A GFP-based assay to quantify the impact of effectors on the ex planta development of the slowly growing barley pathogenRhynchosporium commune

Abstract

A growth assay was established for the barley pathogen Rhynchosporium commune with EGFP-tagged fungal mutants. This assay was used to study the effect of four antibiotics (hygromycin B, nourseothricin, kanamycin, phleomycin) and a herbicide (phosphinothricin) on fungal development. Fitting the growth curves to the modified Gompertz model allowed calculating growth parameters, such as lag periods of fungal colony formation and mycelial growth rates as well as EC(50) values. Except kanamycin all compounds were efficient inhibitors so that the corresponding resistance-conferring genes can be used as markers for selection of fungal transformation-based mutants. In addition the assay was used to quantify the inhibitory activity of a barley secondary metabolite, the indole alkaloid gramine.

PFP1 , a Gene Encoding an Epc-N Domain-Containing Protein, Is Essential for Pathogenicity of the Barley Pathogen Rhynchosporium commune

“…Hyphal morphology is wild type-like, except for the occurrence of chlamydospore-like structures/hyphal swellings. Similar swellings were described previously for fungal mycelia grown in the presence of gramine and were interpreted as a stress response (11). In contrast to the only minor growth deviation observed ex planta, the deletion mutant differs drastically from the wild type in planta.…”

“…After 14 and 21 days, the media were removed by filtration and the mycelia were lyophilized and weighed. To estimate fungal growth on solid lima bean agar (11), colonies were initiated with 5,000 conidia on agar-coated microscope slides and grown at 17°C in the dark. The germination frequencies at 24 and 48 h and the number of germ tubes at 48 h were counted under a microscope (AxioVision LE; Carl Zeiss MicroImaging, Jena, Germany) using 3 groups of 100 spores each in 4 biological replicates.…”

“…Culture of fungal isolate UK7 and of susceptible barley cultivar 'Ingrid' and inoculations was as described previously (9,11,36).…”

“…Initially, thin hyphae with distantly spaced septae grow predominantly in the pectinrich layer of the outer epidermis cell walls (7). Later, thick shortseptate hyphae appear, and they eventually form a dense stroma that supports the formation of new spores (8)(9)(10)(11). The typical disease symptoms, necrotic lesions, occur after a latent period lasting from a few days up to several weeks, when mesophyll cells in leaf regions that are heavily colonized by the fungus collapse (12,13).…”

Rhynchosporium commune: a persistent threat to barley cultivation

“…More recently, Agrobacterium tumefaciens-mediated transformation (ATMT) was used to generate R. commune transformants expressing the green fluorescent protein (GFP) or DsRed fluorescent protein (Kirsten et al, 2011;Linsell et al, 2011;Thirugnanasambandam et al, 2011). These transformants were utilized to compare R. commune growth using confocal microscopy in both the susceptible and resistant cultivars.…”