Fig 1.
MoEnd3 interacts with MoArk1 and F-actin.
(A) Yeast two-hybrid assay for examining the interaction between MoEnd3 and MoArk1. The yeast transformants were isolated from SD-Leu-Trp plates. Their β-galactosidase activity was assayed on SD-Leu-Trp-His-Ade plates containing X-Gal. The transformants expressing AD-MoEnd3 and empty BD, empty AD and BD-MoArk1, and empty AD and BD were used as negative control. (B) In vitro protein binding assay for the MoEnd3-MoArk1 interaction. Ni-NTA beads were used to bind His protein (6 kDa) as a negative control and His-tagged MoArk1 protein (115 kDa), respectively, and incubated with the GST-tagged MoEnd3 protein (69 kDa). The total proteins eluted from beads (output) were separated by 12% SDS-PAGE and immunoblotted with GST and His antibodies. (C) BiFC assay for the MoEnd3-MoArk1 interaction in vivo. Conidia and 24 h appressoria were examined by DIC and fluorescence microscopy. The strains expressing the MoArk1-YFPC and empty YFPN, MoEnd3-YFPN and empty YFPC, empty YFPN and empty YFPC constructs were used as negative controls, Bars = 10 μm. (D) MoEnd3:GFP is co-localized with F-actin. The localization pattern of MoEnd3:GFP was displayed in 6 h and 24 h-appressoria, conidium and hyphal tip region. F-actin was labeled with Lifeact:RFP. Bars = 10 μm. (E) Yeast two-hybrid assay was used to examine the interaction between MoEnd3 and actin. Transformants were isolated from SD-Leu-Trp plates and their β-galactosidase activity was assayed on SD-Leu-Trp-His-Ade plates containing X-Gal. Transformants expressing AD and BD, BD-MoAct1 and AD, and BD and AD-MoEnd3 were used as negative controls. (F) Protein binding assay for MoEnd3-MoAct1 interaction in vitro. GST-beads were used to bind GST protein (24 kDa) or GST-tagged MoEnd3 protein (68 kDa), respectively, and incubated with His-tagged MoAct1 protein (42 kDa). Total eluted fractions from the beads (output) were immunoblotted with the His and GST antibodies. MoEnd3 is important for sexual reproduction and normal endocytosis.
Fig 2.
MoEnd3 is involved in endocytosis and F-actin assembly.
(A) Time course-images of FM4-64 uptake at the hyphal tips. Hyphae stained by FM4-64 were examined by using fluorescence microscopy at different time points. The regions where fluorescence intensity was measured by ImageJ software were labeled by ellipse frame. Bars = 5 μm. (B) The bar chart shows the mean fluorescence intensity at the hyphal tip region. At least 15 hyphae of each strain were measured by applying ImageJ software at each time point. Error bars represent standard deviation (SD) and asterisks represent significant differences (P < 0.01). a.u., arbitrary unites. (C) F-actin network in appressoria (24 h) of Guy11 and ΔMoend3. Line-scan graphs show Lifeact:RFP fluorescence in a transverse section of individual appressorium. Bars = 5 μm. (D) F-actin in conidia of Guy11 and ΔMoend3. Bars = 10 μm. (E) Actin patches in hyphal tip regions. Bars = 10 μm.
Fig 3.
MoEnd3 is important for appressorium formation and virulence.
(A) Appressorium formation assay. Conidia were incubated on hydrophobic surfaces and the samples were observed at different time points. Bar = 10 μm. (B) Appressorium formation rates at different time points were calculated and statistically analyzed. The percentage at a given time was recorded by observing at least 200 conidia for each strain and the experiment was repeated three times. Error bars represent SD and asterisks represent significant differences (P < 0.01). (C) Images show appressoria after 24 h incubation on hydrophobic surfaces. Bar = 10 μm. (D) Mean appressorium diameter. The values were recorded by observing at least 100 appressoria for each strain and the experiment was repeated three times. Error bars represent SD and asterisk represents significant difference (P < 0.01). (E) Appressorium turgor was measured by an incipient cytorrhysis (cell collapse) assay. The percentage of collapsed appressoria was recorded by observing at least 100 appressoria and the experiment was repeated three times. Error bars represent SD and asterisks represent significant differences (P< 0.01). (F) Conidial suspensions of strains were sprayed onto 2-week old rice seedlings (CO-39) and 7-day old barley. Diseased rice and barley leaves were photographed after 7 and 5 days of inoculation, respectively. (G) Penetration assay with rice sheath. Excised rice sheath from 4-week-old rice seedlings was inoculated with conidial suspension. Images show invasive growth in rice sheath epidermal cells at 36 hpi. Bar = 10 μm. (H) Pmk1 phosphorylation level analysis with proteins extracted from mycelium, conidia, and conidia or appressoria incubated on hydrophobic surfaces for 3 h, 8 h and 16 h. The phosphorylation levels of Pmk1 (42-kDa) were detected using a phosphor-MAPK antibody (upper panel). The endogenous Pmk1 was detected using a MAPK antibody (lower panel). (I) Appressorium formation assay on the hydrophobic surfaces. (J) Appressorium formation rates were calculated and statistically analyzed. Asterisks represent significant differences (P<0.01). (K) Pathogenicity assay on rice (CO-39). (L) Quantification of the lesion numbers per 5 cm length of rice leaf. Error bars represent SD and asterisk represents significant difference (P<0.01). (M) Penetration assays in rice sheath. IH growth on rice cells was observed at 36 hpi and 4 types of IH were quantified and statistically analyzed. Error bars represent SD. Micrographs show 4 types of IH in rice cells. Bar = 10 μm. MoEnd3 is involved in endocytosis of Pth11 and MoSho1.
Fig 4.
Pth11 and MoSho1 are transported by MoEnd3-mediated endocytosis.
(A) Pth11:GFP was co-localized with FM4-64 in cytoplasm of germ tubes at 3 h. Merged image shows the GFP channel and FM4-64. Bar = 5 μm. (B) MoSho1:GFP was co-localized with FM4-64 in cytoplasm of germ tubes at 3 h. Merged image shows the GFP channel and FM4-64. Bar = 5 μm. (C) MoMsb2:GFP punctuate structures were not co-localized with FM4-64 marked endosomes in germ tubes. Merged image shows the GFP channel and FM4-64. Bar = 5 μm. (D) Appressorium formation assay with addition of LatB. Guy11 conidia significantly reduced appressorium formation after exposure to LatB for 30 min. The appressorium formation rates were recorded by observing 100 conidia for each sample and the experiment was repeated three times. (E) Pth11:GFP localization patterns with DMSO solvent and LatB treatment in germ tubes of Guy11 at 3 h. Insets highlight areas analyzed by line-scan. Bars = 5 μm. (F) MoSho1:GFP localization patterns with DMSO solvent and LatB treatment in germ tubes of Guy11 at 3 h. Insets highlight areas analyzed by line-scan. Bars = 5μm. (G) Pth11:GFP localization pattern in germ tubes of Guy11 and ΔMoend3 at 3 h. Insets highlight areas analyzed by line-scan. Bars = 5 μm. (H) MoSho1:GFP localization pattern in germ tubes of Guy11 and ΔMoend3 at 3 h. Insets highlight areas analyzed by line-scan. (I) Representative images of FRAP analysis for diffusion at Pth11:GFP localized regions in germ tubes of Guy11 and ΔMoend3. The fluorescence of Pth11:GFP significantly recovered at 35 s post-photobleaching in Guy11 but not in ΔMoend3. (J) Normalized FRAP curves of Pth11:GFP localized regions in Guy11 and ΔMoend3. 20 regions from different cells were subjected to FRAP analysis for each strain. Intervals: 5 s. (K) Representative images of FRAP analysis for diffusion at MoSho1:GFP localized regions in germ tubes of Guy11 and ΔMoend3. The fluorescence of MoSho1:GFP significantly recovered at 35 s post-photobleaching in Guy11 but not in ΔMoend3. (L) Normalized FRAP curves of MoSho1:GFP localized regions in Guy11 and ΔMoend3. 20 regions from different cells were subjected to FRAP analysis for each strain. Intervals: 5 s.
Fig 5.
MoEnd3 functions in nuclear degradation and autophagy.
(A) Nuclei were visible during appressorium development using H1:RFP. The merged image shows H1:RFP and DIC. Bars = 10 μm. (B) Electron micrographs of vacuoles in hyphae following 4 h nitrogen starvation condition. Arrows indicate autophagosomes. Bars = 0.5μm. (C) Hyphae from the strains expressed GFP:MoAtg8 were exposed to nitrogen starvation for 4 h in the presence of 4 mM PMSF. Arrows indicate vacuoles. Merged image shows the GFP:MoAtg8 and DIC. Bar = 10μm. (D) The percentages of vacuoles containing GFP:MoAtg8 were recorded by observing at least 100 vacuoles for each sample, and the experiment was repeated three times. Error bars represent SD and asterisk represents significant difference (P < 0.01). (E) GFP:MoAtg8 proteolysis assay. Total proteins were extracted from the GFP:MoAtg8 expressed strains exposed to nitrogen starvation condition for 0, 2 and 5 h. The full-length GFP:MoAtg8 and free GFP were detected using GFP antibodies. Protein contents were analyzed using the actin antibody.
Fig 6.
MoEnd3 phosphorylation requires MoArk1.
(A) MoEnd3:GFP proteins treated with phosphatase Inhibitor or phosphatase were separated by Mn2+-Phos-tag SDS-PAGE and normal SDS-PAGE respectively, and were probed with GFP antibody. (B) MoEnd3 phosphopeptide (PASLRASFERNKI) in the strain expressing MoARK1 was identified by mass spectrometer analysis and the phosphorylated site was Ser-222. (C) MoEnd3:GFP protein was extracted from the strain expressing MoArk1 and not expressing MoArk1, respectively. MoEnd3S222A:GFP protein was extracted from the strain expressing MoArk1. Then these proteins were separated by Mn2+-Phos-tag SDS-PAGE and normal SDS-PAGE, respectively, and probed with GFP antibody. (D) Hyphae were examined by fluorescence microscopy following 5 min FM4-64 staining. The selected regions where fluorescence was measured by ImageJ software were labeled by ellipse frame. Bars = 5 μm. (E) The bar chart shows mean fluorescence intensity at the hyphal tip region calculated using ImageJ software. At least 15 hyphae were measured for each strain. Asterisks represent significant differences (P < 0.01). (F) Pmk1 phosphorylation level was detected by applying phosphor-Pmk1 antibody. Endogenous Pmk1 level was detected by the Pmk1 antibody. (G) Pathogenicity assay on rice with the MoEnd3 phosphorylation site mutants. Photographs were taken following 7 days of inoculation.
Fig 7.
MoEnd3 is involved in secretion of effectors Avr-Pia and AvrPiz-t.
(A) Pathogenicity of Guy11 and ΔMoend3 was assayed on rice LTH, LTH-Pib and LTH-Pi9. (B) Effector AVR-Pia gene was expressed in Guy11, ΔMoend3, and ΔMoend3/MST7S212DT216E. Pathogenicity of these strains was assayed on rice LTH and LTH-Pia. (C) Effector AVRPiz-t gene was expressed in Guy11, ΔMoend3, and ΔMoend3/MST7S212DT216E. Pathogenicity of these strains was assayed on rice LTH and LTH-Piz-t. (D) The bar chart shows quantification of the virulent-type lesions per 5 cm length of leaf. Error bars represent SD. Asterisk represent significant difference and NS represent no significant difference. (E) Images of BICs in the rice sheath cells infected by strains expressing Avr-Pia:GFP. Merged images show DIC and GFP channel. White arrows indicate the BICs. The percentage ± SD of the types of BIC showed were recorded from three independent experiments. In each experiment, 40 BICs were observed for each strain at 24 hpi. Bar = 10 μm. (F) Images of BICs in the rice sheath cells infected by strains expressing AvrPiz-t:GFP. Merged images show DIC and GFP channel. White arrows indicate the BICs. The percentage ± SD of the types of BIC showed was recorded from three independent experiments. In each experiment, 40 BICs were observed for each strain at 24 hpi.
Fig 8.
A proposed model for MoEnd3 function.
During germ tube development, GPCR Pth11 and membrane sensor MoSho1 are regulated by MoEnd3-mediated endocytosis. MoEnd3 function in endocytosis is negatively regulated by MoArk1-initiated phosphorylation, leading to an efficient endocytosis. Following transport to endosomal systems by endocytic vesicles, Pth11 and MoSho1 can trigger a downstream MAPK cascade, consisting of Mst11, Mst7, Pmk1 and adaptor Mst50. The MAPK cascade facilitates successful nuclear degradation/autophagy, appressorium formation, penetration and pathogenicity. In addition, MoEnd3 is also involved in an endocytosis/exocytosis coupling pathway to facilitate effector secretion and biotrophic growth.