Plants evolved intracellular immune receptors that belong to the NOD-like receptor (NLR) family to recognize the presence of pathogen-derived effector proteins. NLRs possess an N-terminal Toll-like/IL-1 receptor (TIR) or a non-TIR domain [some of which contain coiled coils (CCs)], a central nucleotide-binding (NB-ARC) domain, and a C-terminal leucine-rich repeat (LRR). Activation of NLR proteins results in a rapid and high-amplitude immune response, eventually leading to host cell death at the infection site, the so-called hypersensitive response. Despite their important contribution to immunity, the exact mechanisms of NLR activation and signaling remain unknown and are likely heterogenous. We undertook a detailed structure-function analysis of the plasma membrane (PM)-localized CC NLR Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1) using both stable transgenic Arabidopsis and transient expression in Nicotiana benthamiana. We report that immune signaling is induced only by activated full-length PM-localized RPM1. Our interaction analyses demonstrate the importance of a functional P-loop for in planta interaction of RPM1 with the small host protein RPM1-interacting protein 4 (RIN4), for constitutive preactivation and postactivation self-association of RPM1 and for proper PM localization. Our results reveal an additive effect of hydrophobic conserved residues in the CC domain for RPM1 function and RPM1 self-association and their necessity for RPM1–RIN4 interaction. Thus, our findings considerably extend our understanding of the mechanisms regulating NLR activation at, and signaling from, the PM. Significance Pathogen recognition first occurs at the plasma membrane, where receptor-like kinases perceive pathogen-derived molecules and initiate immune responses. To abrogate this immune response, pathogens evolved effector proteins that act as virulence factors, often following delivery to the host cell. Plants evolved intracellular receptors, known as NOD-like receptors (NLRs), to detect effectors, thereby ensuring activation of effector-triggered immunity. However, despite their importance in immunity, the molecular mechanisms underlying effector recognition and subsequent immune activation by membrane-localized NLRs remain to be fully elucidated. Our analyses reveal the importance of and need for self-association and the coordinated interplay of specific domains and conserved residues for NLR activity. This could provide strategies for crop improvement, contributing to effective, environmentally friendly, and sustainable solutions for future agriculture.

The nucleotide-binding domain leucine-rich repeat (NLR) immune receptors play important roles in innate plant immunity. The activation of NLRs is specifically induced by their cognate effectors released from pathogens. Autoactive NLRs are expected to confer broad-spectrum resistance because they do not need cognate effectors to activate their immune responses. In this study, we demonstrated that the NLR genes RPS2 and RPM1(D505V) from Arabidopsis were autoactive in Oryza sativa and conferred broad-spectrum resistance to fungal pathogen Magnaporthe oryzae, bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), and pest brown planthopper (BPH, Nilaparvata lugens Stål). These results revealed that interfamily transfer of dicot NLRs to monocot species could be functional. The transgenic plants displayed early and strong induction of reactive oxygen species (ROS), callose deposition, and expression of defense-related genes after challenged with M. oryzae. The transcriptome analysis showed that the expressions of some defense-related genes were primed to adapt the transformed autoactive NLRs in the transgenic plants. This study indicates that autoactive NLRs are a promising resource for breeding crops with broad-spectrum resistance and provides new insights for engineering disease resistance.

RPM1 is a plant immune receptor that specially recognizes pathogen-released effectors to activate effector-triggered immunity (ETI) in Arabidopsis thaliana. RPM1 triggers ETI and hypersensitive response (HR) for disease resistance. Previous reports indicated that Phospholipase D (PLD) positively regulated RPM1-mediated HR. However, single, double, and triple pld knock-out mutants of 12 members of the PLD family in A. thaliana did not show suppressed RPM1-mediated HR, indicating the functional redundancy among PLD members. In this study, we revealed that PLD could negatively regulate the function of RPM1. We found that RPM1 interacted with PLDδ, but did not interact with PLDβ1, PLDβ2, and PLDγ3. Overexpression of PLDδ conducted to a reduction of protein level and corresponding activity of RPM1. We found that abscisic acid (ABA) reduced the protein level of RPM1, and the ABA-induced RPM1 reduction required PLD activity and PLD-derived phosphatidic acid (PA). Our study shows that PLD plays both negative and positive roles regulating the protein level and activity of RPM1 during stress responses in plants. PLD proteins are regulating points to integrate the abiotic and biotic responses of plants.

Lodging reduces rice yield, but increasing lodging resistance (LR) usually limits yield potential. Stem strength and leaf type are major traits related to LR and yield, respectively. Hence, understanding the genetic basis of stem strength and leaf type is of help to reduce lodging and increase yield in LR breeding. Here, we carried out an association analysis to identify quantitative trait locus (QTLs) affecting stem strength-related traits (internode length/IL, stem wall thickness/SWT, stem outer diameter/SOD, and stem inner diameter/SID) and leaf type-associated traits (Flag leaf length/FLL, Flag leaf angle/FLA, Flag leaf width/FLW, leaf-rolling/LFR and SPAD/Soil, and plant analyzer development) using a diverse panel of 550 accessions and evaluated over two years. Genome-wide association study (GWAS) using 4,076,837 high-quality single-nucleotide polymorphisms (SNPs) identified 89 QTLs for the nine traits. Next, through “gene-based association analysis, haplotype analysis, and functional annotation”, the scope was narrowed down step by step. Finally, we identified 21 candidate genes in 9 important QTLs that included four reported genes (TUT1, OsCCC1, CFL1, and ACL-D), and seventeen novel candidate genes. Introgression of alleles, which are beneficial for both stem strength and leaf type, or pyramiding stem strength alleles and leaf type alleles, can be employed for LR breeding. All in all, the experimental data and the identified candidate genes in this study provide a useful reference for the genetic improvement of rice LR.

Grain shape is an important agronomic character of rice, which affects the appearance, processing, and the edible quality. Screening and identifying more new genes associated with grain shape is beneficial to further understanding the genetic basis of grain shape and provides more gene resources for genetic breeding. This study has a natural population containing 623 indica rice cultivars. Genome-wide association studies/GWAS of several traits related to grain shape (grain length/GL, grain width/GW, grain length to width ratio/GLWR, grain circumferences/GC, and grain size/grain area/GS) were conducted by combining phenotypic data from four environments and the second-generation resequencing data, which have identified 39 important Quantitative trait locus/QTLs. We analyzed the 39 QTLs using three methods: gene-based association analysis, haplotype analysis, and functional annotation and identified three cloned genes (GS3, GW5, OsDER1) and seven new candidate genes in the candidate interval. At the same time, to effectively utilize the genes in the grain shape-related gene bank, we have also analyzed the allelic combinations of the three cloned genes. Finally, the extreme allele combination corresponding to each trait was found through statistical analysis. This study’s novel candidate genes and allele combinations will provide a valuable reference for future breeding work.