RNA-mediated gene silencing of ToxB in Pyrenophora tritici-repentis

Abstract

The fungus Pyrenophora tritici-repentis causes tan spot, a wheat leaf disease of worldwide importance. The pathogen produces three host-selective toxins, including Ptr ToxB, which causes chlorophyll degradation and foliar chlorosis on toxin-sensitive wheat genotypes. The ToxB gene, which codes for Ptr ToxB, was silenced in a wild-type race 5 isolate of the fungus thorough a sense- and antisense-mediated silencing mechanism. Toxin production by the silenced strains was evaluated in culture filtrates of the fungus via Western blotting analysis, and plant bioassays were conducted to test the virulence of the transformants in planta. The chlorosis-inducing ability of the silenced strains was correlated with the quantity of Ptr ToxB, and transformants in which toxin production was strongly decreased also caused very little disease on toxin-sensitive wheat genotypes. Cytological analysis of the infection process revealed that, in addition to a reduced capacity to induce chlorosis, the silenced strains with the greatest decrease in the levels of Ptr ToxB produced significantly fewer appressoria than the wild-type isolate, 12 and 24 h after inoculation onto wheat leaves. The results provide strong support for the suggestion that the amount of Ptr ToxB protein produced by fungal isolates plays a significant role in the quantitative variation in the virulence of P. tritici-repentis.

Figure 1

Characterization of ToxB‐silenced transformants of Pyrenophora tritici‐repentis. (A) Morphology of colonies of the wild‐type isolate Alg3‐24 and the hygromycin‐resistant transformants tf1, tf2, tf4, tf5 and tf6 on V8‐potato dextrose agar medium after 6 days of growth. (B) Growth rate of all tested strains expressed in colony diameter in millimetres per day. Data points represent the mean of four replications with bars indicating the standard deviation of the mean.

Figure 2

Western blot analysis of protein from ToxB‐silenced transformants and the wild‐type isolate Alg3‐24 of Pyrenophora tritici‐repentis with polyclonal antibodies specific to Ptr ToxB. Lane 1, kaleidoscope prestained standards (Bio‐Rad, Mississauga, ON, Canada; cat. no. 161‐0324); lane 2, transformant tf1; lane 3, tf2; lane 4, tf4; lane 5, tf5; lane 6, tf6; lane 7, wild‐type isolate Alg3‐24. Samples consisted of 5 µg of total protein from concentrated 21‐day‐old culture filtrates of each fungal strain, which were resolved via sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) prior to transfer to a polyvinylidene fluoride membrane. The number under each band represents its intensity relative to the wild‐type isolate, as measured using the densitometry function of Alpha Imager software (Alpha Innotec, San Leandro, CA, USA).

Figure 3

Fluorescence micrographs of infection of leaves of the Ptr ToxB‐sensitive/race 5‐susceptible wheat line 6B662 by ToxB‐silenced transformants and the wild‐type isolate Alg3‐24 of Pyrenophora tritici‐repentis. Samples were stained with aniline blue. (A) Germinated conidium (c) of silenced strain tf5 on the leaf surface, 3 h after inoculation. Note the presence of appressoria (a) at the end of the germ tube (g). (B) Multiple germ tubes with appressoria developing from a single conidium of silenced strain tf2, 3 h after inoculation. (C) Germinated conidium of Alg3‐24, 6 h after inoculation. Note the formation of multiple appressoria from a single germ tube; v, vesicle. (D) Germinated conidium of silenced strain tf4, 24 h after inoculation. Note the germ tube growing over the leaf surface without the formation of any appressoria. (E) Germinated conidium of silenced strain tf1 on the leaf surface, 6 h after inoculation. Note the presence of an appressorium on a stomatal complex (s). (F) Formation of an intracellular vesicle (v) in a host epidermal cell, 6 h after inoculation with tf2; intracellular hyphae (ih) are also visible within the epidermal cell. (G) Intercellular hyphae (ih/me) of silenced strain tf6 in the host mesophyll layer, 6 h after inoculation. (H) Further ingress of hyphae of tf6 in the host mesophyll, 12 h after inoculation. (I) Damaged mesophyll cells (dme), 24 h after inoculation with the wild‐type isolate Alg3‐24. Note the deep staining of the cells with aniline blue, which is indicative of cell damage.

Figure 4

Reactions of wheat line 6B662 and cv. Salamouni to inoculation with ToxB‐silenced transformants and the wild‐type race 5 isolate Alg3‐24 of Pyrenophora tritici‐repentis. (A) Appearance of the Ptr ToxB‐sensitive/race 5‐susceptible line 6B662 and the Ptr ToxB‐insensitive/race 5‐resistant cv. Salamouni 7 days after inoculation with the silenced strains tf1, tf2, tf4, tf5 and tf6, and the wild‐type isolate Alg3‐24, of the fungus. Note the varying levels of chlorosis on line 6B662; the necrotic flecking on ‘Salamouni’ is a typical resistance reaction. (B) Percentage of total leaf area turning chlorotic in line 6B662 in response to infection by the wild‐type isolate and the silenced transformants. Leaves were harvested 7 days after inoculation, and total leaf and total lesion areas were measured using Assess 2.0 Image Analysis Software (Lamari, 2008). Error bars indicate the standard deviation from five repetitions of one run of the experiment.