The ZtvelB Gene Is Required for Vegetative Growth and Sporulation in the Wheat Pathogen Zymoseptoria tritici

Abstract

The ascomycete fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB), a major disease of wheat across Europe. Current understanding of the genetic components and the environmental cues which influence development and pathogenicity of this fungus is limited. The velvet B gene, velB, has conserved roles in development, secondary metabolism, and pathogenicity across fungi. The function of this gene is best characterised in the model ascomycete fungus Aspergillus nidulans, where it is involved in co-ordinating the light response with downstream processes. There is limited knowledge of the role of light in Z. tritici, and of the molecular mechanisms underpinning the light response. We show that Z. tritici is able to detect light, and that the vegetative morphology of this fungus is influenced by light conditions. We also identify and characterise the Z. tritici velB gene, ZtvelB, by gene disruption. The ΔztvelB deletion mutants were fixed in a filamentous growth pattern and are unable to form yeast-like vegetative cells. Their morphology was similar under light and dark conditions, showing an impairment in light-responsive growth. In addition, the ΔztvelB mutants produced abnormal pycnidia that were impaired in macropycnidiospore production but could still produce viable infectious micropycnidiospores. Our results show that ZtvelB is required for yeast-like growth and asexual sporulation in Z. tritici, and we provide evidence for a role of ZtvelB in integrating light perception and developmental regulation in this important plant pathogenic fungus.

Keywords: Aspergillus nidulans; Septoria Tritici Blotch; Zymoseptoria tritici; ascomycete; light; velvet.

FIGURE 1

(A) Zymoseptoria tritici IPO323 vegetative growth on PDA, YPDA, and CDV8 agar at 20°C, under LD or DD conditions, at 10 days post-inoculation (dpi). Cultures grown under LD develop as yeast-like cells which start to melanise after 7–10 dpi Under DD conditions, the Z. tritici cultures melanise and form grey and white aerial hyphae. (B) Neighbour-joining phylogenetic trees of A. nidulans velvet proteins and their relatedness to potential Z. tritici homologues. Bar represents 0.5 amino acid substitutions per site. Blue boxes indicate the candidate orthologues identified in Z. tritici. The previously described Z. tritici MVE-1 protein (Choi and Goodwin, 2011) was identified as a close relative to the A. nidulans VeA protein. Z. tritici protein ID numbers 103692, 86705, and 108742 were identified as potential homologues for the A. nidulans VelC, VelB, and VosA proteins, respectively. (C) Clustal Omega alignment of velB protein sequences from Z. tritici (ZT), A. nidulans (AN), M. oryzae (MO), B. cinerea (Bc), and V. mali (Vm). The characteristic velvet superfamily domains are highlighted in yellow, asterisks identify identical residues, colons identify conserved residues and periods identify semi-conserved residues.

FIGURE 2

Comparison of vegetative growth of the Z. tritici IPO323 and the ΔztvelB mutant strains. Images representative of three independent ΔztvelB strains tested across three independent experiments. (A–D) Z. tritici IPO323 strain and ΔztvelB mutant growth in PDB at 20°C on a rotary shaker at 200 rpm, 7 days post-inoculation (dpi) stained with lactophenol cotton blue. (A,B) The IPO323 strain forms a cloudy suspension composed of short yeast-like cells and branched hyphal cells. (C,D) The ΔztvelB mutants form mycelial clumps composed of branched hyphal cells and no yeast-like cells. (E) IPO323 and ΔztvelB mutant growth on solid media at 20°C under LD conditions, at 7 dpi. The IPO323 strain grows as yeast-like cells which melanise on PDA and CDV8. The ΔztvelB mutants only grow as grey and white aerial hyphae from a melanised base. (F) IPO323 and ΔztvelB mutant growth under LD or DD conditions at 10 dpi. Under LD, the IPO323 colonies melanise and produce grey and white aerial hyphae by 10 dpi. (G) Under DD the IPO323 strain melanises and forms grey aerial hyphae on PDA. On YPDA, DD colonies produce fewer aerial hyphae than under LD. On CDV8, DD colonies produce more white aerial hyphae than under LD. In contrast, the ΔztvelB mutant only grow as grey and white aerial hyphae which are phenotypically similar under both LD and DD conditions.

FIGURE 3

Zymoseptoria tritici IPO323 and the ΔztvelB mutant pycnidia on wheat extract agar (WEA) at 20°C under LD conditions at 69 days post-inoculation. Images representative of three independent ΔztvelB strains tested across two experiments. (A) The IPO323 strain forms white aerial hyphae from a melanised based from which hyaline cirrhus is produced (indicated by arrowheads). (B) Develops spherical black/brown pycnidia beneath the surface of the agar (indicated by arrowheads). (C) The ΔztvelB mutants form white aerial hyphae with large abnormal pycnidia-like structures on the surface of the agar. The pycnidia-like structures exude clear droplets (indicated by arrowheads). (D) The ΔztvelB mutants also form pycnidia-like structures similar to IPO323 beneath the surface of the agar (indicated by arrowheads).

FIGURE 4

Average timing of Z. tritici infection of the IPO323 strain compared to the ΔztvelB mutants at 0–28 dpi. Three independent ΔztvelB strains (dotted lines) were tested against the parental IPO323 strain (straight line), and experiments were repeated in triplicate with 7–12 technical repeats each time. Graph representative of three biological replicates. Wheat leaves (cv. Riband) were inoculated with a plug extracted from a 10-day-old CDV8 agar plate dipped in 0.1% Tween 20. Symptoms were scored on a scale of 1–5 ever 2–3 days. Bars represent standard error and black arrows indicate the point at which the IPO323 strain and first ΔztvelB strain reached the fifth and final stage of infection. In all three experiments, the IPO323 strain produced symptoms before all three ΔztvelB strains.

FIGURE 5

Infection symptoms of wheat leaves (cv. Riband) infected with the Z. tritici IPO323 strain and the ΔztvelB mutants at 28 dpi. Images representative of three independent ΔztvelB mutants tested against IPO323. Experiments were carried out in triplicate with 7–12 technical repeats each time. Leaves were inoculated with a plug extracted from a 10-day-old CDV8 agar plate dipped in 0.1% Tween 20. Samples were collected at 28 dpi and incubated under high humidity to induce cirrhus release. (A) IPO323 produces necrotic lesions after 28 dpi. (B,C) dark brown pycnidia on the infected wheat leaves which produce hyaline cirrhus from the ostiole (indicated by arrowheads). (D) the ΔztvelB mutants produce necrotic lesions on wheat leaves. (E,F) pycnidia are dark brown in colour with clear cirrhus and some have white aerial hyphae on the exterior, developing from the ostiole region (indicated by arrowheads).

FIGURE 6

Comparison of the average pycnidia/mm² and micropycnidiospores/ml produced by IPO323 compared to the three ΔztvelB mutant strains at 28 dpi. Experiments were carried out in triplicate with 7–12 technical replicates each time. Bars represent standard error and an asterisk (^∗) signifies strains significantly different to the IPO323 strain. (A) The ΔztvelB 2 and ΔztvelB 3 mutants produced significantly fewer pycnidia than the IPO323 strain at the 5% level. (B) The three ΔztvelB mutants do not produce statistically significantly numbers of micropycnidiospores compared to the IPO323 strain at the 5% level.

FIGURE 7

Images comparing macropycnidospores and micropycnidospores obtained from wheat leaves (cv. Riband) infected with the IPO323 strain and ΔztvelB mutants. Spores were harvested from infected wheat leaves at 28 days post-inoculation and stained with lactophenol cotton blue. Images representative of all three ΔztvelB knock-out mutant strains tested against the IPO323 strain. Experiments were carried out in triplicate with 7–12 technical replicates each time. Arrowheads indicate micropycnidiospores (A) IPO323 produces both macropycnidiospores and micropycnidiospores. (B) ΔztvelB mutants only produce micropycnidospores. (C) Aggregation of micropycnidiospores obtained from the ΔztvelB mutants.