Discovery of Two Novel Phthalide Phytotoxins from the Wheat Tan Spot Fungal Pathogen Pyrenophora tritici-repentis

Abstract

The Dothideomycete fungal pathogen Pyrenophora tritici-repentis (Ptr) is the causal agent of the tan spot disease of wheat. The proteinaceous necrotrophic effectors ToxA and ToxB are well characterized. A nonproteinaceous effector called ToxC has also been partially characterized. Ptr produces a number of other small molecular weight compounds, but these remain poorly characterized. In this study, two novel compounds, designated ToxE1 and ToxE2, capable of inducing chlorotic symptoms on wheat leaves in a cultivar-specific manner, were purified from Ptr liquid cultures. There is no evidence that these compounds correspond to ToxC. Most isolates produced ToxE1, ToxE2, or both, and both compounds were detected in infected wheat leaves. The structures of both analogues were elucidated by NMR spectroscopy and comprise a phthalide core structure with an amide moiety. We postulate that these compounds have a general phytotoxic effect and may have an ancillary role in disease development.

Keywords: ToxC; ToxE; phthalide; phytotoxin; specialized metabolites; tan spot; yellow leaf spot.

Figure 1

Structures of the ToxE analogues identified from Pyrenophora tritici-repentis.

Figure 2

Key 2D NMR correlations for ToxE1.

Figure 3

Response of wheat leaves (cultivar “Eagle Rock”) to purified ToxE1 and ToxE2. Concentrations of ToxE compounds infiltrated are displayed on the left. Photographs of representative leaves were taken 7 days post infiltration.

Figure 4

ToxE1 and ToxE2 elicited cultivar-specific chlorosis. Leaves of wheat cultivars exhibiting varying degrees of chlorosis upon infiltration with ToxE1 and ToxE2 at the concentration of 200 μg/mL. Cultivar names and their resistance ratings to tan spot disease are indicated (MRMS = moderately resistant to moderately susceptible, MS = moderately susceptible, MSS = moderately susceptible to susceptible, and SVS = susceptible to very susceptible).

Figure 5

ToxE1 and ToxE2 induced light-dependent chlorosis on wheat. The second leaves of cultivar “Eagle Rock” were infiltrated with ToxE compounds at the concentration of 200 μg/mL and incubated under two different light–dark regimes. Top panel shows leaves with chlorosis symptoms induced by ToxE1 and ToxE2 under the 12 h photoperiod. Wheat leaves failed to develop chlorosis when kept in the dark throughout the incubation period (bottom panel).

Figure 6

ToxE2 inhibits leaf growth. (A) Effect of ToxE2 on the leaf length (cm) of wheat seedlings. The length of the third leaf was measured at a weekly time course upon infiltrations of ToxE1 and ToxE2 at the concentrations of 2, 20, and 200 μg/mL. Data points represent the mean length of minimum four biological replicates for each treatment (mean ± SE). B) Growth phenotypes of ToxE2 infiltrated (200 μg/mL) and untreated (control) wheat seedlings.

Figure 7

Response of leaves from nonhost monocots and dicots upon infiltration with ToxE1 and ToxE2. (A, B) Leaves of monocots (barley, Brachypodium, and ryegrass) and Nicotiana benthamiana, displaying absence of symptom, while (C) Arabidopsis “Columbia” and (D) canola varieties exhibited varying degrees of symptoms for ToxE2.