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. 2010 May 19;11(5):613–624. doi: 10.1111/j.1364-3703.2010.00632.x

Inadvertent gene silencing of argininosuccinate synthase (bcass1) in Botrytis cinerea by the pLOB1 vector system

RISHA M PATEL 1, JAN A L VAN KAN 2, ANDY M BAILEY 1,✉, GARY D FOSTER 1,✉
PMCID: PMC6640230  PMID: 20696000

SUMMARY

For several years, researchers working on the plant pathogen Botrytis cinerea and a number of other related fungi have routinely used the pLOB1 vector system, based on hygromycin resistance, under the control of the Aspergillus nidulans oliC promoter and what was reported to be the β‐tubulin (tubA) terminator. Recently, it has been demonstrated that this vector contains a 446‐bp portion of the B. cinerea argininosuccinate synthase gene (bcass1) rather than the tubA terminator. As argininosuccinate synthase is essential for the production of l‐arginine, inadvertent gene silencing of bcass1 may result in partial l‐arginine auxotrophy and, indeed, may lead to altered phenotypes in planta. In this article, we report our findings relating to possible problems arising from this incorrect plasmid construction. As an absolute baseline, gene disruption of bcass1 was carried out and generated a strict auxotroph, unable to grow without exogenous arginine supplementation. The knockout displayed an alteration in host range in planta, showing a reduction in pathogenicity on strawberries, French bean leaves and tomatoes, but maintained wild‐type growth on grape, which is in accordance with the reported arginine availability in such tissues. Deliberate gene silencing of bcass1 mirrored these effects, with strongly silenced lines showing reduced virulence. The degree of silencing as seen by partial auxotrophy was correlated with an observed reduction in virulence. We also showed that inadvertent silencing of bcass1 is possible when using the pLOB1 vector or derivatives thereof. Partial arginine auxotrophy and concomitant reductions in virulence were triggered in approximately 6% of transformants obtained when expressing enhanced green fluorescent protein, luciferase, monomeric red fluorescent protein or β‐glucuronidase using the pLOB1‐based expression system, which inadvertently contains 446 bp of the bcass1 coding sequence. We recommend the testing of transformants obtained using this vector system for arginine auxotrophy in order to provide assurance that any observed effects on the development or virulence are a result of the desired genetic alteration rather than accidental bcass1 silencing.

INTRODUCTION

Botrytis cinerea is an opportunistic necrotrophic fungus with a broad host range, capable of infecting over 200 plant species worldwide, including ornamentals, vegetables and fruits (Williamson et al., 2007). Various biological and chemical control measures have been developed to limit the damage caused by B. cinerea. Because fungicide effectiveness declines soon after field introduction, new targets for control are being sought (Leroux et al., 2002; Smith, 1998). Once potential targets are identified through gene knockout or silencing methodologies, they must be validated by in planta growth assays. Molecular genetic approaches have been deployed in the last decade to identify important virulence determinants of B. cinerea that may serve as targets for novel control measures (van Kan, 2006; Tudzynski and Siewers, 2004). Gene function can be studied by targeted or random insertional mutagenesis and the evaluation of whether the mutation affects virulence.

pLOB1 (GenBank Accession No. AJ439603) (Fig. 1A, B) is a hygromycin resistance vector that is frequently utilized for B. cinerea molecular manipulations. Since its first publication (van Kan et al., 1997), this hygromycin cassette has been used to generate random insertion mutants and over 30 targeted gene‐specific mutants (for example: Brito et al., 2006; 2005, 2006a, 2006b; Espino et al., 2005; ten Have et al., 2010; 2002a, 2002b; van Kan et al., 1997; 2005a, 2005b; Reis et al., 2005; Rui and Hahn, 2007a, 2007b; Schoonbeek et al., 2002; 2002a, 2002b). When the sequence of pLOB1 was compared with the genome sequence of B. cinerea strain B05.10 (http://www.broadinstitute.org/), it became apparent that the terminator sequence downstream of the hygromycin phosphotransferase (hph) gene in pLOB1 was identical to a partial B. cinerea endogene (argininosuccinate synthase, AS) instead of the β‐tubulin terminator. AS is the enzyme that catalyses the rate‐limiting step in the conversion of l‐citrulline to l‐arginine and has been recognized as one of the key factors regulating l‐arginine metabolism. Because the AS sequence present in pLOB1 and its derivatives is identical to that of the endogenous B. cinerea gene, transcriptional readthrough into this region could inadvertently induce gene silencing mechanisms. This could result in partial auxotrophy and, potentially, a reduction in virulence in vivo. In this article, we investigate the possibility of inadvertent bcass1 silencing in pLOB1‐based transformants, and identify phenotypes associated with partial AS down‐regulation in vitro and in vivo.

Figure 1.

Figure 1

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Diagrams of the pLOB1 and pLOB1‐MCS vectors. pLOB1 (A) and pLOB1‐MCS (B) contain the Aspergillus nidulans oliC promoter and bcass1 partial gene fragment. pLOB1 contains the hygromycin resistance gene (hph), whereas pLOB‐MCS contains a 120‐bp multiple cloning site (MCS). Transcript and regulatory sequence sizes are shown above each segment, and construct sizes and names are denoted at the end of each plasmid.

RESULTS

Sequence analysis pLOB1‐based vectors (Fig. 1A, B) are controlled by the Aspergillus nidulans oliC promoter and what was previously reported to be the B. cinerea tubA terminator (van Kan et al., 1997). Sequence analysis indicates that bases 25–471 of the terminator region in pLOB1 (GenBank Accession No. AJ439603) are identical to bases 771–1218 of the B. cinerea AS gene (bcass1) coding region (locus BC1G_00121.1, http://www.broad.mit.edu). Transcriptional readthrough into the pLOB1 ‘terminator’ region may result in the generation of hybrid mRNAs, including a substantial part of bcass1, potentially inducing the gene silencing pathway. The principal aim of this study was to investigate the possibility of inadvertent bcass1 silencing in pLOB1‐based transformants and to identify phenotypes associated with (partial) bcass1 down‐regulation in vitro and in planta.

bcass1 gene replacement

In order to assess the maximum phenotypic effect of the downregulation of AS expression in vitro and in vivo, bcass1 was disrupted with the pΔbcass1 knockout vector (Fig. 2A). Two mutants, designated Δbcass1‐1 and Δbcass1‐2, were found to be incapable of growing on plates without l‐arginine, but demonstrated wild‐type growth rates with l‐arginine supplementation (Fig. 3A). The wild‐type displayed a small (3.2%) but significant (t‐test, F‐value significant at P= 0.006) decrease in radial growth in the presence of l‐arginine (Table 1). Southern analysis confirmed the knockout of the bcass1 gene through homologous recombination of knockout vector pΔbcass1 (Fig. 2B). Mutant Δbcass1‐2 contained a second ectopic integration of the pΔbcass1 plasmid. Integration of the complete pΔbcass1 knockout vector was confirmed by polymerase chain reaction (PCR) (Fig. 2C).

Figure 2.

Figure 2

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(A) bcass1 loci before and after disruption by homologous recombination of a hygromycin resistance knockout cassette. Start of translation (black arrows), stop codons (asterisk), introns (grey boxes) and restriction sites are also indicated. The hygromycin resistance (hph), oliC promoter and trpC terminator are also labelled on the hygromycin resistance knockout cassette. Southern (B) and polymerase chain reaction (PCR) (C) analyses confirm the integration of the knockout cassette. Wild‐type (WT) and two knockout mutants (Δbcass1‐1 and Δbcass1‐2) were digested with restriction enzymes SacI and ApaI and probed with partial bcass1 (B, I) and hygromycin (B, II) gene fragments, respectively. The arrows indicate the size of the ∼5.2‐kb SacI wild‐type fragment and the ∼9.3‐kb ApaI gene disruption fragment. (C) The amplification of genomic DNA with primers spanning the entire gene disruption vector resulted in a gene fragment of ∼6.0 kb for both knockout mutants (Δbcass1‐1 and Δbcass1‐2).

Figure 3.

Figure 3

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(A) Examples of the l‐arginine plate‐based assay. Mutants and wild‐type were plated on minimal medium supplemented with (+Arg) and without (–Arg) l‐arginine. The wild‐type (WT) maintains its growth under both treatment schemes. The Δbcass1‐1 disruption mutant is incapable of growing without l‐arginine, but recovers with l‐arginine supplementation. (B) In planta testing showing reduced lesion size of l‐arginine auxotropic mutants, Δbcass1‐1 and Δbcass1‐2. (a)The left side of French bean leaves is infected with the wild‐type, whereas the right side is the knockout mutant. Strawberries (b), tomatoes (c) and grapes (d) were sliced in half before being inoculated. The left halves of the strawberry and grape are infected with the wild‐type, and the right sides are the knockout mutants. Infections were photographed 3 days post‐inoculation and repeated at least 20 times.

Table 1.

Degree of arginine auxotrophy of pLOB1‐MCS(t‐tubA)‐based transformants. Transformants were grown on minimal medium without or with l‐arginine supplementation, in all cases in three replicates. Colony diameters were recorded after 3 days. The difference in colony diameter was used to determine the level of auxotrophy, where high auxotrophy was defined as >50% reduction (in growth on medium without relative to with arginine), intermediate as 30%–49% reduction and low as 10%–29% reduction.

Transformant Average radial growth without l‐arginine (mm) Average radial growth with l‐arginine (mm) Relative growth in the absence or presence of arginine Level of auxotrophy
WT 34 ± 2 33 ± 2 103 N/A
 51 29 ± 3 35 ± 2 83 Low
 54 25 ± 5 28 ± 1 89 Low
 56 31 ± 1 34 ± 0 90 Low
 59 25 ± 4 32 ± 0 78 Low
 60 24 ± 4 30 ± 2 80 Low
 61 22 ± 2 33 ± 0 67 Intermediate
 62 9 ± 1 27 ± 2 33 High
 63 12 ± 2 35 ± 3 31 High
 64 29 ± 1 32 ± 2 90 Low
 65 27 ± 1 30 ± 1 90 Low
 66 18 ± 3 32 ± 3 56 Intermediate
 67 30 ± 6 33 ± 1 90 Low
 68 18 ± 3 33 ± 1 55 Intermediate
 70 27 ± 2 34 ± 0 79 Low
 75 16 ± 4 32 ± 1 50 High
 77 11 ± 1 29 ± 1 38 High
 79 21 ± 6 31 ± 2 68 Intermediate
 80 18 ± 2 32 ± 2 56 Intermediate
400 27 ± 2 29 ± 2 90 Low
408 26 ± 1 29 ± 4 90 Low
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Wild‐type and l‐arginine auxotrophic mutants, Δbcass1‐1 and Δbcass1‐2, were inoculated onto French bean leaves, strawberries, tomatoes and grapes (20 replicates) to determine whether AS gene knockout resulted in a decrease in the observed virulence (Fig. 3B). Lesion sizes on bean leaves were compared at 3 days post‐inoculation. On average, lesions from knockout mutants Δbcass1‐1 and Δbcass1‐2 were 88.4% and 70.2% smaller, respectively, than those observed for the wild‐type (data not shown). Mutants also exhibited a reduction in growth on strawberries and tomatoes, but appeared to be unaltered in virulence on grapes.

bcass1 complementation

As knockout mutants Δbcass1‐1 and Δbcass1‐2 demonstrated a significant reduction in growth during plate‐based analysis and virulence on French bean leaves, strawberries and tomatoes, gene complementation was performed to validate the observed phenotypes. The complete bcass1 region was amplified and used to complement Δbcass1‐1 and Δbcass1‐2 mutants. As knockout strains Δbcass1‐1 and Δbcass1‐2 are hygromycin B resistant, a second selection marker (pNR2) conferring nourseothricin resistance was used during co‐transformation (Hayashi et al., 2002a; Malonek et al., 2004). Regeneration agar and subsequent subculturing events were not supplemented with l‐arginine to ensure that pBCASS1 had integrated into the fungal genome. The recovered transformants were then plated in triplicate on minimal medium with and without l‐arginine. Four recovered complemented transformants (three generated from Δbcass1‐1 and one generated from Δbcass1‐2) displayed wild‐type growth rates with and without l‐arginine supplementation (results not shown). Wild‐type and complemented transformants were also grown in triplicate on strawberries and French bean leaves, confirming wild‐type growth (results not shown).

Induction of bcass1 silencing

The reduction in virulence by bcass1 gene knockout mutants further corroborated our concerns about the inadvertent generation of partial l‐arginine auxotrophs through gene silencing of bcass1 when using pLOB1‐based vectors. As a result, it became necessary to test whether pLOB1‐based vectors were able to induce bcass1 silencing by co‐transformation of the unmodified pLOB1‐MCS expression vector and pOT‐HYG hygromycin resistance vector into strain B05.10. As pLOB1‐MCS contains a multiple cloning site (120 bp) flanked by the Aspergillus nidulans oliC promoter and bcass1 partial gene fragment, it serves as a sense silencing construct (Fig. 1B). Transformants (49 in total) were tested in vitro in triplicate to screen for the occurrence of silencing‐induced auxotrophy. Transformants showing a reduction in radial growth of at least 10% on minimal medium lacking l‐arginine (in comparison with minimal medium supplemented with l‐arginine) were considered to be partially auxotrophic and potentially demonstrating a level of bcass1 silencing (Table 1; Fig. 4A). Overall, 20 transformants were found to exhibit bcass1 silencing, averaging a decrease in radial growth by 26.5% in the absence of l‐arginine. Integration of the pLOB1‐MCS silencing cassette was confirmed in transformants exhibiting bcass1 silencing by PCR analysis with primers OLI2 and TUB2 (Table S1, see Supporting Information).

Figure 4.

Figure 4

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In vitro (A) and in planta (B) analysis of pLOB1‐MCS‐based transformants. (A) Plate‐based assays show regeneration of growth for select transformants with l‐arginine supplementation (bottom row). Plates without supplementation display a gradient of silencing efficiency/partial auxotrophy (top row). The same transformants were also subjected to in planta virulence assays (B). Plate and leaf photographs were taken at 3 days post‐inoculation.

Transformants exhibiting a range of bcass1 silencing‐induced auxotrophy were selected (three high, three intermediate and six low, as judged from plate‐based analysis). Transformants exhibiting a 10%–29% decrease in radial growth without l‐arginine were considered to demonstrate low levels of silencing. Intermediate levels of silencing were defined as transformants exhibiting a 30%–49% increase in radial growth with nutrient supplementation, whereas highly silenced lines demonstrated a 50%–69% increase in radial growth. Subsequent Northern analysis (probe amplified by primer pair BCASS 1 F and BCASS 1R; Table S1, see Supporting Information) revealed that 11 of the 12 samples exhibited reduced bcass1 transcript levels in comparison with the wild‐type (Fig. 5). Unexpectedly, transformant 54 produced bcass1 transcript levels higher than that of the wild‐type. Northern blots were probed with an actin probe as an indicator of equal loading. Films were overexposed to highlight the degree of silencing displayed by each transformant.

Figure 5.

Figure 5

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Northern analysis confirming reduced bcass1 transcript levels in pLOB1‐MCS‐based transformants. Transformants were probed with a partial bcass1 gene fragment (A), whereas bcactin (probe amplified with primers ACTIN F and ACTIN R) was used to confirm equal loading of mRNA (B). (C) Levels of auxotrophy as determined by plate‐based analysis [low (‘L’), intermediate (‘I’) and high (‘H’)] are also indicated.

Six transformants (61, 62, 63, 68, 70 and 80) with varying degrees of silencing were subjected to in planta analysis to determine whether bcass1 silencing had an impact on virulence (Fig. 4B). The tested transformants exhibited a reduction in lesion area on French bean leaves in comparison with the wild‐type (area determined using ImageJ analysis). The reduction in virulence correlated significantly (F‐value significant at R 2= 0.9338) with the level of silencing‐induced auxotrophy observed during plate‐based analysis. Correlation was calculated by comparing the lesion areas in planta with the area of growth in vitro. The reduction in virulence associated with bcass1 mRNA silencing calls into question the utility/reliability of the pLOB1‐based transformation system.

A clear correlation between the levels of silencing and auxotrophy was not observed in all transformants tested, in contrast with the strict correlation between auxotrophy and reduced virulence.

Inadvertent bcass1 silencing

The lower bcass1 transcript levels observed in the pLOB1‐MCS‐based transformants can be attributed to gene silencing triggered by the partial AS sequence located downstream of the multiple cloning site. Following these observations, concerns arose regarding previous experiments involving pLOB1, as many of these studies have generated mutants displaying an alteration in phenotype in vitro and/or a reduction in virulence. Therefore, the phenotypic screening of previously generated pLOB1‐based transformants may determine the potential effects caused by the bcass1 region in pLOB1‐based vectors. Such investigations may prove to be valuable for future molecular manipulations, as well as aid in the re‐examination of the results presented previously. To determine the potential for inadvertent bcass1 silencing, previously developed pLOB1‐based transformants (2008a, 2008b) were re‐assessed (Table 2). The reporter gene transformants enhanced green fluorescent protein (eGFP), luciferase (LUC), β‐glucuronidase (GUS) and monomeric red fluorescent protein (mRFP) (Patel et al., 2008a) and superoxide dismutase (SOD) silencing transformants (Patel et al., 2008b) were obtained by co‐transformation with a modified pLOB1‐MCS expression vector and pLOB1 hygromycin resistance cassette. All transformants were plated on minimal medium supplemented with and without l‐arginine. In total, 7% of reporter gene (three eGFP, two LUC, two GUS and one mRFP) and 6% of SOD‐silenced (three sense and three antisense) transformants demonstrated bcass1 silencing‐induced auxotrophy (Fig. 6), representing 6.6% of all tested pLOB1‐based transformants.

Table 2.

Summary of transformants used during analysis. Samples include SSOD (sense superoxide dismutase), ASOD (antisense superoxide dismutase), eGFP (enhanced green fluorescent protein), GUS (β‐glucuronidase), LUC (luciferase) and mRFP (monomeric red fluorescent protein). Vectors used in transformation, total number of transformants made (n) and references are also given.

Samples Vectors transformed Total (n) Reference
SSOD pLOB1 + pLOB1‐SSOD 52 Patel et al., (2008b)
ASOD pLOB1 + pLOB1‐ASOD 48 Patel et al., (2008b)
eGFP pLOB1 + pLOB1‐eGFP 24 Patel et al., (2008a)
GUS pLOB1 + pLOB1‐GUS 28 Patel et al., (2008a)
LUC pLOB1 + pLOB1‐LUC 28 Patel et al., (2008a)
mRFP pLOB1 + pLOB1‐mRFP 32 Patel et al., (2008a)
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Figure 6.

Figure 6

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Examples of the l‐arginine plate‐based assay. Transformants were plated on minimal medium supplemented with (+Arg) or without (–Arg) l‐arginine. The wild‐type (WT) maintains its growth under both treatment schemes. The Δbcass1‐1 disruption mutant is incapable of growing without l‐arginine, but recovers with l‐arginine supplementation. Samples GFP1 and GUS163 exhibit stunted growth in comparison with the wild‐type without l‐arginine, but regain colonizing capabilities on plates with l‐arginine.

Transformants that exhibited bcass1 silencing were also tested in planta. Primary leaves of dwarf French bean plants were inoculated with the wild‐type, two bcass1 knockout mutants (Δbcass1‐1 and Δbcass1‐2), eight reporter gene transformants and six SOD‐silenced transformants exhibiting bcass1 silencing (Fig. 7A and data not shown). Leaves were also inoculated with Δbcass1‐1 and each transformant to compare directly the consequences of partial auxotrophy (Fig. 7B). A decrease in virulence was observed in both bcass1 knockout mutants, as well as all SOD and reporter gene transformants, in comparison with the wild‐type. As expected, pLOB1‐based transformants were still more virulent than the knockout Δbcass1‐1. The analysis of lesion area revealed that the wild‐type lesion was at least 42% larger than those of all tested transformants (Table 3). A significant correlation was observed between in planta and plate‐based analysis (F‐value significant at R 2= 0.8967). Analysis was conducted by comparing the area of the lesion in planta (determined using ImageJ analysis) with the area of growth observed during plate‐based analysis. These results indicate that bcass1 down‐regulation may be induced by any pLOB1‐based vectors.

Figure 7.

Figure 7

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(A) Examples of in planta testing showing reduced lesion size in partial l‐arginine auxotrophic transformants (GUS163, GFP155, GFP1 and LUC 84) in comparison with the wild‐type. The left side of the leaf is the wild‐type (WT) sample, and the right side is the transformant. (B) Transformants demonstrated growth equal to or greater than that of the knockout mutant (Δbcass1‐2). The left side of the leaf is the knockout mutant (KO), and the right side is the transformant. The transformants shown contain the β‐glucuronidase (GUS) (GUS163), green fluorescent protein (GFP) (GFP155, 1) or luciferase (LUC) (LUC84) exogenous gene.

Table 3.

Effect of arginine auxotrophy on virulence and radial growth in vitro. Lesion areas of transformants and the wild‐type strain B05.10 on bean leaves were quantified using ImageJ, and are presented in arbitrary units (±standard error of the mean). The relative virulence of the transformants is shown as the proportion of the average lesion area of a transformant relative to the wild‐type (100%). N indicates the number of lesions measured. The relative colony diameter of the transformants in vitro is also indicated. n indicates the number of colonies measured.

Strain Lesion area of mutant Lesion area of wild‐type Relative virulence of mutant Relative colony diameter in vitro
Δbcass1 4300 ± 1100 (N= 17) 56700 ± 7800 (N= 17) 7.5% 0 (n= 3)
GFP1 15300 ± 900 (N= 5) 81200 ± 6300 (N= 5) 19% 77 ± 4% (n= 3)
GFP155 7500 ± 1000 (N= 7) 37700 ± 4300 (N= 7) 20% 83 ± 2% (n= 3)
GUS163 6700 ± 1500 (N= 5) 48000 ± 18000 (N= 5) 14% 85 ± 5% (n= 3)
LUC84 33500 ± 3900 (N= 5) 58700 ± 1600 (N= 5) 57% 87 ± 2% (n= 3)
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Northern analysis was performed to determine bcass1 transcript levels (Fig. 8). All reporter gene transformants showed a degree of bcass1 silencing in comparison with the wild‐type, averaging a c. 50% decrease in bcass1 transcript. These results suggest that the decrease in virulence is caused by the inadvertent silencing of bcass1. The majority of SOD‐silenced transformants (Patel et al., 2008b) displayed a lesser degree of bcass1 silencing, as indicated by the levels of bcass1 transcript compared with the wild‐type.

Figure 8.

Figure 8

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Northern analysis of bcass1 mRNA in reporter gene (A) and superoxide dismutase (SOD) (B) transformants exhibiting bcass1 silencing. All reporter gene transformants showed a degree of bcass1 silencing in comparison with the wild‐type (WT). Green fluorescent protein (GFP) samples (G1 and G152) showed the least amount of silencing, whereas GFP (G155), luciferase (LUC) (L80 and L84), β‐glucuronidase (GUS) (B23 and B163) and monomeric red fluorescent protein (mRFP) (M33) transformants exhibited high levels of silencing. SOD transformants displayed a lesser degree of bcass1 silencing. S44, S160, S165, sense SOD transformants; A76, A100, A101, antisense SOD transformants (Patel et al., 2008b). (A1, B1) Transformants were probed with a partial bcass1 gene fragment (probe amplified by primer pair BCASS 1 F and BCASS 1R; Table S1); (A2, B2) bcactin (probe amplified with primers ACTIN F and ACTIN R) was used to confirm total mRNA levels.

DISCUSSION

Recent sequence annotation of the B. cinerea genome (B05.10, Broad Institute) has increased the need for highly efficient expression and resistance vectors for rapid fungal manipulations and gene screening. pLOB1 is one of the most utilized vector systems for B. cinerea (Espino et al., 2005; ten Have et al., 2010; van Kan et al., 1997; Reis et al., 2005; Rui and Hahn, 2007a; Schouten et al., 2002b). Until recently, the terminator region downstream of the hph gene was thought to be the B. cinerea tubA terminator. However, analysis has revealed that the terminator region is a 446‐bp region of the B. cinerea AS gene, which is located adjacent to the tubA gene. In retrospect, it appears that an error was made in the restriction mapping of the lambda phage on which the tubA gene and the bcass1 gene are located. A 2‐kbp EcoRV fragment that was judged to be a single fragment in reality appeared to be a doublet, and the fragment containing the true tubA terminator region was missing from the phage sequence assembly (J. A. L. van Kan, personal observations). As the fragment that serves as terminator in pLOB1 is a substantial portion of the endogenous bcass1 gene, any transcriptional readthrough may result in unintentional gene silencing of bcass1. The generation of a bcass1 gene replacement mutant helped to characterize the magnitude of the complete auxotrophic effects in vitro and in planta. This ‘maximum phenotype’ could then be compared with bcass1‐silenced lines, where a range of levels of auxotrophy were likely to occur.

The results demonstrated a reduction in growth on French bean leaves, strawberries and tomatoes. Previous studies in other fungi have demonstrated the importance of amino acid biosynthesis to fungal virulence (Divon and Fluhr, 2007). Reduction in virulence has been reported in Fusarium oxysporum f. sp. melonis mutants defective in AS (Namiki et al., 2001), Cladosporium fulvum and Fusarium graminearum defective in methionine biosynthesis and Magnaporthe grisea defective in histidine biosynthesis (Seong et al., 2005; Solomon et al., 2001; Sweigard et al., 1998). The host‐related variation in virulence may be caused by differences in l‐arginine concentrations present in each host tissue. In particular, strawberries and tomatoes contain substantially less l‐arginine (0.2617 and 0.2195 mg/g of fruit, respectively) than grapes (0.4875 mg/g of fruit) (Agricultural Handbook, 1–23, US Department of Agriculture). It may be hypothesized that the concentrations of free l‐arginine present in strawberries, tomatoes and French bean leaves are not adequate for complete knockout rehabilitation. On the other hand, grapes contain a higher concentration of free l‐arginine, which may allow infection by the mutant. Thus, the virulence of Δbcass1‐1 and Δbcass1‐2 may be heavily reliant on host free l‐arginine concentrations and may be exploited to discover nutrient uptake complexities during ensuing fungal infections. The addition of arginine to the conidial suspension used for plant inoculation temporarily relieved the phenotype of bcass1 auxotrophic transformants in a concentration‐dependent manner and allowed the formation and outgrowth of lesions into plant tissue where the level of free arginine was insufficient to sustain growth of the auxotrophic transformant. The higher the arginine concentrations provided in the inoculum, the longer the transformant developed lesions as normal, but lesion development eventually always slowed down or stopped entirely (J. A. L. van Kan, unpublished data).

Although it is difficult to link l‐arginine auxotrophy to fungal virulence, it has been hypothesized that a high concentration of l‐arginine in Gibberella zeae is necessary to shift vegetative growth to sexual reproduction (Kim et al., 2007). Linkages between fungal sexual and asexual development, as well as virulence, have also been observed in Schizosaccharomyces pombe, Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus nidulans, Ustilago maydis, Cryophonectria parasitica, Magnaporthe grisea and Cryptococcus neoformans (Bolker, 1998; Kim et al., 2007). As Δbcass1‐1 and Δbcass1‐2 displayed a severe decrease in pathogenicity on French bean leaves, the induction of bcass1 silencing may also result in transformants exhibiting reduced levels of virulence. The pLOB1‐MCS silencing vector was utilized as a sense silencing construct for the induction of AS down‐regulation, with the frequency of sense transformants exhibiting bcass1 silencing‐induced auxotrophy at 41%. In planta infection assays were assessed and found to produce a range of reductions in virulence, correlating with those observed during plate‐based (in vitro) and Northern analysis. Thus, evidence shows that bcass1 is essential for fungal growth when external l‐arginine concentrations are limiting, and, when silenced, results in a reduction in hyphal radial growth in vitro and host colonization in planta.

As the pLOB1‐MCS silencing cassette was able to induce bcass1 silencing, the employment of any pLOB1‐based expression or hygromycin resistance vector during B. cinerea molecular manipulations may inadvertently induce bcass1 silencing, and our results on plate‐based assays suggest that inadvertent silencing occurs at c. 7% frequency using a wide range of vectors expressing marker genes (eGFP, GUS, LUC and mRFP) or targeting genes for silencing, such as SOD.

The transformants exhibiting bcass1 silencing were tested in planta. All reporter and SOD transformants exhibiting bcass1 silencing showed reduced lesion development in in planta assays. As reporter genes are generally nontoxic exogenous genes, the virulence of reporter strains should be unaffected after transformation. Northern analysis of these transformants demonstrating bcass1 silencing also exhibited reduced levels of bcass1 transcript. Hence, in planta observations are a result of partial l‐arginine auxotrophy. These results also correlate with those observed during plate‐based analysis. As bcsod1 has been found to be a pathogenicity factor (Rolke et al., 2004), the observed reduction in virulence in SOD‐silenced lines (Patel et al., 2008b) may be caused by a combination of bcsod1 and bcass1 silencing. However, our investigations have shown that SOD knockout mutants exhibit wild‐type growth capabilities on French bean leaves (Patel et al., 2008b). As a result, the reduction in virulence by SOD‐silenced transformants may be attributed to inadvertent bcass1 down‐regulation.

Our investigations therefore confirm that pLOB1‐based vectors have the potential to trigger the silencing of bcass1. Some of these silenced lines appear to have significantly reduced levels of virulence, raising the possibility that knockout phenotypes showing reduced levels of virulence could be a result of inadvertent silencing of bcass1, not target disruption. REMI or T‐DNA mutants, obtained using pLOB1‐based vectors, which display interesting phenotypes, could also be caused by inadvertent bcass1 silencing rather than insertional mutagenesis. We recommend that previously established transformants based on pLOB1 should be re‐evaluated for l‐arginine‐dependent growth, and that a different vector series should be developed for all subsequent manipulations in B. cinerea, such as the pOT vector system (2008a, 2008b).

EXPERIMENTAL PROCEDURES

Fungal strains and growth conditions

Experimental analyses and transformation were performed on B. cinerea strain B05.10. Botrytis cinerea conidia were stored as glycerol stocks at −80 °C. Two bcass1 (Δbcass1‐1 and Δbcass1‐2) and three bcsod1 (Δbcsod1‐1, Δbcsod1‐2 and Δbcsod1‐3) knockout mutants served as controls during in vitro and in planta experimentation. The fungus was grown on malt agar containing 5% (w/v) malt extract and 1.8% (w/v) agar and incubated at 20 °C under near‐UV light to induce sporulation. Conidia were harvested 7–14 days post‐inoculation in 10 mL of water containing 0.05% (v/v) Tween‐80, and collected by centrifugation at 3000 g for 5 min. For DNA extraction, 5% (w/v) malt extract broth was inoculated with 5 × 108 spores/L and incubated at 20 °C and 180 r.p.m. for 24 h. Mycelia were harvested by centrifugation and lyophilized.

Transformation of B. cinerea

Transformants were produced using the methodology described by Hamada et al. (1994) with modifications described by van Kan et al. (1997). Auxotrophic mutants and silenced transformants were recovered by supplementation of regeneration agar with 1 mm l‐arginine. Emerging fungal colonies were subjected to several subculturing events (with l‐arginine supplementation) to select against heterokaryons containing mixtures of transformed and untransformed nuclei.

bcass1 gene replacement

The left arm (representing 1591 bases of the 5′ untranslated region and 360 bases of the bcass1 open reading frame) of the knockout vector was amplified with the primer pair LA SACI F and LA SACI R containing SacI restriction sites. The right arm (representing 801–2352 bases past the translational stop codon) containing HindIII and XhoI restriction sites was amplified with the primer pair RA HINDIII F and RA XHOI R (Table S1, see Supporting Information). Fragments were independently ligated into the hygromycin resistance vector pOT‐HYG (controlled by the Aspergillus nidulans oliC promoter and Aspergillus nidulans trpC terminator) and digested with the corresponding restriction enzymes (Fig. 2A). The complete knockout vector, pΔbcass1, was used to transform strain B05.10. The recovered transformants were plated in triplicate on minimal medium with and without l‐arginine supplementation.

For the complementation of the knockout mutants, the complete bcass1 region was amplified from the wild‐type strain B05.10 by PCR with primers COMP BCASS1 F and COMP BCASS1 R (Table S1, see Supporting Information) and cloned into the TOPO TA Cloning pCR®2.1‐TOPO® vector. The subsequent expression cassette, pBCASS1, was used for complementing Δbcass1‐1 and Δbcass1‐2 mutants.

Standard nucleic acid techniques

Fungal genomic DNA was isolated as described by Schouten et al. (2002b). PCR analysis was performed as described by Saiki et al. (1988) using Reddy Mix (Bioline), and amplification products were separated by gel electrophoresis. The primers used during PCR analysis are listed in Table 1. Purified products (Wizard Gel Extraction Kit, Promega) were cloned using the pCR 2.1 TOPO Cloning Kit (Invitrogen). Ligation strategies were designed using Clone Manager Suite (Sci Ed Central). Sequence analysis and alignment were aided by blast analysis at the National Center for Biotechnology Information (NCBI) (Altschul et al., 1990).

Northern and Southern analysis of total RNA and genomic DNA was completed according to Sambrook et al. (1989) with slight amendments (Heneghan et al., 2007; Kilaru et al., 2009). Fungal genomic DNA (5 µg) was digested to completion with SacI or ApaI (50 units). Digested fragments were separated on a 1.0% (w/v) agarose gel. The gel was depurinated with 0.25 m HCl for 15 min and denatured with 0.5 m NaOH for 30 min before being blotted onto Hybond‐N membrane (GE Healthcare). Membranes were rinsed with 2 × standard saline citrate (SSC; 0.3 m NaCl and 0.03 m sodium citrate, pH 7) after RNA or DNA transfer. The RNA or DNA was immobilized by UV cross‐linker.

The membrane containing the SacI‐digested DNA was hybridized with a partial bcass1 gene fragment (amplified with primers BCASS1 F and BCASS1 R; Table S1, see Supporting Information), and the filter containing ApaI‐digested DNA was hybridized with a partial hygromycin resistance gene fragment (amplified with primers HYG F and HYG R; Table S1, see Supporting Information). Integration of the complete pΔbcass1 knockout vector was confirmed by PCR with primers LA SACI F and RA XHOI R (Table S1, see Supporting Information).

l‐Arginine auxotrophy measurements

l‐Arginine auxotrophy was assayed on Gamborg's minimal agar [GM, 3.16 g/L Gamborg's B5 (DUCHEFA Biochemie B.V., Haarlem, the Netherlands), 100 mm sucrose, 100 mm sodium phosphate, 18 g/L agar] supplemented with or without filter‐sterilized 200 µm l‐arginine. Plates were incubated at 20 °C for 3 days before radial measurements were taken. Silencing frequencies were calculated by comparing the colony diameters in the presence and absence of l‐arginine. Transformants exhibiting a ≥10% decrease in radial growth without l‐arginine (in comparison with minimal medium with l‐arginine) were considered to be demonstrating a level of bcass1 silencing.

Infection assay on French bean, strawberry, tomato and grape

Conidia of sporulating B. cinerea wild‐type and transformants were harvested and resuspended in H2O at a working concentration of 1 × 106 conidia/mL. Infection studies were conducted on 14‐day‐old dwarf French bean plants (Tendergreen) in triplicate. Punctured plant leaves were then inoculated with a single droplet containing 5 × 103 conidia from the wild‐type and a transformant. Leaves were incubated over a period of 5 days with diurnal cycles of 16 h light and 8 h darkness and 100% humidity, before being photographed. Areas of infection were then determined with ImageJ software (Abramoff et al., 2004).

Commercially sourced strawberries and grapes were sliced in half and surface sterilized for 5 min in a 5% (v/v) bleach solution and sterilized distilled water. Fruit halves were then arranged in Petri dishes with 10 mL of sterilized distilled water before being punctured with a sterile plastic pipette tip and inoculated with 5 µL of a spore suspension of the B05.10 wild‐type strain (left half of fruit) and appropriate transformant or knockout mutant (right half of fruit). The Petri dishes were incubated at room temperature for 3 days.

Supporting information

Table S1 Primer sequences utilized during experimentation.

Please note: Wiley‐Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Supporting info item

Click here for additional data file. (45KB, doc)

ACKNOWLEDGEMENTS

We would like to thank Professor Paul Tudzynski (University of Munster, Germany) for providing the SOD cDNA and knockout mutants. We would also like to acknowledge Dr Michael Pearson (University of Auckland, New Zealand) for his ongoing collaborations and discussions. This research was made possible by the generosity of the LESARS (University of Bristol, UK) funding body. This work was carried out under DEFRA Licence PHL 247A/6129.

REFERENCES

  1. Abramoff, M.D. , Magelhaes, P.J. and Ram, S.J. (2004) Image Processing with ImageJ. Biophotonics Int. 11, 36–42. [Google Scholar]
  2. Altschul, S.F. , Gish, W. , Miller, W. , Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410. [DOI] [PubMed] [Google Scholar]
  3. Bolker, M. (1998) Sex and crime: heterotrimeric G proteins in fungal mating and pathogenesis. Fungal Genet. Biol. 25, 143–156. [DOI] [PubMed] [Google Scholar]
  4. Brito, N. , Espino, J.J. and Gonzalez, C. (2006) The endo‐beta‐1,4‐xylanase xyn11A is required for virulence in Botrytis cinerea . Mol. Plant–Microbe Interact. 19, 25–32. [DOI] [PubMed] [Google Scholar]
  5. Divon, H.H. and Fluhr, R. (2007) Nutrition acquisition strategies during fungal infection of plants. FEMS Microbiol. Lett. 266, 65–74. [DOI] [PubMed] [Google Scholar]
  6. Doehlemann, G. , Molitor, F. and Hahn, M. (2005) Molecular and functional characterization of a fructose specific transporter from the gray mold fungus Botrytis cinerea . Fungal Genet. Biol. 42, 601–610. [DOI] [PubMed] [Google Scholar]
  7. Doehlemann, G. , Berndt, P. and Hahn, M. (2006a) Trehalose metabolism is important for heat stress tolerance and spore germination of Botrytis cinerea . Microbiology-SGM, 152, 2625–2634. [DOI] [PubMed] [Google Scholar]
  8. Doehlemann, G. , Berndt, P. and Hahn, M. (2006b) Different signalling pathways involving a Gα protein, cAMP and a MAP kinase control germination of Botrytis cinerea conidia. Mol. Microbiol. 59, 821–835. [DOI] [PubMed] [Google Scholar]
  9. Espino, J.J. , Brito, N. , Noda, J. and Gonzalez, C. (2005) Botrytis cinerea endo‐beta‐1,4‐glucanase Cel5A is expressed during infection but is not required for pathogenesis. Physiol. Mol. Plant Pathol. 66, 213–221. [Google Scholar]
  10. Hamada, W. , Reignault, P. , Bompeix, G. and Boccara, M. (1994) Transformation of Botrytis cinerea with the Hygromycin B resistance gene, hph . Curr. Genet. 26, 251–255. [DOI] [PubMed] [Google Scholar]
  11. Ten Have, A. , Espino, J.J. , Brito, N. , Dekkers, E. , Van Sluyter, S. , Kay, J. , Gonzalez, C. and Van Kan, J.A.L. (2010) The Botrytis cinerea Aspartic Proteinase gene family. Fungal Genet. Biol. 47, 53–65. [DOI] [PubMed] [Google Scholar]
  12. Hayashi, K. , Schoonbeek, H.J. and De Waard, M.A. (2002a) Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides. Appl. Environ. Microbiol. 68, 4996–5004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hayashi, K. , Schoonbeek, H.J. and De Waard, M.A. (2002b) Expression of the ABC transporter BcatrD from Botrytis cinerea reduces sensitivity to sterol demethylation inhibitor fungicides. Pestic. Biochem. Physiol. 73, 110–121. [Google Scholar]
  14. Heneghan, M.N. , Costa, A.M.S.B. , Challen, M.P. , Mills, P.R. , Bailey, A. and Foster, G.D. (2007) A comparison of methods for successful triggering of gene silencing in Coprinus cinereus . Mol. Biotechnol. 35, 283–296. [DOI] [PubMed] [Google Scholar]
  15. Van Kan, J.A.L. (2006) Licensed to kill: the lifestyle of a necrotrophic plant pathogen. Trends Plant Sci. 11, 247–253. [DOI] [PubMed] [Google Scholar]
  16. Van Kan, J.A.L. , Van't Klooster, J.W. , Wagemakers, C.A. , Dees, D.C. and Van Der Vlugt‐Bergmans, C.J. (1997) Cutinase A of Botrytis cinerea is expressed, but not essential, during penetration of gerbera and tomato. Mol. Plant–Microbe Interact. 10, 30–38. [DOI] [PubMed] [Google Scholar]
  17. Kars, I. , Wagemakers, C.A.M. , McCalman, M. and Van Kan, J.A.L. (2005a) 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, 641–652. [DOI] [PubMed] [Google Scholar]
  18. Kars, I. , Krooshof, G. , Wagemakers, C.A.M. , Joosten, R. , Benen, J.A.E. and Van Kan, J.A.L. (2005b) Necrotising activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris . Plant J. 43, 213–225. [DOI] [PubMed] [Google Scholar]
  19. Kilaru, S. , Collins, C.M. , Hartley, A.J. , Bailey, A.M. and Foster, G.D. (2009) Establishing molecular tools for genetic manipulation of the pleuromutilin producing fungus Clitopilus passeckerianus . Appl. Environ. Microbiol. 75, 7196–7204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kim, J.E. , Myong, K. , Shim, W.B. , Yun, S.H. and Lee, Y.W. (2007) Functional characterization of acetylglutamate synthase and phosphoribosylamine‐glycine ligase genes in Gibberella zeae . Curr. Genet. 51, 99–108. [DOI] [PubMed] [Google Scholar]
  21. Leroux, P. , Fritz, R. , Debieu, D. , Albertini, C. , Lanen, C. , Bach, J. , Gredt, M. and Chapeland, F. (2002) Mechanisms of resistance to fungicides in field strains of Botrytis cinerea . Pest Manag. Sci. 58, 876–888. [DOI] [PubMed] [Google Scholar]
  22. Malonek, S. , Rojas, M.C. , Hedden, P. , Gaskin, P. , Hopkins, P. and Tudzynski, B. (2004) The NADPH‐cytochrome P450 reductase gene from Gibberella fujikuroi is essential for gibberellin biosynthesis. J. Biol. Chem. 279, 25075–25084. [DOI] [PubMed] [Google Scholar]
  23. Namiki, F. , Matsunaga, M. , Okuda, M. , Inoue, I. , Nishi, K. , Fujita, Y. and Tsuge, T. (2001) Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxysporum f. sp melonis . Mol. Plant–Microbe Interact. 14, 580–584. [DOI] [PubMed] [Google Scholar]
  24. Patel, R.M. , Heneghan, M.H. , Van Kan, J.A.L. , Bailey, A.M. and Foster, G.D. (2008a) The pOT and pLOB vector systems: improving ease of transgene expression in Botrytis cinerea . J. Gen. Appl. Microbiol. 54, 367–376. [DOI] [PubMed] [Google Scholar]
  25. Patel, R.M. , Van Kan, J.A.L. , Bailey, A.M. and Foster, G.D. (2008b) RNA‐mediated gene silencing of superoxide dismutase (bcsod1) in Botrytis cinerea . Phytopathology, 98, 1334–1339. [DOI] [PubMed] [Google Scholar]
  26. Reis, H. , Pfiffi, S. and Hahn, M. (2005) Molecular and functional characterization of a secreted lipase from Botrytis cinerea . Mol. Plant Pathol. 6, 257–267. [DOI] [PubMed] [Google Scholar]
  27. Rolke, Y. , Liu, S. , Quidde, T. , Williamson, B. , Schouten, A. , Weltring, K.‐M. , Siewers, V. , Tenberge, K.B. , Tudzynski, B. and Tudzynski, P. (2004) Functional analysis of H2O2 generating systems in Botrytis cinerea: the major Cu‐Zn‐superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable. Mol. Plant Pathol. 5, 17–27. [DOI] [PubMed] [Google Scholar]
  28. Rui, O. and Hahn, M. (2007a) The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits. Microbiology-SGM 153, 2791–2802. [DOI] [PubMed] [Google Scholar]
  29. Rui, O. and Hahn, M. (2007b) 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, 173–184. [DOI] [PubMed] [Google Scholar]
  30. Saiki, R.K. , Gelfand, D.H. , Stoffel, S. , Scharf, S.J. , Higuchi, R. , Horn, G.T. , Mullis, K.B. and Erlich, H.A. (1988) Primer‐directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487–491. [DOI] [PubMed] [Google Scholar]
  31. Sambrook, J. , Fritsch, E.J. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. [Google Scholar]
  32. Schoonbeek, H.J. , Raaijmakers, J.M. and De Waard, M.A. (2002) Fungal ABC transporters and microbial interactions in natural environments. Mol. Plant–Microbe Interact. 15, 1165–1172. [DOI] [PubMed] [Google Scholar]
  33. Schouten, A. , Tenberge, K.B. , Vermeer, J. , Stewart, J. , Wagemakers, L. , Williamson, B. and Van Kan, J.A.L. (2002a) Functional analysis of an extracellular catalase of Botrytis cinerea . Mol. Plant Pathol. 3, 227–238. [DOI] [PubMed] [Google Scholar]
  34. Schouten, A. , Wagemakers, L. , Stefanato, F.L. , Van Der Kaaij, R.M. and Van Kan, J.A.L. (2002b) Resveratrol acts as a natural profungicide and induces self‐intoxication by a specific laccase. Mol. Microbiol. 43, 883–894. [DOI] [PubMed] [Google Scholar]
  35. Seong, K. , Hou, Z.M. , Tracy, M. , Kistler, H.C. and Xu, J.R. (2005) Random insertional mutagenesis identifies genes associated with virulence in the wheat scab fungus Fusarium graminearum . Phytopathology, 95, 744–750. [DOI] [PubMed] [Google Scholar]
  36. Smith, B.J. (1998) Botrytis blossom blight of southern blueberries: cultivar susceptibility and effect of chemical treatments. Plant Dis. 82, 924–927. [DOI] [PubMed] [Google Scholar]
  37. Solomon, P.S. , Nielsen, P.S. , Clark, A.J. and Oliver, R.P. (2001) Methionine synthase, a gene required for methionine synthesis, is expressed in planta by Cladosporium fulvum . Mol. Plant Pathol. 1, 315–323. [DOI] [PubMed] [Google Scholar]
  38. Sweigard, J.A. , Carroll, A.M. , Farrall, L. , Chumley, F.G. and Valent, B. (1998) Magnaporthe grisea pathogenicity genes obtained through insertional mutagenesis. Mol. Plant–Microbe Interact. 11, 404–412. [DOI] [PubMed] [Google Scholar]
  39. Tudzynski, P. and Siewers, V. (2004) Approaches to molecular genetics and genomics of Botrytis In: Botrytis: Biology, Pathology and Control (Elad Y., Williamson B., Tudzynski P. and Delen N., eds), pp. 53–66. London: Kluwer Academic Publishers. [Google Scholar]
  40. Williamson, B. , Tudzynski, B. , Tudzynski, P. and Van Kan, J.A.L. (2007) Botrytis cinerea: the cause of grey mould disease. Mol. Plant. Pathol. 8, 561–580. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Table S1 Primer sequences utilized during experimentation.

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Supporting info item

Click here for additional data file. (45KB, doc)

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