Verticillium dahliae

Research Paper Acta Microbiologica Sinica 51 7 906-913 4 July 2011 ISSN 0001-6209 CN 11-1995 / Q http / / journals. im. ac. cn / actamicrocn Verticillium dahliae * 100081 Verticillium dahliae PCR T-DNA T-DNA 5-87% 44% PCR 5- Q933 A 0001-6209 2011 07-0906-08 Verticillium dahliae DNA Verticillium wilt T-DNA 1 T-DNA T- DNA DNA T-DNA DNA 2 T- DNA DNA herpes simplex 3 virus thymidine kinase HSVtk 5-5-fluoro-2'- 4 deoxyuridine F2dU SB2007FY027 2008ZX08005-002 * Tel + 86-10-62815976 Fax + 86-10-62895382 E-mail dxf@ caas. net. cn 1984 - E-mail tianlicaas@ gmail. com 2011-01-18 2011-03-07

Verticillium dahliae. / 2011 51 7 907 T-DNA 4 1 amido-phosphoribosyl transferase ADE4 chitin synthase V ChsV 1. 1 1. 1. 1 1 Table 1 1 Strains and plasmids used in this study Strains and plasmids Characteristics Source Strains Vd991 Verticillium dahliae strain This lab AGL-1 Agrobacterium tumefaciens Guangxi University Top10 Escherichia coli competent cell TransGen Biotech Company Plasmids puc-hyg Plasmid carrying Hyg resistant cassette Guangxi University pgko2-gateway Gene knockout plasmid Pennsylvania State University 4 pbht2-tk Plasmid containing HSVtk in T-DNA Pennsylvania State University 1. 1. 2 LB YEB ADE4 DNA CM PDA 12 94 30 s 58 2 min 5 MM IM 6 1. 1. 3 5- Nest-F / R DNA Sigma B 30 94 30 s 58 2 min Merck 72 4 min Gateway BP PCR Biometra pgko2-gateway T-DNA Bio-Rad 1. 2 pko-ade4 pko-chsv pko-ade4 ChsV ADE4 DNA 3 VDAG_00420 Broad Institute pko- PCR 1 A-C 1 PCR PCR puc-hyg Hyg-F / R 2 1. 8 kb 30 94 30 s 58 1 min 72 2 min Mullins 7 5F /5R 3F /3R 5R /3F 5' 1. 4 20 bp Hyg-F / R PCR 1 kb ADE4 VDAG _00128 Broad Institute DNA pbht2-tk T-DNA 3 PCR HSVtk 2 30 94 30 s 58 1 min 72 1 min 2 PCR PCR 1 72 4 min 3 PCR PCR 2 PCR Gateway BP T-DNA HSVtk 1 ChsV 1. 3 pko2-ade4 pko2- ChsV AGL-1 pbht2-tk 30 μg / ml T-DNA Δtk F2dU PCR DNA 0. 005 0. 05 0. 5 5 50 μmol / L PDA 5 d

908 Li Tian et al. / Acta Microbiologica Sinica 2011 51 7 Primer name Primers for gene disruption of ADE4 5F 5R 3F 3R Nest-F Nest-R Hyg-F Hyg-R Test-1 Test-2 Test-3 Test-4 GAPDHF GAPDHR Primers for gene disruption of ChsV 5F 5R 3F 3R Nest-F Nest-R Test-1 Test-2 Test-3 Test-4 Primers for real-time PCR ChsVI-1 ChsVI-2 β-tubulin-1 Table 2 2 Primers used in the present study Sequences 5' 3' GTAATGTAGCGAATGCCTGTG GCCCAAAAATGCTCCTTCAAGTTGACGCTCATCGACAGA CCCTGGGTTCGCAAAGATAATAGGTTTCCGACTCCGATGC TAGGCTGCACAAGATTGGTG GGGGACAAGTTTGTACAAAAAAGCAGGCTAGCATGGGTACATGGCACT GGGGACCACTTTGTACAAGAAAGCTGGGTGGCTTTCAGCAAAGGACATG TTGAAGGAGCATTTTTGGGC TTATCTTTGCGAACCCAGGG CACTGACAGCGTTCTCTGCCAC CATCCACTTCAACGCTCGCAT CTGCCTTCAGACTCAACATCTTCG AGGTCGGGCGTGTAGGACTTCA CAAGGACTGGAGAGGTGG TTCACTCGTTGTCGTACC CAGGTCTTGCCCTTTATTG GCCCAAAAATGCTCCTTCAACAGGACGCTGATTTGGTAT CCCTGGGTTCGCAAAGATAAATACCCTTTCCTACAAGACGA CCTAACGCATTTACTGTCAAAC GGGGACAAGTTTGTACAAAAAAGCAGGCTAGCGACTCTACTAGAACTGC GGGGACCACTTTGTACAAGAAAGCTGGGTTGAGTTTGTGCTTGGGTC TTGCTTCCGTAACGGCGTCT TTCGGGTCCAGCATGACAGT CCTGACCACAACCAAAACGG CTTGAAAACCTGGAAGCCCT CTGGAAGATGTTTGCGAAGACG GGTGATGACCCAATCGACGAG GGCCGCCTCTGACTTCCGTAAC β-tubulin-2 CTCGACCTCCTTCATGGCAACCTT The underlined regions are the attached tail sequence complementary to the primer HygF and HygR respectively. Waved lines refer to attb1 and attb2 for Gateway BP reaction. - 1. 5 ADE4 ChsV 2 Ct ChsVI ΔChsV 50 μg / ml 30 μg / ml 9 3 F2dU 25 5 d 2 PCR RT-PCR 2. 1 PCR 1. 6 ADE4 ChsV 1 PCR 1 1. 8 1. 6. 1 ADE4 ADE4 ADE4 DNA 1-D 3 2 MM MM + ade PCR 3 DNA 5 d 1 kb ADE4 1. 6. 2 ChsV Elson- ADE4 DNA Morgan 8 3 2 β-tubulin DNA

Verticillium dahliae. / 2011 51 7 909 smear 1-D 4 3 PCR 3. 8 1 + 1. 8 + 1 kb 5 μmol / L F2dU PDA F2dU 50μmol / L ADE4 1 D 5 50μmol / L F2dU Gateway BP ADE4 HSVtk T-DNA pgko2-gateway pko-ade4 pko-chsv T-DNA HSVtk pgko2-gateway 50 μmol / L F2dU 2. 3 ADE4 ChsV 15 ADE4 3-B test-1 /2 1. 2 kb DNA 15 DNA 13 1 2 3 4 5 7 8 9 10 12 13 14 15 16 1. 8 kb DNA 13 ADE4 1. 2 kb 1. 8 kb test-1 /2 1. 8 kb 2 6 11 1. 2 kb ADE4 ORF 1 Fig. 1 pko-ade4 Schematic representation A-C and electrophoresis pattern D of the construction of gene replacement cassette Mutant-allele- ADE4. A First round PCR amplification of the components using the specific and chimeric primers. The chimeric primers 5R /3F contain the white box at the beginning of each primer indicating the attached tail sequence complementary to the primer Hyg-F and Hyg- R respectively. B Second round PCR the overhanging chimeric extensions fusion with each other. C Third round PCR amplification of the final product Mutant-allele-ADE4 using nested primers with attb site at 5' end of each primer to generate PCR 1. 8 kb T-DNA T- DNA ADE4 13 /15 = 87% cdna test-3 /4 RT-PCR 0. 5 kb 13 cdna RT-PCR 3 B 1 2 ADE4 ΔADE4 products suitable for use as substrates in following Gateway BP recombination D The photograph of the agarose gel electrophoresis of the resulting products of each PCR step in constructing a deletion cassette of ADE4. 2. 2 50 μmol / L F2dU 7 /16 = 44% RT-PCR 7 ChsV 16 ChsV 7 3 4 6 8 9 13 16 1. 8 kb DNA 3 C 3 4 Δtk 2. 4 ADE4 ChsV F2dU PDA 2 F2dU ADE4 HSVtk T-DNA Δtk ChsV

910 Li Tian et al. / Acta Microbiologica Sinica 2011 51 7 2 F2dU T-DNA HSVtk Fig. 2 Lethal effect of F2dU against ectopic transformant bearing HSVtk. A Schematic diagram of generation of Δtk bearing HSVtk by transformation of pbht2-tk B Growth of V. dahliae wild-type strains and Δtk in the presence of F2dU at concentrations ranging from 0. 005 to 50 μmol / L. 3 ADE4 ChsV Fig. 3 Gene targeting replacement for the ADE4 and ChsV gene in Verticillium dahliae. A Partial map of the target gene fragment on the wild type chromosome and the targeting vector. The 1. 2 kb ORF of target gene was replaced by the 1. 8 kb hyg resistence cassette through homologous recombination between the genomic DNA and the deletion plasmid. B Characterization of the ADE4 deletion mutant with PCR primers Test-1 and Test-2. Line 1-15 represent electrophoresis band of 15 putative ADE4 mutant strains and Line 16 represents wild-type V. dahliae. The ADE4 gene transcript was detected by RT-PCR using the primer pair Test-3 and Test-4. The glyceraldehyde phosphate dehydrogenase GAPDH transcript was used as an internal control. C Characterization of the ChsV deletion mutant using the methods mentioned above.

Verticillium dahliae. / 2011 51 7 911 3 4 5 7 8 9 10 12 13 14 15 16 MM + ade MM 2. 4. 1 ADE4 4 ADE4 MM 6 11 MM + ade 13 ADE4 15 13 1 2 4 Fig. 4 ADE4 Adenine auxotrophic mutants obtained by targeted replacement of ADE4. The same transformants growing on minimal medium B and minimal medium supplemented with 1 mm adenine A. Wild-type is located at top left corners white square. 2. 4. 2 ChsV 7 ChsV 3 Elson-Morgan 3 12% 5-A ChsV ChsVI PCR ΔChsV ChsVI 9. 6 5-B ChsVI ChsV 3 5 ChsV Fig. 5 Pheonotype analysis of ΔChsV. A Relative chitin PCR DNA content between wild type and mutation strains. B Relative ChsVI expression between wild type and mutation strains.

912 Li Tian et al. / Acta Microbiologica Sinica 2011 51 7 aspects of Verticillium wilt diseases caused by V. dahliae 2 d and V. albo-atrum. Molecular Plant Pathology 2006 7 2 71-86. 2 Vasquez KM Marburger K Intody Z Wilson JH. Manipulating the mammalian genome by homologous recombination. Proceedings of the National Academy of Sciences 2001 98 15 8403-8410. 3 Gao F Zhou BJ Li GY Jia PS Li H. A Glutamic 87% 44% acid-rich protein identified in Verticillium dahliae from an F2dU insertional mutagenesis affects. Public Library of Science 50 μmol / L One 2010 5 12 15319-15329. 4 Khang CH Park SY Lee YH Kang S. A dual selection F2dU 0. 5 μmol / L 4 based targeted gene replacement tool for Magnaporthe F2dU grisea and Fusarium oxysporum. Fungal Genetics and Biology 2005 42 6 483-492. 5 Sambrook J Fritsch EF Maniatis T.... 2002 PCR Southern 1595-1605. PCR 6 Hooykaas P Roobol C Schilperoort R. Regulation of PCR the transfer of Ti plasmids of Agrobacterium tumefaciens. Southern Microbiology 1979 110 1 99-109. 7 Mullins ED Kang S. Transformation a tool for studying fungal pathogens of plants. Cellular and Molecular Life PCR Sciences 2001 58 14 2043-2052. 8 Soulie MC Perino C Piffeteau A Choquer M Malfatti T-DNA 11 P Cimerman A Kunz C Boccara M Vidal-Cros A. Botrytis cinerea virulence is drastically reduced after disruption of chitin synthase class III gene Bcchs3a. T-DNA Cellular Microbiology 2006 8 8 1310-1321. ADE4 ChsV 2 /15 = 13% 9 Livak KJ Schmittgen TD. Analysis of relative gene 9 /16 = 56% T-DNA expression data using real-time quantitative PCR and the truncate HSVtk HSVtk 10. MgORP1. Acta Microbiologica HSVtk T-DNA HSVtk 11 Yu JH Hamari Z Han KH Seo JA Reyes-Dominguez Y Scazzocchio C. Double-joint PCR a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genetics and Biology 2004 41 11 1 Fradin EF Thomma BP. Physiology and molecular - 2 Ct Method. Methods 2001 25 4 402-408. Sinica 2008 48 9 1160-1167. 973-981.

Verticillium dahliae. / 2011 51 7 913 High efficient gene knockout in Verticillium dahliae Li Tian Jieyin Chen Jiani Wang Jinlong Wang Xiaofeng Dai * Institute of Crop Science Chinese Academy of Agricultural Sciences Beijing 100081 China Abstract Objective We developed an efficient method of gene knockout in Verticillium dahliae an important soil-borne fungal pathogen that causes cotton vascular wilt diseases. Methods By using fusion PCR we constructed gene knockout vectors. By using Agrobacterium tumefaciens-mediated transformation and applying a herpes simplex virus thymidine kinase HSVtk gene in T-DNA as a conditional lethal gene to counter-select against ectopic transformants we developed an efficient method to select gene knockout transformants. Results Gene knockout frequency for ADE4 and ChsV was 87% and 44% respectively. Conclusion We developed an efficient tool for gene knockout in Verticillium dahliae which would help clarify the infection mechanism of this fungal pathogen. Keywords Verticillium dahliae gene knockout fusion PCR Agrobacterium tumefaciens-mediated transformation lethal gene 5-fluoro-2'-deoxyuridine F2dU Supported by the Basic Program of Science and Technology SB2007FY027 and by the National Transgenic Major Program 2008ZX08005-002 * Corresponding author. Tel + 86-10-62815976 E-mail dxf@ caas. net. cn Received 18 January 2011 / Revised 7 March 2011 檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶 / 978-7-5615-3848-7 48. 00 2011 4 / / - 39 361005 0592-2188509 0592-2184866 www. dangdang. com www. joy. com www. amazon. cn www. beifabook. com http / / www. lifescience. com. cn