Nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes form a major line of defense in plants, acting in both pathogen recognition and resistance machinery activation. NLRs are reported to form large gene clusters in limber pine (Pinus flexilis), but it is unknown how widespread this genomic architecture may be among the extant species of conifers (Pinophyta). We used comparative genomic analyses to assess patterns in the abundance, diversity, and genomic distribution of NLR genes. Chromosome-level whole genome assemblies and high-density linkage maps in the Pinaceae, Cupressaceae, Taxaceae, and other gymnosperms were scanned for NLR genes using existing and customized pipelines. The discovered genes were mapped across chromosomes and linkage groups and analyzed phylogenetically for evolutionary history. Conifer genomes are characterized by dense clusters of NLR genes, highly localized on one chromosome. These clusters are rich in TNL-encoding genes, which seem to have formed through multiple tandem duplication events. In contrast to angiosperms and nonconiferous gymnosperms, genomic clustering of NLR genes is ubiquitous in conifers. NLR-dense genomic regions are likely to influence a large part of the plant\'s resistance, informing our understanding of adaptation to biotic stress and the development of genetic resources through breeding.

Pathogen recognition receptors encoded by R genes play a key role in plant protection. Nowadays, R genes are a basis for breeding many crops, including potato. Many potato R genes have been discovered and found suitable for breeding thanks to the studies of a wide variety of wild potato species. The use of primitive cultivated potato species (PCPS) as representatives of the primary gene pool can also be promising in this respect. PCPS are the closest to the early domesticated forms of potato; therefore, their investigation could help understand the evolution of R genes. The present study was aimed at identifying and analyzing R genes in PCPS listed in the open database of NCBI and Solomics DB. In total, the study involved 27 accessions belonging to three species: Solanum phureja Juz. & Bukasov, S. stenotomum Juz. & Bukasov and S. goniocalyx Juz. & Bukasov Materials for the analysis were the sequencing data for the said three species from the PRJNA394943 and PRJCA006011 projects. An in silico search was carried out for sequences homologous to 26 R genes identified in potato species differing in phylogenetic distance from PCPS, namely nightshade (S. americanum), North- (S. bulbocastanum, S. demissum) and South-American (S. venturii, S. berthaultii) wild potato species, as well as the cultivated potato species S. tuberosum and S. andigenum. Homologs of all investigated protein-coding sequences were discovered in PCPS with a relatively high degree of similarity (85-100 %). Homologs of the Rpi-R3b, Rpi-amr3 and Rpi-ber1 genes have been identified in PCPS for the first time. An analysis of polymorphism of nucleotide and amino acid sequences has been carried out for 15 R genes. The differences in frequencies of substitutions in PCPS have been demonstrated by analysis of R genes, the reference sequences of which have been identified in different species. For all the studied NBS-LRR genes, the proportion of substituted amino acids in the LRR domain exceeds this figure for the NBS domain. The potential prospects of using PCPS as sources of resistance to Verticillium wilt have been shown.
Ключевую роль в защите растений от патогенов играют рецепторы, кодируемые R-генами. Они являются генетической основой для селекции многих сельскохозяйственных культур, в том числе картофеля. Множество генов устойчивости у картофеля стало известно и было вовлечено в селекцию благодаря изучению широкого разнообразия диких сородичей картофеля. Использование примитивных культурных видов (ПКВ), относящихся к первичному генофонду картофеля, также перспективно. Как наиболее близкие к ранним доместицированным формам картофеля, ПКВ представляют особый интерес для исследования эволюции генов устойчивости. Целью настоящего исследования стали поиск и анализ R-генов у ПКВ картофеля, геномы которых с различным качеством сборки представлены в базе данных NCBI. Исследовано 27 образцов, относящихся к трем видам: Solanum phureja Juz. & Bukasov, S. stenotomum Juz. & Bukasov и S. goniocalyx Juz. & Bukasov. Проведен in silico поиск последовательностей, гомологичных 26 R-генам, идентифицированных у различных по филогенетической отдаленности от ПКВ картофеля видов: паслёна (S. americanum Mill.), североамериканских (S. bulbocastanum Dunal., S. demissum Lindl.) и южноамериканских (S. venturii Hawkes &Hjert., S. berthaultii Hawkes) диких видов, а также видов культурного картофеля (S. tuberosum L., S. andigenum Juz. & Bukasov). Гомологи кодирующих последовательностей всех исследованных генов обнаружены у ПКВ картофеля с относительно высокой степенью сходства (85–100 %). Впервые у примитивных культурных видов картофеля найдены гомологи генов R3b, Rpi-amr3 и Rpi- ber1. Для 15 R-генов проведен анализ полиморфизма нуклеотидных и аминокислотных последовательностей. Приведены отличия в частоте замен у ПКВ картофеля при анализе R-генов, референсные последовательности которых идентифицированы у разных видов. Для всех изученных NBS-LRR генов доля замещенных аминокислот в LRR-домене превосходит этот показатель для NBS-домена. Показана потенциальная перспективность использования ПКВ картофеля в качестве источников устойчивости к вертициллёзному увяданию.

Plants are frequently attacked by a variety of pathogens and thus have evolved a series of defense mechanisms, one important mechanism is resistance gene (R gene)-mediated disease resistance, but its expression is tightly regulated. NBS-LRR genes are the largest gene family of R genes. microRNAs (miRNAs) target to a number of NBS-LRR genes and trigger the production of phased small interfering RNAs (phasiRNAs) from these transcripts. phasiRNAs cis or trans regulate NBS-LRR genes, which can result in the repression of R gene expression. In this study, we screened for upregulated miR482 in the susceptible apple cultivar \'Golden Delicious\' (GD) after inoculation with the fungal pathogen Alternaria alternata f. sp. mali (ALT1). Additionally, through combined degradome sequencing, we identified a gene targeted by miR482, named MdTNL1, a gene encoding a TIR-NBS-LRR (Toll/interleukin1 receptor-nucleotide binding site-leucine-rich repeat) protein. This gene exhibited a significant down-regulation post ALT1 inoculation, suggesting an impact on gene expression mediated by miRNA regulation. miR482 could cleave MdTNL1 and generate phasiRNAs at the cleavage site. We found that overexpression of miR482 inhibited the expression of MdTNL1 and thus reduced the disease resistance of GD, while silencing of miR482 increased the expression of MdTNL1 and thus improved the disease resistance of GD. This work elucidates key mechanisms underlying the immune response to Alternaria infection in apple. Identification of the resistance genes involved will enable molecular breeding for prevention and control of Alternaria leaf spot disease in this important fruit crop.

BACKGROUND: Most disease resistance (R) genes in plants encode proteins that contain leucine-rich-repeat (LRR) and nucleotide-binding site (NBS) domains, which belong to the NBS-LRR family. The sequenced genomes of Fusarium wilt-susceptible Vernicia fordii and its resistant counterpart, Vernicia montana, offer significant resources for the functional characterization and discovery of novel NBS-LRR genes in tung tree.
RESULTS: Here, we identified 239 NBS-LRR genes across two tung tree genomes: 90 in V. fordii and 149 in V. montana. Five VmNBS-LRR paralogous were predicted in V. montana, and 43 orthologous were detected between V. fordii and V. montana. The orthologous gene pair Vf11G0978-Vm019719 exhibited distinct expression patterns in V. fordii and V. montana: Vf11G0978 showed downregulated expression in V. fordii, while its orthologous gene Vm019719 demonstrated upregulated expression in V. montana, indicating that this pair may be responsible for the resistance to Fusarium wilt in V. montana. Vm019719 from V. montana, activated by VmWRKY64, was shown to confer resistance to Fusarium wilt in V. montana by a virus-induced gene silencing (VIGS) experiment. However, in the susceptible V. fordii, its allelic counterpart, Vf11G0978, exhibited an ineffective defense response, attributed to a deletion in the promoter\'s W-box element.
CONCLUSIONS: This study provides the first systematic analysis of NBS-LRR genes in the tung tree and identifies a candidate gene that can be utilized for marker-assisted breeding to control Fusarium wilt in V. fordii.

The nucleotide-binding site-leucine-rich repeat (NBS-LRR) gene family is the largest group of disease resistance (R) genes in plants and is active in response to viruses, bacteria, and fungi usually involved in effector-triggered immunity (ETI). Pangenome-wide studies allow researchers to analyze the genetic diversity of multiple species or their members simultaneously, providing a comprehensive understanding of the evolutionary relationships and diversity present among them. The draft pan-genome of three Mangifera indica cultivars (Alphonso, Hong Xiang Ya, and Tommy atkins) was constructed and Presence/absence variants (PAVs) were filtered through the ppsPCP pipeline. As a result, 2823 genes and 5907 PAVs from H. Xiang Ya, and 1266 genes and 2098 PAVs from T. atkins were added to the reference genome. For the identification of CC-NBS-LRR (CNL) genes in these mango cultivars, this draft pan-genome study has successfully identified 47, 27, and 36 members in Alphonso, H. Xiang Ya, and T. atkins respectively. The phylogenetic analysis divided MiCNL proteins into four distinct subgroups. All MiCNL genes are unevenly distributed on chromosomes. Both tandem and segmental duplication events played a significant role in the expansion of the CNL gene family. These genes contain cis-elements related to light, stress, hormone, and development. The analysis of protein-protein interactions (PPI) revealed that MiCNL proteins interacted with other defense-responsive proteins. Gene Ontology (GO) analysis indicated that MiCNL genes play a role in defense mechanisms within the organism. The expression level of the identified genes in fruit peel was observed under disease and cold stress which showed that Mi_A_CNL13 and 14 were up-regulated while Mi_A_CNL15, 25, 30, 31, and 40 were down-regulated in disease stress. On the other hand, Mi_A_CNL2, 14, 41, and 45 were up-regulated and Mi_A_CNL47 is down-regulated in cold stress. Subsequently, the Random Forest (RF) classifier was used to assess the multi-stress response of MiCNLs. It was found that Mi_A_CNL14 is a gene that responds to multiple stress conditions. The CNLs have similar protein structures which show that they are involved in the same function. The above findings provide a foundation for a deeper understanding of the functional characteristics of the mango CNL gene family.

Plants coordinately use cell-surface and intracellular immune receptors to perceive pathogens and mount an immune response. Intracellular events of pathogen recognition are largely mediated by immune receptors of the nucleotide binding and leucine rich-repeat (NLR) classes. Upon pathogen perception, NLRs trigger a potent broad-spectrum immune reaction, usually accompanied by a form of programmed cell death termed the hypersensitive response. Some plant NLRs act as multifunctional singleton receptors which combine pathogen detection and immune signaling. However, NLRs can also function in higher order pairs and networks of functionally specialized interconnected receptors. In this article, we cover the basic aspects of plant NLR biology with an emphasis on NLR networks. We highlight some of the recent advances in NLR structure, function, and activation and discuss emerging topics such as modulator NLRs, pathogen suppression of NLRs, and NLR bioengineering. Multi-disciplinary approaches are required to disentangle how these NLR immune receptor pairs and networks function and evolve. Answering these questions holds the potential to deepen our understanding of the plant immune system and unlock a new era of disease resistance breeding.

UNASSIGNED: Cold stress adversely affects the growth and development of plants and limits the geographical distribution of many plant species. Accumulation of spontaneous mutations shapes the adaptation of plant species to diverse climatic conditions.
UNASSIGNED: The genome-wide association study of the phenotypic variation gathered by a newly designed phenomic platform with the over six millions single nucleotide polymorphic (SNP) loci distributed across the genomes of 417 Arabidopsis natural variants collected from various geographical regions revealed 33 candidate cold responsive genes.
UNASSIGNED: Investigation of at least two independent insertion mutants for 29 genes identified 16 chilling tolerance genes governing diverse genetic mechanisms. Five of these genes encode novel leucine-rich repeat domain-containing proteins including three nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins. Among the 16 identified chilling tolerance genes, ADS2 and ACD6 are the only two chilling tolerance genes identified earlier.
UNASSIGNED: The 12.5% overlap between the genes identified in this genome-wide association study (GWAS) of natural variants with those discovered previously through forward and reverse genetic approaches suggests that chilling tolerance is a complex physiological process governed by a large number of genetic mechanisms.

The nucleotide-binding site and leucine-rich repeat (NBS-LRR) genes, one of the largest gene families in plants, are evolving rapidly and playing a critical role in plant resistance to pathogens. In this study, a genome-wide search in 12 Rosaceae genomes screened out 2188 NBS-LRR genes, with the gene number varied distinctively across different species. The reconciled phylogeny revealed 102 ancestral genes (7 RNLs, 26 TNLs, and 69 CNLs), which underwent independent gene duplication and loss events during the divergence of the Rosaceae. The NBS-LRR genes exhibited dynamic and distinct evolutionary patterns in the 12 Rosaceae species due to independent gene duplication/loss events, which resulted the discrepancy of NBS-LRR gene number among Rosaceae species. Specifically, Rubus occidentalis, Potentilla micrantha, Fragaria iinumae and Gillenia trifoliata, displayed a \"first expansion and then contraction\" evolutionary pattern; Rosa chinensis exhibited a \"continuous expansion\" pattern; F. vesca had a \"expansion followed by contraction, then a further expansion\" pattern, three Prunus species and three Maleae species shared a \"early sharp expanding to abrupt shrinking\" pattern. Overall, this study elucidated the dynamic and complex evolutionary patterns of NBS-LRR genes in the 12 Rosaceae species, and could assist further investigation of mechanisms driving these evolutionary patterns.

The soybean Rpp1 locus confers resistance to Phakopsora pachyrhizi, causal agent of rust, and resistance is usually dominant over susceptibility. However, dominance of Rpp1-mediated resistance is lost when a resistant genotype (Rpp1 or Rpp1b) is crossed with susceptible line TMG06_0011, and the mechanism of this dominant susceptibility (DS) is unknown. Sequencing the Rpp1 region reveals that the TMG06_0011 Rpp1 locus has a single nucleotide-binding site leucine-rich repeat (NBS-LRR) gene (DS-R), whereas resistant PI 594760B (Rpp1b) is similar to PI 200492 (Rpp1) and has three NBS-LRR resistance gene candidates. Evidence that DS-R is the cause of DS was reflected in virus-induced gene silencing of DS-R in Rpp1b/DS-R or Rpp1/DS-R heterozygous plants with resistance partially restored. In heterozygous Rpp1b/DS-R plants, expression of Rpp1b candidate genes was not significantly altered, indicating no effect of DS-R on transcription. Physical interaction of the DS-R protein with candidate Rpp1b resistance proteins was supported by yeast two-hybrid studies and in silico modeling. Thus, we conclude that suppression of resistance most likely does not occur at the transcript level, but instead probably at the protein level, possibly with Rpp1 function inhibited by binding to the DS-R protein. The DS-R gene was found in other soybean lines, with an estimated allele frequency of 6% in a diverse population, and also found in wild soybean (Glycine soja). The identification of a dominant susceptible NBS-LRR gene provides insight into the behavior of NBS-LRR proteins and serves as a reminder to breeders that the dominance of an R gene can be influenced by a susceptibility allele.

Crop yield and global food security are under constant threat from plant pathogens with the potential to cause epidemics. Traditional breeding for disease resistance can be too slow to counteract these emerging threats, resulting in the need to retool the plant immune system using bioengineered made-to-order immune receptors. Efforts to engineer immune receptors have focused primarily on nucleotide-binding domain and leucine-rich repeat (NLR) immune receptors and proof-of-principles studies. Based upon a near-exhaustive literature search of previously engineered plant immune systems we distil five emerging principles in the design of bioengineered made-to-order plant NLRs and describe approaches based on other components. These emerging principles are anticipated to assist the functional understanding of plant immune receptors, as well as bioengineering novel disease resistance specificities.