Abstract
Pathogens like Puccinia triticina, the causal organism for leaf rust, extensively damages wheat production. The interaction at molecular level between wheat and the pathogen is complex and less explored. The pathogen induced response was characterized using mock- or pathogen inoculated near-isogenic wheat lines (with or without seedling leaf rust resistance gene Lr28). Four Serial Analysis of Gene Expression libraries were prepared from mock- and pathogen inoculated plants and were subjected to Sequencing by Oligonucleotide Ligation and Detection, which generated a total of 165,767,777 reads, each 35 bases long. The reads were processed and multiple k-mers were attempted for de novo transcript assembly; 22 k-mers showed the best results. Altogether 21,345 contigs were generated and functionally characterized by gene ontology annotation, mining for transcription factors and resistance genes. Expression analysis among the four libraries showed extensive alterations in the transcriptome in response to pathogen infection, reflecting reorganizations in major biological processes and metabolic pathways. Role of auxin in determining pathogenesis in susceptible and resistant lines were imperative. The qPCR expression study of four LRR-RLK (Leucine-rich repeat receptor-like protein kinases) genes showed higher expression at 24 hrs after inoculation with pathogen. In summary, the conceptual model of induced resistance in wheat contributes insights on defense responses and imparts knowledge of Puccinia triticina-induced defense transcripts in wheat plants.
Highlights
The advent of high-throughput next-generation sequencing (NGS) technologies in the past decade resulted in massive drop in cost of sequencing-per-base that steered the expansion of its PLOS ONE | DOI:10.1371/journal.pone.0148453 February 3, 2016Leaf Rust Responsive Wheat Transcriptome Analysis
Four Serial Analysis of Gene Expression (SAGE) libraries were prepared from wheat plants treated as follows: (i) HD2329 mock inoculated (S-M), (ii) HD2329 pathogen inoculated (S-PI; compatible interaction), (iii) HD2329+Lr28 mock inoculated (R-M) and (iv) HD2329+Lr28 pathogen inoculated (R-PI; incompatible interaction)
Sequencing of the four Sequencing by Oligonucleotide Ligation and Detection (SOLiD)-SAGE libraries generated a total of 165,767,777 reads, each 35 nucleotides long with a total of 8 GB data in Ã.csfasta format
Summary
Growth and pathogen inoculation of plantsNear-isogenic lines of wheat (Triticum aestivum L.) cultivar HD2329 with [seedling leaf rust resistant, nest immune, infection type 0–0; (no uredia or dead flecks)] or without [seedling leaf rust susceptible, infection type 3+; (uredia somewhat larger than normal size and may be associated with chlorosis or rarely necrosis)] the seedling leaf rust resistant gene Lr28 were used in this study [31]. The seedling were grown to single leaf stage (~7 days after germination) in a growth chamber under ideal conditions (22°C, Relative Humidity 80%, 16h light at 300 lux; and 8h of darkness) at National Phytotron Facility, Indian Agricultural Research Institute, New Delhi. Each treatment combination was applied to 15 plants. After inoculation, misting of the growth chamber was performed to create conditions of high humidity (>90%) for 24 hours post inoculation (hpi) in the dark to facilitate infection. Thereafter, the plants were maintained in a standard growth chamber as mentioned earlier [32]. The wheat leaf samples (5 leaves from separate plants of each treatment) were collected at 24 hpi and immediately dipped in liquid nitrogen and kept for RNA isolation (Fig 1). Infectivity studies and its statistical analysis were performed as mentioned in earlier study [32]
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