Abstract
To determine if DNA configuration, gene locus, and flanking sequences will affect homologous recombination in the phytopathogenic fungus Cercospora nicotianae, we evaluated and compared disruption efficiency targeting four cercosporin toxin biosynthetic genes encoding a polyketide synthase (CTB1), a monooxygenase/O-methyltransferase (CTB3), a NADPH-dependent oxidoreductase (CTB5), and a FAD/FMN-dependent oxidoreductase (CTB7). Transformation of C. nicotianae using a circular plasmid resulted in low disruption frequency. The use of endonucleases or a selectable marker DNA fragment flanked by homologous sequence either at one end or at both ends in the transformation procedures, increased disruption efficiency in some but not all CTB genes. A split-marker approach, using two DNA fragments overlapping within the selectable marker, increased the frequency of targeted gene disruption and homologous integration as high as 50%, depending on the target gene and on the length of homologous DNA sequence flanking the selectable marker. The results indicate that the split-marker approach favorably decreased ectopic integration and thus, greatly facilitated targeted gene disruption in this important fungal pathogen.
Abbreviations
- CSPD:
-
Disodium 3-[4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo [3.3.1.1]decan}-4-yl]phenyl phosphate
- BAR :
-
Acetyltransferase gene conferring phosphinothricin resistance
- HYG :
-
Phosphotransferase B gene conferring hygromycin resistance
- dUTP:
-
2′-Deoxyuracil 5′-triphosphate
- DIG:
-
Digoxigenin
References
Chen H, Lee MH, Daub ME, Chung K (2007a) Molecular analysis of the cercosporin biosynthetic gene cluster in Cercospora nicotianae. Mol Microbiol 64:755–770
Chen H, Lee MH, Chung K (2007b) Functional characterization of three genes encoding putative oxidoreductases required for cercosporin toxin biosynthesis in the fungus Cercospora nicotianae. Microbiology 153:2781–2790
Cho Y et al (2006) A high throughout targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs. Mol Plant Microbe Interact 19:7–15
Choquer M, Dekkers K, Ueng PP, Daub ME, Chung KR (2005) The CTB1 gene encoding a fungal polyketide synthase is required for cercosporin biosynthesis and fungal virulence of Cercospora nicotianae. Mol Plant Microbe Interact 18:468–476
Chung KR (2003) Involvement of calcium/calmodulin signaling in cercosporin toxin biosynthesis by Cercospora nicotianae. Appl Environ Microbiol 69:1187–1196
Chung KR, Jenns AE, Ehrenshaft M, Daub ME (1999) A novel gene required for cercosporin toxin resistance in the fungus, Cercospora nicotianae. Mol Gen Genet 262:382–389
Chung KR, Shilts T, Li W, Timmer LW (2002) Engineering a genetic transformation system for Colletotrichum acutatum, the causal fungus of lime anthracnose and postbloom fruit drop. FEMS Microbiol Lett 213:33–39
Chung KR, Daub ME, Kuchler K, Schüller C (2003a) The CRG1 gene required for resistance to the singlet oxygen-generating cercosporin toxin in Cercospora nicotianae encodes a putative fungal transcription factor. Biochem Biophys Res Comm 302:302–310
Chung KR, Ehrenshaft M, Wetzel DK, Daub ME (2003b) Cercosporin toxin deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae. Mol Genet Genomics 270:103–113
Chung KR, Daub ME, Ehrenshaft M (2003c) Expression of the cercosporin toxin resistance gene (CRG1) as a discistronic mRNA in the filamentous fungus Cercospora nicotianae. Curr Genet 43:415–424
da Silva Ferreira ME, Kress MR, Savoldi M, Goldman MH, Hartl A, Heinekamp T, Brakhage AA, Goldman GH (2006) The akuB (KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot Cell 5:207–211
Daub ME, Ehrenshaft M (2000) The photoactivated Cercospora toxin cercosporin: contributions to plant disease and fundamental biology. Annu Rev Phytopathol 38:461–490
Daub ME, Leisman GB, Clark RA, Bowden EF (1992) Reduced detoxification as a mechanism of fungal resistance to singlet-oxygen-generating photosensitizers. Proc Natl Acad Sci USA 89:9588–9592
Daub ME, Herrero S, Chung KR (2005) Photoactivated perylenequinone toxins in fungal pathogenesis of plants. FEMS Microbiol Lett 252:197–206
Dekkers KL et al (2007) The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis. Fungal Genet Biol 44:444–454
Ehrenshaft M, Daub ME (2001) Isolation of PDX2, a second novel gene in the pyridoxine biosynthesis pathway of eukaryotes, archaebacteria, and a subset of eubacteria. J Bacteriol 183:3383–3390
Ehrenshaft M, Jenns AE, Chung KR, Daub ME (1998) SOR1, a gene required for photosensitizer and singlet oxygen resistance in Cercospora fungi, is highly conserved in divergent organisms. Mol Cell 1:603–609
Ehrenshaft M, Bilski P, Li M, Chignell CF, Daub ME (1999) A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis. Proc Natl Acad Sci USA 96:9374–9378
Fairhead C, Llorente B, Denis F, Soler M, Dujon B (1996) New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using ‘split-marker’ recombination. Yeast 12:1439–1457
Fu J, Hettler E, Wickes BL (2006) Split marker transformation increases homologous integration frequency in Cryptococcus neoformans. Fungal Genet Biol 43:200–212
Haarmann T, Lorenz N, Tudzynski P (2008) Use of a nonhomologous end joining deficient strain (∆ku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthase gene involved in ergotamine biosynthesis. Fungal Genet Biol 45:35–44
Herrero S, Amnuaykanjanasin A, Daub ME (2007) Identification of genes differentially expressed in the phytopathogenic fungus Cercospora nicotianae between cercosporin toxin-resistant and -susceptible strains. FEMS Microbiol Lett 275:326–337
Idnurm A, Warnecke DC, Heinz E, Howlett BJ (2003) Characterisation of neutral trehalase and UDP-glucose:sterol glucosyltransferase genes from the plant pathogenic fungus Leptosphaeria maculans. Physiol Mol Plant Pathol 62:305–313
Nelson RT, Pryor BA, Lodge JK (2003) Sequence length required for homologous recombination in Cryptococcus neoformans. Fungal Genet Biol 38:1–9
Ninomiya Y, Suzuki K, Ishii C, Inoue H (2004) Highly efficient gene replacements in Neurospora strains deficient for non-homologous end joining. Proc Natl Acad Sci USA 101:12248–12253
Pratt RJ, Aramayo R (2002) Improving the efficiency of gene replacements in Neurospora crassa: a first step towards a large-scale functional genomics project. Fungal Genet Biol 37:56–71
Segers GC, Bradshaw N, Archer D, Blissett K, Oliver RP (2001) Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum but aldehyde dehydrogenase is dispensable. Mol Plant Microbe Interact 14:367–377
Sollod CC, Jenns AE, Daub ME (1992) Cell surface redox potential as a mechanism of defense against photosensitizers in fungi. Appl Environ Microbiol 58:444–449
Wetzel DK, Ehrenshaft M, Denslow SA, Daub ME (2004) Functional complementation between the PDX1 vitamin B6 biosynthetic gene of Cercospora nicotianae and pdxJ of Escherichia coli. FEBS Lett 564:143–146
You BJ, Lee MH, Chung KR (2008) Production of cercosporin toxin by the phytopathogenic Cercospora fungi is affected by diverse environmental signals. Can J Microbiol 54:259–269
Acknowledgment
The authors thank Dr. Daryl Henderson for the initial editing.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by Axel Brakhage.
The GenBank/EMBL/DDBJ accession numbers for the sequence data reported in this article are: CTB1, AY649543, CTB3, DQ355149, CTB5, DQ991507, and CTB7, DQ991509.
Rights and permissions
About this article
Cite this article
You, BJ., Lee, MH. & Chung, KR. Gene-specific disruption in the filamentous fungus Cercospora nicotianae using a split-marker approach. Arch Microbiol 191, 615–622 (2009). https://doi.org/10.1007/s00203-009-0489-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00203-009-0489-4