MoVam7, a Conserved SNARE Involved in Vacuole Assembly, Is Required for Growth, Endocytosis, ROS Accumulation, and Pathogenesis of Magnaporthe oryzae

Xianying Dou,Zhengguang Zhang,Qi Wang,Wenwen Song,Min Guo,Zhongqiang Qi,Haifeng Zhang,Wei Wang,Xiaobo Zheng,Ping Wang,Robert Arkowitz

doi:10.1371/journal.pone.0016439

Abstract

Soluble NSF attachment protein receptor (SNARE) proteins play a central role in membrane fusion and vesicle transport of eukaryotic organisms including fungi. We previously identified MoSce22 as a homolog of Saccharomyces cerevisiae SNARE protein Sec22 to be involved in growth, stress resistance, and pathogenicity of Magnaporthe oryzae. Here, we provide evidences that MoVam7, an ortholog of S. cerevisiae SNARE protein Vam7, exerts conserved functions in vacuolar morphogenesis and functions in pathogenicity of M. oryzae. Staining with neutral red and FM4-64 revealed the presence of abnormal fragmented vacuoles and an absence of the Spitzenkörper body in the ΔMovam7 mutant. The ΔMovam7 mutant also exhibited reduced vegetative growth, poor conidiation, and failure to produce the infection structure appressorium. Additionally, treatments with cell wall perturbing agents indicated weakened cell walls and altered distributions of the cell wall component chitin. Furthermore, the ΔMovam7 mutant showed a reduced accumulation of reactive oxygen species (ROS) in the hyphal apex and failed to cause diseases on the rice plant. In summary, our studies indicate that MoVam7, like MoSec22, is a component of the SNARE complex whose functions in vacuole assembly also underlies the growth, conidiation, appressorium formation, and pathogenicity of M. oryzae. Further studies of MoVam7, MoSec22, and additional members of the SNARE complex are likely to reveal critical mechanisms in vacuole formation and membrane trafficking that is linked to fungal pathogenicity.

Highlights

Exocytosis and endocytosis are essential for fungal growth, and virulence
The Nterminus of MoVam7 is predicted to have a PHOX homology domain (PX), which was originally identified as a common motif of about 120 amino acids found in the P40phox and P47phox subunits of the neutrophil NADPH oxidase (PHOX) [28]
The C terminus of MoVam7 contains a Soluble NSF attachment protein receptor (SNARE) motif arranged in heptad repeats, which is predicted to encode a vSNARE, but further examination of the C-terminal SNARE motif revealed that the central position (0-layer) of the heptad repeats is a Gln (Q) residue, which is conserved in all t-SNARE proteins, including Vam7 of S. cerevisiae and Yup1 of U. maydis (Figure 1C)

Summary

Introduction

Exocytosis and endocytosis are essential for fungal growth, and virulence. Like in other eukaryotic organisms the secretory processes in fungi require many steps of vesicular traffic between distinct membrane-bound organelles. Secretory materials include extracellular enzymes and others leave the endoplasmic reticulum (ER) in vesicles to Golgi apparatus (GA). Following passage through the GA the cargos are packaged into vesicles destined for the plasma membrane (PM). These vesicles either fuse to the PM or exocytose their contents. Complementary to the secretory pathway, there is an endocytic pathway that internalizes extracellular materials and retrieves membrane and proteins from the PM. Some of the internalized material is delivered to the degradative vacuole after passage through early and late endosomes. Several studies support the existence of endocytosis in filamentous fungi, with FM4-64, a specific tracer of endocytosis is incorporated into the Spitzenkorper, suggesting this organelle may be involved in endocytic membrane recycling [1,2,3,4,5,6]

Methods

Results

Conclusion