BcDR1, a putative gene, regulates the development and pathogenicity of Botrytis cinerea

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

References

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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, Peñalva 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-Peña 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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[27] van Kan J A (2006). Licensed to kill: the lifestyle of a necrotrophic plant pathogen. Trends Plant Sci, 11(5): 247–253

[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

[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

[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

[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

[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

Acknowledgements

This research was financially supported by the Natural Science Foundation of Hebei, China (No. 08B021).

RIGHTS & PERMISSIONS

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg