The fungal pathogen,Raffaelea lauricola, is the causative agent of laurel wilt, a devastating disease affecting theLauraceaefamily. The fungus is vectored by ambrosia beetles that carry the fungus in specialized structures (mycangia), with the fungus acting as a symbiont and food source for beetle larvae growing in tree galleries. In order to probe the molecular basis for plant pathogenicity and insect symbiosis of the laurel wilt fungus, molecular tools including establishment of efficient transformation protocols are required. Resistance marker profiling revealed susceptibility ofR. lauricolato phosphinothricin, chlorimuron ethyl, hygromycin, and benomyl.Agrobacterium-mediated transformation using either thebarorsurmarker resulted in 1-200 transformants/10(5)spores. A second protocol using lithium acetate-polyethylene glycol (LiAc-PEG) treatment of fungal blastospores yielded 5-60 transformants/mu g DNA/10(8)cells. Transformants were mitotically stable (at least 5 generations), and > 95% of transformants showed a single integration event.R. lauricolastrains expressing green and red fluorescent proteins (EGFP and RFP), as well as glucuronidase (GUS), were constructed. Using theAgrobacterium-mediated method, a random T-DNA insertion library was constructed, and genetic screens led to the isolation of developmental mutants as well as mutants displaying enhanced resistance to sodium dodecyl sulfate (SDS) or fluconazole, and those showing decreased susceptibility to biphenol. These results establish simple and reliable genetic tools for transformation ofR. lauricolaneeded for genetic dissection of the symbiotic and virulent lifestyles exhibited by this fungus and establish a library of insertion mutants that can be used in various genetic screens to dissect molecular pathways.