RECOVERY OF A COLLETOTRICHUM ACUTATUM MUTANT WITH REDUCED PATHGENICITY BY TRANSPOSON TAGGING

In this study, impala, a DNA transposable element isolated from the phytopathogenic fungus Fusarium oxysporum, was shown to be active in Colletotrichum acutatum, the causal agent of olive anthracnose. A C. acutatum mutant impaired in nitrate reductase function was co-transformed with the plasmids pNI160, carrying the nidDr::impala construction, and pAN7.1, that confers resistance to hygromycin. Seventy six hygromycin-resistant transformants were obtained and among them 55 impala-transformants were selected by PCR with impala-spedfic primers. The first evidence of impala transposition was obtained by selecting niaD super(+) revertants by the phenotypic excison assay. Two out of three co-transformants analysed gave rise to niaD super(+) revertants. Transposon excision from the niaD gene and its reinsertion in a new genomic site in revertants were shown by Southern blotting analysis. Further evidence of impala transposition was obtained by sequencing the typical transposon footprints left at the niaD site of excision. Twenty four mono-conidic revertant strains were tested for their pathogenicity and a mutant with reduced virulence on olive drupes was recovered. Isolation and analysis of impala flanking regions in this mutant revealed that the transposon was inserted in a coding region containing a conserved domain of the kinesin family, motor proteins associated with microtubules, thus suggesting a role of this putative kinesin in C. acutatum pathogenicity. Moreover, the mutant impaired in pathogenicity showed a peculiar morphology of condia, possibly as a a consequence of altered cell division. This is in agreement with previous studies indicating that kinesin mutations can affect cell division in filamentous fungi.