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PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae.

Ramanujam R, Naqvi NI - PLoS Pathog. (2010)

Bottom Line: 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.A pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 10 fold) cAMP levels relative to the wild type, and was completely defective in virulence.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.

View Article: PubMed Central - PubMed

Affiliation: Fungal Patho-Biology Group, Temasek Life Sciences Laboratory, Singapore.

ABSTRACT
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 pdeHDelta 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 pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 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 pdeHDelta 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.

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Localization and dynamic nature of the PROMpg1-GFP-PdeH during various stages of development in M. oryzae.(A) The PROMpg1-GFP-PdeH localized to the cytosol in the vegetative hyphae and (B) developing aerial structures (conidiophore). Scale Bar = 10 micron. (C), (D) and (E) Snapshots extracted from time-lapse movies (supplemental movies), showing the highly dynamic PROMpg1-GFP-PdeH, during different stages (specified sequentially) of pathogenic development. Scale Bar = 10 micron.
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ppat-1000897-g011: Localization and dynamic nature of the PROMpg1-GFP-PdeH during various stages of development in M. oryzae.(A) The PROMpg1-GFP-PdeH localized to the cytosol in the vegetative hyphae and (B) developing aerial structures (conidiophore). Scale Bar = 10 micron. (C), (D) and (E) Snapshots extracted from time-lapse movies (supplemental movies), showing the highly dynamic PROMpg1-GFP-PdeH, during different stages (specified sequentially) of pathogenic development. Scale Bar = 10 micron.

Mentions: In order to better visualize the intracellular distribution and dynamics of PdeH, we expressed a GFP-PDEH translational fusion construct driven by the MPG1 promoter [82] in the pdeHΔ strain. Similarly, we generated a strain expressing GFP-PdeL fusion protein under the MPG1 promoter. We examined the expression and localization patterns of the PROMpg1-GFP-PdeH fusion protein during different stages of asexual and pathogenic development. Compared to the weak RFP-PdeH signal, the PROMpg1-GFP-PdeH showed a relatively strong cytoplasmic signal in the vegetative mycelia (Figure 11A). During asexual development, the GFP-PdeH fusion protein was predominantly cytosolic (Figure 11B), a pattern comparable to that displayed by the RFP-PdeH strain at a similar stage of development. To gain further insight into the dynamics, we made time-lapse observations of the PROMpg1-GFP-PdeH at different stages of pathogenic development encompassing the following time points: 2–3 hpi (conidial germination; Video S1 and Figure 11C), 4–5 hpi (hooking stage; Video S2 and Figure 11D), and 6–7 hpi (appressorium development; Video S3 and Figure 11E). Time-lapse analysis revealed that the GFP-PdeH fusion protein was associated with vesicular structures, which were highly dynamic and mobile (Video S1 and Figure 11C). Furthermore, co-staining with a nuclear dye (Hoechst 33342) confirmed a peri- and extra- nuclear localization of the GFP-PdeH foci (Figure S5B). At 4–5 hpi, GFP-PdeH localized to regions of the plasma membrane of the hooking germ tube in addition to being associated with highly mobile vesicles shuttling between the conidium and the germ tube (Video S2 and Figure 11D). In addition, GFP-PdeH was vesicular and enriched at the plasma membrane of the appressorium at 6 hpi (Video S3 and Figure 11E).


PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae.

Ramanujam R, Naqvi NI - PLoS Pathog. (2010)

Localization and dynamic nature of the PROMpg1-GFP-PdeH during various stages of development in M. oryzae.(A) The PROMpg1-GFP-PdeH localized to the cytosol in the vegetative hyphae and (B) developing aerial structures (conidiophore). Scale Bar = 10 micron. (C), (D) and (E) Snapshots extracted from time-lapse movies (supplemental movies), showing the highly dynamic PROMpg1-GFP-PdeH, during different stages (specified sequentially) of pathogenic development. Scale Bar = 10 micron.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865543&req=5

ppat-1000897-g011: Localization and dynamic nature of the PROMpg1-GFP-PdeH during various stages of development in M. oryzae.(A) The PROMpg1-GFP-PdeH localized to the cytosol in the vegetative hyphae and (B) developing aerial structures (conidiophore). Scale Bar = 10 micron. (C), (D) and (E) Snapshots extracted from time-lapse movies (supplemental movies), showing the highly dynamic PROMpg1-GFP-PdeH, during different stages (specified sequentially) of pathogenic development. Scale Bar = 10 micron.
Mentions: In order to better visualize the intracellular distribution and dynamics of PdeH, we expressed a GFP-PDEH translational fusion construct driven by the MPG1 promoter [82] in the pdeHΔ strain. Similarly, we generated a strain expressing GFP-PdeL fusion protein under the MPG1 promoter. We examined the expression and localization patterns of the PROMpg1-GFP-PdeH fusion protein during different stages of asexual and pathogenic development. Compared to the weak RFP-PdeH signal, the PROMpg1-GFP-PdeH showed a relatively strong cytoplasmic signal in the vegetative mycelia (Figure 11A). During asexual development, the GFP-PdeH fusion protein was predominantly cytosolic (Figure 11B), a pattern comparable to that displayed by the RFP-PdeH strain at a similar stage of development. To gain further insight into the dynamics, we made time-lapse observations of the PROMpg1-GFP-PdeH at different stages of pathogenic development encompassing the following time points: 2–3 hpi (conidial germination; Video S1 and Figure 11C), 4–5 hpi (hooking stage; Video S2 and Figure 11D), and 6–7 hpi (appressorium development; Video S3 and Figure 11E). Time-lapse analysis revealed that the GFP-PdeH fusion protein was associated with vesicular structures, which were highly dynamic and mobile (Video S1 and Figure 11C). Furthermore, co-staining with a nuclear dye (Hoechst 33342) confirmed a peri- and extra- nuclear localization of the GFP-PdeH foci (Figure S5B). At 4–5 hpi, GFP-PdeH localized to regions of the plasma membrane of the hooking germ tube in addition to being associated with highly mobile vesicles shuttling between the conidium and the germ tube (Video S2 and Figure 11D). In addition, GFP-PdeH was vesicular and enriched at the plasma membrane of the appressorium at 6 hpi (Video S3 and Figure 11E).

Bottom Line: 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.A pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 10 fold) cAMP levels relative to the wild type, and was completely defective in virulence.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.

View Article: PubMed Central - PubMed

Affiliation: Fungal Patho-Biology Group, Temasek Life Sciences Laboratory, Singapore.

ABSTRACT
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 pdeHDelta 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 pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 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 pdeHDelta 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.

Show MeSH
Related in: MedlinePlus