An asexual flower of Silene latifolia and Microbotryum lychnidis-dioicae promoting its sexual-organ development

Silene latifolia is a dioecious flowering plant with sex chromosomes in the family Caryophyllaceae. Development of a gynoecium and stamens are suppressed in the male and female flowers of S. latifolia, respectively. Microbtryum lychnidis-dioicae promotes stamen development when it infects the female flower. If suppression of the stamen and gynoecium development is regulated by the same mechanism, suppression of gynoecium and stamen development is released simultaneously with the infection by M. lychnidis-dioicae. To assess this hypothesis, an asexual mutant, without gynoecium or stamen, was infected with M. lychnidis-dioicae. A filament of the stamen in the infected asexual mutant was elongated at stages 11 and 12 of the flower bud development as well as the male, but the gynoecium did not form. Instead of the gynoecium, a filamentous structure was suppressed as in the male flower. Developmental suppression of the stamen was released by M. lychnidis-dioicae, but that of gynoecium development was not released. It is thought, therefore, that the suppression of gynoecium development was not released by the infection of M. lychnidis-dioicae. M. lychnidis-dioicae would have a function similar to SPF since the elongation of the stamen that is not observed in the healthy asexual mutant was observed after stage 8 of flower bud development. Such an infection experiment also that the Y chromosome of the asexual mutant has genes related to the differentiation of archesporial cells, but none related to maturation of the tapetal cells.


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The basidiomycetous genus Microbotryum contains a species-rich member of smut 4 fungi that infects a wide range of the host plant belonging to Caryophyllaceae, Dipsacaceae, 5 Lamiaceae, and Lentibulariaceae in the dicotyledonous plants [1]. The smut fungus M. autosomes, one X chromosome, and one Y chromosome. It is known that S. latifolia is a 19 model for the study of the evolution of plant sex chromosomes and ecology [9]. We require 4 1 sex-chromosome-linked markers to better understand plant sex chromosomes. Therefore, 2 several Y chromosome-linked markers were made using Amplified Fragment Length 3 Polymorphism (AFLP) [10], Random Amplified Polymorphic DNA (RAPD) [11], and a 4 technique that combines laser microdissection and polymerase chain reaction (PCR) [12]. 5 The male flowers of S. latifolia with Y chromosomes were irradiated with γ rays or heavy ion 6 beams to produce hermaphrodites, an asexual mutant, and a pollen-defect mutant with 7 deletions to a part of the Y chromosome [13], [14], [15]. Using the deletion status of these what differences exist in stamens between the infected asexual mutant, the infected male, and 9 the infected female? We infer that we are able to search for genes related to the anther 10 development, which should exist in the deleted region of the Y chromosome, by comparing 11 pollen sacs of stamens in the infected male, infected female, and infected asexual mutant.

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In this study, progeny of the asexual mutant could be successively produced by 13 crossing the female-like flowers in the asexual mutant with the male flower in the wild-type 14 male [24]. The infected asexual mutant was found as 5 individuals due to inoculation with M.    xylene and embedded in paraffin. Embedded flowers in paraffin were cut into 10-μm sections 8 1 using a microtome (RV-240, Yamato, Japan). Cutting sections were de-paraffinized in xylene 2 and rehydrated in an ethanol series (100,95,90,80,70,50, and 30%; each step for 10 min at 3 room temperature). Rehydrated sections were stained Schiff's reagent. The sections were 4 observed with a microscope (BX60, Olympus, Tokyo, Japan). of each primer at 5 mM. The reaction products were electrophoresed on 1.5% agarose gels.

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After staining with ethidium bromide, fragments were visualized on a UV illuminator (Atto,19 Tokyo, Japan). Wild-type male and female genomic DNA was used as a control. gynoecia of the female-like flowers in the asexual mutant were fertile. As a result, we 11 1 obtained 234 seeds, and those seeds were sowed. Of the germinated seeds, 188 S. latifolia 2 sprouts were inoculated with M. lychnidis-dioicae. We genotyped mock or infected plants 3 three months after infection, based on genotyping by PCR using four markers: MK17, ScQ14, 4 SlAP3, and MS4 (Fig. 2). with the X chromosome and the Y chromosome with deletion of the SPF region (Table 1). In 16 these plants, the infected female was found as 48 individuals, the infected male was found as 17 54 individuals, and the infected asexual mutant was found as 5 individuals (Table 1). Flower 18 bud development in the infected asexual mutant was divided into 12 stages (Fig. 3 a-l). The 19 morphology of the infected asexual mutant appeared to be similar to that of the male and 12 1 healthy asexual mutant at stages 1 to 6 (Fig. 3 a-f). However, extension of the stamen 2 filament in the infected asexual mutant was confirmed, as well as in the male, at stages 7 and 3 8 of flower bud development, and developmental suppression of the stamens did not occur at 4 stage 8 in the asexual mutant (Fig. 3 g, h).  The filamentous gynoecium was extended only in the upward direction and became 9 thinner at stages 8 to 10. The anther in stamens of the infected asexual mutant had four anther 10 locules, as well as the wild-type male (Fig. 3 h-j). The stamen filament of the infected 11 asexual mutant was extended at stages 11 and 12 of flower bud development, as well as the 12 male ( Fig. 3 k-l), but anthers were filled by smut spores instead of pollen. In addition, the 13 gynoecium of the infected asexual mutant did not develop. Instead, the filamentous structure 14 formed in the center of the flowers as in the male flowers (Fig. 3 g-l). It was found that 15 developmental suppression of the stamen was released by M. lychnidis-dioicae, but that of 16 the gynoecium was not released.
14 1 Morphological changes of anther locule caused by M.
In this study, we observed anther locules of the healthy male, the infected male, as 4 well as those of the infected female and infected asexual mutants (Fig. 3, Fig. 4 a-y; S1 Fig.).

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Each of the infected plants was observed and compared at stages II to VI of anther 6 development. Archesporial cells and epidermal cells formed at stage II of anther development 7 in the healthy male (Fig. 4 a). The anther locule at stage V of anther development was 8 composed of a layer of epidermis, a layer of endothecium, a middle layer, a tapetum layer 9 and pollen mother cells as a result of the differentiation of archesporial cells (Fig. 4 a-d, f-i). 10 The middle layer and the tapetum layer caused programmed cell death at stage VI of anther 11 development (Fig. 4 e, j).

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In the infected males, the primary parietal cell layer was divided into two layers at 13 stage III of anther development (Fig. 4 l). Furthermore, the secondary parietal cell layer was 14 divided into two layers at stage IV of the anther development (Fig. 4 m). In the infected 15 males, the endothecium was present in the two layers, unlike in the healthy males.

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Furthermore, the tapetum and the middle layer in infected males showed different 17 morphology from those in the healthy males at stage V of anther development (Fig. 4 n). The 18 tapetum was rapidly disintegrated at stage VI of anther development in the infected male, 19 unlike in the healthy male, where the hyphae of M. lychnidis-dioicae grew (Fig. 4 o).

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In the infected female, only archesporial-like-cells and epidermal cells existed, but 2 primary parietal cells and primary sporogenous cells did not exist in stage III of anther 3 development (Fig. 4 q). Differentiation of the archesporial-like-cells did not occur at later 4 stages (Fig. 4 p-s). Growth of M. lychnidis-dioicae was also observed until stage VI of anther 5 development in the infected female (Fig. 4 t). In the infected asexual mutant, development of 6 the anther locules in developed stamens caused by M. lychnidis-dioicae was similar to that in 7 the infected male at stages II to IV of anther development (Fig. 4 u-w), whereas the 8 morphology of the tapetum at stage V of anther development was different from that in the 9 infected male (Fig. 4 x). Growth of M. lychnidis-dioicae was also observed at stage VI of 10 anther development (Fig. 4 y). Thus, it was found that M. lychnidis-dioicae in the infected Therefore, it is thought that development of the stamen does not occur after stage 7 of flower 2 development. This is due to the suppression of gynoecium development caused by GSF 3 function and a lack of promotion of stamen development due to the deletion of the SPF 4 region. Development of the gynoecium is suppressed and replaced by the filamentous 5 structure because the GSF on the Y chromosome is intact ( Fig. 1; Fig. 2). When M.
6 lychnidis-dioicae infects the asexual mutant, the gynoecium displays a filamentous structure 7 as in the healthy asexual flower mutant. Therefore, it is suggested that the suppression of the stamen did not occur at stage 8 of flower bud development (Fig. 3). The growth area of M.
3 lychnidis-dioicae in the anther locule is different among the infected male, the infected 4 asexual mutant, and the infected female (Fig. 4 n, s, x). We suggest that M. lychnidis-dioicae 5 was able to recognize the tapetum, such that the growth of M. lychnidis-dioicae started with 6 tapetum dissolution and was observed at the position where the tapetum cells were present 7 (Fig. 4 x). Therefore, we suggest that the different growth areas of M. lychnidis-dioicae 8 observed in infected males, infected asexual mutants and infected females was due to the 9 absence of the tapetum.