• Open Access

Functional variability of the Lr34 durable resistance gene in transgenic wheat

Authors


  • Accession numbers for EMBL seq database: FJ436983.

(fax +41 44 634-8204; emails evans.lagudah@csiro.au; bkeller@botinst.uzh.ch)

Summary

Breeding for durable disease resistance is challenging, yet essential to improve crops for sustainable agriculture. The wheat Lr34 gene is one of the few cloned, durable resistance genes in plants. It encodes an ATP binding cassette transporter and has been a source of resistance against biotrophic pathogens, such as leaf rust (Puccinina triticina), for over 100 years. As endogenous Lr34 confers quantitative resistance, we wanted to determine the effects of transgenic Lr34 with specific reference to how expression levels affect resistance. Transgenic Lr34 wheat lines were made in two different, susceptible genetic backgrounds. We found that the introduction of the Lr34 resistance allele was sufficient to provide comparable levels of leaf rust resistance as the endogenous Lr34 gene. As with the endogenous gene, we observed resistance in seedlings after cold treatment and in flag leaves of adult plants, as well as Lr34-associated leaf tip necrosis. The transgene-based Lr34 resistance did not involve a hypersensitive response, altered callose deposition or up-regulation of PR genes. Higher expression levels compared to endogenous Lr34 were observed in the transgenic lines both at seedling as well as adult stage and some improvement of resistance was seen in the flag leaf. Interestingly, in one genetic background the transgenic Lr34-based resistance resulted in improved seedling resistance without cold treatment. These data indicate that functional variability in Lr34-based resistance can be created using a transgenic approach.

Introduction

Fungal pathogens are a major cause of yield losses in wheat. The Food and Agricultural Organization of the United Nations estimates 676 million tonnes of wheat will be harvested in 2011 (GIEWS, 2011), and resistance to fungal pathogens is fundamental to global food security. To reduce crop losses, wheat production is dependent on new and improved cultivars with resistance to the rapidly evolving biotrophic wheat rust diseases, such as leaf rust, stripe rust (Puccinia striiformis f. sp. tritici) and stem rust (Puccinia graminis).

There are two distinct groups of genes providing resistance to biotrophic pathogens such as wheat rusts: (i) R genes, which confer complete, but mostly race-specific resistance based on effector-triggered immunity (Jones and Dangl, 2006) and (ii) genes providing partial or quantitative resistance (QR) (Singh et al., 1991). QR genes are usually more durable than R genes and can confer race nonspecific and broad-spectrum resistance (Kou and Wang, 2010). In contrast to the immunity triggered by R proteins, QR genes do not abolish, but rather slow down disease progression against compatible infection types (Singh et al., 2005). The quantitative effect of QR genes, and the fact that they are acting predominantly at the adult plant stage, make these genes difficult to work with. As a result, R genes have traditionally been the focus in breeding programmes. However, cultivars with resistance based solely or predominantly on R genes are particularly susceptible to the emergence of highly virulent pathotypes. A recent prominent example is the wheat stem rust race, Ug99 (TTKS) (Pretorius et al., 2000). ‘Pyramiding’ QR and R genes into plants provides a method of maintaining a high level of disease resistance for longer periods, making it an increasingly attractive option for breeders and farmers alike.

To date, only a few durable rust resistance genes have been genetically identified in wheat. These include the leaf rust resistance genes Lr34 (Yr18/Pm38) (Dyck et al., 1966; McIntosh, 1992; Singh, 1992; Spielmeyer et al., 2005), Lr46 (Yr29/Pm39) (Lillemo et al., 2007, 2008; Suenaga et al., 2003) and potentially Lr67 (Yr46) (Herrera-Foessel et al., 2010; Hiebert et al., 2010), and the stripe rust resistance gene Yr36 (WKS1) (Uauy et al., 2005). The broad-spectrum resistance of Lr34 and Lr46 to stripe rust and powdery mildew (Blumeria graminis—observed in field trials) and of Lr67 to stripe rust suggests that they are involved in a more general plant defence response than R genes. To date, only Yr36 and Lr34 have been cloned (Fu et al., 2009; Krattinger et al., 2009). Yr36 encodes a kinase-START protein and Lr34 an ATP binding cassette (ABC) transporter belonging to the ABCG (formerly PDR) subclass. Although the diverse nature of these two proteins fails to identify a common mechanism for QR genes, the resistance based on both genes seems to be regulated by environment or development: Lr34 is most effective late in the plant development, and Yr36 requires high-temperature induction for full resistance.

Lr34 has proven to be a durable source of resistance against fungal pathogens, such as leaf rust, stripe rust and powdery mildew, for over 100 years (Kolmer et al., 2008). Hexaploid wheat contains three homoeologous copies of Lr34 on chromosomes 7A, 7D and 4A (which contains a translocation from chromosome 7B). The 7A copy is disrupted by multiple retroelement insertions, and the 7D-copy of Lr34, named Lr34-D, occurs in multiple allelic forms (Dakouri et al., 2010; Lagudah, 2011). There are two predominant forms, Lr34sus-D and Lr34res-D (Krattinger et al., 2011), with the latter being responsible for the resistance (subsequently referred to as Lr34res). Lr34res is typically described as a slow rusting gene that is most effective at adult stage; however, seedlings can also exhibit resistance when exposed to cold temperatures (Rubiales and Niks, 1995; Singh and Gupta, 1992). Lr34res was found to enhance the expression of some race-specific resistance genes and act additively in combination with other slow rusting genes (German and Kolmer, 1992; Singh and Rajaram, 1992). Although the mechanism of quantitative resistance provided by Lr34res is poorly defined, the response is non-hypersensitive and results in decreased infection frequency, increased latency period, higher rates of abortion and smaller colonies. The reduction in haustorium formation associated with Lr34res is not because of papillae formation but results from reduced intercellular hyphal growth (Rubiales and Niks, 1995; Singh and Huerta-Espino, 2003). Lr34res expression also induces a leaf tip necrosis (LTN) phenotype in flag leaves of adult wheat plants in some environments (Dyck, 1991; Lagudah et al., 2006), which was shown to involve senescence-like processes (Krattinger et al., 2009).

In this study, we have made transgenic wheat lines with Lr34res under the control of the native promoter that show the introduction of Lr34res is sufficient to confer rust resistance in a susceptible background. Furthermore, LTN developed in the flag leaves of transgenic lines and involved a senescence-like process. The transgenic lines were used to investigate the histopathological and transcriptional changes of Lr34res and three pathogenesis-related (PR) genes occurring during infection with rust and were compared with Thatcher (Th or Th + Lr16) and Th + Lr34, which carry the endogenous Lr34sus-D and Lr34res respectively. As expected, the highest level of Lr34res expression was seen in flag leaves and the resistance response was non-hypersensitive. Moreover, the insertion of the Lr34res transgene into wheat cv. Bobwhite resulted in increased gene expression in some lines, and we present evidence of the introduced Lr34res working additively with other resistance genes. This generally improved the levels of resistance exhibited by the plants at a range of developmental stages and temperatures, most notably in seedlings without cold treatment, and highlights the importance of genetic background in Lr34res breeding programmes.

Results

Transgenic wheat with the Lr34res gene is resistant to leaf rust and shows LTN

Two selections of the wheat cultivar Bobwhite SH 98 26, subtypes BW26AUS and BW26SUI, were transformed by particle bombardment with a construct containing the full-length Lr34res gene (11 868 bp). The coding sequence has 24 exons and was extended by 2356 bp of promoter sequence and 1758 bp of 3′UTR sequence. This sequence was released as a single fragment by NotI digestion from the plasmid pWGEM-NZf(+)Lr34res. Transformation was either carried out with the complete plasmid (BW26AUS background) or with the NotI-released gene cassette (BW26SUI background). A total of 271 plantlets were regenerated, and eight transformants with the full-length Lr34res construct were obtained—five in the BW26AUS background (#5, 98, 171, 175, 190) and three in the BW26SUI background (#49, 54, 87). Much of the difficulty in obtaining T0 transformants containing the full-length insert can be attributed to the biolistic transformation process used for the insertion of this large construct. Full-length insertions in BW26AUS transgenic lines were identified by genomic DNA blots. There, NotI digested transformant DNA was probed with Lr34 promoter and 3′UTR probes consecutively, thus allowing selection of transformants with full-length insertions. In the BW26SUI transgenic lines, full-length transgene transcript was amplified, subcloned and sequenced to confirm expression of the complete transgene and to distinguish it from the endogenous Lr34sus-D transcript. Analysis of subsequent generations used a combination of PCR screens and genomic blots.

To test resistance to leaf rust in the eight transgenic lines, infected seedlings were exposed to a cold treatment as this induces Lr34res resistance in seedlings (Singh and Gupta, 1992). The varieties Thatcher, BW26AUS and BW26SUI were all susceptible to the leaf rust pathotype Mackellar, with BW26SUI also showing some chlorosis around the pustule margins (Figure 1a and b). All lines identified as containing the full-length Lr34res construct showed resistance to leaf rust in cold-treated seedlings (Figure 1a and b). As endogenous Lr34res is often described as an adult plant resistance gene, a subset of these lines was then analysed for leaf rust resistance on flag leaves. Rust colonization was reduced in the Th + Lr34 line compared with the Thatcher control (Figure 1c). The difference in resistance was even more pronounced in the Lr34res transgenic lines when compared with the negative control 87 and 190 sibling (sib) lines without the transgene (Figure 1d and e). Interestingly, in addition to leaf rust resistance, line 171 showed chlorotic spotting on the leaves (Figure 1e). This chlorotic phenotype occurred even in the absence of pathogen exposure and plants also exhibited a substantially reduced seed set (data not shown).

Figure 1.

 Transgenic wheat lines show increased resistance to leaf rust. Seedlings were infected with leaf rust, pathotype Mackellar and the second leaf assessed after 35 dpi at 4 °C (Switzerland) (a) or 28 dpi at 10 °C (Australia) (b). Flag leaves were grown under standard glasshouse conditions, inoculated with leaf rust and pictures taken 31 dpi (c) and (e) or 26 dpi (d), scale bar represents 10 mm. In all cases, transgenic lines showed resistance to leaf rust when compared with BW26 and Th + Lr34 leaves showed less sporulating pustules compared to Thatcher. Microscopic analysis of leaf rust infection (7 dpi, standard glasshouse conditions) using WGA-alexa staining (f), scale bar represents 100 μm. Quantitative analysis of colony size on 3–4 flag leaves per line (g). Boxplot whiskers depict data range, grey boxes depict data within the upper and lower quartile, and horizontal black bars show median values. The tested lines containing Lr34res showed significantly smaller colony size compared with the Thatcher and 190 sib controls. Letters a, b and c indicate lines with equivalent colony size (least significant difference, < 0.05— Table S1).

Microscopic analysis of flag leaves 7 days post infection (dpi) showed reduced colony growth in Th + Lr34 and the Lr34res transgenic lines corroborating the macroscopic findings (Figure 1f). Quantification of this reduction in growth revealed that all lines have some colonies that fail to grow following penetration of the leaf (Figure 1g). However, both negative control lines (Thatcher and 190 sib) show a greater number of larger, established colonies. As the colony area did not follow a normal distribution, the nonparametric Mann–Whitney U test was used to test for significance. Statistical analysis of colony area showed that those lines containing Lr34res had significantly smaller infection sites than the control lines (< 0.05, Table S1). Interestingly, the colonies on line 190 were also significantly smaller than those of Th + Lr34.

Endogenous Lr34res expression is known to induce LTN in flag leaves of adult wheat plants (Dyck, 1991; Lagudah et al., 2006). LTN was found to be a reliable marker for the detection of Lr34res in wheat cultivars grown in the field (Shah et al., 2011). However, the phenotypic expression of this trait depends strongly on genetic background and the environment, both in the field and in the glasshouse. As the LTN phenotype of breeding lines with an endogenous Lr34res is strongly expressed under greenhouse conditions in Zürich, experiments related to LTN were made under these conditions. Lr34res-induced LTN was consistently observed in the genetic background of Th + Lr34, but not its near-isogenic line, Thatcher (Figure 2a). The flag leaves of the Lr34res transgenic lines 54, 171 and 190 all developed strong LTN when compared to the phenotype of non-transgenic 190 sib and 54 sib lines at the equivalent developmental stage (Figure 2b and c). LTN was also induced in line 49, but not in line 87 (data not shown). Interestingly, Lr34res transgenics challenged with rust and exposed to a cold temperature treatment at the seedling stage showed a much stronger LTN than plants grown in the glasshouse without any treatment, suggesting that elevated Lr34res levels increase the extent of LTN (results not shown).

Figure 2.

 Transgenic wheat lines exhibit LTN. The LTN phenotype is present in the flag leaves of Th + Lr34 and transgenic lines 54, 190 and 171, but not in Thatcher or the sib lines at the same developmental stage (a–c). (d) Analytical HPLC trace (recorded at 320 nm) on leaves showing LTN (190) and control leaves (190 sib); inset: UV/Vis-traces of the fraction containing the NCC with the characteristic absorption spectra at 211 and 315 nm.

It was earlier suggested that the LTN phenotype was the result of premature leaf senescence (Krattinger et al., 2009). Evidence for this hypothesis was provided by the presence of non-fluorescent chlorophyll catabolites (NCCs) (Oberhuber et al., 2003) in Th + Lr34 flag leaves, but not in Thatcher (Krattinger et al., 2009). High pressure liquid chromatography (HPLC) analysis of flag leaf extracts from transgenic line 190 showed a peak at 21-min retention time (Figure 2d), as seen in the Th + Lr34 flag leaves. This peak had the characteristic UV/Vis-spectra, with wavelength maxima at 214 and 315 nm (inset), previously detected in Th + Lr34 flag leaves indicating the presence of chlorophyll catabolites. As expected, this peak was absent in the 190 sib trace. These data indicate that the LTN observed in the transgenic lines corresponds to the LTN induced by Lr34res in the Thatcher background and provide further evidence for senescence being associated with Lr34res expression.

In conclusion, these complementation experiments demonstrate that expression of introduced Lr34res in a susceptible wheat background reproduces the resistance and LTN phenotypes observed in the endogenous Lr34res control line Th + Lr34.

Lr34res gene expression is elevated in flag leaves and is typically enhanced by infection

Quantitative analysis of Lr34res expression was performed to answer the following questions: (i) How do the levels of Lr34res expression in transgenic lines compare with lines containing endogenous Lr34res? (ii) Do age and cold treatment alter expression levels? (iii) Does infection promote Lr34res expression? Analysis was carried out using transgenic lines 171 and 190 with 190 sib, Thatcher and Th + Lr34 as controls. Resistance in wheat containing the endogenous Lr34res gene is age dependent and temperature sensitive (Singh and Gupta, 1992). Therefore, comparative analysis of temperature and developmental stage on Lr34res transcript levels was assessed, as was the effect of pathogen infection. Samples were taken 72 hours post infection (hpi), allowing a 48-h cold treatment (10 °C) starting 24 h after pathogen infection where required. To determine Lr34res gene expression, quantitative real-time PCR (qPCR) assays used Lr34res allele specific primers (Table S2, Figure S1).

As expected, both the negative controls, Thatcher and 190 sib, did not express Lr34res (Figure 3). The expression in transgenic lines followed a similar overall trend to Th + Lr34. The transgenic seedling expression levels were typically higher when compared to the Th + Lr34 seedling expression levels. However, no consistent infection or cold-dependent up-regulation was observed in the two transgenic lines analysed (the statistical analysis is shown in Table S3). Of particular interest was the level of expression in the cold-treated Th + Lr34 infected seedlings, as this treatment results in resistance to pathogen attack (Figure 1a). These Th + Lr34 samples showed significant up-regulation of Lr34res transcript compared with mock-infected, cold-treated controls, but not with infected seedlings grown at 20 °C. Therefore, we conclude that cold temperatures do not affect Lr34res transcript levels.

Figure 3.

Lr34res expression in seedlings and adult plants 72 h after infection with leaf rust. Quantitative measurements of Lr34res expression were taken in five different wheat lines ± infection. Infection/mock infection was carried out under three conditions; seedlings grown in the glasshouse (∼20 °C), seedlings with cold treatment (∼10 °C) and flag leaves (∼20 °C). Thatcher and 190 sib do not contain the Lr34res allele and were included as negative controls. sl refers to the second leaf of the seedling and fl to flag leaf. An asterisk (*) depicts expression levels that were significantly different from the mock-infected control (as assessed using REST 2009). Statistical analysis of the data is presented in Table S3. Expression levels were normalized to the GAPDH and Tubb4 genes. Seedling leaf expression levels, n (number of biological replicates) = 3. Flag leaf expression levels, n = 4. Bars = 1 standard error (SE). Results are relative to an internal control (mock-infected Th + Lr34a, 10 °C = 1).

Although infection generally enhanced Lr34res expression across all environmental and developmental stages, the variability seen in the transgenic lines could also be due to the positional effects of transgene integration and/or the absence of Lr34res-specific regulatory mechanisms in seedlings. The most consistent finding of the quantitative expression analysis was the significantly elevated level of Lr34res expression in the mock-infected and infected flag leaves when compared to all seedling samples across all Lr34res containing lines, with the exception of mock-infected 190 (Table S3). Line 171 had the highest level of expression in both mock and rust-infected flag leaves, which may contribute to the chlorotic leaf spots (see Figure 1e) and reduced seed set observed in this line. In conclusion, quantitative expression analysis showed a very good correlation of the increased resistance to pathogen attack seen in adult plants with elevated Lr34res expression, whereas factors other than expression levels must control cold-induced resistance in seedlings. We hypothesize that the resistance seen in cold-treated seedlings is caused by slower growth of the fungus or the Lr34res-dependent accumulation of a growth inhibiting substance under cold conditions.

Genetic backgrounds affect Lr34res-mediated seedling resistance without cold treatment

At the seedling stage endogenous Lr34res-mediated resistance shows a high degree of phenotypic variability and is difficult to detect (Wamishe and Milus, 2004). As low temperature and low light intensity intensify the effect of Lr34res in seedlings, we hypothesized that, compared to endogenous Lr34res in Th + Lr34, the elevated Lr34res transcript levels in the transgenic lines may provide seedling resistance. We therefore assessed seedling resistance in the Lr34res transgenic lines 171, 190, 49, 54 and 87. These plants were infected 10 days after planting (two-leaf stage) and incubated for 12 days at 20 °C. The first leaf was used to assess infection phenotype and the second leaf was used for quantitative Lr34res expression analysis. Control leaves of Thatcher and Th + Lr34 were similarly susceptible to Mackellar at 12 dpi (Figure 4a), confirming previous findings (Wamishe and Milus, 2004). Macroscopic analysis of the transgenic lines 171 and 190 (BW26AUS background) revealed an infection phenotype similar to the 190 sib line. Sporulating pustules were also visible on all non-transgenic sib lines generated in the BW26SUI background. In contrast, the first leaves of transgenic lines 54, 87 and 49 were resistant, showing chlorosis but no sporulating pustules (Figure 4a). The effect was strongest in lines 54 and 87, whereas some very small pustules were visible in line 49. Interestingly, BW26SUI and the sib lines with this background showed a different infection phenotype and higher background resistance than the 190 sib line generated in the BW26AUS background. This was apparent as a strong chlorosis around pustules on the first leaf (Figure 4a) indicating that the genetic background of BW26SUI and BW26AUS is different and that genetic factors specific to BW26SUI are acting additively with Lr34res to give seedling resistance.

Figure 4.

 Genetic background affects Lr34res-mediated seedling resistance at ambient temperatures. (a) Levels of resistance observed in first leaves of seedlings at 12 dpi with pathotype Mackellar under standard glasshouse conditions. As expected, the two control lines Thatcher and Th + Lr34 showed similar levels of susceptibility when grown at 20 °C. Likewise, transgenic lines 171 and 190 (BW26AUS background) were comparable to the 190 sib line. In contrast, transgenic lines 54, 87 and 49 (BWS26SUI background) showed greater resistance than their corresponding sib line. (b) Quantitative Lr34res expression in leaf 2 compared to the endogenous transcript in the Th + Lr34 control leaf. Expression levels were normalized to the GAPDH and Tubb4 genes and shown relative to Th + Lr34 = 1, n = 3, Error bars = 1 SE.

Quantitative measurements of Lr34res expression in the second leaf of transgenic seedlings and controls grown at 20 °C at 12 dpi revealed large variations between the analysed transgenic lines (Figure 4b). These ranged from 2.4-fold (line 87) to 21.9-fold (line 54) when compared to the endogenous Th + Lr34 Lr34res transcript level. There is no clear correlation between the observed resistance and the expression level, for example, line 49 has higher expression than 87 but is slightly less resistant at seedling stage. Expression levels of infected 171 and 190 seedlings at 20 °C were found to be in the same range as lines 49, 54 and 87.

Taken together, our results confirm previous studies that have shown variable effects of Lr34res on seedling resistance, with subtle differences in uredinium size, latency period and infection frequency (Rubiales and Niks, 1995; Singh and Gupta, 1992; Wamishe and Milus, 2004). However, our results also demonstrate that Lr34res can provide resistance in seedlings provided a genetic background with additive factors is chosen.

Lr34res transgenic lines exhibit characteristics of non-hypersensitive resistance

To analyse the resistance response in the transgenic wheat lines, several biochemical and cytological approaches were taken using the wheat genotypes Thatcher, Th + Lr34, 190 sib, 171 and 190. First, flag leaves were analysed for quantitative expression of three PR genes, known to be up-regulated in wheat following infection with both necrotrophic and biotrophic fungi: PR-1 (function unknown), PR-2 (β-1,3-glucanase) and PR-3 (chitinase) (Bozkurt et al., 2010; Desmond et al., 2006; Hulbert et al., 2007). These studies had revealed that PR transcripts levels are elevated at 72 hpi.

We found that in the infected Th + Lr34 samples, transcript levels of PR-1, PR-2 and PR-3 were significantly elevated compared to the mock-infected controls, whereas in infected Thatcher, only PR-1 and PR-3 transcripts were significantly different from the mock-infected control. However, there was no significant difference in PR gene expression levels if Th + Lr34 and Thatcher were compared, neither in mock-infected nor in infected samples (Figure 5a and Table S4).

Figure 5.

 Transgenic Lr34res lines show non-hypersensitive disease resistance to leaf rust. (a) PR-1, PR-2 and PR-3 expression in flag leaves 72 hpi with leaf rust. An * depicts any expression level that is significantly different from the mock-infected control (as assessed using REST 2009). For full significance assessment, refer to Table S4. Expression levels were normalized to GAPDH and Tubb4. n = 4, error bars = 1 SE Results are relative to an internal control (mock-infected Th + Lr34 a, 10 °C = 1). (b, c) Graphical representation of callose deposition relative to colony area (logarithmic scale) shows Lr34res does not alter the proportion of callose deposited at infection sites. Individual data points are shown; solid lines (in the corresponding colour) depict trend lines. (d, e, f) Representative pictures of infection sites in negative control (sib) lines at 24 h, 48 h and 72 hpi, respectively. (g, h and i) Representative pictures of infection sites in lines containing Lr34res at 24 h, 48 h and 72 hpi, respectively. Dead cells are stained blue and those cells containing H2O2 are stained brown. There was no difference between the levels of ROS or cell death at any time point between the Lr34res line and the negative controls.

PR-1 and PR-2 expression levels in the infected 171 samples were higher compared to mock-infected 171, but this increase was not significant. Pathogen development was slightly delayed in the BW26 cultivars when compared to the Thatcher lines (data not shown). This could explain the observed lack of differentiation in PR gene expression between the mock-infected and infected samples. These results suggest that Lr34res conferred resistance does not require the induction of PR genes, but up-regulation of the latter occurs as a consequence of pathogen infection, at least in some genotypes.

Next we assessed whether callose deposition is associated with infection sites in rust-infected flag leaves. Callose deposition in plants is a basal defence mechanism associated with pathogen infection (Maor and Shirasu, 2005). PEN3, which provides non-host resistance in Arabidopsis and shows homology to LR34res, was shown to be responsible for limiting fungal penetration by encasing haustoria with callose or papillae (Stein et al., 2006). As the level of resistance in some of the transgenic lines appears to be stronger than in the Th + Lr34 line (Figure 1c–e), an increase in callose deposition by LR34res should be particularly apparent in these lines. Therefore, flag leaves were analysed 7 dpi and colony area correlated with the number of cells containing callose. The resulting trend lines showed no statistical difference between those lines with Lr34res and the corresponding susceptible controls, suggesting that all the assayed lines have an equivalent capacity to deposit callose (Figures 5b and c, S2a and b).

To test whether transgenic lines containing Lr34res exhibited increased production of reactive oxygen species (ROS), a typical feature of the hypersensitive response (Levine et al., 1994; Sutherland, 1991), flag leaves were stained for H2O2 with 3-3′diaminobenzidine (DAB). Simultaneously, cell death was examined by trypan blue staining. Leaves were harvested 24, 48 and 72 hpi, stained, and analysed under visible light. Both control and Lr34res transgenic lines showed similarly low levels of ROS production (brown cells) at all three time points and an equivalent cell death response (blue cells—Figure 5d–i).

Discussion

The identification of the quantitative resistance gene Lr34res provides an excellent opportunity to gain insights into the poorly understood molecular mechanisms underlying durable disease resistance. In this study, the Lr34res gene was transformed into two susceptible BW26 genetic backgrounds. This allowed investigation into the effects of Lr34res in a transgenic system, as well as comparative phenotypic studies with the endogenous Lr34res gene in the Th + Lr34 background. As a result of this study, a number of endogenous Lr34res-associated characteristics have been identified in the transgenic lines. In addition, these lines have highlighted some key differences which may provide important insights into maximizing the effectiveness of Lr34res resistance in future breeding programmes.

Analysis of eight independent transgenic lines revealed that the introduction of the Lr34res transgene reproduced two endogenous, Lr34res-associated traits: firstly, leaf rust resistance was observed under the same conditions as for the endogenous Lr34res gene, specifically in adult plants (Dyck, 1991) (Figure 1c–e) and in seedlings grown at low temperatures (Singh and Gupta, 1992) (Figure 1a and b). Microscopic analysis of rust-infected flag leaves showed a reduction in colony size 7 dpi in both Th + Lr34 and the transgenic lines, typical of resistance conferred by slow rusting genes (Figure 1f and g), as previously reported by Rubiales and Niks (1995). There was also no apparent induction of ROS production, localized cell death or increased callose deposition in the Lr34res-containing transgenic lines (BW26AUS background) when challenged with leaf rust (Figure 5b–i). Therefore, the resistance exhibited in Lr34res transgenic wheat is non-hypersensitive as it is in endogenous Lr34res lines. LTN, the second phenotype associated with endogenous Lr34res expression and first described by Dyck (1991), was also induced in the Lr34res transgenics, but not the sib lines grown to the equivalent developmental stage (Figure 2a–c). The presence of the same NCCs in flag leaves of LTN-exhibiting transgenic plants, as well as Th + Lr34 flag leaves (Figure 2d), indicates that the Lr34res transgene induces an equivalent physiological response in the BW26 background and provides further support for the senescence hypothesis raised by Krattinger et al. (2009). The observation that LTN was stronger in plants that had been infected suggests that Lr34res may be involved in the export of a senescence-related compound, especially as infection generally resulted in increased transcript levels (Figure 3).

Despite the majority of endogenous Lr34res-associated characteristics being observed in the transgenic lines, some interesting differences were identified in this study. Lr34res expression levels were shown to be elevated in the transgenic lines compared to the endogenous Lr34res of Th + Lr34. This was particularly apparent in the seedlings (Figures 3 and 4b). Nevertheless, seedling expression was consistently lower than flag leaf expression in both transgenic and endogenous Lr34res-containing lines (Figure 3). An earlier semi-quantitative expression analysis experiment looking at endogenous Lr34-D found an increase in transcript levels correlated with plant development (Krattinger et al., 2009). This could, for example, involve development-associated transcription factors that are inactive in seedlings and are induced at a later developmental stage, thus explaining the greater variability observed in transcript levels of Lr34res transgenic seedlings. This hypothesis is supported by earlier studies suggesting that the regulated activation of pre-existing transcription factors provides an efficient way of manipulating the expression of target genes (Century et al., 2008; Seo et al., 2008).

A notable difference found in the transgenic lines is the variation in resistance conferred to the seedlings at 20 °C (Figure 4a). The variation in the infection-phenotypes of the two genetic backgrounds—BW26AUS and BW26SUI—suggests differences in genes modifying LR34res activity at the seedling stage. Most likely, the additive effects of these genes with the Lr34res transgene account for the resulting resistance. Several studies have already looked into the best combination of QR genes to reach resistance levels of near immunity (Lillemo et al., 2008; Singh et al., 1991, 2005, 2007). Endogenous Lr34res was found to enhance not only the effects of other leaf rust (Lr) genes at both seedling and adult stage (German and Kolmer, 1992; Kloppers and Pretorius, 1997), but also of other QR genes conferring resistance to stem rust (Kolmer et al., 2011). The transformed BW26AUS and BW26SUI cultivars both originated from a CIMMYT line designated as Bobwhite SH 98 26, identified as super-transformable and thus distributed to the research community (Pellegrineschi et al., 2002). Most likely, this line originally exhibited a high level of heterozygosity and independent selfing resulted in distinct genetic backgrounds of the BW26 material used for transformation in different laboratories. It is possible that seedling R genes, which confer some resistance to Mackellar, are present in the BW26SUI background. Mackellar (Pt 10-1,3,9,10,11,12) was identified as an exotic pathotype exhibiting combined virulence to Lr13 and other Lr genes, but responded differently depending on the Lr gene combinations (Park and Williams, 2005). The partial chlorosis observed on first leaves of the BW26SUI sib lines could have arisen from an avirulent interaction of a seedling Lr gene and Mackellar, which was even more effective in combination with the introduced Lr34res. We therefore postulate that Lr34res alone is unable to confer resistance in seedlings in the absence of cold treatment but, in a suitable genetic background, can enhance Lr gene resistance.

To further study the mechanism by which Lr34res confers resistance, PR transcript levels were analysed (Figure 5a). PR gene induction in wheat upon infection with fungal pathogens has been frequently described (Desmond et al., 2006; Moldenhauer et al., 2008; Molina et al., 1999; Pritsch et al., 2000), and two previous microarray studies on Lr34res-containing lines showed induction of several PR genes: Bolton et al. (2008) studied mock-infected and infected leaves of Th + Lr34 3 dpi and found a significant up-regulation of PR-1, PR-2 and PR-3 upon infection. Hulbert et al. (2007) examined gene expression patterns by comparing mock-infected and infected samples from both the tip and base of flag leaves in Th + Lr34 and Thatcher. They found that infection induced PR gene expression in both a resistant and a susceptible background, but PR-1, PR-2 and particularly PR-3 had significantly higher expression levels post infection in the Lr34res background. This up-regulation was specific to leaf tips for PR-1 and PR-2 and correlates with the increased resistance seen at the leaf tip compared with the leaf base (Schnurbusch et al., 2004), correlating PR-1 and PR-2 with Lr34res-associated resistance. On the basis of these earlier findings, we therefore hypothesized that Lr34res expression might induce an increase in PR gene transcripts.

Our analysis showed that infection induced the expression of the three target PR genes in the Thatcher and Th + Lr34 lines but, although the levels were enhanced within the Th + Lr34 background, this was not significant (Figure 5a, Table S4). Thus, our findings in cv. Thatcher show PR gene expression is dependent on infection.

Of particular interest were the PR transcript levels in the mock-infected samples of transgenic line 171 (BW26AUS background), which had significantly elevated Lr34res transcript levels (Figure 3, Table S3) and exhibited pathogen-independent chlorotic leaf spotting and a reduced seed set (Figure 1e). This leaf-spotting phenotype has been associated with increased pathogen resistance in a number of cereals including wheat, covered in detail by Li and Bai (2009). Previously, Anand et al. (2003) showed that overexpression of the PR-2 and PR-3 genes could not only induce a chlorotic lesion response in the absence of infection, but could also promote the expression of other PR genes including PR-1b. However, our results suggest that Lr34res does not directly regulate PR genes, and these in turn are not responsible for the chlorotic lesion phenotype observed. More likely, elevated Lr34res expression directly accounts for the chlorotic lesions, as was found for the quantitatively acting Rp1-DJ in maize (Hu et al., 1997) and the recessive, negative regulator of defence, mlo, in barley (Wolter et al., 1993). If the observed LTN is attributed to Lr34res being involved in the export of a senescence-related compound as hypothesized above, this could explain the chlorotic spotting seen in line 171.

Comparative analysis showed that transgenic Lr34res lines show characteristics typically found in endogenous Lr34res lines. Specifically, a slow rusting, non-hypersensitive resistance and LTN phenotype were reproduced, both particularly apparent in flag leaves. Future field experiments will allow transgenic lines to be analysed for resistance against stripe rust and powdery mildew, where they can be reliably scored. Our results indicate that Lr34res does not confer resistance through hyperactivation of basal defence, but through an unknown mechanism. Transgenic Lr34res lines confirmed the previously reported phenotypic variability in seedlings, but suggested that additive effects of genetic background components can improve Lr34res-conferred resistance in seedlings. In summary, the development and characterization of the transgenic lines has highlighted some important aspects of Lr34res-based resistance and given valuable insight into how this gene can be used for maximum effect in future breeding programmes.

Experimental procedures

Construction of a vector construct containing the full-length genomic Lr34res gene

The genomic Lr34res (resistant allele—FJ436983) sequence was cloned in two fragments from the Lr34 PDR BAC (1) (clone 1803A14) obtained from the ‘Chinese Spring’ BAC library (INRA, Toulouse, France) (Krattinger et al., 2009). The first 6771 bp was released from Lr34 PDR BAC (1) with ApaI and cloned into ApaI-cut pWGEM-NZf(+) (Wang et al., 1998). The remaining 9188 bp was also obtained from Lr34 PDR BAC (1) and cloned into pWGEM-NZf(+) using ApaI and BamHI. The complete genomic Lr34res construct was made by subcloning the 6771 bp ApaI-ApaI fragment into the ApaI-digested pWGEM-NZf(+) vector containing the second fragment. The correct insert orientation was assessed using a BamHI diagnostic digest. The complete Lr34res insertion from pWGEM-NZf(+)Lr34res could be excised with NotI.

Plant material and genetic transformation

Wheat lines were the Canadian cultivar Thatcher (Th) (CSIRO, Australia), Thatcher + Lr16 (Institute of Plant Biology, University of Zürich, Switzerland) and the near-isogenic derivative Thatcher + Lr34res (Th + Lr34RL6058) (Dyck et al., 1994).

Plant transformation was carried out in the Triticum aestivum line Bobwhite SH 98 26 (BW26) subtypes BW26AUS and BW26SUI (Australia and Switzerland respectively). Transformation was performed by particle bombardment of immature embryos, according to Pellegrineschi et al. (2002) with some modifications. Transformation experiments carried out in Australia were as reported in detail by Ryan et al. (2010) and used 8 μg of pWGEM-NZf(+)Lr34res and 2 μg of pCMneoSTLS2 DNA (for geneticin selection) (Maas et al., 1997) in each co-bombardment experiment. Transformations carried out in Switzerland relied on phosphomannose isomerase (Pmi) selection (Reed et al., 2001). The selectable marker gene Pmi was cloned under the maize ubiquitin promoter and the cassette (Ubi : Pmi) released from the vector pAHC17 as described by Brunner et al. (2011). 50 ng of this Ubi : Pmi cassette was co-bombarded with 50 ng of NotI-released Lr34res per shot.

Genotypic characterization of transgenic Lr34res lines

Transgenic T0 and T1 plants obtained in Switzerland were characterized genotypically by Southern blot of the Lr34res transgene and PCR-based amplification of the transgene transcript. Transgenic T0 plants obtained in Australia were genotypically characterized for the NPTII marker using a dot blot (McDonnell et al., 1987) and for Lr34res by genotypic DNA blot. Experiments were carried out on homozygous T3 and T4 generations. Refer to supplementary experimental procedures for information on Southern blot analysis and primer combinations. Primer sequences as described in Table S5.

Artificial leaf rust infection

Leaf rust infections were carried out using pathoype Mackellar, identified as virulent on BW26AUS (Park et al., 2009). Plants were grown in glasshouses (20 °C/16 °C, 16 h/8 h light/dark) and were inoculated at two-leaf stage (seedling) or once the primary flag leaf was fully unfurled from the ear. Synchronization of seed germination was attained by pre-germination on wet filter paper at 4 °C for 1 week in constant darkness before being transplanted into soil. Leaves were sprayed with water and then inoculated with a 1 : 10 homogeneous mixture of Mackellar spores : talcum powder (Australia) or by spraying a spore suspension using 3M™ Fluorinert™ FC-43 (3M) (Switzerland). Plants were then kept in a humidity chamber (16–20 °C) for 24 h before being transferred back to the glasshouse or to a cold chamber (4–10 °C, 12 h light and dark). Infected leaf samples were taken at indicated time points.

Microscopic analysis

The central 5 cm of 3–4 flag leaf samples, taken at 7 dpi, were used to determine colony size. A maximum of 25 infection sites were photographed and analysed per leaf and were selected based on the germinated urediospore penetrating the plant stomates. Microscopic visualization of fungal structures was achieved using wheat germ agglutininin (WGA) conjugated to the fluorophore alexa 488 (Invitrogen, San Diego, CA) staining according to Ayliffe et al. (2011) and Deshmukh et al. (2006). Prior to staining, leaf samples were submerged in 1M KOH, 1 μL Vac-In-Stuff (Lehle Seeds, Round Rock, TX) and incubated at 37 °C with gentle shaking for 24–48 h. Microscopic analysis and subsequent colony measurements were carried out according to Ayliffe et al. (2011). Colony area was calculated using the formula for an ellipse = (π X W X L)/4 (Lee and Shaner, 1985).

Callose deposition was visualized under UV light after addition of 0.005% aniline blue (Hood and Shew, 1996). The number of callose-containing cells was recorded and associated with the corresponding colony area. Analysis of plant H2O2 production in response to fungal colonization was measured using 3-3′diaminobenzidine (DAB) stain (Sigma-Aldrich, St. Louis, MO) (Thordal-Christensen et al., 1997). Localized cell death was measured using trypan blue staining (Belenghi et al., 2003) with modifications from Desmond et al. (2008). Briefly, flag leaf sections were incubated in 1 mg/mL DAB for 18 h at 24 °C in light. Dead cells were stained by boiling in trypan blue and cleared in chloral hydrate. Samples were mounted in 50% glycerol and analysed with a light microscope.

Quantitative PCR-based expression analysis

Expression analysis was carried out using quantitative PCR (qPCR) analysis. RNA was extracted from leaf tissue following mock infection or infection with Mackellar. All qPCR analysis was carried out using a Bio-Rad CFX96 Real-Time System C1000™ Thermal cycler (at CSIRO, Australia) or Applied Biosystems™ 7500 Fast Real-Time PCR System (at University of Zürich, Switzerland) according to the manufacturer’s instructions. The samples for qPCR analysis were prepared manually to a total volume of 10 μL using 5 μL Bio-Rad iQ™ SYBR® Green Supermix, 3 μL or 4 μL diluted cDNA (Australia and Switzerland respectively) and forward and reverse primers at a final concentration of 500 nm. GAPDH and Tubb4 were used as control genes and all data normalized to these. For information about RNA and cDNA preparation, primer design and sequences, and subsequent qPCR analysis and validation, refer to supplementary experimental procedures.

Analysis of non-fluorescent chlorophyll catabolites (NCCs)

Plants were grown in a growth chamber under standard glasshouse conditions until leaf tip necrosis started to develop. Dead tissue of the flag leaves exhibiting LTN was removed, and 2 cm of the yellowing tissue below the necrotic tip was excised for NCC extraction and immediately frozen in liquid nitrogen. The corresponding green tissue from the control flag leaves was collected similarly. Leaf material from three flag leaves was pooled. NCC extraction (MeOH/0.1 potassium phosphate, v/v 3 : 1) was followed by a two-step centrifugation procedure at 4 °C for 15 min. 50 μL of the supernatant was analysed by HPLC (Krattinger et al., 2009).

Acknowledgements

This work was supported by the Grains Research and Development Corporation grant #CSP000063, Australia, an Advanced Investigator grant of the European Research Council (ERC-2009-AdG 249996, Durableresistance), the Swiss National Science Foundation grant 3100A-127061 and a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme (PIOF-GA-2009-252731, Dures). We also thank M. Ayliffe, L. Tabe, for assistance with histology, S. Chandramohan for technical support, J. Taylor for assistance with statistical analysis and S. Hörtensteiner and B. Christ for NCC analysis. Mackellar rust culture was kindly supplied by D. Singh and R. Park.

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