Assays for MAP Kinase Activation in Magnaporthe oryzae and Other Plant Pathogenic Fungi

Abstract

Mitogen-activated protein (MAP) kinases have the hallmark motif TXY and function in key signal transduction pathways in eukaryotic organisms. Most ascogenous plant pathogenic fungi have three MAPK pathways that regulate different developmental and infection processes. In the rice blast fungus Magnaporthe oryzae, the Pmk1 and Mps1 MAP kinases with the TEY motif are essential for appressorium formation, penetration, and invasive growth. Osm1 is the third MAP kinase that has the TGY motif and functions in osmoregulation. Although orthologs of Pmk1 and Mps1 are important for pathogenesis in all the plant pathogens studied, Osm1 orthologs have species-specific roles in stress responses and pathogenesis. Because of their functions in fungal development and pathogenesis, it is important to determine the expression and activation of MAP kinases under different growth conditions or infection stages. In this chapter, we describe methods for protein extraction and detection of the activation of the three MAP kinases in M. oryzae with the commercially available anti-TpEY or anti-TpGY phosphorylation-specific antibodies. Similar approaches can be used to monitor MAP kinase activation in other plant pathogenic fungi.

Mucin Msb2 cooperates with the transmembrane protein Sho1 in various plant surface signal sensing and pathogenic processes in the poplar anthracnose fungus Colletotrichum gloeosporioides

“…Protein isolation and CgMk1 MAPK activation assays were performed following the protocol in Zhang et al ( 2018a ). Vegetative hyphae were harvested from 2‐day‐old cultures in 5× YEG liquid medium (5 g yeast extract and 10 g glucose in 1 L double distilled water) grown at 150 rpm at 25 °C.…”

“…Protein isolation and CgMk1 MAPK activation assays were performed following the protocol in Zhang et al (2018a). Vegetative A putative constitutively active Ste7 allele was constructed by changing Ser-218 to Glu and Thr-222 to Glu using the fast sitedirected mutagenesis kit (TianGen) (Cowley et al, 1994;Zhao et al, 2005) following the manufacturer's instructions.…”

Deletion of FgHOG1 Is Suppressive to the mgv1 Mutant by Stimulating Gpmk1 Activation and Avoiding Intracellular Turgor Elevation in Fusarium graminearum

“…Total proteins were isolated from hyphae harvested from 18 h YEPD cultures as described (Zhang et al, 2018). For assays with different stresses, hyphae harvested from 18 h YEPD were further incubated with 10 μg/ml CR or 2 μg/ml fludioxonil for another 10 min before protein extraction.…”

PoRal2 Is Involved in Appressorium Formation and Virulence via Pmk1 MAPK Pathways in the Rice Blast Fungus Pyricularia oryzae

“…Total proteins were isolated from 2-day-old vegetative hyphae grown in 5 x YEG culture (5g yeast extract, 10g glucose, 1L ddH 2 O) using TCA-acetone precipitation methods, and total protein concentrations were determined using the Enhanced BCA Protein Assay Kit (Beyotime, China). Protein extracts (50μg) were subjected to 12% SDS-PAGE (EpiZyme, China) and transferred to a PVDF membrane as described in previous research ( Zhang et al, 2018 ). Primary antibodies, including anti-Phospho-p44/42 MAPK antibody #4370 (Cell Signaling Technology, Inc.), anti-ERK1/2 MAPK antibody (C-9): sc-514,302 (Santa Cruz Biotechnology, Inc.), and anti-GAPDH R1208-3 antibody (HUABIO, China), along with peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Beyotime, China), were used in this study.…”