Trichoderma harzianum is a biological control agent applied against plant pathogenic fungi causing economical losses worldwide. We are using proteomics to identify key proteins involved in host recognition and biological control in a strain of T. harzianum with well-established biocontrol properties. Isolation of the corresponding genes will become a vital tool for developing genetically improved strains. One of the challenges with filamentous fungi is the solubilisation of proteins especially from the fungal cell envelope. We have found that an alkaline extraction pH does not favour the solubility of alkaline proteins, because they are almost uncharged at the extraction solution pH. Therefore, we have developed a method of treating a sample to extract proteins under acidic conditions. The method increases the solubilisation of alkaline proteins and eliminates acidic cell wall artifacts from microorganisms in general. Using this technique, combined with the use of protease inhibitors during sample preparation, thousands of proteins from T. harzianum have been extracted and separated using two-dimensional gel electrophoresis (2D PAGE). These proteins were identified using a combination of MALDI MS (matrix assisted laser desorption ionization mass spectrometry) and LC MS/MS (liquid chromatography mass spectrometry). Manual de novo sequencing was conducted to obtain sequence tags on unidentified proteins. Using these techniques, 24 protein spots were positively identified derived from 17 gene products.
|Number of pages||1|
|Publication status||Published - 2003|
|Event||XXII Fungal Genetics Conference - Asilomar, California, USA|
Duration: 18 Mar 2003 → 23 Mar 2003
|Conference||XXII Fungal Genetics Conference|
|City||Asilomar, California, USA|
|Period||18/03/03 → 23/03/03|