Activation of Mst11 and Feedback Inhibition of Germ Tube Growth in Magnaporthe oryzae

Abstract

Appressorium formation and invasive growth are two important steps in the infection cycle of Magnaporthe oryzae that are regulated by the Mst11-Mst7-Pmk1 mitogen-activated protein kinase (MAPK) pathway. However, the molecular mechanism involved in the activation of Mst11 MAPK kinase kinase is not clear in the rice blast fungus. In this study, we functionally characterized the regulatory region of Mst11 and its self-inhibitory binding. Deletion of the middle region of Mst11, which contains the Ras-association (RA) domain and two conserved phosphorylation sites (S453 and S458), blocked Pmk1 activation and appressorium formation. However, the MST11(ΔRA) transformant MRD-2 still formed appressoria, although it was reduced in virulence. Interestingly, over 50% of its germ tubes branched and formed two appressoria by 48 h, which was suppressed by treatments with exogenous cAMP. The G18V dominant active mutation enhanced the interaction of Ras2 with Mst11, suggesting that Mst11 has stronger interactions with the activated Ras2. Furthermore, deletion and site-directed mutagenesis analyses indicated that phosphorylation at S453 and S458 of Mst11 is important for appressorium formation and required for the activation of Pmk1. We also showed that the N-terminal region of Mst11 directly interacted with its kinase domain, and the S789G mutation reduced their interactions. Expression of the MST11(S789G) allele rescued the defect of the mst11 mutant in plant infection and resulted in the formation of appressoria on hydrophilic surfaces, suggesting the gain-of-function effect of the S789G mutation. Overall, our results indicate that the interaction of Mst11 with activated Ras2 and phosphorylation of S453 and S458 play regulatory roles in Mst11 activation and infection-related morphogenesis, possibly by relieving its self-inhibitory interaction between its N-terminal region and the C-terminal kinase domain. In addition, binding of Mst11 to Ras2 may be involved in the feedback inhibition of cAMP signaling and further differentiation of germ tubes after appressorium formation.

Phosphorylation-mediated Regulatory Networks in Mycelia of Pyricularia oryzae Revealed by Phosphoproteomic Analyses

“…To our surprise, all these detected phosphoproteins function upstream of Pmk1, whereas phosphorylation was not detected for Pmk1 (23) and its downstream targets (Mst12 and Sfl1) (61,62). Moreover, Mkk1, Mst11, and Mst7 were detected to be phosphorylated at Ser 127 , Ser 557 , and Ser 358 in mycelia, respectively, which differed from those sites that have been previously reported to be important for activation of the downstream proteins (23,55,63). (Fig.…”

“…However, this study in- showing phosphorylation of Mps1 and Pmk1 in mycelia of the wild-type stain P131, ⌬pmk1, ⌬mps1, ⌬pmp1, ⌬pmp1 complemented strain cPmp1 and a Pmp1 S240D allele transformant of ⌬pmp1. Total proteins were extracted from mycelia of the strains and phosphorylation of Mps1 (46 kDa) and Pmk1 (42 kDa) were detected with anti-TpEYp antibody as previously described (55,56). C, Yeast two-hybrid assay showing Pmp1 interacts Mps1 but not Pmk1.…”

“…Western Blot Assay-Total proteins were isolated from mycelia and separated on a 12% SDS-PAGE gel and transferred to PVDF Membrane (Merck Millipore, Shanghai, China). Phosphorylation of the Pmk1 and Mps1 MAP kinases was detected with the PhophoPlus p44/42 MAP kinase antibody (Cell Signaling Technology, Beverly, MA) as described previously (55,56). The anti-actin (Abmart, Shanghai, China) was used as a loading control.…”

Magnaporthe oryzae SMO1encodes a Ras GTPase-activating protein required for spore morphology, appressorium function and rice blast disease

“…Mst50 functions as an adapter protein of the Mst7-Mst11-Pmk1 cascade involved in activating Pmk1 in M. oryzae (Zhao et al 2005). Both Mst50 and Mst11 have been shown to interact with MoRas1 and MoRas2, by yeast two-hybrid assays (Park et al 2006) and deletion of the Rasassociation (RA) domain of Mst11 blocked Pmk1 activation and appressorium formation (Qi et al 2015), supporting a role for Ras signalling in activation of the Pmk1 pathway. It has been shown recently that the transmembrane mucins, Msb2…”

“…Ras-association domain was not important for response to oxidative stress (Li et al 2017). M.oryzae Ras2 is therefore essential for cellular viability, and a key mediator between both the Pmk1 MAPK and cAMP signalling cascades (Qi et al 2015;Zhou et al 2014). Activation and deactivation of Ras2 regulates developmental switches/pathways necessary for growth and pathogenicity of the fungus (Zhou et al 2014).…”