Abstract
When Microbotryum lychnidis-dioicae infects a male Silene latifolia, M. lychnidis-dioicae smut spores develop in the pollen sac instead of pollen. In contrast, when M. lychnidis-dioicae infects a female S. latifolia, the female flowers become male-like, promoting stamen formation. However, it is unclear when and how M. lychnidis-dioicae invades the anther. It is important to investigate not only whether hyphae exist when the apical meristem tissue differentiates into flowers and anthers, but also whether hyphae exist when stamen filaments form. We used Grocott’s methenamine silver stain and lectin stain, which stain chitin in the fungal cell wall, to search for M. lychnidis-dioicae in flower tissues. A few M. lychnidis-dioicae hyphae were observed intercellularly in the center of the connective of vascular bundles at the early anther developmental stage. Subsequently, large numbers of deeply stained M. lychnidis-dioicae hyphae were observed intercellularly in the cells surrounding the pollen sac, as well as in the center of the pollen sac. Hyphae stained with lectin were observed intercellularly in all of the stamen filaments at flower development stages. Hyphae were observed in the peduncle connecting the flower and stem. It is thought that M. lychnidis-dioicae invaded the anther via the stamen filament over a long period. Additionally, in total, 163 sections of connective were obtained, and the cell structure of each anther was colored and subjected to three-dimensional reconstruction. The M. lychnidis-dioicae hyphae observed in the connective were mainly old hyphae with large vacuoles or dead hyphae (S1 Fig). These hyphae branched out, towards the pollen sac, while growing between the cells. We also observed that the host cells that collapsed near the hyphae had thick cell walls and teliospores. Cell wall collapse and cell degeneration were observed only around hyphae with thick cell walls.
Highlights
The stamen primordia are formed in developmental stage 5, and four pollen sacs are formed in flower developmental stage 9
To clarify when and how M. lychnidis-dioicae invades the pollen sac, we comprehensively searched the hyphae with an optical microscope after staining the hyphae
We concluded that hyphae invade from the connective into the pollen sac after the pollen sac is formed, because hyphae of M. lychnidis-dioicae were confirmed only in connective, not in the pollen sac locule in anther developmental stage III (Fig 2)
Summary
Three-dimensional ultrastructural study of S. latifolia and M. lychnidis-dioicae. Microbotryum lychnidis-dioicae is a smut fungus isolated from Silene latifolia [2]. Microbotryum lychnidis-dioicae has long been used as a model system for the study of ecology and the genetics and propagation of sexual infection [3, 4, 5]. Silene latifolia plants infected with M. lychnidis-dioicae show effects in the floral organs [6, 7, 8]. Different phenotypes appear when M. lychnidis-dioicae infects female or male S. latifolia plants. When M. lychnidis-dioicae infects a male of S. latifolia, M. lychnidis-dioicae smut spores are formed instead of pollen in the pollen sac. When M. lychnidis-dioicae infects a female of S. latifolia, stamen formation is promoted. Smut spores are formed instead of pollen in the anthers [6,9]
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