Tracking Fungal Growth: Establishment of Arp1 as a Marker for Polarity Establishment and Active Hyphal Growth in Filamentous Ascomycetes

Abstract

Polar growth is a key characteristic of all filamentous fungi. It allows these eukaryotes to not only effectively explore organic matter but also interact within its own colony, mating partners, and hosts. Therefore, a detailed understanding of the dynamics in polar growth establishment and maintenance is crucial for several fields of fungal research. We developed a new marker protein, the actin-related protein 1 (Arp1) fused to red and green fluorescent proteins, which allows for the tracking of polar axis establishment and active hyphal growth in microscopy approaches. To exclude a probable redundancy with known polarity markers, we compared the localizations of the Spitzenkörper (SPK) and Arp1 using an FM4-64 staining approach. As we show in applications with the coprophilous fungus Sordaria macrospora and the hemibiotrophic plant pathogen Colletotrichum graminicola, the monitoring of Arp1 can be used for detailed studies of hyphal growth dynamics and ascospore germination, the interpretation of chemotropic growth processes, and the tracking of elongating penetration pegs into plant material. Since the Arp1 marker showed the same dynamics in both fungi tested, we believe this marker can be broadly applied in fungal research to study the manifold polar growth processes determining fungal life.

Hyphal Fusions Enable Efficient Nutrient Distribution in Colletotrichum graminicola Conidiation and Symptom Development on Maize

“…Prior to transformation in CgM2, all plasmids were linearized (pCgso_KO: HindIII and NotI, pCgso_c_nat: PvuI, peGFP-Cgatg8_gen: MunI). Protoplasts were obtained from oval conidia of wildtype CgM2 (transformation of pCgso_KO and peGFP-Cgatg8) or ∆Cgso (transformation of pCgso_c_nat) by cell wall digestion using lysis enzyme of Trichoderma harzianum as described previously [33]. To obtain homokaryotic transformants, colonies that had developed on CM plates supplemented with 500 µg/mL hygromycin B (pCgso_KO), 150 µg/mL nourseothricin-dihydrogen sulphate (pCgso_c_nat), and 400 µg/mL geneticin disulphate (peGFP-Cgatg8_gen) were allowed to conidiate on OMA.…”

Maize diterpenoid sensing via Ste3 a-pheromone receptor and rapid germination ofColletotrichum graminicolaoval conidia facilitating root infection

“…(a) oval and falcate conidia of C. graminicola expressing red-labeled Arp1 (CgM2::Arp1-TagRFP-T, 33 ) after 6 h of incubation facing a gradient of maize root exudate (MRE). Germination is indicated by a white asterisk, scale bar = 20 μm.…”

The Small Ras Superfamily GTPase Rho4 of the Maize Anthracnose Fungus Colletotrichum graminicola Is Required for β-1,3-glucan Synthesis, Cell Wall Integrity, and Full Virulence

“…Apical growth and the ability to form infection hyphae with rigid cell walls are pre-requisites for plant invasion and pathogenicity [ 48 ]. To compare invasive growth rates of the WT, Δ rho4 , and ectopic strains of C. graminicola , we employed race tubes ( Figure 5 A) [ 49 ] containing PDA supplemented with 0.15 M KCl and increasing agar concentrations. The highest invasive growth rates of the WT and ectopic strains were measured at agar concentrations of 1–2% ( w / v ), and hyphae of the WT and ectopic strains were even able to invade PDA containing 4% ( w / v ) agar, although at low growth rates ( Figure 5 B).…”