Date Published: August 1, 2013
Publisher: Public Library of Science
Author(s): Ravikrishna Ramanujam, Meredith E. Calvert, Poonguzhali Selvaraj, Naweed I. Naqvi, Jin-Rong Xu.
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.
Eukaryotes, ranging from yeasts to multicellular metazoans, interact with their environment, constantly sampling it for physico-chemical signals or cues for proper growth and development. Extracellular ligands or stimuli detected by membrane bound GPCR (G- protein coupled receptors) are transmitted to the cell interior by heterotrimeric (αβγ) guanine nucleotide binding proteins (G-proteins), which function as intrinsic molecular switches. Ligand activated receptors promote the exchange of GDP to GTP on cognate GαS subunit, triggering its dissociation from the βγ, thereby rendering it active to signal downstream . Both GαS·GTP and Gβγ moieties subsequently propagate the signal through a host of downstream effectors, which include ion channels, adenylate cyclases, phosphodiesterases and phospholipases , . The foremost of these is adenylate cyclase that synthesizes the second messenger Cyclic AMP (cAMP) from ATP. Active signaling by the GαS·GTP persists until the bound GTP is hydrolyzed to GDP, by the intrinsic GTPase activity of GαS, permitting GαS to re-associate with Gβγ to form an inactive complex, and thereby commencing a fresh cycle of signaling , .
Cell shape and the spatial segregation of signaling components therein collectively influence how such molecules interact to produce a timely cellular response. Spatial separation of interacting molecules, by localization to different subcellular compartments/organelles, is a widespread mechanism of regulating pathway activity , . Such a mechanism has been found to widely operate in mammalian cells, wherein signal compartmentalization is established via the anchoring of key molecules on scaffolding proteins called AKAPs.