Figure 1.
Subcellular localization and spatio-temporal dynamics of the negative regulator of G-protein signaling (Rgs1) in M.oryzae.
(A) A schematic of the time line and key morphogenic transitions exhibited by M. oryzae in response to inductive cues. Gt: developing germ tube; and Ap: incipient appressorium at the hooking stage. Lower panels are maximum intensity Z-projections of five confocal slices, 0.5 µm each, of Rgs1-mCherry (pseudocolored yellow) strain at the corresponding time points. Arrowheads indicate punctate (0 h) and tubulo-vesicular compartments of Rgs1 in the developing germ tube (2 h) and the incipient appressorium (4 h). Scale bar, 10 µm. (B) Bi-directional mobility of Rgs1-mCherry tubulo-vesicular compartments, 4 hpi. Red arrows indicate the direction of movement of Rgs1 compartments towards either the appressorium or the terminal cell of the conidium (asterisk). Elapsed time indicated in seconds. Images are in maximum intensity Z-projections of five confocal stacks, 0.5 µm each. See also Video S1. Scale bar = 10 µm.
Figure 2.
Rgs1 co-localizes with active GαS/MagA during appressorium initiation or early pathogenic development.
(A) Subcellular localization of MagAG187S-GFP and MagA-GFP during vegetative growth. Mycelia from the indicated strains were subjected to confocal microscopy. Single plane images are depicted. Scale bar, 10 µm. (B) Colocalization of constitutive active MagAG187S-GFP (green) and Rgs1-mCherry (red) compartments during the hooking stage. MagAG187S-GFP localizes to cytoplasmic vesicular structures and the plasma membrane (asterisk). The arrow highlights compartments co-populated by MagAG187S-GFP and Rgs1-mCherry and the yellow asterisk highlights the same compartment in the inset and the corresponding 3D surface rendering (panels on the far right). The dotted line demarcates the respective conidium. Images in panel B are maximum intensity Z-projections of six confocal stacks, 0.5 µm each. Scale bar equals 10 µm. (C) Single plane images of merged channels and corresponding orthogonal view of entire stack showing the co-localization of MagAG187S-GFP and Rgs1-mCherry. Representative intensity profiles were obtained for each channel from a single slice along the indicated axes (X and Y) and from the corresponding transverse section of the stack (Z) using Fiji software (D) MagAG187S physically interacts with Rgs1. Western blot analysis from a GFP-trap based pull down experiment showing the specific interaction between MagAG187S and Rgs1 during pathogenic differentiation. After probing for MagAG187S -GFP with α-GFP antibody, the same membrane was stripped and re-probed using α-Rgs1 antibody. Total protein from a cytosolic-GFP strain served as a negative control. Equal concentrations of protein from both the strains were used for the pull down experiment. Scale bar = 10 µm.
Figure 3.
Localization of Pth11 and its interaction with Rgs1 in M. oryzae.
(A) Conidia expressing Pth11-mCherry (pseudocolored yellow) were inoculated on an inductive surface and imaged at the indicated time-points. The inset in the 2 h panel highlights a tubulo-vesicular structure containing Pth11-mCherry. The asterisk depicts the association of Pth11 with the plasma membrane. The dotted line delineates the conidial boundary. Images are maximum intensity Z-projections of five confocal stacks, measuring 0.5 µm each. Scale bar, 10 µm. (B) Pth11 physically interacts with Rgs1. Immunoblots from a pull down experiment depicting the specific interaction between Pth11 and Rgs1 during appressorium initiation. Pth11 physically interacted with Rgs1 during pathogenic differentiation and failed to interact with Rgs1 during vegetative growth. In each case, equal concentrations of protein from both the strains were used for the pull down experiment. Total protein from an untagged wild-type strain served as a negative control in B. S1 and S2 represent two independent Pth11-GFP expressing strains. (C) An RFP-trap experiment depicting the specific interaction between Pth11-mCherry and Rgs1. After probing for Pth11-mCherry with α-mCherry antibody, the same membrane was stripped and re-probed using α-Rgs1 antibody. Total protein from a cytosolic-mCherry expressing strain served as a negative control in C. (D) Confocal microscopy based imaging of a BiFC experiment illustrating the in-vivo interaction between Pth11-nYFP and Rgs1-cYFP in the vegetative mycelium and at 4 h post inoculation on an inductive surface (right; DIC in left panel and YFP in right panel). Asterisk indicates YFP signal and thus likely interaction at the plasma membrane. Images are single plane images captured by a confocal microscope. Scale bar, 10 µm.
Figure 4.
Identity and integrity of the tubulo-vesicular compartments that harbor Rgs1.
(A) Rgs1-mCherry colocalizes with certain PI3P-rich endosomal compartments (marked by GFP-FYVE probe). Arrowheads mark the regions magnified in the inset and surface rendered in 3D. (B) Inhibition of PI3 kinase activity by LY294002 results in the loss of Rgs1-mCherry (pseudocoloured yellow) association with tubulo-vesicular structures. Asterisk indicates Rgs1 accumulation along the plasma membrane of the incipient appressorium. DMSO treated sample was used a control. The regions of the germ tube lacking the Rgs1-mC compartments structures are bracketed. LY294002 treatment was carried out 4 hpi. The elapsed time is indicated in seconds (C) GFP-Rab7 colocalizes with Rgs1-mCherry during early stages of pathogenesis. The arrowhead depicts the region magnified in the inset and surface rendered in 3D. (D) The early endosomal marker Rab5 does not colocalize with Rgs1-mCherry. Arrowheads mark the regions magnified in the inset and surface rendered in 3D. Images are maximum intensity Z-projections of five confocal stacks (0.5 µm each) and the yellow asterisk, in the colocalization panels, marks the same compartment in the inset and the surface renderings. Scale bar, 10 µm.
Figure 5.
Rgs1 utilizes microtubules to traverse along the germ tube.
Destabilization of the microtubule cytoskeleton disrupts Rgs1 dynamics. MBC treatment leads to Rgs1-mC (pseudocolored yellow) accumulation along the plasma membrane in the developing appressorium (asterisk) and cytoplasmic aggregation or clustering (arrowhead). The region of the germ tube lacking the Rgs1-mC tubulo-vesicular structures is bracketed. DMSO-treated sample served as a negative control. Elapsed time (in seconds) is recorded in panels D and E. Scale bar, 10 µm.
Figure 6.
The late endosomal/HOPS component Vps39 is essential for proper cAMP signaling and pathogenesis.
(A) Time course depicting delayed appressorium formation and multiple hooking defects (white arrowheads) in the vps39Δ strain on an inductive surface (row one). A vps39Δ-like phenotype in the wild type upon treatment with Bafilomycin A1 (Baf A1) on an inductive surface (row two). Rescue of the vps39Δ phenotype by exogenous cAMP (row three). Wild-type strain served as a control (row four). Images are DIC from a single slice; elapsed time (hpi) displayed. Scale bars depict 10 µm. (B) Bar graph illustrating the efficiency of appressorium formation in the vps39Δ strain. Values represent mean ± S.E from three independent replicates, 300 conidia per sample. (C) Blast infection assays. Conidia from the indicated strains were inoculated on the susceptible rice (CO39) seedlings and imaged 14 d post inoculation.
Figure 7.
The Late Endosomal scaffolding function is necessary for proper cAMP signaling.
Subcellular localization of Adenylate cyclase Mac1 (Adc-GFP), Rgs1-mCherry (mC, pseudo colored yellow) and GFP-Rab7 in the wild type and vps39Δ at germ tube hooking. (A) Adenylate cyclase Mac1 (Adc-GFP), arrowhead illustrates the localization of Adc-GFP to tubular cytosolic compartments in the wild type. The dotted line marks the boundary of the conidium (B) Rgs1-mCherry (mC, pseudo colored yellow); region devoid of Rgs1 tubulo-vesicular structures is bracketed and the arrowheads highlight cytoplasmic aggregates and vacuolar accumulation of Rgs1-mC in the vps39Δ. (C) GFP-Rab7; Inset highlights the morphology of GFP-Rab7 compartments in the wild type and vps39Δ backgrounds. All images are single plane confocal images. Scale bars, 10 µm. (D) A simplified model of late endosomal compartments functioning as signaling scaffolds that anchor key activators and regulators of G-protein signaling in the rice-blast fungus M. oryzae. This model does exclude the possible contribution of G-protein signaling/signal initiation occurring at the plasma membrane and also the possibility that signaling components are actively trafficked to the vacuole either for sequestration or for degradation to maintain cellular homeostasis.