Present address: Boyce Thompson Institute for Plant Research, Room# 403, Ithaca, NY 14853, USA.
Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation
Article first published online: 29 JUL 2010
© 2010 The Authors. Molecular Plant Pathology © 2010 BSPP and Blackwell Publishing Ltd
Molecular Plant Pathology
Volume 12, Issue 1, pages 43–55, January 2011
How to Cite
MÜNCH, S., LUDWIG, N., FLOSS, D. S., SUGUI, J. A., KOSZUCKA, A. M., VOLL, L. M., SONNEWALD, U. and DEISING, H. B. (2011), Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation. Molecular Plant Pathology, 12: 43–55. doi: 10.1111/j.1364-3703.2010.00651.x
- Issue published online: 1 DEC 2010
- Article first published online: 29 JUL 2010
- Top of page
- EXPERIMENTAL PROCEDURES
- Supporting Information
A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5′-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5′-flank of an ORF. In both cases, the ORFs have unknown function.
- Top of page
- EXPERIMENTAL PROCEDURES
- Supporting Information
The fungus Colletotrichum graminicola (Ces.) Wilson [teleomorph Glomerella graminicola (Politis)] is the causal agent of leaf anthracnose and stem rot of corn (Bergstrom and Nicholson, 1999). In order to invade the first epidermal cell and to establish a compatible parasitic relationship with the host plant, the pathogen differentiates an elaborate infection cell, called an appressorium (Deising et al., 2000; Tucker and Talbot, 2001; Wang et al., 2005). During maturation, appressoria form rigid cell walls which melanize, and the synthesis of high concentrations of compatible solutes (De Jong et al., 1997) results in the generation of enormous appressorial turgor pressure (Howard et al., 1991). Turgor is translated into force, which is exerted at the appressorial base, supporting penetration into the host (Bastmeyer et al., 2002; Bechinger et al., 1999; Küster et al., 2008). In the host, C. graminicola forms biotrophic infection structures, i.e. the infection vesicle and primary hyphae, which develop without causing macroscopically visible damage to the host. Subsequently, the formation of secondary hyphae is initiated, which grow rapidly (Krijger et al., 2008), are highly destructive and represent the necrotrophic lifestyle of the pathogen (Bergstrom and Nicholson, 1999; Horbach et al., 2009; Münch et al., 2008).
Depending on the lifestyle and genome size of the fungus, Idnurm and Howlett (2001) estimated that plant pathogenic fungi probably harbour between 60 and 360 virulence or pathogenicity genes. However, only some of these have been identified. Several of these genes are involved in infection structure differentiation or the synthesis of toxins or cell wall-degrading enzymes (Idnurm and Howlett, 2001; Madrid et al., 2003; Möbius and Hertweck, 2009; Werner et al., 2007). Other genes play a role in compromising host defence responses in order to establish a compatible pathogenic interaction. Successful suppression of the host defence depends on the secretion of fungal effector molecules and, consequently, involves the secretory machinery, as indicated by mutants of the rice blast fungus Magnaporthe oryzae deficient in a P-type ATPase, which are unable to suppress the formation of reactive oxygen species in their host, and thus show reduced virulence (Gilbert et al., 2006). In Colletotrichum gloeosporioides, secretion of the effector molecule encoded by the gene CgDN3 is required to avert a hypersensitive-like response of a compatible host during the establishment of a biotrophic interaction (Stephenson et al., 2000). So far, a number of fungal and oomycete effector molecules have been identified (Kamoun, 2009).
Random mutagenesis has been exploited extensively as a tool to tag the fungal genes required for the establishment of a pathogenic interaction with the host plant (Hamer et al., 2001; Kahmann and Basse, 1999; Mullins and Kang, 2001). In restriction enzyme-mediated integration (REMI) mutagenesis experiments performed with the plant pathogens Ustilago maydis, M. oryzae and Cochliobolus heterostrophus, the percentage of transformants with virulence defects ranged from 0.5 to 2% (Bölker et al., 1995; Lu et al., 1994; Sweigard et al., 1998) and, in C. graminicola, only 0.3% of the transformants were affected in virulence (Epstein et al., 1998; Thon et al., 2000). Importantly, analyses of REMI mutants of different fungi have shown that up to 50% of the mutations may not be tagged (Kahmann and Basse, 1999; Maier and Schäfer, 1999). These findings raise doubts about whether REMI mutagenesis is a suitable tool for the efficient identification of virulence genes.
Agrobacterium tumefaciens-mediated transformation (ATMT) is thought to avoid most of the problems associated with REMI mutagenesis and related protoplast transformation techniques. For several years, ATMT has been well established for plant transformation (Escobar and Dandekar, 2003; Gelvin, 2003). After the initial application to yeast (Bundock et al., 1995), de Groot et al. (1998) used this technique to transform filamentous fungi. ATMT has since been used to transform several plant pathogenic fungi, e.g. Fusarium oxysporum, Verticillium dahliae, Botrytis cinerea, Calonectria morganii, M. oryzae, Colletotrichum falcatum and Colletotrichum acutatum (Dobinson et al., 2004; Jeon et al., 2007; Khang et al., 2006; Malonek and Meinhardt, 2001; Maruthachalam et al., 2008; Rolland et al., 2003; Shen et al., 2008), and the oomycete Phytophthora infestans (Vijn and Govers, 2003). Recently, Flowers and Vaillancourt (2005) developed an efficient ATMT protocol for C. graminicola. However, because of the high rates of tandem integration of the T-DNA and multiple integrations of the entire Ti-plasmid, problems in rescuing flanking genomic regions and identification of the disrupted genes occurred. Therefore, these authors considered ATMT to be unsuitable for the identification of pathogenicity or virulence genes in C. graminicola.
Here, we report a modified ATMT protocol and the generation of a transformant collection. Approximately 70% of the transformants showed T-DNA integration at one single site. In virulence assays on whole plants, we identified 10 transformants with virulence defects from 500 independent transformants. Seven transformants differing in virulence were studied in detail and, in all of these transformants, T-DNA integration sites were identified. In six transformants, T-DNA integration occurred into the 5′-flanks or coding regions of putative genes with unknown function. In one transformant, T-DNA integrated into the 5′-flank of a gene with similarity to allantoicase genes of other Ascomycota, such as M. oryzae or Neurospora crassa.
- Top of page
- EXPERIMENTAL PROCEDURES
- Supporting Information
Generation of a transformant library by ATMT and the mode of T-DNA integration into the genome of C. graminicola
Using a modified protocol for ATMT, we generated a collection of approximately 2000 transformants of the corn pathogen C. graminicola. In contrast with other protocols, in which acetosyringone was added to in vitro cultures of A. tumefaciens, we added this substance during the co-cultivation of germinating falcate conidia of C. graminicola with A. tumefaciens. On average, 3.5 transformants were obtained from 10 000 conidia. No transformants were obtained in the absence of acetosyringone, indicating that this compound is indispensable for the induction of T-DNA transfer into fungal cells. The integrated T-DNA was mitotically stable, as shown by analyses of 10 randomly chosen transformants. Four successive 14-day passages of these transformants on oatmeal agar (OMA) medium without the selection marker hygromycin B did not result in the loss of integrated T-DNA, as indicated by genomic Southern blot analysis and the maintenance of the ability of all 10 transformants to grow on potato-dextrose-agar (PDA) containing hygromycin B.
In random mutagenesis experiments, single-copy T-DNA integration into one chromosomal locus of the recipient allows the efficient identification of the tagged gene and circumvents time-consuming analyses of the integration event mediating an altered phenotype in transformants harbouring two or more T-DNA insertions at different chromosomal loci. In order to characterize T-DNA integration events in our transformant collection, we investigated 105 randomly chosen transformants by genomic Southern blot analyses. Blots of HindIII-digested genomic DNA were probed with a digoxigenin (Dig)-labelled polymerase chain reaction (PCR) fragment corresponding to 688 bp of the hph gene and 321 bp of the 5′-flanking T-DNA (Fig. 1B). More than two-thirds, i.e. 69.5%, of the transformants tested showed a single-site integration of the T-DNA. In 26.7% of the transformants, two integration events occurred at different sites of the genome, and only 3.8% of the transformants exhibited more than two T-DNA integration sites. A representative selection of 40 transformants is shown in Fig. 1C. Southern blot experiments with Eco32I- or XbaI-digested genomic DNA confirmed these results. As expected, genomic DNA of the nontransformed wild-type (WT) strain did not hybridize with the probe (data not shown).
Southern blot analyses also allowed us to determine at what frequency the entire Ti-plasmid was integrated into the fungal genome, and if single or multiple copies integrated (Flowers and Vaillancourt, 2005; Li et al., 2007). The integration of the entire Ti-plasmid into the fungal genome led to the detection of a fragment of at least 10.5 kb when DNA was restricted with HindIII. The size of the fragments exceeding 10.5 kb depends on the distance to the next HindIII restriction site in the flanking genomic region. In 33% of the transformants, the fragment size was sufficiently large to harbour the entire Ti-plasmid. However, this number overestimates the fraction of transformants harbouring the entire Ti-plasmid, as it also includes transformants with T-DNAs integrated into sites with long distances to the next HindIII restriction site. Transformants with fragment sizes smaller than 10.5 kb do not harbour the entire Ti-plasmid, which was the case in 67% of all transformants analysed.
Only in 3% of all transformants were analysed fragments of exactly 10.7 kb in HindIII-digested DNA and 10.0 kb in Eco321-digested DNA detected, indicating that tandem integration of the entire Ti-plasmid had occurred into a single genomic site.
Identification and characterization of transformants with reduced virulence
Using detached corn leaves, we screened 500 transformants for reduced virulence. Sixty-one transformants with reduced virulence were identified and further analysed on intact living plants. Ten transformants of these 61 exhibited either moderately or strongly reduced virulence in whole-plant assays. Three of these transformants (AT402, AT405 and AT416), exhibiting a strongly reduced virulence on intact corn plants, have been reported previously by Münch et al. (2008). The other seven candidates, also exhibiting a strongly reduced virulence phenotype (AT396, AT397, AT399, AT420, AT425) or moderately reduced virulence (AT039, AT171), are presented here (Fig. 2).
For further characterization, we chose transformants that were nonpathogenic (AT399) or moderately and strongly impaired in virulence (AT171 and AT039) (Fig. 2). Microscopic analyses of infection structures formed in planta at different time points after inoculation indicated at which stage the infection process was hampered in the transformants analysed. At 4 days post-inoculation (dpi), 85% of the conidia of the WT isolate had germinated, and 99% of the germlings had formed appressoria, the vast majority of which (97%) had melanized (Fig. 3, WT, asterisks). At this time point, 92% of the appressoria formed had penetrated the host epidermal cell and differentiated biotrophic infection vesicles and primary hyphae (Fig. 3, WT, p), as well as necrotrophic secondary hyphae (Fig. 3, WT, s).
Transformant AT399 did not cause any visible disease symptoms in detached corn leaves or on intact plants (Fig. 2). When compared with the WT isolate, germination rates on the leaf surface (66%) and rates of appressorium formation (3% of the germlings) were significantly reduced in this transformant (Fig. 3, AT399). Although melanized appressoria showed a normal morphology (Fig. 3, AT399, asterisk), host penetration rates were drastically reduced (6% vs. 92% of WT appressoria). Microscopic analyses of infected leaves (Fig. 3) showed that infection structures of AT399, formed pre- (germ tubes and appressoria) and post-penetration (infection vesicles, primary and secondary hyphae), had no morphological defects (Fig. 3, AT399, p).
Transformants AT039 and AT171 germinated and differentiated appressoria at rates indistinguishable from those of the WT isolate. Appressoria exhibited normal shape and melanization (Fig. 3, AT039 and AT171, asterisks), but host penetration rates were severely reduced (39% in AT039 and 49% in AT171 vs. 92% in WT). When penetration occurred, these mutants were able to form both primary and secondary hyphae (Fig. 3, AT039 and AT171, p and s). These counts of infection structures thus suggest that the reduced severity of disease symptoms caused by these two mutants (Fig. 2) may be a result of reduced penetration competence. Reduced penetration competence, in turn, may be caused by reduced appressorial turgor pressure (Wang et al., 2005).
The osmolyte concentration causing incipient cytorrhysis can be taken as a measure of appressorial turgor pressure. Appressoria formed on plastic sheets at 24 h post-inoculation (hpi) were incubated with different concentrations of glycerol, and rates of collapsed appressoria were determined (Fig. 4A). Incipient appressorial cytorrhysis in the WT isolate CgM2 and the mutants AT039, AT171 and AT399 occurred at very similar glycerol concentrations, indicating similar appressorial turgor pressures (Fig. 4B). Therefore, reduced penetration rates and symptom severity are probably not a result of insufficient turgor pressure in these three mutants.
A reduction in mutant efficiency with regard to infection structure formation could be a result of the disruption of a virulence gene or a gene involved in general growth processes. To address this question, we investigated AT039, AT171 and AT399 in radial growth assays on differently complex carbon sources, and compared the growth with that of WT. As presented in Fig. 5, AT039 and AT399 can grow as efficiently as WT, irrespective of the type of carbon source present in the medium. AT171 showed a retardation of growth compared with WT for all different carbon sources. In addition, for all mutants and WT, we observed no difference in growth between carbon sources of low and high complexity. In summary, our data suggest that the reduction in virulence of AT039 and AT399 does not result from a retardation of growth in general. The affected genes are dispensable for normal axenic growth, but indispensable for full virulence. In contrast, the disrupted gene in AT171 is needed for both general growth processes and full virulence.
Identification of T-DNA integration sites
Genomic Southern blot experiments showed that the transformants AT039, AT171 and AT399 had single T-DNA integrations (Fig. 6A). T-DNA integration sites were identified by the amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing, using a modification of the method described by Liu and Baird (2001). This approach allowed the amplification of 390-bp and 600-bp fragments from DNA extracted from transformant AT399. Comparisons of the sequences flanking the T-DNA with fungal genome sequences showed no significant similarities. However, the gene prediction tool augustus (http://augustus.gobics.de/) predicted an open reading frame (ORF) of 1302 bp with four introns, and suggested that the T-DNA had integrated 240 bp downstream of the start codon into the second exon (Fig. 6B). The scores for the five different exons in this hypothetical ORF range between 0.8 and 1.0.
Southern blot experiments with HindIII-digested DNA showed that transformants AT171 and AT039 carry single T-DNA integrations, and that the T-DNA integration sites clearly differ from those of the transformant described above (Fig. 6A). However, as transformants AT171 and AT039 were difficult to discriminate on the basis of blots with HindIII-digested DNA, corresponding experiments were performed with XbaI-digested DNA. Different fragment sizes clearly showed that integration sites differed in these transformants. Amplification and sequencing of a 184-bp fragment flanking the T-DNA integration site of transformant AT171 was sufficient to assemble the entire gene sequence from the whole genome shotgun sequence of C. graminicola (Fig. S1A, B, see Supporting Information). In this mutant, T-DNA integration occurred 83 bp upstream of a gene with significant similarity to the allantoicase genes of other Ascomycota, i.e. N. crassa (Acc. No. XP_956494.1; E value: 3e−171) and M. oryzae (Acc. No. XP_001406661.1; E value: 7e−162). On the protein level, the amino acid sequences of allantoicase of N. crassa, and of the corresponding proteins of M. oryzae and C. graminicola, showed a homology of 70% using the clustal 2.0.12 multiple sequence alignment tool (http://www.ebi.ac.uk) (Fig. S1C). Using N. crassa or M. oryzae as reference organisms, one intron of 77 bp, starting 95 bp downstream of the start codon, was predicted for the putative allantoicase gene of C. graminicola (Fig. 6B). Furthermore, amplification and sequencing of a 263-bp DNA fragment adjacent to the right border and a 62-bp fragment flanking the left border of the T-DNA revealed that the integration in transformant AT039 had occurred into the promoter region 450 bp upstream of the start codon of a hypothetical ORF. The target gene encodes a hypothetical protein, is 555 bp long and has similarities with ORFs of unknown function known in N. crassa (E-value: 2e−28), Giberella zea (E-value: 3e−24) and M. oryzae (E-value: 3e−20).
Photosynthetic activity in infected tissue
Screening for a lack of macroscopically visible disease symptoms allows the identification of the pathogenicity mutants, but mutants affected in virulence are often overlooked. In order to introduce an independent and quantitative measure for fungal virulence, the quantum efficiency of photosystem II (ΦPSII) was determined by chlorophyll fluorescence imaging of developing anthracnose lesions at 4 and 6 dpi (Fig. 7), which were rare for mutants AT039 and AT399. ΦPSII was recorded at distances of 250 and 500 µm from the lesion border to assess spatial differences in photosynthetic efficiency. Measurements at leaf areas devoid of disease symptoms and in mock-inoculated leaves served as controls. Leaf areas without disease symptoms, which were scrutinized by acid fuchsin staining, either lacked fungal infection structures or displayed single dispersed penetration events.
In comparison with mock controls, maize leaves infected with C. graminicola WT exhibited a more than 35% decline in ΦPSII within a distance of 250 µm to the lesion border at both 4 and 6 dpi (Fig. 7). Leaf tissue more than 500 µm from the margin of the lesion did not exhibit significant differences in photosynthetic activity relative to mock controls. In leaves infected with the C. graminicola transformants AT039, AT171 and AT399, no effect on photosynthetic performance was detectable at 4 dpi at all distances to the lesion relative to mock controls (Fig. 7A). At 6 dpi, however, ΦPSII adjacent to lesions caused by transformant AT171 was reduced to a similar degree as in the C. graminicola WT infection. In contrast, lesion borders in AT039- and AT399-infected leaves displayed only a slightly reduced ΦPSII (Fig. 7B). To exclude the possibility that the changes in ΦPSII between WT- and transformant-infected leaves were caused by the abundance of fungal hyphae at the margins of the lesion, we again checked the fungal colonization of these regions by acid fuchsin staining. Secondary fungal hyphae of all mutants were abundant within 500 µm of the lesion border, comparable with that observed for WT (data not shown).
These data demonstrate that the differences in virulence between the mutant strains and WT are reflected by changes in ΦPSII. Thus, chlorophyll fluorescence measurements provide an additional tool to evaluate fungal aggressiveness in the C. graminicola–maize pathosystem.
- Top of page
- EXPERIMENTAL PROCEDURES
- Supporting Information
ATMT has been exploited extensively in random mutagenesis experiments to identify the genes involved in the pathogenic interactions of fungi with plants, insects, mammals and other fungi (Covert et al., 2001; Fitzgerald et al., 2003; Leclerque et al., 2004; Mullins et al., 2001; Rho et al., 2001; Sugui et al., 2005; Talhinhas et al., 2008; Tsuji et al., 2003; White and Chen, 2006). The frequency and mode of T-DNA integration into the genome of the recipient are the most critical factors for the subsequent identification of the genes affected. In plants, it is well known that ATMT leads to the integration of multiple copies of T-DNA into one locus, and junctions with at least one right T-DNA border occur frequently (De Neve et al., 1997). Moreover, as shown recently, not only T-DNA- and Ti-plasmid sequences, but, in rare cases, also chromosomal fragments of A. tumefaciens are transferred into Arabidopsis thaliana (Ülker et al., 2008). Also, in fungi, T-DNA integration may occur as single or multiple copies of either T-DNA or the entire Ti-plasmid at the same or different loci of the genome. In the rice blast fungus M. oryzae, for example, head-to-tail integration of two T-DNA copies and even of the entire Ti-plasmid were found, but single-copy T-DNA integration into one genomic site occurred predominantly (Li et al., 2007; Meng et al., 2007). In general, ATMT has been described as a method feasible for random mutagenesis in many plant pathogenic fungi, including different Colletotrichum species (Maruthachalam et al., 2008; Mullins et al., 2001; Talhinhas et al., 2008; Tsuji et al., 2003). In contrast, in C. graminicola, ATMT is considered to be unsuitable for random mutagenesis because of tandem integrations of entire Ti-plasmids in the large majority of transformants, causing significant problems in the rescue of regions flanking the integration site, and thus in the identification of the gene affected.
Using the modified ATMT protocol presented here, we were able to demonstrate that this method is fully suitable for the identification of novel virulence genes in C. graminicola. When compared with the report by Flowers and Vaillancourt (2005), the transformation efficiency was considerably lower in our experiments, which may be a result of the reduced presence times of acetosyringone or of the A. tumefaciens strain and binary vector used (Flowers and Vaillancourt, 2005). In our experiments, 69.5% of the transformants had single-site T-DNA integrations, when compared with the inexactly determined maximum rate of single-site integrations of 64% reported by Flowers and Vaillancourt (2005). These transformants, however, had multi-copy integrations of the entire Ti-plasmid in approximately 70% of all cases, with only a minor fraction of single-site T-DNA integrations. In our experiments, multi-copy integration of the entire Ti-plasmid occurred in only approximately 3% of all transformants investigated, and therefore represented rare events. The modified ATMT method, in combination with the simple amplification of genomic DNA flanking the T-DNA integration site, makes forward genetics suitable for the identification of the genes contributing to the virulence of C. graminicola.
A systematic analysis of T-DNA insertion events in 175 independent transformants of M. oryzae showed that the distribution of T-DNA inserts among the chromosomes was not random. Moreover, significant insertion bias was detected, with promoters receiving twofold more T-DNA hits than expected (Meng et al., 2007). A similar pattern was detected in Arabidopsis thaliana (Alonso et al., 2003). However, although it was not the intention of this work to characterize the randomization of integration events, the analysis of the small number of transformants of C. graminicola with virulence defects presented here is consistent with the findings by Meng et al. (2007), as the integration site was preferentially the promoter region of a gene (Fig. 6B). Interestingly, microscopic analyses of the infection process (Fig. 3) revealed that the mutants analysed had defects at the pre-penetration stages of pathogenicity. As ATMT was performed with germinating spores, one may speculate that T-DNA integration primarily occurred into the genes required during germination, and these genes may also play a role during the pre-penetration stages of plant infection. This may imply, however, that the genes specifically required during biotrophic or necrotrophic development in planta would be tagged by ATMT in rare cases only.
The modified ATMT protocol presented here allowed the identification of mutants clearly affected in the genes required for the infection process (Fig. 2) (Münch et al., 2008). However, spatially resolved quantitative assessment of the virulence of the mutants was possible by the determination of ΦPSII at different distances from the inoculation site. In WT-infected leaves, this method revealed the gradual destruction of the host tissue, as ΦPSII increased with increasing distance from the centre of infection. Interestingly, transformant AT171 was affected at the appressorial penetration stage (Fig. 3), and therefore showed delayed invasion and host colonization. In contrast with the WT strain, which caused a significant reduction of 35% in ΦPSII at a distance of 250 µm from the inoculation site at 4 dpi, a significant reduction in ΦPSII of 30% caused by transformant AT171 could be measured at a distance of 250 µm from the inoculation site only at 6 dpi. In contrast, transformant AT039 only displayed a reduction in ΦPSII of 17% compared with mock control leaves at 6 dpi. The transformant AT399 was nonpathogenic in the leaf segment and whole-plant infection assays, and caused only a very slight reduction (10%) in ΦPSII in rare cases in which lesions were produced.
After C-tailing of completely digested DNA and the amplification of fragments with a T-DNA/genomic DNA junction, even short genomic sequences of 117 bp flanking T-DNA, as obtained from transformant AT171, were fully sufficient to locate this sequence in the genome of C. graminicola. The observed virulence defect in mutant AT171 is probably a result of the insertion of T-DNA into the promoter region of a gene encoding the enzyme allantoicase (Lee et al., 1990). In filamentous fungi, such as N. crassa, purines are converted to allantoate, which is subsequently used as a substrate by the enzyme allantoicase, producing (S)-ureidoglycolate. The final products of the pathway are carbon dioxide and ammonia, which can be re-utilized as a nitrogen source (Yoo and Cooper, 1991; Yoo et al., 1985).
The regulation of fungal pathogenicity by nitrogen limitation is clearly established (Talbot et al., 1997). Indeed, during pre-penetration development, e.g. germ tube growth on the plant cuticle, nitrogen is hardly available and the allantoicase pathway may be involved in the provision of ammonia and compensation for nitrogen limitation. In M. oryzae, multiple genes, involved in both nitrogen metabolism and pathogenicity, have been identified (Meng et al., 2007). For example, a gene encoding the vacuolar serine protease, SPM1, was shown to be indispensable for full sporulation and appressorial development (Meng et al., 2007), and a mutation in one of the two regulatory genes NPR1 or NPR2 (for nitrogen pathogenicity regulation) resulted in a failure to use different nitrogen sources and a lack of pathogenicity (Lau and Hamer, 1996). Gene expression data also support the idea that the allantoicase pathway may play a role in pathogenicity. In M. oryzae, Donofrio et al. (2006) identified four genes in this pathway, encoding an allantoate permease, an MFS allantoate transporter, an allantoinase and an allantoin permease, which are upregulated under nitrogen starvation conditions, and may thus be required during plant infection.
In summary, the work described here shows that the utilization of the modified ATMT protocol is well suited for the identification of novel genes required for virulence in the corn pathogen C. graminicola. Thus, also in this fungus, ATMT represents an excellent alternative to REMI mutagenesis (Epstein et al., 1998; Thon et al., 2000).
- Top of page
- EXPERIMENTAL PROCEDURES
- Supporting Information
Cultivation and ATMT of C. graminicola
The WT isolate CgM2 of Colletotrichum graminicola (Ces.) Wils. (teleomorph Glomerella graminicola (Politis)) was grown on OMA (50 g oat meal, 15 g agar per litre) under constant black light irradiation (lamps: TL-D 36 W/08; Philips, Hamburg, Germany) as described previously (Sugui and Deising, 2002). Conidia from 14-day-old cultures were used for ATMT.
In the A. tumefaciens strain LBA1100 (Beijersbergen et al., 1992), which already contained the vir-plasmid pAL1100 carrying a spectinomycin resistance cassette, the binary vector pPK2 (Covert et al., 2001) (Fig. 1A) was introduced by electroporation (2500 V, 5 ms, multiporator; Eppendorf, Hamburg, Germany). The T-plasmid pPK2 confers kanamycin resistance as a bacterial selection marker and carries the hygromycin resistance cassette between the right and left border. Agrobacterium tumefaciens strain LBA1100-pPK2 was grown overnight at 28 °C and 120 rpm in 7 mL of Luria–Bertani (LB) medium (Sambrook et al., 1989) containing 50 µg/mL each of kanamycin and spectinomycin. Cells were diluted into induction medium (IM; Bundock et al., 1995) to an optical density at 600 nm (OD660) of 0.15, and were grown to an OD660 of 0.6–0.8.
Aliquots of 100 µL of bacteria and 100 µL of conidia suspended in sterile distilled water (105–106 conidia/mL) were mixed and plated onto cellulose membranes (filter no. 595; Ø= 90 mm; Schleicher & Schuell GmbH, Dassel, Germany) placed on solidified IM containing 5 mm glucose and 200 µm acetosyringone. After incubation in the dark for 3 days, the membranes were transferred to PDA (24 g potato dextrose, 15 g agar per litre) containing 200 µg/mL cefotaxim and 100 µg/mL hygromycin B, and incubated at room temperature for 14 days. Mycelia of putative transformants were transferred to selection plates [SYC, 1 m sucrose; 0.1% (w/v) yeast extract; 0.1% (w/v) casein-hydrolysate; 1.5% agar-agar] containing 100 µg hygromycin B/mL.
To generate homokaryotic strains, mycelia were allowed to form mononucleate conidia on OMA and plated out at low densities on SYC plates containing 100 µg/mL hygromycin B (Werner et al., 2007). Single colonies were transferred to OMA plates, and clonal conidia were used directly or stored in the presence of 40% (v/v) glycerol at −70 °C.
To test for the mitotic stability of the integrated hygromycin resistance cassette, conidia of 10 transformants were cultivated on OMA without hygromycin B, with biweekly transfer to new plates. After four passages in the absence of hygromycin B, transformants were grown on OMA plates containing hygromycin B (100 µg/mL) and, for DNA isolation and Southern blot analyses, in liquid complete medium (CM; Leach et al., 1982) without hygromycin B at 23 °C and 100 rpm (Unitron Shaker; Infors, Bottmingen, Switzerland).
Radial growth assays were performed on solid media [0.2% (w/v) yeast extract, 6 mm Ca(NO3)2, 1.5 mm KH2PO4, 2 mm MgSO4, 1 mm NaCl, 1.5% (w/v) agarose] containing either 2% (w/v) of different carbon sources (glucose, maltose, sucrose, starch, cellulose, pectin) or no carbon source (minimal medium; MM). In addition, a complex medium (CM) was used as described by Sugui and Deising (2002). For the quantification of growth, 5 µL of a conidial suspension (104 conidia/mL) were pipetted onto the centre of the plate, the plates were incubated at 23 °C in the dark and radial growth was measured.
Isolation of genomic DNA and Southern hybridization
Fungal genomic DNA was isolated using the phenol–chloroform extraction procedure, combined with RNase A and proteinase K treatment, as described previously (Sugui and Deising, 2002). The digestion of DNA with HindIII, Eco32I and XbaI, electrophoresis on 0.7% agarose, depurination and transfer onto nylon membranes (Hybond-N+; Amersham Pharmacia Biotech, Freiburg im Breisgau, Germany) followed standard protocols (Brown, 1999; Sambrook et al., 1989).
The Dig-labelled probe used in hybridization experiments, corresponding to 1 kb of the T-DNA (Fig. 1B), was amplified from plasmid pPK2 by PCR. In a total volume of 10 µL, the PCR mixture consisted of 1 × reaction buffer containing 1.5 mm MgCl2, 0.025 U/µL Taq DNA Polymerase (Amersham Pharmacia Biotech), 0.2 mm deoxynucleoside triphosphate (dNTP), 0.7 mm alkali-labile Dig-dUTP (Roche, Mannheim, Germany), 0.5 µm each of forward (5′-GTCCCTCAGTCCCTGGTAGG-3′) and reverse (5′-TGTTGGCGACCTCTATTGG-3′) primers, and 10 pg of plasmid DNA. Hybridization and visualization of the hybridized Dig-labelled probe were performed as suggested by the manufacturer (Roche, Mannheim, Germany).
Identification of T-DNA integration sites
The identification of genomic regions flanking T-DNA integration sites was performed by the polynucleotide tailing method, as described previously (Liu and Baird, 2001), with some modifications. Genomic DNA of the transformants was completely digested with KpnI, PvuI or AatII. Depending on the restriction enzyme used, one degenerated primer (degKpn, 5′-GGAGACTGACATGGACTGAAGGAGTAAAGGGIIGGGIIGGGIIGGGGTACC-3′; degPvu, 5′-GGAGACTGACATGGACTGAAGGAGTAAAGGGIIGGGIIGGGIIGGGATCG-3′; degAat, 5′-GGAGACTGACATGGACTGAAGGAGTAAAGGGIIGGGIIGGGIIGGGACGTC-3′) was used in combination with a primer specific for either the left (pPK2-rev7, 5′-TCACTGCCCGCTTTCCAGTC-3′) or the right (CgSQ8, 5′-CAGATCAACGGTCGTCAAGAG-5′) T-DNA border. One microgram of tailed genomic DNA was used as template in a total volume of 20 µL of PCR. The PCR conditions were 95 °C for 2 min, followed by five cycles of 95 °C for 30 s, 70 °C for 30 s and 72 °C for 5 min; five cycles of 95 °C for 30 s, 65 °C for 30 s, 72 °C for 5 min, and 20 cycles of 95 °C for 30 s, 60.5 °C for 30 s, 72 °C for 5 min. Final elongation was at 72 °C for 5 min. In the subsequent nested PCR, the anchor primer (Tn, 5′-GGAGACTGACATGGACTGAAGGAGT-3′) was used in combination with either the right (CgSQ8n, 5′-ACTGAGGAATCCGCTCTTGG-3′) or left (CgSQrev20, 5′-GGTTTGCGTATTGGCTAGAG-3′) nested T-DNA border primer. One microlitre of the first PCR served as template. The reaction volume was again 20 µL. The annealing temperature was 59 °C, the extension time was 5 min, and buffer and nucleotide concentrations were as described above.
Sequencing of the PCR products was performed as described previously (Krijger et al., 2008). Nucleotide sequences were compared with known protein sequences by the Basic Local Alignment Search Tool (blastx) (Altschul et al., 1990) provided by the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov), and further analysed by the gene prediction program augustus (http://augustus.gobics.de) (Stanke et al., 2004).
Corn (Zea mays cv. Nathan) leaf segments and plants were used to assess the virulence of the WT isolate and transformants of C. graminicola generated by ATMT.
Corn leaf segments (10 cm) of the third leaf of 14-day-old plants were inoculated with 10-µL droplets containing 5 × 103 conidia in 0.02% (v/v) Tween 20 (Mercure et al., 1994). Inoculated leaf segments were incubated in the dark at 25 °C in a sealed moist Petri dish. Leaves were photographed and subjected to microscopy at 1, 2, 3, 4 and 7 dpi. After bleaching in acetate–ethanol (1:3, v/v) overnight, leaf segments were stained by acid-fuchsin (de Neergaard, 1997). Microscopy was performed using a Nikon Eclipse 600 epifluorescence microscope (Nikon, Düsseldorf, Germany), as described previously (Werner et al., 2007). Digital images were taken with a CCD-1300 camera (VDS Vosskühler GmbH, Osnabrück, Germany) and processed with the software package NS Elements (Nikon).
For whole-plant virulence assays, 14-day-old corn plants were evenly spray inoculated with 1.7 × 106 conidia per plant and cultivated in a growth chamber (Percival-36HIL) at 16 h/8 h day/night rhythm and a light flux of 400 µE (MASTER PL-L 55W/840/4P, Phillips, Poland and Radium-25W lamps, Radium, Frankfurt, Germany). During the first 24 hpi, the plants were kept at 25 °C and 100% relative humidity, followed by day/night conditions of 25 °C/20 °C and 50%/70% relative humidity, respectively. Symptoms were photographed daily from 3 to 7 dpi.
Plastic sheets (No. 3558, Avery Dennison, Holzkirchen, Germany) of 1 cm × 1 cm were inoculated with 10 µL of water droplets containing 2000 spores and stored in a sealed moist Petri dish in the dark at 23 °C. After 24 h, each sheet was incubated for 10 min in a solution of 0, 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 m glycerol, and collapsed and noncollapsed appressoria were counted by light microscopy (Eclipse 600; Nikon).
Chlorophyll fluorescence imaging
Chlorophyll fluorescence of leaves infected with mutant and WT C. graminicola, and of control leaves, was recorded with a combined gas exchange/chlorophyll fluorescence imaging system (GFS-3000/MINI Imaging-PAM chlorophyll fluorometer; Walz, Effeltrich, Germany) starting at 4 dpi in daily intervals. Data analysis was performed with ImagingWin v2.30 software (Walz). Prior to the measurements, the leaves were dark adapted for 20 min and then placed into the cuvette of the GFS-3000 IRGA. CO2 and H2O partial pressures were set to 350 µg/g and 10 000 µg/g, respectively, to obtain identical conditions for all measurements. An initial saturating pulse was applied to determine the maximal fluorescence (Fv/Fm) before the absorptivity (abs.) was determined using the formula abs. = 1 −RR/RNIR, where RR is the signal intensity for reflected red light (660 nm) and RNIR is the intensity for reflected near-infrared light (780 nm). Subsequently, the leaf was illuminated with 400 µE/m2/s of actinic light, and chlorophyll fluorescence images were taken in 60-s intervals on application of saturating light pulses, until ΦPSII reached its maximum. Fluorescence parameters were calculated according to Horst et al. (2010).
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The A. tumefaciens strain LBA1100 and the plasmid pPK2 were kindly supplied by P. J. J. Hooykaas (University of Leiden, the Netherlands). In addition, we thank E. Vollmer, A. Beutel and D. Jany for skilful technical assistance, and B. Sode, A. Mickel and W. Kummer for experimental support. Financial support by the Deutsche Forschungsgemeinschaft (DFG) (FOR666, DE 403/16-1 and SO 300/11-1) is gratefully acknowledged.
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Fig. S1 T-DNA disruption in AT171 leads to the identification of an allantoicase gene in Colletotrichum graminicola. (A) Scheme of the disrupted region in AT171. (B) Partial genomic sequence of C. graminicola wild-type including the 5′ flank (bp 1–184), the T-DNA integration site in AT171 and the allantoicase gene (start and stop codons are framed, intron with grey background). (C) Alignment of the protein sequences of allantoicase of C. graminicola (Cg) with the sequences of Neurospora crassa (Nc) and Magnaporthe oryzae (Mo). The protein sequence of N. crassa is annotated as an allantoicase and that of M. oryzae includes several regions of an allantoicase. The sequence homology between C. graminicola and N. crassa is 80%, between C. graminicola and M. oryzae 79%, and between all three sequences 70%.
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