Plasmids used in this work.

a<p><i>Erwinia uredovora</i> genes are: <i>crtE</i> (geranylgeranyl pyrophosphate synthase), <i>crtB</i> (phytoene synthase), <i>crtI</i> (phytoene dehydrogenase), and <i>crtY</i> (lycopene cyclase). <i>Cm</i>: chloramphenicol resistance. <i>Amp</i>: ampicillin resistance. <i>P. blakesleeanus carRA</i> and <i>carB</i> cDNAs in pCS19 and 6pCS16 plasmids are cloned in the multiple cloning site of vector pUC19.</p

<i>E. coli</i> complementation assays.

<p>A: Phytoene synthase activity assay (<i>crtB⁻</i> strain; pAVB5). In the negative control (pUC19), bacteria do not accumulate any carotenoid and show a normal, whitish color. In the positive control (pAVB13), bacteria accumulate β-carotene and show a yellow phenotype. After transformation with pCS19, a light yellow color was detected (due to the β-carotene production as the result of the phytoene synthase activity from the <i>carRA</i> gene). B: Lycopene cyclase activity assay (<i>crtY⁻</i> strain; pAVB12). In the negative control (pUC19), bacteria accumulate lycopene and show a pink color. In the positive control (pAVB2), lycopene is converted into β-carotene resulting in a bright yellow color. Transformation with pCS19 led to a light-orange phenotype due to the β-carotene production as the result of the lycopene cyclase activity of the <i>carRA</i> gene. Data of HPLC analyses are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023102#pone-0023102-t002" target="_blank">Table 2</a>.</p

Carotenogenesis pathway.

<p>Enzymatic steps and structural genes involved in the biosynthesis of β-carotene from GGPP in <i>P. blakesleeanus</i>, <i>M. circinelloides</i> and <i>E. uredovora</i>. PS, phytoene synthase; PD, phytoene dehydrogenase; LC, lycopene cyclase.</p

Northen blot and RT-RCR analyses of the <i>Mucor</i> transformants.

<p>A: Northern blot hybridization of total RNA from <i>M. circinelloides</i> transformants T7, T8 and T21 with a <i>carRA</i> cDNA probe. The untransformed <i>Mucor</i> MS7 strain and the <i>P. blakesleeanus</i> wild-type (Pwt) strain were used as negative and positive controls, respectively. B: Ethidium bromide staining after gel electrophoresis of RNA samples. C: Detection by RT-PCR of the <i>P. blakesleeanus carRA</i> cDNA in the same RNA samples. D: Quality control of the same RNA samples by RT-PCR detection of the <i>M. circinelloides pyrG</i> cDNA. M: 100 bp marker.</p

HPLC elution profiles of the carotenoids produced in the <i>Mucor</i> transformants.

<p>Carotenoids were detected at 450 nm. Profiles obtained from <i>M. circinelloides</i> wild-type, MS7 (<i>leuA1</i>, <i>carP4</i>) and MS21 (<i>leuA1</i>, <i>carR9</i>) strains, as well as T7 and T21 transformants (MS7 and MS21, respectively, transformed with the <i>P. blakesleeanus carRA</i> gene) are shown. β: β-carotene, L: Lycopene. Y-axis not scaled.</p

Southern blot analysis of the <i>Mucor</i> transformants.

<p>Genomic DNA from <i>M. circinelloides</i> untransformed MS7 strain was employed as negative control and plasmid DNA from pCS5.1(3) (P5 lane) was employed as positive control. DNA samples were digested with <i>Sac</i>I or <i>Sal</i>I enzymes. Hybridizations were performed with a <i>P. blakesleeanus carRA</i> probe (A) and a <i>M. circinelloides leuA</i> probe (B).</p

Restriction map of plasmid pCS5.1(3).

<p>This plasmid includes the <i>M. circinelloides leuA</i> and the <i>P. blakesleeanus carRA</i> genes. It was employed in the transformation of the <i>M. circinelloides</i> strains MS7, MS8 and MS21. Digestion with <i>Sac</i>I results in plasmid linearization (12-kb single band). Digestion with <i>Sal</i>I gives rise to two fragments of 7.3 kb (including the <i>leuA</i> gene) and 4.7 kb (including the <i>carRA</i> gene).</p

Carotenoid accumulation in <i>E. coli</i> co-transformants.

a<p>Cm, chloramphenicol resistance; Amp, ampicillin resistance. Genes in plasmid are shown in brackets. “Carotenoid mutant” genotype is shown in square brackets.</p>b<p>-, carotenoid not detected. All data are given in ng per gram dry weight. Data are averages and standard errors. Phytoene was not detected in any co-transformation.</p>c<p>The gene for phytoene dehydrogenase from <i>E. uredovora</i> (<i>crtI</i>) has been replaced by the gene from <i>P. blakesleeanus</i> (<i>carB</i>), so this plasmid would be the same kind of “carotenoid mutant” as pAVB16.</p

Schematic organization of carotenogenesis structural genes.

<p>The organization of the structural genes involved in the carotenoid biosynthesis pathway in <i>P. blakesleeanus</i>, <i>M. circinelloides</i> and <i>E. uredovora</i> is shown. The function of these genes is described in the main text.</p

Genomic Analysis of the Necrotrophic Fungal Pathogens <i>Sclerotinia sclerotiorum</i> and <i>Botrytis cinerea</i>

<div><p><i>Sclerotinia sclerotiorum</i> and <i>Botrytis cinerea</i> are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of <i>S. sclerotiorum</i> and two strains of <i>B. cinerea</i>. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the <i>S. sclerotiorum</i> assembly to 16 chromosomes and found large-scale co-linearity with the <i>B. cinerea</i> genomes. Seven percent of the <i>S. sclerotiorum</i> genome comprises transposable elements compared to <1% of <i>B. cinerea</i>. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of <i>B. cinerea</i>–specific secondary metabolites relative to <i>S. sclerotiorum</i>. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between <i>S. sclerotiorum</i> and <i>B. cinerea</i>. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.</p></div