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Thus, pSec3- s 1 contained the entire Ses locus present in the s 1 mutant strain. Among 17 tested Hyg R transformants, seven could not differentiate Secteurs. For three transformants, the expected wild-type Stu I fragment was absent, suggesting that integration altered the Ses locus, thus accounting for the lack of Secteur formation. For the two other transformants, the wild-type Stu I fragment was present, as was an additional band larger than 15 kb. As this band hybridized with psec3-s 1 and the hph hygromycin resistance marker data not shown , these transformants could be interpreted as bearing a co-integration of pBC-Hygro and pSecs 1 at an ectopic position.

Sequencing of the Ses locus in these two transformants that do not display Secteurs confirmed the presence of both alleles, i. Total genomic DNAs of wild-type wt Nectria haematococca and five s transformants Tr1-Tr5 that were obtained after co-transformation of wt with psec3-s 1 plasmid and pBC-Hygro vector, and probed with psec3-s 1.

Tr3 and Tr5 contain both the wild type and the s 1 allele. Although the Secteur and Anneau phenomena were described about 30 years ago [ 17 ], the molecular mechanisms able to generate these fascinating bistable differentiations are still mysterious. Early studies suggested peculiar epigenetic mechanisms [ 24 ].


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In this study, we have initiated the molecular characterization of Ses , the primary locus controlling the development of Secteurs. The experimental design, based on the differences in the growth rate between modified and normal cells, was very efficient in finding mutations that block the propagation of Secteurs after the action of both UV and NG N -methyl- N '-nitro- N -nitrosoguanidine. A vast collection of mutants affected in the Secteur expression was easily obtained.

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The recessivity of s 1 , deduced from the heterokaryon tests was the initial point of our cloning strategy, i. The screening of two representative cosmid libraries failed, and we examined various hypotheses to explain this failure, especially the possibility of the dominance of s 1. In order to easily clone the Ses locus, we used an insertional mutagenesis strategy, a powerful method for gene isolation in filamentous fungi [ 25 ].

This frequency is similar to the 0. Among them, 9 nas mutants and 1 s mutant were obtained. Although we may have been lucky, the recovery of this transformant may reflect a preference for plasmid integration in the region of the Ses locus, which is compatible with the high recombination frequency observed at Ses. Cloning of Ses permitted the construction by transformation of a partial diploid containing both the wild-type and s 1 mutant alleles.

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The fact that this partial diploid has a s phenotype indicates that s 1 allele is indeed dominant over the wild-type allele in partial diploids. Similar contradictions have been observed in A. These data were interpreted as being due to a limitation of regulatory gene products to the nucleus or by a stringent dose effect when combined with a nonrandom distribution of nuclei in heterokaryons [ 26 , 27 ].

In our study, a dose effect is also probable.

In addition, phase-contrast microscopic observation of the prototrophic mycelia revealed the presence of rows of uninucleate cells disrupting the heterokaryotic association. This structure does not prevent the stability of balanced heterokaryons with regard to metabolic requirements hence the complementation of the auxotrophic mutations , but might severely affect the dosage of the SesA products expressed from the wild-type and s 1 nuclei in different portions of the thallus.

Because s 1 is dominant and corresponds to a missense mutation, it is likely that the product expressed from SesA in s 1 is a dominant negative. The molecular characterization of Ses shows that it is composed of two linked and expressed genes, SesA and SesB , both of which are necessary for Secteur expression. Each has a different role. The s 1 and s 2 mutants map to SesA. Because s 2 is an integration of a large segment of DNA at the beginning of SesA, the phenotype of the s 2 mutant probably results from a complete loss of function of SesA.

The s 1 mutant carries a dominant missense mutation and therefore synthesizes the SesA mRNA, and probably a dominant negative protein. The SesAp protein has no evident homologue in the databanks yielding a clue to its function. The function of SesB and its homologue remains unknown in fungi; however the inactivation by RNA interference of the most similar gene in C. To date, this situation has not been described for any of the previously reported systems responsible for bistable or multistable switches in fungi.

Previous systems implicated prions [ 23 , 29 ], chromatin silencing [ 12 ], hysteresis in a MAP kinase cascade [ 16 ], and possible membrane inheritance [ 30 , 31 ]. At present, it is premature to propose a molecular model to explain the Secteur, because SesA and SesB display weak similarity only with proteins of unknown function. Two formal models could explain how the regulation might work.

Firstly, the product of SesA SesAp could exist in two states: The transition A N to A S may be initiated by a rare event in cells of the growing margin of the thallus. Once formed, the A S state becomes predominant by directing other A N molecules to adopt the same state.

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This positive feedback loop allows A S propagation from hyphae to hyphae through anastomoses. Models integrating functional elements of Secteur expression in Nectria haematococca. A S determines overproduction of pigments and B S is responsible for growth alteration. The product of SesA , SesAp, is responsible for pigmentation. The product of SesB , SesBp, has a catalytic activity necessary for the normal growth.

These two products negatively regulate each other, and the equilibrium constants favor SesAp. In juvenile thalli, only SesBp is produced, but SesAp can be randomly produced, switching to a new state with a majority of SesAp. In the normal mycelium, SesBp, which probably has a catalytic activity, would be active in promoting healthy growth. The appearance in some cells from the growing margin of a sufficient amount of SesAp would displace the equilibrium, resulting in the inhibition of SesBp.

In order for this to happen, the equilibrium constants must be in favor of SesAp. This would result in growth impairment because SesBp would not be present and in a red pigmentation because SesAp would be present. In the s mutants, SesAp would never be present, thus making the presence of SesBp constitutive hence the absence of Secteurs and the healthy growth.

This model could explain the dynamic equilibrium found in the ZiS area, as well as the dose effect for SesAp, explaining the difference of heterokaryons and partial diploids. Secteur, one of the two bistable and hysteretic differentiations exhibited by the filamentous fungus N. Each gene has a distinct role in defining bistability, and atypical regulatory relations between the two proteins coded by the two genes may account for the hysteresis of Secteur differentiation. The data exemplify the diversity of mechanisms that generate differentiation in fungi, and, more generally, in eukaryotes.

FFF, very fertile; F, fertile; St, sterile. The a 1 s 1 I 4 mod and 61a 1 strains used as recipients in transformation experiments were constructed by crossing the s 1 I 4 mod strain with a 1 mutant and the 61 mutant with a 1 I 4 mod strain, respectively unpublished data. The marker segregation was recorded after three weeks. General culture conditions and manipulations were as described by Daboussi-Bareyre [ 20 ]. The medium selective for transgenes was PH8, i. The general methods for crossing the homothallic N. Since the strains used in this study were homothallic, detection of hybrid perithecia in crosses were performed using as a partner in crosses the double mutant strain I 4 mod.

This strain developed white, self-sterile perithecia whereas wild-type and fertile mutants developed self-fertile, red perithecia [ 32 ]. Thus, all the white, fertile perithecia, which appeared on the crossing plates, were hybrid. The progeny of crosses was analyzed in accordance with the following procedure: A sample of about germinating ascospores from each dish was transferred to PDA.

Heterokaryons were constructed as described in [ 20 ]. Auxotrophic marked strains, which were recovered in the progeny, were paired on a cellophane membrane in the combinations appropriate to restore prototrophy. After 48 hours of growth on complete medium, the membrane was transferred to minimal medium. Widely inclusive inocula from prototrophic mycelia observed at the junction of the two partners were transferred to minimal medium and examined for their pigmentation and their ability to express the two modifications, either spontaneously or after inoculation.

For NG mutagenesis, 0. After 1 hour, the treated cultures were exposed to light for NG degradation. Fast-growing sectors were clearly visible a week after mutagenesis. For the estimation of the number of sectors recovered from each treated thallus, only sectors harboring different phenotypes were considered. The majority of the recovered mutants corresponded to nas mutants, defined by 1 the inability to differentiate both Anneaux and Secteurs, 2 an exuberant white mycelium, and 3 the inability to differentiate perithecia.

Some of these mutants could express the modifications at other temperatures, as was previously observed for mutants and [ 33 , 34 ]. A unique s mutant was selected as a wild-type-growing sector. This mutant displayed a wild-type phenotype, except for the inability to express the Secteur spontaneously or after inoculation. Both vectors carried a hygromycin resistance gene. Southern analysis on 10 independent hygromycin-resistant transformants Hyg R indicated that the transforming DNA inserted generally at one or two genomic sites, in some cases in a tandem fashion data not shown.

This pattern of integration was suitable for recovery of tagged genes.

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These most likely resulted from the plasmid integrations, since a sample of regenerating protoplasts treated in the same way but without the transforming DNA did not show any phenotypic variation. As control, a sample of transformants with an altered morphology was purified through single conidial isolation. Data showed that the mutant phenotype was stable.

Potential candidates affected in the expression of the Secteur were recovered as thalli that did not form spontaneous Secteurs. Inoculation experiments were used to confirm the mutant phenotypes. Protoplasts were prepared as described in [ 36 ], with the following modifications. Mycelia were collected 0. Protoplasts were separated from cell debris by addition of 5 ml of ST buffer 0. The protoplasts collected at the interface of the two solutions were harvested by centrifugation at g for 5 min, washed twice in ice-cold buffers, once in ST and then in STC 0.

Aliquots of the protoplasts were mixed with 2. Numerous small colonies stopped growing after 2—3 days. These were interpreted as abortive transformants. Only the colonies that grew after this delay were considered resistant to hygromycin. All the nucleic acid manipulations were performed using standard methods [ 37 ].

For Southern blot analysis, genomic DNA was extracted as described in [ 38 ]. The cosmid libraries were made in the pMoCosX vector as recommended by Orbach [ 39 ]. Each tested gene was represented by 2 to 5 independent clones, indicating that the library contained at least four N. The genBank accession no. SG performed all the experimental work and contributed to the interpretation of the data. PS and MJD performed part of the phylogenetic analysis, contributed to the interpretation of the data, and supervised the work. The writing of the manuscript was done in teamwork. All authors read and approved the final manuscript.

We thank Emilie Robillard for expert technical assistance. The work was done in compliance with the current laws governing genetic experimentations in France. National Center for Biotechnology Information , U. Published online Aug Author information Article notes Copyright and License information Disclaimer. Received May 5; Accepted Aug This article has been cited by other articles in PMC. Abstract Background Bistability and hysteresis are increasingly recognized as major properties of regulatory networks governing numerous biological phenomena, such as differentiation and cell cycle progression.

Conclusions The cloning of Ses provides evidence that a system encoded by two linked genes directs a bistable and hysteretic switch in a eukaryote. Open in a separate window. Table 1 Mutagenesis efficiency and spectrum of mutants. Table 2 Results of crosses involving mutants of the Secteur. Crosses Progency analyzed Wild-type progeny No. Table 3 Phenotypes of heterokaryotic mycelia from various pairings.

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Insertional mutagenesis identified the Ses locus Since the s 1 mutant allele appears recessive to the wild type in balanced heterokaryons, we tried to clone the Ses locus by complementation using the sib selection strategy [ 21 ]. The s2 transformant carries a DNA fragment integrated in the Ses locus In order to prove that s 2 carries integration in the Ses locus, we crossed it with the wild-type wt and s 1 strains. Gene organization of the Ses locus A 9-kb-long region surrounding the integration site in the s 2 strain was sequenced GenBank Accession no.

Discussion Although the Secteur and Anneau phenomena were described about 30 years ago [ 17 ], the molecular mechanisms able to generate these fascinating bistable differentiations are still mysterious. The impact of mutagenesis procedures The experimental design, based on the differences in the growth rate between modified and normal cells, was very efficient in finding mutations that block the propagation of Secteurs after the action of both UV and NG N -methyl- N '-nitro- N -nitrosoguanidine. Relationships between s1 and wt alleles: Integrated model of functional elements at the Ses locus The molecular characterization of Ses shows that it is composed of two linked and expressed genes, SesA and SesB , both of which are necessary for Secteur expression.

Conclusions Secteur, one of the two bistable and hysteretic differentiations exhibited by the filamentous fungus N. Methods Fungal strains and growth conditions The homothallic N. Table 4 Nectria haematococca strains used. Mating and ascospore recovery The general methods for crossing the homothallic N.

Heterokaryon test Heterokaryons were constructed as described in [ 20 ]. Transformation procedure Protoplasts were prepared as described in [ 36 ], with the following modifications.

DNA manipulation All the nucleic acid manipulations were performed using standard methods [ 37 ]. GenBank accession The genBank accession no. Authors' contributions SG performed all the experimental work and contributed to the interpretation of the data. Acknowledgements We thank Emilie Robillard for expert technical assistance. Paris — CNRS; Non-nucleic acid inheritance and epigenetic phenomena. Silar P, Daboussi MJ. Non-conventional infectious elements in filamentous fungi. Hysteresis drives cell-cycle transitions in Xenopus laevis egg extracts.

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