Evidence for a diffusible factor that induces susceptibility in the Colletotrichum-maize disease interaction

Abstract

Colletotrichum graminicola, the causal agent of maize anthracnose, is a hemibiotrophic fungus that initially infects living host cells via primary hyphae surrounded by a membrane. A nonpathogenic mutant disrupted in a gene encoding a component of the signal peptidase complex, and believed to be deficient in protein processing and secretion, regained pathogenicity when it was inoculated onto maize leaf sheaths close to the wild-type fungus. Evidence is presented suggesting that the wild-type produces a diffusible factor(s) that induces the localized susceptibility of host cells at the borders of expanding colonies, causing them to become receptive to biotrophic invasion. The induced susceptibility effect is limited to a distance of approximately eight cells from the edge of the wild-type colony, is dosage dependent and is specific to C. graminicola.

Figure 1

Development of wild‐type (WT) and cpr1 mutant strains on maize leaf sheaths. Percentage of ungerminated spores (white bar), appressoria (light grey bar) and invasive primary hyphae (dark grey bar) at 12, 24 and 48 h post‐inoculation (hpi).

Figure 2

(a) Symptom development in inoculated and mock‐inoculated maize leaf sheaths 96 h post‐inoculation (hpi). (b) Phenotypes of the wild‐type (WT) and cpr1 mutant on maize leaf sheaths at 20, 48 and 60 hpi (for WT only). (c) Penetration of living cells at the edges of a necrotrophic colony. Plasmolysis and neutral red uptake were used to determine host cell viability. Cells that had not yet been colonized or that had just been invaded by biotrophic hyphae usually remained alive, indicated by the presence of plasmolysis, or plasmolysis combined with neutral red staining (asterisks). AP, appressoria; BH, biotrophic primary infection hyphae; NH, necrotrophic hyphae. Scale bars, 50 μm.

Figure 3

Pattern of reactive oxygen species (ROS) accumulation in maize leaf sheaths inoculated with the wild‐type (WT) (a–c) or cpr1 mutant (d–f), indicated by 3,3′‐diaminobenzidine (DAB) staining. AP, appressoria; BH, biotrophic primary infection hyphae; hpi, hours post‐inoculation. Scale bars, 20 μm.

Figure 4

Diagrams of leaf sheath inoculations (a) and co‐inoculations (d, g, h, m). Colonization by WT‐mRFP (b) and cpr1‐Zsgreen (c), at 72 h post‐inoculation (hpi), on maize leaf sheaths. (e) cpr1‐Zsgreen colonization in co‐inoculations with WT‐mRFP. (f) Confocal image showing cpr1‐Zsgreen crossing intact plant cell walls (arrows) in co‐inoculations. Viability of newly invaded plant cells (arrows) and of cells surrounding wild‐type (WT) (i, k) and cpr1 (j, l) colonies in co‐inoculations, demonstrated by plasmolysis and neutral red staining (asterisks), at 60 hpi. (m–o) Triple inoculations with WT‐mRFP, cpr1‐Zsgreen and nonpathogen (NP) CgSl1‐GFP. (n) Nonpathogen failed to penetrate maize tissue, and cells underneath appressoria continued to plasmolyse (asterisks). (o) cpr1‐Zsgreen invading adjacent cells (arrows) that are still alive (asterisks). Scale bars (except f), 50 μm; (f) 20 μm. AP, appressoria; BH, biotrophic primary hyphae.

Figure 5

The effect of timing of co‐inoculations (‘predisposition time’) on induced susceptibility. Wild‐type (WT) inoculum was added 0, 12, 24 or 36 h after the mutant inoculum (light gray bars). In the controls, cpr1 and the WT (black bars), or cpr1 and water (grey bars), were applied to the tissue simultaneously, 0, 12, 24 and 36 h after the sheaths had been detached. All treatments were evaluated 60 h after the addition of WT. Treatments with different letters are significantly different from each other (P < 0.05).