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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

Front Agric Chin    2011, Vol. 5 Issue (3) : 338-343    https://doi.org/10.1007/s11703-011-1090-6
RESEARCH ARTICLE
BcDR1, a putative gene, regulates the development and pathogenicity of Botrytis cinerea
Bin ZHAO1, Meng ZHENG2, Zhiying SUN1, Zhiyong LI3, Jihong XING1(), Jingao DONG1()
1. Molecular Plant Pathology Laboratory, Agricultural University of Hebei, Baoding 071001, China; 2. Agriculture Bureau of Langfang, Langfang 065000, China; 3. Millet Institute, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang 050031, China
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Abstract

Botrytis cinerea is one of the important phytopathogenic fungi. Cloning of the genes related to their development and pathogenicity is fundamental to the pathogen control. A mutant (BCt160), which produces abnormal conidia and no sclerotia, was identified from Botrytis cinerea mutant library generated by Agrobacterium tumefaciens-mediated transformation (ATMT). Southern blotting analysis showed that one T-DNA insertion occurred in the genome of the mutant. TAIL-PCR (thermal asymmetric interlaced PCR) and bioinformatic analysis indicated that the exogenous T-DNA insertion occurred in the second exon of a putative gene BC1G_12388.1, named as BcDR1 (B. cinerea development-related gene 1). The function analysis of BcDR1 gene showed that the BcDR1 was related to development, morphological differentiation, and pathogenicity of B. cinerea, suggesting that BcDR1 gene was required for the development and pathogenicity of B. cinerea.

Keywords Botrytis cinerea      T-DNA mutagenesis      BcDR1      functional analysis     
Corresponding Author(s): XING Jihong,Email:xingjihong2000@126.com; DONG Jingao,Email:shmdjg@hebau.edu.cn   
Issue Date: 05 September 2011
 Cite this article:   
Bin ZHAO,Meng ZHENG,Zhiying SUN, et al. BcDR1, a putative gene, regulates the development and pathogenicity of Botrytis cinerea[J]. Front Agric Chin, 2011, 5(3): 338-343.
 URL:  
https://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1090-6
https://academic.hep.com.cn/fag/EN/Y2011/V5/I3/338
Fig.1  PCR analysis and Southern blotting of hygromycin resistance gene.
Note: (B) Southern blotting of BCt160 mutant with hygromycin B resistant gene. M1 is DNA marker DL2000. M2 is DNA molecular-weight marker DIG labeled, 0.12–23.1 kb.
Fig.2  Bioinformatics analysis of .
Note: A means illustration of the BC1G12388.1 locus with the T-DNA insertion in mutant BCt160, and primers used for identifying mutant BCt160 and mutant gene. B means phylogenetics analysis of from different fungi species retrieved from GenBank database including (A6SIW8; BC1G_12388; ), (A7ERY9; SS1G_08094) and (C8VQP5, C8V280, C8V4S7, C8VRR4, C8VTY5; Putative Zn(II)Cys transcription factor).
Fig.3  PCR analysis results of gene.
Note: A represents amplification using LB3 and P4. B represents amplification using gene specific primers.
Fig.4  RT-PCR analysis results of .
Fig.5  Phenotypic analysis of BCt160 mutant.
Note: A is phenotypic characteristics of BCt160 mutant cultured in PDA medium. B is conidium characteristics of BCt160 mutant. C is pathogenicity assay of BCt160 mutant (1, BCt160; 2, BC22; 3, needle inoculation BC22; 4, needle inoculation BCt160).
StrainsThe area of sclerotias (mm2)The quantity of sporulation (10/cm2)The size of conidiaLesion diameter with inoculation no wound (mm)Lesion diameter with wound inoculation (mm)
BC22>2000 a12.1±0.3 a(8.5±0.3) μm × (9.7±0.3) μm a15.4±1.3 a31.7±2.1a
BCt1600 b30.0±0.4 b(8.7±0.4) μm × (19.2±0.2) μm b7.2±0.9 b23.7±1.7 b
Tab.1  Phenotypic analysis between mutant strain BCt160 and wild-type BC22
1 Bahn Y S, Xue C, Idnurm A, Rutherford J C, Heitman J, Cardenas M E (2007). Sensing the environment: lessons from fungi. Nat Rev Microbiol , 5(1): 57–69
doi: 10.1038/nrmicro1578
2 Balhadère P V, Foster A J, Talbot N J (1999). Identification of pathogenicity mutants of the rice blast fungus Magnaporthe grisea by insertional mutagenesis. Mol Plant Microbe Interact , 12(2): 129–142
doi: 10.1094/MPMI.1999.12.2.129
3 Brito N, Espino J J, Gonzalez C (2006). The endo-beta-1,4-xylanase xyn11A is required for virulence in Botrytis cinerea. Mol Plant Microbe Interact , 19(1): 25–32
doi: 10.1094/MPMI-19-0025
4 Choquer M, Fournier E, Kunz C, Levis C, Pradier J M, Simon A, Viaud M (2007). Botrytis cinerea virulence factors: new insights into a necrotrophic and polyphageous pathogen. FEMS Microbiol Lett , 277(1): 1–10
doi: 10.1111/j.1574-6968.2007.00930.x
5 Cui Z, Ding Z, Yang X, Wang K, Zhu T (2009). Gene disruption and characterization of a class V chitin synthase in Botrytis cinerea. Can J Microbiol , 55(11): 1267–1274
doi: 10.1139/W09-076
6 de Groot M J, Bundock P, Hooykaas P J, Beijersbergen A G (1998). Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol , 16(9): 839–842
doi: 10.1038/nbt0998-839
7 Drenth A, Goodwin S B, Fry W E, Davidse L C (1993). Genotypic diversity of Phytophthora infestans in the Netherlands revealed by DNA polymorphisms. Phytopathology , 83(10): 1087–1092
doi: 10.1094/Phyto-83-1087
8 Elad Y, Williamson B, Tudzynski P, Delen N (2004) Botrytis spp. and Diseases They Cause in Agricultural Systems---An Introduction. In: Elad Y, Williamson B, Tudzynski P, Delen N, eds. Botrytis: Biology, Pathology and Control . the Netherlands: Kluwer Academic Publishers
9 Fillinger S, Chaveroche M K, Shimizu K, Keller N, D’Enfert C (2002). cAMP and ras signalling independently control spore germination in the filamentous fungus Aspergillus nidulans. Mol Microbiol , 44(4): 1001–1016
doi: 10.1046/j.1365-2958.2002.02933.x
10 Jurick W N II, Rollins J A (2007). Deletion of the adenylate cyclase (sac1) gene affects multiple developmental pathways and pathogenicity in Sclerotinia sclerotiorum. Fungal Genet Biol , 44(6): 521–530
doi: 10.1016/j.fgb.2006.11.005
11 Kars I, McCalman M, Wagemakers L, van Kan J A (2005). Functional analysis of Botrytis cinerea pectin methylesterase genes by PCR-based targeted mutagenesis: Bcpme1 and Bcpme2 are dispensable for virulence of strain B05.10. Mol Plant Pathol , 6(6): 641–652
doi: 10.1111/j.1364-3703.2005.00312.x
12 Lazo G R, Stein P A, Ludwig R A (1991). A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nat Biotechnol , 9(10): 963–967
doi: 10.1038/nbt1091-963
13 Liebmann B, Gattung S, Jahn B, Brakhage A A(2003). cAMP signaling in Aspergillus fumigatus is involved in the regulation of the virulence gene pksP and in defense against killing by macrophages. Mol Genet Genomics , 269(3): 420–435
doi: 10.1007/s00438-003-0852-0
14 Michielse C B, Hooykaas P J J, Hondel C A M J J, Ram A F J(2005). Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet , 48(1): 1–17
doi: 10.1007/s00294-005-0578-0
15 Mullins E D, Chen X, Romaine P, Raina R, Geiser D M, Kang S (2001). Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology , 91(2): 173–180
doi: 10.1094/PHYTO.2001.91.2.173
16 Nierman W C, Pain A, Anderson M J, Wortman J R, Kim H S, Arroyo J, Berriman M, Abe K, Archer D B, Bermejo C, Bennett J, Bowyer P, Chen D, Collins M, Coulsen R, Davies R, Dyer P S, Farman M, Fedorova N, Fedorova N, Feldblyum T V, Fischer R, Fosker N, Fraser A, García J L, García M J, Goble A, Goldman G H, Gomi K, Griffith-Jones S, Gwilliam R, Haas B, Haas H, Harris D, Horiuchi H, Huang J, Humphray S, Jiménez J, Keller N, Khouri H, Kitamoto K, Kobayashi T, Konzack S, Kulkarni R, Kumagai T, Lafton A, Latgé J P, Li W, Lord A, Lu C, Majoros W H, May G S, Miller B L, Mohamoud Y, Molina M, Monod M, Mouyna I, Mulligan S, Murphy L, O’Neil S, Paulsen I, Pe?alva M A, Pertea M, Price C, Pritchard B L, Quail M A, Rabbinowitsch E, Rawlins N, Rajandream M A, Reichard U, Renauld H, Robson G D, de Córdoba S R, Rodríguez-Pe?a J M, Ronning C M, Rutter S, Salzberg S L, Sanchez M, Sánchez-Ferrero J C, Saunders D, Seeger K, Squares R, Squares S, Takeuchi M, Tekaia F, Turner G, de Aldana C R V, Weidman J, White O, Woodward J, Yu J H, Fraser C, Galagan J E, Asai K, Machida M, Hall N, Barrell B, Denning D W(2005). Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature , 438(7071): 1151–1156
doi: 10.1038/nature04332
17 Piers K L, Heath J D, Liang X, Stephens K M, Nester E W (1996). Agrobacterium tumefaciens-mediated transformation of yeast. Proc Natl Acad Sci USA , 93(4): 1613–1618
doi: 10.1073/pnas.93.4.1613
18 Rivera M C, Lopez M V, Lopez S E (2009). Mycobiota from Cyclamen persicum and its interaction with Botrytis cinerea. Mycologia , 101(2): 173–181
doi: 10.3852/08-122
19 Rolland S, Jobic C, Fevre M, Bruel C (2003). Agrobacterium-mediated transformation of Botrytis cinerea, simple purification of monokaryotic transformants and rapid conidia-based identification of the transfer-DNA host genomic DNA flanking sequences. Curr Genet , 44(3): 164–171
doi: 10.1007/s00294-003-0438-8
20 Rui O, Hahn M (2007). The Slt2-type MAP kinase Bmp3 of Botrytis cinerea is required for normal saprotrophic growth, conidiation, plant surface sensing and host tissue colonization. Mol Plant Pathol , 8(2): 173–184
doi: 10.1111/j.1364-3703.2007.00383.x
21 Segmuller N, Ellendorf U, Tudzynski B, Tudzynski P (2007). BcSAK1, a stress-activated mitogen-activated protein kinase, is involved in vegetative differentiation and pathogenicity in Botrytis cinerea. Eukaryot Cell , 6(2): 211–221
doi: 10.1128/EC.00153-06
22 Takano Y, Komeda K, Kojima K, Okuno T (2001). Proper regulation of cyclic AMP-dependent protein kinase is required for growth, conidiation, and appressorium function in the anthracnose fungus Colletotrichum lagenarium. Mol Plant Microbe Interact , 14(10): 1149–1157
doi: 10.1094/MPMI.2001.14.10.1149
23 Tellier F, Fritz R, Kerhoas L, Ducrot P H, Einhorn J, Carlin-Sinclair A, Leroux P (2008). Characterization of metabolites of fungicidal cymoxanil in a sensitive strain of Botrytis cinerea. J Agric Food Chem , 56(17): 8050–8057
doi: 10.1021/jf8010917
24 Thevelein J M, Gelade R, Holsbeeks I, Lagatie O, Popova Y, Rolland F, Stolz F, Van de Velde S, Van Dijck P, Vandormael P, Van Nuland A, Van Roey K, Van Zeebroeck G, Yan B (2005). Nutrient sensing systems for rapid activation of the protein kinase A pathway in yeast. Biochem Soc Trans , 33(1): 253–256
doi: 10.1042/BST0330253
25 Tudzynski P, Siewers V (2004). Approaches to Molecular Genetics and Genomics of Botrytis. In: Elad Y, Williamson B, Tudzynski P, Delen N, eds. Botrytis: Biology, Pathology and Control . The Netherlands: Kluwer Academic Press, 53–66
26 van der Vlugt-Bergmans C J, Wagemakers C A, van Kan J A (1997). Cloning and expression of the cutinase A gene of Botrytis cinerea. Mol Plant Microbe Interact , 10(1): 21–29
doi: 10.1094/MPMI.1997.10.1.21
27 van Kan J A (2006). Licensed to kill: the lifestyle of a necrotrophic plant pathogen. Trends Plant Sci , 11(5): 247–253
doi: 10.1016/j.tplants.2006.03.005
28 Vienken K, Fischer R (2006). The Zn(II)2Cys6 putative transcription factor NosA controls fruiting body formation in Aspergillus nidulans. Mol Microbiol , 61(2): 544–554
doi: 10.1111/j.1365-2958.2006.05257.x
29 Williamson B, Tudzynski B, Tudzynski P, van Kan J A (2007). Botrytis cinerea: the cause of grey mould disease. Mol Plant Pathol , 8(5): 561–580
doi: 10.1111/j.1364-3703.2007.00417.x
30 Yamauchi J, Takayanagi N, Komeda K, Takano Y, Okuno T (2004). cAMP-pKA signaling regulates multiple steps of fungal infection cooperatively with Cmk1 MAP kinase in Colletotrichum lagenarium. Mol Plant Microbe Interact , 17(12): 1355–1365
doi: 10.1094/MPMI.2004.17.12.1355
31 Zhao W, Panepinto J C, Fortwendel J R, Fox L, Oliver B G, Askew D S, Rhodes J C (2006). Deletion of the regulatory subunit of protein kinase A in Aspergillus fumigatus alters morphology, sensitivity to oxidative damage, and virulence. Infect Immun , 74(8): 4865–4874
doi: 10.1128/IAI.00565-06
32 Zheng L, Campbell M, Murphy J, Lam S, Xu J R (2000). The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea. Mol Plant Microbe Interact , 13(7): 724–732
doi: 10.1094/MPMI.2000.13.7.724
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