title: Transient transformation of Podosphaera xanthii by electroporation of conidia.
abstract: BACKGROUND
  Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew.
  
  
  RESULTS
  To introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B.
  
  
  CONCLUSIONS
  This is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.
authors: Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Departamento de Microbiología, Universidad de Málaga, Bulevar Louis Pasteur 31 (Campus Universitario de Teatinos), 29071, Málaga, Spain. dvela@uma.es.
authors: David
authors: D
authors: Vela-Corcía
authors: Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Departamento de Microbiología, Universidad de Málaga, Bulevar Louis Pasteur 31 (Campus Universitario de Teatinos), 29071, Málaga, Spain. diego_romero@uma.es.
authors: Diego
authors: D
authors: Romero
authors: Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Estación Experimental "La Mayora", 29750 Algarrobo-Costa, Málaga, Spain. tores@eelm.csic.es.
authors: Juan Antonio
authors: JA
authors: Torés
authors: Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Departamento de Microbiología, Universidad de Málaga, Bulevar Louis Pasteur 31 (Campus Universitario de Teatinos), 29071, Málaga, Spain. adevicente@uma.es.
authors: Antonio
authors: A
authors: De Vicente
authors: Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Departamento de Microbiología, Universidad de Málaga, Bulevar Louis Pasteur 31 (Campus Universitario de Teatinos), 29071, Málaga, Spain. aperez@uma.es.
authors: Alejandro
authors: A
authors: Pérez-García
mesh_terms: D001203:Ascomycota / Q000235:genetics* / Q000254:growth & development; D001233:Aspergillus nidulans / Q000737:chemistry / Q000235:genetics; D001562:Benzimidazoles / Q000378:metabolism; D002219:Carbamates / Q000378:metabolism; D018548:Cotyledon / Q000382:microbiology; D028464:Cucurbita / Q000382:microbiology; D004560:Electricity; D018274:Electroporation / Q000379:methods*; D017930:Genes, Reporter; D005819:Genetic Markers; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006921:Hygromycin B / Q000378:metabolism; D009492:Neurospora crassa / Q000737:chemistry / Q000235:genetics; D010935:Plant Diseases / Q000382:microbiology; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional; D013172:Spores, Fungal / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic
chemical_list: D001562:Benzimidazoles; D002219:Carbamates; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; C006698:carbendazim
references: Curr Genet. 2007 Mar;51(3):209-19
references: 17256172
references: Fungal Genet Biol. 2009 Feb;46(2):137-45
references: 19100853
references: Mol Plant Pathol. 2009 Mar;10(2):153-60
references: 19236565
references: Curr Genet. 2009 Oct;55(5):537-42
references: 19629488
references: Plant Physiol. 2005 Aug;138(4):2155-64
references: 16040663
references: FEMS Microbiol Lett. 2006 Jun;259(2):165-73
references: 16734775
references: Plant J. 2010 Jan;61(2):364-9
references: 19874543
references: Appl Microbiol Biotechnol. 2010 Jul;87(3):1151-6
references: 20449738
references: Plant Cell. 2010 Sep;22(9):3130-41
references: 20884801
references: J Microbiol Methods. 2011 Mar;84(3):442-6
references: 21256886
references: Fungal Biol. 2011 Jul;115(7):633-42
references: 21724169
references: Ultrasound Med Biol. 2012 Jun;38(6):1078-84
references: 22502888
references: Curr Genet. 2012 Jun;58(3):129-38
references: 22388868
references: Fungal Biol. 2014 Feb;118(2):228-41
references: 24528644
references: Mol Gen Genet. 2000 Jan;262(6):911-5
references: 10660051
references: Nat Biotechnol. 2000 Feb;18(2):205-7
references: 10657129
references: Antisense Nucleic Acid Drug Dev. 2001 Feb;11(1):7-14
references: 11258623
references: Appl Environ Microbiol. 2001 May;67(5):1987-94
references: 11319072
references: Mol Plant Microbe Interact. 2002 Feb;15(2):172-9
references: 11878320
references: Science. 2002 Jul 26;297(5581):537-41
references: 12142520
references: Trends Plant Sci. 2002 Aug;7(8):352-6
references: 12167330
references: Curr Opin Microbiol. 2003 Jun;6(3):310-6
references: 12831909
references: Methods Mol Biol. 2004;267:297-313
references: 15269433
references: Nature. 1967 Jul 15;215(5098):321-2
references: 6059533
references: Gene. 1987;56(1):117-24
references: 2824287
references: Gene. 1987;57(1):21-6
references: 3322945
references: Microbiol Rev. 1989 Mar;53(1):148-70
references: 2651864
references: Cell. 1991 Aug 9;66(3):507-17
references: 1651171
references: Can J Microbiol. 1991 Nov;37(11):858-63
references: 1838030
references: Biotechnology. 1992;24:384-6
references: 1422046
references: Curr Genet. 1995 Dec;29(1):100-2
references: 8595653
references: Methods Mol Biol. 1998;102:285-98
references: 9680628
references: Mol Gen Genet. 1998 Aug;259(3):327-35
references: 9749676
references: Fungal Genet Biol. 1999 Feb;26(1):19-32
references: 10072317
references: Can J Microbiol. 1998 Dec;44(12):1137-41
references: 10347861
references: Curr Genet. 1999 Aug;36(1-2):98-104
references: 10447601
references: J Biosci Bioeng. 2007 Dec;104(6):517-20
references: 18215641
references: Int J Food Microbiol. 2007 Nov 1;119(3):230-5
references: 17765991