A conserved fungal glycosyltransferase facilitates pathogenesis of plants by enabling hyphal growth on solid surfaces
The F. graminearum gene FGRRES_00702 (http://fungi.ensembl.org/Fusarium_graminearum/Info/Index) orthologous to ZtGT2 was functionally characterized by gene deletion using a selection step in liquid rather than solid medium. (A) Genomic left border and right border regions (white bars) were amplified with primers and fused to parts of the hph hygromycin resistance gene. Fused PCR fragments were used in a split-marker strategy to replace FGRRES_00702. Horizontal black bars represent genomic areas outside the replacement construct, vertical black bars represent 3 introns. (B) Anticipated diagnostic PCR for successful gene replacement of FGRRES_00702. (C) Results of diagnostic PCR and expected sizes indicated in (A) and (B). Loadings are: M—λ DNA-BstEII digest, 1–5 transformants MU424 to MU428, WT, no DNA. MU426 and MU427 have the ΔFGRRES_00702 null allele and lost the 1.0 kb GT2 fragment. (D) Severely reduced growth for the FgGT2 null allele mutants on the surface of potato dextrose agar plates after 8 days incubation compared to the wild-type PH-1 strain. (E) Wheat ears inoculated with FgGT2 null mutant and wild-type strain 13 days post-inoculation. Spore-droplet inoculated spikelets are marked with black dots. For the mock inoculation, only water was used.