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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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Subcellular distribution of RFP-PdeH fusion protein during different stages of pathogenic and asexual development.(A) Vegetative hyphae from RFP-PdeH strain were imaged after 3 d growth on PA medium. (B) Mycelial blocks of the RFP-PdeH strain were exposed to constant illumination on fresh agar medium, and developing aerial (conidiophore) structures imaged after 24 h. Scale Bar = 10 micron. (C) Conidia harvested from the RFP-PdeH strain were inoculated on plastic cover slips and incubated in a moist chamber prior to microscopic observations. Bright field and epifluorescence images were captured at the indicated time points, using the requisite filter sets. The arrows highlight the localization pattern of RFP-PdeH fusion protein at 2 hpi (punctate) and 4 hpi (plasma membrane). Scale Bar = 10 micron. (D) RFP-PdeH conidia were inoculated on rice leaf sheath and the infection hyphae imaged after 36 h using epifluorescence microscopy. Scale Bar = 10 micron.
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ppat-1000897-g010: Subcellular distribution of RFP-PdeH fusion protein during different stages of pathogenic and asexual development.(A) Vegetative hyphae from RFP-PdeH strain were imaged after 3 d growth on PA medium. (B) Mycelial blocks of the RFP-PdeH strain were exposed to constant illumination on fresh agar medium, and developing aerial (conidiophore) structures imaged after 24 h. Scale Bar = 10 micron. (C) Conidia harvested from the RFP-PdeH strain were inoculated on plastic cover slips and incubated in a moist chamber prior to microscopic observations. Bright field and epifluorescence images were captured at the indicated time points, using the requisite filter sets. The arrows highlight the localization pattern of RFP-PdeH fusion protein at 2 hpi (punctate) and 4 hpi (plasma membrane). Scale Bar = 10 micron. (D) RFP-PdeH conidia were inoculated on rice leaf sheath and the infection hyphae imaged after 36 h using epifluorescence microscopy. Scale Bar = 10 micron.

Mentions: The expression and localization of RFP-PdeH was then examined at different stages of asexual and pathogenic development. Vegetative hyphae and developing conidiophores showed a predominantly weak cytosolic distribution of RFP-PdeH (Figure 10A and 10B). Next, we looked at the distribution of RFP-PdeH at different stages of pathogenic development (Figure 10C). Conidia were harvested from the RFP-PDEH strain, inoculated on plastic cover slips and observed using epifluorescence microscopy. In freshly harvested conidia (0 h), RFP-PdeH localized as cytosolic punctae mostly in the terminal cell of the conidium. At 2 hpi, RFP-PdeH foci were predominant in the terminal cell as well as in the developing germ tube (Figure 10C; arrow). After 4 hpi, RFP-PdeH sustained its distinct punctate localization throughout the terminal cell of the conidium and the germ tube. Furthermore, there was a notable signal although weak, from the rim of the hooking germ tube, indicating a probable association with the plasma membrane (Figure 10C; arrow). The RFP-PdeH signal was weak and indiscernible in the conidia at 6 hpi and 8 hpi, but localized as randomly distributed punctae throughout the developing appressorium, and also showed a possibly weak association with the appressorial membrane. We excluded the possibility that the membrane localization was an artifact of melanization, since tricyclazole treated RFP-PdeH appressoria retained the weak association with the plasma membrane in addition to the distinct cytosolic punctae. (Figure S5A; arrows). In mature melanized appressoria (21 hpi; Figure 10C), the RFP-PdeH displayed a predominantly vacuolar localization. The RFP-PdeH was uniformly distributed through out the cytosol in the infection hyphae within the rice leaf sheath at 36 hpi (Figure 10D). Thus, RFP-PdeH displays a predominantly cytosolic distribution during asexual differentiation, whereas it localizes as distinct cytosolic foci, (and weakly to the plasma membrane of the germ tubes) during pathogenic development. RFP-PdeH was uniformly distributed throughout the cytosol in the infection hyphae during the biotrophic phase.


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)

Subcellular distribution of RFP-PdeH fusion protein during different stages of pathogenic and asexual development.(A) Vegetative hyphae from RFP-PdeH strain were imaged after 3 d growth on PA medium. (B) Mycelial blocks of the RFP-PdeH strain were exposed to constant illumination on fresh agar medium, and developing aerial (conidiophore) structures imaged after 24 h. Scale Bar = 10 micron. (C) Conidia harvested from the RFP-PdeH strain were inoculated on plastic cover slips and incubated in a moist chamber prior to microscopic observations. Bright field and epifluorescence images were captured at the indicated time points, using the requisite filter sets. The arrows highlight the localization pattern of RFP-PdeH fusion protein at 2 hpi (punctate) and 4 hpi (plasma membrane). Scale Bar = 10 micron. (D) RFP-PdeH conidia were inoculated on rice leaf sheath and the infection hyphae imaged after 36 h using epifluorescence microscopy. Scale Bar = 10 micron.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865543&req=5

ppat-1000897-g010: Subcellular distribution of RFP-PdeH fusion protein during different stages of pathogenic and asexual development.(A) Vegetative hyphae from RFP-PdeH strain were imaged after 3 d growth on PA medium. (B) Mycelial blocks of the RFP-PdeH strain were exposed to constant illumination on fresh agar medium, and developing aerial (conidiophore) structures imaged after 24 h. Scale Bar = 10 micron. (C) Conidia harvested from the RFP-PdeH strain were inoculated on plastic cover slips and incubated in a moist chamber prior to microscopic observations. Bright field and epifluorescence images were captured at the indicated time points, using the requisite filter sets. The arrows highlight the localization pattern of RFP-PdeH fusion protein at 2 hpi (punctate) and 4 hpi (plasma membrane). Scale Bar = 10 micron. (D) RFP-PdeH conidia were inoculated on rice leaf sheath and the infection hyphae imaged after 36 h using epifluorescence microscopy. Scale Bar = 10 micron.
Mentions: The expression and localization of RFP-PdeH was then examined at different stages of asexual and pathogenic development. Vegetative hyphae and developing conidiophores showed a predominantly weak cytosolic distribution of RFP-PdeH (Figure 10A and 10B). Next, we looked at the distribution of RFP-PdeH at different stages of pathogenic development (Figure 10C). Conidia were harvested from the RFP-PDEH strain, inoculated on plastic cover slips and observed using epifluorescence microscopy. In freshly harvested conidia (0 h), RFP-PdeH localized as cytosolic punctae mostly in the terminal cell of the conidium. At 2 hpi, RFP-PdeH foci were predominant in the terminal cell as well as in the developing germ tube (Figure 10C; arrow). After 4 hpi, RFP-PdeH sustained its distinct punctate localization throughout the terminal cell of the conidium and the germ tube. Furthermore, there was a notable signal although weak, from the rim of the hooking germ tube, indicating a probable association with the plasma membrane (Figure 10C; arrow). The RFP-PdeH signal was weak and indiscernible in the conidia at 6 hpi and 8 hpi, but localized as randomly distributed punctae throughout the developing appressorium, and also showed a possibly weak association with the appressorial membrane. We excluded the possibility that the membrane localization was an artifact of melanization, since tricyclazole treated RFP-PdeH appressoria retained the weak association with the plasma membrane in addition to the distinct cytosolic punctae. (Figure S5A; arrows). In mature melanized appressoria (21 hpi; Figure 10C), the RFP-PdeH displayed a predominantly vacuolar localization. The RFP-PdeH was uniformly distributed through out the cytosol in the infection hyphae within the rice leaf sheath at 36 hpi (Figure 10D). Thus, RFP-PdeH displays a predominantly cytosolic distribution during asexual differentiation, whereas it localizes as distinct cytosolic foci, (and weakly to the plasma membrane of the germ tubes) during pathogenic development. RFP-PdeH was uniformly distributed throughout the cytosol in the infection hyphae during the biotrophic phase.

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