Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases) and hydrolysis (by cAMP phosphodiesterases). We functionally characterized gene-deletion mutants of a high-affinity (PdeH) and a low-affinity (PdeL) cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHΔ showed enhanced conidiation (2–3 fold), precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHΔ pdeLΔ mutant showed reduced conidiation, exhibited dramatically increased (∼10 fold) cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHΔ defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling during M. oryzae development is crucial for successful establishment and spread of the blast disease in rice.

The interaction of Magnaporthe oryzae, the rice blast fungus, and rice begins when M. oryzae establishes contact with the host plant surface. On perception of appropriate surface signals, M. oryzae forms appressoria and initiates host invasion. Pth11, an important G-protein-coupled receptor necessary for appressorium formation in M. oryzae, contains seven transmembrane regions and a CFEM (common in several fungal extracellular membrane proteins) domain with the characteristic eight cysteine residues. We focused on gaining further insight into the role of the CFEM domain in the putative surface sensing/response function of Pth11. Increased/constitutive expression of CFEM resulted in precocious, albeit defective, appressoria formation in wild-type M. oryzae. The Pth11 mutant, probably with disrupted disulfide bonds in the CFEM, showed delayed appressorium formation and reduced virulence. Furthermore, the accumulation of reactive oxygen species (ROS) was found to be altered in the pth11Δ strain. Strikingly, antioxidant treatment induced appressorium formation in pth11Δ. The Gα subunit MagB and the mitogen-activated protein (MAP) kinase Pmk1 were required for the formation of antioxidant-induced appressoria. We conclude that the CFEM domain of Pth11 is required for proper development of the appressoria, appressoria-like structures and pathogenicity. Highly regulated ROS homeostasis is important for Pth11-mediated appressorium formation in M. oryzae.

In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate) highly dynamic tubulo-vesicular network, scaffold active MagA/GαS, Rgs1 (a GAP for MagA), Adenylate cyclase and Pth11 (a non-canonical GPCR) in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation) of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.

Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gαi1(G42R) binding to GDP·AlF4 − or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gβγ and GoLoco motifs in any nucleotide state. The corresponding Gαq(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.

BackgroundRgs1, a prototypical Regulator of G protein Signaling, negatively modulates the cyclic AMP pathway thereby influencing various aspects of asexual development and pathogenesis in the rice-blast fungus Magnaporthe oryzae. Rgs1 possesses tandem DEP motifs (termed DEP-A and DEP-B; for Dishevelled, Egl-10, Pleckstrin) at the N-terminus, and a Gα-GTP interacting RGS catalytic core domain at the C-terminus. In this study, we focused on gaining further insights into the mechanisms of Rgs1 regulation and subcellular localization by characterizing the role(s) of the individual domains and the full-length protein during asexual development and pathogenesis in Magnaporthe.Methodology/Principal FindingsUtilizing western blot analysis and specific antisera against the N- and C-terminal halves of Rgs1, we identify and report the in vivo endoproteolytic processing/cleavage of full-length Rgs1 that yields an N-terminal DEP and a RGS core domain. Independent expression of the resultant DEP-DEP half (N-Rgs1) or RGS core (C-Rgs1) fragments, failed to complement the rgs1Δ defects in colony morphology, aerial hyphal growth, surface hydrophobicity, conidiation, appressorium formation and infection. Interestingly, the full-length Rgs1-mCherry, as well as the tagged N-terminal DEP domains (individually or in conjunction) localized to distinct punctate vesicular structures in the cytosol, while the catalytic RGS core motif was predominantly vacuolar.Conclusions/SignificanceBased on our data from sequence alignments, immuno-blot and microscopic analysis, we propose that the post-translational proteolytic processing of Rgs1 and the vacuolar sequestration of the catalytic RGS domain represents an important means of down regulating Rgs1 function and thus forming an additional and alternative means of regulating G protein signaling in Magnaporthe. We further hypothesize the prevalence of analogous mechanisms functioning in other filamentous fungi. Furthermore, we conclusively assign a specific vesicular/membrane targeting function for the N-terminal DEP domains of Rgs1 in the rice-blast fungus.

Cell polarity is the asymmetric compartmentalization of cellular components. An opposing gradient of partitioning-defective protein kinases, atypical protein kinase C (aPKC) and PAR-1, at the cell cortex guides diverse asymmetries in the structure of metazoan cells, but the mechanism underlying their spatial patterning remains poorly understood. Here, we show in Caenorhabditis elegans zygotes that the cortical PAR-1 gradient is patterned as a consequence of dual mechanisms: stabilization of cortical dynamics and protection from aPKC-mediated cortical exclusion. Dual control of cortical PAR-1 depends on a physical interaction with the PRBH-domain protein PAR-2. Using a reconstitution approach in heterologous cells, we demonstrate that PAR-1, PAR-2, and polarized Cdc42-PAR-6-aPKC comprise the minimal network sufficient for the establishment of an opposing cortical gradient. Our findings delineate the mechanism governing cortical polarity, in which a circuit consisting of aPKC and the PRBH-domain protein ensures the local recruitment of PAR-1 to a well-defined cortical compartment.

The cAMP-dependent PKA signalling plays a central role in growth, asexual development and pathogenesis in fungal pathogens. Here, we functionally characterised RPKA, the regulatory subunit of cAMP/PKA and studied the dynamics and organisation of the PKA subunits in the rice blast pathogen Magnaporthe oryzae. The RPKA subunit was essential for proper vegetative growth, asexual sporulation and surface hydrophobicity in M. oryzae. A spontaneous suppressor mutation, SMR19, that restored growth and conidiation in the RPKA deletion mutant was isolated and characterised. SMR19 enhanced conidiation and appressorium formation but failed to suppress the pathogenesis defects in rpkAΔ. The PKA activity was undetectable in the mycelial extracts of SMR19, which showed a single mutation (val242leu) in the highly conserved active site of the catalytic subunit (CPKA) of cAMP/PKA. The two subunits of cAMP/PKA showed different subcellular localisation patterns with RpkA being predominantly nucleocytoplasmic in conidia, while CpkA was largely cytosolic and/or vesicular. The CpkA anchored RpkA in cytoplasmic vesicles, and localisation of PKA in the cytoplasm was governed by CpkA in a cAMP-dependant or independent manner. We show that there exists a tight regulation of PKA subunits at the level of transcription, and the cAMP signalling is differentially compartmentalised in a stage-specific manner in rice blast.

Cell polarity arises through the spatial segregation of polarity regulators. PAR proteins are polarity regulators that localize asymmetrically to two opposing cortical domains. However, it is unclear how the spatially segregated PAR proteins interact to maintain their mutually exclusive partitioning. Here, single-molecule detection analysis in Caenorhabditis elegans embryos reveals that cortical PAR-2 diffuses only short distances, and, as a result, most PAR-2 molecules associate and dissociate from the cortex without crossing into the opposing domain. Our results show that cortical PAR-2 asymmetry is maintained by the local exchange reactions that occur at the cortical-cytoplasmic boundary. Additionally, we demonstrate that local exchange reactions are sufficient to maintain cortical asymmetry in a parameter-free mathematical model. These findings suggest that anterior and posterior PAR proteins primarily interact through the cytoplasmic pool and not via cortical diffusion.