Localization of Ptr ToxA Produced by Pyrenophora tritici-repentis Reveals Protein Import into Wheat Mesophyll Cells

Abstract

The plant pathogenic fungus Pyrenophora tritici-repentis secretes host-selective toxins (HSTs) that function as pathogenicity factors. Unlike most HSTs that are products of enzymatic pathways, at least two toxins produced by P. tritici-repentis are proteins and, thus, products of single genes. Sensitivity to these toxins in the host is conferred by a single gene for each toxin. To study the site of action of Ptr ToxA (ToxA), toxin-sensitive and -insensitive wheat (Triticum aestivum) cultivars were treated with ToxA followed by proteinase K. ToxA was resistant to protease, but only in sensitive leaves, suggesting that ToxA is either protected from the protease by association with a receptor or internalized. Immunolocalization and green fluorescent protein tagged ToxA localization demonstrate that ToxA is internalized in sensitive wheat cultivars only. Once internalized, ToxA localizes to cytoplasmic compartments and to chloroplasts. Intracellular expression of ToxA by biolistic bombardment into both toxin-sensitive and -insensitive cells results in cell death, suggesting that the ToxA internal site of action is present in both cell types. However, because ToxA is internalized only in sensitive cultivars, toxin sensitivity, and therefore the ToxA sensitivity gene, are most likely related to protein import. The results of this study show that the ToxA protein is capable of crossing the plant plasma membrane from the apoplastic space to the interior of the plant cell in the absence of a pathogen.

Figure 1.

ToxA Is Protected from PK Degradation in ToxA-Sensitive Leaves. (A) PK treatment of leaves 2 h after ToxA treatment does not reduce ToxA-induced necrosis. Leaves of sensitive (Sen) and insensitive (Ins) wheat cultivars infiltrated with ToxA alone (ToxA) or ToxA followed by proteinase K (ToxA+PK) were harvested 2 d after infiltration. Black dots on leaves define the treatment area. (B) ToxA can be isolated from sensitive, but not insensitive, wheat cultivars treated with ToxA/PK. The immunoblot was probed with anti-ToxA antibody. Sizes of proteins in the molecular mass markers are noted on the left.

Figure 2.

Immunolocalization of ToxA in ToxA-Sensitive and -Insensitive Wheat. (A) to (D) Confocal imaging of thin sections of fixed/methacrylate-embedded leaf tissue immunostained with anti-ToxA antibody and Alexa-488–tagged secondary antibody. Chloroplast autofluorescence (magenta) and Alexa-488 fluorescence (green) images are merged. Green autofluorescence of mesophyll cell membrane ([A] to [D]), vascular tissue ([B] and [C]), and epidermal tissue ([A], [B], and [D]) is present in both treated and untreated tissue. (A) Water-treated insensitive leaves. (B) Water-treated sensitive leaves. (C) ToxA-treated insensitive leaves. (D) ToxA-treated sensitive leaves. Closed arrowheads indicate ToxA-specific signal present in aggregates. Open arrowheads indicate ToxA-specific signal associated with chloroplasts.

Figure 3.

Localization of GFP-ToxA Fusion Protein in ToxA-Sensitive and -Insensitive Wheat. (A) GFP-ToxA fusion protein induces symptoms comparable to native ToxA. Leaves were treated with proteins indicated on the right and harvested 24 h after infiltration. Black dots on leaves define the treatment area. (B) to (G) Epifluorescence microscopy on live, whole leaf tissue of ToxA-sensitive and -insensitive wheat treated with ToxA + GFP (nonfused) or GFP-ToxA fusion protein. (B) Insensitive leaves treated with ToxA + GFP. (C) Insensitive leaves treated with GFP-ToxA. (D) Sensitive leaves treated with ToxA + GFP. (E) Sensitive leaves treated with GFP-ToxA. (F) and (G) Enlargements of boxed sections in (E). (H) to (M) Confocal imaging of paraformaldehyde-fixed, ToxA-insensitive ([H] to [J]) and ToxA-sensitive ([K] to [M]) leaves treated with GFP-ToxA. Chloroplast autofluorescence is shown in (H) and (K), GFP fluorescence is shown in (I) and (L), and merged autofluorescence and GFP fluorescence is shown in (J) and (M). Note GFP fluorescence in sensitive leaves is compartmentalized (e.g., cell marked “a”) and associated with chloroplasts (arrows).

Figure 4.

ToxA Activity Is Light Dependent. Sensitive leaves were treated with 5 μM ToxA or GFP-ToxA fusion protein and covered with a light-exclusion clip. Black dots on leaves define the treatment area. The solid line below the leaves corresponds to the area covered. Leaves were harvested and photographed 48 h after treatment.

Figure 5.

ToxA Expression in Planta in Both ToxA-Sensitive and -Insensitive Wheat Cultivars Leads to Cell Death. Average GUS expression (dots/per leaf) for cobombardment of 1 μg each of pBS/35S:GUS (control) or 35S:ToxA/35S:GUS (ToxA). Graph represents the average of four independent experiments. The error bars represent standard error. P values were calculated by Student's paired, one-tailed t test. One asterisk indicates P < 0.05, and two asterisks indicates P < 0.01. GUS expression was decreased by 63 and 48% in sensitive and insensitive leaves, respectively.