Barley lesion mimics, supersusceptible or highly resistant to leaf rust and net blotch

Authors

  • S. A. I. Wright,

    1. Dipartimento di Scienze Animali, Vegetali e dell'Ambiente, Università degli Studi del Molise, Campobasso, Italy
    2. Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg
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  • M. Azarang,

    1. Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg
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  • A. B. Falk

    Corresponding author
    1. Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden
    • Dipartimento di Scienze Animali, Vegetali e dell'Ambiente, Università degli Studi del Molise, Campobasso, Italy
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E-mail: anders.falk2@comhem.se

Abstract

Lesion mimic mutants of plants have the feature of spontaneously displaying necrotic spots or bands on their leaves. Lesion mimics have often displayed enhanced resistance to biotrophic pathogens whilst showing increased susceptibility to necrotrophs. This paper identifies three novel, non-allelic mutants of barley (Hordeum vulgare), which spontaneously form necrotic leaf lesions: Necrotic leaf spot 9.3091 (nec9.3091), Mottled leaf 8.1661 (mtt8.1661) and Mottled leaf 9.2721 (mtt9.2721). The Necrotic leaf spot 8.3550 mutant (nec8.3550), formerly known as bst1, was included in the study because it is a lesion mimic mutant belonging to the same original pool. The reactions of the mutants to the biotroph Puccinia hordei and the necrotroph Pyrenophora teres f. sp. teres were investigated. Mutants nec8.3550 and mtt8.1661 were more resistant than the parental Bowman near-isogenic line with the Rph3.c gene (Bowman Rph3.c, NGB 22452) to leaf rust, caused by P. hordei. Mutants nec8.3550, mtt8.1661 and mtt9.2721 were more susceptible than Bowman Rph3.c to net blotch, caused by P. teres f. sp. teres. Autofluorescence was detected in leaf tissues of all mutants. Based on the high expression of the PR1 and Hv-HIR genes, combined with the low susceptibility to P. hordei, nec8.3550 appears to have entered a state of systemic acquired resistance, which is quite distinct from the resistance expressed in mtt8.1661. The latter mutant has low or no expression of PR1 and Hv-HIR genes, yet it is highly resistant to rust. It is also extremely susceptible to net blotch. These mutants can serve as genetic sources of novel disease resistance for barley improvement.

Introduction

When pathogenic microorganisms come into physical contact with their plant hosts, they encounter a multitude of defence strategies, which plants have developed to prevent infection. As a first step to activate defence reactions, plants have developed membrane-localized pattern-recognition receptors (PRR), which detect microbe-associated molecular patterns (MAMP; Jones & Dangl, 2006). An additional receptor class, which is mainly intracellular, consists of resistance (R) proteins, which detect isolate-specific pathogen effectors. The activation of receptors can induce localized cell death, the so-called hypersensitive reaction (HR), which is associated with the rapid production of very reactive molecules, in particular reactive oxygen species and nitrous oxide (Besson-Bard et al., 2009). These molecules play a role in resistance mechanisms by stimulating the reinforcement of cell walls and by acting as direct antimicrobial agents. They also partake in local and systemic signalling, which leads to the activation of defence systems, even in the absence of a disease-causing agent. Additional compounds, such as salicylic acid, jasmonic acid and ethylene, participate in the signalling, which eventually leads to the full activation of the resistance response and is often associated with the production of fluorescing compounds, such as the phenolic lignin precursor, cinnamoyl alcohol (Hammond-Kosack & Jones, 1996), or phytoalexins, exemplified by coumaroyl-agmatine in barley (von Röpenack et al., 1998). Salicylic acid, being a positive regulator of HR, is also probably involved in systemic acquired resistance and expression of pathogenesis-related genes (PR). Expression of the PR1 gene has been found to be associated with the expression of systemic acquired resistance, particularly in Arabidopsis (Dietrich et al., 1994). In several plant species, a group of genes called HIR (hypersensitive induced reaction) have been suggested to be involved in the formation of HR (Rostoks et al., 2003). In rice and pepper, the HIR proteins interact with small leucine-rich repeat (LRR) proteins, which appear to modulate the function of HIR (Jung & Hwang, 2007; Zhou et al., 2010). The HIR proteins are part of an evolutionary conserved family of plasma membrane-associated proteins, which form so-called ‘lipid rafts’, a protein–lipid complex, which may be involved in transport across the plasma membrane (Green & Young, 2008). HR and salicylic acid are clearly associated with plant resistance to biotrophic pathogens. HR or programmed cell death is not a very efficient defence response against necrotrophic pathogens, because they use dead or dying plant tissues as infection ports (Govrin & Levine, 2000).

Lesion mimic mutants (LMMs) are plants that spontaneously develop necrotic lesions in the absence of plant-pathogenic organisms. This phenotype has been observed in many plant species, for example in maize, rice, barley and Arabidopsis. Many LMMs display changes in leaf pigment levels. The nomenclature of LMMs is based on their visual appearance, such as: necrotic leaf or necrotic leaf spot (nec), zebra stripe (zeb) and mottled leaf (mtt). Mottled leaves have uneven blotches, spots or bands, usually chlorotic in colour, which in some cases become necrotic or in part necrotic with time. If the lesions become necrotic with time, the mottled leaf mutants are considered to be LMMs. This is the case for barley mutants mtt1, mtt5 and mtt6 (for examples, visit: http://ace.untamo.net/).

In Arabidopsis, lesion mimic phenotypes result from mutations in genes coding for cyclic-nucleotide-gated ion channels, copine proteins, enzymes in biosynthetic pathways of porphyrins and chlorophyll metabolism, disease resistance, and others (reviewed by Lorrain et al., 2003). A screen for revertants, called lazarus (laz) of the acd11 LMM revealed that the LAZ2 gene encodes a histone lysine methyltransferase, supporting the notion of chromatin remodelling as a part of plant defence reactions (reviewed by Berr et al., 2012). Interestingly, the LAZ5 gene encodes an R protein, thus suggesting the existence of a more direct link between acd11 and disease resistance (Palma et al., 2010). Moreover, mutations in the ADR1 R gene were found to suppress the lesion mimic phenotype of the Arabidopsis lsd1 LMM (Bonardi et al., 2011). The lsd1 LMM was originally identified from its ‘runaway cell death’ phenotype that is triggered by various external stimuli.

In many cases, LMMs display resistance against particular pathogens. However, the use of LMMs in breeding for plant disease resistance is often limited by their displaying increased susceptibility to other plant pathogens. In barley, the mlo mutants have received particular attention as a source of resistance to the biotrophic powdery mildew (Blumeria graminis f. sp. hordei; von Röpenack et al., 1998). However, cultivars with mlo mutants are particularly susceptible to spot blotch disease caused by the hemibiotrophic fungus Bipolaris sorokiniana (teleomorph: Cochliobolus sativus; Kumar et al., 2001). Jarosch et al. (1999) reported that mlo mutants are attacked by the hemibiotrophic rice blast fungus, Magnaporthe oryzae, and in this case, the lesion mimic phenotype leads to a loss of non-host resistance. Other examples of LMMs in barley with increased resistance to certain pathogens are albostrians (Jain et al., 2004), nec1 (Keisa et al., 2011) and bst1 (Persson et al., 2009). A trend of these particular mutants is that they are highly resistant to biotrophic pathogens, exemplified by powdery mildew (von Röpenack et al., 1998; Jain et al., 2004), but they vary in their susceptibility to hemibiotrophic and necrotrophic pathogens (Kumar et al., 2001; Schäfer et al., 2004; Persson et al., 2009).

The LMM, Necrotic leaf spot 8 mutant 3550 (nec8.3550), formerly known as Bipolaris sorokiniana tolerant mutant 1 (bst1), had been identified as a potential source of resistance to spot blotch, caused by Bipolaris sorokiniana (Persson et al., 2008, 2009). This study investigates the level of resistance of the nec8.3550 mutant to other pathogens. In addition, three other LMMs from the same genetic background were included in the study. As the mutants can be of importance in plant breeding, further characterization was required with regard to the phenotypes of the mutants, in particular to determine the expression of the PR1 and the HIR genes, which have previously been shown to be associated with the increased resistance to pathogens. The results show that LMMs can be extremely susceptible, or much more resistant than the original Bowman Rph3.c line.

Materials and methods

Plant material and mutagenic treatment

Bowman (PI 483237) was the recurrent parent for generation of the near-isogenic barley line Bowman Rph3.c (Bowman*11//Estate/3.2 uz als MM), BW746 or NGB 22452, which was obtained from Dr Jerome D. Franckowiak at North Dakota State University, Fargo, ND, USA. It was constructed by introgression of the Rph3.c resistance gene from cv. Estate into cv. Bowman (Bowman*11//Estate (CIho 3410)/R.I. Wolfe's second Multiple Marker Stock for chromosome 3), and represents the 10th backcross to Bowman. Mutation was performed with fast neutrons at the International Atomic Energy Agency (IAEA), Vienna, Austria, with a dose of 5 Gy. Five thousand M2 spikes were sown and the mutant plants were screened for those displaying spontaneous cell death or other aberrant phenotypes. Selected mutants were backcrossed twice to the original Bowman Rph3.c line, and their phenotypes were analysed using the backcrossed material. The phenotype of the F1 and F2 plants indicated that the four mutants selected were inherited as monogenic recessives. Previously described barley mutants Mottled leaf 1 (mtt1), Mottled leaf 2 (mtt2), Mottled leaf 5 (mtt5), Mottled leaf 6 (mtt6), Necrotic leaf spot 1 (nec1), Necrotic leaf spot 4 (nec4) and Necrotic leaf spot 5 (nec5) were obtained from the USDA-ARS collection (Aberdeen, ID, USA). These mutants were crossed to the newly generated mutants with similar phenotypes. Allelism was tested by studying the phenotype in F1. If the Bowman phenotype (non-lesion phenotype) was observed in F1, the two mutants that had been crossed were considered to be mutated in different genes. Plants were grown either in a greenhouse or in caged outdoor compartments during the summer. Experiments were carried out in controlled growth chambers at 22°C with 16/8 h of light/dark (long-day conditions), or 8/16 h of light/dark (short-day conditions). Seeds of the mutants were deposited in the USDA-ARS collection (Aberdeen, ID, USA) and in the NordGen seed collection (Alnarp, Sweden), and GSHO and NGB numbers were assigned.

Mutant necrotic leaf spot 8.3550 (nec8.3550) has previously been demonstrated to confer an increased level of resistance to the barley spot blotch fungus (Persson et al., 2008). The objective of the present work was to test the level of resistance of this mutant to other important fungal pathogens of barley. This is particularly desirable because it is possible that enhanced resistance to one pathogen could lead to enhanced susceptibility to other pathogens. Another aim was to compare the response to pathogens of nec8.3550 to that of other barley mutants. The study therefore set out to find additional mutants from the same mutated population as was used for the isolation of nec8.3550. It was considered relevant to compare nec8.3550 to mutants from the same genetic background rather than to mutants from other barley cultivars, because different genetic backgrounds may display different levels of interaction with pathogens.

Maintenance and inoculation procedures for Puccinia hordei

The leaf rust pathogen P. hordei was maintained on, and periodically retransferred to, living barley plants of cv. WW Ingrid (W. Weibull AB, Landskrona, Sweden). For maintenance of the rust isolate and for experiments, plants were inoculated by transferring uredospores from infected plants to the first leaves of healthy plants. After inoculation, the plants were maintained in a dark, moist chamber for 18 h. The P. hordei isolate ND9721, which is avirulent on plants carrying the rust resistance gene Rph3, was obtained from Dr B. J. Steffenson, North Dakota, USA. The P. hordei isolate D2, which is virulent on plants carrying the Rph3 gene, was obtained from Dr Roland Jönsson, Svalöf Weibull AB, Svalöv, Sweden. Rust pustules were counted on 1 cm2 leaf areas at different time points after inoculation. The rust experiment was repeated three times.

Maintenance and inoculation procedures for Pyrenophora teres

Isolates of the net blotch pathogen Pyrenophora teres f. sp. teres were obtained from Morten Rasmussen, Svalöf Weibull AB, Svalöv, Sweden, and Hans Lyngs Jørgensen, University of Copenhagen, Frederiksberg, Denmark. Cultures were grown on grass agar for spore production, essentially as described (Weiergang et al., 2002b). Spores were harvested, suspended in water and diluted to a concentration of 13 000 mL−1. The spore suspension was either sprayed on the plants or leaves were submerged into it. Inoculated plants were maintained in plastic bags for 5 days, and then grown in a moist chamber for an additional 3 days before scoring according to a visual scoring scale (Tekauz, 1985). Quantification of spores on the leaf surfaces was done by collecting infected leaves 10–12 days after inoculation. Leaf tissue was weighed and then agitated in water with 0·1% Tween 20. The spore suspension was applied to a Burkner chamber for counting. Spore counts were expressed as the number of spores per milligram fresh leaf tissue. Presence of the fungus in the leaves was confirmed by placing leaves on grass agar and examining the growth of P. teres on the agar surface. The infection experiment with spore counting was repeated three times.

Molecular techniques

RNA preparation and northern blot hybridization, including loading control by hybridization with a rRNA-specific probe, was carried out as described (Falk et al., 1992), with a molecular weight marker R7020 (Sigma-Aldrich). A probe for the barley pathogenesis-related protein 1 gene (PR1) was amplified from barley genomic DNA using primers designed from the sequence of the barley PR1 gene (Bryngelsson et al., 1994). Quantitative PCR (qPCR) of the genes Hv-hir1, Hv-hir2, Hv-hir3, Hv-hir4 and the reference gene ubiquitin 1 (Ubi1) was carried out using previously described primers (Rostoks et al., 2003). qPCR was run as triplicates on three different leaf samples collected at 12 days after sowing. The resulting nine data points were used to calculate a standard deviation and mean fold difference in the amount of transcript compared to the original Bowman Rph3.c line. An anova analysis was used to test for differences between the means of the mutants and Bowman Rph3.c for each gene that was analysed. RNA samples for qPCR were prepared as described (Falk et al., 1992) with a DNase treatment step included (RQ1 RNase-free DNase, Promega Corporation).

Genetic mapping of mutants was done essentially as described (Castiglioni et al., 1998; Persson et al., 2009).

Microscopic examination of the leaves of barley mutants

Mtt8.1661, mtt9.2721, nec9.3091 and nec8.3550 were tested for autofluorescence using a fluorescence microscope with a dichroic mirror of 395 nm, an excitation filter of 365 nm and a barrier filter of 420 nm. Leaf material was harvested 12 days after having been cultivated under long day conditions and was cleared by boiling for 5 min in alcoholic lactophenol (95% ethanol + lactophenol, 2:1). Cleared leaves were mounted on slides in 70% glycerol.

Statistical analysis

Analysis of the data generated from trials with pathogens was performed using the minitab statistics analysis program (MINITAB Inc.) and the statistica program (StatSoft). Student's t-test and one-way anova was used for testing the difference in means between populations. Means of populations with different letters in the figures are significantly different at a level of < 0·05. Segregating F2 populations were analysed using the chi-squared test.

Results

Isolation and initial characterization of barley lesion mimic mutants

From a screen of about 5000 M2 spikes, three mutants were found that were characterized by the presence of necrotic or chlorotic leaf areas in the form of spots or bands. The mutants were originally designated 1661, 2721 and 3091 (Fig. 1), but were renamed Mottled leaf 8.1661 (mtt8.1661), Mottled leaf 9.2721 (mtt9.2721) and Necrotic leaf spot 9.3091 (nec9.3091) based on current nomenclature and allelism tests. The mutants were characterized with respect to their visual appearance, expression of PR1 and the Hv-HIR genes and their interaction with P. hordei and P. teres. In order to relate these mutants properly to previously described barley lesion mimic mutants, previously described barley mutants with aberrant leaf phenotypes (Lundqvist et al., 1997) were cultivated and the phenotypes were compared to those of the newly obtained mutants (Fig. 2).

Figure 1.

The near-isogenic barley Bowman Rph3.c and four lesion mimic mutants grown under long-day conditions (16/8 h light/dark). (a) Leaf phenotypes. Scale bar top left = 0·7 cm. (b) Autofluorescence of leaf tissue with lesions of each respective genotype was examined by using a fluorescence microscope with a dichroic mirror of 395 nm, an excitation filter of 365 nm and a barrier filter of 420 nm. Each fluorescence image insert contains a leaf area with a lesion. Scale bar = 200 μm. BW Rph3.c, Bowman Rph3.c; mtt8.1661, Mottled leaf 8.1661; mtt9.2721, Mottled leaf 9.2721; nec9.3091, Necrotic leaf spot 9.3091; and nec8.3550, Necrotic leaf spot 8.3550.

Figure 2.

Leaf phenotypes of nine mutants and near-isogenic barley Bowman Rph3.c grown under long-day conditions (16/8 h light/dark). Scale bar top left = 0·7 cm. mtt1, mutant mottled leaf 1; mtt2, mutant mottled leaf 2; mtt5, mutant mottled leaf 5; mtt6, mutant mottled leaf 6; nec1, mutant necrotic leaf spot 1. BW Rph3.c, Bowman Rph3.c; mtt8.1661, Mottled leaf 8.1661; mtt9.2721, Mottled leaf 9.2721; nec9.3091, Necrotic leaf spot 9.3091; and nec8.3550, Necrotic leaf spot 8.3550.

Mottled leaf 8.1661 (mtt8.1661), previously described as accession 1661 (Wright et al., 2007), displays chlorotic bands. These bands are most pronounced on the first leaf. Under long day conditions, the bands do not appear on the later emerging leaves and the plants eventually seem to recover from the phenotype conferred by the mutation. Under short-day conditions the mutation is semilethal as all leaves develop the characteristic chlorotic bands (Fig. 1a) and the plants fail to reach maturity and produce seeds. However, the phenotype initially proves to be more severe under long-day conditions. Mtt8.1661 belongs to the propagation class of lesion mimic mutants (Dangl et al., 1996). The cell walls of the leaf tissues that display the bleached phenotype are highly autofluorescent and the fluorescence is more pronounced in the vascular tissues (Fig. 1b). The green tissues of the same mutant show little or no autofluorescence (data not shown). The cell walls are clearly thickened in lesioned tissue. The PR1 gene is expressed at a low but detectable level in mtt8.1661 (Fig. 3). The Hv-hir4 gene is significantly more expressed in mtt8.1661 compared to the Bowman Rph3.c line (Fig. 4). In addition, the Ubi1 (ubiquitin 1) gene, which is included as a reference, is significantly more expressed in mtt8.1661 than in the Bowman Rph3.c line. Mtt8.1661 is phenotypically similar to the previously described mutants Mottled leaf 1 and Mottled leaf 5, which have clearly marked white bands across the leaves (Fig. 2; Lundqvist et al., 1997). Allelism was tested between mtt8.1661 and Mottled leaf 1 and Mottled leaf 5 by performing crosses to the mutants and studying the phenotype in F1. Absence of mutant phenotype in F1 plants indicated that the mutants were non-allelic.

Figure 3.

Expression in leaves of pathogenesis-related protein 1 gene (PR1) in four barley lesion mimic mutants. Total barley RNA was hybridized to a radioactively labelled probe derived from the barley gene for PR1 (a) and to an rRNA-specific probe (b) as a loading control. Lane 1, Bowman Rph3.c; lane 2, Mottled leaf 8.1661 (mtt8.1661); lane 3, Mottled leaf 9.2721 (mtt9.2721); lane 4, Necrotic leaf spot 9.3091 (nec9.3091); and lane 5, Necrotic leaf spot 8.3550 (nec8.3550). Molecular weights derived from a molecular weight standard are indicated to the right.

Figure 4.

Quantification of expression of Hv-HIR genes and the ubiquitin 1 gene (Ubi1) in four barley lesion mimic mutants by real-time PCR. H1, Hv-hir1; H2, Hv-hir2; H3, Hv-hir3; H4, Hv-hir4. Expression was determined as fold of difference in the amount of transcript of mutants compared to the original near-isogenic Bowman Rph3.c, which was assigned the numeric value 1. The results were analysed by t-tests, using Bonferroni corrections. Means sharing the same letter were not significantly different. 1, Bowman Rph3.c; 2, Mottled leaf 8.1661 (mtt8.1661); 3, Mottled leaf 9.2721 (mtt9.2721); 4, Necrotic leaf spot 9.3091 (nec9.3091); and 5, Necrotic leaf spot 8.3550 (nec8.3550).

Mottled leaf 9.2721 (mtt9.2721), previously described as accession 2721 (Wright et al., 2007), is characterized by chlorotic leaf spots and bands that coalesce and eventually form large white patches on the leaves (Fig. 1a). The phenotype is displayed on all leaves. As the leaves mature, the white regions gradually become darker, seeming to undergo necrosis and death. Often the necrosis affects the leaf edges, resulting in wrinkled leaf edges. Under short-day conditions, the leaf phenotype is delayed by several days and is reduced in severity. Eventually all leaves display the phenotype even under short-day conditions. Mtt9.2721 belongs to the propagation class of lesion mimic mutants (Dangl et al., 1996). Autofluorescence is clearly noticeable in the cell walls or apoplastic compartments of the bleached leaf tissues, and is more pronounced in the vascular tissues (Fig. 1b). The green tissues display little or no autofluorescence (data not shown). The cell walls of leaf lesions are clearly thickened. Expression of PR1 is clearly detected (Fig. 3). The Hv-hir1, Hv-hir3 and Hv-hir4 genes are significantly more expressed in mtt9.2721 than in the Bowman line with Rph3.c (Fig. 4). The mutant initially appeared to be similar to Mottled leaf 2 and Mottled leaf 6, which display yellow bands on the leaves (Lundqvist et al., 1997; Fig. 2). However, these mutants do not display the necrosis of mtt9.2721. Allelism was tested between mtt9.2721 and Mottled leaf 2 and Mottled leaf 6 by performing crosses to the mutants and studying the phenotype in F1. Absence of mutant phenotype in F1 plants indicated that the mutants were non-allelic.

Necrotic leaf spot 9.3091 (nec9.3091), previously described as accession 3091 (Wright et al., 2007), has brown spots towards the leaf tips and leaf edges, particularly on the first leaf (Fig. 1a). Short days do not significantly alter the phenotype. Nec9.3091 belongs to the initiation class of lesion mimic mutants (Dangl et al., 1996). There is a relatively low level of autofluorescence in the leaves of nec9.3091 (Fig. 1b). The autofluorescence does not seem to be restricted to the cell walls of the vascular tissues, as for mtt8.1661 and mtt9.2721, but also occurs in the green, non-lesioned tissue. Cell walls also show some thickening in non-lesioned tissues. The PR1 gene is strongly expressed (Fig. 3). The Hv-hir3 and Hv-hir4 genes are expressed significantly more in mutant nec9.3091 than in the Bowman line with Rph3.c (Fig. 4). The leaf phenotype resembles those of mutants Necrotic leaf spot 4 and Necrotic leaf spot 5 (Lundqvist et al., 1997). However, allelism tests were performed between nec9.3091 and Necrotic leaf spot 4 and Necrotic leaf spot 5 by performing crosses to the mutants and studying the phenotype in F1. Absence of mutant phenotype in F1 plants indicated that the mutants were non-allelic.

Necrotic leaf spot 8.3550 (nec8.3550), previously known as accession 3550 (Wright et al., 2007) or Bipolaris sorokiniana tolerant mutant 1 (bst1; Persson et al., 2008, 2009), was renamed according to the accepted nomenclature of barley mutants (Franckowiak & Lundqvist, 2010), in which the two previous names are shown in the new name in the order of: mutant name and its symbol. The proposed nomenclature is considered to be easier to follow through the literature in the future. Nec8.3550 is preferred to bst1 in part because the originally published symbol does not follow the barley nomenclature rules. It is also considered too narrow to name a mutant according to its interaction with a single plant pathogen. When Barley Genetics Stock descriptions of the new loci are published, the other names and suggested gene symbols can be listed in those publications as ‘Previous nomenclature and gene symbolization’. Thus, various names used for the same mutants can be identified and traced to a specific publication. This mutant derives from the same screening of 5000 M2 spikes as the other three LMMs; a brief description is given here including some new observations. Nec8.3550 has conspicuous black or brown spots on the leaves (Fig. 1a). The spots usually do not coalesce. They appear on all above-ground parts of the plant including the awns. Nec8.3550 is delayed in maturation and ripening by about 4 weeks compared to Bowman Rph3.c. Short-day conditions lead to a slightly less pronounced phenotype. Nec8.3550 belongs to the initiation class of lesion mimic mutants (Dangl et al., 1996). Autofluorescence is clearly noticeable (Fig. 1b) and does not seem to be restricted to the cell walls of the vascular bundles, but occurs in all parts of the leaves, also non-lesioned tissue. Cell walls also show some thickening in non-lesioned tissue. The PR1 gene is highly expressed (Fig. 3). The Hv-hir1, Hv-hir2, Hv-hir3 and Hv-hir4 genes are significantly more expressed in nec8.3550 than in Bowman Rph3.c (Fig. 4). Nec8.3550 is similar to Necrotic leaf spot 1 in appearance (Fig. 2; Lundqvist et al., 1997), but allelism can be ruled out due to different mapping positions, nec1 mapping on chromosome 1HL, whereas nec8.3550 maps on chromosome 5HL (Persson et al., 2009).

All mutants were exposed to the artificial inducer of systemic resistance, benzothiadiazole. This treatment did not produce any significant change in leaf phenotype.

The mutants have been made available through the USDA germplasm collection (www.ars-grin.gov/npgs) under the stock numbers GSHO 3597 for Mottled leaf 8.1661 (mtt8.1661), GSHO 3598 for Mottled leaf 9.2721 (mtt9.2721), GSHO 3599 for Necrotic leaf spot 9.3091 (nec9.3091) and GSHO 3600 for Necrotic leaf spot 8.3550 (nec8.3550). They were also deposited in the seed collection of the Nordic Genetic Resource Center (NordGen), Alnarp, Sweden (www.nordgen.org), with the following numbers: NGB 23523 for mtt8.1661, NGB 23524 for mtt9.2721, NGB 23525 for nec9.3091 and NGB 20834 / NGB 23526 for nec8.3550. The mutants mtt8.1661, mtt9.2721 and nec8.3550 were backcrossed a number of times to cv. Bowman to create the near-isogenic lines BW598, BW597 and BW626 (Druka et al., 2011). They were designated the allele names mtt.k, mtt.l and nec.w, respectively (Druka et al., 2011), and were deposited in the NordGen bank with the numbers NGB 22164, NGB 22163 and NGB 22192. The near-isogenic line (NIL) of Bowman with Rph3.c (the third gene identified in barley that confers resistance to P. hordei, allele c) received the number BW746 (Druka et al., 2011), and has been deposited at NordGen with the numbers NGB 22452 and NGB 23527.

Molecular mapping of mutants

Segregation was analysed in the F2 generation of the crosses to cvs Nudinka and Proctor. Chi-squared analysis for all four mutants showed that the test hypothesis for F2 segregation (H0: 3:1 segregation, Bowman Rph3.c:mutant) could not be rejected, suggesting that all mutants are inherited as monogenic recessives. An AFLP-based method was used to map the mutants based on polymorphisms between barley lines Proctor and Nudinka (Castiglioni et al., 1998). However, none of the premapped Proctor–Nudinka AFLP polymorphisms were linked to mutants mtt8.1661 or mtt9.2721. For the mutation in nec9.3091, linkage was detected to AFLP marker E37M33-6 on barley chromosome 3H. The mutation in nec8.3550 is located to chromosome 5HL, distal to the centromere (Persson et al., 2009).

Interactions of nec8.3550, mtt8.1661, mtt9.2721 and nec9.3091 with the biotrophic fungal pathogen Puccinia hordei

The lesion mimic mutants were tested for susceptibility to two different isolates of P. hordei. In mtt8.1661 and nec8.3550 enhanced resistance was detected to the P. hordei isolate that was virulent on Rph3.c (Fig. 5), as significantly fewer pustules formed on these mutants than on Bowman Rph3.c. This was especially obvious in mtt8.1661, where no pustules at all were noted in the bleached tissues, and very few pustules were present in the surrounding green tissue of the first leaf. There was a tendency for fewer pustules to be formed on mtt9.2721 and nec9.3091 than on Bowman Rph3.c, but this difference was not statistically significant. The mutants were also inoculated with a P. hordei isolate that was avirulent for the Rph3 resistance gene. It was found that the mutant phenotypes did not influence the resistance phenotype conferred by the race-specific resistance gene Rph3.

Figure 5.

Infection level of barley leaf rust, caused by Puccinia hordei, in four lesion mimic mutants of barley and the original near-isogenic Bowman Rph3.c. The plants were inoculated by direct application of uredospores by hand. The growth of the pathogen was recorded by counting pustules on the leaf surface. The results were analysed by t-tests, using Bonferroni corrections (Table S1). Means sharing the same letter are not significantly different. BW Rph3.c, Bowman Rph3.c; mtt8.1661, Mottled leaf 8.1661; mtt9.2721, Mottled leaf 9.2721; nec9.3091, Necrotic leaf spot 9.3091; and nec8.3550, Necrotic leaf spot 8.3550. < 0·001 for all pairwise comparisons that showed significant differences between means, α = 0·05.

Interactions of nec8.3550, mtt8.1661, mtt9.2721 and nec9.3091 with the necrotrophic barley pathogen Pyrenophora teres

The lesion mimic mutants were analysed with respect to their interaction with the necrotrophic pathogen, P. teres f. sp. teres. Tests revealed that Bowman Rph3.c was mildly susceptible to P. teres and that the reaction was similar in all 84 Bowman Rph3.c plants tested. The susceptibility was confirmed by counting the number of spores produced by P. teres on the leaf surfaces of Bowman Rph3.c and the mutants (Fig. 6). Especially high spore accumulations were present on leaves of mtt8.1661. Stereomicroscopic investigation confirmed the profuse sporulation of P. teres on leaf surfaces of mtt8.1661 and mtt9.2721 (data not shown). However, no spores or other fungal structures were observed through the stereomicroscope on leaf surfaces of Bowman Rph3.c, nor on nec9.3091 or nec8.3550. Nevertheless, spore counts revealed the presence of the fungus on these mutants and on Bowman Rph3.c (Fig. 6). Nec8.3550 supported spore levels comparable to those of mtt9.2721, which were both significantly higher than those supported by Bowman Rph3.c. Infected leaves were also placed on grass agar plates 8 days after inoculation to examine whether P. teres was present. After a few days on a grass agar surface, P. teres mycelium had emerged from all leaf pieces, essentially showing that the fungus was present in all the mutants and in Bowman Rph3.c (data not shown).

Figure 6.

Infection level of the barley net blotch pathogen, Pyrenophora teres f. sp. teres, in four lesion mimic mutants of barley and the original near-isogenic Bowman Rph3.c. Spores from leaves were suspended in water supplemented with 0·1% Tween 20, 10–12 days after inoculation. The number of spores per cm2 leaf tissue was calculated. The results were analysed by t-tests, using Bonferroni corrections (Table S2). Means sharing the same letter were not significantly different. BW Rph3.c, Bowman Rph3.c; mtt8.1661, Mottled leaf 8.1661; mtt9.2721, Mottled leaf 9.2721; nec9.3091, Necrotic leaf spot 9.3091 and nec8.3550, Necrotic leaf spot 8.3550. < 0·01 for all pairwise comparisons that showed significant differences between means, α = 0·05.

Initially, attempts were also made to quantify P. teres infection of barley leaves according to a visual scoring scale of disease symptoms (Tekauz, 1985). The scoring indicated that mtt8.1661 and mtt9.2721 were more susceptible than Bowman Rph3.c, whereas the responses of nec9.3091 and nec8.3550 were essentially indistinguishable from that of Bowman Rph3.c. However, because the symptoms caused by P. teres infection resembled the mutant phenotypes of nec9.3091 and nec8.3550 (Fig. 7), spore counts and microscopic investigations of the leaf surfaces were carried out as described.

Figure 7.

Leaves of four lesion mimic mutants of barley and the original near-isogenic Bowman Rph3.c, which are infected with the barley net blotch pathogen, Pyrenophora teres f. sp. teres. Scale bar top left = 0·7 cm. The insert displays an enlarged leaf area of Necrotic leaf spot 8.3550 (nec8.3550), originating from the area boxed in black on the large photo. The top arrow points to a long-stretched dark brown lesion, indicative of barley net blotch symptoms. The bottom arrow indicates a brown circular lesion, caused by the mutant phenotype. BW Rph3.c, Bowman Rph3.c; mtt8.1661, Mottled leaf 8.1661; mtt9.2721, Mottled leaf 9.2721; nec9.3091, Necrotic leaf spot 9.3091 and nec8.3550, Necrotic leaf spot 8.3550.

Table 1. Summary of characteristics of barley Bowman line Rph3.c and four lesion mimic mutants
Barley genotypeaMutation in chromosome numberbAppearancecAutofluorescence in leaf tissuecPR-1 expressionSporulation of P. hordei race D2dSporulation of P. tereseExpression of Hv-hir1, 3 and 4fExpression of Ubi1g
  1. a

    Abbreviated names of mutants: mtt8.1661, Mottled leaf 8.1661; mtt9.2721, Mottled leaf 9.2721; nec9.3091, Necrotic leaf spot 9.3091; and nec8.3550, Necrotic leaf spot 8.3550.

  2. b

    Chromosome number 3 is also known as 3H; chromosome 7 is also known as 5H, depending on the nomenclature system used.

  3. c

    See Figure 1 for visual appearance. Autofluorescence in leaf tissue was determined by fluorescence microscopy.

  4. d

    P. hordei, Puccinia hordei. Spores from infected leaves were suspended in H2O and the spore concentration was determined as spores/mg leaf. +++, >1·5 × 103; ++, 0·8–1·5 × 103; +, <0·8 × 103 spores/mg leaf.

  5. e

    P. teres, Pyrenophora teres. Spores from infected leaves were suspended in H2O and the spore concentration was determined as spores/mg leaf. +++, >1·0 × 103; ++, 0·09–1·0 × 103; +, <0·09 × 103 spores/mg leaf.

  6. f

    Hv-hir1, 3 and 4, the Hordeum vulgare hypersensitive induced reaction (HIR) genes. Hv-hir2 expression is not included, because there were few significant differences. Expression was quantified by quantitative PCR (qPCR).

  7. g

    Ubi1, the ubiquitin 1 gene from H. vulgare. Expression was quantified by qPCR.

Bowman Rph3.cN/AGreen leavesNoneNone++++
mtt8.1661 NDChlorotic bandsIn bleached tissueVery low(+)++++++
mtt9.2721 NDChlorotic, white spots and bandsIn bleached tissueHigh+++++++
nec9.3091 3(3H)Brown spotsLowHigh++++
nec8.3550 7(5H)Black or brown spotsLow; mainly in necrotic spotsVery high++++++/−

Discussion

Three novel barley lesion mimic mutants are described and their interaction with pathogens is characterized. Nec8.3550, a previously described LMM from the same mutant batch, was included in the study. The characteristics of the mutants are summarized in Table 1. Barley mutants with leaf spots and bands have been isolated by others (Lundqvist et al., 1997; Rostoks et al., 2003, 2006), and some of these mutants, such as mlo and nec1, have been tested with respect to their interaction with pathogens (Kumar et al., 2001; Keisa et al., 2011). The present mutants are not identical to the mlo mutants for several reasons. First, the leaf lesions of mlo mutants have a different appearance from those of the mutants of this study. Secondly, the mutants described here are susceptible to powdery mildew (unpublished observations), whereas the mlo mutants are highly resistant to powdery mildew. In addition, the mlo locus is situated on chromosome 4H, whereas the mutations of nec9.3091 and nec8.3550 map to chromosomes 3H and 5H, respectively. The leaves of nec1 are very similar in appearance to those of nec8.3550. However, the nec1 mutation is located on yet another chromosome (1H). Similarities to the Arabidopsis HLM1 gene led to the finding that the nec1 locus encodes the cyclic nucleotide-gated ion channel 4 protein (Rostoks et al., 2006). The cyclic nucleotide-gated ion channel 2 gene maps to the same region as nec8.3550 but it does not map to, the nec8.3550 locus.

Mtt8.1661 was unique among the mutants by being extremely susceptible to P. teres. This pathogen is usually considered to be necrotrophic (Jørgensen et al., 1998). As necrotrophs preferentially invade the plants through dead or dying tissue, mutants that display wilted leaves would be expected to be more susceptible to necrotrophs, particularly if the outer wax layer barrier is absent or thin, as it appears to be in mtt8.1661. These results are similar to those obtained with the albostrians mutant, which was supersusceptible to the hemibiotroph Bipolaris sorokiniana (Schäfer et al., 2004). Necrotrophs are usually considered to be dependent on cell death of the host for infection. Initially, cell death may be caused by such means as physical wounds, attack from other pathogens or may even occur spontaneously, thus providing an initial entrance port for the pathogen, facilitating the colonization of plant tissues (Govrin & Levine, 2000). For the ensuing stages of infection, necrotrophs seem to be dependent on toxins that cause cell death (Weiergang et al., 2002a). In some cases, the toxins produced by necrotrophs may cause cell death by stimulating the plant's own immunity towards biotrophs (Hammond-Kosack & Rudd, 2008; Deller et al., 2011). This implies a dichotomy in plant defence against biotrophs versus necrotrophs, such that a pre-existing state of defence against biotrophs may predispose the plant for attack by a necrotroph (Lorang et al., 2007; Hammond-Kosack & Rudd, 2008; Oliver & Solomon, 2010). This difference is also implicated in the different reactions of mtt8.1661 to the necrotrophic P. teres as opposed to the biotrophic P. hordei, although the molecular reason for this is currently not known. One possible explanation is that the photobleached leaf tissues in mtt8.1661 are depleted of readily available nutrients and therefore not readily accessible to the biotrophic P. hordei. Again, similar results were reported from the albostrians mutant, which displayed enhanced resistance to Blumeria graminis f. sp. hordei in photobleached leaves (Jain et al., 2004), whereas green leaves still supported growth of the pathogen. Similarly, mtt8.1661 supported some growth of P. hordei in the segments of green tissue that were alternating the portions of photobleached tissue. The lower susceptibility of mtt8.1661 to rust does not appear to be connected to its expression of PR1, because PR1 is expressed at approximately the same levels in this mutant as in Bowman line with Rph3.c.

In mtt9.2721, significant growth of P. hordei was observed in photobleached tissues that were still green at the time of inoculation. Bleaching is therefore not by itself sufficient for inhibiting leaf rust growth, because mutant mtt9.2721 showed significant bleaching while supporting leaf rust levels comparable to those of Bowman Rph3.c. The photobleaching phenotype of mutant mtt9.2721 was not as severe and extensive as that of mutant mtt8.1661 and was also delayed by at least 3 days compared to mtt8.1661. This raises the hypothesis that if bleaching occurs several days after inoculation, the pathogen may still be able to complete its infection. Mtt9.2721 allowed a clearly observable and profuse leaf surface sporulation by P. teres, although not quite as profuse as in mtt8.1661. An interesting observation is that both the mutants that showed a strong bleaching phenotype (mtt9.2721 and mtt8.1661) allowed profuse leaf surface sporulation by P. teres. The autofluorescence in mtt9.2721 and mtt8.1661 was restricted to the apoplastic compartment. It is suggested that cell wall reinforcing compounds, such as lignin precursors, may be responsible for the autofluorescence in mtt9.2721 and mtt8.1661. This interpretation also seems consistent with the thick cell walls that were observed especially in mtt9.2721 and mtt8.1661.

Nec9.3091 was slightly less susceptible to leaf rust than Bowman Rph3.c. The reason for the relative susceptibility of nec9.3091 and mtt9.2721 compared to mtt8.1661 and nec8.3550 is probably that their leaves are green, i.e. free from mutant lesions, for a longer time during early development. The rust could infect them while the tissue was still free from lesions, and as infection progressed the fungus was apparently not obstructed in its further development, although the leaf tissues started to form spontaneous lesions. The autofluorescence of nec9.3091 and nec8.3550 was not restricted to the apoplastic compartment. It is hypothesized that barley phytoalexins such as coumaroyl-agmatine may be responsible for the autofluorescence in these two mutants.

For nec8.3550, it is estimated that about 10% of the total leaf area displayed necrotic lesions. As the leaf rust in nec8.3550 grew to only 26% of the levels in Bowman Rph3.c, it is unlikely that this effect would be due only to the fungus avoiding the necrotic areas. A more plausible explanation is the presence of a state of continuous defence, similar to systemic acquired resistance, SAR. Nec8.3550, and also mtt9.2721 and nec9.3091, display an increase in autofluorescence and expression of PR1. It is therefore hypothesized that they have entered SAR, a condition known to occur in many lesion mimic mutants (Lorrain et al., 2003). SAR is often accompanied by autofluorescing tissues and high expression of PR1, traits that are normally absent in Bowman Rph3.c plants (Figs 1 and 3). Mtt8.1661 did not show a significant expression of PR1 or the Hv-HIR genes, thus suggesting that mechanisms other than SAR may be active in its defence response. However, a problem with linking SAR to expression of particular defence genes is that the correlation between gene expression and SAR seems to be unclear, at least in cereals (Molina et al., 1999). The expression of Hv-HIR genes were previously tested in barley lesion mimic mutants (Rostoks et al., 2003). The results presented here support the conclusion that the Hv-hir3 gene is especially prone to being highly expressed in lesion mimic mutants. In nec8.3550, Hv-hir3 reached an expression level that is about 200-fold higher than that found in Bowman Rph3.c. Nec8.3550 displays significant expression differences for all Hv-HIR genes and also for the reference Ubi1, in comparison to Bowman Rph3.c. This could indicate a global alteration of gene expression in nec8.3550 and should prompt a more comprehensive expression analysis of this mutant. The Hv-HIR genes encode proteins with a SPFH domain, which is found in stomatins and prohibitins and is thought to be involved in control of membrane ion channels (Tavernarakis et al., 1999; Nadimpalli et al., 2000; Rostoks et al., 2003). Over-expression of Hv-hir3 may lead to aberrant regulation of membrane ion channels that may lead to the lesion mimic phenotype (Rostoks et al., 2003). Another possibility is that over-expression of Hv-HIR genes is more directly causing the observed changes in resistance or susceptibility to pathogens. In rice (Zhou et al., 2010) and pepper (Jung & Hwang, 2007), it has been shown that HIR proteins interact physically with proteins containing extracellular leucine-rich repeats. Similar proteins have been implicated as possible pathogen recognition receptors. A hypothesis for further research could therefore be that over-expression of the Hv-HIR genes leads to defence responses via activation of pathogen recognition receptors.

A connection between LMM and plant defence is indicated by the fact that some LMM suppressor mutant genes, in particular LAZ5 and ADR1, encode R proteins (Palma et al., 2010; Bonardi et al., 2011), albeit with unknown pathogen specificity. ADR1 was identified from the phoenix21 suppressor mutant of the Arabidopsis lsd1 LMM, which reacts with a ‘runaway cell death’ in response to various stimuli (Bonardi et al., 2011). ADR1 appears to regulate accumulation of the defence hormone salicylic acid. Clearly, screenings for LMM suppressor mutants can be a very constructive way of elucidating the function of the gene that has been mutated in the original LMM, as well as enhancing the understanding of plant defence functions. To this end, the nec8.3550 mutant was remutagenized and a set of suppressor mutants were identified (Persson et al., 2008). The suppressor mutants showed varying levels of resistance to the barley spot blotch pathogen, Bipolaris sorokiniana. However, the increased resistance to B. sorokiniana seen in nec8.3550 was essentially lost in the suppressor mutant that most greatly suppressed spontaneous lesion development in nec8.3550.

The mutants described in this work could be of interest in breeding barley for disease resistance. However, the strong lesion phenotype would most likely limit their direct usefulness due to the lowered photosynthetic and biomass production potential. The remutagenesis approach that was applied to nec8.3550 could possibly resolve the problem. Double mutants would be selected that had lost undesirable phenotypic traits whilst maintaining their level of pathogen resistance. In a similar fashion, the disease resistance expressed in the three novel barley mutants could be exploited and analysed further through remutagenesis.

Acknowledgements

Dr Jerome D Franckowiak is acknowledged for discussions concerning barley lesion mimic mutants and for critical reading of the manuscript. Dr Hans Lyngs Jørgensen has kindly provided us with a virulent isolate of P. teres f. sp. teres and protocols for pathogenicity testing. The authors are indebted to Dr Udda Lundqvist for her contribution regarding the changed and consistent nomenclature of mutants (Franckowiak & Lundqvist, 2010). Maselaboratorierna AB, Uppsala, Sweden is gratefully acknowledged for kindly having provided laboratory space and access to equipment. The work was funded by the Swedish Research Council for Forestry and Agriculture (SJFR, now Formas) through a grant to ABF. SAIW and MA received financial support from the Swedish Farmers' Foundation for Agricultural Research (SLF). The authors state that they have no conflicts of interest to declare.

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