Comparative determination of cerato-ulmin on cell surface and in mycelial extracts of pathogenic and non-pathogenic Ophiostoma species

Scala, Felice; Bertelli, Emanuela; Coppola, L.; Sorbo, G. Del; Tegli, Stefania; Scala, Aniello

doi:10.1017/s0953756296003358

Cited by 12 publications

(11 citation statements)

References 19 publications

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“…CU is known to be produced by these pathogens at different levels in liquid cultures (Scheffer et al 1987). Besides being secreted in cultures, CU is also found in the cell wall, on the cell surface and in mycelial extracts of O. novo -ulmi and O. ulmi (Takai and Hiratsuka 1980;Svircev et al 1988;Scala et al 1997). It has been hypothesized that the expression of the single copy cu gene coding for this protein is controlled at the level of transcription (Jeng et al 1996;Pipe et al 1997).…”

Section: Discussionmentioning

confidence: 99%

“…This may explain the abundant secretion of cerato-ulmin during pathogenesis and its indirect role in pathogenicity. O. ulmi and O. novo-ulmi are dimorphic fungi that are capable of growing as either yeast or mycelium, and CU is produced in both developmental stages (Takai and Hiratuska 1980;Puhalla and Bell 1981;Scala et al 1997). The data produced to date would suggest that the yeastlike stage is involved in disease dissemination.…”

Section: Introductionmentioning

confidence: 99%

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Real time RT-PCR quantification and Northern analysis of cerato-ulmin ( CU) gene transcription in different strains of the phytopathogens Ophiostoma ulmi and O. novo-ulmi

et al. 2003

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Cerato-ulmin is a surface protein that belongs to the class of fungal proteins known as hydrophobins. This class II hydrophobin is produced throughout the life cycle and in all developmental stages of Ophiostoma novo-ulmi and O. ulmi; the aggressive and non-aggressive pathogens responsible for Dutch elm disease. Since yeast/mycelial transitions are often important to pathogenesis in dimorphic fungi such as Ophiostoma, we have examined the levels and abundance of cu mRNA in the yeast and mycelial stages of this fungus. The fungus contains one copy of the cu gene per haploid genome, located on chromosome IV. Our studies have been done using phosphoimager-based Northern analysis and real-time quantitative RT-PCR (qRT-PCR) to measure levels of cu mRNA. These measurements were made in both yeast-like and mycelial stages of the pathogen. Two wild-type, aggressive, strains of O. novo-ulmi (VA30 and H327) and one wild type non-aggressive strain of O. ulmi (H5) were analysed. As controls, we have utilized two types of mutants that we had previously generated, the null cu mutants THEK5-8 and THEK5-8-1, and a cu over-expression mutant, H5-tf16. Data generated by both Northern hybridization and real-time qRT-PCR analyses demonstrate that there is no cu mRNA transcription in the null mutants. The Northern analysis clearly showed that the over-expressing mutant H5-tf16 produces much more cu mRNA than the non-aggressive or aggressive strains. The quantitative data generated using qRT-PCR demonstrated that mycelium generally had 20-60% more cu mRNA than the yeast form. The non-aggressive strain of O. ulmi (H5) produces one-tenth as much cu mRNA as the aggressive strains (VA30 and H327). When transformed with additional copies of the cu gene, this same non-aggressive strain (H5-tf16) expressed about 20 times more cu mRNA than the wild type H5 strain. These data were consistently generated in multiple real-time qRT-PCR experiments with different RNA preparations, clearly demonstrating that the quantitative abundance values obtained were reproducible. Our study represents the first report on the use of real-time qRT-PCR to compare and quantify gene transcription in different growth phases of a fungal pathogen.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Real time RT-PCR quantification and Northern analysis of cerato-ulmin ( CU) gene transcription in different strains of the phytopathogens Ophiostoma ulmi and O. novo-ulmi

et al. 2003

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“…No explanation is as yet available for this observation, even if Scala et al (1997) suggested that it could depend on differences in the regulation of CU synthesis and in sorting and secretion mechanisms between the Ophiostoma species.…”

Section: Analysis Of the Deduced Geo1 Proteins In Geosmithia Speciesmentioning

confidence: 94%

“…Also species in the genus Ophiostoma showed a different behaviour with respect to the secretion of the class II hydrophobin cerato-ulmin (CU) in the culture medium: Ophiostoma novo-ulmi and Ophiostoma himal-ulmi secreted high amounts of CU, Ophiostoma ulmi nil or low amount, while no CU was detected in Ophiostoma quercus culture medium (Brasier & Mehrotra 1995;Scala et al 1997). No explanation is as yet available for this observation, even if Scala et al (1997) suggested that it could depend on differences in the regulation of CU synthesis and in sorting and secretion mechanisms between the Ophiostoma species.…”

Section: Analysis Of the Deduced Geo1 Proteins In Geosmithia Speciesmentioning

confidence: 99%

Interspecific variability of class II hydrophobin GEO1 in the genus Geosmithia

et al. 2014

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a b s t r a c tThe genus Geosmithia Pitt (Ascomycota: Hypocreales) comprises cosmopolite fungi living in the galleries built by phloeophagous insects. Following the characterization in Geosmithia species 5 of the class II hydrophobin GEO1 and of the corresponding gene, the presence of the geo1 gene was investigated in 26 strains derived from different host plants and geographic locations and representing the whole phylogenetic diversity of the genus. The geo1 gene was de-tected in all the species tested where it maintained the general organization shown in Geosmithia species 5, comprising three exons and two introns. Size variations were found in both introns and in the first exon, the latter being due to the presence of an intragenic tandem repeat sequence corresponding to a stretch of glycine residues in the deduced proteins.At the amino acid level the deduced proteins had 44.6 % identity and no major differences in the biochemical parameters (pI, GRAVY index, hydropathy plots) were found. GEO1 release in the fungal culture medium was also assessed by turbidimetric assay and SDS-PAGE, and showed high variability between species. The phylogeny based on the geo1 sequences did not correspond to that generated from a neutral marker (ITS rDNA), suggesting that sequence similarities could be influenced by other factors than phylogenetic relatedness, such as the intimacy of the symbiosis with insect vectors. The hypothesis of a strong selection pressure on the geo1 gene was sustained by the low values (<1) of non synonymous to synonymous nucleotide substitutions ratios (Ka/Ks), which suggest that purifying selection might act on this gene. These results are compatible with either a birth-and-death evolution scenario or horizontal transfer of the gene between Geosmithia species.

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“…Cerato-ulmin (CU) is a class II hydrophobin of about 8 kDa present on the cell surface and/or excreted in culture by the phytopathogenic fungi Ophiostoma ulmi, O. novo-ulmi, and Ophiostoma himal-ulmi, and by the nonpathogen Ophiostoma quercus (Svircev et al 1988;Scala et al 1997;Carresi et al 2008). Ophiostoma ulmi and O. novo-ulmi are responsible, respectively, for the first and the second DED pandemics that decimated elm populations in Europe, North America, and Asia since the last century.…”

Section: Introductionmentioning

confidence: 99%

Widespread horizontal transfer of the cerato-ulmin gene between Ophiostoma novo-ulmi and Geosmithia species

et al. 2014

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a b s t r a c tPrevious work had shown that a sequence homologous to the gene encoding class II hydrophobin cerato-ulmin from the fungus Ophiostoma novo-ulmi, the causal agent of Dutch Elm Disease (DED), was present in a strain of the unrelated species Geosmithia species 5 (Ascomycota: Hypocreales) isolated from Ulmus minor affected by DED. As both fungi occupy the same habitat, even if different ecological niches, the occurrence of horizontal gene transfer was proposed. In the present work we have analysed for the presence of the cerato-ulmin gene 70 Geosmithia strains representing 29 species, isolated from different host plants and geographic locations. The gene was found in 52.1 % of the strains derived from elm trees, while none of those isolated from nonelms possessed it. The expression of the gene in Geosmithia was also assessed by real time PCR in different growth conditions (liquid culture, solid culture, elm sawdust, dual culture with O. novo-ulmi), and was found to be extremely low in all conditions tested. On the basis of these results we propose that the cerato-ulmin gene is not functional in Geosmithia, but can be considered instead a marker of more extensive transfers of genetic material as shown in other fungi.

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Comparative determination of cerato-ulmin on cell surface and in mycelial extracts of pathogenic and non-pathogenic Ophiostoma species

Cited by 12 publications

References 19 publications

Real time RT-PCR quantification and Northern analysis of cerato-ulmin ( CU) gene transcription in different strains of the phytopathogens Ophiostoma ulmi and O. novo-ulmi

Real time RT-PCR quantification and Northern analysis of cerato-ulmin ( CU) gene transcription in different strains of the phytopathogens Ophiostoma ulmi and O. novo-ulmi

Interspecific variability of class II hydrophobin GEO1 in the genus Geosmithia

Widespread horizontal transfer of the cerato-ulmin gene between Ophiostoma novo-ulmi and Geosmithia species

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