Ernesto P. Benito

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum and Botrytis cinerea 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 S. sclerotiorum and two strains of B. cinerea. 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 S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. 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 B. cinerea–specific secondary metabolites relative to S. sclerotiorum. 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 S. sclerotiorum and B. cinerea. 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.

Plant Defense Mechanisms Are Activated during Biotrophic and Necrotrophic Development of Colletotricum graminicola in Maize

Hemibiotrophic plant pathogens first establish a biotrophic interaction with the host plant and later switch to a destructive necrotrophic lifestyle. Studies of biotrophic pathogens have shown that they actively suppress plant defenses after an initial microbe-associated molecular pattern-triggered activation. In contrast, studies of the hemibiotrophs suggest that they do not suppress plant defenses during the biotrophic phase, indicating that while there are similarities between the biotrophic phase of hemibiotrophs and biotrophic pathogens, the two lifestyles are not analogous. We performed transcriptomic, histological, and biochemical studies of the early events during the infection of maize (Zea mays) with Colletotrichum graminicola, a model pathosystem for the study of hemibiotrophy. Time-course experiments revealed that mRNAs of several defense-related genes, reactive oxygen species, and antimicrobial compounds all begin to accumulate early in the infection process and continue to accumulate during the biotrophic stage. We also discovered the production of maize-derived vesicular bodies containing hydrogen peroxide targeting the fungal hyphae. We describe the fungal respiratory burst during host infection, paralleled by superoxide ion production in specific fungal cells during the transition from biotrophy to a necrotrophic lifestyle. We also identified several novel putative fungal effectors and studied their expression during anthracnose development in maize. Our results demonstrate a strong induction of defense mechanisms occurring in maize cells during C. graminicola infection, even during the biotrophic development of the pathogen. We hypothesize that the switch to necrotrophic growth enables the fungus to evade the effects of the plant immune system and allows for full fungal pathogenicity.

The phytoene dehydrogenase gene of Phycomyces : regulation of its expression by blue light and vitamin A

Abstract By using a polymerase chain reaction based cloning strategy we isolated the gene (carB) encoding the enzyme phytoene dehydrogenase from Phycomyces blakesleeanus. The deduced protein, a 583 residue polypeptide, showed great similarity to carotenoid dehydrogenases from other fungi and bacteria, especially in the amino-terminal region. The main conserved regions found in other phytoene dehydrogenases, which are thought to be essential for the enzymatic activity, are present in the sequence from Phycomyces. Heterologous expression of the Phycomyces gene in Escherichia coli showed that, as in other fungi and bacteria, a single polypeptide catalyzes the four dehydrogenations that convert phytoene to lycopene. RNA measurements indicated that the level of expression of the phytoene dehydrogenase gene in wild-type mycelia increased in response to blue light. The kinetics of this increase in transcription of the gene after blue light induction (0.1 and 0.4 W/m2) exhibit a two-step (biphasic) dependence on fluence rate, suggesting that there could be two separate components involved in the reception of the low and high blue light signal. The presence of vitamin A in the medium stimulated transcript accumulation in the wild type and in some carotenogenic mutant strains. Diphenylamine, a phytoene dehydrogenase inhibitor, did not affect the level of transcription of this gene.

Heterologous transformation of Mucor circinelloides with the Phycomyces blakesleeanus leu1 gene.

SummaryThe leu1 gene of Phycomyces blakesleeanus was isolated within a HindIII-HindIII genomic DNA fragment by heterologous hybridization screening of a cosmid library, making use of the Mucor circinelloides leuA gene as a probe. The complete nucleotide sequence of this fragment reveals a single 2070 bp ORF with no introns, which presents at least 68% homology with that of the leuA gene. The P. blakesleeanus leu1 gene has also been expressed in the M. circinelloides mutant R7B (leu-), which was used to isolate the leuA gene by complementation. The homology with other known sequences shows that the leu1 gene encodes the P. blakesleeanus α-IPM (isopropylmalate) isomerase.

ISOLATION, CHARACTERIZATION AND TRANSFORMATION, BY AUTONOMOUS REPLICATION,OF MUCOR CIRCINELLOIDES OMPDECASE-DEFICIENT MUTANTS

Pyrimidine auxotrophs ofMucor circinelloides were isolated after mutagenesis with nitrosoguanidine and selected for resistance to 5-fluoroorotate. These mutants were genetically and biochemically characterized and found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase) activity or in orotate phosphoribosyl transferase (OPRTase) activity. Different circular DNA molecules containing the homologouspyrG gene were used to transform a representative OMPdecase-deficient strain to uracil prototrophy. Southern analysis, as well as mitotic stability analysis of the transformants, showed that the transforming DNA is always maintained extrachromosomally. The smallest fragment tested that retained both the capacity to complement thepyrG4 mutation and the ability to be maintained extrachromosomally when cloned in a suitable vector is a 1.85 kbM. circinelloides genomic DNA fragment. This fragment consists of thepyrG coding region flanked by 606 nucleotides at the 5′ and 330 nucleotides at the 3′ ends, respectively. Sequence analysis reveals that it does not share any element in common with anotherM. circinelloides genomic DNA fragment which also promotes autonomous replication in this organism, except those related to transcription. Furthermore, it differs from elements which have been shown to be involved in autonomous replication in other fungal systems. An equivalent plasmid harbouring the heterologousPhycomyces blakesleeanus pyrG gene yielded lower transformation rates, but the transforming DNA was also maintained extrachromosomally. Our results suggest that autonomous replication inM. circinelloides may be driven by elements normally present in nuclear coding genes.

ISOLATION AND MOLECULAR ANALYSIS OF THE OROTIDINE-5'-PHOSPHATE DECARBOXYLASE GENE (PYRG) OF PHYCOMYCES BLAKESLEEANUS

SummaryThe pyrG gene of Phycomyces was isolated from a Phycomyces genomic library, constructed in the cosmid pHS255, by hybridization with a 170 bp fragment of the pyrG gene of Aspergillus niger. This fragment includes a consensus sequence found in almost all species in which the orotidine-5′-phosphate decarboxylase (OMPdecase) gene has been sequenced. The complete nucleotide sequence of the cloned pyrG gene from Phycomyces was determined and the transcription start sites mapped. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Saccharomyces cerevisiae, A. niger, Schizophyllum commune and Homo sapiens. Analysis of the sequence revealed the presence of two introns. The precise length and location of these introns was determined by sequencing the pyrG cDNA and comparing it with the genomic clone. Non-coding flanking regions showed obvious homology to the consensus TATA and CAAT boxes, and the polyadenylation signal “AATAAA”. The pyrG gene is the second Phycomyces gene that has been cloned and analysed. This is the first time that introns have been reported in Phycomyces.

Flux of nitric oxide between the necrotrophic pathogen Botrytis cinerea and the host plant

Nitric oxide (NO) production by Botrytis cinerea and the effect of externally supplied NO were studied during saprophytic growth and plant infection. Fluorescence analysis with 4,5-diaminofluorescein diacetate and electrochemical studies were conducted in vitro between 4 and 20 h of incubation and in planta between 15 and 75 h post-inoculation. The production of NO by B. cinerea in vitro was detected inside the germinating spores and mycelium and in the surrounding medium. In planta production of NO showed a large variation that was dependent on the host plant and developmental stage of the infection. The induced production of NO was detected from 16 h of in vitro incubation in response to externally added NO. The production of NO by B. cinerea is probably modulated to promote fungal colonization of the plant tissue. The production of NO which diffuses outside the fungal cells and the induction of NO production by exogenous NO open up the possibility of NO cross-talk between the fungus and the plant. Finally, the existence of an NO concentration threshold is proposed, which may increase or reduce the plant defence against necrotrophic fungal pathogens.

Double-stranded RNA and virus-like particles in the grass endophyte Epichloë festucae

Two double-stranded ribonucleic acid (dsRNA) elements were detected in an isolate of the clavicipitaceous grass endophyte Epichloe festucae obtained from an asymptomatic Festuca rubra plant. No dsRNA was detected in three other isolates of E. festucae. The size of the two dsRNA elements isolated from mycelium was of 5.2 and 3.2 kbp. Hybridization experiments with a cDNA probe complementary to the 5.2 kbp dsRNA showed that there was no sequence similarity between the dsRNA elements. Isometric virus-like particles of about 50 nm in diameter were observed by electron microscopy in sucrose-gradient purified virus preparations from the isolate containing dsRNA. The 5.2 kbp dsRNA element was present in the partially purified virus preparations, suggesting that this element is encapsidated. There was no difference with respect to growth rate or colony morphology between the dsRNA containing and dsRNA-free isolates. The presence of dsRNA and virus-like particles suggests the presence of a mycovirus in the Vit5 strain of E. festucae.

The flavohemoglobin BCFHG1 is the main NO detoxification system and confers protection against nitrosative conditions but is not a virulence factor in the fungal necrotroph Botrytis cinerea.

Flavohemoglobins constitute a group of proteins involved in the metabolism of nitric oxide (NO). Botrytis cinerea was shown to have a single flavohemoglobin coding gene, Bcfhg1. Its expression was developmentally regulated, with maximum expression levels during germination of conidia, and was enhanced very quickly upon exposure to NO of germinating conidia, but not of mycelium growing and branching actively. Expression in planta paralleled the expression pattern during saprophytic growth with maximal expression occurring during the very early stages of the infection process. Bcfhg1 complemented the Saccharomyces cerevisiae yhb1 mutation, indicating that the encoded enzyme has NO dioxygenase activity. Biochemical and functional characterization of DeltaBcfhg1 mutants in comparison with the wild type strain demonstrated that, although BCFHG1 showed a high affinity for its substrate, appeared to represent the main inducible NO detoxification system and conferred protection against nitrosative stress in B. cinerea, the ability of the DeltaBcfhg1 mutant strains to infect different hosts was not affected.

Heterologous expression of the Phycomyces blakesleeanus phytoene dehydrogenase gene (carB) in Mucor circinelloides

Abstract. A phytoene dehydrogenase-deficient mutant of Mucor circinelloides accumulating only phytoene was transformed with the gene encoding the corresponding enzyme (carB gene) of Phycomyces blakesleeanus. Carotenoids derived from phytoene were detected in the transformants showing that the P. blakesleeanus carB gene complements the M. circinelloides carB mutation. These newly formed carotenoids accumulated in low quantities, indicating that functional complementation was poor. carB mRNA molecules correctly transcribed were detected in the transformants, but they represented a small proportion of the total population of carB-derived mRNAs, mostly constituted by truncated transcripts and by transcripts longer than the transcript that is functional in Phycomyces. These results showed that the P. blakesleeanus carB gene was expressed in M. circinelloides and suggested that the poor complementation observed was owing, at least in part, to the lack of specificity in the recognition of the transcription initiation and termination signals of the P. blakesleeanus carB gene by the M. circinelloides transcriptional machinery.