Chromosome-based molecular characterization of pathogenic and non-pathogenic wheat isolates of Pyrenophora tritici-repentis

Fungal Genetics and Biology 37 (2002) 180–189 www.academicpress.com

Chromosome-based molecular characterization of pathogenic and non-pathogenic wheat isolates of Pyrenophora tritici-repentis Amnon Lichter,1 Janey M. Gaventa, and Lynda M. Ciuffetti* Department of Botany and Plant Pathology, Oregon State University, 2082 Cordley Hall, Corvallis, OR 97331-2902, USA Received 19 October 2001; accepted 22 May 2002

Abstract The ToxA gene of Pyrenophora tritici-repentis encodes a host-selective toxin (Ptr ToxA) that has been shown to confer pathogenicity when used to transform a non-pathogenic wheat isolate. Major karyotype polymorphisms between pathogenic and nonpathogenic strains, and to a lesser extent among pathogenic strains, and among non-pathogenic strains were identified. ToxA was localized to a 3.0 Mb chromosome. PCR-based subtraction was carried out with the ToxA chromosome as tester DNA and genomic DNA from a non-pathogenic isolate as driver DNA. Seven of 8 single-copy probes that originated from the 3.0 Mb chromosome could be assigned to a 2.75 Mb chromosome of a non-pathogenic isolate. Nine different repetitive DNA probes originated from the 3.0 Mb chromosome, including sequences that correspond to known fungal transposable elements. Two additional single-copy probes that originated from a 3.4 Mb chromosome were unique to the pathogens and they correspond to a peptide synthetase gene. Our findings suggest substantial differences between pathogenic and non-pathogenic isolates of P. tritici-repentis. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Karyotype; ToxA; Pyrenophora; Ptr necrosis toxin; Ptr toxin; Host-selective toxins

1. Introduction Pyrenophora tritici-repentis (Died.) Drechs. (syn. P. trichostoma (Fr.) Fckl.), anamorph: Helminthosporium tritici-repentis (syn. Drechslera tritici-repentis (Died.) Shoem.), causal agent of tan spot of wheat, is a serious disease of worldwide and economic importance. The complex race structure and symptoms (Lamari et al., 1995) elicited by P. tritici-repentis suggest that this fungus produces multiple host-selective toxins (HSTs). This has been supported by our work and that of others which has, thus far, resulted in the identification and characterization of two HSTs produced by this fungus, Ptr2 ToxA (Ballance et al., 1989; Tomas et al., 1990; Tuori et al., 1995), (syn. Ptr necrosis toxin, Ptr toxin, ToxA) (Ciuffetti et al., 1998), and Ptr ToxB (Orolaza *

Corresponding author. Fax: 1-541-737-3573. E-mail address: ciuff[email protected] (L.M. Ciuffetti). 1 Present address: Department of Postharvest Sciences, ARO the Volcani Center, Bet Dagan, POB 6, 50250 Israel. 2 Abbreviations used: Ptr, Pyrenophora tritici-repentis; CHEF–PFGE, clamped homogeneous electric field–pulsed-field gel electrophoresis.

et al., 1995; Strelkov et al., 1998), (syn. Ptr chlorosis toxin) (Ciuffetti et al., 1998). Recently, Effertz et al. (2002) partially purified a third host-selective toxin produced by P. tritici-repentis and designated this toxin, Ptr ToxC (syn. Ptr chlorosis toxin). Ptr ToxC appears to be a polar, non-ionic, low-molecular weight molecule (Effertz et al., 2002). Based on race complexity, it is likely that this fungus produces additional toxins. Also, it is clear that some isolates produce more than one toxin (Effertz et al., 2002; Tuori et al., 1995). In contrast to most other host-selective toxins (HSTs) (Walton, 1996; Wolpert et al., 2002), the characterized HSTs (Ptr ToxA and Ptr ToxB) produced by P. tritici-repentis are proteins. Because the ToxA locus is a simple gene, we were able to demonstrate through transformation of a non-pathogenic, Ptr ToxA
isolate that expression of ToxA is both necessary and sufficient for pathogenesis on the toxin-sensitive wheat cultivars tested (Ciuffetti et al., 1997). Southern analysis indicated that ToxA is absent from Ptr ToxA
isolates of P. tritici-repentis (Ballance et al., 1996; Ciuffetti et al., 1997), a finding consistent with the molecular genetic analyses of other HST-

1087-1845/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 7 - 1 8 4 5 ( 0 2 ) 0 0 5 0 0 - 5

A. Lichter et al. / Fungal Genetics and Biology 37 (2002) 180–189

producing fungi (Ahn and Walton, 1996; Yang et al., 1996). We were interested in conducting comparative analyses of tox
(non-pathogenic, Ptr ToxA
) and toxþ (pathogenic, Ptr ToxAþ ) isolates of P. tritici-repentis in an effort to gain insights into the possible origin(s) of toxin production and consequently, the evolution of pathogenic potential. With the exception of toxin production (and consequently, pathogenesis) and the presence of the ToxA gene, toxþ and tox
isolates appear morphologically identical. It was of interest to address the following questions: 1. Do the isolates have different karyotypes; 2. Does the chromosome that contains ToxA have a similar counterpart in the non-pathogenic isolates; 3. If so, would ToxA define the only molecular difference between the homologous chromosome of the pathogenic and nonpathogenic isolates? As will be discussed below the answers to these questions are, for the most part, positive but complex. Subtraction hybridization (Lisitsyn and Wigler, 1995) as modified by Lichter and Mills (1997) was employed to generate chromosome-specific probes linked to ToxA. By Southern hybridization it can be concluded that most of the genetic content of the chromosome that harbors ToxA is contained within a smaller chromosome of the non-pathogenic isolate. In addition, many unique and repetitive genetic components make up differences between the pathogenic and non-pathogenic isolates throughout the genome.

2. Materials and methods 2.1. Fungal strains P. tritici-repentis, Pt-1C-BFP (Ptr ToxAþ ) (Tuori et al., 1995) and SD20 (Ptr ToxA
) (Ciuffetti et al., 1997) were used for molecular analysis of the differences between pathogenic and non-pathogenic isolates. Other pathogenic isolates that were used in this study were MA1, MA2, SD2, SD3, SD8, SD15, SD16, and the nonpathogenic isolates SD13 and SD11. Pt-1C was obtained from William Bockus, Kansas State University, MA1 and MA2 were collected in Oregon (Putnam and Ciuffetti, unpublished), and the SD isolates were obtained from Gary Buchenau, South Dakota State University. Conidial suspensions and mycelial production in liquid culture were as described in Tuori et al. (1995). 2.2. DNA extraction For DNA extractions, cultures were grown in modified Fries medium at room temperature in circulation at 120 rpm. After 24 h, mycelium was collected on miracloth, washed with water, lyophilized, frozen with liquid nitrogen and ground with mortar and pestle. Efficient extraction of fungal DNA was hampered by polysac-

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charide contamination and the problem was circumvented using the Easy-DNA genomic DNA isolation kit (Invitrogen, Carlsbad, CA). Typical yields were 1–2 lg DNA/mg of ground mycelia. 2.3. Karyotype analysis Spheroplasts for CHEF–PFGE analysis were prepared as described in Ciuffetti et al. (1997). Embedding and lysis of spheroplasts was essentially as described in Miao et al. (1991b). Spheroplasts were embedded in low-gelling-temperature agarose at ca. 108 /ml. Electrophoresis was performed on the CHEF DR II system (BIO-RAD, Hercules, CA) with 0.5 Tris Borate EDTA (TBE) buffer cooled to 14 °C and 1% agarose (BIO-RAD, ultra-pure DNA grade). Initially, the different isolates were separated using the following parameters defined as Method I: 0.8% agarose, voltage of 60 for 120 h with a pulse length of 20 min, followed by a voltage of 70 for 24 h with a pulse length of 680–220 s. For optimal separation of chromosomes at the 2.5–3 Mb range the following parameters were adopted from Mills et al. (1995): 1% agarose, pulse length of 960–480 s over 96 h; initial voltage of 75 V with 5 V increments at 24 h intervals (Method II). Chromosome plugs of H. wingii and S. pombe (BIO-RAD) were used as size markers. Chromosomal DNA was eluted from the gel with the QIAex II kit (Qiagen, Valencia, CA) after in-gel digest with the restriction endonuclease TaqI according to the manufacturerÕs recommendations (New England Biolabs, Beverly, MA). 2.4. Construction of chromosome-specific subtraction library An agarose plug containing the 3.0 Mb chromosome of BFP that was identified to harbor ToxA, was isolated from a CHEF gel and defined as the tester DNA source. After its in-gel digestion with TaqI (New England Biolabs), the DNA was isolated by the QIAex II extraction kit and the products were further digested to completion with TaqI. Driver DNA was prepared by TaqI digestion of genomic DNA of the Ptr ToxA
isolate, SD20. Purified fragments from the tester and driver DNAs were amplified as described (Lichter and Mills, 1997) in reference to the RDA technology (Lisitsyn and Wigler, 1995). Briefly, the DNAs were ligated to an adaptor and amplified with a corresponding primer. After adaptor removal by TaqI, the tester DNA (5 ng) was combined with the driver DNA (10 lg). Amplification of self-annealed tester DNA was performed in two stages, which included an intermediate digest of single-strand sequences and imperfectly matched DNA hybrids with Mung Bean nuclease (New England Biolabs) (10 U) for 1 h. After heat-inactivation of the enzyme, the reaction products were purified with a Wizard column (Promega,

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Madison, WI). Amplification of subtracted tester DNA was followed by BamHI digestion and ligation of the products to pBluescript, which had been linearized with BamHI and dephosphorylated with Shrimp alkaline phosphatase (Amersham Pharmacia Biotech, Piscataway, NJ). Ligates were electroporated into XL-Blue cells (Stratagene, La Jolla, CA).

Computer Group. Sequence data was submitted to GenBank and given the following accession numbers: 2541-AZ877699; 2542-AZ877698; 1221-AZ877703; 1222-AZ877702; 2991-AZ877701; 2162-AZ877700; 2163AZ877705; 1063-AZ877704; 1061-AZ15846.

3. Results 2.5. Differentiating between abundant and non-abundant cloned DNA fragments The tester DNA is comprised of single copy sequences and repetitive DNA, the latter of which may account for a large proportion of the chromosome. Thus, a probe composed of the tester DNA contains repetitive DNA, which is expected to provide a strong signal compared to non-abundant sequences that should fail to yield a signal above a certain detection limit. Three hundred clones from the subtraction library were lifted onto nylon membranes (Schleicher and Schuell, Keene, MA) according to the manufacturerÕs recommendations. Approximately 100 ng of tester DNA was labeled with [32 P] dCTP (DuPont NEN, Boston, MA) using the DECAprimer II DNA labeling kit (Ambion Inc. Austin, TX). Colony hybridization was performed in 0.25 M Na2 HPO4, pH 7.2; 7% SDS with 1% blocking powder (Schleicher and Schuell) at 65 °C and differential signal intensity was used to identify clones with single copy and repetitive DNA. 2.6. Hybridization analyses Fungal DNA (5–7 lg) was digested with the restriction endonucleases BamHI or PvuII and electrophoresis was conducted in 1.2% agarose and 20 cm-long gels. Gels were blotted onto a GeneScreen Plus nylon membrane (NEN) after upward alkaline transfer with 0.4 N NaOH. DNA probes (10 ng) were labeled and hybridized to the blots as described for colony hybridization. Each probe was also assayed to determine its chromosomal origin by hybridization to CHEF gel blots. A ToxA probe was made of a 1 kb fragment from the plasmid pCT53 (Ciuffetti et al., 1997) after BamHI and HindIII digestion and isolation from an agarose gel with the QIAquick kit (Qiagene). A telomere probe was made of a HindIII–EcoRI digest of the plasmid pNL17 (Kistler and Benny, 1992). Plasmid DNA from the subtraction process was prepared for sequence analysis by purification of alkali DNA minipreps using the QIAex II kit (Qiagen). Sequencing was preformed at the Central Services Laboratory, Center for Gene Research and Biotechnology (Oregon State University, Corvallis, OR). Database searches were performed using BlastX analysis (default mode) at the NCBI site and data analysis was performed using the GCG 9.0 software package of the University of Wisconsin Genetic

The ability to transform a non-pathogenic isolate of P. tritici-repentis with the ToxA gene and to create a transgenic strain that displays the typical tan spot symptoms (Ciuffetti et al., 1997) raised interest in the genomic and chromosomal differences between the isolates. Morphologically, Ptr ToxA
isolates are indistinguishable from the Ptr ToxAþ isolates, each has the typical snake-head structure of the conidium. To compare these isolates, their karyotypes were analyzed and the chromosome that contains the ToxA gene was subjected to molecular analyses. 3.1. Karyotypes of Pyrenophora tritici-repentis The karyotype of 6 Ptr ToxAþ isolates and 3 nonpathogenic isolates was determined by CHEF gel analysis with special attention to the karyotype of strains BFP (Ptr ToxAþ , pathogenic) and SD20 (Ptr ToxA
, non-pathogenic (Fig. 1, BFP (+) and SD20 ()) are compared side by side in panels C and D). Major size differences were observed between chromosomes of the pathogenic and non-pathogenic isolates and to a lesser extent among the pathogenic isolates. The extent of variability between Ptr ToxAþ and Ptr ToxA
isolates made it impossible to assign homologies between chromosomes according to their size. Chromosomal sizes (in Mb) were determined from different gels as compared to the chromosomes of H. wingii and S. pombe. The chromosomes of BFP were assigned the following sizes: 3.4, 3.15, 3.0, 2.85, 2.4, 2.15, and 2 (data compiled from Fig. 1 and additional CHEF gels). The size of the largest chromosome in Figs. 1A–C, which does not appear in Fig. 1D, could not be determined. The 2.0 Mb complex band (Fig. 1D) corresponds to 2 or 3 chromosomes in the range of 2.05–2.15 Mb, as indicated from the intensity and width of the band. Attempts to assess the number of chromosome ends within the 2.0 Mb complex of BFP were made by extracting the DNA from the gel plug and digesting it with endonucleases. Corresponding Southern blots were probed with the telomere probe of Fusarium oxysporum (Kistler and Benny, 1992) but these attempts failed, because of surprisingly low restriction size variability for 6 restriction endonucleases (not shown). Chromosomal sizes for SD20 are: 3.5, 3.2, 2.75, 2.6, 2.35, 2.15, 1.9, and 1.1. As for BFP, the largest chromosome of SD20 (Fig. 1A, lane 5) is not always visible (e.g., Fig. 1D, ‘‘)’’ lane). The wide 3.2 Mb band of SD20

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Fig. 1. Karyotype analysis of P. tritici-repentis and identification of the chromosome that contains ToxA. (A) CHEF gel analysis of chromosomes from selected isolates: pathogenic isolates SD2, SD8, BFP, and PTR4 (lanes 1–4), and non-pathogenic isolates, SD20 and SD13 (lanes 5–6). (B) CHEF gel (stained with ethidium bromide, left) and Southern blot analysis (right) of the pathogenic isolates SD2, SD3, and SD15 (lanes 1–3). The membrane was probed with the ToxA gene. (C) CHEF gel (left) and Southern blot analysis (right) of the pathogenic and non-pathogenic isolates, BFP and SD20 (designated as ‘‘+’’ and ‘‘),’’ respectively) hybridized with a telomere probe (Kistler and Benny, 1992). (D) CHEF gel (left) and Southern blot analysis (right) of isolates BFP and SD20 probed with ToxA. The electrophoretic conditions for panels A and B were as specified for method I in Section 2 and method II for panels C (67 h) and D (96 h). Hw and Sp denote chromosomes of Hansenula wingei and Schizosaccharomyces pombe, respectively. The sizes of H. wingei chromosomes are specified in panels A and C and the sizes of the chromosomes of S. pombe in panel B are 5.7, 4.8, and 3.5 Mb (top to bottom).

(Fig. 1D, ‘‘)’’ lane) may correspond to 2 chromosomes. The intensity of the 1.9 Mb band in the same lane also indicates the presence of more than one chromosome. Note the presence of a 1.1 Mb chromosome that was detected only in karyotype of SD20. CHEF gel blot hybridization with the telomere probe yielded signal with all the chromosomes of BFP and SD20 (Fig. 1C). Size variation between the Ptr ToxA
isolates, SD20 and SD13, was observed for the 3.2 Mb complex of SD20 (Fig. 1A, lanes 5 and 6, respectively). Hybridization of a ToxA probe to Southern blots of CHEF gels demonstrated that ToxA resided on a 3.0 Mb chromosome of isolates SD2, SD3 and BFP (Fig. 1B, lanes 1–2 and Fig. 1D, +1, respectively) and a slightly larger chromosome of SD15 (Fig. 1B, lane 3). To create a chromosome-specific subtraction library from the 3.0 Mb chromosome containing ToxA, it was necessary to further separate it from the 2.85 Mb band of BFP (Fig. 1D, ‘‘+’’ lane). These separation conditions confirm that ToxA resides on the 3.0 Mb chromosome of BFP.

3.2. Subtraction of the 3.0 Mb chromosome with DNA from a Ptr ToxA
isolate The absence of ToxA in non-pathogenic wheat isolates of P. tritici-repentis could result from a gene deletion or the loss of a B chromosome (Miao et al., 1991a; Mills and McCluskey, 1990) in a pathogenic isolate, or from an acquisition event during the evolution of the pathogen. Thus, the genes that reside on the 3.0 Mb chromosome could be either absent in non-pathogenic strains, scattered among different chromosomes, or located on a partially homologous chromosome. One way of discerning among these possibilities was to generate a chromosome-specific library of the 3.0 Mb chromosome and compare the distribution of unique sequences among the chromosomes of the non-pathogenic strain. One problem with this approach is the presence of repetitive DNA sequences in the chromosome, which hampers attempts to isolate unique sequences that are used as chromosomal markers (Mills and McCluskey, 1990). Another interesting question was whether the 3.0 Mb

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ToxA chromosome contained additional pathogen-specific genes. To help resolve these questions a DNA subtraction method was employed between fragments of the 3.0 Mb chromosome and the whole genome of a nonpathogenic isolate. Subtraction was carried out for one instead of the three rounds typically used in order to retain single-copy homologous sequences that were essential for mapping a putative homologous chromosome in Ptr ToxA
isolates. Amplicons made of the 3.0 Mb chromosome were subtracted with driver amplicons made of SD20 DNA at a ratio of 1:200. Southern blot analysis was performed with ToxA as a probe and hybridized to TaqI digests from amplified driver and tester DNAs, as well as subtracted products cleaved with BamHI. The subtraction products were cloned as BamHI fragments into pBluescript and colony hybridization of 300 clones with tester DNA as a probe was performed in order to discern between low and high copy number DNA. Approximately 30% of the clones tested displayed strong or medium signals, whereas the remaining 70% of the clones displayed low or no signals (Table 1). Upon stripping and re-probing with ToxA, one of 300 colonies yielded a positive signal (results not shown). 3.3. Probes that identified single copy loci Of 22 probes that were selected for further analysis (Table 1), 11 corresponded to single copy loci, as determined by Southern blot hybridizations with DNA of Table 1 Summary of the products obtained from the subtraction process between amplicons of the 3.0 Mb chromosome (ToxA chromosome) and amplicons made of the genome of a non-pathogenic strain Analysis of the subtraction products Number of clones tested after subtraction Strong signal Medium signal Low or no signal ToxA positive

300 32/200a 26/200 141/200 1/300

Single copy probes Non-polymorphic Polymorphic Verified to originate from the 3.0 Mb chromosome Mapped to the 2.75 Mb chromosome of SD20 Present only in the pathogenb

11 7 2 8 7 4

Repetitive probes Redundantc Exclusive to Ptr ToxAþ 1–2 copies in Ptr ToxA
Low copy number in Ptr ToxA
, as compared to Ptr ToxAþ

11 2 1 2 6

a

Of the 300 clones analyzed, 200 clones were used for calculation of signal intensity. b Two of the 4 probes were not derived from the 3.0 Mb chromosome of BFP. c Redundant probes displayed identical pattern in Southern blot analyses.

BFP (Ptr ToxAþ ). Seven of the 11 probes (e.g., probes 1221 and 123, Fig. 2) showed an identical restriction pattern with SD20 (Ptr ToxA
) DNA and 2 probes identified a polymorphic pattern for at least one restriction site (probes 287 and 1222, Fig. 2). Eight singlecopy probes were verified to originate from the 3.0 Mb chromosome of BFP and 7 of these probes mapped to a 2.75 Mb chromosome of SD20 (Fig. 3). These results suggest that, for the most part, the 2.75 Mb chromosome of SD20 should be considered homologous to the 3.0 Mb chromosome of BFP. Three of the 11 probes identified pathogen-specific loci but only one of the three (2162) originated from the 3.0 Mb chromosome (Figs. 2 and 3) and this probe hybridized weakly to the 2.35 Mb chromosome of SD20 (Fig. 3) and to restricted fragments of SD20 (Fig. 2). 3.4. Probes that identified repetitive loci The subtraction process was expected to enrich for repetitive DNA that was unique to the tester chromosome or strain. From this point of view it is not entirely surprising that the repetitive probes were either absent or present at a reduced number in the non-pathogenic isolate SD20 (Fig. 4). Of the 11 repetitive sequences that were analyzed, two were redundant and none of the 9 probes showed identical pattern for BFP (Ptr ToxAþ ) and SD20 (Ptr ToxA
). Of the probes shown in Fig. 4, one probe (2541) did not hybridize to DNA of SD20, three probes (284, 267, and 1061) were present at single or low copy number or hybridized with low intensity to DNA of SD20. Probe 2991 showed a partially similar pattern with low abundance in SD20, whereas probe 263 was present at intermediate copy number and displayed a polymorphic-low intensity pattern. None of the re-

Fig. 2. Southern blot analyses of selected single copy probes derived from the 3.0 Mb chromosome of the Ptr ToxAþ isolate of P. triticirepentis, BFP. Genomic DNA prepared from the Ptr ToxAþ isolate, BFP (+) or the Ptr ToxA
isolate, SD20 ()) was digested with the restriction endonucleases, PvuII (P) and BamHI (B). A number identifies the probe used in each panel. A size marker (Kb) is aligned to the left panel.

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Fig. 3. Southern blot analyses of CHEF gels with selected single copy probes derived from the 3.0 Mb chromosome of the Ptr ToxAþ isolate of P. tritici-repentis, BFP. CHEF-gel blots corresponded to chromosomes of the Ptr ToxAþ isolate, BFP (+) or the Ptr ToxA
isolate, SD20 ()). A number identifies the probe used in each panel. The calculated sizes (in Mb) of chromosomes hybridizing with the probes are denoted to the left.

Fig. 4. Southern blot analyses of selected multiple copy probes derived from the 3.0 Mb chromosome of the Ptr ToxAþ isolate of P. triticirepentis, BFP. Genomic DNA prepared from the Ptr ToxAþ isolate, BFP (+) or the Ptr ToxA
isolate, SD20 ()) was digested with the restriction endonuclease PvuII. A number identifies the probe used in each panel. A size marker (in Kb) is aligned to the left panel.

petitive probes hybridized exclusively to the 3.0 Mb chromosome of BFP; rather they hybridized to most if not all of the chromosomes of BFP (Fig. 5). In contrast, probe 2991 mapped only to the 2.75 Mb chromosome of SD20 and probe 2542 hybridized weakly to 3 chromosomes (2.35, 2.75, and 3.2 Mb). Hence, the repetitive DNA gives the impression of marked differences between pathogenic and non-pathogenic isolates while single copy probes imply much more similarity.

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Fig. 5. Southern blot analyses of selected multiple copy probes derived from the 3.0 Mb chromosome of the Ptr ToxAþ isolate of P. triticirepentis, BFP. CHEF-gel blots corresponded to chromosomes of the Ptr ToxAþ isolate, BFP (+) or the Ptr ToxA
isolate, SD20 ()). A number identifies the probe for each panel. The sizes of chromosomes hybridizing with the probes are denoted to the left of the first panel to identify chromosomes of BFP or to right of the last panel, to identify chromosomes of SD20. A denotes the high molecular weight chromosome of BFP and 2C corresponds to a complex of chromosomes of BFP ranging between 2.05 and 2.15 Mb.

Fig. 6. Southern blot analyses of selected probes derived from the 3.0 Mb chromosome of the Ptr ToxAþ isolate of P. tritici- repentis, BFP. Genomic DNAs were prepared from the Ptr ToxAþ isolates MA1, MA2, SD16, SD3, SD8, and BFP (lanes 1–6, respectively) and the Ptr ToxA
isolates, SD20, SD13, and SD11 (lanes 7–9, respectively). The DNA was digested with the restriction endonuclease, PvuII. The blots in panels A and B were probed with two different probes each: ÔaÕ denotes the signal generated by a ToxA probe, ÔbÕ denotes the signal generated by probe 1221, ÔcÕ denotes the signal generated by probe 1222, and ÔdÕ denotes the signal generated from probe 1063. The blot in panel C was probed with probe 2991. Size reference is aligned to the left of panel A.

3.5. Comparative analysis of selected probes and isolates Selected probes were hybridized to Southern blots containing PvuII-digested genomic DNA of 6 Ptr ToxAþ and 3 Ptr ToxA
isolates. Probe 1221 (signal

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Table 2 Sequence similarities for selected probes used in this study Similarity toa

e-value/scoreb

Accession of target

Fungal a-amylase Zinc finger protein

9e-17/86 0.11/35

S77586 NM076905

Sequence similarity for repetitive probes (to fungal transposons) 2991 AZ877701 Grasshopper (Gypsy-like retroelement) 2542 AZ877698 Maggy (Gypsy-like retroelement) 2541 AZ877699 Tfo1 (Ac-like element) 1061 AZ915846 Tc1/Mariner-like element

0.4/34 5e-8/57 4e-3/40 1.1/33

M77661 L35053 AB008746 Z29098

Sequence similarity for peptide synthetase 2163 AZ877704 1063 AZ877705

2e-7/55 3e-8/57

X98442 X98442

Probe number

Probe accession

Sequence similarity for single copy probes 1221 AZ877703 2162 AZ877700

a b

Fungal peptide synthetase Fungal peptide synthetase

Similarity to the fungal gene with the highest score. Expect e-value/score refer to statistical data obtained following BlastX analysis.

labeled as ÔbÕ, Fig. 6A) yielded a uniform PvuII pattern among all the isolates (lanes 1–6 are pathogenic isolates, lanes 7–9 are non-pathogenic isolates) while ToxA (signal labeled as ÔaÕ, Fig. 6A) displayed a polymorphic pattern among pathogenic isolates (lanes 1–6). Note that two isolates from Oregon (MA1 and MA2, lanes 1 and 2, respectively) had a distinct pattern for ToxA. Another single-copy marker, 1222 (signal labeled as ÔcÕ, Fig. 6B), was absent from isolates SD16 and SD3 (lanes 3 and 4, respectively) and had a different pattern for Ptr ToxA
isolates (lanes 7–9). The probe 1063 (signal labeled as ÔdÕ, Fig. 6B) was non-polymorphic among the Ptr ToxAþ isolates (lanes 1–6) and was absent from Ptr ToxA
isolates (SD20, SD13, and SD11, lanes 7-9). Probe 2991 (Fig. 6C), which was a low copy number repetitive probe, shared 2 common-size bands for all the isolates although pathogenic strains (lanes 1–6) had additional bands and a higher intensity for the lower band of the PvuII digest.

3.6. Sequence similarity of selected probes that originated from the 3.0 Mb chromosome Among the sequenced probes that corresponded to single copy clones, the most significant similarity (60% identity for 43 amino acids) in BLAST analysis was of clone 1221 to fungal a-amylases (Table 2). The sequence of probe 2162 had low similarity to a zinc finger protein. The sequence of clone 2991 had low similarity to a segment of the polyprotein of the retrotransposon Grasshopper from Magnaporthe grisea (Table 2). Another probe, 2542, displayed 60% identity over 42 aa to the retroelement MAGGY from M. grisea or REAL of Alternaria alternata. Probe 2541, which hybridized only to the Ptr ToxAþ isolate, BFP, displayed 34% identity over 44 aa to TfoI, the Ac-like element from Fusarium oxysporum. Probe 1061 hybridized to many bands of Ptr ToxAþ isolate, BFP, but hybridized to only one band of Ptr ToxA
isolate, SD20. It corresponded, by low

Fig. 7. Hybridization with probes corresponding to the putative peptide synthetase of P. tritici-repentis. (A) Southern blot analyses of CHEF gels with probes 2163 and 1063. CHEF-gel blots corresponded to chromosomes of the Ptr ToxAþ isolate, BFP (+) or the Ptr ToxA
isolate, SD20 ()). The calculated sizes (in Mb) of chromosomes of BFP are aligned to the left lane that shows the hybridization pattern of the multicopy probe 2541 with all the chromosomes of BFP. (B) Southern blot analyses of probes 2163 and 1063. Genomic DNA prepared from the Ptr ToxAþ isolate, BFP (+) or the Ptr ToxA
isolate, SD20 ()) was digested with the restriction endonucleases, PvuII. Size reference (in Kb) is aligned to the left panel.

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sequence similarity, to the transposable element Tc1/ Mariner of Drosophila hydei (Table 2). 3.7. Identification of a peptide synthetase gene among the single copy probes Of the 11 single-copy probes that were identified in Southern blot analysis, probes 2163 and 1063 showed sequence similarity to fungal peptide synthetases (Table 2). Both sequences displayed the highest similarity to various segments of the peptide synthetase of Metarhizium anisopliae (Bailey et al., 1996). According to the results shown in Fig. 7A these probes originated from a 3.4 Mb chromosome, which is probably the second largest chromosome of strain BFP (Ptr ToxAþ , Fig. 1). Southern blot analysis of genomic DNA digested with two different restriction endonucleases revealed hybridization with single predominant bands (Fig. 7B). In order to verify that the two probes are part of the same gene it was necessary to demonstrate linkage. The modular structure of peptide synthetases makes it difficult to predict the exact relative space and order between the two probes by homology. Thus, forward and reverse primers were prepared according to the sequence of probes 2163 and 1063. The combinations of primers FP1063 and RP-2163 yielded a fragment of 4.5 kb and sequence analysis of both ends proved that the amplified fragment was part of a peptide synthetase (not shown).

4. Discussion The non-pathogenic isolates of P. tritici-repentis are morphologically indistinguishable from the pathogenic isolates. Moreover, a single gene, encoding the proteinaceous toxin, Ptr ToxA, can transform a non-pathogenic isolate to a pathogen (Ciuffetti et al., 1997). Therefore, it was of interest to determine whether the chromosome that contains the ToxA gene is unique to the pathogenic isolates, and if not, if its genetic content is scattered among the chromosomes of the non-pathogenic strains. Although variation was apparent, electrophoretic karyotype analyses of the pathogenic isolates revealed similarity in the basic pattern. Such findings are consistent with numerous reports in the literature (Frank, 1995; Kistler and Miao, 1992; McCluskey and Mills, 1990; McCluskey et al., 1994; Plummer and Howlett, 1993; Skinner et al., 1991; Zolan, 1995). The localization of ToxA to the 3.0 Mb chromosome suggested that it may be a B-like supernumerary chromosome (Kistler and Miao, 1992; Miao et al., 1991a; Zolan, 1995) that is entirely lacking in the non-pathogenic isolates. In addition, it was of interest to determine if other simple (e.g., deletions) or complex (e.g., multiple polymorphism) differences could be readily identified.

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The advances in PCR technology make it relatively straightforward to amplify the contents of individual chromosomes by adapter ligation (Lichter and Mills, 1997). The amplified DNA can be used for preparation of chromosome-specific libraries and the unique markers generated can be employed to identify homologous chromosomes from related strains by hybridization (McCluskey and Mills, 1990). However, often this objective is hampered by the presence of repetitive DNA, and this can lead to loss of information on the unique composition of the chromosome. For that reason, we chose to take additional efforts to enrich the chromosome-specific library for unique sequences by employing a PCR-based subtraction process (Lichter and Mills, 1997; Lisitsyn and Wigler, 1995). The 3.0 Mb ToxA chromosome was used as the tester DNA and the genome of a non-pathogenic isolate as the driver DNA. However, this process was not driven to completion in order to obtain molecular markers common to both strains to identify their distribution in the non-pathogenic strain. The fact that approximately 30% of the clones after subtraction reacted strongly with a probe made of random fragments from the 3.0 Mb chromosome indicated that our products contained a relatively high proportion of repetitive DNA, yet still contained an abundance of single-copy clones. Both the single copy and repetitive markers that were isolated with the subtraction process were useful for characterizing the differences among the strains. The fact that most of the randomly selected single copy probes were non-polymorphic for two adjacent restriction sites among the pathogenic and non-pathogenic isolates, indicates their relatedness. Of the 11 markers, 7 were shown to originate from the 3.0 Mb chromosome and to hybridize to the 2.75 Mb chromosome in the non-pathogenic isolate. These results suggest that the 2.75 Mb in the ToxA
isolate is the counterpart of the 3.0 Mb chromosome in the ToxAþ isolate and the difference in size could be mediated by deletion or insertion. An indication for such an event may be represented by probe 2162. This probe originated from the 3.0 Mb chromosome of the pathogen but did not have a homolog in the 2.75 Mb chromosome of the non-pathogen. Instead, probe 2162 hybridized weakly to the 2.35 Mb chromosome of SD20 (Fig. 3). The presence of an a-amylase-like gene (probe 1221) in the 3.0 Mb ToxA-chromosome and its non-polymorphic pattern among pathogenic and non-pathogenic isolates, is to be expected among closely related isolates. It represents a marker that escaped subtraction and is present on both the 3.0 Mb and 2.75 Mb chromosomes (not shown). A different example is probe 1222, which is present in non-pathogenic isolates and is lacking in two out of six pathogenic isolates (signal labeled as ÔcÕ, Fig. 6B, lanes 3 and 4). Based on our results, we can determine that substantial portions of the 3.0 Mb

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chromosome are present in the non-pathogenic isolate. However, we have no genetic evidence or correlative data that the 3.0 Mb chromosome is a B-type supernumerary chromosome. It is probably not coincident that all the 11 repetitive probes, analyzed by Southern blot hybridization, were either absent in the non-pathogenic isolate SD20 or present at very low copy number or low similarity (intensity). This is because the subtraction process is more efficient in amplifying multi-copy sequences that are unique to the tester, compared to single-copy sequences. As opposed to the single-copy markers, the repetitive markers reveal major genomic differences between the pathogenic and non-pathogenic strains. These sequences were not confined to the 3.0 Mb chromosome and it is not surprising that most of them were homologous to 4 fungal transposable elements, MAGGY, Grasshopper, Tfo1, and Fot1/Pot2 (Dobinson et al., 1993; Farman et al., 1996; Okuda et al., 1998). These belong to 3 types of transposable elements: retroelements, AC-type, and Tc1/Mariner-like elements. It is noteworthy that the sequence of clone 1061 was similar to the insect transposable element Tc1/Mariner, and only indirectly to the fungal members of this family, Fot1 or Pot2 (Daboussi et al., 1992; Kachroo et al., 1994). Transposable elements have been suggested to be responsible, in part, for karyotype polymorphisms (Daboussi, 1997; Rachidi et al., 1999; Zolan, 1995). It is evident that there is substantial difference between the pathogenic and nonpathogenic isolates in the transposable element content. However, unless a reverse subtraction is performed, one can not conclude that the non-pathogenic isolates contain fewer transposable elements. Two additional pathogen-specific probes were identified among the 11 single-copy probes and these originated from the 3.4 Mb chromosome. The presence of these probes in the 3.0 Mb fraction was probably the result of fragmentation of the larger chromosome. Both probes showed sequence similarity to fungal peptide synthetases, and current data support the notion that they are part of the same gene. Their independent isolation suggests that the subtraction process was effective. Genes for peptide synthetases are typically very large and encode multifunctional enzymes that can link amino acids by a non-ribosomal mechanism. The amino acids may undergo cyclization and unique modifications. The products of these genes may be antibacterial toxins (antibiotics), surfactants, siderophores, immunosuppressive drugs, insect toxins, non-selective and hostselective toxins (Bailey et al., 1996; Challisa and Ravel, 2000; Johnson et al., 2000; Scott-Craig et al., 1992; van Liempt et al., 1989; Weber et al., 1994). The identification of a pathogen-specific peptide synthetase in P. tritici-repentis raises questions of its function. Clearly, it is not essential for Ptr ToxA-induced symptom development because ToxA alone was shown to be a sufficient

pathogenicity determinant on the wheat cultivars tested (Ciuffetti et al., 1997). However, it is possible that the pathogen-specific peptide synthetase could be involved in another cultivar-specific activity. The product of this gene, as well as its function will be explored further. We have investigated genomic differences between pathogenic and non-pathogenic isolates of P. tritici-repentis. We can suggest that the differences between the isolates are more complex than can be explained by a simple molecular event. For example, deletion of ToxA and adjacent sequences could not have led directly to the generation of the non-pathogenic isolates we examined because they also lack the peptide synthetase gene as well as several transposable elements that are scattered throughout the chromosomes. Alternatively, formation of a pathogenic strain due to acquisition of the ToxA gene by a previously non-pathogenic strain requires a compatible genetic source to occupy the same niche and the ability to undergo a reproductive process or a horizontal gene transfer event. Such a simple acquisition event seems unlikely if we assume that the recipient of ToxA was related to the non-pathogenic isolates we analyzed because it could not account for all the differences observed among the strains. Instead, we can only conclude that the capability to transform a nonpathogen to a pathogen with a single gene such as ToxA, does not imply strict genetic relatedness, but rather must reflect the pathogenic potential of the isolate and the gene. Acknowledgments We wish to thank Mr. Blaine Baker for his excellent technical assistance in preparation of the figures and Drs. Dallice Mills and Thomas Wolpert for critical review of the manuscript. This research was supported by a U.S. Department of Agriculture Grant (# 97-353034563). References Ahn, J.H., Walton, J.D., 1996. Chromosomal organization of TOX2, a complex locus controlling host-selective toxin biosynthesis in Cochliobolus carbonum. Plant Cell 8, 887–897. Bailey, A.M., Kershaw, M.J., Hunt, B.A., Paterson, I.C., Charnley, A.K., Reynolds, S.E., Clarkson, J.M., 1996. Cloning and sequence analysis of an intron-containing domain from a peptide synthetaseencoding gene of the entomopathogenic fungus Metarhizium anisopliae. Gene 173, 195–197. Ballance, G., Lamari, L., Bernier, C., 1989. Purification and characterization of a host-selective necrosis toxin from Pyrenophora tritici-repentis. Physiol. Molec. Plant Pathol. 35, 203–213. Ballance, G., Lamari, L., Kowatsch, R., Bernier, C., 1996. Cloning, expression and occurrence of the gene encoding the Ptr necrosis toxin from Pyrenophora tritici-repentis. Mol. Plant Pathol. Available from: . Challisa, G.L., Ravel, J., 2000. Coelichelin, a new peptide siderophore encoded by the Streptomyces coelicolor genome: structure predic-

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