Physiological and Molecular Plant Pathology 63 (2003) 191–199 www.elsevier.com/locate/pmpp The utilisation of di/tripeptides by Stagonospora nodorum is dispensable for wheat infection Peter S. Solomona, Stephen W. Thomasb, Pietro Spanuc, Richard P. Olivera,* a Australian Centre for Necrotrophic Fungal Pathogens, SABC, DSE, Murdoch University, Perth 6150, Australia b Division of Plant Industries, Department of Agriculture, Orange 2800, NSW, Australia c Biological Sciences, Imperial College, London SW7 2AZ, UK Accepted 17 December 2003 Abstract A gene required for di/tripeptide transport in the necrotrophic wheat pathogen Stagonospora nodorum has been cloned, characterised and inactivated by homologous recombination. Recent genome sequencing projects have revealed the presence of fungal homologues though Ptr2 is the first di/tripeptide transporter to be cloned and function characterised from a fungus. Analysis of Ptr2 expression in vitro revealed strong expression in the absence of nitrogen and in the presence of carbon; interestingly there was very low expression in the absence of both nitrogen and carbon. The expression of Ptr2 during infection showed the gene was significantly up-regulated during the initial stages of infection before decreasing to a lower constitutive level suggesting the fungus may be nitrogen starved during the pre-penetration stage of the infection. Ptr2 was inactivated by homologous gene recombination resulting in the strain S. nodorum ptr2. Peptide uptake studies of S. nodorum ptr2 suggest that Ptr2 is solely responsible for the uptake of di/tripeptides. The ability of S. nodorum ptr2 to cause infection was also examined. Pathogenicity assays revealed that the mutant strain was fully pathogenic. As the gene has been shown to be fully responsible for di/tripeptide transport, this implies that the uptake of these small peptides is not required for S. nodorum pathogenicity on wheat leaves. q 2004 Elsevier Ltd. All rights reserved. Keywords: Stagonospora nodorum; di/tripeptides; In planta nutrition 1. Introduction To successfully colonise a host plant, a fungal phytopathogen must overcome a series of physical and chemical barriers. The primary physical barrier the fungus encounters is the surface of the host. Once inside the leaf the host can often respond with various means of chemical defence such as the production of reactive oxygen species, fungal cell wall degrading enzymes and phytoalexins. After successfully overcoming these, the fungus can then go into accessing the nutrients provided by the plant. It had long been assumed that once the fungus had penetrated and was established internally, there was an abundance of nutrients to feed on. However, several key reports around this time though discovered that the expression of various genes required for pathogenicity were only expressed in vitro under nutrient limiting conditions, particularly nitrogen * Corresponding author. Tel.: þ 61-8-9360-7404; fax: þ61-8-9360-6303. E-mail address: roliver@central.murdoch.edu.au (R.P. Oliver). 0885-5765/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2003.12.003 [1 –4]. This led to the counter-assumption that the infecting fungus was instead growing under starvation conditions during infection. Very few reports have since directly measured the nutrient composition of the host during infection. One recent report that measured the nitrogen composition of the tomato leaf apoplast during infection by Cladosporium fulvum revealed the presence of abundant amino acids and other nitrogen sources [5]. However this organism is a unique biotroph and may not be representative of other fungal phytopathogens [6]. Phaeosphaeria nodorum (Müller) Hejaroude (anamorph Stagonospora nodorum (Berk.) Castellani and Germano) [7] is a necrotrophic phytopathogen that is the causal agent of leaf and glume blotch on wheat and is responsible for $60 M (AUD) of crop loss in Australia each year. Surprisingly, though being an economically important pathogen and comparatively simple to work with, very little is known at a molecular level about how the fungus infects wheat. As with other fungal phytopathogens, S. nodorum must be able to acquire nutrients during colonisation of wheat to 192 P.S. Solomon et al. / Physiological and Molecular Plant Pathology 63 (2003) 191–199 complete its lifecycle. A previous report has shown that S. nodorum does produce extracellular proteases during infection [8] and consequently peptides could play a role in nutrition. During the sequencing of a cDNA library of S. nodorum, a clone was sequenced with strong similarity to the peptide transporter, PTR2 from the yeast Saccharomyces cerevisiae. Molecular and biochemical evidence has been gathered showing that PTR2 is required for the uptake of di/ tripeptide in yeast [9,10]. The acquisition of a putative S. nodorum homologue of PTR2 provided the opportunity to determine if di/tripeptides played any role in the pathogenicity of S. nodorum on wheat. Peptide transporters have been previously cloned and characterised from several sources including yeasts, plants and mammals [9 – 18]. Interestingly though while orthologues of Ptr2 have been sequenced in fungal genome sequencing projects, none have been functionally characterised from a filamentous fungus. In this study, we have examined the possibility that small peptides act as a nitrogen source for S. nodorum during infection by isolating and characterising a di/tripeptide transporter gene Ptr2. the inoculum using a Paasche airbrush paint sprayer (Paasche Airbrush Co., USA). The plants were then allowed to sit for 15 min before the spraying was repeated. After the second spraying, the plants were covered and incubated at 20 8C in the dark for 2 days at which point the cover was removed and the plants were grown at the normal growth conditions. Seven days after infection the plants were scored for disease severity. The infections were given a score between 0 and 10 depending on the severity of the infection with 0 being uninfected and no symptoms of disease and 10 being a completely necrotic dead plant. Pathogenicity of the mutants was also investigated using a detached leaf assay. This involved placing 2-week-old wheat leaves on 0.15% benzimadazol plates with the ends of the leaves embedded into the agar. The leaves were inoculated with 5 ml drops of inoculum containing 104 spores in 0.02% tween 20. The plates wrapped in parafilm and incubated at 20 8C in a 12 h day/night cycle. Disease severity was assessed by measuring the size of necrotic tissue 6 days after inoculation. 2.3. Cloning of insertional inactivation construct 2. Materials and methods 2.1. Fungal strains and media S. nodorum SN15 was provided by the Department of Agriculture, Western Australia. The fungus was routinely grown on CzV8CS (Czapek Dox agar (Oxoid) 45.4 g l21, agar 10.0 g l21, CaCO3 3.0 g l21, Campbell’s V8 juice 200 ml l21, casamino acids 20.0 g l, peptone 20 g l21, yeast extract 20 g l21, adenine 3 g l21, biotin 0.02 g l21, nicotinic acid 0.02 g l21, p-aminobenzoic acid 0.02 g l21, pyrodoxine 0.02 g l21, thiamine 0.02 g l21) containing 1.5% agar. Plates were incubated at 22 8C in 12 h cycles of darkness and near-UV light (Phillips TL 40W/05). Liquid cultures were started with the addition of 107 spores to 100 ml CzV8Cs and also grown at 22 8C shaking at 130 rpm in the dark. For experiments that required defined growth conditions, S. nodorum SN15 was used to inoculate minimal media that consisted of 30 g l21 sucrose, 2 g l21 NaNO2 3, 1.0 g l21 K2HPO4, 0.5 g l21 KCl, 0.5 g l21 MgSO4·7H2O, 0.01 g l21 ZnSO 4 ·7H2 O, 0.01 g l 21 FeSO 4 ·7H 2O, 0.0025 g l21 CuSO4·5H2O. 15 g l21 agarose was added when plates were required. A modified form of the vector pGPS3 (New England Biolabs) was used to create insertional inactivation constructs. A phleomycin cassette from pAN8.1 was cloned into the SwaI-SpeI sites within the transprimer of pGPS3 creating pGPS-phleo. The transposition reaction was then undertaken as per the pGPS3 protocol. Briefly, 20 ng of pGPS-phleo and 80 ng of Ptr2 cDNA were incubated together in the presence of the TnsABC transposase complex (New England Biolabs). The reaction was followed by a PI-SceI restriction enzyme digest that destroyed any remaining pGPS-phleo vector. The products of the transposon reaction were transformed into E. coli strain XL1Blue (Stratagene) and the resulting colonies screened by restriction enzyme digests to determine whether the transposon had inserted into the cDNA or the bluescript backbone. Those that appeared to have inserted within the cDNA were then sequenced using primers homologous to the ends of the transposon. This process is illustrated in Fig. 1. 2.2. Plant material and infection conditions Ten centimetre diameter pots containing Perlite (P500) and Grade 2 vermiculite (The Perlite and Vermiculite Factory, WA, Australia) were seeded with five seeds of the wheat variety Amery and grown at 20 8C in a 12 h day/night cycle. For the infections, an inoculum was prepared consisting of 106 spores per ml in 0.02% tween 20. Twoweek-old plants were sprayed twice for 30 s each time with Fig. 1. Graphical representation depicting the result of a homologous recombination event between the pGPSP-Ptr1 construct and Ptr2. P.S. Solomon et al. / Physiological and Molecular Plant Pathology 63 (2003) 191–199 2.4. Preparation of Stagonospora nodorum protoplasts Preparation of S. nodorum protoplasts was essentially as described elsewhere [19]. Mycelium grown by inoculating 100 ml of CzV8CS liquid medium with 5 £ 107 spore suspension and shaking at 140 rpm overnight at 22 8C was harvested by centrifuging at 10,000g for 10 min at 4 8C. The supernatant was discarded and the cells washed once with 600 mM magnesium sulphate before being resuspended in 20 ml of filter-sterilized 1.2 M magnesium sulphate buffered to pH 5.8 with 10 mM phosphate buffer and containing 15 mg ml21 glucanex (Novo Nordisk). The solution was incubated without agitation in a glass petri dish for 2 h at 28 8C, transferred to a sterile centrifuge tube and gently overlaid with 5 ml of 600 mM sorbitol, 10 mM Tris (pH 7.5). After centrifugation at 4000g for 25 min at 4 8C, the protoplasts, which settled at the interface, were removed and mixed with an equal volume of 1 M sorbitol, 10 mM Tris (pH 7.5). The protoplasts were pelleted at 1500g for 10 min at 4 8C, washed with 3 ml STC (1.2 M sorbitol, 10 mM calcium chloride, 10 mM Tris, pH 7.5) buffer and gently resuspended in 0.5 ml of the same buffer. The concentration of the protoplast suspension was estimated using a haemocytometer and adjusted as required. The protoplasts were now ready for use in transformation experiments. Note that all solutions used for the preparation of S. nodorum protoplasts were freshly made the day prior and were filter sterilised. 2.5. Transformation of Stagonospora nodorum to phleomycin resistance Transformation was as essential as previously described [5]. Up to 7.5 mg of transforming DNA was dissolved in a maximum volume of 25 ml STC buffer and added to 100 ml of protoplast suspension. For reliable transformation, a concentration of at least 5 £ 108 protoplasts per ml was required. A negative control was also prepared where protoplasts received an equivalent volume of STC buffer. Mixtures were incubated at room temperature for 20 min before the addition of 200 ml of a filter-sterilized solution containing 60% (w/v) polyethylene glycol 4000, 10 mM calcium chloride and 10 mM Tris (pH 7.5). The solution was mixed by gentle inversion and a further two additions (200 ml and 800 ml) of the same solution was made followed by gentle mixing after each addition. After another incubation of 20 min at room temperature, the mixture was centrifuged at 12,000g for 5 min, the supernatant discarded and the protoplasts gently resuspended in 1 ml STC buffer. Selection of phleomycin-resistant transformants was accomplished by adding 250 ml of the transformation mixture to 5 ml soft Cz-top proto (Czapek-Dox liquid 45.4 g l21, Oxoid agar 7.5 g l21, sorbitol 182.2 g l21, filtered V8 juice 200 ml l21, pH 6.0) and pouring onto Cz plates (Czapek-Dox agar 45.4 g l21, Oxoid agar 10 g l21, sorbitol 182.2 g l21, filtered V8 juice 200 ml l21, pH 6.0). 193 This was repeated four times using the full 1 ml of the transformed protoplasts. After 40 h, the protoplasts were overlaid with a further 5 ml soft Cz containing enough phleomycin to give a final concentration of 50 mg ml21 for the whole plate. All incubations were at 22 8C in the dark. Approximately 9 days after the phleomycin overlay, colonies were cut from the agar and placed on fresh CzV8CS þ phleomycin for 3 weeks. Spores from these were collected and diluted to single spores. After 1 week’s growth, pycnidia from single spore growth was plated out and incubated for a further 3 weeks. These resulting pycnidia were used for further analysis. 2.6. Molecular techniques Genomic DNA was isolated using a Nucleon Phytopure genomic DNA extraction kit (Amersham Pharmacia Biotech). For Southern analysis, 5 mg of genomic DNA was digested with the appropriate restriction enzyme in a volume of 300 ml at 37 8C for 16 h. Upon completion, the digest was ethanol precipitated and resuspended in 20 ml sterile water. The digested genomic DNA was then transferred to Hybond N þ membrane as previously described [20]. The blot was air-dried and the DNA crosslinked using a UV GS Genelinker (BioRad). Hybridisation and detection was performed using the DIG system (Roche). The Ptr2 probe was generated by excising the cDNA from pBluescript using XhoI and PstI and labelled as outlined in the manufacturer’s instructions. Total RNA was isolated from fungal mycelium and plant material using TRIzol Reagent (Invitrogen). Five micrograms of RNA was reverse transcribed using Superscript II RNase H2 reverse transcriptase using a oligo dT(18) primer and performed as per the manufacturer’s protocol. The resulting cDNA was then quantified using a ABI PRISMe 770 Sequence detector (Applied Biosystems). The PCR was as follows; 0.5 ml cDNA, 1.5 mM MgCl2, 0.4 mM dNTPs, 2 mM of each primer, 2 U Taq polymerase, 1 ml ROX reference dye (Invitrogen) and 1 ml of 1 £ SYBRwGreen nucleic acid stain (Molecular Probes Inc.) with the reaction made up to 25 ml with sterile H2O. The conditions used were 95 8C/2 min, (95 8C/30 s, 55 8C/30 s, 72 8C/1 min) for 40 cycles, 72 8C/5 min. The primers used for Ptr2 cDNA amplification were 50 ACA ACA CCA TCG TCA TCT TC 30 (PtrF2) and 50 TTC ATC TCA TCC TCC GTC TC 30 (PtrR2). Amplification of an actin control fragment was performed as per Ptr2. The primers used were as follows; 50 CTG CTT TGA GAT CCA CAT 30 (actinF) and 50 GTC ACC ACT TTC AAC TCC 30 (actinR). For the quantitation of gene expression, the expression of Ptr2 was normalised against the actin expression for that particular time point or growth condition. To ensure statistical confidence, each sample was assayed five times and the data processed students t-test. All sequencing was performed using a Perkin Elmer PCR sequencing premix and analysed on an ABI 373 or 377 automatic DNA sequencer. 194 P.S. Solomon et al. / Physiological and Molecular Plant Pathology 63 (2003) 191–199 2.7. Stagonospora nodorum spore PCR Preliminary screening of fungal transformants was performed by spore PCR. A full loopful of transformant growth (including spores and mycelia) was scraped into 500 ml of DNA extraction buffer (0.5 M NaCl, 10 mM Tris–HCl pH 7.5, 10 mM EDTA, 1% SDS). The solution was then placed at 220 8C and allowed to freeze before being thawed at 37 8C. After repeating the freeze–thawing, the suspension was then centrifuged at 12,000g for 15 min to remove cellular debris and the supernatant was added to an equal volume of isopropanol. The precipitated DNA was collected by centrifugation at 12,000g for 10 min and washed once with 70% ethanol before being air-dried and resuspended in 10 ml sterile water. Two micro litre of the DNA solution was used in the resulting PCR using the conditions listed above. 2.8. Construction of libraries Genomic DNA from S. nodorum SN15 was partially digested with Sau3A and fractionated by centrifugation on sucrose gradients as described [18]. DNA fragments (12 – 20 kb) were pooled and the ends partially filled with dATP and dGTP to make them compatible with the Xho1 digested vector lambda Bluestar (Novagen). Ligation to the lambda arms, packaging of phage using Phagemaker (Novagen) extract and plating were performed according to the manufacturers instructions. The resulting library consisted of 1.57 £ 106 pfu ml21 and represents a 3 –4-fold representation of the S. nodorum genome assuming a genome size of 30 Mb. Genomic clones containing the Ptr2 open reading frame were isolated using the labelled Ptr2 cDNA as described (Novagen). The cDNA library from which the Ptr2 cDNA clone was obtained was created from S. nodorum grown on wheat cell walls and was a generous gift from Dr. Richard Cooper (University of Bath, UK). 2.9. Dipeptide growth studies All growth studies of the fungus were performed in 96well microtitre plates. The growth medium used was a minimal medium minus nitrate and with the appropriate nitrogen source added. 100 ml of media was inoculated with 103 spores and the plates were incubated at 22 8C shaking at 130 rpm. Growth was measured daily on a microplate reader (Bio-Rad, Model 3550-UV) at 595 nm. All dipeptides used during this study were purchased from Sigma. 3. Results 3.1. Nucleotide analysis A cDNA orthologous to the di/tripeptide transporter PTR2 from S. cerevisiae was identified during sequencing of the S. nodorum wheat cell-wall library. A genomic library was screened with the Ptr2 cDNA clone. Four clones were isolated and one (pPtrg1) was used for subsequent nucleotide analysis. Sequencing revealed a Ptr2 open reading frame of 2026 bases that has been subsequently submitted to Genbank (Accession no. AY187281) (Fig. 2). Comparison of the genomic and cDNA sequences revealed the presence of two introns from bases 538– 611 and 1065– 1120. Translation initiation of Ptr2 is predicted to occur at ATAAGAATGG, which compares moderately well with the Kozak consensus sequence for higher eukaryotes, G44C39C53M76C55A100T100G100G46 [21]. A TATA motif, required for RNA polymerase II transcription initiation was identified five bases upstream of the predicted start codon while a potential polyadenylation site was predicted by computer analysis 200 bases downstream from the stop codon (http://angis.murdoch.edu.au/). Genomic Southern analysis was used to determine the copy number of the Ptr2 gene in S. nodorum genome. Restriction enzyme digested genomic DNA was probed with an internal Ptr2 fragment. Only one band was evident at various stringency conditions indicating that Ptr2 exists in a single copy (data not shown). 3.2. Analysis of predicted Ptr2 amino acid sequence The Ptr2 gene was predicted to encode a protein of 626 amino acids. No obvious N terminal signal peptide sequence was evident. Computer analysis predicted the presence of 9 – 12 plasma membrane spanning domains, depending on the parameters used. A comparison to the NCBI nonredundant protein database using the BlastX algorithm revealed Ptr2 from S. nodorum to be most similar to PTR2, Pt2A and Ptr2 from S. cerevisiae, A. thaliana and S. pombe, respectively. 3.3. Construction of Ptr2 insertional inactivation construct The development of the insertional inactivation construct was undertaken using the Ptr2 cDNA as the target for a novel transposon containing the phleomycin selectable marker. After the transposition reaction and subsequent transformation in to E. coli XL1Blue, 30 colonies were obtained. Twelve of these colonies were selected at random for plasmids extraction. Two of the 12 plasmids appeared to have the transposon inserted into the Ptr2 cDNA as determined by restriction enzyme analysis. Sequencing of these two plasmids using primers homologous to the end of the transposon confirmed that both had inserted into the Ptr2 cDNA, albeit at different regions. In one of the constructs, the transposon had integrated into the Ptr2 cDNA 507 bases downstream of the predicted translation initiation sequence. This construct was named pGPSP-Ptr1 and chosen for the subsequent transformation. P.S. Solomon et al. / Physiological and Molecular Plant Pathology 63 (2003) 191–199 195 Fig. 2. The nucleotide and predicted protein sequence of Ptr2. Underlined sequence represents non-coding introns whilst the sequence in bold denotes the peptide transporter putative consensus sequence FYXXINXGSL. 3.4. Inactivation of Ptr2 in Stagonospora nodorum The pGPSP-Ptr1 construct was transformed into S. nodorum as described in Section 2. Thirty-seven phleomycin resistant transformants were recovered and analysed by PCR. All 37 transformants tested positive for the presence of phleomycin resistance cassette with 13 of these testing positive for the presence of an intact Ptr2 gene. This indicated that the Ptr2 gene in 24 of the transformants had undergone homologous recombination with the phleomycin construct. Six of these transformants and a potential ectopic transformant were chosen for further characterisation by Southern analysis. In all six transformants there was an identical band shift from the wild type Ptr2 corresponding to the size of the insertional construct (Fig. 3A). This band shift was not evident for the ectopic strain, which retained both the band corresponding to the wild type as well as two additional bands suggesting three separate integration events. The insertion of the phleomycin cassette in Ptr2 was further confirmed by the probing of the membrane with a homologous phleomycin probe (Fig. 3B). No hybridisation was evident in the lane containing 196 P.S. Solomon et al. / Physiological and Molecular Plant Pathology 63 (2003) 191–199 Fig. 3. Southern analysis of S. nodorum SN15 (lane 1), six potential Ptr mutants lanes 2–7) and one ectopic mutant (lane 8). (A) Digested genomic DNA probed with a Ptr2 specific probe. (B) Digested genomic DNA probed with a phleomycin resistance cassette specific probe. the digested wild type gDNA while single bands were present in the transformants at the same migration distance as observed when probed with the Ptr2 probe. The mutants named S. nodorum SNPtr1 and S. nodorum SNPtr7, and the ectopic strain S. nodorum SNPtr3, were selected for further analysis. 3.5. Dipeptide uptake Previous studies in yeast have analysed dipeptide transport by making use of highly specialized toxic dipeptides. Unfortunately these were not available during the course of this study and consequently dipeptide uptake was analysed by studying the ability of the fungus to grow on dipeptides as a sole nitrogen source. The S. nodorum strains SN15, SNPtr1, SNPtr7 and SNPtr3 were compared for their ability to grow on dipeptides as described in Section 2. Each strain was grown on nitrate, the nitrogen-rich Arg-Leu dipeptide and the comparatively poor Glu-Glu dipeptide. All four strains appear to have grown equally well in minimal media with nitrate supplied as a nitrogen source (Fig. 4). The growth of the strains, however, differed markedly when grown on dipeptides as a sole nitrogen source. During the first 4 days, essentially no growth was observed for the mutants when grown on either of the dipeptides. After 4 days, limited growth was observed in mutants at rates much lower than the wild type and ectopic strains. As expected, higher growth was observed for the wild type and ectopic strains when grown on Arg-Leu compared to Glu-Glu due to the much higher amount of nitrogen present. Fig. 4. Growth studies of the wild type and mutants on various nitrogen sources. (A) 0.5 mM nitrate, (B) 1 mM Glu-Glu, (C) 1 mM Arg-Leu. S, S. nodorum SN15; A, S. nodorum SnPtr1; B, S. nodorum SnPtr7; X, S. nodorum SnPtr3. was not affected when the fungus was starved either of carbon or of carbon and nitrogen together. When the fungus was starved of nitrogen alone, the expression of Ptr2 was seen to increase over 100-fold when compared to expression on nitrogen supplemented minimal media suggesting that the expression of Ptr2 was up-regulated in the absence of external nitrogen. 3.6. In vitro Ptr2 expression The expression of Ptr2 was examined under various starvation conditions by growing S. nodorum SN15 for 3 days in full minimal media prior to exposure to a particular starvation condition for a further 6 h. The RNA was extracted and expression analysed using real-time RT-PCR as described in Section 2. Fig. 5 displays the normalized expression of Ptr2 under three different growth starvation conditions compared to the expression observed on full minimal media. The expression of Ptr2 Fig. 5. Analysis of Ptr2 expression in S. nodorum during growth under various starvation conditions. C2, minimal media minus carbon; N2, minimal media minus nitrogen; C2N2, minimal media minus carbon and nitrogen. The experiment was performed in duplicate on two independent RNA populations. P.S. Solomon et al. / Physiological and Molecular Plant Pathology 63 (2003) 191–199 197 3.7. Ptr2 expression in planta 4. Discussion To determine the pattern of expression of Ptr2 in planta, S. nodorum spores were used to inoculate detached wheat leaves. Lesions were excised at various time points, the RNA extracted and Ptr2 mRNA was quantified. The normalized Ptr2 expression at different stages during infection is shown in Fig. 6. Statistical analysis of the normalized expression at each time point using students t-test revealed that the transcription of Ptr2 was significantly higher at the 6 h and 1 dpi when compared to the other time points suggesting that the peptide transporter was up-regulated during the beginning of the infection. No significant change in regulation was observed for the remainder of the infection including at 10 dpi when sporulation was evident. Since the observation that some genes required for pathogenicity were expressed in vitro only under nitrogen limiting conditions, the nitrogen status of the infecting fungus has received considerable attention. The aim of this study was further to characterize how fungal phytopathogens acquire nutrients in planta. A comprehensive review of peptide transporters has been previously undertaken [22]. The review described how, peptide transporters could be grouped into two distinct families based predicted protein sequences. The first of these belong to the ATP-binding cassette (ABC) family. These are predominantly of prokaryotic origin and are usually composed of multiple proteins with one or more components that are members of the ABC family [23]. They require ATP hydrolysis for energy and have the ability to take up peptides up to six amino acids in length. To date, no ABC type peptide transporters have been described from eukaryotes. More recently, a second group of peptide transporters have been identified originating from mammals, plants and yeasts. The transport of di/tripeptides in these eukaryotic systems is Hþ-dependent and Naþindependent, and in contrast to the prokaryotic systems, is not energetically coupled to ATP hydrolysis for energy. Further, these eukaryotic transporters appear to take up only di/tripeptides. Sequence analysis of this second group revealed a well-conserved motif, FYXXINXGSL, which is unique to this group. Examination of the Ptr2 sequence has identified the sequence FYFCINVGCL at position 264 – 274 which, nearly perfectly matches the consensus sequence. The predicted amino acid sequence had significant similarity (41%) with the S. cerevisiae gene PTR2 and we therefore named the S. nodorum gene Ptr2. Database searches revealed similar sequences in Schizosaccharomyces pombe (PTR2) and Arabidoposis thalania (Pt2A), Candida albicans, Magnaporthe grisea, Neurospora crassa and various animals. To gain further insight into the function of the peptide transporter and to determine its requirement during infection, the gene was inactivated by homologous recombination with an insertional mutation construct. A novel method was used to generate the construct whereby a transposon containing a selectable marker was transposed into a cDNA clone. The cDNA containing the transposon could then be introduced into the genome by homologous recombination thus rendering the gene inactive. Traditional methods of developing inactivation constructs have typically involved isolating genomic clones, mapping restriction sites and then either removing part of the gene of interest and replacing it with a selectable marker or simply inserting the marker in the gene thus disrupting translation. The method employed in this study was considerably quicker. The size of the homologous flanking sequence surrounding the marker is often the limitation to generating an adequate number of successful transformants [24]. Many fungal 3.8. Pathogenicity assay The ability of the transformants to cause disease was first assessed using a detached leaf assay. Leaves on benzimadazol plates were inoculated with 105 spores each of S. nodorum SN15, SNPtr1, SNPtr3 and SNPtr7, and incubated using conditions described in Section 2. Microscopic analysis of germination and growth on the surface of the leaf revealed no obvious differences between the four strains inoculated. At 6 days post inoculation, the lengths of the necrotic lesions were measured with all strains appearing to cause the same size lesions (results not shown). Further microscopic analysis revealed the lesions caused by the four strains to be identical. The infections were allowed to continue sporulation where no significant difference was obvious in lesion size or spore numbers produced by the four strains. The transformants were also tested for their ability to cause disease on intact wheat seedlings. A spore suspension containing 106 spores per ml was sprayed onto 2-week-old wheat plants and grown under conditions described in Section 2. The plants were then scored blindly for disease after 7 days. Both the Ptr2 inactivated strains and the ectopic mutant were found to be fully pathogenic suggesting that the inactivation of Ptr2 did not affect pathogenicity (results not shown). Fig. 6. Analysis of Ptr2 expression during infection. The experiment was performed in duplicate on two independent RNA populations. 198 P.S. Solomon et al. / Physiological and Molecular Plant Pathology 63 (2003) 191–199 phytopathogens, such as M. grisea and C. fulvum require up to 10 kb of homologous sequence. In this study, 2 kb of homologous sequence used resulted in a total of 37 transformants recovered. Of these, approximately 65% appeared to have undergone homologous single copy integrations. The generation of S. nodorum strains lacking the Ptr2 gene provided the opportunity to examine the requirement for the gene for S. nodorum to utilize di/tripeptides. The uptake of di/tripeptides has been well characterised in yeast through the use of toxic dipeptide analogues [9,10,25]. Unfortunately these toxic analogues are not available commercially and Ptr2 was characterised by examining the ability of the mutant to grow on dipeptides as sole nitrogen sources. The results revealed that the Ptr2 inactive S. nodorum strains grew extremely poorly compared to the wild type when growing on Arg-Leu and Glu-Glu, whilst growth on nitrate was not affected. The lack of growth was particularly striking during the first days of growth when virtually no growth measurable. This strongly indicates that Ptr2 is responsible for the uptake of dipeptides and that no other specific uptake system exists in S. nodorum. After 4 days, some limited growth was observed on the dipeptides. Given the extremely poor rate of growth after 4 days, it is unlikely that it is due to a second dipeptide transporter but more probably the presence of secreted peptidases and promiscuous amino acid transporters. These results, along with only one band observed in the low stringency Southern blot, are consistent with what is observed in yeast, which has only one gene for di/tripeptide uptake. In contrast, higher eukaryotes contain at least two genes for di/tripeptide uptake [11 –18]. It has been demonstrated previously that the expression of some fungal genes, including genes required for virulence, is influenced by the nutritional state [1 – 4]. Consequently, the expression of Ptr2 was examined under different in vitro starvation conditions. No significant difference was observed in expression where the fungus was starved of carbon or starved of carbon and nitrogen. However a large increase in expression was observed when the fungus was starved of nitrogen. This was consistent with the fact the Ptr2 gene used for this study was isolated from a cDNA library cloned from S. nodorum growing on wheat cell walls, presumably carbon rich but very little nitrogen. Unfortunately no expression analysis has been done on PTR2 in yeast growing under starvation conditions so comparisons are not possible. Analysis of Ptr2 expression during infection revealed that the gene is up-regulated during the first 24 h of infection. After this, the expression quickly declines to lower constitutive levels for the remainder of the infection. Initially it was thought that the increased expression observed at the beginning of the infection maybe due to the very low levels of actin present in the samples, however, the expression of several other genes has since been analysed with these cDNA samples with none demonstrating high relative expression at the initial stages of the infection. The up-regulation of the di/tripeptide transporter at the beginning of the infection was not unexpected. During this period, the fungus has germinated on the leaf and has begun penetrating the surface. It is during this phase of the infection cycle that the fungus would be predominantly reliant on intracellular stores prior to gaining access to the relatively nutrient-rich intracellular environment. Consequently, it would also be likely that other forms of transporters would be up-regulated in an attempt to scavenge any possible nutrient source available. After the fungus has successfully penetrated, it would have access to the probable vast array of nutrients present within the leaf and consequently would have no need to expend energy transporting less preferred nutrient sources such as peptides. This was depicted in the lower constitutive level of Ptr2 expression observed throughout the remainder of the infection. The idea that the expression is up-regulated during the beginning of infection due to starvation conditions also compares well with the in vitro expression results which imply that during the initial stages of infection nitrogen may be limiting whilst carbon is not. The generation of an S. nodorum strain lacking Ptr2 also provided the opportunity to determine the requirement of di/tripeptide uptake for S. nodorum pathogenicity. Interestingly, even though the expression of Ptr2 was significantly up-regulated during the initial stages of infection, the inactivation of the gene had no effect on the ability of the fungus to infect wheat leaves. Wheat plants infected with the wild type, the ptr2 mutants and an ectopic strain were all scored nearly identically towards the end of infection. One thought was that maybe as Ptr2 expression was up-regulated during the initial stages of infection, then maybe Ptr2 is more critical at this time, and consequently, pathogen development could be hindered during this period in the mutant compared to the wild type. Microscopic analysis of both mutants and of the wild type during the early stages of infection revealed though that this was not the case and the growth of the mutants appeared no different compared to the wild type. 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