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Keywords:

  • chlorosis;
  • Drechslera tritici-repentis;
  • host-selective toxins;
  • tan spot of wheat;
  • ToxB;
  • Triticum aestivum

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Tan spot, caused by the fungus Pyrenophora tritici-repentis, is an important foliar disease of wheat. The pathogen produces at least three host-specific toxins, including Ptr ToxB, a 6·6 kDa protein that causes chlorosis in sensitive wheat genotypes and is encoded by the ToxB gene. Using quantitative reverse transcriptase (qRT)-PCR, transcript levels of ToxB homologues found in virulent (≡ pathogenic) and weakly virulent (≡ weakly pathogenic) race 5 isolates and an avirulent (≡ nonpathogenic) race 4 isolate of the fungus were compared in conidia, mycelia, and in host tissues after inoculation of resistant and susceptible wheat genotypes. Abundance of the ToxB transcript was greatest in the virulent isolate, followed by the weakly virulent and avirulent isolates, and was positively correlated with the development of chlorosis in the susceptible wheat genotype; transcript was detectable in mycelia and conidia of all isolates tested, and levels peaked at 24 h after inoculation of the race 5 isolates onto resistant and susceptible wheat hosts, before disease symptoms developed. Although all isolates could penetrate host epidermal cells to varying degrees, transcript abundance was positively correlated with the greater and more rapid development of appressoria. Therefore Ptr ToxB may have other roles in the basic pathogenic fitness of the fungus and/or in pre-penetration processes, suggesting that quantitative variation in the virulence of P. tritici-repentis is related to the extent of ToxB gene expression.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Tan spot, caused by the homothallic ascomycete Pyrenophora tritici-repentis (anamorph: Drechslera tritici-repentis), is an important foliar disease of wheat throughout the major wheat growing regions of the world. The pathogen induces two distinct symptoms on susceptible wheat genotypes: tan necrosis and/or extensive chlorosis. Isolates of P. tritici-repentis were initially classified into four pathotypes based on their ability to induce these symptoms on specific wheat differential lines (Lamari & Bernier, 1989b). However, the pathotype classification system allowed for a theoretical maximum of only four groups (pathotype 1: nec+chl+, pathotype 2: nec+chl, pathotype 3: necchl+, and pathotype 4: necchl) and was later extended to accommodate isolates that induced the same symptom on different wheat lines/cultivars (Lamari et al., 1995). A race classification system was adopted, which was based on the reaction of isolates on individual wheat genotypes (Lamari et al., 1995). This system can accommodate any number of races and is limited only by the uniqueness and number of the differential wheat genotypes used (Lamari et al., 1995). To date, eight races of the pathogen have been identified according to their virulence on three effective wheat differentials, namely line 6B365, line 6B662, and cv Glenlea (Lamari & Bernier, 1989b; Lamari et al., 1995, 1998; Strelkov et al., 2002; Lamari et al., 2003).

The development of necrosis and chlorosis symptoms on affected wheat leaves results from the differential production of host-specific toxins (HSTs) by isolates of P. tritici-repentis. At least three HSTs are produced, termed Ptr ToxA, Ptr ToxB and Ptr ToxC, all of which appear to be pathogenicity factors (Lamari & Bernier, 1989c; Orolaza et al., 1995; Effertz et al., 2002). In addition, there are preliminary reports of the production of at least two other toxins by the pathogen (Ali et al., 2002; Manning et al., 2002). Ptr ToxA (≡ Ptr toxin, Ptr necrosis toxin, and ToxA [Ciuffetti et al., 1998]) is a 13·2 kDa protein (Ballance et al., 1989; Tomás et al., 1990; Tuori et al., 1995; Zhang et al., 1997) that induces necrosis on sensitive wheat genotypes. Ptr ToxB (≡ Ptr chlorosis toxin [Ciuffetti et al., 1998]) induces chlorosis on sensitive wheat lines/cultivars, and is also a protein, with a 6·61 kDa mass (Strelkov et al., 1999). Ptr ToxC, which has only been partially characterized and appears to be a low molecular mass molecule, also induces chlorosis, but not on the same wheat genotypes as Ptr ToxB (Effertz et al., 2002).

The genes encoding Ptr ToxA and Ptr ToxB, termed ToxA and ToxB, respectively, have been cloned and characterized by several independent research groups (Ballance et al., 1996; Martinez et al., 2001; Strelkov & Lamari, 2003; Martinez et al., 2004; Strelkov et al., 2006). However, while ToxA is found as a single copy only in isolates of P. tritici-repentis that produce Ptr ToxA (Ballance et al., 1996; Ciuffetti et al., 1997; Lamari et al., 2003), ToxB is a multiple copy gene, forms of which are also found in isolates that lack Ptr ToxB toxin activity (Strelkov & Lamari, 2003; Martinez et al., 2004; Strelkov et al., 2006). Thus, while the ToxB gene has been identified in isolates representing races 5, 6, 7 and 8 of P. tritici-repentis, all of which produce chlorosis on the Ptr ToxB-sensitive wheat line 6B662, homologues of the gene have also been found in race 3 and 4 isolates, which possess no toxin activity (Lamari et al., 2003; Martinez et al., 2004; Strelkov et al., 2006). In race 4, the ToxB homologue (referred to as toxb by Martinez et al. [2004]) occurs as a single copy, and independent analysis of this gene from two different isolates indicated that it possesses only 86% similarity to ‘wild-type’ToxB from race 5 isolates (Strelkov & Lamari, 2003; Martinez et al., 2004). However, the role of ToxB homologues in race 4 isolates is not clear, since Ptr ToxB does not appear to control vital biological functions in P. tritici-repentis, aside from its role in pathogenesis (Strelkov & Lamari, 2003).

Martinez et al. (2004) reported that the form of ToxB (toxb) that they identified in a race 4 isolate from North Dakota was not expressed in mycelia under conditions favouring expression of wild-type ToxB in race 5 isolates. Strelkov et al. (2006) confirmed this apparent lack of expression of the gene in mycelia of a race 4 isolate from the Canadian prairies, and provided preliminary evidence that the gene was expressed in conidia of the same isolate. Similarly, Strelkov et al. (2006) also reported that a form of ToxB was expressed in conidia, but not 3- to 9-day-old mycelia, of a Canadian race 5 isolate that had previously been shown to be weakly virulent (Strelkov et al., 2002). However, there are no data available on expression of ToxB and its homologues in planta, and expression of the different forms of the gene has not been quantified or compared under any conditions. Such information is critical to understanding the role of this gene in avirulent isolates of P. tritici-repentis, as well as in quantitative variation in the virulence of the fungus. Hypothetically, differences in the level of ToxB gene expression may contribute to reduced virulence in race 4 and certain race 5 isolates of the fungus. Therefore, the objective of the present study was to examine transcript abundance of the different forms of the ToxB gene in virulent, weakly virulent and avirulent isolates of P. tritici-repentis in conidia, in culture and in planta, using quantitative reverse transcriptase (qRT)-PCR, which is a specific and sensitive method for reproducible quantification of mRNA (Bustin, 2002). In this context and throughout this paper, virulence describes the degree of pathogenicity on specific host genotypes. In addition, formation of appressoria and penetration of the host by the fungal isolates was examined microscopically, in order to evaluate their general pathogenic ability separately from their ability to produce Ptr ToxB.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fungal isolates

Three isolates of P. tritici-repentis were used in this study, representing races 4 and 5 of the pathogen. Race 4 isolate 90-2 was obtained from the Canadian prairies and is an avirulent (≡ nonpathogenic) isolate (Lamari et al., 1998) lacking Ptr ToxB activity. Race 5 isolates Alg3-24 and 92-171R5 were collected in eastern Algeria, North Africa and the Saskatchewan-Manitoba border, Canada (Lamari et al., 1995; Strelkov et al., 2002), respectively, and while both possess Ptr ToxB activity, that activity is much weaker in 92-171R5 (Strelkov et al., 2002). Isolates were grown on V8-potato dextrose agar (V8-PDA) in Petri dishes and conidial inoculum was produced as previously described (Lamari & Bernier, 1989c). To produce mycelia in liquid culture, five plugs (1 cm in diameter) were cut from 4–5 cm diameter colonies of the fungus and transferred to Erlenmeyer flasks, containing 150 mL of Fries medium (Dhingra & Sinclair, 1986) amended with 0·1% yeast extract, and incubated for 5, 10, 15 or 20 days in the dark at room temperature with no agitation.

Plant material and inoculation

Two hexaploid wheat genotypes, 6B662 and cv. Erik, were inoculated with P. tritici-repentis. Line 6B662 is Ptr ToxB-sensitive, resistant to race 4 and susceptible to race 5, while cv. Erik is Ptr ToxB-insensitive and resistant to both races. Five to six evenly spaced seeds were sown in 10 cm plastic pots filled with Metro-Mix 220 soil (W.R. Grace and Co.), and seedlings were maintained in a greenhouse at 20°C/18°C (day/night) with a 16 h photoperiod at 250 µmol m−2s−1 (natural light supplemented with artificial lighting) until inoculation at the 2–3 leaf stage. The seedlings were inoculated with a suspension of 3500 conidia mL−1 (to which 10 drops of Tween 20 [polyoxyethylene sorbitol monolaurate] per L were added) using a sprayer connected to an air line. Leaves were sprayed until run-off. Negative controls were inoculated with sterile distilled H2O containing the same concentration of Tween 20. Immediately following inoculation, the plants were placed in darkness in a misting chamber (relative humidity ≥ 95%) for a 24 h period, with continuous wetness provided by an ultrasonic humidifier. After incubation under high humidity, the plants were transferred to a growth chamber and kept at 20°C/18°C (day/night) with a 16 h photoperiod (180 µmol m−2s−1) and 60% relative humidity. Tissue from the centre of the second leaves (at least 2 cm from the base and tip) was sampled for analysis at 0, 12, 24, 48, 72, 120, 144 and 168 h after inoculation. To confirm infection by P. tritici-repentis, conidia of the fungus were re-isolated as described by Lamari et al. (1995) from leaves harvested 168 h after inoculation. Experiments were independently repeated three times, with 3 to 4 technical replicates at each time point.

Chlorophyll/carotenoid concentration

To estimate chlorophyll and carotenoid concentration in inoculated wheat leaves, 3 to 4 leaf segments (total weight 40 mg) were randomly chosen in each treatment and processed as previously described (Strelkov et al., 1998). Briefly, leaves were cut, weighed, and ground in a mortar and pestle in the presence of liquid nitrogen. Homogenates were extracted 3× with 1 mL of 80% acetone (v/v), the extracts were combined, and the total volume was adjusted to 4 mL with 80% acetone (v/v), corresponding to a final concentration of 10 mg leaf tissue per millilitre of acetone (Witham et al., 1971). The optical density of the extracts was measured at 470, 647 and 663 nm with a spectrophotometer (Hewlett Packard) and the concentration of chlorophyll and carotenoid pigments was calculated using the equations of Lichtenthaler (1987).

RNA extraction

For total RNA extraction, conidia and 5-, 10-, 15- and 20-day-old mycelia of P. tritici-repentis were harvested as described previously (Strelkov et al., 2002), lyophilized and stored at –80°C until processing. Leaf samples were flash frozen in liquid nitrogen at the time of harvest and processed immediately. Total RNA was extracted from leaf and fungal samples using the RNeasy Plant Mini Kit (Qiagen), following the manufacturer's protocol. After extraction, RNA samples were treated with RNase-free DNase I (Ambion) to remove any DNA contamination. The total RNA concentration of each sample was quantified spectrophotometrically using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies), and confirmed by comparing band intensities on 1·2% denaturing agarose gels using a Gel Doc 1000 documentation system (Bio-Rad).

Endogenous control for qRT-PCR

The actin housekeeping gene was selected as an endogenous control to normalize ToxB expression data obtained using qRT-PCR (Suzuki et al., 2000; Kim et al., 2003; Yan & Liou, 2006). Since no actin sequence information was available for P. tritici-repentis, the gene was sequenced from isolates Alg3-24, 92-171R5 and 90-2 of the fungus. Genomic DNA was extracted from 10-day-old mycelial mats of each fungal isolate according to the protocol of Rogers & Bendich (1994) and subjected to PCR using the universal actin forward primer Act-1 (5′-TGGGACGATATGGAIAAIATCTGGCA-3′) and the reverse primer Act-5ra (5′-TTAGAAGCACTTNCGGTG-3′) (Voigt & Wöstemeyer, 2000; Voigt et al., 2005). PCR-reaction conditions were as previously reported (Voigt & Wöstemeyer, 2000; Voigt et al., 2005), except that the temperature profile was slightly modified and consisted of 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 1 min at 52°C, 1 min at 72°C, and a final extension of 7 min at 72°C. The PCR products were visualized on agarose gels and subsequently purified using the QIAquick PCR Purification Kit (Qiagen) as per the manufacturer's instructions. DNA sequencing was performed on a CEQ2000XL DNA Analysis System (Beckman Coulter) in the Department of Biochemistry, University of Alberta.

Preparation of TaqMan probe and primers

All primers and probes used in qRT-PCR were designed using Primer Express Software (Applied Biosystems). The primers and probe for quantifying ToxB expression were based on conserved regions of the gene from P. tritici-repentis isolates Alg3-24, 92-171R5 and 90-2 (GenBank Accession Nos. AF483831, AF483834 and AF483832, respectively). Forward primer ToxBqF2 (5′-CATGCTACTTGCTGTGGCTATCC-3′) and reverse primer ToxBqR2 (5’-GGACACAGCCAGTCGCAAT-3′) were used in conjunction with the TaqMan MGB (minor groove-binding) probe ToxBq2 (5′-CTTGTTTCGGCCAACTG-3′) to amplify a 104 bp product from the ToxB open reading frame (ORF). The primers and probe for the endogenous control were based on the actin gene sequences obtained from isolates Alg3-24, 92-171R5 and 90-2 (see above) and included the forward primer ACTqF1 (5′-CTACGAGCTTCCCGACGGT-3′), reverse primer ACTqR1 (5′-TCTGGAGCACGGAAACGC-3′), and the TaqMan MGB probe ACTq1 (5′-AAGTCATACCCATTGGC-3′). The probe and primers, which amplified a 60 bp product, were designed to bind to regions of the actin sequence specific to P. tritici-repentis and not wheat, so that they could be used to quantify expression of the gene by the fungus in the host. Thus, while they were identical to the corresponding regions of the fungal actin sequence, ACTqF1, ACTqR1 and ACTq1 shared only 79, 61 and 82% similarity, respectively, with the gene from wheat. To ensure that there was no amplification of the wheat actin gene, controls containing only host cDNA were included in the qRT-PCR assays. The ToxBq2 probe was labelled with the fluorescent reporter dye FAMTM at its 5′-end, while the ACTq1 probe was labelled with VIC® (Applied Biosystems).

Quantitative reverse transcriptase-PCR

Total RNA (1 µg) from each sample was reverse-transcribed with the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen), using an oligo(dT)12–18 primer, according to the manufacturer's instructions. Quantitative RT-PCR amplifications were conducted as uniplex reactions in Micro Amp 96 well reaction plates in an ABI PRISM® 7700 Sequence Detector (Applied Biosystems). Reaction mixtures consisted of 10 µL TaqMan Universal Master Mix (2×) (Applied Biosystems), 0·225 µL each of 100 µm forward and reverse primers, 0·05 µL fluorogenic probe (100 µm), and either 20 ng cDNA in the in vitro and conidial studies, or 50 ng cDNA in the in planta studies. Volumes were adjusted to 20 µL with DEPC-treated water and reactions were conducted under the following conditions: 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. Fluorescent emissions were recorded in real time at 518 nm and 554 nm for the FAM and VIC reporter dyes, respectively, and data were collected using Sequence Detector V.1·7 software (Applied Biosystems). Controls which had not been subjected to treatment with reverse transcriptase or to which no template had been added were included in all qRT-PCR assays, as were controls containing only host cDNA. As noted above, the actin gene was used as the endogenous control for normalizing ToxB transcript profiles in all experiments. When examining ToxB expression in vitro or in conidia, the data were calibrated relative to ToxB transcript levels in 5-day-old mycelia of isolate 90-2, according to the comparative CT method for relative quantification of gene expression (Livak & Schmittgen, 2001). In the in planta studies, qRT-PCR data from each isolate/host combination were calibrated relative to ToxB levels at 0 h for isolate 90-2 on the same host, following the comparative CT method (Livak & Schmittgen, 2001). After qRT-PCR, the amplicons obtained were visualized on 3% agarose gels.

Microscopic analysis

For cytological observations, tissue from the middle of the second leaf of individual wheat plants was sampled for analysis at 3, 6, 12, 24, 48 and 72 h after inoculation. Leaf segments (1–2 cm in length) were stored in alcoholic lactophenol (3 parts 95% ethanol, 1 part lactophenol) until examined (Tuite, 1969; Larez et al., 1986; Lamari & Bernier, 1989a). Samples were cleared by boiling in alcoholic lactophenol for 3 min, and then stained in lactophenol-cotton blue for 30 min to 1 h, or in 0·2% Fluorescent Brightener 28 (Sigma) in 0·1 M Tris-HCl buffer (pH 8·5) for 10 min (Rohringer et al., 1977; Dushnicky et al., 1996). After staining, the leaf segments were washed with distilled water and mounted in 100% glycerol for observation. A total of 30 randomly selected conidia per sample were examined for percentage germination, number of germ tubes, number of appressoria, and papilla formation underneath the appressoria. Material was examined with a Leitz Wetziar Dialux 20 bright-field microscope and a Leica DM-RXA fluorescent microscope using blue light excitation. Brightfield and fluorescence images were recorded with a CoolSNAP cf Digital Camera (Photometrics), or a MacroFire LM CCD Digital Camera (Optronics), respectively. Analysis of variance for multiple comparisons (Tukey's studentized range test) was conducted using SAS 9·1 software (SAS Institute Inc., 2004).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Disease development

Chlorosis symptoms were induced by race 5 isolates Alg3-24 and 92-171R5 on the Ptr ToxB-sensitive wheat genotype 6B662 (Fig. 1). Chlorotic lesions began to appear 72 h after inoculation with isolate Alg3-24, and 96 h after inoculation with 92-171R5. In response to Alg3-24, chlorosis spread throughout the affected leaves, and by 168 h after inoculation, approximately 50% of the total leaf area was chlorotic. In contrast, isolate 92-171R5 induced weaker symptoms, and the development of chlorosis was not as extensive. Neither isolate produced chlorosis on the Ptr ToxB-insensitive cv. Erik, although small, localized necrotic lesions typical of a resistant reaction (Lamari & Bernier, 1989a) were observed (Fig. 1). While inoculation with race 4 isolate 90-2 caused the development of a few small chlorotic flecks on line 6B662, this isolate failed to induce any significant symptoms on either genotype, confirming its avirulence (nonpathogenicity) (Fig. 1). No symptoms were observed on control plants inoculated with only water (data not shown). Single spore isolates of P. tritici-repentis were successfully re-isolated from leaves of line 6B662 and cv. Erik 168 h after inoculation with Alg3-24, 92-171R5 or 90-2, in all repetitions of the experiment. However, isolates of 90-2 were recovered at much lower frequencies, since this isolate produced very few conidia on infected hosts (only on the chlorotic flecks).

image

Figure 1. Reaction of wheat line 6B662 and cv. Erik to inoculation with race 5 isolates Alg3-24 and 92-171R5, and race 4 isolate 90-2 of Pyrenophora tritici-repentis. Leaves are shown 168 h after inoculation with the respective isolates. Line 6B662 is sensitive to Ptr ToxB and susceptible to isolates Alg3-24 and 92-171R5. Erik is insensitive to Ptr ToxB and resistant to isolates Alg3-24 and 92-171R5. Both wheat genotypes are resistant to isolate 90-2.

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Pigment concentration

Total chlorophyll concentration in the leaves of wheat genotypes 6B662 and cv. Erik was in the 2·0 to 2·5 mg per g tissue range at the time of inoculation with P. tritici-repentis (Fig. 2a). In line 6B662, chlorophyll levels began to decrease 72 h after inoculation with race 5 isolates Alg3-24 and 92-171R5, and continued to decline throughout the time-course of the study in the case of Alg3-24 (Fig. 2a). By 168 h, the level of total chlorophyll had dropped from 2·21 ± 0·10 (SE) to 0·987 ± 0·063 mg per g tissue in response to inoculation with Alg3-24, and from 2·23 ± 0·064 to 1·77 ± 0·20 mg per g tissue in response to inoculation with 92-171R5. Similar rates of decline were observed for chlorophyll a and b, and the ratio between the two pigments remained constant (data not shown). A decline in total chlorophyll concentration was also observed in cv. Erik in response to inoculation with isolates Alg3-24 and 92-171R5, but it was of a smaller magnitude (Fig. 2a). Total chlorophyll concentration in cv. Erik decreased from 2·30 ± 0·057 to 1·51 ± 0·044 mg per g tissue at 168 h after inoculation with Alg3-24, while a decrease from 2·27 ± 0·051 to 1·81 ± 0·10 mg per g tissue was observed in response to 92-171R5 (Fig. 2a). Inoculation with race 4 isolate 90–2 did not cause any detectable decline in total chlorophyll concentration in either wheat genotype (Fig. 2a). Similarly, chlorophyll content in water-inoculated seedlings also remained constant at all time points examined.

image

Figure 2. Total chlorophyll (a) and carotenoid (b) concentration over time after inoculation of wheat line 6B662 and cultivar Erik with race 5 isolates Alg3-24 and 92-171R5, and race 4 isolate 90-2, of Pyrenophora tritici-repentis. Controls were inoculated with water only. Tissue from the centre of the second leaves (at least 2 cm from the base and tip) was sampled for analysis at 0, 12, 24, 48, 72, 120, 144 and 168 h after inoculation. Pigment concentrations were calculated using the equations of Lichtenthaler (1987), and the means from three biological repetitions of the experiment are shown. Error bars indicate the standard error of the mean.

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The trends in carotenoid concentration were similar to those observed with respect to chlorophyll. On line 6B662, the carotenoid concentration declined from 0·622 ± 0·023 mg per g tissue at 0 h to 0·397 ± 0·027 mg per g tissue at 168 h after inoculation with Alg3-24, and from 0·661 ± 0·017 to 0·498 ± 0·082 mg per g tissue after inoculation with 92-171R5 (Fig. 2b). A smaller decrease was observed in cv. Erik in response to inoculation with these two isolates. In this genotype, carotenoid concentration declined from 0·686 ± 0·023 to 0·550 ± 0·020 mg per g tissue at 168 h after inoculation with Alg3-24, and from 0·659 ± 0·016 to 0·575 ± 0·022 mg per g tissue after inoculation with 92-171R5 (Fig. 2b). As was observed with respect to chlorophyll, inoculation of 6B662 or cv. Erik with isolate 90-2 or water had no significant effect on carotenoid concentration. The decline of both total chlorophyll and carotenoid concentration in cv. Erik, in response to inoculation with Alg3-24 and 92-171R5, is likely to reflect the development of necrotic flecks in this cultivar, since total leaf area was sampled as opposed to only the chlorotic zones. Consistent with this hypothesis, inoculation with 90-2, which did not cause necrotic flecks in cv. Erik, did not result in a decline in either pigment.

Actin gene sequences

Amplification of genomic DNA from P. tritici-repentis isolates Alg3-24, 92-171R5 and 90-2 with the actin-specific primers Act-1 and Act-5ra (Voigt & Wöstemeyer, 2000; Voigt et al., 2005) yielded a single PCR product approximately 900 bp in size for each isolate. Nucleotide sequences of 893, 726 and 872 bp in length were obtained for the amplicons from Alg3-24 (GenBank Accession No. EF180087), 92-171R5 (EF180088) and 90-2 (EF180089), respectively. Sequence analysis revealed that the gene shared 99% homology over 721 bp amongst the three isolates. In contrast, comparison with an actin gene sequence from wheat available in GenBank (Accession No. AY423548) revealed only 72% similarity over 545 bp.

Quantitative reverse transcriptase-PCR

Analysis of ToxB expression by qRT-PCR allowed comparison of relative transcript levels among the three P. tritici-repentis isolates included in this study. This analysis revealed that while expression of ToxB was greatest in 5- to 20-day-old mycelia of Alg3-24, a very low level of transcript was also detectable in mycelia of isolates 90-2 and 92-171R5 (Table 1). The amount of mycelial transcript increased in all isolates during the time course of the study, but the level in Alg3-24 was nearly 20 000-fold greater than in 92-171R5 and 25 000-fold greater than in 90-2 by 20 days. The qRT-PCR analysis also revealed that the amount of ToxB transcript was greater in conidia of isolate 90-2 than in liquid-grown mycelia, at all time-points tested. In isolate 92-171R5, expression in conidia was greater than in mycelia at 5 and 10 days, nearly the same at 15 days, and about half at 20 days. In contrast, ToxB expression in Alg3-24 was consistently greater in mycelia than conidia, particularly after day 5. Nevertheless, the abundance of the transcript was 2 to 3 orders of magnitude greater in conidia of Alg3-24 than in conidia of 90-2 or 92-171R5.

Table 1.  Quantitative reverse transcriptase-PCR analysis of the relative expression of the ToxB gene in mycelia and conidia of Pyrenophora tritici-repentis isolates Alg3-24, 92-171R5 and 90-2
Age of mycelia (days)Relative expressiona
Alg3-2492-171R590-2
  • a

    ToxB expression data was normalized using an actin endogenous control; relative expression was calibrated against ToxB transcript levels in 5-day-old mycelia of isolate 90-2 according to the comparative CT method for relative quantification of gene expression (Livak & Schmittgen, 2001). The range of values obtained for each treatment is indicated in brackets and represents variation among three biological replicates.

55·00 × 1041·871·00
(4·63 × 104–5·39 × 104)(1·51–2·29)(0·74–1·34)
104·76 × 1056·361·59 × 101
(3·56 × 105–6·35 × 105)(5·28–7·67)(1·29 × 101–1·94 × 101)
158·34 × 1053·50 × 1011·62 × 101
(6·37 × 105–1·09 × 106)(2·22 × 101–5·53 × 101)(8·51–3·09 × 101)
201·56 × 1067·82 × 1016·31 × 101
(1·37 × 106–1·76 × 106)(7·35 × 101–8·32 × 101)(5·49 × 101–7·25 × 101)
conidia1·90 × 1043·68 × 1012·44 × 102
(1·49 × 104–2·41 × 104)(2·79 × 101–4·85 × 101)(2·08 × 102–2·86 × 102)

The ToxB expression pattern was also quantified in planta after inoculation of the Ptr ToxB-sensitive and insensitive wheat genotypes 6B662 and cv. Erik with isolates Alg3-24, 92-171R5 and 90-2. Abundance of the ToxB transcript was much higher in leaves inoculated with Alg3-24 than in leaves inoculated with 92-171R5 or 90-2 (Tables 2 and 3). Transcript abundance was also higher in leaves inoculated with 92-171R5 than with 90-2 from 12 to 48 h after inoculation. In the case of the race 5 isolates Alg3-24 and 92-171R5, relative transcript abundance increased rapidly after inoculation, peaking at 24 h. In 6B662 leaves inoculated with Alg3-24, relative ToxB transcript abundance increased 2500-fold from 0 to 24 h (Table 2), while in cv. Erik it increased 3360-fold over the same period (Table 3). Similarly, after inoculation with 92-171R5, abundance of the ToxB transcript increased 37 and 143-fold in 6B662 and cv. Erik, respectively, from 0 to 24 h. After 24 h, relative expression of the ToxB gene by isolate Alg3-24 declined slowly in 6B662, while it remained relatively stable until 148 h in cv. Erik. A more rapid decline was observed in transcript levels in both wheat genotypes after inoculation with 92-171R5 (Tables 2 and 3). By 168 h after inoculation, ToxB transcript abundance in 92-171R5-inoculated tissue was only a fraction of that at 0 h, while in Alg3-24-inoculated tissue, it was still several hundred-fold greater. The expression pattern for the ToxB gene in race 4 isolate 90-2 was markedly different. Unlike in tissues inoculated with isolates Alg3-24 and 92-171R5, there was no large increase in the level of ToxB transcript after inoculation with 90-2, with relative abundance remaining very low throughout the time-course of the study (Tables 2 and 3). Indeed, with 90-2, the greatest increase in ToxB transcript level was not observed until 168 h after inoculation in line 6B662, when it was only 12-fold greater than at time 0 h. On cv. Erik the highest abundance was also observed at 168 h, but the increase was only 2·2-fold relative to 0 h. No amplification signals were detected in the negative controls.

Table 2.  Quantitative reverse transcriptase-PCR analysis of the relative expression of the ToxB gene by isolates Alg3-24, 92-171R5 and 90-2 of Pyrenophora tritici-repentis, at various times after inoculation onto the wheat line 6B662
Hours after inoculationRelative expressiona
Alg3-2492-171R590-2
  • a

    ToxB expression data was normalized using an actin endogenous control; relative expression was calibrated against ToxB transcript levels at 0 h for isolate 90-2 according to the comparative CT method for relative quantification of gene expression (Livak & Schmittgen, 2001). The range of values obtained for each treatment is indicated in brackets and represents variation among three biological replicates.

05·243·711·00
(4·25–6·45)(2·46–5·57)(0·810–1·23)
121·18 × 1035·02 × 1010·189
(7·34 × 102 –1·88 × 103)(3·42 × 101–7·35 × 101)(0·151–0·234)
241·31 × 1041·36 × 1020·102
(8·66 × 103–1·99 × 104)(1·15 × 102–1·60 × 102)(0·0799–0·110)
485·60 × 1039·190·415
(5·07 × 103–6·08 × 103)(5·98–1·41 × 101)(0·342–0·521)
728·60 × 1032·161·13
(2·49 × 103 –1·01 × 104)(1·58–2·94)(0·860–1·33)
963·04 × 1030·4208·88
(2·48 × 103–3·72 × 103)(0·244–0·715)(5·73–1·28 × 101)
1203·33 × 1030·3301·05
(2·80 × 103–3·95 × 103)(0·190–0·569)(0·824–1·33)
1481·63 × 1030·08991·87
(1·34 × 103 –1·98 × 103)(0·0605–0·121)(1·01–3·43)
1721·39 × 1030·06011·20 × 101
(1·10 × 103 –1·75 × 103)(0·0411–0·0800)(5·66–2·56 × 101)
Table 3.  Quantitative reverse transcriptase-PCR analysis of the relative expression of the ToxB gene by isolates Alg3-24, 92-171R5 and 90-2 of Pyrenophora tritici-repentis, at various times after inoculation onto the wheat cv. Erik
Hours after inoculationRelative expressiona
Alg3-2492-171R590-2
  • a

    ToxB expression data was normalized using an actin endogenous control; relative expression was calibrated against ToxB transcript levels at 0 h for isolate 90-2 according to the comparative CT method for relative quantification of gene expression (Livak & Schmittgen, 2001). The range of values obtained for each treatment is indicated in brackets and represents variation among three biological replicates.

01·330·2701·00
(0·850–2·07)(0·202–0·361)(0·621–1·61)
121·35 × 1031·30 × 1010·120
(1·07 × 103–1·69 × 103)(9·25–1·82 × 101)(0·111–0·140)
244·43 × 1033·87 × 1010·212
(3·01 × 103–6·03 × 103)(2·86 × 101–4·98 × 101)(0·114–0·547)
485·08 × 1037·460·415
(4·12 × 103–6·25 × 103)(5·06–1·10 × 101)(0·132–0·520)
724·88 × 1031·520·443
(4·09 × 103–5·79 × 103)(1·23–1·89)(0·371–0·519)
962·50 × 1031·071·73
(1·91 × 103–3·28 × 103)(0·830–1·37)(1·10–2·69)
1205·79 × 1030·4110·704
(5·04 × 103–6·65 × 103)(0·267–0·620)(0·450–1·07)
1483·57 × 1030·07051·34
(3·15 × 103–4·03 × 103)(0·0420–0·122)(0·942–1·89)
1726·13 × 1020·03872·17
(5·26 × 102–7·14 × 102)(0·0240–0·0595)(0·880–5·35)

Fungal development in compatible and incompatible interactions

On the leaf surface of resistant and susceptible wheat genotypes, conidial germination in the three isolates ranged from 80 to 97% at 3 h after inoculation, with the highest percentage of germinated spores observed in 90-2. However, by 6 h, germination of conidia approached 100% in all isolate/genotype combinations (data not shown). The average number of germ tubes formed per conidium on the leaf surface also did not differ significantly (P < 0·05) between isolates at most time points, and no significant differences were observed in terms of the germ tube number when the same isolate was inoculated onto the different host genotypes.

The number of appressoria produced by each isolate was similar on both wheat genotypes, but differed between isolates at most time-points (Table 4a). By 3 h after inoculation, Alg3-24 had produced an average of 0·81 or 1·26 appressoria per conidium on Erik and 6B662, respectively. In contrast, appressoria formed by 92-171R5 or 90-2 were rare or absent at this time. Throughout the remainder of the time-course, the number of appressoria produced was consistently higher in Alg3-24 than in 92-171R5 or 90-2, with the lowest values usually observed for 90-2 (Table 4a). Therefore, while the mean number of appressoria produced by 90-2 on either host ranged from 0·47 to 0·80 at 24 to 72 h, in Alg3-24 these values ranged from 1·10 to 1·93. The differences between Alg3-24 and 90-2 were significantly different (P < 0·05) at all time-points on both wheat genotypes, with the values for 90-2 always lower (Table 4a). The mean number of appressoria produced per conidium by 92-171R5 was usually somewhere in between those observed for Alg3-24 and 90-2.

Table 4.  Mean number of (a) appressoria produced and (b) papillae induced per conidium of Pyrenophora tritici-repentis, after inoculation of wheat line 6B662 and cv. Erik with isolates Alg3-24, 92-171R5 and 90-2 of the pathogen.
IsolateHost3 h6 h12 h24 h48 h72 h
  • a

    Mean number of appressoria and papillae was assessed per 30 randomly selected conidia at different times after inoculation of 6B662 and cv. Erik with the various isolates.

  • b

    Means followed by a common letter in the same column are not significantly different (P < 0·05) as determined by Tukey's Studentized Range (HSD) test.

(a) Mean number of appressoria produceda
Alg3-246B6621·26 ab0·55 a1·23 a1·37 ab1·17 a1·83 a
Erik0·81 b0·93 a1·23 a1·93 a1·47 a1·10 b
92-171R56B6620 c0·48 ab0·73 ab0·30 c1·23 a0·97 bc
Erik0 c0·43 ab0·90 ab0·87 bc1·07 ab1·03 bc
90-26B6620 c0·07 b0·53 b0·47 c0·50 b0·80 bc
Erik0·07 c0·27 b0·37 b0·60 c0·47 b0·43 c
(b) Mean number of papillae induceda
Alg3-246B6620·27 a0·17 a0·43 a0·37 b0·47 a0·63 a
Erik0·20 a0·17 a0·10 ab0·80 a0·40 a0·33 b
92-171R56B6620 b0 b0·10 ab0·07 c0·10 b0·03 c
Erik0 b0·07 ab0·20 ab0·40 b0·17 b0 c
90-26B6620 b0 b0 b0·07 c0 b0 c
Erik0 b0 b0 b0·07 c0 b0 c

Following penetration by the fungal isolates, the host epidermal cells stained deeply with cotton blue (data not shown), suggesting that major physiological changes had occurred. Penetrations often resulted in the formation of papillae beneath the appressoria. The number of papillae formed by 6B662 and cv. Erik in response to inoculation with Alg3-24 was significantly different (P < 0·05) only at 24 h (higher in Erik) and 72 h (higher in 6B662), and fluctuated substantially at the various time-points (Table 4b). Similarly, inoculation with 90-2 did not induce differential responses with respect to papilla formation in the hosts. In response to inoculation with 92-171R5, no significant differences were observed except at 24 h, at which time cv. Erik produced significantly greater numbers of papilla than 6B662 (P < 0·05). As papillae were counted based on the number induced per conidium, rather than per appressorium, direct comparisons cannot be made in terms of papilla formation in response to the different isolates, since the isolates produced significantly different numbers of appressoria (Table 4a). Nevertheless, the development of papillae appeared greatest after inoculation with Alg3-24 and least in response to 90-2 (Table 4b).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The data show that the ToxB gene is transcribed, albeit at very different levels, in isolates Alg3-24, 92-171R5 and 90-2 of P. tritici-repentis. In mycelia grown in liquid culture, relative transcript abundance was greatest in Alg3-24 at all time-points examined. Levels were many folds lower in 92-171R5 and 90-2, and transcript abundance in mycelia of these two isolates was within the same order of magnitude. This very low abundance in mycelia probably explains previous reports suggesting that ToxB was not expressed in mycelia of race 4 isolates (Martinez et al., 2004; Strelkov et al., 2006), or in mycelia of the weakly virulent race 5 isolate 92-171R5 (Strelkov et al., 2006). In those reports, gene expression was examined by conventional RT-PCR and Northern blotting, which are less sensitive techniques. Although expression analysis in culture is important in demonstrating that the ToxB homologues in avirulent and weakly virulent isolates are active genes, the expression pattern of these genes in conidia and in toxin-sensitive and insensitive host tissues is of greater relevance with respect to disease development.

A number of fungal species, such as Botryodiplodia theobromae (Knight et al., 1976), Mucor racemosus (Linz et al., 1982) and Neurospora tetrasperma (Plesofsky-Vig et al., 1992), are known to produce stored mRNA that is translated upon germination. Given the importance of Ptr ToxB in establishing a compatible interaction between host and pathogen, its presence in conidia presumably enables faster toxin production during the early stages of infection. As expected, the greatest amount of transcript was found in conidia of the most virulent isolate, Alg3-24, with levels in 90-2 and 92-171R5 several orders of magnitude lower. Similarly, in planta analysis of the relative abundance of the ToxB transcript revealed that expression of the gene was greatest in Alg3-24, followed by 92-171R5 and 90-2. Since the region encoding the mature toxin in isolates 92-171R5 and Alg3-24 is identical, the observed differences in ToxB expression may largely explain the decreased virulence associated with 92-171R5 and manifested as a reduced ability to induce chlorosis, which provides a measure of Ptr ToxB activity (Strelkov et al., 1999). Thus, while chlorophyll concentration declined by about 55% over 168 h after inoculation of the toxin-sensitive genotype with Alg3-24, this decline was only 21% after inoculation with 92-171R5. Similarly, the decline in carotenoid concentration was also greater in Alg3-24 than 92-171R5.

The situation with respect to the avirulent race 4 isolate 90-2 appears more complex. In planta expression of the ToxB homologue was lowest in this isolate, although since it was still detectable, a low level of chlorosis development would have been expected in the toxin-sensitive wheat genotype. However, no declines in either chlorophyll or carotenoid concentrations were observed, and pigment levels were nearly identical to water-treated controls. The form of ToxB in race 4 isolates exhibits only 86% similarity to ‘wild-type’ToxB from race 5 isolates (Strelkov & Lamari, 2003; Martinez et al., 2004), and the protein it encodes is less active (Kim & Strelkov, 2007). Thus, it seems that a lower level of ToxB expression, combined with a less active form of the toxin, results in the avirulence of 90-2 and other nonpathogenic race 4 isolates; this is reflected in their inability to induce chlorosis. Nevertheless, the fact that the ToxB homologue from race 4 is an active gene, which is expressed in planta, is consistent with the hypothesis of Martinez et al. (2004) that it may have a function with respect to infection of other hosts.

The differences observed among the isolates in ToxB expression level and pattern most likely reflect the significant differences that have been reported upstream of the open reading frame in these isolates (Martinez et al., 2004; Strelkov et al., 2006). However, in addition to the differences in regulation of expression, the differential expression of this gene may also be attributable to variation in ToxB copy number. In the virulent isolate Alg3-24, the ToxB gene is found as 8–10 copies, while in 92-171R5, only two copies are found, and in 90-2, the ToxB homologue exists as a single copy (Strelkov et al., 2006). While transcript levels were highest in Alg3-24, the general in planta expression pattern of the ToxB gene was quite similar in Alg3-24 and 92-171R5 (race 5), but differed from 90-2 (race 4). In the race 5 isolates, transcript abundance peaked at 24 h after inoculation, preceding the development of chlorosis by approximately 48 h in the toxin-sensitive genotype 6B662. Although high titre antibodies were not available to correlate gene expression with protein levels, the delayed effect of the toxin in inducing chlorosis could explain why the development of this symptom occurred after transcript levels had reached their maximum. When toxin-sensitive leaf tissue is infiltrated with pure or partially purified Ptr ToxB, chlorosis does not develop until 48 to 72 h after treatment (Strelkov et al., 1998, 1999). It is also possible that while ToxB transcript levels reach their maximum in the early phases of infection, toxin activity does not peak until later. Indeed, previous research with Ptr ToxA indicated that while maximum toxin activity in culture occurs at 16 to 22 days (Ballance et al., 1989; Tomás et al., 1990), the ToxA transcript is most abundant at 6 and 7 days (Ciuffetti et al., 1997).

The ToxB expression profiles in all three isolates were similar in the Ptr ToxB-sensitive and insensitive hosts. Therefore, it was the toxin-insensitivity of cv. Erik that ensured that Ptr ToxB-induced chlorosis did not develop in this cultivar (at least in response to the race 5 isolates), resulting in greatly reduced lesion development. The formation of papillae did not appear to be an effective defense mechanism against infection, since the number of papillae formed by cv. Erik and 6B662 were generally not significantly different. Previous research has demonstrated that P. tritici-repentis can germinate, form appressoria, penetrate epidermal cells and grow into the intercellular space of the mesophyll in both resistant and susceptible wheat genotypes, with further growth in the mesophyll stopped in the incompatible reaction, but continuing in the compatible reaction (Larez et al., 1986; Lamari & Bernier, 1989b). However, differences between the compatible and incompatible reactions are not observed until 48 h after inoculation, and in the earlier phases of infection, the extent of pathogen ingress is comparable in resistant and susceptible genotypes (Lamari & Bernier, 1989b). The similar ToxB expression profiles detected in 6B662 and cv. Erik are consistent with these reports; in the race 5 isolates, the amount of ToxB transcript peaked at 24 h, prior to the onset of host resistance. It is possible that P. tritici-repentis can produce a flux of ToxB transcript early in the infection process, before resistance mechanisms in the host are fully activated.

The observation that isolates of P. tritici-repentis can form appressoria and penetrate the epidermal cells of resistant host genotypes (Larez et al., 1986; Lamari & Bernier, 1989b), combined with a study in which the acquisition of toxin-producing ability (Ptr ToxA) was shown to be a sufficient condition for virulence on a Ptr ToxA-sensitive wheat genotype (Ciuffetti et al., 1997), suggests that the virulence conferred by the Ptr toxins is superimposed on the general pathogenic ability of the fungus (Lamari et al., 1998; Strelkov & Lamari, 2003). However, in the current study, the number of appressoria formed differed among isolates, while it was generally similar when the same isolate was inoculated onto resistant and susceptible wheat genotypes. The highly virulent isolate Alg3-24 produced significantly higher numbers of appressoria per conidium than avirulent isolate 90-2, whilst isolate 92-171R5 formed intermediate numbers of appressoria, which is consistent with its classification as weakly virulent (Strelkov et al., 2002). The speed of appressorium formation was also significantly different. While significant numbers of appressoria were formed by Alg3-24 at 3 h after inoculation, isolates 92-171R5 and 90-2 developed few if any appressoria until 6 h. In contrast, features not exclusively associated with parasitic ability, such as the conidial germination rate and number of germ tubes per conidium, were similar in all isolates (data not shown).

These results suggest that the underlying pathogenic ability may not be the same among isolates of P. tritici-repentis. Indeed, previous cytological studies have described the incompatible interaction between virulent (toxin-producing) isolates and resistant or toxin-insensitive wheat genotypes, while the present comparison is between a truly avirulent isolate (i.e. producing no known toxins) and virulent and weakly virulent isolates. However, a strong correlation between ToxB expression and appressorium formation was also observed, with the isolate producing the most ToxB transcript also producing the most appressoria, and vice versa. This suggests that there may be a relationship between the ability to produce Ptr ToxB and form appressoria, and that the toxin could have other roles in the pre-penetration process and/or in bestowing basic pathogenic fitness to isolates of P. tritici-repentis. The detection of ToxB transcript in conidia and in the early phases of infection is consistent with this hypothesis, but additional research is required to examine these possibilities. Regardless of any other mechanisms that may be involved, the results presented here indicate that quantitative variation in the virulence of P. tritici-repentis is related to the extent of ToxB gene expression.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Financial support was provided by the Natural Sciences and Engineering Research Council of Canada and the Alberta Ingenuity Fund (to S.E. Strelkov). We thank Dr Lakhdar Lamari (University of Manitoba) for providing isolates Alg3-24, 92-171R5 and 90-2, and Drs Randy Currah (University of Alberta) and T. Kelly Turkington (Agriculture and Agri-Food Canada, Lacombe, AB) for helpful suggestions. The technical assistance of Ms Shirley Brezden is also gratefully acknowledged.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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