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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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Loss of PdeH function advances appressorium formation and maturity.Comparative time-lapse observation of germination and appressorium formation in the wild-type or pdeHΔ conidia. Equivalent number of conidia from the indicated strains were inoculated on cover slips and incubated in a moist chamber at room temperature. The samples were analysed and micrographed every hour over an 8 h period. White arrows indicate the stage of appressorium initiation/germ tube hooking, whereas black arrows depict melanized appressoria. Scale bar = 10 micron.
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ppat-1000897-g005: Loss of PdeH function advances appressorium formation and maturity.Comparative time-lapse observation of germination and appressorium formation in the wild-type or pdeHΔ conidia. Equivalent number of conidia from the indicated strains were inoculated on cover slips and incubated in a moist chamber at room temperature. The samples were analysed and micrographed every hour over an 8 h period. White arrows indicate the stage of appressorium initiation/germ tube hooking, whereas black arrows depict melanized appressoria. Scale bar = 10 micron.

Mentions: We observed that the loss of PdeH derails cAMP-associated surface signaling and significantly accelerated appressorium formation. Time-course analysis revealed that pdeHΔ is significantly advanced in all stages of pathogenic development. On inductive surfaces, the wild type conidia underwent germ tube hooking at 3–4 hpi (Figure 5; white arrow), followed by tip swelling and growth into an immature appressoria by 5–6 hpi. By 8 hpi, the wild type formed melanized appressoria (Figure 5; black arrow). Under identical conditions, the pdeHΔ conidia initiated germ tube hooking as early as 2 hpi (Figure 5; white arrow), formed immature appressoria by 3–4 hours and finally melanized appressoria in about 5 hours (Figure 5; black arrow). Thus, the pdeHΔ not only initiated appressoria earlier but also formed melanized appressoria more rapidly, at least 3 h in advance compared to the wild type. The pdeLΔ behaved similar to the wild type taking about 8 hours to form melanized appressoria. Our findings suggest that precocious elevated cAMP levels can disrupt the temporal regulation of the processes that lead to proper initiation, development and maturation of appressoria in M. oryzae.


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)

Loss of PdeH function advances appressorium formation and maturity.Comparative time-lapse observation of germination and appressorium formation in the wild-type or pdeHΔ conidia. Equivalent number of conidia from the indicated strains were inoculated on cover slips and incubated in a moist chamber at room temperature. The samples were analysed and micrographed every hour over an 8 h period. White arrows indicate the stage of appressorium initiation/germ tube hooking, whereas black arrows depict melanized appressoria. Scale bar = 10 micron.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000897-g005: Loss of PdeH function advances appressorium formation and maturity.Comparative time-lapse observation of germination and appressorium formation in the wild-type or pdeHΔ conidia. Equivalent number of conidia from the indicated strains were inoculated on cover slips and incubated in a moist chamber at room temperature. The samples were analysed and micrographed every hour over an 8 h period. White arrows indicate the stage of appressorium initiation/germ tube hooking, whereas black arrows depict melanized appressoria. Scale bar = 10 micron.
Mentions: We observed that the loss of PdeH derails cAMP-associated surface signaling and significantly accelerated appressorium formation. Time-course analysis revealed that pdeHΔ is significantly advanced in all stages of pathogenic development. On inductive surfaces, the wild type conidia underwent germ tube hooking at 3–4 hpi (Figure 5; white arrow), followed by tip swelling and growth into an immature appressoria by 5–6 hpi. By 8 hpi, the wild type formed melanized appressoria (Figure 5; black arrow). Under identical conditions, the pdeHΔ conidia initiated germ tube hooking as early as 2 hpi (Figure 5; white arrow), formed immature appressoria by 3–4 hours and finally melanized appressoria in about 5 hours (Figure 5; black arrow). Thus, the pdeHΔ not only initiated appressoria earlier but also formed melanized appressoria more rapidly, at least 3 h in advance compared to the wild type. The pdeLΔ behaved similar to the wild type taking about 8 hours to form melanized appressoria. Our findings suggest that precocious elevated cAMP levels can disrupt the temporal regulation of the processes that lead to proper initiation, development and maturation of appressoria in M. oryzae.

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