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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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PDEH transcript is differentially regulated during infection-related development.Real Time RTPCR analysis and quantification of PDEH and PDEL transcript abundance in wild type and the indicated PDE mutants. (A) Total RNA from wild-type conidia inoculated on inductive Gelbond membrane for the indicated time points was subjected to Real-Time RTPCR-based quantification of PDEH (black bar) or PDEL (white bar) transcript. The expression levels at 0 h were considered as baseline and set as 1.0 (B) The levels of the PDEH (black bar) or PDEL (white bar) transcript was analyzed at the late stage of pathogenesis (24 h, 29 h and 48 h) in wild type M. oryzae inoculated on barley leaf explants. MGG_04795 was used as a positive control (gray bar). The expression levels at 21 h were set as 1.0 (C) Comparative quantitative analysis of PDEH transcript in the wild type (black bar) and pdeLΔ (white bar) using Real Time RTPCR at the specified time points. (D) PDEL transcript levels assessed in the wild type (black bar) and pdeHΔ (white bar) using Real Time RTPCR. In addition, the Real Time expression data was normalized to an endogenous control transcript (β-tubulin gene, MGG_00604). Each Real Time RTPCR reaction was repeated thrice independently with three biological replicates per data set.
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ppat-1000897-g008: PDEH transcript is differentially regulated during infection-related development.Real Time RTPCR analysis and quantification of PDEH and PDEL transcript abundance in wild type and the indicated PDE mutants. (A) Total RNA from wild-type conidia inoculated on inductive Gelbond membrane for the indicated time points was subjected to Real-Time RTPCR-based quantification of PDEH (black bar) or PDEL (white bar) transcript. The expression levels at 0 h were considered as baseline and set as 1.0 (B) The levels of the PDEH (black bar) or PDEL (white bar) transcript was analyzed at the late stage of pathogenesis (24 h, 29 h and 48 h) in wild type M. oryzae inoculated on barley leaf explants. MGG_04795 was used as a positive control (gray bar). The expression levels at 21 h were set as 1.0 (C) Comparative quantitative analysis of PDEH transcript in the wild type (black bar) and pdeLΔ (white bar) using Real Time RTPCR at the specified time points. (D) PDEL transcript levels assessed in the wild type (black bar) and pdeHΔ (white bar) using Real Time RTPCR. In addition, the Real Time expression data was normalized to an endogenous control transcript (β-tubulin gene, MGG_00604). Each Real Time RTPCR reaction was repeated thrice independently with three biological replicates per data set.

Mentions: In M. oryzae, cAMP signaling is known to regulate appressorium initiation and development [69], [71]. Extensive work carried out on PDEs in mammalian cells and in Dictyostelium, has described the existence of feedback loops (positive and negative) between varying cAMP levels and PDE gene expression [77], [78]. We were interested in elucidating if PDE transcripts were differentially regulated in response to fluctuating cAMP levels during pathogenic development in M. oryzae. Quantative Real-Time RTPCR analysis was used to measure the PDE transcript levels during different developmental stages during infection. The time points and tissues used were 0 h (freshly harvested conidia), 3 h (conidial germination and growth) and 6 h (appressorium initials). In addition, we also performed Real-Time RTPCR analysis on inoculated barley leaves at different stages of in planta growth. We included MGG_04795 (BAS1) as a positive control, since it has been shown to be highly upregulated during infection [79]. Comparative analysis of the Real-Time RTPCR data showed that PDEH transcript levels were significantly down regulated during the early stages of pathogenic development at 3 h (2.5 fold) and at 6 h (2 fold) compared to freshly harvested wild-type conidia (Figure 8A). A similar trend of reduction, 1.4 fold at 3 h and 2.5 fold at 6 h, was evident for the PDEL transcript. Compared to the 21 h time point, the PDEH transcript levels were consistently higher across all other time points tested: 1.5 fold at 24 h, 3 fold at 29 h and 5 fold at 48 h (Figure 8B). However, the levels of PDEL transcript did not show significant changes at similar time points. This suggests that the PDEH transcript levels are differentially regulated during early as well as the late stages of pathogenic differentiation in M. oryzae, likely in response to the varying cAMP levels at these stages of development.


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 transcript is differentially regulated during infection-related development.Real Time RTPCR analysis and quantification of PDEH and PDEL transcript abundance in wild type and the indicated PDE mutants. (A) Total RNA from wild-type conidia inoculated on inductive Gelbond membrane for the indicated time points was subjected to Real-Time RTPCR-based quantification of PDEH (black bar) or PDEL (white bar) transcript. The expression levels at 0 h were considered as baseline and set as 1.0 (B) The levels of the PDEH (black bar) or PDEL (white bar) transcript was analyzed at the late stage of pathogenesis (24 h, 29 h and 48 h) in wild type M. oryzae inoculated on barley leaf explants. MGG_04795 was used as a positive control (gray bar). The expression levels at 21 h were set as 1.0 (C) Comparative quantitative analysis of PDEH transcript in the wild type (black bar) and pdeLΔ (white bar) using Real Time RTPCR at the specified time points. (D) PDEL transcript levels assessed in the wild type (black bar) and pdeHΔ (white bar) using Real Time RTPCR. In addition, the Real Time expression data was normalized to an endogenous control transcript (β-tubulin gene, MGG_00604). Each Real Time RTPCR reaction was repeated thrice independently with three biological replicates per data set.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865543&req=5

ppat-1000897-g008: PDEH transcript is differentially regulated during infection-related development.Real Time RTPCR analysis and quantification of PDEH and PDEL transcript abundance in wild type and the indicated PDE mutants. (A) Total RNA from wild-type conidia inoculated on inductive Gelbond membrane for the indicated time points was subjected to Real-Time RTPCR-based quantification of PDEH (black bar) or PDEL (white bar) transcript. The expression levels at 0 h were considered as baseline and set as 1.0 (B) The levels of the PDEH (black bar) or PDEL (white bar) transcript was analyzed at the late stage of pathogenesis (24 h, 29 h and 48 h) in wild type M. oryzae inoculated on barley leaf explants. MGG_04795 was used as a positive control (gray bar). The expression levels at 21 h were set as 1.0 (C) Comparative quantitative analysis of PDEH transcript in the wild type (black bar) and pdeLΔ (white bar) using Real Time RTPCR at the specified time points. (D) PDEL transcript levels assessed in the wild type (black bar) and pdeHΔ (white bar) using Real Time RTPCR. In addition, the Real Time expression data was normalized to an endogenous control transcript (β-tubulin gene, MGG_00604). Each Real Time RTPCR reaction was repeated thrice independently with three biological replicates per data set.
Mentions: In M. oryzae, cAMP signaling is known to regulate appressorium initiation and development [69], [71]. Extensive work carried out on PDEs in mammalian cells and in Dictyostelium, has described the existence of feedback loops (positive and negative) between varying cAMP levels and PDE gene expression [77], [78]. We were interested in elucidating if PDE transcripts were differentially regulated in response to fluctuating cAMP levels during pathogenic development in M. oryzae. Quantative Real-Time RTPCR analysis was used to measure the PDE transcript levels during different developmental stages during infection. The time points and tissues used were 0 h (freshly harvested conidia), 3 h (conidial germination and growth) and 6 h (appressorium initials). In addition, we also performed Real-Time RTPCR analysis on inoculated barley leaves at different stages of in planta growth. We included MGG_04795 (BAS1) as a positive control, since it has been shown to be highly upregulated during infection [79]. Comparative analysis of the Real-Time RTPCR data showed that PDEH transcript levels were significantly down regulated during the early stages of pathogenic development at 3 h (2.5 fold) and at 6 h (2 fold) compared to freshly harvested wild-type conidia (Figure 8A). A similar trend of reduction, 1.4 fold at 3 h and 2.5 fold at 6 h, was evident for the PDEL transcript. Compared to the 21 h time point, the PDEH transcript levels were consistently higher across all other time points tested: 1.5 fold at 24 h, 3 fold at 29 h and 5 fold at 48 h (Figure 8B). However, the levels of PDEL transcript did not show significant changes at similar time points. This suggests that the PDEH transcript levels are differentially regulated during early as well as the late stages of pathogenic differentiation in M. oryzae, likely in response to the varying cAMP levels at these stages of development.

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