To localize Cmu1 during biotrophic growth, plants were infected with SG200Dcmu1-cmu1–HA, which carries a cmu1–HA fusion gene inserted in single copy under control of its native promoter. Plants infected with SG200 or with SG200 Pcmu1GFP–HA expressing cytoplasmic green fluorescent protein (GFP) under the cmu1 promoter served as negative controls. Freeze-substituted and resin-embedded sections of maize tissue harvested 3 days after infection with these strains were incubated with anti-HA antibodies and gold markers. Cmu1–HA could be detected inside the fungal hyphae, in the biotrophic interface as well as inside the plant cytoplasm but rarely in the plant cell wall (Fig. 2A and Supplementary Fig. 7). The distribution of gold particles was quantified (Fig. 2B). Gold labelling of plant tissue infected with the parental strain SG200 was negligible (Supplementary Fig. 8), whereas non-secreted GFP–HA was absent from the biotrophic interphase, showed strong accumulation in the fungal cytosol and weak background labelling in the plant cytosol (Supplementary Fig. 9 and Fig. 2B).
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