title: Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit.
abstract: The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post-harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P. digitatum genes up-regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)-based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall-degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P. digitatum genes, including those coding for proteases, cell wall-related enzymes, redox homoeostasis and detoxification processes, confirmed their up-regulation at varying time points during the infection process. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P. digitatum Δndo1 mutants were as virulent as the wild-type. However, the two Δpnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P. digitatum.
authors.affiliation: Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Calle Catedrático Agustín Escardino 7, Paterna, 46980, Valencia, Spain.
authors.forename: Mario
authors.initials: M
authors.lastname: López-Pérez
authors.forename: Ana-Rosa
authors.initials: AR
authors.lastname: Ballester
authors.forename: Luis
authors.initials: L
authors.lastname: González-Candelas
mesh_terms: D002957:Citrus / Q000254:growth & development / Q000382:microbiology*; D003001:Cloning, Molecular; D020224:Expressed Sequence Tags; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D063990:Gene Ontology; D018390:Gene Targeting; D005800:Genes, Fungal; D058977:Molecular Sequence Annotation; D009154:Mutation; D010407:Penicillium / Q000235:genetics* / Q000472:pathogenicity*; D017422:Sequence Analysis, DNA; D066251:Subtractive Hybridization Techniques; D014170:Transformation, Genetic; D014774:Virulence / Q000235:genetics
chemical_list: D005656:Fungal Proteins
keywords: cell wall-degrading enzymes; fruit-pathogen interaction; gene expression; gene knockout; phytopathogenic fungi; proteases
references.citation: Fungal Genet Biol. 2006 Sep;43(9):605-17
references.pmid: 16731015
references.citation: Fungal Genet Biol. 2010 Jan;47(1):53-65
references.pmid: 19853057
references.citation: Mol Plant Microbe Interact. 2013 Apr;26(4):431-41
references.pmid: 23252459
references.citation: Mol Plant Pathol. 2003 Nov 1;4(6):447-57
references.pmid: 20569404
references.citation: Mol Plant Pathol. 2011 Dec;12(9):879-97
references.pmid: 21726388
references.citation: Appl Microbiol Biotechnol. 2013 Jun;97(12):5433-45
references.pmid: 23296496
references.citation: Mol Plant Pathol. 2004 Jan 1;5(1):17-27
references.pmid: 20565578
references.citation: Plant Cell. 2006 Nov;18(11):3332-45
references.pmid: 17138696
references.citation: Plant Physiol. 1994 Jun;105(2):467-472
references.pmid: 12232215
references.citation: Mol Plant Microbe Interact. 1998 Oct;11(10):1009-16
references.pmid: 9768518
references.citation: Mol Plant Pathol. 2000 May 1;1(3):169-78
references.pmid: 20572963
references.citation: Int J Plant Genomics. 2008;2008:619832
references.pmid: 18483572
references.citation: Mol Plant Microbe Interact. 2001 Jun;14(6):749-57
references.pmid: 11386370
references.citation: Mol Plant Pathol. 2002 Sep 1;3(5):347-58
references.pmid: 20569342
references.citation: Int J Food Microbiol. 2011 Aug 2;148(2):135-40
references.pmid: 21663991
references.citation: Appl Microbiol Biotechnol. 2013 Oct;97(20):9087-98
references.pmid: 23917633
references.citation: BMC Genomics. 2009 Sep 11;10:428
references.pmid: 19747386
references.citation: Plant J. 2005 Jul;43(2):213-25
references.pmid: 15998308
references.citation: Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6025-30
references.pmid: 8650213
references.citation: Fungal Genet Biol. 2011 Feb;48(2):144-53
references.pmid: 20887796
references.citation: Appl Environ Microbiol. 2000 Aug;66(8):3421-6
references.pmid: 10919801
references.citation: Microbiology (Reading). 2004 Jul;150(Pt 7):2475-2489
references.pmid: 15256589
references.citation: FEMS Microbiol Rev. 2005 Sep;29(4):719-39
references.pmid: 16102600
references.citation: Nucleic Acids Res. 1992 Nov 25;20(22):6115-6
references.pmid: 1461751
references.citation: Microbiology (Reading). 2001 Mar;147(Pt 3):717-726
references.pmid: 11238979
references.citation: Microbiol Res. 2013 May 6;168(4):211-22
references.pmid: 23238263
references.citation: PLoS Genet. 2011 Aug;7(8):e1002230
references.pmid: 21876677
references.citation: Mol Plant Microbe Interact. 2001 Aug;14(8):988-95
references.pmid: 11497471
references.citation: BMC Plant Biol. 2010 Aug 31;10:194
references.pmid: 20807411
references.citation: Mol Plant Pathol. 2012 Dec;13(9):1135-9
references.pmid: 22512872
references.citation: BMC Genomics. 2012 Nov 21;13:646
references.pmid: 23171342
references.citation: Nat Genet. 2013 Jan;45(1):59-66
references.pmid: 23179022
references.citation: BMC Mol Biol. 2008 Aug 01;9:70
references.pmid: 18673530
references.citation: FEMS Microbiol Lett. 2013 Nov;348(1):11-8
references.pmid: 23952944
references.citation: BMC Genomics. 2013 Apr 23;14:274
references.pmid: 23617724
references.citation: Pest Manag Sci. 2006 Mar;62(3):195-207
references.pmid: 16475240
references.citation: Annu Rev Phytopathol. 2008;46:149-87
references.pmid: 18680426
references.citation: Phytopathology. 2005 Jul;95(7):744-50
references.pmid: 18943005
references.citation: Nucleic Acids Res. 1992 May 11;20(9):2380
references.pmid: 1594460
references.citation: Phytopathology. 2007 Nov;97(11):1491-500
references.pmid: 18943520
references.citation: Mol Plant Pathol. 2002 Jul 1;3(4):227-38
references.pmid: 20569330