The rice blast fungus MoRgs1 functioning in cAMP signaling and pathogenicity is regulated by casein kinase MoCk2 phosphorylation and modulated by membrane protein MoEmc2

Abstract

GTP-binding protein (G-protein) and regulator of G-protein signaling (RGS) mediated signal transduction are critical in the growth and virulence of the rice blast pathogen Magnaporthe oryzae. We have previously reported that there are eight RGS and RGS-like proteins named MoRgs1 to MoRgs8 playing distinct and shared regulatory functions in M. oryzae and that MoRgs1 has a more prominent role compared to others in the fungus. To further explore the unique regulatory mechanism of MoRgs1, we screened a M. oryzae cDNA library for genes encoding MoRgs1-interacting proteins and identified MoCkb2, one of the two regulatory subunits of the casein kinase (CK) 2 MoCk2. We found that MoCkb2 and the sole catalytic subunit MoCka1 are required for the phosphorylation of MoRgs1 at the plasma membrane (PM) and late endosome (LE). We further found that an endoplasmic reticulum (ER) membrane protein complex (EMC) subunit, MoEmc2, modulates the phosphorylation of MoRgs1 by MoCk2. Interestingly, this phosphorylation is also essential for the GTPase-activating protein (GAP) function of MoRgs1. The balance among MoRgs1, MoCk2, and MoEmc2 ensures normal operation of the G-protein MoMagA-cAMP signaling required for appressorium formation and pathogenicity of the fungus. This has been the first report that an EMC subunit is directly linked to G-protein signaling through modulation of an RGS-casein kinase interaction.

Rgs1 is a regulator of effector gene expression during plant infection by the rice blast fungus Magnaporthe oryzae

“…This led to identification of a mutant, cer7 , which carries a single point mutation in a gene called RGS1 . We show that the Rgs1 protein– which has been previously implicated as a regulator of G-protein signalling during appressorium development by M. oryzae (15–17) –also acts as a transcriptional regulator of effector gene expression. We provide evidence that Rgs1 is necessary for repressing the expression of at least 60 temporally co-regulated effector-encoding genes during the pre-penetration stages of development and that these genes are subsequently de-repressed during invasive growth by the fungus enabling their specific deployment in plant tissue.…”

“…Appressorium development requires heterotrimeric G-protein signalling, to transmit surfacesensing cues to downstream modules that facilitate morphogenesis, controlled by the mitogenactivated protein kinase Pmk1 (12,13), and cAMP-dependent protein kinase A pathways (14). Gprotein subunits in M. oryzae are controlled by Regulator of G-protein Signalling (RGS) proteins, which are important for appressorium development (15)(16)(17).…”

Homeostasis of cell wall integrity pathway phosphorylation is required for the growth and pathogenicity of Magnaporthe oryzae

“…Previous studies showed that continuous activation of MoRgs1 attenuates the virulence of M. oryzae . Constitutively activated MoRgs1 causes protein mislocalization, leading to its functional defects (Yu et al, 2021). Nevertheless, the mechanisms by which MAPK kinase overphosphorylation affects its function remain unknown.…”

“…For protein kinase reaction, 2 μg of MoPtc1 or MoPtc2 was mixed with MoMkk1, MoMck1 in a kinase reaction buffer (100 mM phosphate‐buffered saline, pH 7.5, 10 mM MgCl 2 , 1 mM ascorbic acid; Sigma‐Aldrich), with the addition of 50 mM ATP (Sigma Aldrich). The subsequent experiments were performed according to the protocol (Feng et al, 2021; Jin & Gou, 2016; Yin et al, 2020; Yu et al, 2021).…”

Co-evolved plant and blast fungus ascorbate oxidases orchestrate the redox state of host apoplast to modulate rice immunity

“…The MoAO1 gene deletion mutant was obtained by the one-step gene replacement strategy (Yu et al, 2021). Sequence fragments (1.0 kb) located in the CDSs and upstream and downstream regions of target genes were amplified by PCR using the primer pairs listed in Supplemental Table 3.…”