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Time course of the fertilization of a protoperithecium by a single male nucleus (Movie S6). Confocal microscopy of the fertilization of an H1-sGFP female with H1-RFP conidial suspension. The (Continued on next page)
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Sexual reproduction is a key process influencing the evolution and adaptation of animals, plants, and many eukaryotic microorganisms, such as fungi. However, the sequential cell biology of fertilization and the associated nuclear dynamics after plasmogamy are poorly understood in filamentous fungi. Using histone-fluorescent parental isolates, we tr...
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... where the next steps of sexual reproduction, such as karyogamy, meiosis, and ascosporogenesis, take place. In N. crassa, macroconidia are typically multinucleate. We reproducibly observed that fertilization by macroconidia led to the discharge of several nuclei from a single conidium into a trichogyne (i.e., three nuclei per conidium in Fig. 6, 2 nuclei in Fig. 2, and 2 nuclei in Movies S7 and S8). Furthermore, we observed that trichogynes can branch and that those branches are capable of binding, coiling, and fusing with at least two conidia (A and B; Fig. 6 and Movie S6). In the trichogyne of Fig. 6, male nuclei were tracked from the conidia to the protoperithecium over a ...
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... macroconidia led to the discharge of several nuclei from a single conidium into a trichogyne (i.e., three nuclei per conidium in Fig. 6, 2 nuclei in Fig. 2, and 2 nuclei in Movies S7 and S8). Furthermore, we observed that trichogynes can branch and that those branches are capable of binding, coiling, and fusing with at least two conidia (A and B; Fig. 6 and Movie S6). In the trichogyne of Fig. 6, male nuclei were tracked from the conidia to the protoperithecium over a distance of up to 574 mm ( Fig. 6; conidium A). Three male RFP nuclei entered the trichogyne from conidium A in 35 min (Fig. 6, labeled 1, 2, and 3; from t = 46 min to t = 1 h 21 min), followed by three additional nuclei ...
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... nuclei from a single conidium into a trichogyne (i.e., three nuclei per conidium in Fig. 6, 2 nuclei in Fig. 2, and 2 nuclei in Movies S7 and S8). Furthermore, we observed that trichogynes can branch and that those branches are capable of binding, coiling, and fusing with at least two conidia (A and B; Fig. 6 and Movie S6). In the trichogyne of Fig. 6, male nuclei were tracked from the conidia to the protoperithecium over a distance of up to 574 mm ( Fig. 6; conidium A). Three male RFP nuclei entered the trichogyne from conidium A in 35 min (Fig. 6, labeled 1, 2, and 3; from t = 46 min to t = 1 h 21 min), followed by three additional nuclei from conidium B, which were released in ...
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... 2, and 2 nuclei in Movies S7 and S8). Furthermore, we observed that trichogynes can branch and that those branches are capable of binding, coiling, and fusing with at least two conidia (A and B; Fig. 6 and Movie S6). In the trichogyne of Fig. 6, male nuclei were tracked from the conidia to the protoperithecium over a distance of up to 574 mm ( Fig. 6; conidium A). Three male RFP nuclei entered the trichogyne from conidium A in 35 min (Fig. 6, labeled 1, 2, and 3; from t = 46 min to t = 1 h 21 min), followed by three additional nuclei from conidium B, which were released in only 6 min (Fig. 6, labeled 4, 5, and 6; from t = 1 h 25 min to t = 1 h 31 min). By carefully tracking the ...
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... and that those branches are capable of binding, coiling, and fusing with at least two conidia (A and B; Fig. 6 and Movie S6). In the trichogyne of Fig. 6, male nuclei were tracked from the conidia to the protoperithecium over a distance of up to 574 mm ( Fig. 6; conidium A). Three male RFP nuclei entered the trichogyne from conidium A in 35 min (Fig. 6, labeled 1, 2, and 3; from t = 46 min to t = 1 h 21 min), followed by three additional nuclei from conidium B, which were released in only 6 min (Fig. 6, labeled 4, 5, and 6; from t = 1 h 25 min to t = 1 h 31 min). By carefully tracking the different male nuclei within the trichogyne, we determined that the nucleus entering the ...
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... 46 min to t = 1 h 21 min), followed by three additional nuclei from conidium B, which were released in only 6 min (Fig. 6, labeled 4, 5, and 6; from t = 1 h 25 min to t = 1 h 31 min). By carefully tracking the different male nuclei within the trichogyne, we determined that the nucleus entering the protoperithecium was nucleus 4 from conidium B ( Fig. 6; t = 1 h 53 min). Strikingly, although the remaining male nuclei continued their migration up the trichogyne, all of them stalled and accumulated upstream of the now-fertilized perithecium ( Fig. 6; t = 4 h 24 min; Movie S6). It was also the case in Movie S7, where two conidial male nuclei migrated up the trichogyne and only one ...
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... tracking the different male nuclei within the trichogyne, we determined that the nucleus entering the protoperithecium was nucleus 4 from conidium B ( Fig. 6; t = 1 h 53 min). Strikingly, although the remaining male nuclei continued their migration up the trichogyne, all of them stalled and accumulated upstream of the now-fertilized perithecium ( Fig. 6; t = 4 h 24 min; Movie S6). It was also the case in Movie S7, where two conidial male nuclei migrated up the trichogyne and only one entered the protoperithecium. Accordingly, immobile RFP male nuclei in trichogynes were frequently observed late in the experiment (between 10 and 14 h), where presumably, nuclei were somehow blocked from ...
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... the trichogyne and only one entered the protoperithecium. Accordingly, immobile RFP male nuclei in trichogynes were frequently observed late in the experiment (between 10 and 14 h), where presumably, nuclei were somehow blocked from migrating after protoperithecium fertilization (data not shown). Note that the two full fertilization events imaged (Fig. 6, Movies S6 and S7) were the only recordings where we tracked multiple male nuclei migrating into a trichogyne from plasmogamy to entry into a protoperithecium. These live-cell recordings led us to hypothesize that entry of a first male nucleus into a protoperithecium inhibits entry of the following ...
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... second important feature provoked by entry of male nuclei into prothoperithecia was the quantified increase of volume of the latter ( Fig. 6; from t = 1 h 53 min; Fig. 7). Finally, we detected a second RFP focal signal in the core of the protoperithecium twice ( Fig. 6; t = 2 h 25 min to 4 h 27 min; Movie S7; 4 h 22 min 52 sec). Careful analysis of the images did not indicate any entry of any of the remaining nuclei. Thus, the appearance of this second RFP focal signal 32 ...
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... second important feature provoked by entry of male nuclei into prothoperithecia was the quantified increase of volume of the latter ( Fig. 6; from t = 1 h 53 min; Fig. 7). Finally, we detected a second RFP focal signal in the core of the protoperithecium twice ( Fig. 6; t = 2 h 25 min to 4 h 27 min; Movie S7; 4 h 22 min 52 sec). Careful analysis of the images did not indicate any entry of any of the remaining nuclei. Thus, the appearance of this second RFP focal signal 32 min after initial protoperithecium entry in Fig. 6 (Movie S6) may indicate that this male nucleus 4 has divided. However, we ...
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... Finally, we detected a second RFP focal signal in the core of the protoperithecium twice ( Fig. 6; t = 2 h 25 min to 4 h 27 min; Movie S7; 4 h 22 min 52 sec). Careful analysis of the images did not indicate any entry of any of the remaining nuclei. Thus, the appearance of this second RFP focal signal 32 min after initial protoperithecium entry in Fig. 6 (Movie S6) may indicate that this male nucleus 4 has divided. However, we cannot exclude that the appearance of a second focus within the protoperithecium may be due to the stretching of the nucleus. These data suggest that entry of male nuclei into protoperithecia may trigger at least two signals, a first one in order to avoid ...
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... interesting feature of trichogynes is their ability to fuse with several conidia and, as a consequence, to allow the discharge of nuclei belonging to different conidia ( Fig. 6 and Movie S6). It is interesting to note that nuclei from conidium A stopped migration, especially nucleus 1, remaining immobile for 23 min (from t = 1 h 25 min 58 s to t = 1h 52 min 59 s), while the nuclei from conidium B were released into the tricogyne. We measured the average speed of nuclei 1 and 2 before the pause and compared it ...
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... to fully understanding sexual reproduction from the initial plasmogamy of sexual cells to the final production of sexual spores. In previous Neurospora studies, following the work of B. O. Dodge, who identified the sexual reproductive organs in the Analysis of segmented images of H1-sGFP fertilization by H1-RFP conidial suspension in Movie S6 (Fig. 6). The green signal of the H1-sGFP nuclei composing the protoperithecium was segmented in order to evaluate the volume of the perithecium in the course of fertilization. The time of entry of the first H1-RFP male nucleus into the trichogyne (plasmogamy) and the time of entry of the nucleus number 4 into the protoperithecium ...
Citations
... This hormonal chemotropism is controlled by the mating type genes on a pheromone/receptor recognition basis and occurs only between cells of opposite mating type in heterothallic species (Bistis, 1983). The contact between the trichogyne and the spermatia triggers cell fusion, i.e. plasmogamy, after which the spermatia nucleus migrates across the trichogyne into the ascogonium (Brun et al., 2021). Plasmogamy is controlled by mating type genes (Bistis, 1996;Peraza-Reyes and Malagnac, 2016). ...
... Neurospora crassa (Maheshwari, 1999). In these two species, however, microconidia designate the male gametes, are able to germinate, and are distinct from macroconidia (often called conidia) which are asexual spores but can also be fertilizing elements (Brun et al., 2021); hence both microconidia and macroconidia are spermatia in B. cinerea and N. crassa. ...
Sexual reproduction in Ascomycetes is well described in several model organisms such as Neurospora crassa or Podospora anserina. Deciphering the biological process of sexual reproduction (from the recognition between compatible partners to the formation of zygote) can be a major advantage to better control sexually reproducing pathogenic fungi. In Pyricularia oryzae, the fungal pathogen causing blast diseases on several Poaceae species, the biology of sexual reproduction remains poorly documented. Besides the well-documented production of asexual macroconidia, the production of microconidia was seldom reported in P. oryzae, and their role as male gamete (i.e., spermatia) and in male fertility has never been explored. Here, we
characterised the morphological features of microconidia and demonstrated that they are bona fide spermatia. Contrary to macroconidia, microconidia are not able to germinate and seem to be the only male gametes in P. oryzae. We show that fruiting body (perithecium) formation requires microconidia to get in contact with mycelium of strains of opposite mating type, to presumably fertilise the female gametes.
... The physiological mechanisms occurring during sexual reproduction have been described in some model ascomycetes (e.g. Neurospora crassa; Brun et al., 2021), but the interaction with the host plant remains poorly understood for biotrophic and hemibiotrophic plant pathogens. ...
Little is known about the impact of host immunity on sexual reproduction in fungal pathogens. In particular, it is unclear whether crossing requires both sexual partners to infect living plant tissues. We addressed this issue in a three-year experiment investigating different scenarios of Zymoseptoria tritici crosses according to the virulence (‘vir’) or avirulence (‘avr’) of the parents against a qualitative resistance gene. Co-inoculations (‘vir × vir’, ‘avr × vir’, ‘avr × avr’) and single inoculations were performed on a wheat cultivar carrying the Stb16q resistance gene (Cellule) and a susceptible cultivar (Apache), in the greenhouse. We assessed the intensity of asexual reproduction by scoring disease severity, and the intensity of sexual reproduction by counting the ascospores discharged from wheat residues. As expected, disease severity was more intense on Cellule for ‘vir × vir’ co-inoculations than for ‘avr × vir’ co-inoculations, with no disease for ‘avr × avr’. However, all types of co-inoculation yielded sexual offspring, whether or not the parental strains caused plant symptoms. Parenthood was confirmed by genotyping (SSR markers), and the occurrence of crosses between (co-)inoculated and exogenous strains (other strains from the experiment, or from far away) was determined. We showed that symptomatic asexual infection was not required for a strain to participate in sexual reproduction, and deduced from this result that avirulent strains could be maintained asymptomatically “on” or “in” leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually. In two of the three years, the intensity of sexual reproduction did not differ between the three types of co-inoculation in Cellule, suggesting that crosses involving avirulent strains are not anecdotal. We discuss the possible mechanisms explaining the maintenance of avirulence in Z. tritici populations and the potential impact of particular resistance deployments such as cultivar mixtures for limiting resistance breakdown.
... This hormonal chemotropism is controlled by the mating type genes on a pheromone/receptor recognition basis and occurs only between cells of opposite mating type in heterothallic species (Bistis, 1983). The contact between the trichogyne and the spermatia triggers cell fusion, that is, plasmogamy, after which the spermatia nucleus migrates across the trichogyne into the ascogonium (Brun et al., 2021). Plasmogamy is controlled by mating-type genes (Bistis, 1996;Peraza-Reyes & Malagnac, 2016). ...
... Microconidia were proved to be spermatia in several Ascomycete species, including Botrytis cinerea (Fukumori et al., 2004) and Neurospora crassa (Maheshwari, 1999). In these two species, however, microconidia designate the male gametes, are able to germinate, and are distinct from macroconidia (often called conidia) which are asexual spores but can also be fertilising elements (Brun et al., 2021); hence both microconidia and macroconidia are spermatia in B. cinerea and N. crassa. ...
Sexual reproduction in Ascomycetes is well described in several model organisms such as Neurospora crassa or Podospora anserina. Deciphering the biological process of sexual reproduction (from the recognition between compatible partners to the formation of zygote) can be a major advantage to better control sexually‐reproducing pathogenic fungi. In Pyricularia oryzae, the fungal pathogen causing blast diseases on several Poaceae species, the biology of sexual reproduction remains poorly documented. Besides the well‐documented production of asexual macroconidia, the production of microconidia was seldom reported in P. oryzae, and their role as male gamete (i.e. spermatia) and in male fertility has never been explored. Here we characterized the morphological features of microconidia and demonstrate that they are bona fide spermatia. Contrary to macroconidia, microconidia are not able to germinate and seem to be the only male gametes in P. oryzae. We show that fruiting body (perithecium) formation requires microconidia to get in contact with mycelium of strains of opposite mating type, to presumably fertilize the female gametes. This article is protected by copyright. All rights reserved.
... The physiological and cellular mechanisms occurring during sexual reproduction have been described in some model ascomycetes (e.g. Neurospora crassa; Brun et al., 2021), but the interaction with the host plant remains poorly understood for biotrophic and hemibiotrophic plant pathogens. ...
Little is known about the impact of host immunity on sexual reproduction in fungal pathogens. In particular, it is unclear whether crossing requires both sexual partners to infect living plant tissues. We addressed this issue in a three-year experiment investigating different scenarios of Zymoseptoria tritici crosses on wheat according to the virulence (‘vir’) or avirulence (‘avr’) of the parents against a qualitative resistance gene. Co-inoculations (‘vir × vir’, ‘avr × vir’, ‘avr × avr’) and single inoculations were performed on a cultivar carrying the resistance gene (Cellule) and a susceptible cultivar (Apache), in the greenhouse. We assessed the intensity of asexual multiplication by scoring disease severity, and the intensity of sexual reproduction by counting the ascospores discharged from wheat residues. As expected, disease severity was more intense on Cellule for ‘vir × vir’ co-inoculations than for ‘avr × vir’ co-inoculations, with no disease for ‘avr × avr’. However, all types of co-inoculation yielded sexual offspring, whether or not the parental strains caused plant symptoms. Parenthood was confirmed by genotyping (SSR markers), and the occurrence of crosses between (co-)inoculated and exogenous strains (other strains from the experiment, or from far away) was determined. We found that symptomatic asexual infection was not required for a strain to participate in sexual reproduction, and that avirulent strains could be maintained asymptomatically “on” or “in” leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually. In two of the three years, the intensity of sexual reproduction did not differ significantly between the three types of co-inoculation in Cellule, suggesting that crosses involving avirulent strains are not anecdotal. We discuss the possible mechanisms explaining the maintenance of avirulence in Z. tritici populations and supporting the potential efficacy of cultivar mixtures for limiting resistance gene breakdown.
Highlights
Avirulent Zymoseptoria tritici strains can reproduce sexually in wheat plants carrying the corresponding resistant gene.
Symptomatic infection of plant tissues is not essential for a strain to reproduce sexually.
Avirulent strains can be maintained asymptomatically “on” or “in” leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually.
Crosses of virulent strains with virulent and avirulent strains in a plant host carrying the corresponding resistance gene can produce offspring with similar population sizes.
Several possible scenarios for sexual crosses can explain the maintenance of avirulence in Zymoseptoria tritici populations evolving in a wheat canopy, particular in cultivar mixtures.
This paper addresses the stability of mycelial growth in fungi and differences between ascomycetes and basidiomycetes. Starting with general evolutionary theories of multicellularity and the role of sex, we then discuss individuality in fungi. Recent research has demonstrated the deleterious consequences of nucleus-level selection in fungal mycelia, favoring cheaters with a nucleus-level benefit during spore formation but a negative effect on mycelium-level fitness. Cheaters appear to generally be loss-of-fusion (LOF) mutants, with a higher propensity to form aerial hyphae developing into asexual spores. Since LOF mutants rely on heterokaryosis with wild-type nuclei, we argue that regular single-spore bottlenecks can efficiently select against such cheater mutants. We then zoom in on ecological differences between ascomycetes being typically fast-growing but short-lived with frequent asexual-spore bottlenecks and basidiomycetes being generally slow-growing but long-lived and usually without asexual-spore bottlenecks. We argue that these life history differences have coevolved with stricter nuclear quality checks in basidiomycetes. Specifically, we propose a new function for clamp connections, structures formed during the sexual stage in ascomycetes and basidiomycetes but during somatic growth only in basidiomycete dikaryons. During dikaryon cell division, the two haploid nuclei temporarily enter a monokaryotic phase, by alternatingly entering a retrograde-growing clamp cell, which subsequently fuses with the subapical cell to recover the dikaryotic cell. We hypothesize that clamp connections act as screening devices for nuclear quality, with both nuclei continuously testing each other for fusion ability, a test that LOF mutants will fail. By linking differences in longevity of the mycelial phase to ecology and stringency of nuclear quality checks, we propose that mycelia have a constant and low lifetime cheating risk, irrespective of their size and longevity.
