Graziani S et al. (2004), Bistability and hysteresis of the 'Secteur' dif...

ECB-ART-39125

BMC Biol 2004 Aug 16;2:18. doi: 10.1186/1741-7007-2-18.

Show Gene links Show Anatomy links

Bistability and hysteresis of the ''Secteur'' differentiation are controlled by a two-gene locus in Nectria haematococca.

Graziani S , Silar P , Daboussi MJ .

???displayArticle.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. The full scope of the underlying molecular mechanisms leading to bistability and hysteresis remains elusive. Nectria haemaotcocca, a saprophytic or pathogenic fungus with sexual reproduction, exhibits a bistable morphological modification characterized by a reduced growth rate and an intense pigmentation. Bistability is triggered by the presence or absence of sigma, a cytoplasmic determinant. This determinant spreads in an infectious manner in the hyphae of the growing margin, insuring hysteresis of the differentiation. RESULTS: Seven mutants specifically affected in the generation of sigma were selected through two different screening strategies. The s1 and s2 mutations completely abolish the generation of sigma and of its morphological expression, the Secteur. The remaining five mutations promote its constitutive generation, which determines an intense pigmentation but not growth alteration. The seven mutations map at the same locus, Ses (for ''Secteur-specific''). The s2 mutant was obtained by an insertional mutagenesis strategy, which permitted the cloning of the Ses locus. Sequence and transcription analysis reveals that Ses is composed of two closely linked genes, SesA, mutated in the s1 and s2 mutant strains, and SesB, mutated in the s* mutant strains. SesB shares sequence similarity with animal and fungal putative proteins, with potential esterase/lipase/thioesterase activity, whereas SesA is similar to proteins of unknown function present only in the filamentous fungi Fusarium graminearum and Podospora anserina. CONCLUSIONS: The cloning of Ses provides evidence that a system encoded by two linked genes directs a bistable and hysteretic switch in a eukaryote. Atypical regulatory relations between the two proteins may account for the hysteresis of Secteur differentiation.

???displayArticle.pubmedLink??? 15312233
???displayArticle.pmcLink??? PMC516043
???displayArticle.link??? BMC Biol

Genes referenced: hoxa9 LOC100893907 LOC115919910 LOC115925415 pir

???attribute.lit??? ???displayArticles.show???

	Figure 1. The Secteur and its transmission through contamination tests in Nectria haematococca. (A) Wild-type strain exhibiting three spontaneous Secteurs (1, 2, 3). (B) s789 mutant. (C) Contamination tests, using plugs from a Secteur or from s789, on wild type (wt), result in one Secteur at each infection point. (D)Contamination tests on s1 mutant did not result in the induction of any Secteur at the infection points.
	Figure 2. Selection of Nectria haematococca mutants affected in the expression of the Secteur. (A) Compares the morphology of a Normal growing culture (N) and of a ZiS culture (modified culture obtained by excision from a Secteur). (B) A 4-day ZiS culture submitted to UV mutagenesis. Note the emergence of four fast-growing sectors, which correspond to nas mutants.
	Figure 3. Phenotypic characterization of transformants. (A) Detection of a nas mutant phenotype among 12 transformants. Arrows indicate some Secteurs. (B) Summary of the different mutants recovered among the 5000 transformants: 152 were altered in pigmentation (99 reddish, 16 white/beige/yellow, 37 greenish), 45 were altered in colony morphology (12 irregular, 12 compact, 19 colonial, 2 fluffy), and 10 were affected in the expression of the Secteur (9 nas and 1 s).
	Figure 4. Analysis of pAN7-1 integration pattern in s2.(A) Southern blot analysis of total genomic DNA of s2 digested with ClaI, which does not cut the plasmid (lane 1), and BamHI, which cuts once in pAN7-1 (lane 2), and probed with the pAN7-1 vector. Sizes are indicated in kilobases (kb). (B) Restriction map of the pAN7-1 integration point in s2. Wild-type genomic DNA is shown as a thick line, pAN7-1 sequences are represented by the open bar, and the dotted line stands for unknown sequences. The fragment LI is inserted at the bacterial replication origin (ORI). SI is truncated and carries a part of hph and of tTrpC.
	Figure 5. Types of transformants recovered in transformation experiments using s* mutants with XN31E6 and psecX5. As the respective proportion of each type of transformant was similar with both vectors, the proportion observed is the combined percentage computed with 60 tested transformants. S, transformants differentiating Secteurs as wild type; mild S, transformants differentiating a Secteur that propagates more slowly than wild type and that frequently reverts to a normal morphology. S → s, transformants differentiating the Secteurs but showing an s phenotype beyond the modified area; s, transformants unable to differentiate Secteurs.
	Figure 6. Southern blot analysis of XN31E6 transformants. XN31E6 cosmid DNA along with total genomic DNAs of wild type (wt) s2, and five transformants obtained by transforming s*789 with XN31E6 were digested by XhoI and hybridized with an XN31E6 probe. Wild type, Tr1, Tr2, Tr3, and Tr4 differentiate wild-type Secteurs (S), Tr5 has a mild S phenotype, and s2 is not able to differentiate Secteur(s). The 7-kb band corresponding to pMoCosX is marked by dark connecting lines. The arrow points to the 5.3-kb fragment, which shifts in s2.
	Figure 7. Schematic representation of the Ses locus of Nectria haematococca. (A) Open reading frames larger than 200 aa or showing sequence similarity to other genes are represented by gray arrows. Black arrows represent sequences in inverted repeated orientation: repeat 1 is 290 bp long; repeat 2 is 79 bp long; repeat 3 is 32 bp long; and repeat 4 is 93 bp long. Thick lines: subclones able to restore the Secteur modification in s* mutants. (B) Detailed map of the region between oligonucleotides im317 and ip2393c. Vertical bold lines: position of the indicated mutations. Insertion: integration point of pAN7-1 in s2. Black lozenge: polyadenylation site.
	Figure 8. SesA and SesB conservation during evolution. (A) Alignment of SesAp with its homologues in Fusarium graminearum (F. gra 214 and F. gra 201) and Podospora anserina (P. ans). The amino acid change in s1 is boxed. (B) Alignment of SesBp with representative homologues from F. graminearum (Fg1 and Fg2; the sequence was obtained from the Fungal Genome Initiative, ), P. anserina (Pa; ), Homo sapiens (Hs; GENBANK: BC001705), Drosophila melanogaster (Dm; GENBANK: AAF5369), and Caenorhabditis elegans (Ce; PIR: T21079). Arrowheads indicate the amino acid changed in s4, s789, and s18; arrow indicates the position of the frameshift in s27. (C) Conservation of Ses organization in F. graminearum. SesA corresponds to F. gra 214 of (A) and SesB corresponds to Fg1 of (B). Percentages of identity with Nectriahaematococca SesAp and SesBp are 41% and 79%, respectively. In (A) and (B), conserved amino acids are shaded. Color reflects the degree of conservation.
	Figure 9. Southern blot analysis of StuI-digested DNAs of wild-type Nectria haematococca and five transformants. 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-s1 plasmid and pBC-Hygro vector, and probed with psec3-s1. All the transformants share a large band (>15 kb), absent from wild type. Tr3 and Tr5 contain both the wild type and the s1 allele. The faint bands noted * correspond to repeated sequences homologous to those present at the Ses locus.
	Figure 10. Models integrating functional elements of Secteur expression in Nectria haematococca. (A) First model: In the wild-type strain, the product of the SesA and SesB genes can exist in a normal state (AN and BN) and in a modified state (AS and BS). AS determines overproduction of pigments and BS is responsible for growth alteration. (B) Second model: 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.

References [+] :

Akins, General method for cloning Neurospora crassa nuclear genes by complementation of mutants. 1985, Pubmed