Nucleotide-binding sites and leucine-rich repeat proteins (NLRs) act as critical intracellular immune receptors. Previous studies reported an Arabidopsis-resistant gene L3 (AT1G15890), which encoded a coiled-coil (CC) NLR that conferred cell death in bacteria; however, its function in planta remains unclear. This study describes a comprehensive structure-function analysis of L3 in Nicotiana benthamiana. The results of the transient assay showed that the L3 CC domain is sufficient for cell-death induction. The first 140 amino acid segment constituted the minimal function region that could cause cell death. The YFP-labeled L3 CC domain was localized to the plasma membrane, which was considered crucial for the function and self-interaction of the L3 CC domain. The results of point mutations analysis showed that L3 CC domain function is affected by mutations in some specific residues, and loss-of-function mutations in the CC domain affected the function of full-length L3. These study results offered considerable evidence to understand the activation mechanism of L3.

Plant proteins with domains rich in leucine repeats play important roles in detecting pathogens and triggering defense reactions, both at the cellular surface for pattern-triggered immunity and in the cell to ensure effector-triggered immunity. As intracellular parasites, viruses are mostly detected intracellularly by proteins with a nucleotide binding site and leucine-rich repeats but receptor-like kinases with leucine-rich repeats, known to localize at the cell surface, have also been involved in response to viruses. In the present review we report on the progress that has been achieved in the last decade on the role of these leucine-rich proteins in antiviral immunity, with a special focus on our current understanding of the hypersensitive response.

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.

Plants have evolved two layers of protection against biotic stress: PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). The primary mechanism of ETI involves nucleotide-binding leucine-rich repeat immune receptors (NLRs). Although NLR genes have been studied in several plant species, a comprehensive database of NLRs across a diverse array of species is still lacking. Here, we present a thorough analysis of NLR genes across 100 high-quality plant genomes (PlantNLRatlas). The PlantNLRatlas includes a total of 68,452 NLRs, of which 3,689 are full-length and 64,763 are partial-length NLRs. The majority of NLR groups were phyletically clustered. In addition, the domain sequences were found to be highly conserved within each NLR group. Our PlantNLRatlas dataset is complementary to RefPlantNLR, a collection of NLR genes which have been experimentally confirmed. The PlantNLRatlas should prove helpful for comparative investigations of NLRs across a range of plant groups, including understudied taxa. Finally, the PlantNLRatlas resource is intended to help the field move past a monolithic understanding of NLR structure and function.

Nucleotide-binding domain leucine-rich repeat (NLR) immune receptors are important components of plant and metazoan innate immunity that can function as individual units or as pairs or networks. Upon activation, NLRs form multiprotein complexes termed resistosomes or inflammasomes. Although metazoan paired NLRs, such as NAIP/NLRC4, form hetero-complexes upon activation, the molecular mechanisms underpinning activation of plant paired NLRs, especially whether they associate in resistosome hetero-complexes, is unknown. In asterid plant species, the NLR required for cell death (NRC) immune receptor network is composed of multiple resistance protein sensors and downstream helpers that confer immunity against diverse plant pathogens. Here, we show that pathogen effector-activation of the NLR proteins Rx (confers virus resistance), and Bs2 (confers bacterial resistance) leads to oligomerization of their helper NLR, NRC2. Activated Rx does not oligomerize or enter into a stable complex with the NRC2 oligomer and remains cytoplasmic. In contrast, activated NRC2 oligomers accumulate in membrane-associated puncta. We propose an activation-and-release model for NLRs in the NRC immune receptor network. This points to a distinct activation model compared with mammalian paired NLRs.

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.

Cyst nematodes are considered a dominant threat to yield for a wide range of major food crops. Current control strategies are mainly dependent on crop rotation and the use of resistant cultivars. Various crops exhibit single dominant resistance (R) genes that are able to activate effective host-specific resistance to certain cyst nematode species and/or populations. An example is the potato R gene Gpa2, which confers resistance against the potato cyst nematode (PCN), Globodera pallida population D383. Activation of Gpa2 results in a delayed resistance response, which is characterized by a layer of necrotic cells formed around the developing nematode feeding structure. However, knowledge about the Gpa2-induced defense pathways is still lacking. Here, we uncover the transcriptional changes and gene expression network induced upon Gpa2 activation in potato roots infected with G. pallida. To this end, in vitro-grown Gpa2-resistant potato roots were infected with the avirulent population D383 and virulent population Rookmaker. Infected root segments were harvested at 3 and 6 dpi and sent for RNA sequencing. Comparative transcriptomics revealed a total of 1,743 differentially expressed genes (DEGs) upon nematode infection, of which 559 DEGs were specifically regulated in response to D383 infection. D383-specific DEGs associated with Gpa2-mediated defense mainly relates to calcium-binding activity, salicylic acid (SA) biosynthesis, and systemic acquired resistance (SAR). These data reveal that cyst nematode resistance in potato roots depends on conserved downstream signaling pathways involved in plant immunity, which are also known to contribute to R genes-mediated resistance against other pathogens with different lifestyles.

Nucleotide binding, leucine-rich repeat (NB-LRR) proteins are critical for disease resistance in plants, while we do not know whether S-acylation of these proteins plays a role during bacterial infection. We identified 30 Arabidopsis mutants with mutations in NB-LRR encoding genes from the Nottingham Arabidopsis Stock Center and characterized their contribution to the plant immune response after inoculation with Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Of the five mutants that were hyper-susceptible to the pathogen, three (R5L1, R5L2 and RPS5) proteins contain the conserved S-acylation site in the N-terminal coiled-coil (CC) domain. In wild-type (WT) Arabidopsis plants, R5L1 was transcriptionally activated upon pathogen infection, and R5L1 overexpression lines had enhanced resistance. Independent experiments indicated that R5L1 localized at the plasma membrane (PM) via S-acylation of its N-terminal CC domain, which was mediated by PROTEIN S-ACYL TRANSFERASE 13/16 (PAT13, PAT16). Modification of the S-acylation site reduced its affinity for binding the PM, with a consequent significant reduction in bacterial resistance. PM localization of R5L1 was significantly reduced in pat13 and pat16 mutants, similar to what was found for WT plants treated with 2-bromopalmitate, an S-acylation-blocking agent. Transgenic plants expressing R5L1 in the pat13 pat16 double mutant showed no enhanced disease resistance. Overexpression of R5L1 in WT Arabidopsis resulted in substantial accumulation of reactive oxygen species after inoculation with Pst DC3000; this effect was not observed with a mutant R5L1 carrying a mutated S-acylation site. Our data suggest that PAT13- and PAT16-mediated S-acylation of R5L1 is crucial for its membrane localization to activate the plant defense response.

Plant Nucleotide binding-Leucine rich repeat (NLR) proteins play a significant role in pathogen detection and the activation of effector-triggered immunity. NLR regulation has mainly been studied at a protein level, with large knowledge gaps remaining regarding the transcriptional control of NLR genes. The mis-regulation of NLR gene expression may lead to the inability of plants to recognize pathogen infection, lower levels of immune response activation, and ultimately plant susceptibility. This highlights the importance of understanding all aspects of NLR regulation. Three main mechanisms have been shown to control NLR expression: epigenetic modifications, cis elements which bind transcription factors, and post-transcriptional modifications. In this review, we aim to provide an overview of these mechanisms known to control NLR expression, and those which contribute toward successful immune responses. Furthermore, we discuss how pathogens can interfere with NLR expression to increase pathogen virulence. Understanding how these molecular mechanisms control NLR expression would contribute significantly toward building a complete picture of how plant immune responses are activated during pathogen infection-knowledge which can be applied during crop breeding programs aimed to increase resistance toward numerous plant pathogens.

Avocado is an important agricultural food crop in many countries worldwide. Phytophthora cinnamomi, a hemibiotrophic oomycete, remains one of the most devastating pathogens within the avocado industry, as it is near impossible to eradicate from areas where the pathogen is present. A key aspect to Phytophthora root rot disease management is the use of avocado rootstocks partially resistant to P. cinnamomi, which demonstrates an increased immune response following infection. In plant species, Nucleotide binding-Leucine rich repeat (NLR) proteins form an integral part of pathogen recognition and Effector triggered immune responses (ETI). To date, a comprehensive set of Persea americana NLR genes have yet to be identified, though their discovery is crucial to understanding the molecular mechanisms underlying P. americana-P. cinnamomi interactions. In this study, a total of 161 PaNLR genes were identified in the P. americana West-Indian pure accession genome. These putative resistance genes were characterized using bioinformatic approaches and grouped into 13 distinct PaNLR gene clusters, with phylogenetic analysis revealing high sequence similarity within these clusters. Additionally, PaNLR expression levels were analyzed in both a partially resistant (Dusa®) and a susceptible (R0.12) avocado rootstock infected with P. cinnamomi using an RNA-sequencing approach. The results showed that the partially resistant rootstock has increased expression levels of 84 PaNLRs observed up to 24 h post-inoculation, while the susceptible rootstock only showed increased PaNLR expression during the first 6 h post-inoculation. Results of this study may indicate that the partially resistant avocado rootstock has a stronger, more prolonged ETI response which enables it to suppress P. cinnamomi growth and combat disease caused by this pathogen. Furthermore, the identification of PaNLRs may be used to develop resistant rootstock selection tools, which can be employed in the avocado industry to accelerate rootstock screening programs.