Mochizuki S, Minami E, Nishizawa Y

Microbiologyopen. 2015 Oct 15. doi: 10.1002/mbo3.304. [Epub ahead of print]

http://www.ncbi.nlm.nih.gov/pubmed/26472068

Abstract

The rice blast fungus Magnaporthe oryzae grows inside living host cells. Cytological analyses by live-cell imaging have revealed characteristics of the biotrophic invasion, particularly the extrainvasive hyphal membrane (EIHM) originating from the host plasma membrane and a host membrane-rich structure, biotrophic interfacial complex (BIC). Here, we observed rice subcellular changes associated with invasive hyphal growth using various transformants expressing specifically localized fluorescent proteins. The invasive hyphae did not penetrate across but were surrounded by the host vacuolar membrane together with EIHM even after branching. High-resolution imaging of BICs revealed that the host cytosol was accumulated at BIC with aggregated EIHM and a symplastic effector, Pwl2, in a punctate form. The vacuolar membrane did not aggregate in but closely surrounded the BIC. A good correlation was observed between the early collapse of vacuoles and damage of invasive hyphae in the first-invaded cell. Furthermore, a newly developed, long-term imaging method has revealed that the central vacuole gradually shrank until collapse, which was caused by the hyphal invasion occurring earlier in the neighboring cells than in the first-invaded cells. These data suggest that M. oryzae may suppress host vacuole collapse during early infection stages for successful infection.

Figure 1.Visualization of host cytosol during Magnaporthe oryzae infections. Leaf sheaths of transgenic rice plants constitutively expressing AcGFP1 (cyto- GFP line) were inoculated with a conidial suspension of a compatible strain (Ina86-  137) transformed with tefp::mCherry(TmC1 line) and observed using a laser confocal microscope. Representative data of the 8 (A), 15 (B), and 12 (C) similar images are shown. Cyto-  GFP signals are detected along the primary invasive hyphae both in the first-invaded (A and B) and second-  invaded cells (C), in addition to the predicted positions of the BIC (yellow arrows). GFP, stacked z- series confocal fluorescence images of GFP signals corresponding roughly to the surface half of rice epidermal cells. mCherry + GFP, mergers of the GFP image, and stacked z- series confocal fluorescence images of mCherry signals. Wedge, appressorial penetration site. Size bar = 20 μm. BIC, biotrophic interfacial complex; GFP, green fluorescent protein; DIC, differential interference contrast images.