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Aspergillus oxylipin signaling and quorum sensing pathways depend on g protein-coupled receptors.

Affeldt KJ, Brodhagen M, Keller NP - Toxins (Basel) (2012)

Bottom Line: Here, we present evidence that oxylipins stimulate a burst in cAMP in A. nidulans, and that loss of an A. nidulans GPCR, gprD, prevents this cAMP accumulation.A. flavus undergoes an oxylipin-mediated developmental shift when grown at different densities, and this regulates spore, sclerotial and aflatoxin production.The finding of GPCRs that regulate production of survival structures (sclerotia), inoculum (spores) and aflatoxin holds promise for future development of anti-fungal therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology and Department of Medical Microbiology and Immunology, 1550 Linden Drive, Madison, WI 53706, USA.

ABSTRACT
Oxylipins regulate Aspergillus development and mycotoxin production and are also involved in Aspergillus quorum sensing mechanisms. Despite extensive knowledge of how these oxylipins are synthesized and what processes they regulate, nothing is known about how these signals are detected and transmitted by the fungus. G protein-coupled receptors (GPCR) have been speculated to be involved as they are known oxylipin receptors in mammals, and many putative GPCRs have been identified in the Aspergilli. Here, we present evidence that oxylipins stimulate a burst in cAMP in A. nidulans, and that loss of an A. nidulans GPCR, gprD, prevents this cAMP accumulation. A. flavus undergoes an oxylipin-mediated developmental shift when grown at different densities, and this regulates spore, sclerotial and aflatoxin production. A. flavus encodes two putative GprD homologs, GprC and GprD, and we demonstrate here that they are required to transition to a high-density development state, as well as to respond to spent medium of a high-density culture. The finding of GPCRs that regulate production of survival structures (sclerotia), inoculum (spores) and aflatoxin holds promise for future development of anti-fungal therapeutics.

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Related in: MedlinePlus

(a) The general scheme for deleting A. flavus gprC and gprD by replacing with pyrG is shown here. The light blue bar represents the region amplified for the Southern probe; (b) The plasmid used to deplete A. flavus gprC and gprD transcripts, pKJA27, is depicted here. The light blue bar represents the region amplified for the Southern probe; (c) The ∆gprC::pyrG strain, TKJA10.1, was confirmed by Southern analysis. Genomic DNA was digested with SacI (WT expected bands: 5.2 and 2.1 kb; ∆gprC expected bands: 4.9, 2.1, and 1.1 kb) and XhoI (WT expected band: 5.2 kb; ∆gprC expected bands: 3.8 and 2.1 kb); (d) The ∆gprD::pyrG strain, TKJA8.1, was confirmed by Southern analysis. Genomic DNA was digested with XhoI (WT expected bands: 6.1 and 2.3 kb; ∆gprD expected bands: 5.8, 2.3, and 1.1 kb) and SphI (WT expected bands: 6.5, 6.1 (faint), and 1.3 kb; ∆gprD expected bands: 8.6 and 6.1 (faint) kb); (e) The KD::gprCD strain, TKJA14.2, was confirmed by Southern analysis. The gpdA promoter is derived from A. nidulans, so the probe will only hybridize if pKJA27 is present. Genomic DNA was digested with StuI (WT and parental strain 3357.5 (denoted “P”) should have no bands; transformants should have one band for each copy of the plasmid they integrated); (f) The KD::gprCD strain, TKJA14.2, was confirmed by Northern analysis. Probes within the coding regions of gprC and gprD were used, and correct transformants were identified by the smear of degraded transcripts, seen for transformants #14 and #15.
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toxins-04-00695-f009: (a) The general scheme for deleting A. flavus gprC and gprD by replacing with pyrG is shown here. The light blue bar represents the region amplified for the Southern probe; (b) The plasmid used to deplete A. flavus gprC and gprD transcripts, pKJA27, is depicted here. The light blue bar represents the region amplified for the Southern probe; (c) The ∆gprC::pyrG strain, TKJA10.1, was confirmed by Southern analysis. Genomic DNA was digested with SacI (WT expected bands: 5.2 and 2.1 kb; ∆gprC expected bands: 4.9, 2.1, and 1.1 kb) and XhoI (WT expected band: 5.2 kb; ∆gprC expected bands: 3.8 and 2.1 kb); (d) The ∆gprD::pyrG strain, TKJA8.1, was confirmed by Southern analysis. Genomic DNA was digested with XhoI (WT expected bands: 6.1 and 2.3 kb; ∆gprD expected bands: 5.8, 2.3, and 1.1 kb) and SphI (WT expected bands: 6.5, 6.1 (faint), and 1.3 kb; ∆gprD expected bands: 8.6 and 6.1 (faint) kb); (e) The KD::gprCD strain, TKJA14.2, was confirmed by Southern analysis. The gpdA promoter is derived from A. nidulans, so the probe will only hybridize if pKJA27 is present. Genomic DNA was digested with StuI (WT and parental strain 3357.5 (denoted “P”) should have no bands; transformants should have one band for each copy of the plasmid they integrated); (f) The KD::gprCD strain, TKJA14.2, was confirmed by Northern analysis. Probes within the coding regions of gprC and gprD were used, and correct transformants were identified by the smear of degraded transcripts, seen for transformants #14 and #15.

Mentions: Individual deletion mutants of both gprC and gprD were created by replacing each gene with pyrG (Table 1, Figure S3). A third strain, KD::gprCD (for “knock-down of gprC and gprD”) with both genes down-regulated by RNAi [52], was also created with the thought that the two proteins may have overlapping function due to their high identity (Table 1, Figure S3). All mutants were confirmed with Southern blots, and in the case of KD::gprCD, Northern blots as well (Figure S3).


Aspergillus oxylipin signaling and quorum sensing pathways depend on g protein-coupled receptors.

Affeldt KJ, Brodhagen M, Keller NP - Toxins (Basel) (2012)

(a) The general scheme for deleting A. flavus gprC and gprD by replacing with pyrG is shown here. The light blue bar represents the region amplified for the Southern probe; (b) The plasmid used to deplete A. flavus gprC and gprD transcripts, pKJA27, is depicted here. The light blue bar represents the region amplified for the Southern probe; (c) The ∆gprC::pyrG strain, TKJA10.1, was confirmed by Southern analysis. Genomic DNA was digested with SacI (WT expected bands: 5.2 and 2.1 kb; ∆gprC expected bands: 4.9, 2.1, and 1.1 kb) and XhoI (WT expected band: 5.2 kb; ∆gprC expected bands: 3.8 and 2.1 kb); (d) The ∆gprD::pyrG strain, TKJA8.1, was confirmed by Southern analysis. Genomic DNA was digested with XhoI (WT expected bands: 6.1 and 2.3 kb; ∆gprD expected bands: 5.8, 2.3, and 1.1 kb) and SphI (WT expected bands: 6.5, 6.1 (faint), and 1.3 kb; ∆gprD expected bands: 8.6 and 6.1 (faint) kb); (e) The KD::gprCD strain, TKJA14.2, was confirmed by Southern analysis. The gpdA promoter is derived from A. nidulans, so the probe will only hybridize if pKJA27 is present. Genomic DNA was digested with StuI (WT and parental strain 3357.5 (denoted “P”) should have no bands; transformants should have one band for each copy of the plasmid they integrated); (f) The KD::gprCD strain, TKJA14.2, was confirmed by Northern analysis. Probes within the coding regions of gprC and gprD were used, and correct transformants were identified by the smear of degraded transcripts, seen for transformants #14 and #15.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475224&req=5

toxins-04-00695-f009: (a) The general scheme for deleting A. flavus gprC and gprD by replacing with pyrG is shown here. The light blue bar represents the region amplified for the Southern probe; (b) The plasmid used to deplete A. flavus gprC and gprD transcripts, pKJA27, is depicted here. The light blue bar represents the region amplified for the Southern probe; (c) The ∆gprC::pyrG strain, TKJA10.1, was confirmed by Southern analysis. Genomic DNA was digested with SacI (WT expected bands: 5.2 and 2.1 kb; ∆gprC expected bands: 4.9, 2.1, and 1.1 kb) and XhoI (WT expected band: 5.2 kb; ∆gprC expected bands: 3.8 and 2.1 kb); (d) The ∆gprD::pyrG strain, TKJA8.1, was confirmed by Southern analysis. Genomic DNA was digested with XhoI (WT expected bands: 6.1 and 2.3 kb; ∆gprD expected bands: 5.8, 2.3, and 1.1 kb) and SphI (WT expected bands: 6.5, 6.1 (faint), and 1.3 kb; ∆gprD expected bands: 8.6 and 6.1 (faint) kb); (e) The KD::gprCD strain, TKJA14.2, was confirmed by Southern analysis. The gpdA promoter is derived from A. nidulans, so the probe will only hybridize if pKJA27 is present. Genomic DNA was digested with StuI (WT and parental strain 3357.5 (denoted “P”) should have no bands; transformants should have one band for each copy of the plasmid they integrated); (f) The KD::gprCD strain, TKJA14.2, was confirmed by Northern analysis. Probes within the coding regions of gprC and gprD were used, and correct transformants were identified by the smear of degraded transcripts, seen for transformants #14 and #15.
Mentions: Individual deletion mutants of both gprC and gprD were created by replacing each gene with pyrG (Table 1, Figure S3). A third strain, KD::gprCD (for “knock-down of gprC and gprD”) with both genes down-regulated by RNAi [52], was also created with the thought that the two proteins may have overlapping function due to their high identity (Table 1, Figure S3). All mutants were confirmed with Southern blots, and in the case of KD::gprCD, Northern blots as well (Figure S3).

Bottom Line: Here, we present evidence that oxylipins stimulate a burst in cAMP in A. nidulans, and that loss of an A. nidulans GPCR, gprD, prevents this cAMP accumulation.A. flavus undergoes an oxylipin-mediated developmental shift when grown at different densities, and this regulates spore, sclerotial and aflatoxin production.The finding of GPCRs that regulate production of survival structures (sclerotia), inoculum (spores) and aflatoxin holds promise for future development of anti-fungal therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology and Department of Medical Microbiology and Immunology, 1550 Linden Drive, Madison, WI 53706, USA.

ABSTRACT
Oxylipins regulate Aspergillus development and mycotoxin production and are also involved in Aspergillus quorum sensing mechanisms. Despite extensive knowledge of how these oxylipins are synthesized and what processes they regulate, nothing is known about how these signals are detected and transmitted by the fungus. G protein-coupled receptors (GPCR) have been speculated to be involved as they are known oxylipin receptors in mammals, and many putative GPCRs have been identified in the Aspergilli. Here, we present evidence that oxylipins stimulate a burst in cAMP in A. nidulans, and that loss of an A. nidulans GPCR, gprD, prevents this cAMP accumulation. A. flavus undergoes an oxylipin-mediated developmental shift when grown at different densities, and this regulates spore, sclerotial and aflatoxin production. A. flavus encodes two putative GprD homologs, GprC and GprD, and we demonstrate here that they are required to transition to a high-density development state, as well as to respond to spent medium of a high-density culture. The finding of GPCRs that regulate production of survival structures (sclerotia), inoculum (spores) and aflatoxin holds promise for future development of anti-fungal therapeutics.

Show MeSH
Related in: MedlinePlus