Appl Microbiol Biotechnol (2014) 98:9413–9424 DOI 10.1007/s00253-014-6087-7

METHODS AND PROTOCOLS

Efficiency of different strategies for gene silencing in Botrytis cinerea José Espino & Mario González & Celedonio González & Nélida Brito

Received: 17 July 2014 / Revised: 5 September 2014 / Accepted: 7 September 2014 / Published online: 8 October 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract The generation of knock-out mutants in fungal pathogens by gene replacement and insertional mutagenesis is the classical method to validate virulence factors. An alternative strategy consists of silencing the candidate virulence gene by making use of the phenomenon of RNA interference (RNAi), adding features such as the possibility of generating knock-down mutants with variable expression levels of the target gene or the ability to simultaneously target multiple genes. Two different approaches have been assayed to generate knock-down mutants by RNAi in the phytopathogenic fungus Botrytis cinerea. In the first one, the single nitrate reductase gene in the B. cinerea genome, niaD, was silenced by transformation with a construct containing a 400-bp niaD fragment between two opposing promoters, so that a dsRNA fragment was generated. As an alternative approach, the mgfp4 gene coding for the green fluorescent protein (GFP) was silenced by transforming two different GFP-expressing strains of B. cinerea with a hairpin RNA (hpRNA)-expressing vector, containing two inverted copies of a 300-bp mgfp4 fragment separated by a spacer DNA. While the opposing dual-promoter strategy produced gene silencing in about half of the transformants assayed, the efficiency of the hpRNAexpressing vector was higher, inducing a decrease in GFP levels in more than 90 % of transformants. The degree of

Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-6087-7) contains supplementary material, which is available to authorized users. J. Espino : M. González : C. González : N. Brito (*) U.D. Bioquímica y Biología Molecular, Universidad de La Laguna, 38206 La Laguna, Tenerife, Spain e-mail: [email protected] Present Address: J. Espino Institut für Biologie und Biotechnologie der Pflanzen, Westf. Wilhelms-Universität, Schlossplatz 8, 48143 Münster, Germany

silencing achieved was high with both methods, but the hpRNA strategy resulted in a higher proportion of strongly silenced transformants. Keywords Botrytis . RNA interference . Post-transcriptional gene silencing . Hairpin RNA . Opposing dual-promoter . niaD . GFP

Introduction The phytopathogenic fungus Botrytis cinerea causes gray mold in more than 200 plant species (Elad et al. 2004), including many economically important crops. With the aim of elucidating the molecular mechanisms underlying plantpathogen interaction, different experimental approaches have been used in order to identify genes/proteins contributing to virulence: differential display RT-PCR (Benito et al. 1996), suppression subtractive hybridization (Gronover et al. 2004), proteomics (Espino et al. 2010), transcriptomics (Leroch et al. 2013), etc. The putative virulence factors identified by these approaches need to be validated, and the generation of the corresponding knock-out mutants is the method most frequently used. Site-specific integration of exogenous DNA in the genome of Botrytis is an efficient process (Noda et al. 2007), and this fungus shows one of the highest rates of homologous recombination among filamentous fungi (Tudzynski and Siewers 2004). Nevertheless, the generation of knock-out mutants is not always possible and is far from being an easy and straightforward technique. Transformation of B. cinerea results always in heterokaryons, which even under selection pressure can maintain a mixture of transformed and untransformed nuclei, i.e., some nuclei carry a modified copy of the target gene and others a wild-type copy. This makes necessary, therefore, to purify homokaryons through single spore isolation or through several subculturing

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events under selection pressure, followed by verification of the correct genotypes by PCR and Southern blot, a timeconsuming and labor-intensive process. On the other hand, the purification of homokaryotic mutants for essential genes is impossible by definition, and in these cases maintaining transformants in the heterokaryon state can be useful. Heterokaryons may display at least partial phenotype pointing to a putative function for the target gene (Giesbert et al. 2014). Nevertheless, variable ratios of untransformed versus transformed nuclei in these strains make it difficult to work with them, and the necessity to maintain selective pressure makes certain experiments impossible, such as those in planta. RNA interference or RNA-mediated silencing has emerged as a powerful tool to analyze gene function. It is triggered by double-stranded RNA molecules (dsRNAs) with sequence homology to the target DNA or RNA, and leads either to DNA methylation and chromatin modification (transcriptional gene silencing) or to mRNA degradation or translational repression (post-transcriptional gene silencing) (Burroughs et al. 2014). The ancestral role of RNA-mediated silencing may have been the defense against viruses and transposable elements, but through evolution small RNAs have become essential as regulatory elements in various developmental and physiological processes (Burroughs et al. 2014). Several recent studies have now demonstrated that RNA silencing also plays a role in plant defense against non-viral pathogens (Nunes and Dean 2012), including the spreading of systemic acquired resistance to the offspring (Luna et al. 2012). In filamentous fungi, RNA silencing was first reported in Neurospora crassa and described as quelling, a phenomenon that occurs exclusively in vegetative cells via the so-called short interfering RNAs (siRNAs) (Chang et al. 2012; Dang et al. 2014). Shiu et al. (2001) reported a related process in this fungus, meiotic silencing by unpaired DNA or MSUD, which silences the copies of unpaired genes during sexual development as a surveillance mechanism. Since then, quelling has been described in diverse fungal species but, besides N. crassa, MSUD has been demonstrated so far only in Gibberella zeae (Son et al. 2011). In N. crassa, both mechanisms evolved independently from a common ancestor, sharing only a few components (Burroughs et al. 2014; Chang et al. 2012; Dang et al. 2014). Proteins containing conserved Argonaute-Piwi, Dicer, or RNA-dependent RNA polymerase (RdRP) domains are key components of these silencing mechanisms and participate in the generation of siRNAs, MSUDassociated small interfering RNAs, and different types of small RNAs with a vast array of RNA interference-related cellular functions (Burroughs et al. 2014). RNA silencing has become an alternative strategy that can be useful to validate candidate virulence factors by a loss-offunction approach (Kück and Hoff 2010), and it has certain advantages that may be relevant to specific circumstances. This strategy does not result usually in complete silencing of

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the target gene but in diminished levels of the target mRNA (knock-down), and the extent of silencing is usually variable from transformant to transformant resulting in a collection of strains silenced in variable degrees (Kück and Hoff 2010). An evident application of these feature is the study of essential genes (Ziv and Yarden 2010), but it is not the only one. Knock-down strains for a given gene may be essential when validating new targets in the design of novel fungicides, for example, since no fungicide will completely inhibit its target in the field and it will be helpful to estimate the minimum effective inhibition rate (Cools and Hammond-Kosack 2013). The use of RNA silencing to study B. cinerea genes was first reported in 2008. The genes coding for a superoxide dismutase (Patel et al. 2008), a phospholipase C component of the BCG1- and calcineurin-dependent signaling pathway (Schumacher et al. 2008), and a phosphatidylethanolaminespecific phospholipase D (Rolland and Bruel 2008) were silenced using different strategies, most often by the expression of the two strands of the target gene to generate a dsRNA molecule. RNA silencing was later used to explain the inadvertent gene silencing of argininosuccinate synthase (bcass1) by the pLOB1 vector system (Patel et al. 2010), and to elucidate the role in virulence, growth, and differentiation of Bcpp2Ac, a PP2A serine/threonine protein phosphatase (Giesbert et al. 2012). Nevertheless, a detailed comparative study of the different silencing strategies has not been carried out, to our knowledge, in this fungus. Here, we report the efficiency of RNA interference as a tool to obtain knock-down transformants in B. cinerea, using two different strategies to generate double-stranded RNA molecules: transformation with a hairpin RNA-expressing plasmid, or with a plasmid containing the target sequence to be silenced between two opposing promoters.

Materials and methods Genome sequence data mining B. cinerea genome databases at URGI (Unité de Recherche Génomique Info, http://urgi.versailles.inra.fr/) and Broad Institute (http://www.broad.mit.edu/annotation/) were searched with the blastp and tblastn programs to identify genes coding for Argonaute-, Dicer-, and RdRP-like proteins. Experimentally characterized proteins were used as query (Table S1 in Online Resource 1). The BLOSUM 50 matrix was used in all the searches, and only the results with an Expect (E) value lower than 10−5 were considered. UniProt (http://www.uniprot.org) accession numbers are indicated for each protein sequence. Conserved domains were defined with reference to the Pfam database (http://pfam.xfam.org). Gene IDs indicate gene codes in the genome databases of the two B.

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cinerea strains with sequenced genomes: B. cinerea B05.10 (codes BC1G …) and B. cinerea T4 (codes BofuT4…). Fungal strains and culture conditions B. cinerea B05.10 strain was obtained from P. Tudzynski (Westfaelische Wilhelms-Universitaet Muenster, Germany) and can be found in public culture collections such as CECT (http://www.cect.org, ref. CECT 20754) and VTT (http:// culturecollection.vtt.fi, ref. D-071295). B05.HYG3 strain contains a 5′ truncated copy of the hygromycin resistance gene and was obtained in previous work (Noda et al. 2007). Fungal strains were maintained as conidial suspensions in 15 % glycerol at −80 °C for long storage and at 4 °C in silica gel for routine use (Delcan et al. 2002). Conidia were prepared as described by Benito and associates (Benito et al. 1998) from cultures on tomato-agar plates (25 % homogenized tomato fruit, 2 % agar, pH 5.4). Liquid cultures were routinely made on Gamborg’s B5 medium (Duchefa Biochemie Bv, Haarlem, The Netherlands) supplemented with 10 mM KH2PO4, 1 % glucose, and 0.05 % yeast extract, inoculated with 106 conidia/ml and incubated at 20 °C with shaking for 3 days. For the induction of nitrate reductase, mycelia were then collected by centrifugation, washed, and transferred to the same fresh medium but with 20 mM sodium nitrate instead of yeast extract. After 3 h at 20 °C with shaking, mycelia were collected by filtration, washed with sterile water, dried with filter paper, and frozen at −20 °C until use. To induce the expression of genes under the control of the xyn11A promoter, the above Gamborg’s B5 medium was supplemented with 1 % xylan (beechwood xylan, Sigma-Aldrich, MO, USA) instead of glucose, inoculated with 106 conidia/ml and, after 3 days at 20 °C, mycelia were filtered, washed, and stored at −20 °C until further use. Fluorescence of the mycelium of the green fluorescent protein (GFP)-expressing strains was observed for whole colonies grown on tomato-agar plates for 2 days, illuminating the plates with near-UV. Standard molecular techniques Recombinant DNA methods were performed as described by Sambrook and Russell (2001). Genomic DNA from B. cinerea was extracted using a method previously developed in our laboratory (González et al. 2008). All cloning PCRs were carried out using the high fidelity Phusion DNA polymerase (Finnzymes, Keilaranta, Finland). Taq DNA polymerase (GenScript, Picataway, NJ, USA) was used for all other PCRs. Oligonucleotides were from Invitrogen (Paisley, Scotland), and their nucleotide sequences are indicated in Table S2 (Online Resource 1). Recombinant plasmids were maintained in Escherichia coli SURE2 (Stratagene, La Jolla,

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CA) cells. DNA sequencing was performed by Sistemas Genómicos S.A. (Valencia, Spain). B. cinerea transformations were carried out according to the protocol of Hamada et al. (1994), modified by van Kan et al. (1997).

Construction of plasmids and B. cinerea strains Two different plasmids, pLOB-MBDSil and pLOBDIMERSil, were constructed containing two different regions of the nitrate reductase gene niaD (BC1G_09772.1) flanked by two inverted copies of its own promoter (Fig. S1a in Online Resource 1). These plasmids were used to transform B. cinerea B05.10, resulting in random integration of the foreign DNA, and the hygromycin-resistant transformants were characterized by PCR to ensure that the silencing construct was intact and also to discard homologous recombination of the exogenous DNA at the niaD locus (Table S3 in Online Resource 1). Plasmids pHYG5-MBDSil and pHYG5-DIMERSil contain the same dual-promoter niaD silencing constructs as the two above but in a vector backbone design for site-specific integration in the B. cinerea genome (Noda et al. 2007) (Fig. S1b in Online Resource 1). These plasmids were used to transform B. cinerea B05.HYG3 (Noda et al. 2007), and the site-specific integration was confirmed by PCR (Table S3 in Online Resource 1). Additional PCR reactions were used to ensure that no additional integrations had occurred at the niaD gene and to ensure the integrity of the silencing construct (Table S3 in Online Resource 1). Two plasmids, pTUBGFP-HYG5 and pXYLGFP-HYG5, were constructed in order to generate B. cinerea strains expressing GFP under the control of the tubA promoter (Benito et al. 1998) or the xylanase xyn11A promoter (Brito et al. 2006), respectively (Fig. S1c in Online Resource 1). The strain B05.HYG3 (Noda et al. 2007) was transformed with them. Transformants were then purified by single-conidia isolation on hygromycin-containing plates to ensure homokaryosis, and the site-directed integration of both plasmids in the B05.HYG3 genome and the integrity of the promoter-GFP hybrid constructs were confirmed by PCR amplifications (Table S3 in Online Resource 1). The resulting strains expressing GFP were named HYG3.TUBGFP and HYG3.XYLGFP, respectively. Plasmid pBOS was designed to express a hairpin RNA (hpRNA) intended to silence the expression of GFP and contains two inverted copies of a 300-bp mgfp4 region flanking a spacer sequence (Fig. S1d in Online Resource 1). The strains HYG3.TUBGFP and HYG3.XYLGFP were transformed with the plasmid pBOS to evaluate the silencing of the mgfp4 gene by inducing the expression of hairpin RNA molecules. The nourseothricin-resistant transformants were characterized by PCR to verify the integrity of the parental

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copy of the mgfp4 gene and the exogenous silencing construct (Table S3 in Online Resource 1). Nitrate reductase assay Protein extracts were obtained by grinding frozen mycelia in a mortar and resuspending the resulting powder in 0.1 M potassium phosphate pH 7, 1 mM EDTA, 20 μM FAD, and 10 % glycerol. The mixtures were then centrifuged for 5 min at 15,500×g, at 4 °C, and the supernatants were collected for assay. Nitrate reductase activity was assayed in a final volume of 500 μl containing 50 mM potassium phosphate pH 7.4, 20 mM NaNO3, 20 μM FAD, and 0.2 mM NADPH. The assay mixture was incubated at 30 °C for 15 min, and the reaction stopped with 500 μl of 0.02 % N-(1-naphthyl)ethylenediamine and 500 μl of 1 % sulfanilamide in 3 N HCl. These reagents served to assay nitrite by a colorimetric reaction (Snell and Snell 1949), and the absorbance was determined at 540 nm after 10 min. Proteins were quantified using the Bradford protein assay (Bradford 1976). Specific activity of nitrate reductase is expressed as nmoles of nitrite produced per minute and per milligram of protein. The assays were done in triplicate for each sample. GFP expression Crude extracts were prepared as for DNA purification (González et al. 2008) with several modifications. Briefly, frozen mycelium was homogenized in microcentrifuge tubes with sea sand (fine-grained, Panreac, Barcelona, Spain) and PBS (10 mM Na2HPO4, 2 mM KH2PO4 pH 7.4, 137 mM NaCl, 2.7 mM KCl), with the aid of a household drill, and the extract was then collected by centrifugation at 15,500×g for 10 min. One milligram of total proteins from each sample was loaded to 12 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to nitrocellulose (Protran BA 85, Whatman, Schleicher & Schuell, Dassel, Germany), and probed with a 1:1000 dilution of anti-GFP monoclonal antibodies (Roche Diagnostics, Basel, Switzerland) and a 1:3000 dilution of antimouse IgG conjugated to horseradish peroxidase (Sigma Chemical Co., St. Louis, MO). The peroxidase was detected with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA), and the intensity of the bands was measured with the software Quantity One (Bio-Rad, CA, USA) on the chemiluminescence signal recorded with a Gel Doc XR+ system (Bio-Rad). To correct protein loading errors, 2 mg of total proteins were loaded to SDS-PAGE gels, stained with Coomassie Brilliant Blue (Neuhoff et al. 1988) and each lane was scanned (Epson Perfection 1240U scanner model) and analyzed with the image analysis software Quantity One (BioRad). The ratio between GFP band intensities in Western blot, on one hand, and the intensity of the whole lane in the

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Coomassie-stained SDS-PAGE, on the other, was then calculated for each sample and taken as a measure of the amount of GFP expressed by each strain. The results were normalized to the value obtained for the control strain and are expressed as percentage. Quantitative real-time PCR Total RNA was isolated with the RNeasy plant mini kit (Qiagen Inc., Valencia, CA, USA), and mRNA was purified with the Oligotex mRNA mini kit (Qiagen). Absence of contaminant genomic DNA was assayed by PCR with the primer pair GFPC/GFPCDNA and, when necessary, it was eliminated by treatment with RNase-free DNaseI. cDNA synthesis was carried out with the First Strand cDNA synthesis kit for RT-PCR (AMV) (Roche, IN, U.S.A.). Amplification was carried out in an iCycler iQ Real-Time PCR system (BioRad), with the Bio-Rad iQ SYBR Green supermix and the primer pair GFPC/GFPCDNA. The B. cinerea actin gene actA was used to correct for sample-to-sample variation in the amount of RNA (primers ACTAFW and ACTARV). The amplification of a single fragment was verified for every PCR reaction by running the final product on a 12 % polyacrylamide gel electrophoresis. The relative mRNA amounts were calculated by the ΔΔCt method from the mean of three independent determinations of the threshold cycle (Schmittgen and Livak 2008). Deviation from the mean was calculated from the standard deviation (SD) in the ΔΔCt value, using the expression 2−(ΔΔCt±SD).

Results The RNA silencing machinery in B. cinerea The genome databases of B. cinerea strains T4 and B05.10 were searched with the BLAST algorithm, using as query sequences representative and experimentally characterized members of the key families involved in the phenomenon of RNA silencing: Dicer, RdRP, and Argonaute (Table S1 in Online Resource 1). Only hits with an expect value lower than 10−5 were considered. Dicer proteins are RNase III-type endonucleases which specifically cut dsRNA and are essential for the biogenesis of siRNAs and other small RNAs (Jaskiewicz and Filipowicz 2008). Proteins in this group have invariably two catalytic RNase III domains in tandem, and some of them may contain one or more dsRNA binding domains (dsRBD), one or more ATP-dependent RNA helicase motifs at the N-terminus, and a domain of unknown function (the DUF283 domain). They may also contain a PAZ domain (for Piwi-Argonaute-Zwille), which acts as an RNA binding module that specifically

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recognizes the 3′ ends with two protruding nucleotides, a feature typical of siRNAs (Jaskiewicz and Filipowicz 2008). Two genes coding for putative Dicer proteins were identified in the B. cinerea genome. The first one, BC1G_10438.1/ BofuT4_P093500.1, is homologous to fungal DCL2 proteins and codes for a 1398-amino acids polypeptide containing two typical RNase III domains, one dsRNA binding domain and two helicase domains (DEAD helicase and helicase C) (Fig. S2 in Online Resource 1). Its sequence identity with DCL2 proteins from N. crassa (Q7SCC1), Magnaporthe oryzae (A4RHU9), and Cryphonectria parasitica (Q2VF18) is in the range 35–38 %. The second gene, BC1G_10104.1/ BofuT4_P068420.1+BofuT4_P068410.1, is annotated as two different genes in B. cinerea T4 due to its location at a gap between contigs bt4ctg_0903 and bt4ctg_0904, in the supercontig bt4_SuperContig_83_1. It codes for a protein of 1842 amino acids that, besides dsRBD and RNase III domains, contains a conserved helicase motif (Fig. S2 in Online Resource 1). It is homologous to fungal DCL1 proteins, with sequence identities in the range 42–45 % with the corresponding proteins in M. oryzae (A4RKC3), C. parasitica (Q2VF19), and N. crassa (Q7S8J7). The role of RNA-dependent RNA polymerases (RdRPs) in RNA silencing is the synthesis of dsRNA from single-stranded RNA templates. They contain a conserved structure with a double-psi β barrel and display the DXDGD motif (where X represents any amino acid), which is conserved in all RdRPs and corresponds to the active site of the enzyme (Wassenegger and Krczal 2006). Three genes were identified in B. cinerea as RdRP homologues (Fig. S2 in Online Resource 1). BC1G_15614.1/BofuT4_P146920.1+BofuT4_P146930.1 codes for an 1173-amino acids protein with the DLDGD motif and is highly similar (48 % amino acid identity) to the suppressor of ascus dominance (SAD-1) protein from N. crassa (Q9C162). In the genome of strain T4, the gene was not completely sequenced and is split in two parts located at the ends of the consecutive contigs bt4ctg_2043 and bt4ctg_2044, in the supercontig bt4_SuperContig_110r_56_1. The second gene, BC1G_09542.1/BofuT4_P130500.1 (BC1G_09542.1 is incomplete due to a gap in the genome sequence at the end of the contig 2182), codes for a protein of 1007 amino acids with an RdRP domain displaying the DYDGD motif. This protein is highly similar (41 % identity) to N. crassa QDE1 polymerase (Q9Y7G6). The third gene, BC1G_15659.1/ BofuT4_P117290.1, codes for a protein of 1080 amino acids also containing the DLDGD motif and highly similar to RNAdependent RNA polymerases from Marssonina brunnea (K1X3W7) and Ajellomyces dermatitidis (C5GLL2), with sequence identities of 44 and 42 %, respectively, and with lower similarity to RDR1 proteins from Arabidopsis thaliana (Q9LQV2) and Oryza sativa (Q0DXS3). Finally, Argonaute family proteins (AGOs) are composed of four domains: the N-terminal domain, the PAZ domain that

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binds the 3′-end of guide RNAs/DNAs, the middle (MID) domain that provides a binding pocket for the 5′-phosphate of guide RNAs/DNAs, and the P-element-induced wimpy testis (PIWI) domain that adopts an RNase H fold and has endonucleolytic activity in some but not all AGOs (Kuhn and JoshuaTor 2013). The PAZ and PIWI domains are considered as the two signatures of this group of proteins, the first one located in the center and the last one near the C-terminus. These proteins are key components in the multiprotein complex RNAinduced silencing complex (RISC), and are required for its silencing activity (Gagnon and Corey 2012). Four AGO-like proteins could be found in the B. cinerea genome, all of them with PIWI and PAZ domains (Fig. S2 in Online Resource 1). The first one is BC1G_00797.1/BofuT4_P019310.1, a protein of 1013 amino acids that has a 34 % identity with the proteins QDE-2 from N. crassa (Q9P8T1) and RDE-1 from Caenorhabditis elegans (G5EEH0). An erroneously annotated intron in the B05.10 gene (BC1G_00797.1) situates the PAZ domain within the intron. The second gene, BC1G_06939.1/BofuT4_P087670.1, codes for a 939-amino acids protein similar (42 % identity) to N. crassa SMS-2 (Q8J276). The third AGO-like gene, BC1G_15122.1+ BC1G_15121.1/BofuT4_P060800.1, codes for a protein similar (29 % identity) to C. parasitica AGL3 (D0U266). The last AGO-like gene is BC1G_06419.1+BC1G_06418.1/ BofuT4_P063040.1 and the protein is homologous (30 % identity) to AGL2 from C. parasitica (D0U265).

Silencing by random integration of a dual-promoter construct The classical strategy to produce RNA silencing is the introduction in the genome of a DNA construct that generates dsRNA molecules homologous to at least part of the target gene to be silenced. This dsRNA is then processed to generate siRNAs that guide the sequencedependent degradation of the target mRNA. One possibility to generate dsRNA is to clone a fragment of the gene to be silenced between two inverted promoters, a strategy known as dual-promoter, which leads to the simultaneous production of sense and antisense RNAs that hybridize inside the cell generating dsRNA molecules. We tried this method to silence the nitrate reductase gene in B. cinerea, an enzyme catalysing the NAD(P)H-dependent reduction of nitrate to nitrite, which is the first step in its conversion to ammonium prior to the incorporation of nitrogen into carbon skeletons. The genome of B. cinerea contains a single nitrate reductase gene, niaD (BC1G_09772.1/ BofuT4_P116590.1), that codes for a 908-amino acids protein with the typical domains of these enzymes (Campbell 2001): the molybdenum-molybdopterin domain, the dimerization interface, the Cyt b domain, and the FAD binding domain.

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Two different regions of the niaD gene were used to construct the silencing vectors, avoiding the use of the FAD and heme domains since that could have led to the simultaneous silencing of additional proteins displaying them. The two regions, one corresponding to the molybdenum cofactor binding domain (amino acids 99–266), and another to the dimerization domain (amino acids 333–465), were cloned between two opposing copies of the niaD promoter (Fig. 1a) in a vector conferring hygromycin resistance. The resulting plasmids, pLOB-MBDSil and pLOB-DIMERSil, were transformed into the B. cinerea strain B05.10. To be functional in silencing, the plasmids should integrate in such a way that the integrity of the silencing construct (the niaD fragment between the two inverted promoters) is maintained, and that the integrity of the original niaD locus in the genome is not altered, i.e., the original niaD gene is not knocked-out by the integration of the transforming plasmid. All the transformants

were analyzed by PCR (Table S3 in Online Resource 1) to ensure that these two conditions were met (not shown) and around 20 from each transformation were selected and further analyzed. Those transformants showing a reduction in nitrate reductase activity of at least 10 % in comparison with the parent strain were considered to be silenced. About half of the transformants obtained were silenced for the target gene, in both transformations (Table 1), but the degree of silencing was more pronounced for the transformants silenced with the MBD fragment: all silenced transformants obtained with pLOB-MBDSil showed less than half the activity of the parent strain, while only two of the ten silenced transformants obtained with pLOB-DIMERSil did (Fig. 1). It is worth noting the high variability of silenced phenotypes obtained by this approach, with a wide range of silencing degrees among the transformants (Fig. 1). These variations may be due to the different epigenetic states of

Fig. 1 Silencing of the niaD gene by the dual-promoter strategy. a Structure of the silencing construct showing the two inverted niaD promoters flanking a niaD region. b–e Level of nitrate reductase activity in each of the silenced transformants, expressed as percentage of the activity shown by the parent strain B05.10 (1.6±0.2 nmoles min−1 mg protein−1) or B05.HYG3 (1.43±0.5 nmoles min−1 mg protein−1). Transforming plasmids used for silencing (indicated) contained a region of niaD coding for

either the dimerization sequence of nitrate reductase (pLOB-DIMERSil, pHYG5-DIMERSil) or the molybdenum cofactor binding domain (pLOBMBDSil, pHYG5-MBDSil), and were introduced in the genome by random (b, c) or site-directed (d, e) integration. The different colors in bars serve to group strains with different degrees of silencing: less than 20 % of the activity of the wild type (black bars), 20–70 % (white bars), or more than 70 % (gray bars)

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Table 1 Comparison of silencing efficiencies obtained by the dual-promoter and the hpRNA strategies niaD silencing by a dual-promoter construct Parent strain Transforming plasmid Mode of integration in the genome Number of transformants analyzed Number of silenced transformantsa a

mgfp4 silencing by expression of hpRNA

B05.10 B05.10 B05.HYG3 B05.HYG3 HYG3. TUBGFP pLOB-MBDSil pLOB-DIMERSil pHYG5-MBDSil pHYG5-DIMERSil pBOS Random Random Site-directed Site-directed Random

HYG3. XYLGFP pBOS Random

21

19

18

99

48

45

12 (57.1 %)

10 (52.6 %)

9 (50.0 %)

43 (43 %)

45 (93.7 %)

43 (95.5 %)

A reduction higher than 10 % in NR activity or GFP level, as compared with the parent strains, was considered significant

the sites where the silencing constructs integrated in each individual transformant. Since the integration site is chosen at random, the transformants may differ greatly in the level of dsRNAs produced. Integration in euchromatin areas, for example, could result in higher levels of expression than integration in areas of heterochromatin, thus resulting in more evident knock-down phenotypes. To eliminate this source of variation, a new approach was designed using the B. cinerea strain B05.HYG3 (Noda et al. 2007), which ensures site-specific integration at a defined genome locus of at least one copy of the transforming DNA.

Silencing by site-directed integration of a dual-promoter construct In this new approach, we made use of a site-directed integration system previously designed in our laboratory (Noda et al. 2007), which consists in a strain (B05.HYG3) containing a 5′ truncated copy of the hygromycin resistance cassette, and a vector (pBS.HYG-5) which contains a 3′ truncated copy of the same resistance marker that overlaps with the one in the strain, so that both copies share a common region of 720 bp of the hph gene (Noda et al. 2007). The only way to obtain hygromycin-resistant transformants is by integration of the plasmid in the HYG-3′ locus of the recipient strain. The dual-promoter silencing constructs for the two niaD fragments described above (Fig. 1a) were cloned in pBS.HYG-5 and the resulting plasmids, pHYG5-MBDSil and pHYG5-DIMERSil, were transformed into strain B05.HYG3 to obtain 18 and 99 hygromycin-resistant transformants, respectively. All the strains tested contained one copy of the transforming plasmid integrated into the locus with the truncated hygromycin resistance cassette, as well as an intact niaD gene (not shown). Similarly to the results obtained for the random integration of the silencing plasmids, about half of the transformants showed nitrate reductase levels significantly lower than the parent strain (Table 1). The level of reduction varied between the silenced strains (Fig. 1d, e) in a way similar to the random

integration strategy, so that a collection of transformants silenced in a wide range of degrees was obtained.

Gene silencing by expression of hairpin RNA A different strategy to generate silencing dsRNAs in the cells, alternative to the dual-promoter, is the expression of a silencing construct composed of two inverted repeat regions of the same gene fragment separated by an unrelated spacer DNA. The transcription of this construct would result in a hairpin RNA with a double strand region homologous to the target gene, which can be processed by the silencing machinery and lead to the degradation of the target mRNA. We tried this method for the silencing of mgfp4, a GFP gene optimized for plants (Haseloff et al. 1997). Using the site-directed integration strategy outlined above and plasmids pXYLGFP-HYG5 and pTUBGFP-HYG5, two B. cinerea strains were constructed expressing the reporter protein, one of them under the control of the xyn11A promoter (Brito et al. 2006) and the other under the control of the tubA promoter (Benito et al. 1998). One GFP-expressing transformant was selected, from each transformation, and characterized by PCR (Table S3 in Online Resource 1) to ensure the integration at the HYG3 locus and the integrity of the mgfp4 gene (not shown). These strains were named HYG3.XYLGFP and HYG3.TUBGFP, respectively. The expression of GFP in these strains was corroborated, in the first place, by direct observation of the colonies under nearultraviolet light and of the hyphae under the fluorescence microscope (Fig. 2a), and also by Western blot with antiGFP antibody (Fig. 2b). In order to silence the gene for GFP, these strains were transformed with plasmid pBOS containing two inverted copies of an mgfp4 region flanking a spacer sequence (Fig. 2c), as well as the nourseothricin resistance cassette to allow selection. Fifty transformants from each transformation were analyzed by PCR (Table S3 in Online Resource 1) to ensure the integrity of the silencing construct and the GFP-expressing cassette, and 93 strains were identified that fulfilled these

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Fig. 2 Silencing of the mgfp4 gene by the hairpin strategy. a Fluorescence of the parent B. cinerea strains used to silence GFP, in colonies grown on tomato-agar plates (left) or under the fluorescence microscope (right). b Western blot showing the linearity of GFP signal versus protein amount. Different volumes of cell-free extracts from the two GFP-expressing B. cinerea strains, with the indicated total protein amounts, were probed with anti-GFP antibody. The strain B05.HYG3 (BH) was used as

a negative control. c Structure of the silencing construct in the transforming plasmid (pBOS) showing the two inverted GFP regions flanking a linker sequence, which results in the silencing hpRNA after transcription. d GFP levels among the mgfp4-silenced transformants obtained from the two parent strains. See legend to Fig. 1 for meaning of bar colors

criteria (48 in the transformation of HYG3.TUBGFP and 45 in the transformation of HYG3.XYLGFP). The level of GFP was determined for each one of them by Western blot with anti-GFP antibodies and a reduction higher than 10 %, as compared with the parent strains, was considered significant. Forty-three of 45 HYG3.XYLGFP transformants showed at least this level of reduction, and similar results were obtained for the transformation of the HYG3.TUBGFP strain (Table 1). For both GFP-expressing strains, a collection of transformants silenced for GFP in very different degrees (Fig. 2d) was obtained, similarly to the dual-promoter strategy (Fig. 1). However, the proportion of transformants with undetectable or low expression of the target gene is higher in the case of the hairpin strategy (Figs. 1 and 2). These results indicate, when compared with those obtained by the dual-promoter strategy, that in B. cinerea, the expression of a hairpin RNA is quite more effective in silencing than the simultaneous expression of the two complementary RNAs. To confirm that the reduced levels of GFP in the silenced transformants were indeed due to decreased levels of mgfp4 mRNA, six silenced strains were chosen and mRNA levels were quantified by quantitative real-

time PCR (Q-RT-PCR). The results showed that in all silenced transformants the levels of mgfp4 mRNA were reduced in more than 60 %, in accordance with the levels of GFP protein observed in the same strains by Western blot (Fig. 3a). These six strains were also grown in tomato-agar plates to verify whether the levels of reduction in the amount of green fluorescent protein detected in the extracts caused a change in the fluorescence of the colonies. Only in those cases where the level of protein was reduced over 90 % (transformants 3 and 12), the fluorescence of the colonies became undetectable, while the rest of the strains showed residual fluorescence at the border of the colonies (Fig. 3b).

Discussion RNA interference is a homology-dependent mechanism highly conserved in eukaryotes involved in gene expression regulation and protection against viruses, non-viral pathogens, and transposons. Members of three key protein families involved in this mechanism, Argonaute, Dicer, and RNA-dependent

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Fig. 3 Levels of GFP mRNA, protein, and fluorescence in selected strains silenced for mgfp4. a mgfp4 mRNA and protein (GFP) levels in six silenced strains. Selected transformants silenced for mgfp4 by the hairpin strategy were used to determine mRNA levels by Q-RT-PCR and GFP levels by semi-quantitative Western blot. mRNA and GFP levels are expressed as percentage of the value obtained for the parent strain HYG3.TUBGFP (T). Inset displays the actual Western blot used for GFP semi-quantification. b Fluorescence of the same six transformants grown on tomato-agar plates for 3 days. Parent strains B05.HYG3 (BH) and HYG3.TUBGFP (T), as well as the wild-type strain B05.10 (B0), were included as controls

RNA polymerase, participate in the biogenesis of different types of small RNAs able to produce transcriptional and posttranscriptional gene silencing. The number of these proteins is quite variable among fungal species, ranging from eight Argonaute proteins and eight RdRPs in Coprinopsis cinerea to a single copy of each in Schizosaccharomyces pombe, or even complete loss of the silencing machinery in some species (Billmyre et al. 2013; Nunes et al. 2011). In this study, we performed BLAST searches for putative RNA silencing proteins in the B. cinerea genome using as query experimentally characterized proteins from different eukaryotes (Table S1 in Online Resource 1) and we have identified two Dicer-like proteins, three RdRPs, and four Argonaute-like proteins (Fig. S2 in Online Resource). Previously, Nunes et al. (2011), analyzing 54 fungal genomes, have reported also 2 Dicer-like proteins, but 2 RdRPs and 3 Argonaute-like proteins for the B. cinerea genome. The role of both Dicer-like proteins of B. cinerea, Bc-DCL1 (BC1G_10104.1) and Bc-DCL2 (BC1G_10438.1), in siRNAs biogenesis has recently been studied (Tauati et al. 2014; Weiberg et al. 2013). Single knock-out mutants in either one of the two genes resulted in slower growth and delayed sporulation, but changes in siRNAs levels were not evident. The double mutant, however, not only showed more obvious defects in growth and sporulation but was completely unable

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to synthesize two different siRNAs. Moreover, as a consequence of B. cinerea-derived siRNAs being able to modulate immunity in plant cells during infection, the double mutant was also severely affected in virulence (Weiberg et al. 2013). This partial redundancy has also been described for DCL-1 and DCL-2 proteins in Mucor circinelloides (Garre et al. 2014) and N. crassa (Dang et al. 2014), but not in M. oryzae where only DCL2 is involved in the production of siRNAs (Kadotani et al. 2004). One of the RdRP identified in our Blast analysis (BC1G_15614.1) shows high homology with N. crassa SAD1, and one of the Argonature-like proteins (BC1G_06939.1) was related to N. crassa SMS-2, both proteins associated with MSUD in this fungus (Billmyre et al. 2013; Dang et al. 2014). On the other hand, Hammond et al. (2013) have also described a putative B. cinerea homologue of N. crassa SAD-4, another protein required for MSUD. These homologies point to the existence of MSUD in B. cinerea, a phenomenon which has never experimentally demonstrated in this fungus. In fact, although proteins related with this process have been identified in different fungal species by genome comparative analysis, MSUD has been observed experimentally only in N. crassa (Shiu et al. 2001) and G. zeae (Son et al. 2011). We have also shown here that the B. cinerea silencing machinery is able to reduce expression of both a transgene (mgfp4) and an endogenous gene (niaD) when a dsRNA homologous to the target gene is produced in the fungal cells. This mechanism was not dependent on the promoter of the gene to be silenced, as the percentage of mgfp4-silenced strains was almost the same (average of 94 %) when the target gene was expressed under the control of the inducible xyn11A promoter (Brito et al. 2006) or the constitutive tubA promoter (Benito et al. 1998). The silencing efficiency did not show a great dependency either on the region of the target gene used in the silencing constructs, as shown by the fact that two different regions of the niaD gene, corresponding to different domains of the protein, were able to silence niaD expression with similar efficiencies (Table 1). While one of the regions (the molybdenum cofactor binding domain) seemed to result in lower niaD expression among silenced transformants obtained by the random integration approach (Fig. 1), this difference was not evident in the other approach (site-directed integration). Moreover, the percentage of silenced transformants was almost the same, about 50 %, regardless of the niaD region used (Table 1). It is worth noting that all silenced transformants were assayed as heterokaryons, so the phenotype is displayed even when there is a mixture of transformed and untransformed nuclei. As already discussed above, this is a great benefit when working with a fungus such as Botrytis since it eliminates the need to purify homokaryons. We analyzed the efficiency of two different strategies to induce gene silencing in B. cinerea. One of them, the dualpromoter system, induced the production of double-stranded

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RNA molecules by introducing in the genome a DNA construct containing the target sequence to be silenced between two opposing promoters. The second one, the hairpin RNA strategy, used constructs designed to express a selfcomplementary RNA able to generate the dsRNA by itself. Although the first of these strategies was quite efficient in generating strains silenced for the target gene (about 50 % of transformants, see Table 1), the efficiency was higher for the hairpin RNA strategy, which resulted in more than 90 % of the transformants having a reduction of more than 10 % in the expression of the target gene, as compared with the parent strain. One interesting feature of the silenced transformants, which is true for the two strategies, is the generation of a collection of strains silenced in very different degrees, a fact which has also been observed in RNA silencing experiments carried out with B. cinerea elsewhere (Patel et al. 2008, 2010; Rolland and Bruel 2008; Schumacher et al. 2008). This variability does not seem to be due, at least exclusively, to the transforming DNA being integrated at different loci for the different transformants, since it did not decrease when a site-directed integration system was used. Similar results were shown by Schumacher et al. (2008), targeting the bcplc1-silencing construct to the niaD locus, indicating that the integration loci of the silencing constructs is not the primary source of variation in the degree of silencing among transformants. The availability of these assortments of knocked-down strains for a given gene may be extremely useful in experiment where different levels of expression are required. These may include, for instance, the evaluation of a given protein as potential target for new fungicides against B. cinerea. If for a given protein, for example, a slight reduction in its amount causes avirulence or loss of viability, this protein would be a very interesting target in the design of novel control strategies. As far as we know, this is the first time that the dualpromoter strategy has been used to silence a gene in B. cinerea. Previous examples of RNA silencing have been done by the expression of sense or antisense fragments of the target gene (Giesbert et al. 2012; Patel et al. 2008, 2010; Schumacher et al. 2008), or by the expression of constructs able to generate RNA stem-loop structures (Giesbert et al. 2012; Rolland and Bruel 2008; Schumacher et al. 2008). Patel et al. (2008) reported silencing of the bcsod1 gene using the sense and antisense-mediated strategy in 36 % of all recovered transformants (27 % of sense and 47 % of antisense). It seems, therefore, that the dual-promoter and the expression of antisense RNA both result in similar silencing frequencies, which is higher than the one obtained by the expression of the sense strand. The best alternative, however, is the hairpin RNA strategy, as exemplified in this work by the silencing of GFP by transformation with plasmid pBOS (Fig. 2), with 90 % of transformants silenced on average.

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Gene silencing in B. cinerea would serve as a powerful tool for the genetic manipulation of this phytopathogenic fungus. It would be particularly suited to study essential genes, which are not amenable to study by gene knock-out methods. The use of inducible promoters in the silencing constructs would allow controlling the expression of the silencing dsRNAs to produce conditional silencing, as has been reported for Aspergillus fumigatus with the cellobiohydrolase gene cbhB promoter (Bromley et al. 2006). Additionally, this strategy has the potential of simultaneously silencing several genes. This has been addressed, firstly, by the expression of short dsRNAs homologous to several different, but related, genes that share the same sequence (Romao-Dumaresq et al. 2012). An alternative strategy consists in the expression of chimeric dsRNAs composed of various discrete regions homologous to different unrelated genes, which has been shown to be very successful in the co-silencing of the CAP59 and ADE2 genes in Cryptococcus neoformans, responsible for the formation of the capsid and the colony color (Liu et al. 2002). The same approach allowed the co-silencing of two genes involved in the synthesis of the cell wall in A. fumigatus (Henry et al. 2007), six hydrophobin genes in Cladosporium fulvum (Lacroix and Spanu 2009), or 10 xylanase genes in M. oryzae (Nguyen et al. 2011). One added advantage of the simultaneous silencing of several genes is the fact that only one transformation event is required and therefore only one marker gene is needed. The alternative knock-out of all genes involved would require one transformation event and one marker gene per knock-out. Two features of B. cinerea make this strategy particularly interesting for this fungus. Its genetic transformation, in first place, is not an easy technique, and gene markers are scarce. Secondly, the ability to silence multiple genes simultaneously is especially appropriate for the high prevalence of redundancy among B. cinerea genes, especially within secreted protein families such as polygalacturanases, proteases, etc. (Espino et al. 2010). The use of gene silencing in B. cinerea could accelerate the analysis of the contribution to virulence of defined biological functions carried out by multiple proteins, such as the degradation of cellulose, xylan, or cutin, among others. Acknowledgments Support was provided by grants from the Ministerio de Educación y Ciencia (BIO2002-02048 and AGL200609300). JJE was supported by Gobierno de Canarias; 85 % of funds received from Gobierno de Canarias came from the European Regional Development Fund.

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Efficiency of different strategies for gene silencing in Botrytis cinerea.

The generation of knock-out mutants in fungal pathogens by gene replacement and insertional mutagenesis is the classical method to validate virulence ...
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