Genetics of chilling response at early growth stage in rice: a recessive gene for tolerance and importance of acclimation.

Low-temperature adaptation in rice is mediated by the ability of a genotype to tolerate chilling temperatures. A genetic locus on chromosome 11 was analysed for chilling tolerance at the plumule stage in rice. The tolerant allele of A58, a japonica landrace in Japan, was inherited as a recessive gene (ctp-1A58), whereas the susceptible alleles from wild rice (Ctp-1W107) and modern variety (Ctp-1HY) were the dominant genes. Another recessive tolerant allele (ctp-1Silewah) was found in a tropical japonica variety (Silewah). Fine-mapping revealed that a candidate gene for the ctp-1 locus encoded a protein similar to the nucleotide-binding domain and leucine-rich repeat (NLR) protein, in which frameshift mutation by a 73 bp-deletion might confer chilling tolerance in ctp-1A58. Analysis of near-isogenic lines demonstrated that ctp-1A58 imparted tolerance effects only at severe chilling temperatures of 0.5 °C and 2 °C, both at plumule and seedling stages. Chilling acclimation treatments at a wide range of temperatures (8 °C-16 °C) for 72 h concealed the susceptible phenotype of Ctp-1W107 and Ctp-1HY. Furthermore, short-term acclimation treatment of 12 h at 8 °C was enough to be fully acclimated. These results suggest that the NLR gene induces a susceptible response upon exposure to severe chilling stress, however, another interacting gene(s) for acclimation response could suppress the maladaptive phenotype caused by the Ctp-1 allele. This study provides new insights for the adaptation and breeding of rice in a low-temperature environment.

Constitutive expression of nucleotide-binding and leucine-rich repeat gene AtRPS2 enhanced drought and salt tolerance in rice.

Drought and salt are major abiotic stresses that severely restricts plant growth and development, leading to serious losses in agricultural production. Therefore, improving crop tolerance to drought and salt stresses is an urgent issue. A previous study showed that overexpression of Arabidopsis NLR gene AtRPS2 conferred broad-spectrum disease resistance in rice. In this study, we demonstrated that constitutive expression of AtRPS2 increased abscisic acid (ABA) sensitivity during seedling stage, the shoot length of transgenic plants were shorter than wild type plants. Exogenous application of ABA markedly induced the expression of stress-related genes and promoted stomatal close in transgenic plants. Overexpression of AtRPS2 also enhanced drought and salt tolerance in rice, transgenic plants exhibited higher survival rates under drought and salt conditions than wild type plants. The activities of catalase (CAT) and superoxide dismutase (SOD) were higher in AtRPS2 transgenic rice than wild type plants. In addition, the expression of stress-related genes and ABA-responsive genes were significantly upregulated in AtRPS2 transgenic plants than wild type plants under drought and salt treatments. Besides, exogenous application of ABA could facilitate drought and salt tolerance in AtRPS2 transgenic plants. Taken together, this study indicated that AtRPS2 could improve drought and salt tolerance in rice, and this phenomenon is likely to be regulated through ABA signaling pathways.

Comparative-genomic analysis reveals dynamic NLR gene loss and gain across Apiaceae species.

Introduction: Nucleotide-binding leucine-rich repeat (NLR) genes play a crucial role in green plants' responding to various pathogens. Genome-scale evolutionary studies of NLR genes are important for discovering and applying functional NLR genes. However, little is known about the evolution of NLR genes in the Apiaceae family including agricultural and medical plants. Methods: In this study, comparative genomic analysis was performed in four Apiaceae species to trace the dynamic evolutionary patterns of NLR genes during speciation in this family. Results: The results revealed different number of NLR genes in these four Apiaceae species, namely, Angelica sinensis (95), Coriandrum sativum (183), Apium graveolens (153) and Daucus carota (149). Phylogenetic analysis demonstrated that NLR genes in these four species were derived from 183 ancestral NLR lineages and experienced different levels of gene-loss and gain events. The contraction pattern of the ancestral NLR lineages was discovered during the evolution of D. carota, whereas a different pattern of contraction after first expansion of NLR genes was observed for A. sinensis, C. sativum and A. graveolens. Discussion: Taken together, rapid and dynamic gene content variation has shaped evolutionary history of NLR genes in Apiaceae species.

Coordinated regulation of plant defense and autoimmunity by paired trihelix transcription factors ASR3/AITF1 in Arabidopsis.

Plants perceive pathogens and induce robust transcriptional reprogramming to rapidly achieve immunity. The mechanisms of how immune-related genes are transcriptionally regulated remain largely unknown. Previously, the trihelix transcriptional factor ARABIDOPSIS SH4-RELATED 3 (ASR3) was shown to negatively regulate pattern-triggered immunity (PTI) in Arabidopsis thaliana. Here, we identified another trihelix family member ASR3-Interacting Transcriptional Factor 1 (AITF1) as an interacting protein of ASR3. ASR3-Interacting Transcriptional Factor 1 and ASR3 form heterogenous and homogenous dimers in planta. Both aitf1 and asr3 single mutants exhibited increased resistance against the bacterial pathogen Pseudomonas syringae, but the double mutant showed reduced resistance, suggesting AITF1 and ASR3 interdependently regulate immune gene expression and resistance. Overexpression of AITF1 triggered autoimmunity dependently on its DNA-binding ability and the presence of ASR3. Notably, autoimmunity caused by overexpression of AITF1 was dependent on a TIR-NBS-LRR (TNL) protein suppressor of AITF1-induced autoimmunity 1 (SAA1), as well as enhanced disease susceptibility 1 (EDS1), the central regulator of TNL signaling. ASR3-Interacting Transcriptional Factor 1 and ASR3 directly activated SAA1 expression through binding to the GT-boxes in SAA1 promoter. Collectively, our results revealed a mechanism of trihelix transcription factor complex in regulating immune gene expression, thereby modulating plant disease resistance and autoimmunity.

Genome-wide identification and analysis of the NLR gene family in Medicago ruthenica.

Medicago ruthenica, important forage in the legume family, possesses high nutritional value and carries abundant tolerance genes. This study used whole-genome data of M. ruthenica to perform a genome-wide analysis of the nucleotide-binding site-leucine-rich repeat receptor (NLR) gene family, which is the largest family of plant disease resistance genes (R genes). A total of 338 NLR genes were identified in the M. ruthenica genome, including 160 typical genes that contained 80 coiled-coil (CC)-NBS-LRR (CNL) genes, 76 toll/interleukin-1 receptor (TIR)-NBS-LRR (TNL) genes, four resistance to powdery mildew 8 (RPW8)-NBS-LRR (RNL) subclass genes, and 178 atypical NLR genes encoding proteins without at least one important domain. Among its eight chromosomes, M. ruthenica chromosomes 3 and 8 contained most of the NLR genes. More than 40% of all NLR genes were located on these two chromosomes, mainly in multigene clusters. The NLR proteins of M. ruthenica had six highly conserved motifs: P-loop, GLPL, RNBS-D, kinase-2, RNBS-C, and MHDV. Phylogenetic analysis revealed that the NLR genes of M. ruthenica formed three deeply separated clades according to the N-terminal domain of the proteins encoded by these genes. Gene duplication and syntenic analysis suggested four gene duplication types in the NLR genes of M. ruthenica, namely, tandem, proximal, dispersed, and segmental duplicates, which involved 189, 49, 59, and 41 genes, respectively. A total of 41 segmental duplication genes formed 23 NLR gene pairs located on syntenic chromosomal blocks mainly between chromosomes 6 and 7. In addition, syntenic analysis between M. truncatula and M. ruthenica revealed 193 gene pairs located on syntenic chromosomal blocks of the two species. The expression analysis of M. ruthenica NLR genes showed that 303 (89.6%) of the NLR genes were expressed in different varieties. Overall, this study described the full NLR profile of the M. ruthenica genome to provide an important resource for mining disease-resistant genes and disease-resistant breeding.

Genome-Wide Analysis of NLR Disease Resistance Genes in an Updated Reference Genome of Barley.

Barley is one of the top 10 crop plants in the world. During its whole lifespan, barley is frequently infected by various pathogens. In this study, we performed genome-wide analysis of the largest group of plant disease resistance (R) genes, the nucleotide binding site-leucine-rich repeat receptor (NLR) gene, in an updated barley genome. A total of 468 NLR genes were identified from the improved barley genome, including one RNL subclass and 467 CNL subclass genes. Proteins of 43 barley CNL genes were shown to contain 25 different integrated domains, including WRKY and BED. The NLR gene number identified in this study is much larger than previously reported results in earlier versions of barley genomes, and only slightly fewer than that in the diploid wheat Triticum urartu. Barley Chromosome 7 contains the largest number of 112 NLR genes, which equals to seven times of the number of NLR genes on Chromosome 4. The majority of NLR genes (68%) are located in multigene clusters. Phylogenetic analysis revealed that at least 18 ancestral CNL lineages were presented in the common ancestor of barley, T. urartu and Arabidopsis thaliana. Among them fifteen lineages expanded to 533 sub-lineages prior to the divergence of barley and T. urartu. The barley genome inherited 356 of these sub-lineages and duplicated to the 467 CNL genes detected in this study. Overall, our study provides an updated profile of barley NLR genes, which should serve as a fundamental resource for functional gene mining and molecular breeding of barley.

The NLR Protein Encoded by the Resistance Gene Ty-2 Is Triggered by the Replication-Associated Protein Rep/C1 of Tomato Yellow Leaf Curl Virus.

The whitefly-transmitted tomato yellow leaf curl virus (TYLCV) is one of the most destructive viral pathogens of cultivated tomato. To combat TYLCV, resistance gene Ty-2 has been introduced into cultivated tomato (Solanum lycopersicum) from wild tomato species Solanum habrochaites by interspecific crossing. Introgression lines with Ty-2 contain a large inversion compared with S. lycopersicum, which causes severe suppression of recombination and has hampered the cloning of Ty-2 so far. Here, we report the fine-mapping and cloning of Ty-2 using crosses between a Ty-2 introgression line and several susceptible S. habrochaites accessions. Ty-2 was shown to encode a nucleotide-binding leucine-rich repeat (NLR) protein. For breeding purposes, a highly specific DNA marker tightly linked to the Ty-2 gene was developed permitting marker-assisted selection. The resistance mediated by Ty-2 was effective against the Israel strain of TYLCV (TYLCV-IL) and tomato yellow leaf curl virus-[China : Shanghai2] (TYLCV-[CN : SH2]), but not against tomato yellow leaf curl Sardinia virus (TYLCSV) and leafhopper-transmitted beet curly top virus (BCTV). By co-infiltration experiments we showed that transient expression of the Rep/C1 protein of TYLCV, but not of TYLCSV triggered a hypersensitive response (HR) in Nicotiana benthamiana plants co-expressing the Ty-2 gene. Our results indicate that the Rep/C1 gene of TYLCV-IL presents the avirulence determinant of Ty-2-mediated resistance.

Alien Domains Shaped the Modular Structure of Plant NLR Proteins.

Plant innate immunity mostly relies on nucleotide-binding (NB) and leucine-rich repeat (LRR) intracellular receptors to detect pathogen-derived molecules and to induce defense responses. A multitaxa reconstruction of NB-domain associations allowed us to identify the first NB-LRR arrangement in the Chlorophyta division of the Viridiplantae. Our analysis points out that the basic NOD-like receptor (NLR) unit emerged in Chlorophytes by horizontal transfer and its diversification started from Toll/interleukin receptor-NB-LRR members. The operon-based genomic structure of Chromochloris zofingiensis NLR copies suggests a functional origin of NLR clusters. Moreover, the transmembrane signatures of NLR proteins in the unicellular alga C. zofingiensis support the hypothesis that the NLR-based immunity system of plants derives from a cell-surface surveillance system. Taken together, our findings suggest that NLRs originated in unicellular algae and may have a common origin with cell-surface LRR receptors.

Population Genetics of the Highly Polymorphic RPP8 Gene Family.

Plant nucleotide-binding domain and leucine-rich repeat containing (NLR) genes provide some of the most extreme examples of polymorphism in eukaryotic genomes, rivalling even the vertebrate major histocompatibility complex. Surprisingly, this is also true in Arabidopsis thaliana, a predominantly selfing species with low heterozygosity. Here, we investigate how gene duplication and intergenic exchange contribute to this extraordinary variation. RPP8 is a three-locus system that is configured chromosomally as either a direct-repeat tandem duplication or as a single copy locus, plus a locus 2 Mb distant. We sequenced 48 RPP8 alleles from 37 accessions of A. thaliana and 12 RPP8 alleles from Arabidopsis lyrata to investigate the patterns of interlocus shared variation. The tandem duplicates display fixed differences and share less variation with each other than either shares with the distant paralog. A high level of shared polymorphism among alleles at one of the tandem duplicates, the single-copy locus and the distal locus, must involve both classical crossing over and intergenic gene conversion. Despite these polymorphism-enhancing mechanisms, the observed nucleotide diversity could not be replicated under neutral forward-in-time simulations. Only by adding balancing selection to the simulations do they approach the level of polymorphism observed at RPP8. In this NLR gene triad, genetic architecture, gene function and selection all combine to generate diversity.

Comparative analysis of targeted long read sequencing approaches for characterization of a plant's immune receptor repertoire.

BACKGROUND: The Oxford Nanopore Technologies MinION™ sequencer is a small, portable, low cost device that is accessible to labs of all sizes and attractive for in-the-field sequencing experiments. Selective breeding of crops has led to a reduction in genetic diversity, and wild relatives are a key source of new genetic resistance to pathogens, usually via NLR immune receptor-encoding genes. Recent studies have demonstrated how crop NLR repertoires can be targeted for sequencing on Illumina or PacBio (RenSeq) and the specific gene conveying pathogen resistance identified. RESULTS: Sequence yields per MinION run are lower than Illumina, making targeted resequencing an efficient approach. While MinION generates long reads similar to PacBio it doesn't generate the highly accurate multipass consensus reads, which presents downstream bioinformatics challenges. Here we demonstrate how MinION data can be used for RenSeq achieving similar results to the PacBio and how novel NLR gene fusions can be identified via a Nanopore RenSeq pipeline. CONCLUSION: The described library preparation and bioinformatics methods should be applicable to other gene families or any targeted long DNA fragment nanopore sequencing project.