Limits...
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.

Show MeSH

Related in: MedlinePlus

PdeH regulates the intracellular cAMP levels during growth and development in M. oryzae.Bar graphs depicting the steady-state levels of cAMP prevalent in the pdeHΔ, pdeLΔ, pdeHΔ pdeLΔ and wild type during asexual development and pathogenesis. (A) Estimation of the cAMP levels in the indicated strains during asexual development (aerial hyphae, conidiophores and conidia). The indicated strains were initially incubated in the dark (black bar; non-conidiating) for 3 d, followed by exposure to light for 24 h (white bar; conidiating) prior to cAMP measurements. Values represent mean ± S.E from two independent experiments with two replicates each. (B) Conidia from the pdeHΔ (black bar), pdeLΔ (white bar) and wild type (gray bar) strains were inoculated on inductive surface of GelBond membranes and harvested at the indicated time points for cAMP estimations. The 0 h time point represents freshly harvested conidia, while 6 h to 21 h represents the early stage of pathogenic development. Values represent mean ± S.E from three independent experiments with three replicates each. (C) The steady-state levels of cAMP were also quantified from conidia inoculated on inductive surfaces for 24, 28, or 30 h representing the late stages of pathogenic development. Values represent mean ± S.E from three separate experiments with three replicates each.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2865543&req=5

ppat-1000897-g003: PdeH regulates the intracellular cAMP levels during growth and development in M. oryzae.Bar graphs depicting the steady-state levels of cAMP prevalent in the pdeHΔ, pdeLΔ, pdeHΔ pdeLΔ and wild type during asexual development and pathogenesis. (A) Estimation of the cAMP levels in the indicated strains during asexual development (aerial hyphae, conidiophores and conidia). The indicated strains were initially incubated in the dark (black bar; non-conidiating) for 3 d, followed by exposure to light for 24 h (white bar; conidiating) prior to cAMP measurements. Values represent mean ± S.E from two independent experiments with two replicates each. (B) Conidia from the pdeHΔ (black bar), pdeLΔ (white bar) and wild type (gray bar) strains were inoculated on inductive surface of GelBond membranes and harvested at the indicated time points for cAMP estimations. The 0 h time point represents freshly harvested conidia, while 6 h to 21 h represents the early stage of pathogenic development. Values represent mean ± S.E from three independent experiments with three replicates each. (C) The steady-state levels of cAMP were also quantified from conidia inoculated on inductive surfaces for 24, 28, or 30 h representing the late stages of pathogenic development. Values represent mean ± S.E from three separate experiments with three replicates each.

Mentions: In order to assess whether the phenotypic defects in pdeHΔ were due to elevated levels of cAMP, we quantified and compared the steady-state levels of cAMP during the conidiation phase in the pdeHΔ, pdeLΔ, pdeHΔ pdeLΔ and the wild type (Figure 3A). First, we measured cAMP levels in cultures grown in the dark for 3 d and compared it to those in cultures exposed to constant illumination for a period 24 h (after a 3 d incubation in the dark). We found that even in the absence of light stimulation (darkness), the pdeHΔ strain accumulated ∼3 fold (p<0.005) and ∼2.6 fold (p<0.005) higher levels of cAMP, compared to the wild type or the pdeLΔ respectively (Figure 3A). Under similar conditions, the double deletion mutant accumulated ∼3 fold (p<0.001) higher cAMP compared to pdeHΔ and ∼10 fold higher levels compared to the wild type or pdeLΔ (p<0.001).


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)

PdeH regulates the intracellular cAMP levels during growth and development in M. oryzae.Bar graphs depicting the steady-state levels of cAMP prevalent in the pdeHΔ, pdeLΔ, pdeHΔ pdeLΔ and wild type during asexual development and pathogenesis. (A) Estimation of the cAMP levels in the indicated strains during asexual development (aerial hyphae, conidiophores and conidia). The indicated strains were initially incubated in the dark (black bar; non-conidiating) for 3 d, followed by exposure to light for 24 h (white bar; conidiating) prior to cAMP measurements. Values represent mean ± S.E from two independent experiments with two replicates each. (B) Conidia from the pdeHΔ (black bar), pdeLΔ (white bar) and wild type (gray bar) strains were inoculated on inductive surface of GelBond membranes and harvested at the indicated time points for cAMP estimations. The 0 h time point represents freshly harvested conidia, while 6 h to 21 h represents the early stage of pathogenic development. Values represent mean ± S.E from three independent experiments with three replicates each. (C) The steady-state levels of cAMP were also quantified from conidia inoculated on inductive surfaces for 24, 28, or 30 h representing the late stages of pathogenic development. Values represent mean ± S.E from three separate experiments with three replicates each.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000897-g003: PdeH regulates the intracellular cAMP levels during growth and development in M. oryzae.Bar graphs depicting the steady-state levels of cAMP prevalent in the pdeHΔ, pdeLΔ, pdeHΔ pdeLΔ and wild type during asexual development and pathogenesis. (A) Estimation of the cAMP levels in the indicated strains during asexual development (aerial hyphae, conidiophores and conidia). The indicated strains were initially incubated in the dark (black bar; non-conidiating) for 3 d, followed by exposure to light for 24 h (white bar; conidiating) prior to cAMP measurements. Values represent mean ± S.E from two independent experiments with two replicates each. (B) Conidia from the pdeHΔ (black bar), pdeLΔ (white bar) and wild type (gray bar) strains were inoculated on inductive surface of GelBond membranes and harvested at the indicated time points for cAMP estimations. The 0 h time point represents freshly harvested conidia, while 6 h to 21 h represents the early stage of pathogenic development. Values represent mean ± S.E from three independent experiments with three replicates each. (C) The steady-state levels of cAMP were also quantified from conidia inoculated on inductive surfaces for 24, 28, or 30 h representing the late stages of pathogenic development. Values represent mean ± S.E from three separate experiments with three replicates each.
Mentions: In order to assess whether the phenotypic defects in pdeHΔ were due to elevated levels of cAMP, we quantified and compared the steady-state levels of cAMP during the conidiation phase in the pdeHΔ, pdeLΔ, pdeHΔ pdeLΔ and the wild type (Figure 3A). First, we measured cAMP levels in cultures grown in the dark for 3 d and compared it to those in cultures exposed to constant illumination for a period 24 h (after a 3 d incubation in the dark). We found that even in the absence of light stimulation (darkness), the pdeHΔ strain accumulated ∼3 fold (p<0.005) and ∼2.6 fold (p<0.005) higher levels of cAMP, compared to the wild type or the pdeLΔ respectively (Figure 3A). Under similar conditions, the double deletion mutant accumulated ∼3 fold (p<0.001) higher cAMP compared to pdeHΔ and ∼10 fold higher levels compared to the wild type or pdeLΔ (p<0.001).

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