The NLR-Annotator Tool Enables Annotation of the Intracellular Immune Receptor Repertoire

Abstract

Disease resistance genes encoding nucleotide-binding and leucine-rich repeat (NLR) intracellular immune receptor proteins detect pathogens by the presence of pathogen effectors. Plant genomes typically contain hundreds of NLR-encoding genes. The availability of the hexaploid wheat (Triticum aestivum) cultivar Chinese Spring reference genome allows a detailed study of its NLR complement. However, low NLR expression and high intra-family sequence homology hinders their accurate annotation. Here we developed NLR-Annotator, a software tool for in silico NLR identification independent of transcript support. Although developed for wheat, we demonstrate the universal applicability of NLR-Annotator across diverse plant taxa. We applied our tool to wheat and combined it with a transcript-validated subset of genes from the reference gene annotation to characterize the structure, phylogeny and expression profile of the NLR gene family. We detected 3,400 full-length NLR loci of which 1,560 were confirmed as expressed genes with intact open reading frames. NLRs with integrated domains mostly group in specific subclades. Members of another subclade predominantly locate in close physical proximity to NLRs carrying integrated domains, suggesting a paired helper-function. Most NLRs (88%) display low basal expression (in the lower 10 percentile of transcripts). In young leaves subjected to biotic stress we found upregulation of 266 of the NLRs. To illustrate the utility of our tool for the positional cloning of resistance genes, we estimated the number of NLR genes within the intervals of mapped rust resistance genes. Our study will support the identification of functional resistance genes in wheat to accelerate the breeding and engineering of disease-resistant varieties.

Multiple wheat genomes reveal global variation in modern breeding

“…NLR repertoire. NLR signatures were annotated using NLR-Annotator 58,59 (https://github.com/steuernb/NLR-Annotator) with the option -a. We estimated redundancy of NLR signatures between genomes at different thresholds of identity: 95%, 98% and 100%.…”

Identification of New Leaf Rust Resistance Loci in Wheat and Wild Relatives by Array-Based SNP Genotyping and Association Genetics

“…These genes play an important role in pathogen recognition and initiation of downstream signaling cascades. In the wheat genome, as many as 661 to 1,560 full-length NBS-LRR genes have been reported, higher than any other plant species (Gu et al, 2015;Steuernagel et al, 2020). Four of the six Lr genes cloned to date encode CC-NBS-LRR proteins (Feuillet et al, 2003;Huang et al, 2003;Cloutier et al, 2007;Thind et al, 2017).…”

A molecular roadmap to the plant immune system

“…Many different types of proteins have been found as IDs in plant NLRs, and likely function in direct perception of effectors ( 193 – 197 ). The integrated HMA domains of the sensor NLRs of the rice Pik and Pia pairs are exemplars of IDs and serve as model systems for understanding the principles of effector perception by these domains ( 11 , 29 , 31 , 178 ).…”