotes recovery of RFP fluorescence of MoRgs7 and MoMagA in late endosomes within 90 sec (Fig 2A and 2B). Furthermore, we used FRAP to bleach the fluorescence in endosomes in the germ tube on the hydrophilic surfaces at 3 h post-inoculation. The recovery of fluorescence of MoRgs7:RFP and MoMagA:RFP in the endosomes was rarely detected (Fig 2A and 2B), suggesting that MoRgs7 and MoMagA are rarely internalized through endocytosis upon the perception of the hydrophilic surface.
Intriguingly, the absence of MoRgs7 and MoMagA endocytosis on the hydrophilic surface did not couple with accumulation of MoRgs7:RFP or MoMagA:RFP signals at the PM of the germ tubes (Fig 2C and 2D). As treating germinated conidia with LatB on hydrophilic surfaces for 1 h still could not cause accumulation of RFP signals at the PM (Fig 2C and 2D), we thus reasoned that in response to exposure to hydrophilic cues MoRgs7 and MoMagA were rarely sent to the PM from intracellular systems.

MoRgs7 localization pattern is different from that of MoRgs8
MoRgs8 also contains a 7-TM domain. To examine whether MoRgs8 undergoes similar endocytosis, we expressed MoRgs8:GFP in Guy11 and observed MoRgs8 localization during appressorium development on the hydrophobic surface. However, MoRgs8:GFP was found to be evenly distributed in the cytoplasm of germ tubes (Fig 3A). When compared with MoRgs7: GFP (Fig 3B), MoRgs8:GFP did not display any obvious endosome-localization patterns in the germ tubes. Further, LatB failed to cause any effects to MoRgs8:GFP distribution (Fig 3A). In contrast, the MoRgs7:GFP signal was enhanced at the PM in response to LatB (Fig 3B). These results revealed that MoRgs8 may function differently from MoRgs7.

MoRgs7 is able to interact with the hydrophobic surface
Since the above results showed that the hydrophobic surface, not the hydrophilic surface, induces the PM localization of MoRgs7 in germ tubes during appressorium development, we hypothesized that MoRgs7 is possibly involved in sensing hydrophobic surfaces and the 7-TM examining the interaction of MoRgs7, MoRgs7 7TM, and MoRgs7 Δ7TM with MoMagA. The total proteins were extracted from the mycelium of the strains co-expressing MoRgs7:GFP and MoMagA:S, 7TM:GFP and MoMagA:S, and MoRgs7 Δ7TM :GFP and MoMagA:S, respectively. The elution contains the proteins precipitated with GFP fusion proteins. These samples were probed using anti-GFP and anti-S antibodies. (C and D) LatB enhanced MoRgs7:RFP and MoMagA signals at the plasma membrane of germ tubes (3 h post-inoculation). The DMSO solvent treatment was used as a control. The sections selected by white arrows were subjected to lines-can analysis for the distributions of MoRgs7:RFP and MoMagA:RFP. Percentage of a pattern showed in image was calculated by observation for 50 germinated conidia that were randomly chosen, and observation was conducted for 3 times. Bars = 5 μm. (E and F) EGA induced the localizations of MoRgs7:RFP and MoMagA:RFP on MoRab5-labeled early endosomes (3 h post-inoculation). The co-localization of MoRgs7:RFP with MoRab7:GFP and DMSO treatment were used as the controls. The values were the mean Pearson's correlation coefficients of MoRgs7:RFP/MoMagA:RFP with MoRab5:GFP/MoRab7:GFP. ImageJ software was used to calculate the Pearson's correlation coefficients, which were generated by analyzing images taken from 5 germinated conidia. Bars = 5 μm.
may have a role in this process. We hypothesized that MoRgs7 at the PM may attach to hydrophobic surfaces in a hydrophobic interactive manner, and formation of such interactions by PM proteins including MoRgs7 is a step in the perception of hydrophobic cues. To test this hypothesis, we first examined whether MoRgs7 has the ability to bind to hydrophobic materials by performing an affinity precipitation assay with phenyl-agarose gel beads. The phenyl groups attached to the beads are highly hydrophobic. The beads were then incubated with MoRgs7:GFP and the GFP protein (a negative control), respectively, in a high concentration of salt solution containing 1.5 M NaCl and 1.5 M MgSO 4 . This allowed proteins to bind to the beads, as at high salt concentration non-polar side chains on the surface upon protein can interact with the hydrophobic groups [20]. Then we washed the beads to remove unbound proteins using a series of aqueous solutions with different salt concentrations. If an intense hydrophobic interaction between the protein and phenyl groups was formed, the protein will be hardly removable from beads even by low salt concentration solution containing 0.3 M NaCl and 0.3 MMgSO 4 , or containing 0.2 M NaCl and 0.2 M MgSO 4 . After washing, we used Western-blot analysis to detect the amount of MoRgs7:GFP or GFP that remained bound to beads. The results indicated that MoRgs7:GFP, but not GFP, remained in the elution (Fig 4A). This suggested that MoRgs7 has astrong ability to interact with hydrophobic materials and this ability may allow MoRgs7 to mediate a hydrophobic interaction between the pathogen and the hydrophobic surface. In addition, the hydrophobicity of 7-TM of MoRgs7 was tested and found to have hydrophobicity as full-length MoRgs7 (Fig 4A), and deletion of 7-TM decreased the level of hydrophobicity of MoRgs7, suggesting that this 7-TM is critical for hydrophobicity of MoRgs7. The results also indicated that MoRgs8 has a weak hydrophobicity compared to MoRgs7 despite having a 7-TM, suggesting that the characteristic of 7-TM is varied from MoRgs7 to MoRgs8.
We then investigated whether MoRgs7 forming a hydrophobic interaction with hydrophobic surfaces is an approach of M. oryzae to detect hydrophobic cues. Given that urea and ethylene glycol can interrupt hydrophobic interactions by causing a disordering of water molecules on hydrophobic regions [21,22], they were applied to germinating conidia on hydrophobic surfaces at 1 h-post inoculation when most of conidia only germinated with a germ tube. In the presence of 0.5 M ethylene glycol or 0.1 M urea, appressorium formation was about 50% lower than that of water treatment at 4 h post-inoculation, even though almost 80% of conidia developed appressorium at 10 h post-inoculation (Fig 4B and 4C). Moreover, in the presence of 1 M ethylene glycol or 0.8 M urea, less than 20% of conidia developed appressoria even at 10 h post-inoculation. Most of conidia only germinated germ tubes with or without swelling at terminals. These results implied that a successful hydrophobic interaction formation is a critical step in hydrophobic surface recognition by M. oryzae.

MoRgs7 and MoMagA do not depend on each other in endocytosis
To examine the nature of MoRgs7-MoMagA endocytosis and whether MoRgs7 internalization is dependent on MoMagA, we determined the rate of MoRgs7 internalization in the wild-type strain Guy11 and the ΔMomagA mutant using FRAP analysis. We found that MoRgs7 internalization had a normal rate in the ΔMomagA as that in Guy11 (S3A and S3B Fig). In addition, the internalization rate of MoMagA was also the same in Guy11 and the ΔMorgs7 strain (S3C and S3D Fig). These results suggested that MoRgs7 and MoMagA do not depend on each other in internalization.

MoCrn1 interacts with MoRgs7 and F-actin, and affects microtubule function
To further understand the endocytosis process of MoRgs7, we searched for additional protein partners of MoRgs7 through a yeast two-hybrid (Y2H) screening and identified a coronin protein homolog, MoCrn1, as two polypeptides of 148 and 273 amino acids, from a cDNA library in the pGADT7 vector. MoRgs7 cDNA was inserted into pGBKT7 as bait. The interaction between MoCrn1 and MoRgs7 was specific, as an interaction between MoCrn1 and other RGS proteins, including MoRgs1, MoRgs3 and MoRgs4, cannot be established ( Fig 5A). The interaction was further validated by co-IP and bimolecular fluorescence complementation (BiFC). The co-IP assay indicated that both the 7-TM and the RGS domains could interact with MoCrn1, independently (Fig 5B and 5C). BiFC revealed that MoCrn1 interacts with MoRgs7  The yeast two-hybrid assay for examining the interaction of MoCrn1 with MoRgs1, MoRgs3, MoRgs4, and MoRgs7. The yeast transformants were isolated from SD-Leu-Trp plates, following growing on SD-Leu-Trp-His-Ade plates containing X-α-Gal for examining β-galactosidase activity. The transformants expressing AD and BD, BD-MoRgs1/3/4/7 and AD, BD and AD-MoCrn1 were used as negative control. (B) Co-IP assay for examining the interaction between MoCrn1 (tagged with GFP) and MoRgs7 (tagged with RFP tag) using anti-GFP beads. The total proteins were extracted from the mycelium of the strain co-expressing MoCrn1-GFP and MoRgs7-RFP. GFP protein was used as negative control. The anti-GFP and anti-RFP antibodies were used to detect GFP, MoCrn1-GFP and MoRgs7-RFP proteins. (C) Co-IP assay for examining the interaction of MoRgs7, 7-TM and MoRgs7 Δ7TM (tagged with GFP) with MoCrn1 (tagged with S-tag) using anti-GFP beads. (D) BiFC assay for examining the interaction of MoCrn1 with MoRgs7. The conidia were allowed to germinate on hydrophobic surfaces for 3 h. YFP signals could be detected from the germ tubes and conidia of the strain co-expressing MoRgs7-N'YFP and MoCrn1-C'YFP. The strains co-expressing empty-N'YFP and MoCrn1-C'YFP, and co-expressing MoRgs7-N'YFP and Empty-C'YFP were used as negative control. Bar = 5 μm. (E) The yeast two-hybrid assay for examining the interaction of MoCrn1 with during appressorium development ( Fig 5D). The YFP signal could be detected at the PM of germ tubes while some weak signals appeared in the cytoplasm (Fig 5D), suggesting that the interaction is more often to occur at the PM. To investigate the interaction at the PM of germ tubes, we further conducted co-localization assay with co-expression of MoCrn1:GFP and MoRgs7:RFP in Guy11. And their co-localization at the PM in germ tubes of conidia on the hydrophobic surface was examined at 3 h post-inoculation (S6A Fig). However, we failed to detect the co-localization at the PM in germ tubes because the small amount of PM-localized MoRgs7:RFP is present (S6A Fig), as shown above ( Fig 1C). MoCrn1:GFP formed actin patches-like structures, as described for coronin in Neurospora crassa [23]. But, from a small number of conidia, slightly obvious PM-localized MoRgs7:RFP was observed in germ tubes and partly co-localized with MoCrn1 patches (S6A Fig). The co-localization result suggested that a small amount of PM-localized MoRgs7 indeed has the opportunity to interact with MoCrn1.
In the eukaryotic cells, coronin proteins act as F-actin binding proteins and regulate actinrelated processes such as membrane trafficking [24]. We tested whether MoCrn1 associates with actin in M. oryzae using Lifeact, a living cell actin marker described previously [17,25,26]. The MoCrn1:GFP and Lifeact:RFP were co-expressed in Guy11 and co-localization of MoCrn1:GFP and Lifeact:RFP was examined under a confocal microscope. We observed that MoCrn1:GFP and actin were dispersed in nascent appressoria after 6 h of incubation (S5A In the budding yeast Saccharomyces cerevisiae, Crn1 interacts with the microtubule [27]. The Δcrn1 mutant cells as well as cells overexpressing Crn1 showed microtubule defects and the mutant Δcrn1 is more sensitive than wild type strains to benomyl [28]. To determine whether MoCrn1 also affects the microtubule, the pYES2 construct containing the full-length MoCrn1 cDNA was expressed in the yeast Δcrn1 mutant. On SD plates containing 10, 20, and 30 μg/ml benomyl, Δcrn1 exhibited most significant inhibition in growth compared to the wild type strain BY4741 (S5G Fig). However, there was no significant difference between the Δcrn1 MoMagA, MoMagB, MoMagC and two active forms of MoMagA, MoMagA G187S and MoMagA Q208L . (F) Co-IP assay for examining the interaction of MoCrn1 (tagged with GFP) with MoMagA (tagged with S-tag) using anti-GFP beads. (G) BiFC assay for examining the interaction of MoCrn1 and MoRgs7. YFP signals could be detected from the germ tubes and conidia of the strain coexpressing MoMagA-N'YFP and MoCrn1-C'YFP. The strains co-expressing empty-N'YFP and MoCrn1-C'YFP, and co-expressing MoMagA-N'YFP and Empty-C'YFP were used as negative control. Bar = 5 μm.

MoCrn1 is important for the internalization of MoRgs7 and MoMagA during appressorium development
As MoCrn1 interacts with MoRgs7 and is localized to the PM associated actin patches that represent endocytic pits [29], we hypothesized that MoCrn1 may function as an adaptor protein to direct MoRgs7 to endocytic pits/vesicles for internalization during appressorium development. To prove this, we investigated whether MoCrn1 affects MoRgs7 endocytosis by observing the spatial distribution of MoRgs7:RFP in germinated conidiaonthe hydrophobic surface at 3 h post-inoculation. Despite of that endosome-localized MoRgs7 was found in both the ΔMocrn1 mutant and Guy11, the ΔMocrn1 mutant displayed a higher concentration of MoRgs7:RFP at the PM of the germ tube than Guy11 did ( Fig 6A). FRAP analysis indicated the fluorescence recovery of MoRgs7:RFP in ΔMocrn1 was evidently delayed than that in Guy11 (Fig 6C), suggesting that the diffusion of MoRgs7:RFP fluorescence into endosomes was impaired. This is consistent with our hypothesis that MoCrn1 is implicated in MoRgs7 internalization during appressorium development.
Since MoRgs7 and MoMagA are both internalized via endocytosis, we also examined if MoCrn1 has a role in the MoMagA internalization through a protein-protein interaction. We first validated the interaction between MoCrn1 and MoMagA. In Y2H, we found that MoCrn1 interacts with MoMagA and this interaction was specific, since MoCrn1 was not found to interact with MoMagB and MoMagC (Fig 5E). In addition, MoCrn1 did not interact with MoMagA G187S and MoMagA Q208L (Fig 5E), the twoactive forms of MoMagA [3]. The interaction between MoCrn1 and MoMagA was again confirmed by co-IP (Fig5F) and BiFC assays (Fig5G). In BiFC assay YFP is observed at the PM of germ tubes, revealing that MoCrn1 can interact with MoMagA at the PM of germ tubes during appressorium development. Moreover, similar to the co-localization of MoCrn1 with MoRgs7, from a small number of conidia the partial co-localizaiton between MoCrn1 and MoMagA was also found at the PM in germ tubes (S6B Fig).
We next tested whether MoCrn1 affects the MoMagA distribution during appressorium development on hydrophobic surfaces. In Guy11, we have observed that MoMagA:RFP displayed the endosome localization pattern in germ tubes and conidia. In ΔMocrn1, we could still observe MoMagA:RFP on late endosomes, but there was a significant increase in the membrane localization of MoMagA:RFP (Fig 6B). We again employed the FRAP assay to determine MoMagA internalization and found that the recovery of fluorescence of MoMagA:RFP in endosomes was slower in ΔMocrn1 than that in Guy11 (Fig 6D). These results confirmed that MoCrn1 is important for MoMagA internalization during appressorium development.

MoCrn1 controls MoCap1 localization
MoCrn1 is co-localized with F-actin so that MoCrn1 is similar to the adenylate cyclase associated protein MoCap1 that functions in cAMP signaling [6]. To examine whether MoCrn1 is required for MoCap1 localization, we expressed MoCap1:GFP in ΔMocrn1 and observed that the actin-like localization pattern of MoCap1 was completely disrupted in appressoria, conidia and hyphae of ΔMocrn1 (S8 Fig). Strikingly, MoCap1 preferred to form cytoplasmic aggregations. Additionally, we found that MoCrn1 interacts with MoCap1 by performing a co-IP assay (Fig 8F), in which the strain co-expressing MoCrn1:GFP and MoCap1:S was used. These results led us to conclude that MoCrn1 has a crucial role in recruiting MoCap1 to actin patches.

MoCrn1 is important for turgor generation, degradation of glycogen and lipid in the appressorium and pathogenicity
MoCrn1 has been associated with MoRgs7, MoMagA, and MoCap1 that all have a role in cAMP signaling. Indeed, we found that the ΔMocrn1 mutant also showed attenuated cAMP levels (S7A Fig) and a delay in appressorium formation (S9 Fig). At 4 h post-germination, nearly 40% of ΔMocrn1 conidia formed appressorium on a hydrophobic surface compared with 80% of Guy11 did. However, over 80% of ΔMocrn1 conidia could still form the appressorium at 6 h post-germination (S9 Fig). An incipient collapse assay indicated that MoCrn1 contributes to full turgor generation, since the collapse rate of the appressorium was significantly higher in ΔMocrn1 than in Guy11 and the complemented strains (S7B Fig). Intracellular cAMP levels regulate the degradation of glycogen and lipid that are required for proper turgor generation in the appressorium [5,30]. We thus compared the degradation of glycogen and lipid betweentheΔMocrn1mutant and Guy11 strains. Conidia were allowed to germinate on hydrophobic surfaces and iodine and Neil Red were used to stain glycogen and lipid, respectively [31]. At 6 h post-inoculation, glycogen appeared in the early appressorium (S7C Fig), and it broke down in 68.4% of the Guy11 appressoria after 16 h and 87% after 24 h, in comparison to 22.4% of ΔMocrn1 appressoria after 16 h and 53% after 24 h (S7E Fig). Resembling to the glycogen, lipid degradation in ΔMocrn1 appressoria was slower than Guy11. Lipid bodies disappeared in 44% of ΔMocrn1 appressoria at 16 h, compared to 86.4% of Guy11 appressoria (S7D and S7F Fig). These results indicated that MoCrn1 is indispensable for an efficient degradation of glycogen and lipid necessary for the appressorial turgor generation.
We further evaluated the ΔMocrn1 mutant for pathogenicity on rice. The conidial suspensions from Guy11, ΔMocrn1, and the complemented strain were sprayed onto the susceptible rice cultivar CO-39. ΔMocrn1 produced fewer lesions than Guy11 and the complemented strain, which were confirmed by lesion quantification (Fig 7A). We also performed rice sheath penetration assays by observing 100 appressoria each strain and classifying invasive hyphae (IH) types as previously described [17]. We observed that over 40% of ΔMocrn1 appressoria were defective in penetration and 55.6% of appressoria that penetrated and formed less extended IH. In contrast, 90% of Guy11 appressoria successfully penetrated rice cells and about 50% of that produced strong IH (Fig 7B).

MoCrn1 is involved in appressorium development and pathogenicity through regulating intracellular cAMP levels
To explore whether MoCrn1 regulates turgor generation involving the process of cAMP signaling, the incipient collapse assay was performed. We found that exogenous 8-Br-cAMP could suppress the defect of ΔMocrn1 in turgor generation (S7B Fig). The numbers of the collapsed appressoria in the ΔMocrn1mutant were reduced by 20% and 10% with 1 and 2 mM cAMP, respectively, compared to those without 8-Br-cAMP. In addition, the ΔMocrn1 mutant appressorium underwent successful glycogen and lipid breakdown following 8 and 16 h, respectively, following treatment with 5 mM 8-Br-cAMP (S7E and S7F Fig). Furthermore, 1 or 2 mM 8-Br-cAMP addition to the conidia suspensions in the inoculation of detached barley leaves could suppress the defect of ΔMocrn1 in infection to some degree ( Fig 7C). This result was also confirmed by the penetration assay, in which 8-Br-cAMP treatment restored the penetration defect to almost 80% of the ΔMocrn1 appressoria in comparison to 43 ± 4.9% of ΔMocrn1 without cAMP (Fig 7B). This is similar to the effect of the ΔMocrn1mutant that expresses the constitutively active form of MoMagA, MoMagA G187S (S7G and S7H Fig and Fig  7A).

MoCrn1 function is dependent on its protein binding ability
To examine the ability of MoCrn1 in binding multiple proteins, we identified putative actin binding domains and characterized their function. Human coronin Arg 29 and Arg 30 are thought to be important for the interaction with F-actin [32,33]. The alignment showed that a majority of coronins contain a conserved basic amino acid at these two positions (Fig 8A). In addition, the C-terminal coiled-coil (CC) domain is important for coronins to interact with the actin nucleation complex Arp2/3 [34]. Accordingly, we mutated His 29 to Asp 29 and deleted the CC domain of MoCrn1, and fused the mutant proteins with GFP ( Fig 8B). We found that MoCrn1 H29D and MoCrn1 ΔCC mutants had completely altered actin-like localization patterns (Fig 8C). To further analyze the effects of these mutant alleles, we performed the co-IP assay  and found that MoCrn1 H29D and MoCrn1 ΔCC mutants failed to interact with MoRgs7, MoMagA, and MoCap1 (Fig 8D, 8E and 8F).
We also expressed MoCrn1 H29D and MoCrn1 ΔCC mutants in ΔMocrn1. FRAP analysis showed that the expression of MoCrn1 H29D and MoCrn1 ΔCC caused no effect on delayed endocytosis of MoRgs7 and MoMagA in ΔMocrn1 (Fig 9). HPLC analysis revealed cAMP levels of the strain expressing MoCrn1 H29D or MoCrn1 ΔCC comparable to that of the ΔMocrn1 mutant ( Fig 7D). Moreover, virulence and the degradation of appressorial glycogen and lipid in the MoCrn1 H29D and MoCrn1 ΔCC strains were also indistinguishable from those of the ΔMocrn1 mutant (Fig 7E, 7F and 7G). Taken together, these results suggested that MoCrn1 function is dependent on its ability to interact with F-actin, MoRgs7, MoMagA, and MoCap1.

Discussion
We here investigated the distinct functional mechanism of RGS and 7-TM-containing protein MoRgs7 beyond its RGS functions.We found that MoRgs7 has a GPCR-like endocytosis pattern and is predominantly localized to late endosomes similar to other signaling proteins, including MoRgs1, MoMagA, and MoMac1. Such late endosome localizations of signaling proteins are critical to GPCR function and for cAMP signal transduction. Our results further showed that MoRgs7 couples with MoMagA to undergo endocytosis. Interestingly, by inhibiting endocytosis, we could observe increased PM localization of MoRgs7 and MoMagA. And by inhibiting trafficking from the early endosomes to the late endosomes, we could observe the early endosome localization of MoRgs7 and MoMagA.
Understanding how pathogen receptors recognizethe plant surface signal has a beneficial effect on the controlling rice disease at early stages. Our results provided evidences that MoRgs7 serves as a GPCR-like receptor to detect environmental hydrophobic cues. The affinity precipitation assay with phenyl-agarose gel beads indicates that MoRgs7 has strong ability to form hydrophobic interaction with hydrophobic materials, revealing that MoRgs7 can form interaction with hydrophobic surface when MoRgs7 is localized to the PM. Importantly, disruption of such hydrophobic interaction during M. oryzae germinating on the hydrophobic surface led to the aberrant appressorium formation. We also noted that the ΔMorgs7 mutant developed defective appressoria, even though no decrease in appressorium formation frequency. Based on these studies, we concluded that forming hydrophobic interactions with hydrophobic surface by MoRgs7 and other membrane proteins is a critical step in recognizing hydrophobic surface cues.
We reasoned that MoRgs7 may undergo a functional process similar to mammalian GPCRs. In mammalian cells, when a ligand binds to a GPCR, ligand can activate GPCR by inducing conformational changes in GPCR, subsequently the active GPCRs can activate the Gα proteins and are transported by endocytosis to sustain downstream signaling, be recycled, or be degraded from endosomes [35]. Considering our studies and previous findings by others in mammalian cells, we proposed a functional model of MoRgs7 (Fig 10). In this model, MoRgs7 acts as a GPCR during appressorium development to interact with the hydrophobic surface. Subsequently, this interaction induces MoRgs7-MoMagA endocytosis that is regulated by MoCrn1. MoRgs7 facilitates activating cAMP signaling from endosomes along with MoMagA. Conversely, MoRgs7 may elevate its GAP activity to regulate MoMagA when cAMP signaling is fully activated. Thus, MoRgs7 has dual roles in regulating signal transduction.
How MoRgs8 that also contains 7-TM domain but lacks sensory functions is not understood. MoRgs8 was distributed in the cytoplasm of germ tubes but did not undergo endocytosis. MoRgs8 could be involved in a mechanism distinct from MoRgs7 and future studies are needed to address such distinct mechanism(s).
There was precedence that endocytosis of RGS proteins plays a role in promoting Gα-mediated signaling. In Arabidopsis thaliana, in response to glucose, RGS protein AtRgs1 internalizes via endocytosis to uncouple itself from Gα protein AtGPA1 anchored in the PM, leading to AtGPA1 sustaining activation. And this process is required for both G-protein-mediated sugar signaling and cell proliferation [36]. However, other details including the initiation of MoRgs7-MoMagA complex disassembly following endocytosis remain not understood. We recently reported a distinct mechanism of how M. oryzae might negatively regulate the GAP activity of MoRgs7. This mechanism implicates the MoMip11 protein that interacts with MoRgs7 and the GDP bound MoMagA, but not the GTP bound MoMagA (Fig 10) [14]. MoMip11 prevents MoRgs7 from interacting with the GTP bound MoMagA, therefore interfering with MoRgs7 GAP function by sustaining MoMagA activation [14].
During endocytic vesicle formation, a series of adaptor proteins in cytoplasm can accumulate at endocytic sites. Those adaptors serve to select endocytic cargos and specifically bind to cargos, recruiting their cargos to endocytic pits/vesicles [29,37]. Since we found that endocytosis of MoRgs7 and MoMagA are independent of each other, we considered that the adaptor MoRgs7 promotes surface perception and cAMP signaling protein(s) for the two proteins can anchor MoRgs7 or MoMagA to endocytic pits even though MoRgs7 and MoMagA do not interact with each other. Despite of that, the MoRgs7-MoMagA interaction is still important for MoRgs7 to regulate MoMagA activity. To further investigate the physiological function of MoRgs7 and MoMagA endocytosis, coronin protein MoCrn1 that emerges as an adaptor protein for MoRgs7 and MoMagA was identified and characterized. MoCrn1 is localized to actin patches that represent endocytic sites, interacting with MoRgs7 and MoMagA and regulating their endocytosis. Disruption of MoCrn1 by gene deletion or point mutations (H29D mutation and CC domain deletion) not only attenuated MoRgs7 and MoMagA endocytosis, but also led to a decreased cAMP level that is lower than the threshold for proper appressorium development. Our results support that MoRgs7 and MoMagA endocytosis regulated by MoCrn1 facilitates initiating cAMP signaling and appressorium development. However, for BiFC assays to test MoCrn1-MoRgs7 and MoCrn1-Mo-MagA interactions we queried why the YFP fluorescence evenly distributes at the PM of germ tubes, not just at the actin patches. A possible explanation is that, before MoCrn1 accumulates at actin patches, the cytoplasm-localized MoCrn1 has already bound to the cytoplasmic peptides of PM-localized MoRgs7 and MoMagA. At later stage, these interactions enable MoCrn1 to direct MoRgs7 and MoMagA to endocytic pits/vesicles.
Coronin proteins are known as regulators of the cytoskeleton and membrane trafficking in a number of species including yeast, Neurospora crassa, Dictyostelium discoideum, Drosophila, and human [23,24]. In D.discoideum and mammalian cells, coronins have evolved to be modulators of signal transduction. Those coronins are critical for Rac1 GTPase activation and Rac1-dependent signaling [33,38]. Additionally, upon cell surface stimulation coronin 1 interacts with and activates Gα to stimulate cAMP/PKA pathway in neuronal cell, even though how coronin 1 activates Gα is less clear [39]. Compared to those studies, our work revealed that MoCrn1 is involved in a distinct mechanism to facilitate Gα-cAMP signaling. MoCrn1 has an adaptor protein-like function by directing MoRgs7 and MoMagA to endocytic pits to promote their internalization. This function, thereby allows MoCrn1 to have a role in facilitating cAMP signaling. However, the function was not found yet for other eukaryote coronins, thus it is not known whether coronin is generally required for endocytosis of RGS and Gα proteins in eukaryotes except M. oryzae. Interestingly, MoCrn1 also interacts with MoCap1 that is thought as one of activators of MoMac1 [6]. Based on the above, we proposed that MoCrn1 is likely to be a hub or organizing protein of the network of MoRgs7-MoMagA-MoCap1.

Strains and culture conditions
The M. oryzae Guy11 strain was used as wild type for transformation in this study. For vegetative growth, small agar blocks were taken from the edge of 7-day-old cultures and cultured in liquid CM medium for 48 h. For conidiation, strains were cultured on SDC plates at 28˚C for 7 days in the dark, followed by constant illumination for 3 days [8,17,31,[40][41][42][43].

Targeted MoCRN1 deletionand the ΔMocrn1 mutant complementation
The MoCRN1 deletion mutant was generated using the standard one-step gene replacement strategy [44]. First, two approximate 1.0 kb of sequences flanking of MoCRN1 (MGG_06389) were amplified with two primer pairs MoCRN1-F1/MoCRN1-R1, MoCRN1-F2/MoCRN1-R2, the resulting PCR products ligated with the HPH cassette released from pCX62. The protoplasts of wild type Guy11 were transformed with the vectors for targeted gene deletion by inserting the hygromycin resistance HPH marker gene cassette into the two flanking sequences of the MoCRN1 gene. For selecting hygromycin-resistant transformants, CM plates were supplemented with 250 μg/ml hygromycin B (Roche, USA).
To generate complementary construct pYF11-MoCRN1, the gene sequence containing the MoCRN1 gene and 1.0 kb native promoter was amplified with MoCRN1-comF/ MoCRN1-comR. Yeast strain XK1-25 was co-transformed with this sequence and XhoI-digested pYF11 plasmid. Then the resulting yeast plasmid was expressed in E. coli. To generate the complementary strain, the pYF11-MoCRN1 construct was introduced into the ΔMocrn1 mutant and pYF11 contains the bleomycin-resistant gene for M. oryzae transformants screen [31,44].

Southern blot analysis
EcoRV was used to digest the genomic DNA from Guy11 and the ΔMocrn1 mutant. The digest products were separated in 0.8% agar gel and were hybridized with the MoCRN1 gene probe. The probe was designed according to the disruption strategy and was amplified from Guy11 genomic DNA using primers MoCRN1-InterF/MoCRN1-InterR. To confirm MoCRN1 replacements, labeled MoCRN1 probe was used to hybridize the EcoRV-digested genomic DNA from the ΔMocrn1 mutant and wild-type Guy11. The copy number of the HPH gene in the ΔMocrn1 mutant was detected using labeled HPH fragments that amplified from the plasmid of pCB1003 with primers FL1111/FL1112. The whole hybridization was carried out according to the manufacturer's instruction for DIG-High Prime.

Pathogenicity assay
The conidia were suspended in a 0.2% (w/v) gelatin solution (5×10 4 spores/ml), then the solutions were sprayed onto 2-week-old seedling of susceptible rice (Oryza sativa cv. CO-39) and also inoculated into 3-week-old rice CO-39 as described. Then the plants were incubated at 25˚C with 90% humidity in the dark for the first 24 h, followed by a 12h/12h light/dark cycle. Lesions were observed after 7 days of incubation [41]. For pathogenicity assay with detached barley leaves [40], three 20 μl droplets of the conidia suspensions (1×10 5 , 1×10 4 , 1×10 3 spores/ ml, respectively) added cAMP solution or not, were placed onto the upper side of the 7-dayold barley (cv. Four-arris) leaves. Then the leaves were incubated at 25˚C with 90% humidity and in the dark for the first 24 h, followed by a 12h/12h light/dark cycle. Lesions were observed after 5 days of incubation.

Glycogen and lipid staining during appressorium development
To visualize glycogen, the samples were stained by iodine solution containing 60 mg/ml KI and 10 mg/ml I2 for 1 min. Nile red solution consisting of 50 mM Tris/maleate buffer (pH 7.5) and 2.5 mg/ml Nile red (9-diethylamino-5H-benzo-a-phenoxazine-5-one, Sigma), was used to treat the samples for 3 min, then the samples were examined under a fluorescence microscope with RFP channel [17,26,30].

Co-IP assay
The DNA fragments for expressing GFP fusion proteins were respectively inserted into the pYF11 construct that contains bleomycin resistant gene and G418 resistancegene, and the DNA fragments for expressing S-tag fusion proteins were respectively inserted into the pXY203 construct hat contains hygromycin gene. Then the constructs for expressing GFP and S-tag fusion proteins were co-transformed into wild-type strain Guy11, and the transformants resistant to hygromycin and bleomycin or G418 were isolated. The total protein of the transformants was extracted from mycelium using protein lysis buffer [1 M Tris-Cl (pH7.4), 1 M NaCl, 0.5 M EDTA, 1% Triton×100] and incubated with anti-GFP agarose beads (GFP-Trap, Chromotek, Martinsried, Germany) for 4 h, followed by washing beads with washing buffer (50 mM Tris HCl, 150 mM NaCl, pH 7.4) for 4 times. The proteins that bind to the beads were eluted by 0.1 M glycine HCl (pH 3.5) and were probed by anti-GFP and anti-S antibodies.

Binding assay for MoCrn1 and MoAct1 interaction
MoCRN1 and MoACT1 full-length cDNAs were cloned and inserted into pGEX4T-2 and pET32a, respectively. These constructs were transformed into E. coli strain BL21 for expressing proteins. Bacterial lysate containing GST:MoCrn1 protein was incubated with 30 μl GST agarose beads for 2 h. Then the beads were washed by washing buffer for 4 times and incubated with His:MoAct1 protein for 2 h, followed by washing beads with using washing buffer (50 mM Tris HCl, 150 mM NaCl, pH7.4) for 4 times again. The beads were boiled to elute proteins, and eluted proteins (output) were probed with anti-GST and anti-His antibodies.

Yeast two-hybrid assay
Constructs of BD:MoMagA, BD:MoMagB and BD:MoMagC were used in previous experiments and kept in our lab. Full-length cDNAs of MoCRN1 was cloned and inserted into pGADT7 (AD) vector. Full-length cDNAs of MoCAP1, MoMagA G187S , MoMagA Q208L and MoACT1 genes were inserted into pGBKT7 (BD) vector. To examine the interaction of proteins, the AD and BD constructs were co-transformed into yeast strain AH109 and the transformants were grown on SD-Trp-Leu medium. Then the Trp+ and Leu+ transformants were isolated and assayed for growth on SD-Trp-Leu-His-Ade medium added X-α-Gal.

FRAP analysis
The germinated conidia with 3 h of incubation on hydrophobic or hydrophilic surfaces were treated with cycloheximide and benomyl as described [17]. FRAP were performed using a fluorescence microscope Zeiss LSM710. Regions containing MoRgs7:RFP and MoMagA:RFP in germ tube were selected for photo-bleaching. Photobleaching was carried out using an Argon-multiline laser at a wavelength of 561 nm with 80% laser power and 80 iterations in ROI. Images were acquired with 2% laser power at a wavelength of 555 nm every 5 sec. For quantitative analyses, fluorescence intensity was measured using the ZEISS ZEN blue software and fluorescence recovery curves were fitted using following formula: F(t) = F min + (F max − F min )(1-exp −kt ), where F(t) is the intensity offluorescence at time t, F min is the intensity of fluorescence immediately post-bleaching, F max is the intensity of fluorescence following complete recovery, and k is the rate constant of the exponential recovery [45]. Mobile Fraction was calculated as the following formula: Mf = (F end − F 0 )/(F pre − F 0 ), where F end is the stable fluorescent intensity of the punctae after sufficient recovery, F 0 is the fluorescent intensity immediately after bleaching, and F pre is the fluorescent intensity before bleaching [46].

Assays with drugs or inhibitors
LatrunculinB (Cayman, USA) is dissolved in DMSO at a concentration of 25 mg/ml. Conidia incubated on the coverslips with hydrophobic surface were treated with LatB (final concentration 0.1 μg/ml) for 30 min, while the controls were treated with 5% DMSO. Then samples were washed with distilled water. Cycloheximide (MedChemExpress, USA) was solved in distilled water and the germinated conidia were treated with a final concentration 10 μg/ml for 10 min. Then samples were washed with distilled water. Benomyl (Aladdin, Shanghai, China) was solved in 0.1% DMSO and added to germinated conidia with a final concentration 1μg/ml. Then the samples were washed with distilled water. EGA (Merck, USA) was solved in 5% DMSO and was applied to samples with concentration 5 μg/ml for 1 h.

Affinity precipitation of MoRgs7:GFP with Phenyl-agarose gel beads
The total proteins were extracted from the Guy11 strain expressing MoRgs7:GFP or GFP, respectively, and were incubated with 100 mg of Phenyl-agarose beads (Senhui Microsphere Tech, Suzhou, China) in 1.5 ml microcentrifuge tubes at 10˚C for 16 h. After incubation, the tubes were centrifuged (13000 g, 5 min) to remove the suspension. The beads were then gently washed with a series of aqueous solutions with different concentrations of NaCl and MgSO4 (1.5/1.0/0.8/0.5/0.3/0.2/0.1 M NaCl and MgSO4, 10 mM HEPES, pH 7.0), respectively, for 3 times to remove the unbound proteins. 100 μl of 1% SDS solution was added to the washed beads, followed by boiling the SDS solution and beads for 10 min to obtain elution, which was examined by western-blot using anti-GFP antibody.

Assays with fluorescence microscope and calculation of Pearson correlation coefficient for co-localization
All the samples were observed under a confocal fluorescence microscope (Zeiss LSM710, 63× oil). The filter cube sets: GFP (excitation spectra: 488 nm, emission spectra: 510 nm), RFP (excitation spectra: 555 nm, emission spectra: 584 nm). Exposure time: 800 ms. ImageJ software was applied to calculate Pearson correlation coefficient for analyzing co-localization of GFP fusion protein with RFP fusion protein. One area of interest was photographed with GFP and RFP channels respectively and photographs were opened using ImageJ software. Picture type was set to 8 bits. The "colocalization finder" in "plugin" section was applied to the pictures and Pearson correlation coefficient was calculated.

cAMP extraction and HPLC analysis
All of the strains were cultured on CM medium at 28˚C, were cut into 1×1 mm squares, and were cultured in liquid CM for another 2 days. Filtering to collect mycelium and quickly ground into powder in liquid N 2 . 1 mg of mycelium was mixed with 20 μl of 6% TCA solution. Samples were centrifuged (1,377 × g, 15 min), the top layers were collected and were washed twice with five times the volume of anhydrous ether. The pellet was collected for HPLC. HPLC analysis was done with a programmable Agilent Technology Zorbax 1200 series liquid chromatograph. The solvent system consisted of methanol (90%)and water (10%), at a flow rate of 1 ml per minute; 0.1 mg of cAMP solution per milliliter was eluted through the column (SBC18, 5 μl, 4.6 × 250 mm) and was detected at 259 nm UV. Each sample was eluted through the column in turn and peak values were detected with the same time as the standard [47].

Construction of vectors used to express fluorescent proteins
For construction of pHZ65:MoMagA vector used to express MoMagA-N'YFP, the N'YFP sequence was inserted into the alphaB-alphaC loop of MoMagA as described [48], then the MoMagA sequence containing N'YFP and the native promoter was fused with the pYF11 plasmid. For construction of vector used to express MoMagA:RFP, the RFP sequence was also inserted into the alphaB-alphaC loop of MoMagA. Then the MoMagA sequence containing the native promoter was fused with pYF11 plasmid. For construction of other vectors used to express proteins tagged with RFP or GFP, RFP or GFP was fused to protein sequence C-terminals, then protein sequences containing their native promoters were fused with the pYF11 plasmid.