Regulation of cyclic AMP signaling during asexual development and pathogenesis in Magnaporthe oryzae.
Date of Issue2013
School of Biological Sciences
Temasek Lifesciences Laboratory
The conidial germ tube in Magnaporthe oryzae, the causal agent of the devastating rice-blast disease, responds to inductive surface cues and switches from polarized to isotropic growth to ultimately form the specialized infection structure known as the appressorium. The critical role played by signaling pathways, especially the G-protein/ cyclic AMP (cAMP) signaling in appressorium initiation was identified almost two decades ago, yet several fundamental questions remain unanswered. The purpose of this study was to 1) identify additional regulatory proteins that facilitate the maintenance of steady-state levels of cAMP; 2) decipher in-vivo mechanisms that function to control the activity of such regulatory proteins; 3) investigate the spatial and temporal dynamics of the cAMP signaling cascade and finally 4) understand how the signals are transmitted through the crowded intracellular network to specific sites of growth and/or signaling. Utilizing a combination of reverse genetics, biochemical analyses and cell biology techniques, in Chapter I, I identified and functionally characterized two cAMP phosphodiesterases (a high affinity- PdeH and a low affinity PdeL) in M. oryzae. I also demonstrate that PdeH is the major enzyme hydrolyzing and regulating intracellular levels of cAMP during asexual, pathogenic and in planta development. More importantly, the study identifies that cAMP levels are modulated by PdeH in a biphasic manner, i.e. up regulation during early stages (appressorium initiation), and down regulation at the late stages (host invasion) of pathogenic development. The data further implicates the existence of two distinct pools of cAMP (cytosolic and nuclear) in M. oryzae, which are differentially modulated by PdeH and PdeL respectively. Given that Regulator of G-protein signaling (RGS) proteins function as key negative regulators that control the intensity and duration of active G-protein signaling, in Chapter II, I sought to address and understand the mechanisms possibly involved in the regulation of a proto-typical member, namely Rgs1(a GAP for MagA). I found that Rgs1 was subjected to an endo-proteolytic cleavage yielding an N-terminal DEP-DEP fragment (that facilitated membrane targeting) and a C-terminal catalytic core RGS domain that was sequestered within vacuole in the absence of a functional DEP motif. Rgs1 cleavage likely represents an important means of ultimately modulating the duration and intensity of G-protein signaling. Based on the physical interaction with Rgs1 specifically during early stages of appressorium initiation, I demonstrate Pth11 to be a bona fide GPCR (albeit non-canonical) for cAMP signaling in M. oryzae. Finally, in Chapter III, I demonstrate that in the likely absence of AKAP-like anchor proteins in M. oryzae, the late endosomal compartments (comprising of a PI3P-rich dynamic tubulo-vesicular network) are capable of serving as scaffolds or platforms to anchor active MagA/GαS, Rgs1, Adenylate cyclase and Pth11. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Exogenous cAMP remarkably rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae, suggesting that the late endosomal HOPS complex potentially serves as a scaffold to anchor, compartmentalize and mobilize G-protein signaling components to sites of active growth and signaling, during early pathogenesis. Taken together, the results presented in this thesis suggest that M. oryzae utilizes a variety of complex cellular mechanisms to tightly regulate the steady-state levels as well as modulate the spatio-temporal dynamics of cAMP signaling in response to inductive cues as well as in the context of changing cellular geometry