Ras2 is important for growth and pathogenicity in Fusarium circinatum
Phasha, Mmatshepho Malekgale; Wingfield, Michael J.; Wingfield, Brenda D.; Coetzee, Martin Petrus Albertus; Hallen-Adams, H.; Fru, Felix F.; Swalarsk-Parry, Benedicta S.; Yilmaz, Neriman; Duong, Tuan A.; Steenkamp, Emma Theodora
In this study, we investigated to possible role of Ras2 in Fusarium circinatum- a fungus that causes pine pitch canker disease on many different pine species and has a wide geographic distribution. This protein is encoded by the RAS2 gene and has been shown to control growth and pathogenicity in a number of fungi in a mitogen-activated protein kinase- and/or cyclic adenosyl monophosphate pathway-dependent manner. The aim was therefore to characterize the phenotypes of RAS2 gene knockout and complementation mutants of F. circinatum. These mutants were generated by transforming protoplasts of the fungus with suitable split-marker constructs. The mutant strains, together with the wild type strain, were used in growth studies as well as pathogenicity assays on Pinus patula seedlings. Results showed that the knockout mutant strain produced significantly smaller lesions compared to the complementation mutant and wild type strains. Growth studies also showed significantly smaller colonies and delayed conidial germination in the knockout mutant strain compared to the complement mutant and wild type strains. Interestingly, the knockout mutant strain produced more macroconidia than the wild type strain. Collectively, these results showed that Ras2 plays an important role in both growth and pathogenicity of F. circinatum. Future studies will seek to determine the pathway(s) through which Ras2 controls these traits in F. circinatum.
Supplementary data 1. Gene knockout and transformation protocol for Fusarium circinatum using split-marker constructs.
Supplementary data 2. Maximum likelihood phylogeny for the RAS2 gene from selected Fusarium and closely related fungal species. Bootstrap values above 60% are shown at the nodes, and the scale bar shows substitutions per site. The tree was rooted using Aspergillus nidulans and Alternaria burnsii as outgroup species. GenBank genome assembly numbers are shown in brackets.