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The Lr34/Yr18/Sr57/Pm38/Ltn1 multi-resistance locus has been deployed and remained effective in wheat cultivars for more than 100 years. The durability and pleiotropic nature makes Lr34 a unique and highly valuable resource for rust resistance breeding. Despite its functional annotation as an ABC transporter, the mode of action is unknown. Considering this, we aimed to decipher molecular factors and signaling components essential for Lr34 function using RNA-seq of Chara resistant (Lr34) and Chara mutant (heavy ion irradiation, HII) susceptible wheat lines. Screening of Chara and Chara HII lines with Lr34-specific markers confirmed the integrity of Lr34 in both lines; however, phenotyping confirmed rust and powdery mildew susceptibility in the Chara HII lines. Plants were grown under controlled conditions and infected with Puccinia triticina pathotype 76-1,3,5,7,9,10,12,13+Lr37 at the flag leaf stage. Flag leaves were sampled at 0, 24, 48, 72, 96 and 168 hours post inoculation (hpi) from mock and infected plants. Based on real-time PCR analysis of basal defense genes and the Lr34 gene, we selected 72 hpi for RNA-seq with four biological replicates per condition. The samples were sequenced on an Illumina Hiseq 4000 at the Beijing Genomics Institute, China. A total of 9.0 Gb of sequence (2.25 Gb/library) from 16 libraries for four conditions was obtained. Differential expression analysis was performed using the Tuxedo analysis pipeline with standard parameters. Analysis revealed deletion of DNA fragments with collinear gene order on chromosomes 1A, 2D, 5A, 5B, 5D and 7D of Chara HII mutants. To determine the significance of the deletions we performed bulk segregant analyses on segregating F2 populations of Chara ? Chara HII crosses. Analyses revealed key genomic regions associated with Lr34-functional resistance and we are in the process of validating candidate genes using qPCR.
Four leaf rust adult plant resistance genes (Lr34, Lr46, Lr67 and Lr68) are known to be associated with leaf tip necrosis (LTN). LTN caused by these genes is different from each other at phenotypic level. LTN associated with APR genes Lr34, Lr46 and Lr67 has been designated as Ltn1, Ltn2 and Ltn3. Seventy-seven CIMMYT genotypes were selected to find out the association between genotypic and phenotypic variability for LTN and its association with yield traits; 1000 grain weight, grain yield, leaf area and percentage of leaf tip necrosis in the flag leaf of main tiller. All the genotypes were screened for the presence of 3 APR genes with linked markers, csLV34 for Lr34; Xwmc44 and Xgwm259 for Lr46 and Xcfd71 for Lr67. The genotypes were grouped into 5 classes; only Lr34, only Lr46, only Lr67, Lr34+L46+Lr67 and genotypes lacking all three genes. Molecular analysis revealed that 7 genotype with Lr34 only, 6 with Lr46 only, 7 with Lr67 only, 13 with all the 3 genes, and 28 without any Lr gene. Phenotypic data of LTN percentage was compared and it was noted that maximum LTN % was observed for Lr67 (7.811%) followed by Lr46 (7.348%) and Lr34 (6.47%). Surprisingly, presence of all three genes reduced the LTN% (4.7055%) as compared with absence of all three genes (6.011%). It was also observed that the three genes simultaneously reduced 1000 grain weight and plot yield. All three genes increased leaf area highly significantly both when they are alone or together (34.7 to 44.7 cm2) in comparison to those genotypes (24.7 cm2) which lacks these Lr genes and also reduced 1000-grain weight and plot yield but non-significantly.
The Lr34 resistance gene from Triticum aestivum encodes a putative ABC transporter protein that confers broad spectrum, partial adult plant resistance to all three rusts species and powdery mildew. It has remained a durable source of resistance for over 100 years in which time no increased virulence towards Lr34 has been observed. This gene is located on chromosome 7D and consequently cannot be readily transferred to durum wheat by traditional breeding. A transgenic approach was used to transfer Lr34 to durum wheat cultivar Stewart by Agrobacterium transformation. Homozygous progeny from a number of independent Stewart lines expressing Lr34 under regulatory control of its endogenous promoter showed high levels of rust resistance at the seedling stage. A correlation between seedling resistance and transgene expression levels was observed in these plants. In contrast seedlings from a near isogenic line of hexaploid wheat cultivar Thatcher containing Lr34 showed only a minor difference in rust growth when compared with Thatcher seedlings, typical of this adult plant resistance gene in hexaploid wheat. Little is known about how the Lr34 gene product functions; however, the seedling resistance of these durum transgenics enables functional assays to be readily undertaken without the need for adult plant material. By infecting seedlings we have shown that day length has an effect on Lr34 resistance to leaf rust, with higher levels of resistance observed under long days (16 h light) compared with short days (8 h light). This study demonstrates that Lr34 provides strong and presumably durable seedling resistance to rust in durum plants that can be used to further understand how this gene confers resistance.
Plant breeders use naturally occurring resistance genes to fight plant diseases. However, new fungal strains rapidly emerge and defeat these genes. For almost a century, the wheat Lr34 gene has conferred a degree of stable resistance to the wheat rusts, making it one of the most important resistance genes. While sequence homology of the cloned Lr34 gene predicted that it encodes a putative ATP binding cassette (ABC) transporter protein belonging to the ABC G subfamily (also known as Pleiotropic Drug Resistance or PDR), its target transport substrate and mechanism of action remains enigmatic. In an effort to understand this transporter we designed several DNA constructs of the Lr34 gene and expressed them in yeast (Saccharomyces cerevisiae). Here we report the successful expression and purification of functional recombinant Lr34 protein. In vitro proteoliposome translocation assays identified the transport substrate of the Lr34Sus protein and demonstrated that the LR34Res protein has the same transport specificity. We also report the identification of related metabolites from flag leaves of Lr34-expressing wheat plants and discuss the functional relevance of these metabolites to the disease resistance and leaf tip necrosis (LTN) phenotypes caused by expression of Lr34Res.