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. 2017 Apr;163(4):541-553.
doi: 10.1099/mic.0.000396. Epub 2017 Apr 6.

Unravelling the biosynthesis of pyriculol in the rice blast fungus Magnaporthe oryzae

Affiliations

Unravelling the biosynthesis of pyriculol in the rice blast fungus Magnaporthe oryzae

Stefan Jacob et al. Microbiology (Reading). 2017 Apr.

Abstract

Pyriculol was isolated from the rice blast fungus Magnaporthe oryzae and found to induce lesion formation on rice leaves. These findings suggest that it could be involved in virulence. The gene MoPKS19 was identified to encode a polyketide synthase essential for the production of the polyketide pyriculol in the rice blast fungus M. oryzae. The transcript abundance of MoPKS19 correlates with the biosynthesis rate of pyriculol in a time-dependent manner. Furthermore, gene inactivation of MoPKS19 resulted in a mutant unable to produce pyriculol, pyriculariol and their dihydro derivatives. Inactivation of a putative oxidase-encoding gene MoC19OXR1, which was found to be located in the genome close to MoPKS19, resulted in a mutant exclusively producing dihydropyriculol and dihydropyriculariol. By contrast, overexpression of MoC19OXR1 resulted in a mutant strain only producing pyriculol. The MoPKS19 cluster, furthermore, comprises two transcription factors MoC19TRF1 and MoC19TRF2, which were both found individually to act as negative regulators repressing gene expression of MoPKS19. Additionally, extracts of ΔMopks19 and ΔMoC19oxr1 made from axenic cultures failed to induce lesions on rice leaves compared to extracts of the wild-type strain. Consequently, pyriculol and its isomer pyriculariol appear to be the only lesion-inducing secondary metabolites produced by M. oryzae wild-type (MoWT) under these culture conditions. Interestingly, the mutants unable to produce pyriculol and pyriculariol were as pathogenic as MoWT, demonstrating that pyriculol is not required for infection.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
Secondary metabolites produced by M. oryzae 70-15 (MoWT). The structures of the major polyketides found in extracts of MoWT after fermentation in MM or in REM are shown.
Fig. 2.
Fig. 2.
qRT-PCR analysis of the expression level from genes encoding reducing non-methylating PKS in MoWT. The M. oryzae cultures were grown in CM for 72 h at 26 °C and 120 r.p.m. The mycelium was then transferred to MM, REM or CM for further submersed cultivation at 26 °C and 120 r.p.m. Samples were taken after 2 and 4 h and RNA was isolated from the mycelium samples for qRT-PCR analysis. The results of transcript abundance in MM or REM are given relative to quantification in CM. The experiments were conducted in triplicate. CM, complete medium; MM, minimal medium; REM, rice-extract medium.
Fig. 3.
Fig. 3.
Time-dependent HPLC analysis of culture filtrate extracts from MoWT in correlation to qRT-PCR analysis of the expression level from the MoPKS19 gene. The M. oryzae cultures were grown as described in Methods. Samples were taken 1, 2, 4, 8 and 24 h after transfer to REM. The HPLC analysis was conducted using extracts of the culture broth, and RNA was isolated from the mycelium samples. The results of transcript abundance in REM are given relative to quantification in CM. The experiments were conducted in triplicate. CM, complete medium; REM, rice-extract medium; mAU, milli-absorption units. Bars represent (+/−) sem.
Fig. 4.
Fig. 4.
Schematic presentation of the MoPKS19 gene cluster in the genome of MoWT. (a) A scheme of 30 ORFs neighbouring the MoPKS19 gene is presented. The genes are shown with their position either on the ‘sense (5′–3′)’ or on the ‘antisense (3′–5′)’ strand. White arrows imply genes with no obvious functions with significant use in this study (e.g. hypothetical proteins without predicted protein domains). (b) Schematic presentation of protein domains of MoPks19p. KS, β-Ketoacyl synthase; AT, acyltransferase; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; ACP, ACP domain.
Fig. 5.
Fig. 5.
Analysis of polyketide in culture filtrate extracts from M. oryzae and phytotoxic activity of extracts and pure compounds. (a) HPLC chromatograms of extracts from MoWT, ΔMopks19, ΔMopks19/MoPKS19, ΔMoC19oxr1 and the overexpression mutant MoEF1 :: C19OXR1. The M. oryzae cultures were grown as described in Methods. Samples were taken 8 h after transfer to REM. The HPLC analysis was conducted using extracts of the culture broth (210 nm wavelength). mAU, Milli-absorption units. Phytotoxic activity of the extracts towards rice leaves is shown below each chromatogram. The assays were conducted as described in Methods. (b) Phytotoxicity was monitored for the pure compounds under equal conditions.
Fig. 6.
Fig. 6.
(a) qRT-PCR analysis of the expression level from the oxidase/reductase-encoding genes in the putative MoPKS19 gene cluster. The M. oryzae cultures were grown for 72 h in CM at 26 °C and 120 r.p.m. The mycelium was transferred for further submersed cultivation to REM at 26 °C and 120 r.p.m. Samples were taken before (CM control) and 8 h after the transfer to REM. The results of transcript abundance are given relative to quantification of the MoEF1 gene in the MoWT. Three replicates were made of each. (b) qRT-PCR analysis of the expression level from the MoPKS19 gene in the mutant strains. The M. oryzae cultures were grown for 72 h in CM at 26 °C and 120 r.p.m. The mycelium was transferred for further submersed cultivation to MM or REM at 26 °C and 120 r.p.m. Samples were taken after 8 h. The RNA was isolated from the mycelium samples and the results of transcript abundance in REM are given relative to quantification in the MoWT. Three replicates were made of each. Bars represent (+/−) sem.

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