Muxing Liu, Jiexiong Hu, Ao Zhang, Ying Dai, Weizhong Chen, Yanglan He, Haifeng Zhang, Xiaobo Zheng, Zhengguang Zhang

New Phytol. 2021 Apr; 230(2):720-736.

Abstract

Plant pathogens exploit the extracellular matrix (ECM) to inhibit host immunity during their interactions with the host. The formation of ECM involves a series of continuous steps of vesicular transport events. To understand how such vesicle trafficking impacts ECM and virulence in the rice blast fungus Magnaporthe oryzae, we characterised MoSwa2, a previously identified actin-regulating kinase MoArk1 interacting protein, as an orthologue of the auxilin-like clathrin uncoating factor Swa2 of the budding yeast Saccharomyces cerevisiae. We found that MoSwa2 functions as an uncoating factor of the coat protein complex II (COPII) via an interaction with the COPII subunit MoSec24-2. Loss of MoSwa2 led to a deficiency in the secretion of extracellular proteins, resulting in both restricted growth of invasive hyphae and reduced inhibition of host immunity. Additionally, extracellular fluid (ECF) proteome analysis revealed that MoSwa2-regulated extracellular proteins include many redox proteins such as the berberine bridge enzyme-like (BBE-like) protein MoSef1. We further found that MoSef1 functions as an apoplastic virulent factor that inhibits the host immune response. Our studies revealed a novel function of a COPII uncoating factor in vesicular transport that is critical in the suppression of host immunity and pathogenicity of M. oryzae.

See: https://pubmed.ncbi.nlm.nih.gov/33423301/

Figure 1: MoSwa2 is subjected to phosphorylation regulation by MoArk1 and is required for normal endocytosis. (a) Yeast‐two‐hybrid assay for the interaction between MoSwa2 and MoArk1. Yeast transformants expressing the prey and bait constructs were assayed for growth on SD−Leu−Trp and SD−Leu−Trp−His plates with β‐galactosidase activities (LacZ). (b) Co‐immunoprecipitation (co‐IP) analyses of the interaction between MoSwa2 and MoArk1. The MoArk1‐3xFlag and MoSwa2‐GFP were co‐expressed in the wild‐type strain Guy11. The co‐IP experiment was performed with the anti‐Flag Affinity Gel and the isolated protein was analysed by western blotting using anti‐FLAG and anti‐GFP antibodies. (c) Domain map of MoSwa2. The protein sequence of MoSwa2 was used to perform the SMART (http://smart.embl‐heidelberg.de) and Motif Scan (https://myhits.sib.swiss/cgi‐bin/motif_scan) analysis service for functional domain prediction. (d) In vitro phosphorylation analysis by FDIT method. Purified proteins of GST‐MoArk1 and His‐MoSwa2 were used for protein kinase reaction and then dyed with Pro‐Q^® Diamond Phosphorylation Gel Stain. The fluorescence signal at 590 nm (excited at 530 nm) was measured in a Cytation3 microplate reader (Biotek, Winooski, VT, USA). The experiments were repeated three times and showed similar results. Asterisks indicate statistical significances (P < 0.01). (e) Time course images of FM4‐64 uptake at the hyphal tips. Hyphae stained by FM4‐64 were examined using fluorescence microscopy at different time points. Insets highlight areas analysed by line scan. Fluorescence intensity was measured using imagej software.