Aims: this PhD thesis has two objectives: (i) The study of GEO1 protein, a new hydrophobin that we identified in the genus Geosmithia; (ii) The study of a hypothetical Horizontal Gene Transfer (HGT) event occurred between Ophiostoma novo-ulmi and Geosmithia spp. Methods and Results: the characterization of the geo1 gene sequence was carried out in Geosmithia pallida species 5 (isolate IVV7), where it consists of 3 exons and 2 introns. The presence of the geo1 gene was analyzed by specific PCR in 90 Geosmithia isolates sampled from elm trees affected by Dutch elm disease (DED) and then on 35 species representing the whole phylogenetic diversity of the genus. The amplification was positive respectively on 58 isolates from elm and on 26 species of the genus, where the gene maintained the general organization shown in IVV7. Results were analyzed by bioinformatic tools like BLAST, ClustalW, MEGA5, Expasy. The variability of the gene is due to differences in nucleotide sequence length and to the presence of a internal tandem repeat (ITR) which corresponds to a stretch of glycine residues in the deduced proteins. Biochemical tests demonstrated that the GEO1 protein belongs to class II hydrophobins. At the amino acid level the deduced proteins had 44,6% homology and no major differences were found in the biochemical parameters (pI, GRAVY index, hydropathy plots). GEO1 production in the fungal culture filtrate was tested by turbidimetric assay and Western blotting. In the same isolates we investigated the presence of the gene encoding the class II hydrophobin cerato-ulmin (cu gene) by PCR. The amplification was successful in 58,8% of the strains isolated from elm trees, while no results were obtained in the other samples. The presence of cu mRNA was also assessed by real time PCR after in IVV7 and then in 7 more species of Geosmithia. The gene showed its maximum expression after 8 days of fungal growth in liquid shaken culture, even if in very low amount. Finally, the induction of cu gene expression was tested in IVV7 grown on elm sawdust and in dual culture with O. novo-ulmi and in both conditions the expression level was increased, even if remaining in all cases extremely low. Conclusions: the phylogenetic analysis based on the geo1 sequences did not correspond to the tree generated with a neutral marker. This suggests that sequence similarities could be influenced by other factors than phylogenetic relatedness, for example the close relationship of symbiosis between some Geosmithia species and their insect vectors. The exclusive presence of the cu gene in isolates derived from elm trees could confirm its recent horizontal gene transfer from O. novo-ulmi. Significance and Impact of the Study: geo1 gene occurred in the whole genetic diversity of Geosmithia spp. Because some hydrophobins are involved in fungal pathogenesis inducing the plant defence response, it would be interesting to continue the studies on GEO1 to verify if different isoforms of the protein could act like elicitors. Geosmithia spp. and O. novo-ulmi share the same habitat, the larval galleries built by their insect vectors, which represent an authentic microbiota. Here, numerous interactions between the different organisms take place. A possible mechanism of cu gene horizontal transfer is the hyphal anastomosis formation and to date are in progress experiments to validate this hypothesis.

Biochemical and genetical identification and characterization of hydrophobins in Geosmithia spp / A.Frascella. - (2014).

Biochemical and genetical identification and characterization of hydrophobins in Geosmithia spp.

FRASCELLA, ARCANGELA
2014

Abstract

Aims: this PhD thesis has two objectives: (i) The study of GEO1 protein, a new hydrophobin that we identified in the genus Geosmithia; (ii) The study of a hypothetical Horizontal Gene Transfer (HGT) event occurred between Ophiostoma novo-ulmi and Geosmithia spp. Methods and Results: the characterization of the geo1 gene sequence was carried out in Geosmithia pallida species 5 (isolate IVV7), where it consists of 3 exons and 2 introns. The presence of the geo1 gene was analyzed by specific PCR in 90 Geosmithia isolates sampled from elm trees affected by Dutch elm disease (DED) and then on 35 species representing the whole phylogenetic diversity of the genus. The amplification was positive respectively on 58 isolates from elm and on 26 species of the genus, where the gene maintained the general organization shown in IVV7. Results were analyzed by bioinformatic tools like BLAST, ClustalW, MEGA5, Expasy. The variability of the gene is due to differences in nucleotide sequence length and to the presence of a internal tandem repeat (ITR) which corresponds to a stretch of glycine residues in the deduced proteins. Biochemical tests demonstrated that the GEO1 protein belongs to class II hydrophobins. At the amino acid level the deduced proteins had 44,6% homology and no major differences were found in the biochemical parameters (pI, GRAVY index, hydropathy plots). GEO1 production in the fungal culture filtrate was tested by turbidimetric assay and Western blotting. In the same isolates we investigated the presence of the gene encoding the class II hydrophobin cerato-ulmin (cu gene) by PCR. The amplification was successful in 58,8% of the strains isolated from elm trees, while no results were obtained in the other samples. The presence of cu mRNA was also assessed by real time PCR after in IVV7 and then in 7 more species of Geosmithia. The gene showed its maximum expression after 8 days of fungal growth in liquid shaken culture, even if in very low amount. Finally, the induction of cu gene expression was tested in IVV7 grown on elm sawdust and in dual culture with O. novo-ulmi and in both conditions the expression level was increased, even if remaining in all cases extremely low. Conclusions: the phylogenetic analysis based on the geo1 sequences did not correspond to the tree generated with a neutral marker. This suggests that sequence similarities could be influenced by other factors than phylogenetic relatedness, for example the close relationship of symbiosis between some Geosmithia species and their insect vectors. The exclusive presence of the cu gene in isolates derived from elm trees could confirm its recent horizontal gene transfer from O. novo-ulmi. Significance and Impact of the Study: geo1 gene occurred in the whole genetic diversity of Geosmithia spp. Because some hydrophobins are involved in fungal pathogenesis inducing the plant defence response, it would be interesting to continue the studies on GEO1 to verify if different isoforms of the protein could act like elicitors. Geosmithia spp. and O. novo-ulmi share the same habitat, the larval galleries built by their insect vectors, which represent an authentic microbiota. Here, numerous interactions between the different organisms take place. A possible mechanism of cu gene horizontal transfer is the hyphal anastomosis formation and to date are in progress experiments to validate this hypothesis.
2014
P.P.Bettini
ITALIA
A.Frascella
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/851896
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