Genetic tools for transformation of Xyleborus ambrosia beetle fungal symbionts

Ambrosia beetles rely on obligate fungal partners for survival, and these associations are emerging as model systems for examining the development and evolution of fungal-animal mutualism. However, genetic tools for the manipulation of such mutualistic fungi remain largely lacking, and consequently the genetic basis employed by these fungi to establish associations with their beetle partners remains almost completely unexplored. Here, we provide methods for protoplast generation and transformation of several major filamentous fungal partners of Xyleborus ambrosia beetles including Raffaela arxii , R. fusca , Harringtonia aguacate , and Graphium ambrosium using reporter constructs driven by the H. lauricola gpd promoter sequence. In addition, we developed inducible expression systems for the beetle symbiont, but plant pathogen, H. lauricola , responsible for laurel wilt disease, and show that these constructs are also functional in G. ambrosium . This work demonstrates the utility of H. lauricola genetic parts in transforming diverse groups of fungi and offers a toolbox for their genetic dissection. Fungal strains showed sensitivity to hygromycin, and protoplasts derived from the fungi were transformed with plasmids expressing the hygromycin selective marker ( HPH ) along with either green- or red-fluorescent proteins (GFP/RFP), with expression driven by the constitutive H. lauricola gpd promoter. Transformed strains for the fungal species described above showing expression of either GFP or RFP were obtained. A series of inducible plasmids using the H. lauricola alcA p - RFP -(ethanol) and glaA p - RFP -(maltose) inducible promoters were also tested and validated. Our data provide methods and tools for genetic manipulation of a wide range of ambrosia beetle fungal symbionts, including marking cells with fluorescent proteins and use of inducible expression systems. Intriguingly, the method/plasmids developed did not result in transformation of the related Xyleborus fungal symbiont, Neocosmospora affinis . The fluorescent strains developed can be used to monitor symbiotic colonization of the beetle host, fungal development in host galleries, host preferences, and a range of other applications, and promoters may be used for further knockout and expression studies.