Fig. 2: Ebg1 is a secreted protein.

a Subcellular localization of Ebg1-GFP in the infection hyphae of M. oryzae. The Ebg1-GFP fluorescence from the Δebg1/EBG1 complemented transformants was distributed mainly around the cell wall and the tip of infection hyphae. Images were captured at 48 h post inoculation (hpi) on barley epidermal leaves. Scale bars = 20 μm. b, c Laser confocal micrographs of M. oryzae strain expressing Ebg1-GFP and Pwl2-mCherry. The Ebg1-GFP/Pwl2-mCherry image shows the overlay of the GFP channel and mCherry channel. A merged image shows the overlay of GFP channel, mCherry channel, and the brightfield channel. Images were captured at 26 hpi (b) and 40 hpi (c) on rice cultivar LTH. Scale bars = 10 μm. d The Ebg1 protein was detected both in vegetative hyphae and cultured medium. A Δebg1/EBG1 complemented transformant and a P131/GFP transformant were cultured in liquid CM, and total proteins extracted from the mycelia and from the culture filtrates were immunoblotted with an anti-GFP antibody. Intact and truncated versions of Ebg1-GFP were labeled with a black solid triangle and an open triangle, respectively. Anti-actin antibody were used to confirm the protein samples of vegetative hyphae. e Ebg1 has a functional signal peptide directing its secretion. The EBG1 full-length sequence (EBG1-FL) and signal peptide deleted sequence (EBG1-ΔSP) were constructed into the pSUC2 vector and transformed into yeast YTK12(SUC2-) for the invertase secretion test. The yeast transformants with EBG1-FL grew well on the YPRAA medium containing raffinose and antimycin A, whereas EBG1-ΔSP failed to grow on the same medium. Yeast strains with Avr1b and Mg87 were used as positive and negative controls, respectively.