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. 2018 Jan 6;19(1):27.
doi: 10.1186/s12864-017-4405-z.

Polyketide Synthases of Diaporthe Helianthi and Involvement of DhPKS1 in Virulence on Sunflower

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Free PMC article

Polyketide Synthases of Diaporthe Helianthi and Involvement of DhPKS1 in Virulence on Sunflower

Michelina Ruocco et al. BMC Genomics. .
Free PMC article

Abstract

Background: The early phases of Diaporthe helianthi pathogenesis on sunflower are characterized by the production of phytotoxins that may play a role in host colonisation. In previous studies, phytotoxins of a polyketidic nature were isolated and purified from culture filtrates of virulent strains of D. helianthi isolated from sunflower. A highly aggressive isolate (7/96) from France contained a gene fragment of a putative nonaketide synthase (lovB) which was conserved in a virulent D. helianthi population.

Results: In order to investigate the role of polyketide synthases in D. helianthi 7/96, a draft genome of this isolate was examined. We were able to find and phylogenetically analyse 40 genes putatively coding for polyketide synthases (PKSs). Analysis of their domains revealed that most PKS genes of D. helianthi are reducing PKSs, whereas only eight lacked reducing domains. Most of the identified PKSs have orthologs shown to be virulence factors or genetic determinants for toxin production in other pathogenic fungi. One of the genes (DhPKS1) corresponded to the previously cloned D. helianthi lovB gene fragment and clustered with a nonribosomal peptide synthetase (NRPS) -PKS hybrid/lovastatin nonaketide like A. nidulans LovB. We used DhPKS1 as a case study and carried out its disruption through Agrobacterium-mediated transformation in the isolate 7/96. D. helianthi DhPKS1 deleted mutants were less virulent to sunflower compared to the wild type, indicating a role for this gene in the pathogenesis of the fungus.

Conclusion: The PKS sequences analysed and reported here constitute a new genomic resource that will be useful for further research on the biology, ecology and evolution of D. helianthi and generally of fungal plant pathogens.

Keywords: Diaporthe helianthi; Pathogen virulence, toxins; Plant pathogen; Polyketide synthases.

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Figures

Fig. 1
Fig. 1
(G): Restriction map (not all restriction sites are included) of the genomic fragment of Diaporthe helianthi containing the DhPKS1 gene with the original sequence isolated by Vergara et al. (2004) (C) plus its flanking gene portions (A and B) used for hygromicin phosphotransferase (HPH) gene replacement cassette construction. (P): Restriction map (not all sites are included) of plasmid pUR5750•ΔDhPKS1 containing hph gene of E. coli under the control of the A. nidulans gpdA promoter (p) and trpc terminator (t). In the construction design, pGEM-T Easy vector system (pG) and pBluescript KS (pB) were used as intermediate vectors. H = HindIII; K = KpnI; E = EcoRI
Fig. 2
Fig. 2
Phylogenetic tree of Diaporthe helianthi PKSs proteins (highlighted in bold) and reference PKSs from other fungi based on Karlsson et al., [34] . DhPKS1 used for molecular characterization is highlighted in red. Numbers next to the nodes represent bootstrap values. Full D. helianthi PKS genes information is provided in Additional file 1
Fig. 3
Fig. 3
PCR analysis of Diaporthe helianthi strain 7/96 (lane 3) and ΔDhPKS1 putative transformants (lanes 4–8) with hph2-for/trpc2-rev primers. Lanes 1 and 2 correspond to 1 kb molecular weight ladder and positive control, respectively
Fig. 4
Fig. 4
PCR analysis of Diaporthe helianthi strain 7/96 (lane 2) and ΔDhPKS1 putative transformants (lanes 4–15) with Dhpks537-for/Dhpks1326-rev primers. Lanes 1 and 3 correspond to 1 kb molecular weight ladder and negative control, respectively
Fig. 5
Fig. 5
Southern blot analysis of ΔDhPKS1 transformants (lanes 4–8) and Diaporthe helianthi strain 7/96 (lane 2) genomic DNA digested with SpeI + BglII, by hybridization with a hygromycin-resistance cassette specific probe. Lanes 1, 2 and 3 correspond to λHindIII molecular weight ladder, negative and positive controls, respectively
Fig. 6
Fig. 6
Phytotoxic effect of ΔDhPKS1 mutant 7/96-Tr1 (a) and Diaporthe helianthi strain 7/96 (b) culture filtrates on sunflower cuttings
Fig. 7
Fig. 7
Pathogenicity bioassay of Diaporthe helianthi strain 7/96 (a) and ΔDhPKS1 mutant 7/96-Tr1 (b) on sunflower stem. c Development of necrotic area (cm2) on leaf blade in sunflower plants inoculated with the mutant (ΔDhPKS1), parental (wild type) and empty vector transformant strains. Observations made at 36, 72 and 84 h after inoculum. Different letters indicate statistically significant differences (p < 0.01) at T3 Dunnett test

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