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.

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Limited phosphorus availability in the soil is one of the major constraints to the growth and productivity of rice across Asian, African and South American countries, where 50% of the rice is grown under rain-fed systems on poor and problematic soils. With an aim to determine novel alleles for enhanced phosphorus uptake efficiency in wild species germplasm of rice Oryza rufipogon, we investigated phosphorus uptake1 (Pup1) locus with 11 previously reported SSR markers and sequence characterized the phosphorus-starvation tolerance 1 (PSTOL1) gene. In the present study, we screened 182 accessions of O. rufipogon along with Vandana as a positive control with SSR markers. From the analysis, it was inferred that all of the O. rufipogon accessions undertaken in this study had an insertion of 90 kb region, including Pup1-K46, a diagnostic marker for PSTOL1, however, it was absent among O. sativa cv. PR114, PR121, and PR122. The complete PSTOL1 gene was also sequenced in 67 representative accessions of O. rufipogon and Vandana as a positive control. From comparative sequence analysis, 53 mutations (52 SNPs and 1 nonsense mutation) were found in the PSTOL1 coding region, of which 28 were missense mutations and 10 corresponded to changes in the amino acid polarity. These 53 mutations correspond to 17 haplotypes, of these 6 were shared and 11 were scored only once. A major shared haplotype was observed among 44 accessions of O. rufipogon along with Vandana and Kasalath. Out of 17 haplotypes, accessions representing 8 haplotypes were grown under the phosphorus-deficient conditions in hydroponics for 60 days. Significant differences were observed in the root length and weight among all the genotypes when grown under phosphorus deficiency conditions as compared to the phosphorus sufficient conditions. The O. rufipogon accession IRGC 106506 from Laos performed significantly better, with 2.5 times higher root weight and phosphorus content as compared to the positive control Vandana. In terms of phosphorus uptake efficiency, the O. rufipogon accessions IRGC 104639, 104712, and 105569 also showed nearly two times higher phosphorus content than Vandana. Thus, these O. rufipogon accessions could be used as the potential donor for improving phosphorus uptake efficiency of elite rice cultivars.

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Leaf rust caused by Puccinia triticina (Pt) is one of the most important diseases of bread wheat globally. Recent advances in sequencing technologies have provided opportunities to analyse the complete transcriptomes of the host as well as pathogen for studying differential gene expression during infection. Pathogen induced differential gene expression was characterized in a near isogenic line carrying leaf rust resistance gene Lr57 and susceptible recipient genotype WL711. RNA samples were collected at five different time points 0, 12, 24, 48, and 72 h post inoculation (HPI) with Pt 77-5. A total of 3020 transcripts were differentially expressed with 1458 and 2692 transcripts in WL711 and WL711+Lr57, respectively. The highest number of differentially expressed transcripts was detected at 12 HPI. Functional categorization using Blast2GO classified the genes into biological processes, molecular function and cellular components. WL711+Lr57 showed much higher number of differentially expressed nucleotide binding and leucine rich repeat genes and expressed more protein kinases and pathogenesis related proteins such as chitinases, glucanases and other PR proteins as compared to susceptible genotype. Pathway annotation with KEGG categorized genes into 13 major classes with carbohydrate metabolism being the most prominent followed by amino acid, secondary metabolites, and nucleotide metabolism. Gene co-expression network analysis identified four and eight clusters of highly correlated genes in WL711 and WL711+Lr57, respectively. Comparative analysis of the differentially expressed transcripts led to the identification of some transcripts which were specifically expressed only in WL711+Lr57. It was apparent from the whole transcriptome sequencing that the resistance gene Lr57 directed the expression of different genes involved in building the resistance response in the host to combat invading pathogen. The RNAseq data and differentially expressed transcripts identified in present study is a genomic resource which can be used for further studying the host pathogen interaction for Lr57 and wheat transcriptome in general.

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1Disease resistance genes encoding intracellular immune receptors of the nucleotide-binding 2 and leucine-rich repeat (NLR) class of proteins detect pathogens by the presence of pathogen 3 effectors. Plant genomes typically contain hundreds of NLR encoding genes. The availability 4 of the hexaploid wheat cultivar Chinese Spring reference genome now allows a detailed study 5 of its NLR complement. However, low NLR expression as well as high intra-family sequence 6 homology hinders their accurate gene annotation. Here we developed NLR-Annotator for in 7 silico NLR identification independent of transcript support. Although developed for wheat, we 8 demonstrate the universal applicability of NLR-Annotator across diverse plant taxa. Applying 9 our tool to wheat and combining it with a transcript-validated subset of genes from the 10 reference gene annotation, we characterized the structure, phylogeny and expression profile of 11 the NLR gene family. We detected 3,400 full-length NLR loci of which 1,540 were confirmed 12 as complete genes. NLRs with integrated domains mostly group in specific sub-clades. 13Members of another subclade predominantly locate in close physical proximity to NLRs 14 carrying integrated domains suggesting a paired helper-function. Most NLRs (88%) display 15 low basal expression (in the lower 10 percentile of transcripts), which may be tissue-specific 16 and/or induced by biotic stress. As a case study for applying our tool to the positional cloning 17 of resistance genes, we estimated the number of NLR genes within the intervals of mapped rust 18 resistance genes. Our study will support the identification of functional resistance genes in 19 wheat to accelerate the breeding and engineering of disease resistant varieties. 20 22All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/339424 doi: bioRxiv preprint first posted online Jun. 5, 2018; 3 Background 1The status of wheat as the world's most widely grown and important food crop [1] is 2 threatened by the emergence and spread of new and old diseases. For example, 3 wheat stem rust, long considered a vanquished foe of the past, has in the last 20 years 4 caused devastating epidemics in Africa [2, 3] and eastern Russia [4], while, in 2013 5and 2016 large outbreaks occurred for the first time in >50 years in western Europe 6 [5, 6]. The spread of disease into new regions can be attributed to the very success of 7 wheat as a globally traded commodity. In that context, wheat blast, a new disease for 8 wheat, which had until recently been confined to Brazil and other countries in South 9America, appearead in 2016 in Bangladesh, possibly as a result of importing 10 contaminated grain [7, 8]. The warm, wet climates in the wheat belts of India and 11China, which supply ~30% of the worlds wheat [1], favour the further proliferation of 12 this deva...

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