Ziyi Yin, Wanzhen Feng, Chen Chen, Jiayun Xu, Ying Li, Lina Yang, Jingzhen Wang, Xinyu Liu, Wenhao Wang, Chuyun Gao, Haifeng Zhang, Xiaobo Zheng, Ping Wang, Zhengguang Zhang.

Autophagy. 2020 May;16(5):900-916.  doi: 10.1080/15548627.2019.1644075.

Abstract

Cells are faced with various stresses during their growth and development, and autophagy is a degradative process in which cells can break down their own components to recycle macromolecules and provide energy under these stresses. For pathogenic fungi that utilize cell wall as the first barrier against external stress, the cell wall integrity (CWI) pathway also provides an essential role in responding to these stresses. However, the specific connection between autophagy and CWI remains elusive in either the model fungi including budding yeast Saccharomyces cerevisiae or the rice blast fungus Magnaporthe oryzae. Here, we provided evidence that the endoplasmic reticulum (ER) stress is highly induced during M. oryzae infection and that CWI MAP kinase kinase MoMkk1 (S. cerevisiae Mkk1/2 homolog) was subject to phosphorylation regulation by MoAtg1, the only identified kinase in the core autophagy machinery. We also identified MoMkk1 serine 115 as the MoAtg1-dependent phosphorylation site and this phosphorylation could activate CWI, similar to that by the conserved MAP kinase kinase kinase MoMck1 (S. cerevisiae Bck1 homolog). Together with the first report of MoMkk1 subjects to phosphorylation regulation by MoAtg1, we revealed a new mechanism by which autophagy coordinates with CWI signaling under ER stress, and this MoAtg1-dependent MoMkk1 phosphorylation is essential for the pathogenicity of M. oryzae.Abbreviations: A/Ala: alanine; Atg: autophagy-related; Bck1: bypass of C kinase 1; co-IP: co-immunoprecipitation; CWI: cell wall integrity;DTT: dithiothreitol; ER: endoplasmic reticulum; GFP: green fluorescent protein; Mo: Magnaporthe oryzae; MAPK: mitogen-activated protein kinase; Mkk1: mitogen-activated protein kinase-kinase 1; MS: mass spectrometry; PAS: phagophore assembly site; RFP: red fluorescent protein; RT: room temperature; S/Ser: serine; Slt2: suppressor of the lytic phenotype 2; T/Thr: threonine; UPR: unfolded protein response; Y2H: yeast two-hybrid screen.

See https://pubmed.ncbi.nlm.nih.gov/31313634/

Figure 1: ER stress is highly induced during M. oryzae infection. (A) RT-PCR analysis of testing UPR-induced MoHAC1 splicing in M. oryzae. RT-PCR was performed using total RNAs extracted from the wild-type stain grown in liquid CM for 2 d and then treated with 10 μM DTT for another 4 h and harvested. For RNA extraction during infection stages, four milliliters of conidial suspension (2 × 10⁵ spores/ml) of Guy11 were sprayed on 2-week-old rice seedlings (Oryza sativa cv. CO39) and the infected leaves were collected and cooled with liquid nitrogen. (B) Expression of uMoHAC1 and sMoHAC1 were analyzed by qRT-PCR in both mycelial and infection stages (2 h and 8 h). ‘HY’: hyphae stage, ‘-’: hyphae without any treatment. (C) The relative expression levels of putative UPR-target genes were analyzed by qRT-PCR and normalized to that of ACTIN. Error bars represent the standard deviations and asterisks represent significant differences (p < 0.01).