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

Abstract

Hyphal and germling fusion is a common phenomenon in ascomycetous fungi. Due to the formed hyphal network, this process enables a coordinated development as well as an interaction with plant hosts and efficient nutrient distribution. Recently, our laboratory work demonstrated a positive correlation between germling fusion and the formation of penetrating hyphopodia on maize leaves outgoing from Colletotrichum graminicola oval conidia. To investigate the probable interconnectivity of these processes, we generated a deletion mutant in Cgso, in which homologs are essential for cellular fusion in other fungal species. However, hyphopodia development was not affected, indicating that both processes are not directly connected. Instead, we were able to link the cellular fusion defect in ∆Cgso to a decreased formation of asexual fruiting bodies of C. graminicola on the leaves. The monitoring of a fluorescent-labelled autophagy marker, eGFP-CgAtg8, revealed a high autophagy activity in the hyphae surrounding the acervuli. These results support the hypothesis that the efficient nutrient transport of degraded cellular material by hyphal fusions enables proper acervuli maturation and, therefore, symptom development on the leaves.

Keywords: Colletotrichum graminicola; autophagy; conidiation; falcate conidia; germling fusion; oval conidia.

Figure 1

Hyphopodia and cellular fusion formation by oval conidia-derived germlings. Inoculation of secondary leaves obtained from 16 d old Z. mays (cv Micado) plants with 10³ and 10² oval conidia of the depicted strains. In addition, 1 dpi, the infection process was stopped. Leaves were parted in four and de-colorized in 100% EtOH for 3 d. (a) overview of typical representation of colony and infection structure development with an inoculum of 10³ at 1 dpi, hp = hyphopodia, scale bar = 10 μm; (b) enlarged depiction of the indicated area of (a), indicating presence or absence of hyphal fusions (black arrow heads), oc = oval conidia, hp = hyphopodia, scale bar = 10 μm; (c,d) quantification of cellular fusions (c) or hyphopodia (d) formed by germlings derived from oval conidia in one inoculation spot. Error bars represent SD calculated from ≥3 experiments, * p < 0.05.

Figure 2

Z. mays leaf infection. Second leaves of 16 d old Z. mays plants (cv Mikado) were inoculated with droplets C. graminicola conidia containing 10³ conidia. Typical appearance of symptoms on intact leaves is depicted after incubation with falcate (fc) or oval (oc) conidia for 5 d. Arrows indicate development of acervuli along vascular bundles, scale bar = 1 mm.

Figure 3

Generation of falcate conidia in C. graminicola strains. C. graminicola CgM2 (wildtype), ΔCgso deletion strain as well as ΔCgso with a re-integrated Cgso gene including native 5’ and 3’ regions (ΔCgso_c) were incubated for 21 d on oatmeal agar (OMA) plates at 23 °C. (a) plate overview, scale bar = 1 cm; (b) cross section, scale bar = 1 mm, ac = acervuli, ah = aerial hyphae; (c) quantification of falcate conidia per plate. Values are depicted in a logarithmic scale, error bars represent SD calculated from 6 experiments, * p < 0.05.

Figure 4

Development of young acervuli in a Cgso deletion strain. C. graminicola CgM2 (wildtype), ΔCgso deletion strain as well as the corresponding complementation strain ΔCgso_c were analyzed for the development of asexual fruiting bodies, the acervuli. Depicted strains were inoculated on microscopic slides covered with reduced oat meal agar (OMA_red) for 5 d, 23 °C, ah = aerial hyphae, s = setae, hyphal fusions are indicated with black arrow heads, empty hyphal compartments with white arrow heads, scale bar = 10 μm. (a) overview of acervuli forming regions; (b) Z-projections of stack images of hyphae with developing setae and conidiophores (levels of 1 μm); (c) total numbers of hyphal fusions on hyphae, which show (ac) or do not show (non-ac) developing acervuli. Error bars represent SD calculated from 30 experiments, * p < 0.05.

Figure 5

Autophagy in developing acervuli and model. (a) quantification of falcate conidia after growth on complex medium (CM) for 21 d, 23 °C. Values are depicted in a logarithmic scale, error bars represent SD calculated from six experiments, ns, p > 0.05; (b) C. graminicola wildtype strain CgM2 and CgM2::eGFP-Cgatg8 expressing green fluorescent autophagy marker CgAtg8 were inoculated on microscopic slides covered with reduced oat meal agar (OMA_red) for 5 d, 23 °C. Selected layers from acervuli recordings with a fixed distance of 1 μm are depicted for each strain. In CgM2, falcate conidia appear green due to autofluorescence, s = setae, c = conidiophores, fc = falcate conidia, hyphal fusions are indicated with black arrow heads, scale bar = 10 μm; (c) optimized distribution of autophagy-recycled cellular components by hyphal fusion bridges allows for proper acervulus maturation and falcate conidia production in C. graminicola.