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

Introduction

Botrytis cinerea, one of the worldwide and important plant-necrotrophic pathogenic fungi, can infect at least 235 dicotyledonous species including a wide range of important crops, fruits, vegetables and ornamental plants, and can cause significant yield losses (Elad et al., 2004). The typical symptoms caused by the pathogen on the leaves and soft fruits include decay, collapse and water soaking of parenchyma tissues and gray conidium groups on the surface of various organs. During different stages of development, B. cinerea is a typical necrotroph that kills plant cells which subsequently serve as nutrient sources (Williamson et al., 2007; Rivera et al., 2009).

In recent years, B. cinerea has become a model organism for molecular plant pathology and developmental biology. Moreover, B. cinerea strain B05.10 (http://www.broad.mit.edu/annotation/genome/botrytis_cinerea/Home.html) and T4 (http://urgi.versailles.inra.fr/index.php/urgi/Species/Botrytis) have been sequenced, which may contribute to understanding of the phenotypic and genotypic variability, the occurrence of virulence and development-related factors of this pathogen. The significant progresses in functional genomics of B. cinerea will build important theoretical basis for the control of gray mold diseases caused by this pathogenic fungus. By now, nearly 30 virulent genes have been obtained in B. cinerea (Choquer et al., 2007). B. cinerea secretes many enzymes and metabolites which are presumed to help kill the host cells subsequently (Vankan, 2006; Tellier et al., 2008). In most cases, B. cinerea enzymes are found to degrade plant cuticle and cell wall components such as hemicellulose (Brito et al., 2006), pectin (Kars et al., 2005), cutin (van der Vlugt-Bergmans et al., 1997) and chitin synthase (Cui et al., 2009). The chemical structures of secondary metabolites have been determined, such as the sesquiterpenes botrydial (PubChem compound: CID: 185781), abscissic acid (CID: 5375200) and botcinic acid for the polyketide (CID: 11509607). Some growth development pathways, involving growth (Zheng et al., 2000; Nierman et al., 2005; Rui and Hahn, 2007; Segmuller et al., 2007), sporulation (Takano et al., 2001), conidial germination (Fillinger et al., 2002; Liebmann et al., 2003; Yamauchi et al., 2004; Zhao et al., 2006), nutrient sensing (Thevelein et al., 2005; Bahn et al., 2007) and sclerotia formation (Jurick and Rollins, 2007), have been reported in some phytopathogenic fungi. In our preliminary studies, the ATMT mutant library of B. cinerea was constructed using Agrobacterium tumefaciens AGL-1 carrying the binary vector plasmid pBHtl.

In this study, a novel mutant (BCt160) which cannot produce sclerotia but abnormal conidia was screened from the ATMT mutant library. PCR, Southern blotting and RT-PCR techniques were used to analyze and identify T-DNA mutant insertion sites and the mutant gene. The results will promote the research of development mechanism and molecular pathogenicity in B. cinerea.

Materials and methods

Strains and plasmids

The wild-type strain BC22 of B. cinerea isolated from diseased tomatoes was used for ATMT mutant library construction. Mutant strain BCt160 was obtained by screening transformation of the BC22 strain previously, using Agrobacterium tumefaciens AGL-1 carrying the plasmid pBHt1 (Mullins et al., 2001). The Agrobacterium tumefaciens strain AGL-1 was kindly provided by Professor Zonghua Wang at the Department of Plant Pathology, Agricultural and Forestry University of Fujian, China. It carried the hygromycin phosphotransferase gene which can resist hygromycin B (Lazo et al., 1991). The B. cinerea strains were cultured on potato dextrose agar (PDA) medium at 20°C in dark.

DNA and RNA manipulations

Genomic DNA of B. cinerea was extracted with modified CTAB (cetyl triethyl ammnonium bromide) method (Drenth et al., 1993). Total RNA was isolated from the frozen fungal mycelia using an RNA extraction kit (Cat. No. SK1322, Sangon, China). The quality and quantity of RNA were measured by the nucleonic acid and protein detection instrument (NanoDrop ND-1000, USA). The first-strand cDNA was synthesized according to the manuscript of promega kit (Cat. No. A3500, promega, China). PCR was carried out in 20 μL reaction volume using first-strand cDNA as template under the following conditions: initial denaturation at 94°C for 5 min, followed by 30 cycles at 94°C for 30 s, at 66°C for 45 s and at 72°C for 30 s, with final 10 min extension at 72°C.

Identification of mutant BCt160

Using the genomic DNA of wild-type BC22 and mutant BCt160 as templates, respectively, PCR amplifications were performed to identify the T-DNA insertion in mutant BCt160 with specific primers (P1: 5′-CGCCCAAGCTGCATCATCGAA-3′, P2: 5′-CGACAGCGTCTCCGACCTGA-3′) of the hygromycin resistance gene. Southern blotting was used to confirm the presence of exogenous hygromycin resistance gene in the genome of mutant BCt160. Genomic DNA of wild-type BC22 and mutant BCt160 were digested by HindIII, then electrophoresed with 0.8% agarose gel and transferred onto a nylon membrane. The transferred DNA was hybridized with DIG-labeled DNA probe for 16 h, and thereafter, the immunological detection was run for more than 16 h (DIG DNA Labeling and Detection Kit, Cat. No. 1093657, Roche Applied Science, Germany).

Acquisition of the flanking sequence of T-DNA insertion site in mutant BCt160

TAIL-PCR was used to amplify the flanking sequence of T-DNA insertion site in mutant BCt160. The thermal cycling settings and reaction conditions of TAIL-PCR were performed according to the previous report (Mullins et al., 2001). Taking genomic DNA of mutant BCt160 as template, PCR products were amplified with the primer combination AD4 (5′-TCGTNCGNACNTAGGA-3′) and LB1/LB2/LB3, respectively. The secondary and tertiary PCR products were analyzed by 1% agarose gel electrophoresis. The tertiary PCR products were purified using QIA quick columns (TIANgel Midi Purification Kit, Cat. No. Dp209-03, Qiagen, Germany) and sequenced by Sangon Co. Ltd., China.

Bioinformatics analysis of the mutant

To determine the T-DNA insertion site and mutant gene, the flanking sequences of T-DNA insertion site obtained by TAIL-PCR were jointed into one sequence according to the common sequence between them and aligned with the genome sequences in B. cinerea genome database (http://www.broad.mit.edu/annotation/genome/botrytis_cinerea) using the BLAST program. To further know about the function of the mutant gene, bioinformatics analysis of mutant gene proceeded. General BLAST programs of EBI (http://www.ebi.ac.uk/Tools/blast/) were used for the nucleotide acid and amino acid sequences homology alignment. DNASTAR software was used for the local sequences homology alignment and phylogenetics analysis.

Gene identification of the mutant

Taking genomic DNA of wild-type BC22 and mutant BCt160 as templates, BcDR1 (BC1G_12388.1) gene-specific primers (P3: 5′-CGTAAACACTTCAGCGAG-3′, P4: 5′-TAAGCGTGCCATACCAGAG-3′) and T-DNA specific primer LB3 were used for verifying T-DNA insertion of BcDR1 gene. Taking the equal aliquots of cDNA of wild-type BC22 and mutant BCt160 as templates, expression levels of BcDR1 were analyzed by RT-PCR with specific primers of BcDR1 (P3, P5: 5′-GCACAACGTGTTGAAGTC-3′); at the same time, Tublin gene (F: 5′-AVTGGGCTAAGGGTCATT-3′, R: 5′-TCTCCGTAAGATGGGTTG-3′) was used for equal loading control.

Phenotype analysis of the mutant

Wild-type BC22 and mutant BCt160 strains were inoculated in PDA medium and cultured in the darkness at 20°C for 10 d, respectively. Conidial morphogenesis was observed using microscope, and the number of conidia was calculated. Mycelium of wild-type BC22 and mutant BCt160 were inoculated on the surface of mature tomatoes for virulence detection. The lesion diameter of tomatoes was measured 3 days after inoculation. The experiment was repeated three times.

Results

Identification of mutant BCt160

A mutant strain BCt160, which produced abnormal conidia, was screened from ATMT mutant library by microscopic observation. By using specific primers of the hygromycin resistance gene for verifying T-DNA insertion of mutant BCt160, a band of 800 bp was amplified from the mutant strain BCt160, and no PCR product in WT (wild-type BC22- no insertion) (Fig. 1A). The copy number of T-DNA in mutant BCt160 was analyzed by Southern blotting using the hygromycin resistance gene as the probe (Fig. 1B). The result of hybridization with single band in mutant BCt160 indicated that T-DNA only had a single copy in vivo.

Bioinformatics analysis of the mutant

The flanking sequence of T-DNA insertion site in mutant BCt160 was obtained by TAIL-PCR technique (date not shown). The comparison of the sequence obtained by TAIL-PCR with B. cinerea database indicated that the T-DNA inserted the second exon of the putative gene BC1G_12388.1 (BcDR1) (Fig. 2 A). The BcDR1 gene contained 4 exons and 3 introns and encoded a putative protein of 414 amino acids. By phylogenetics analysis of BcDR1 from different fungi species retrieved from GenBank database including Botrytis cinerea, Sclerotinia sclerotiorum and Aspergillus nidulans, the phylogenetic tree showed that BcDR1 shared 48% sequence similarity to putative Zn(II)₂Cys₆ transcription factor from Aspergillus nidulans (Fig. 2 B).

Gene identification of the mutant

To identify the mutant gene further, PCR amplifications were performed with BcDR1 specific primer P4 and T-DNA specific primer LB3. Fragment of 750 bp PCR product was amplified in mutant BCt160, with no PCR product in the wild-type strain (Fig. 3 A). Taking genomic DNA of mutant BCt160 and WT as templates, BcDR1 gene-specific primers P3 and P4 were used for PCR amplifications. A band of 700 bp was amplified from WT, with no PCR product in the mutant BCt160 (Fig. 3 B). This result also confirmed that BcDR1 gene was inserted by a T-DNA in the mutant BCt160.

Analysis of the BcDR1 gene expression in mutant BCt160

Using Tublin gene as control, expression levels of BcDR1 in mutant BCt160 and wild type were analyzed using RT-PCR technique. The results showed that expression of BcDR1 gene in mutant BCt160 was significantly lower than that of wild type (Fig. 4). The results further verified that gene BcDR1 was mutated in the mutant BCt160.

Phenotypic analysis of the mutant BCt160

It was found that strain BCt160 did not produce sclerotia and its sporulation significantly increased, compared to wild type (Fig. 5 A, Table 1) through phenotypic analysis. Conidia changed morphologically; they were rod-like and larger than those of the wild type (Fig. 5 B, Table 1). Pathogenicity tests showed that the pathogenicity of the mutant BCt160 significantly decreased (Fig. 5 C, Table 1), suggesting that the BcDR1 genes are involved in the development of B. cinerea and signal transduction regulation of pathogenicity.

Discussion

Agrobacterium tumefaciens-mediated transformation is an important technology for genetic transformation. Since it was established in yeast (Piers et al., 1996) and other filamentous fungi (de Groot et al., 1998), this technique was successfully applied to over 60 species of filamentous fungi (Michielse et al., 2005). Moreover, fungi are mostly with the genetic features of haploid, and mutants can be expressed in phenotype without the homozygous process. However, the classical REMI technique successfully applied in several phytopathogens did not perform well in B. cinerea (Balhadère et al., 1999; Tudzynski and Siewers, 2004). Therefore, more results of mutant gene function obtained by ATMT analysis could be expected.

Pathogen B. cinerea as model organisms of developmental biology and molecular plant pathology, Agrobacterium tumefaciens-mediated transformation was established for B. cinerea in Bruel’s laboratory (Rolland et al., 2003), and also an ATMT mutant library of B. cinerea was built in our early study. In this study, the inserted vector with antibiotic selected marker led to gene deactivation. By screening mutant library of B. cinerea, a spore-morphogenesis-variant, no-sclerotia, virulence-reduced mutant BCt160 was obtained. We assumed that BcDR1 gene was involved in the development and pathogenicity of B. cinerea. BLASTP analysis showed that the protein of BcDR1 displayed high homology to SS1G_08094 of Sclerotinia sclerotiorum and the putative Zn(II)₂Cys₆ transcription factor of Aspergillus nidulans. The putative Zn(II)₂Cys₆ transcription factor of Aspergillus nidulans represses sexual development upon integration of several environmental signals (Vienken and Fischer, 2006), which coincides with our experimental hypothesis. This paper is the first report of the functional analysis of putative gene BcDR1. In-depth study of the gene’s function will clearly provide basis for researching its pathogenic molecular mechanism and controlling B. cinerea. The specific mechanisms of BcDR1 gene regulation, development and pathogenicity, especially the cause of significant virulence reduction by the mutant conidia, remain unclear and will be our further research emphases.

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

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