Identification of genes differentially expressed during the interaction between Trichoderma Harzianum and Rhizoctonia solani by Rapid Subtraction Hybridization

To find key genes of T. harzianum expressed during the antagonistic interaction with Rhizoctonia solani a cDNA subtraction library technique (Rapid Subtraction Hybridization, RaSH) was used. cDNA from T. harzianum grown on complete medium was subtracted from cDNA derived from T. harzianum grown as antagonist in confrontation with R. solani. Differential gene expression of 200 clones was verified by Reverse Northern blot analysis. About 50 clones were sequenced and estimated for similarities by BLAST search of the JGI T. reesei genome database (http://genome.jgi-psf.org/Trire2/Trire2.home.html). A total of 13 fragments showed no hits on the databases; for 37 gene fragments, representing 25 different genes, corresponding counterpart sequences were found with similarities to T. reesei. The 14 most interesting genes were characterised for their differential expression patterns during the time course of interaction between T. harzianum and R. solani by Northern Blot analysis. Four genes seem to be significantly upregulated during antagonism between T. harzianum and R. solani. These include a gene similar to the endoglucanase Cel61b of H. jecorina, a dehydrogenase with similarity to the Fusarium NUBMNEUCR NADH ubiquinone oxydoreductase, a gene similar to T. reesei AXE1 (acetyl xylan esterase) and genes involved in general sugar transport. Present research focuses on the characterisation of the complete gene sequences and their regulatory targets in order to get a detailed knowledge of the gene role in metabolism during the antagonistic phase of Trichoderma. These data will permit the development of new molecular markers for a rapid and efficient identification of new biocontrol agents.Work funded by the Italian Ministry of Foreign Affairs, Buero fuer Akademische Kooperation und Mobilitaet des Oesterreichischen Austauschdienstes, Fondazione Banco di Sardegna, University of Sassari.