Study of two different hydrophobins in Geosmithia spp.

The genus Geosmithia includes several fungal species associated with phloem-feeding bark beetles. In previous studies, we showed that the gene encoding cerato-ulmin (cu) in Ophiostoma novo-ulmi was also present in a Geosmithia isolate obtained from an elm tree affected by Dutch Elm Disease (DED). To explain this result, we hypothesized a horizontal gene transfer (HGT). Thirty-six isolates of Geosmithia, collected from elm trees with DED symptoms, were used in this work to verify if other Geosmithiae contained the cu gene. Sequencing of the PCR products obtained with different primer pairs designed on the cu gene sequence revealed that fragments highly homologous to the cu gene were present in all the analyzed Geosmithia isolates. Culture filtrates of 16 Geosmithia isolates gave a positive response in ELISA assays using anti-CU antibodies; however, Western blotting and mass spectrometry analyses showed that the MW of the protein was higher than that expected for CU. This protein was purified and characterized. Applying Edmann sequencing and Genome Walking techniques, we obtained a partial nucleotide sequence of the gene and an amino acid sequence of the protein, that were different from those known for CU. Our results show that isolates of Geosmithia spp. harbour two different hydrophobin genes: one, homologous to the cu gene from O. novo-ulmi, could have been acquired by HGT between the two fungal species, as they occupy the same habitat in elm trees. The other gene codes for a new hydrophobin, named Geo1, with a good homology level to the O. novo-ulmi CU protein.