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A Putative ABC Transporter Confers Durable Resistance to Multiple Fungal Pathogens in Wheat

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Science  06 Mar 2009:
Vol. 323, Issue 5919, pp. 1360-1363
DOI: 10.1126/science.1166453

Abstract

Agricultural crops benefit from resistance to pathogens that endures over years and generations of both pest and crop. Durable disease resistance, which may be partial or complete, can be controlled by several genes. Some of the most devastating fungal pathogens in wheat are leaf rust, stripe rust, and powdery mildew. The wheat gene Lr34 has supported resistance to these pathogens for more than 50 years. Lr34 is now shared by wheat cultivars around the world. Here, we show that the LR34 protein resembles adenosine triphosphate–binding cassette transporters of the pleiotropic drug resistance subfamily. Alleles of Lr34 conferring resistance or susceptibility differ by three genetic polymorphisms. The Lr34 gene, which functions in the adult plant, stimulates senescence-like processes in the flag leaf tips and edges.

Improved control of fungal rust diseases in cereals through breeding varieties with durable rust resistance is critical for world food security. International attention has been recently drawn to the continuing major threat of fungal rust diseases of cereals, highlighting the need for effective and durable sources of rust resistance. The most profitable and environmentally friendly strategy for farmers to control wheat rusts in both the developing and the developed world is to grow genetically resistant wheat varieties.

The wheat gene Lr34 is associated with resistance to two rust diseases of wheat, leaf rust (caused by Puccinia triticina) (Fig. 1, A and B), and stripe rust (P. striiformis) (13), as well as powdery mildew (Blumeria graminis) (4). Lr34 provides an important source of partial resistance that is expressed in adult plants during the critical grain-filling stage and is most effective in the uppermost leaf, the so-called flag leaf. When deployed with other adult plant resistance genes, near-immunity can be achieved (5). Flag leaves of many wheat cultivars containing Lr34 develop a necrotic leaf tip, a morphological marker described as leaf tip necrosis (Fig. 1C) (6, 7). The gene was first documented in Canada by Dyck although Lr34-containing germplasm has been a part of wheat improvement since the early 20th century. Wheat cultivars containing Lr34 occupy more than 26 million ha in various developing countries alone and contribute substantially to yield savings in epidemic years (8).

Fig. 1.

Lr34 phenotypes and mapping. (A) Lr34 confers a partial slow-rusting resistance. The images show rows of the resistant selection Jupateco R (left) and of the susceptible near-isogenic line Jupateco S (right) infected with leaf rust in Mexico. (B) Progression of leaf rust infection on three successive aging flag leaves of Jupateco R (left) and Jupateco S (right). (C) Lr34 is associated with leaf tip necrosis (Ltn1) that can be observed on the resistant near-isogenic line Arina Lr34 (left) but not on the –Lr34 Swiss winter wheat cultivar Arina (right). (D) Consensus genetic map of Lr34 based on three high-resolution mapping populations defined a 0.15-cM target interval for Lr34. (E) The corresponding physical target interval sequenced on the +Lr34 cultivar Chinese Spring contains eight open reading frames (arrows). Blue lines indicate repetitive regions without genes and numbers refer to the respective positions within the 363-kb interval. Gly, glycosyl transferase; Cyst, cysteine proteinase; Cyp, cytochrome P450; Kin, lectin receptor kinase; ABC, ABC transporter; Hex, hexose carrier; Ψ, pseudogene. (F) Gene structure of Lr34. Open boxes indicate exons; introns are shown as adjoining lines. Red marks indicate the mutation sites of the eight mutants 2B, 2F, 2G, 3E, 4C, 4E, m19, and m21. The three sequence polymorphisms between susceptible and resistant alleles are indicated in blue. (G) Low-temperature–induced seedling resistance of Lr34. (Left) Microscopic visualization of wheat germ agglutinin stained fungal colonization of mesophyll cells between 14 and 31 days post inoculation (DPI) of Lalbahadur Lr34(L34) and derived mutant m19 and m21 seedlings (scale bar, 100 μm). (Right) Progression of leaf rust sporulation on seedling leaf surfaces between 34 and 59 DPI (scale bar, 10 mm).

The Lr34 gene has remained durable, and no evolution of increased virulence toward Lr34 has been observed for more than 50 years. This is in contrast to many other rust resistance genes, the so-called gene-for-gene class, that provide resistance to some but not all strains of a rust species (912). Despite the importance of adult plant resistance genes (13), no such gene has been cloned to date. Understanding the molecular nature of this class of resistance has important implications for long-term control of rust diseases. Previous studies have localized the codominant gene Lr34 on the short arm of chromosome 7D between the two markers gwm1220 and SWM10 (14, 15). We further reduced the target interval in a map-based cloning approach based on three high-resolution populations (16) (table S1). High-resolution mapping revealed a 0.15-cM target interval for Lr34 flanked by XSWSNP3/XcsLVA1 and XcsLVE17 (Fig. 1D). The 363-kb physical interval containing both flanking markers was fully sequenced in the Lr34-containing hexaploid wheat cultivar Chinese Spring (FJ436983). Sequence analysis revealed the presence of a generich island containing eight open reading frames (Fig. 1E) predicted to encode proteins with homologies to a hexose carrier, an ATP-binding cassette (ABC) transporter, two cytochromes P450, two lectin receptor kinases, a cysteine proteinase, and a glycosyl transferase. The latter two genes were interrupted by repetitive elements and were excluded as candidates for Lr34. Molecular markers derived from the coding sequences resembling one of the two lectin receptor kinases (SWDEL3), the ABC transporter (SWDEL2/csLVD2), and the hexose carrier (SWDEL1) were cosegregating with Lr34.

To determine whether one of these cosegregating genes corresponds to Lr34, we examined for sequence differences in their coding regions from the three pairs of +/–Lr34 parental lines of the mapping populations. Consistent sequence polymorphism between the alleles of all parental pairs was found only in the putative ABC transporter gene. Second, we sequenced locus-specific DNA fragments covering parts of the six candidate genes on two γ-irradiation (m19 and m21) and six sodium azide–induced Lr34 mutants (2B, 2F, 2G, 3E, 4C, and 4E) that were selected for loss-of-function of the Lr34 resistance. Each mutant showed sequence alterations in the putative ABC transporter gene (table S2), leading to either splice site mutations resulting in strongly reduced splicing efficiency or mis-splicing (fig. S1), amino acid exchanges, frame shifts, or premature stop codons (Fig. 1F). To test for the presence of additional mutations in the other genes cosegregating with Lr34, we sequenced DNA fragments covering 12 to 15 kb of the other five candidate genes and intergenic regions on the six mutants 2B, 3E, 4C, 4E, m19, and m21 without finding any sequence polymorphism. Hence, we can exclude the possibility that the eight independent mutations found in the putative ABC transporter gene are due to a generally very high mutation frequency in these lines, and we conclude that this gene is responsible for conferring the durable Lr34 disease resistance.

Lr34 cosegregated with partial resistance to adult plant stripe rust (Yr18), powdery mildew (Pm38), as well as leaf tip necrosis (Ltn1). The mutants were more susceptible to leaf rust, stripe rust, and powdery mildew, and they did not show leaf tip necrosis. These observations, based on eight independent mutations within a single putative ABC transporter gene, strongly suggest that the same gene controls resistance based on Lr34, Yr18, and Pm38 as well as leaf tip necrosis. Depending on the genetic background, Lr34 was also shown to confer resistance against stem rust (17), and the tested mutants were more susceptible to stem rust than the wild type. Further evidence that the putative ABC transporter gene confers slow-rusting resistance came from the study of the early infection process of leaf rust in seedlings of mutants. It was shown earlier that Lr34 conferred resistance at the seedling stage to leaf rust at low temperatures (18). Analysis of infection processes in seedlings grown at 4° to 8°C revealed differences in resistance response to leaf rust (Fig. 1G). From the fourth week after infection, the colonized area in mutants m19 and m21 was larger relative to the wild type. External symptoms of sporulation were evident in the mutants by the fifth week, whereas the presence of the active Lr34 gene delayed visible symptoms until after the sixth week after infection.

The nucleotide sequence of Lr34 spans 11,805 base pairs (bp). Sequencing of the full-length cDNA revealed that Lr34 consist of 24 exons (Fig. 1F). The predicted 1401–amino acid protein (fig. S2A) belongs to the pleiotropic drug resistance subfamily of ABC transporters. Pleiotropic drug resistance transporters share a common basic structure containing two cytosolic nucleotide binding domains and two hydrophobic transmembrane domains. Fifteen pleiotropic drug resistance–like genes have been identified in the genome of Arabidopsis, and 23 members were described in rice. The closest LR34 homolog in rice is OsPDR23, showing 86% amino acid identity. In Arabidopsis, the closest homologs are 56% identical to LR34 at the amino acid level (fig. S3). Pleiotropic drug resistance transporters are known to confer resistance to various drugs, but little is known about their substrate specificity (19). In Arabidopsis, it has previously been reported that PEN3/PDR8 contributes to resistance toward non–host pathogens (20). The current model suggests that PEN3 may be involved in translocating toxic compounds derived from glucosinolates into the apoplast (21).

We next determined the sequence differences between the Lr34 alleles in cultivars with or without Lr34-based resistance. Comparison of genomic sequences of the putative pleiotropic drug resistance transporter in the +Lr34 cultivar Chinese Spring and the –Lr34 French winter wheat cultivar Renan (FJ436985) revealed that the gene was present in both wheat varieties. Only three polymorphisms distinguished the alleles of Chinese Spring and Renan (Fig. 1F). One single-nucleotide polymorphism was located in the large intron 4. The other two sequence differences were located in exons. A deletion of 3 bp (ttc) found in exon 11 in Chinese Spring resulted in the deletion of a phenylalanine residue, whereas a second single-nucleotide polymorphism in exon 12 converted a tyrosine to a histidine in the resistant cultivar. Both sequence differences located in exons affect the first transmembrane domain connecting the two nucleotide binding domains and may alter the structure and substrate specificity of the transporter (fig. S2B). Sequence comparison of 2 kb of the putative Lr34 promoter regions did not reveal any differences between the two cultivars. Three breeding lineages of Lr34 in wheat germplasm have been identified: (i) Far-East germplasm; (ii) spring wheat lines from North and South America that were traced back to Lr34 cultivar sources developed in Italy; and (iii) winter wheat material in Europe. The same resistance haplotype was found in these three breeding lineages, suggesting a single origin of Lr34 (Table 1).

Table 1.

Diagnostic value of the three sequence differences between +Lr34 and –Lr34 alleles. The three different origins of Lr34 are indicated. Some lines without Lr34 were included for the American and European material. SNP, single-nucleotide polymorphism.

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Lr34 is more effective during adult growth stage than in seedlings under field conditions. Because leaf tip necrosis develops spontaneously and independent of infection, expression of Lr34 does not depend on the presence of pathogens. To determine whether the difference in resistance between seedlings and adult plants in lines containing Lr34 is related to the expression level of Lr34, we performed reverse transcription–polymerase chain reaction (RT-PCR) using uninfected leaf material of the near-isogenic lines Thatcher and Thatcher Lr34. As expected, the putative pleiotropic drug resistance transporter was expressed at very low levels in 14-day-old seedlings grown at 20°C, whereas the expression level was clearly higher in flag leaves of adult plants before (53-day-old plants) and after the development (63-day-old plants) of leaf tip necrosis (Fig. 2). There was no visible difference in expression between resistant and susceptible plants, which is in agreement with the absence of sequence polymorphisms in the putative promoter region. The unspliced Lr34 gene product also accumulated in adult plants after 63 days. It has been reported that splicing may be very inefficient in some genes that are expressed at low levels (22).

Fig. 2.

Expression analysis of Lr34. RT-PCR was performed on uninfected leaf material with a primer pair amplifying the first three exons of the gene. Leaves of the near-isogenic lines Thatcher and Thatcher Lr34 were harvested at the seedling stage after 14 days and of adult flag leaves before (53-day-old plants) and after development (63-day-old plants) of leaf tip necrosis. Expression levels of leaf base and leaf tip were separately determined from adult leaves, because leaf tips of Thatcher Lr34 are much more resistant than the respective leaf base. The correctly spliced and the unspliced Lr34 gene product are indicated by arrows. Contamination with genomic DNA can be excluded because the GAPDH control did not amplify an unspliced fragment. Different panels represent independent gel runs and distances between spliced and unspliced band vary. Th, Thatcher; Th Lr34, Thatcher Lr34; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

The level of Lr34-mediated resistance for leaf rust infection correlated with the development of leaf tip necrosis (fig. S4). In addition, flag leaf tips of Lr34-containing cultivars are more resistant than their respective leaf bases. Rubiales and Niks reported that Lr34 is associated with reduced intercellular hyphal growth but not with a hypersensitive response or papilla formation (18). Analysis of microarray studies revealed that genes with similar annotations were up-regulated both in uninfected flag leaves of Lr34-containing near-isogenic lines (23) and senescing wheat flag leaves (24). We therefore tested the hypothesis whether leaf senescence, a highly controlled process (25) starting from the leaf tips, may contribute to Lr34 resistance. The barley cDNA HvS40 is known to be up-regulated during leaf senescence (26). Northern blot analysis revealed that HvS40 was highly expressed in uninfected flag leaf tips of Thatcher Lr34 but not of Thatcher and the six azide-induced mutants in 63-day-old plants (Fig. 3). Further evidence of Lr34's involvement in leaf senescence came from the analysis of chlorophyll degradation products (27). Nonfluorescent chlorophyll catabolites, which are hallmarks of leaf senescence, were detected in the flag leaf tip of Thatcher Lr34 but not in Thatcher (fig. S5). Thus, it is possible that Lr34 resistance is the result of senescence-like processes. Alternatively, LR34 may play a more direct role in resistance by exporting metabolites that affect fungal growth, similar to the proposed role for PEN3. Arabidopsis PEN3 is a pleiotropic drug-resistance protein and was shown to be involved in non–host resistance to barley powdery mildew. Thus, LR34 and PEN3 belong to the same protein family, raising the possibility of similar defense mechanisms in non–host resistance and durable resistance to an adapted pathogen.

Fig. 3.

Lr34 stimulates senescence-like processes in flag leaves. Northern blot using the probe HvS40 on 63-day-old flag leaf tips (after development of leaf tip necrosis) of the near-isogenic lines Thatcher and Thatcher Lr34 and the azide-induced Lr34 mutants 2B, 2F, 2G, 3E, 4C, and 4E. Th, Thatcher; Th Lr34, Thatcher Lr34.

The observation that multiple pathogen resistance in wheat, which comprises Lr34, Yr18, Pm38, as well as the phenotypic marker Ltn1, is controlled by the same gene demonstrates the existence in plants of single genetic factors that act durably against several diseases.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1166453/DC1

Materials and Methods

Figs. S1 to S5

Tables S1 to S3

References

References and Notes

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