Fig 1.
Diagrams of the observation area.
The stamen primordia, anther, and filament were observed. Black markings and asterisks indicate the observed areas. 4, 5A, 5B, 5C, and 5D in the anther indicate observation areas in Fig 5. Roman numerals indicate anther developmental stages; Arabic numerals indicate flower developmental stages.
Fig 2.
Modified Grocott’s methenamine silver-stained anther tissues from an infected male, showing the purple hyphae of Microbotryum violaceum invading the male flower anthers of Silene latifolia.
Fungal hyphae and spores stained by modified Grocott’s method were detected in the intercellular region and pollen sacs. Stars in inset indicate the observed area. Black arrows indicate smut fungus. White arrows indicate teliospore. (A) connective in stage III, (B) pollen sac in stage VII, (C) pollen sac in stage IX. Bars = 50 μm.
Fig 3.
Hyphae localization in the stamen filament of plant developmental stages 9–12 and the peduncle.
Fungal hyphae were observed in the stamen filament at stages 9–12. Fungal hyphae were also observed in the peduncle at stage 12. (A, C, E, G, I) Stained hyphae using WGA labeled with Alexa 647; (B, D, F, H, J) Merged images.
Fig 4.
Three-dimensional ultrastructural study of connective in an infected male S. latifolia.
We observed connective using a serial-section method. (A) Low-magnification TEM images, (B) high-magnification of TEM images, (C) 3D reconstructed images. Numbers indicate the same position in b and c. The cells, indicated by numbers, are sieve and companion cells. PCW, plant cell wall; PV, plant vacuole; PN, plant nucleus; HCW, hyphae cell wall; HC, hyphae cytoplasm; HV, hyphae vacuole. Bars = 10 μm.
Fig 5.
3D ultrastructural study of pollen sacs in infected male in stages VI-VIII.
We observed pollen sacs in an infected male using a serial-section method. PCW indicates plant cell wall. (A) 3D reconstructed image of the region between the endothelium and connective, (B) 3D reconstructed images of the region between the tapetum and the middle layer, (C) 3D reconstructed images of the region between the epidermis and endothelium, (D) 3D reconstructed images of the region in the center of the pollen sac. PV, plant vacuole; PM, plant mitochondrion; PN, plant nucleus; PP, plant plastid; HCW, hyphae cell wall; HC, hyphae cytoplasm; HV, hyphae vacuole; HN, hyphae nucleus. Bar = 10 μm.
Fig 6.
Thickness of cell walls in host cells, and inner and outer localized M. lychnidis-dioicae.
We measured the thickness of cell walls of S. latifolia cells and inner and outer hyphae of M. lychnidis-dioicae in the anther. We measure 50 individuals and sections at each organization. Ep, epidermis; En, endothelium; M, middle layer; T, tapetum; Ohy, outer hyphae; Ihy, inner hyphae. N.S.: not significant, * p < 0.01 (by The Mann-Whitney U-test)