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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.
Puccinia triticina, the causal agent of wheat leaf rust, is a devastating disease that can cause up to 40% yield loss. During fungal infection the host plant recognizes pathogen effectors, which trigger a host defense response. Changes in the pathogen effectors due to host selection pressure are responsible for the rapid development of new rust races and make durable resistance hard to obtain. The objectives of this study are to identify and characterize wheat genes that are utilized by races differently throughout infection and to understand functions of these genes using gene silencing. Six races of leaf rust were inoculated on a susceptible wheat variety and tissue was collected at six days post inoculation. RNA was sequenced and 63 wheat genes were identified that showed varying expression in response to the six races. 54 of these genes were evaluated in a time course study from zero days to six days post inoculation with the same six races. Real-time PCR was then used to analyze the timing of expression during early infection. The characterized genes have proposed functions involved in plant defense and stress, energy and metabolism, protein transport, replication, and RNA binding. Majority of the candidate genes showed three main expression patterns. However, race specific expression was found in three wheat genes that are affected by race shifts on Lr2A, Lr2C, and Lr17A. Sixteen potential susceptibility genes were also identified. Host susceptibility genes could be altered to provide durable resistance. RNAi was used to silence seven wheat genes to further understand their roles in leaf rust infection. T0 and T1 plants have been obtained and confirmed for the gene of interest. T2 plants were inoculated and observed for changes in susceptibility.