Protein stability governed by α1-2 helices in Pvr4 is essential for localization and cell death.

Plant nucleotide-binding domain leucine-rich-repeat receptor (NLR) confers disease resistance to various pathogens by recognizing effectors derived from the pathogen. Previous studies have shown that overexpression of the CC domain in several NLRs triggers cell death, implying that the CC domain plays an important role as a signaling module. However, how CC domain transduces immune signals remains largely unknown. A Potyvirus-resistant NLR protein, Pvr4, possesses a CC domain (CCPvr4 ) that induces cell death upon transient overexpression in Nicotiana benthamiana. In this study, loss-of-function mutants were generated by error-prone PCR-based random mutagenesis to understand the molecular mechanisms underlying CCPvr4 -mediated cell death. Cell biology and biochemical studies revealed that M16 and Q52 in the α1 and α2 helices, respectively, are crucial for protein stability, and mutation of these residues disrupts localization to the plasma membrane and oligomerization activity. The increase of the protein stability of these mutants by tagging a green fluorescent protein (GFP) variant led to restoration of cell death-inducing activity and plasma membrane localization. Another mutant, I7E in the very N-terminal region, lost cell death-inducing activity by weakening the interaction with plasma membrane H+ -ATPase compared to CCPvr4 , although the protein remained in the plasma membrane. Moreover, most of the mutated residues are on the outer surface of the funnel shape in the predicted pentameric CCPvr4 , implying that the disordered N-terminal region plays a crucial role in association with PMA as well as targeting to the plasma membrane. This work could provide insights into the molecular mechanisms of cell death induced by NLR immune receptors.

Oomycete effector AVRblb2 targets cyclic nucleotide-gated channels through calcium sensors to suppress pattern-triggered immunity.

Transient and rapid increase in cytosolic Ca2+ plays a crucial role in plant-pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Cyclic nucleotide-gated channels (CNGCs) have been implicated in mediating this Ca2+ influx; however, their regulatory mechanisms remain poorly understood. Here, we have found that AVRblb2 requires the calmodulin (CaM) and calmodulin-like (CML) proteins as co-factors to interact with the NbCNGCs, resulting in the formation of AVRblb2-CaM/CML-NbCNGCs complex. Furthermore, CaM and CML are dissociated from NbCNGC18 during PTI response to increase Ca2+ influx; however, Avrblb2 inhibits calcium channel activation by disrupting the release of CaM and CML from NbCNGC18. Following recognition of PAMP, NbCNGC18 forms active heteromeric channels with other NbCNGCs, which may give selectivity of CNGC complex against diverse signals for fine-tuning of cytosolic Ca2+ level to mediate appropriate responses. Silencing of multiple NbCNGCs compromised the function of AVRblb2 on the pathogenicity of Phytophthora infestans, confirming that AVRblb2 contributes to pathogen virulence by targeting CNGCs. Our findings provide new insights into the regulation of CNGCs in PTI and the role of pathogen effectors in manipulating host cell physiology to promote infection.

Phytophthora infestans RxLR Effector PITG06478 Hijacks 14-3-3 to Suppress PMA Activity Leading to Necrotrophic Cell Death.

Pathogens often induce cell death for their successful proliferation in the host plant. Plasma membrane H+-ATPases (PMAs) are targeted by either pathogens or plant immune receptors in immune response regulation. Although PMAs play pivotal roles in host cell death, the molecular mechanism of effector-mediated regulation of PMA activity has not been described. Here, we report that the Phytophthora infestans RxLR effector PITG06478 can induce cell death in Nicotiana benthamiana but the induced cell death is inhibited by fusicoccin (FC), an irreversible PMA activator. PITG06478, which is localized at the plasma membrane, is not directly associated with the PMA but is associated with Nb14-3-3s, a PMA activator. Immunoblot analyses revealed that the interaction between PITG06478 and Nb14-3-3s was disrupted by FC. PMA activity in PITG06478-expressing plants was eventually inhibited, and cell death likely occurred because the 14-3-3 protein was hijacked. Our results further confirm the significance of PMA activity in host cell death and provide new insight into how pathogens utilize essential host components to sustain their life cycle. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Nucleotide-binding leucine-rich repeat network underlies nonhost resistance of pepper against the Irish potato famine pathogen Phytophthora infestans.

Nonhost resistance (NHR) is a robust plant immune response against non-adapted pathogens. A number of nucleotide-binding leucine-rich repeat (NLR) proteins that recognize non-adapted pathogens have been identified, although the underlying molecular mechanisms driving robustness of NHR are still unknown. Here, we screened 57 effectors of the potato late blight pathogen Phytophthora infestans in nonhost pepper (Capsicum annuum) to identify avirulence effector candidates. Selected effectors were tested against 436 genome-wide cloned pepper NLRs, and we identified multiple functional NLRs that recognize P. infestans effectors and confer disease resistance in the Nicotiana benthamiana as a surrogate system. The identified NLRs were homologous to known NLRs derived from wild potatoes that recognize P. infestans effectors such as Avr2, Avrblb1, Avrblb2, and Avrvnt1. The identified CaRpi-blb2 is a homologue of Rpi-blb2, recognizes Avrblb2 family effectors, exhibits feature of lineage-specifically evolved gene in microsynteny and phylogenetic analyses, and requires pepper-specific NRC (NLR required for cell death)-type helper NLR for proper function. Moreover, CaRpi-blb2-mediated hypersensitive response and blight resistance were more tolerant to suppression by the PITG_15 278 than those mediated by Rpi-blb2. Combined results indicate that pepper has stacked multiple NLRs recognizing effectors of non-adapted P. infestans, and these NLRs could be more tolerant to pathogen-mediated immune suppression than NLRs derived from the host plants. Our study suggests that NLRs derived from nonhost plants have potential as untapped resources to develop crops with durable resistance against fast-evolving pathogens by stacking the network of nonhost NLRs into susceptible host plants.

Ptr1 and ZAR1 immune receptors confer overlapping and distinct bacterial pathogen effector specificities.

Some nucleotide-binding and leucine-rich repeat receptors (NLRs) indirectly detect pathogen effectors by monitoring their host targets. In Arabidopsis thaliana, RIN4 is targeted by multiple sequence-unrelated effectors and activates immune responses mediated by RPM1 and RPS2. These effectors trigger cell death in Nicotiana benthamiana, but the corresponding NLRs have yet not been identified. To identify N. benthamiana NLRs (NbNLRs) that recognize Arabidopsis RIN4-targeting effectors, we conducted a rapid reverse genetic screen using an NbNLR VIGS library. We identified that the N. benthamiana homolog of Ptr1 (Pseudomonas tomato race 1) recognizes the Pseudomonas effectors AvrRpt2, AvrRpm1, and AvrB. We demonstrated that recognition of the Xanthomonas effector AvrBsT and the Pseudomonas effector HopZ5 is conferred independently by the N. benthamiana homolog of Ptr1 and ZAR1. Interestingly, the recognition of HopZ5 and AvrBsT is contributed unequally by Ptr1 and ZAR1 in N. benthamiana and Capsicum annuum. In addition, we showed that the RLCK XII family protein JIM2 is required for the NbZAR1-dependent recognition of AvrBsT and HopZ5. The recognition of sequence-unrelated effectors by NbPtr1 and NbZAR1 provides an additional example of convergently evolved effector recognition. Identification of key components involved in Ptr1 and ZAR1-mediated immunity could reveal unique mechanisms of expanded effector recognition.

The Phytophthora capsici RxLR effector CRISIS2 triggers cell death via suppressing plasma membrane H+-ATPase in the host plant.

Pathogen effectors can suppress various plant immune responses, suggesting that they have multiple targets in the host. To understand the mechanisms underlying plasma membrane-associated and effector-mediated immunity, we screened the Phytophthora capsici RxLR cell death-inducer suppressing immune system (CRISIS). We found that the cell death induced by the CRISIS2 effector in Nicotiana benthamiana was inhibited by the irreversible plasma membrane H+-ATPase (PMA) activator fusicoccin. Biochemical and gene-silencing analyses revealed that CRISIS2 physically and functionally associated with PMAs and induced host cell death independent of immune receptors. CRISIS2 induced apoplastic alkalization by suppressing PMA activity via its association with the C-terminal regulatory domain. In planta expression of CRISIS2 significantly enhanced the virulence of P. capsici, whereas host-induced gene-silencing of CRISIS2 compromised the disease symptoms and the biomass of the pathogen. Thus, our study has identified a novel RxLR effector that plays multiple roles in the suppression of plant defense and in the induction of cell death to support the pathogen hemibiotrophic life cycle in the host plant.

Plasma membrane-localized plant immune receptor targets H+ -ATPase for membrane depolarization to regulate cell death.

The hypersensitive response (HR) is a robust immune response mediated by nucleotide-binding, leucine-rich repeat receptors (NLRs). However, the early molecular event that links activated NLRs to cell death is unclear. Here, we demonstrate that NLRs target plasma membrane H+ -ATPases (PMAs) that generate electrochemical potential, an essential component of living cells, across the plasma membrane. CCA 309, an autoactive N-terminal domain of a coiled-coil NLR (CNL) in pepper, is associated with PMAs. Silencing or overexpression of PMAs reversibly affects cell death induced by CCA 309 in Nicotiana benthamiana. CCA 309-induced extracellular alkalization causes plasma membrane depolarization, followed by cell death. Coimmunoprecipitation analyses suggest that CCA 309 inhibits PMA activation by preoccupying the dephosphorylated penultimate threonine residue of PMA. Moreover, pharmacological experiments using fusicoccin, an irreversible PMA activator, showed that inhibition of PMAs contributes to CNL-type (but not Toll interleukin-1 receptor NLR-type) resistance protein-induced cell death. We suggest PMAs as primary targets of plasma membrane-associated CNLs leading to HR-associated cell death by disturbing the electrochemical gradient across the membrane. These results provide new insight into NLR-mediated cell death in plants, as well as innate immunity in higher eukaryotes.

Admixture of divergent genomes facilitates hybridization across species in the family Brassicaceae.

Hybridization and polyploidization are pivotal to plant evolution. Genetic crosses between distantly related species are rare in nature due to reproductive barriers but how such hurdles can be overcome is largely unknown. Here we report the hybrid genome structure of xBrassicoraphanus, a synthetic allotetraploid of Brassica rapa and Raphanus sativus. We performed cytogenetic analysis and de novo genome assembly to examine chromosome behaviors and genome integrity in the hybrid. Transcriptome analysis was conducted to investigate expression of duplicated genes in conjunction with epigenome analysis to address whether genome admixture entails epigenetic reconfiguration. Allotetraploid xBrassicoraphanus retains both parental chromosomes without genome rearrangement. Meiotic synapsis formation and chromosome exchange are avoided between nonhomologous progenitor chromosomes. Reconfiguration of transcription network occurs, and less divergent cis-elements of duplicated genes are associated with convergent expression. Genome-wide DNA methylation asymmetry between progenitors is largely maintained but, notably, B. rapa-originated transposable elements are transcriptionally silenced in xBrassicoraphanus through gain of DNA methylation. Our results demonstrate that hybrid genome stabilization and transcription compatibility necessitate epigenome landscape adjustment and rewiring of cis-trans interactions. Overall, this study suggests that a certain extent of genome divergence facilitates hybridization across species, which may explain the great diversification and expansion of angiosperms during evolution.

Comparative analysis of de novo genomes reveals dynamic intra-species divergence of NLRs in pepper.

BACKGROUND: Peppers (Capsicum annuum L.) containing distinct capsaicinoids are the most widely cultivated spices in the world. However, extreme genomic diversity among species represents an obstacle to breeding pepper. RESULTS: Here, we report de novo genome assemblies of Capsicum annuum 'Early Calwonder (non-pungent, ECW)' and 'Small Fruit (pungent, SF)' along with their annotations. In total, we assembled 2.9 Gb of ECW and SF genome sequences, representing over 91% of the estimated genome sizes. Structural and functional annotation of the two pepper genomes generated about 35,000 protein-coding genes each, of which 93% were assigned putative functions. Comparison between newly and publicly available pepper gene annotations revealed both shared and specific gene content. In addition, a comprehensive analysis of nucleotide-binding and leucine-rich repeat (NLR) genes through whole-genome alignment identified five significant regions of NLR copy number variation (CNV). Detailed comparisons of those regions revealed that these CNVs were generated by intra-specific genomic variations that accelerated diversification of NLRs among peppers. CONCLUSIONS: Our analyses unveil an evolutionary mechanism responsible for generating CNVs of NLRs among pepper accessions, and provide novel genomic resources for functional genomics and molecular breeding of disease resistance in Capsicum species.

Genome-wide functional analysis of hot pepper immune receptors reveals an autonomous NLR clade in seed plants.

Plants possess hundreds of intracellular immune receptors encoding nucleotide-binding domain leucine-rich repeat (NLR) proteins. Full-length NLRs or a specific domain of NLRs often induce plant cell death in the absence of pathogen infection. In this study we used genome-wide transient expression analysis to identify a group of NLRs (ANLs; ancient and autonomous NLRs) carrying autoactive coiled-coil (CCA ) domains in pepper (Capsicum annuum). CCA -mediated cell death mimics hypersensitive cell death triggered by the interaction between NLRs and pathogen effectors. Sequence alignment and mutagenesis analyses revealed that the intact α1 helix of CCA s is critical for both CCA - and ANL-mediated cell death. Cell death induced by CCA s does not require NRG1/ADR1 or NRC type helper NLRs, suggesting ANLs may function as singleton NLRs. We also found that CCA s localize to the plasma membrane, as demonstrated for Arabidopsis singleton NLR ZAR1. Extended studies revealed that autoactive CCA s are well conserved in other Solanaceae plants as well as in rice, a monocot plant. Further phylogenetic analyses revealed that ANLs are present in all tested seed plants (spermatophytes). Our study not only uncovers the autonomous NLR clade in plants but also provides powerful resources for dissecting the underlying molecular mechanism of NLR-mediated cell death in plants.

Identification of the Capsicum baccatum NLR Protein CbAR9 Conferring Disease Resistance to Anthracnose.

Anthracnose is caused by Colletotrichum species and is one of the most virulent fungal diseases affecting chili pepper (Capsicum) yield globally. However, the noble genes conferring resistance to Colletotrichum species remain largely elusive. In this study, we identified CbAR9 as the causal locus underlying the large effect quantitative trait locus CcR9 from the anthracnose-resistant chili pepper variety PBC80. CbAR9 encodes a nucleotide-binding and leucine-rich repeat (NLR) protein related to defense-associated NLRs in several other plant species. CbAR9 transcript levels were induced dramatically after Colletotrichum capsici infection. To explore the biological function, we generated transgenic Nicotiana benthamiana lines overexpressing CbAR9, which showed enhanced resistance to C. capsici relative to wild-type plants. Transcript levels of pathogenesis-related (PR) genes increased markedly in CbAR9-overexpressing N. benthamiana plants. Moreover, resistance to anthracnose and transcript levels of PR1 and PR2 were markedly reduced in CbAR9-silenced chili pepper fruits after C. capsici infection. Our results revealed that CbAR9 contributes to innate immunity against C. capsici.

Molecular Characterization of a Pathogen-Inducible Bidirectional Promoter from Hot Pepper (Capsicum annuum).

In hot pepper, the sesquiterpene phytoalexin capsidiol is catalyzed by the two final-step enzymes, a sesquiterpene cyclase (EAS) and a hydroxylase (EAH), which are genetically linked and present as head-to-head orientation in the genome. Transcriptomic analysis revealed that a subset of EAS and EAH is highly induced following pathogen infection, suggesting the coregulation of EAS and EAH by a potential bidirectional activity of the promoter (pCaD). A series of the nested deletions of pCaD in both directions verified the bidirectional promoter activity of the pCaD. Promoter deletion analysis revealed that the 226 bp of the adjacent promoter region of EAS and GCC-box in EAH orientation were determined as critical regulatory elements for the induction of each gene. Based on promoter analyses, we generated a set of synthetic promoters to maximize reporter gene expression within the minimal length of the promoter in both directions. We found that the reporter gene expression was remarkably induced upon infection with Phytophthora capsici, Phytophthora infestans, and bacterial pathogen Pseudomonas syringae pv. tomato DC3000 but not with necrotrophic fungi Botrytis cinerea. Our results confirmed the bidirectional activity of the pCaD located between the head-to-head oriented phytoalexin biosynthetic genes in hot pepper. Furthermore, the synthetic promoter modified in pCaD could be a potential tool for pathogen-inducible expression of target genes for developing disease-resistant crops.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Occurrence of land-plant-specific glycerol-3-phosphate acyltransferases is essential for cuticle formation and gametophore development in Physcomitrella patens.

During the evolution of land plants from aquatic to terrestrial environments, their aerial surfaces were surrounded by cuticle composed of cutin and cuticular waxes to protect them from environmental stresses. Glycerol-3-phosphate acyltransferase (GPAT) harboring bifunctional sn-2 acyltransferase/phosphatase activity produces 2-monoacylglycerol, a precursor for cutin synthesis. Here, we report that bifunctional sn-2 GPATs play roles in cuticle biosynthesis and gametophore development of Physcomitrella patens. Land plant-type cuticle was observed in gametophores but not in protonema. The expression of endoplasmic reticulum-localized PpGPATs was significantly upregulated in gametophores compared with protonema. Floral organ fusion and permeable cuticle phenotypes of Arabidopsis gpat6-2 petals were rescued to the wild type (WT) by the expression of PpGPAT2 or PpGPAT4. Disruption of PpGPAT2 and PpGPAT4 caused a significant reduction of total cutin loads, and a prominent decrease in the levels of palmitic and 10,16-dihydroxydecanoic acids, which are major cutin monomers in gametophores. Δppgpat2 mutants displayed growth retardation, delayed gametophore development, increased cuticular permeability, and reduced tolerance to drought, osmotic and salt stresses compared to the WT. Genome-wide analysis of genes encoding acyltransferase or phosphatase domains suggested that the occurrence of sn-2 GPATs with both domains may be a key event in cuticle biogenesis of land plants.

TGFam-Finder: a novel solution for target-gene family annotation in plants.

Whole-genome annotation error that omits essential protein-coding genes hinders further research. We developed Target Gene Family Finder (TGFam-Finder), an alternative tool for the structural annotation of protein-coding genes containing target domain(s) of interest in plant genomes. TGFam-Finder took considerably reduced annotation run-time and improved accuracy compared to conventional annotation tools. Large-scale re-annotation of 50 plant genomes identified an average of 150, 166 and 86 additional far-red-impaired response 1, nucleotide-binding and leucine-rich-repeat, and cytochrome P450 genes, respectively, that were missed in previous annotations. We detected significantly higher number of translated genes in the new annotations using mass spectrometry data from seven plant species compared to previous annotations. TGFam-Finder along with the new gene models can provide an optimized platform for comprehensive functional, comparative, and evolutionary studies in plants.

Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments.

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th ß-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 ß-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.

A human pathogenic bacterium Shigella proliferates in plants through adoption of type III effectors for shigellosis.

Shigella, which infects primates, can be transmitted via fresh vegetables; however, its molecular interactions with plants have not been elucidated. Here, we show that four Shigella strains, Shigella boydii, Shigella sonnei, Shigella flexneri 2a, and S. flexneri 5a, proliferate at different levels in Arabidopsis thaliana. Microscopic studies revealed that these bacteria were present inside leaves and damaged plant cells. Green fluorescent protein (GFP)-tagged S. boydii and S. flexneri 5a colonized leaves only, whereas S. flexneri 2a colonized both leaves and roots. Using Shigella mutants lacking type III secretion systems (T3SSs), we found that T3SSs that regulate the pathogenesis of shigellosis in humans also play a central role in bacterial proliferation in Arabidopsis. Strikingly, the immunosuppressive activity of two T3S effectors, OspF and OspG, was required for proliferation of Shigella in Arabidopsis. Of note, delivery of OspF or OspG effectors inside plant cells upon Shigella inoculation was confirmed using a split GFP system. These findings demonstrate that the human pathogen Shigella can proliferate in plants by adapting immunosuppressive machinery used in the original host human.

HSP70s Enhance a Phytophthora infestans Effector-Induced Cell Death via an MAPK Cascade in Nicotiana benthamiana.

A destructive pathogen, Phytophthora infestans, secretes hundreds of effectors for successful survival in its host plants. The effectors modulate the plant defense system at diverse cellular compartments to take an advantage of pathogen survivals. A few research studies have shown the mode of action of each effector and their interacting proteins in plant cells. Here, we investigated the mode of action of a P. infestans effector, Pi23226, which induces cell death in Nicotiana benthamiana. To identify its host factors, we performed coimmunoprecipitation and liquid chromatography-mass spectrometry, and selected members of heat shock protein 70 (HSP70s) as candidates. These HSP70s, known to function as chaperones, were associated with Pi23226 in planta and accelerated Pi23226-induced cell death. Additionally, they were found to be involved in plant basal defense by suppressing the growth of P. infestans. We also found that specific components of a mitogen-activated protein kinase cascade were involved in Pi23226-induced cell death. Our findings show that HSP70s functions in defense systems by regulating effector-triggered cell death and by suppressing the growth of the pathogen. This suggests that host plants manipulate the ubiquitous proteins to detect pathogen effectors for functioning in the defense system.

Suppression or Activation of Immune Responses by Predicted Secreted Proteins of the Soybean Rust Pathogen Phakopsora pachyrhizi.

Rust fungi, such as the soybean rust pathogen Phakopsora pachyrhizi, are major threats to crop production. They form specialized haustoria that are hyphal structures intimately associated with host-plant cell membranes. These haustoria have roles in acquiring nutrients and secreting effector proteins that manipulate host immune systems. Functional characterization of effector proteins of rust fungi is important for understanding mechanisms that underlie their virulence and pathogenicity. Hundreds of candidate effector proteins have been predicted for rust pathogens, but it is not clear how to prioritize these effector candidates for further characterization. There is a need for high-throughput approaches for screening effector candidates to obtain experimental evidence for effector-like functions, such as the manipulation of host immune systems. We have focused on identifying effector candidates with immune-related functions in the soybean rust fungus P. pachyrhizi. To facilitate the screening of many P. pachyrhizi effector candidates (named PpECs), we used heterologous expression systems, including the bacterial type III secretion system, Agrobacterium infiltration, a plant virus, and a yeast strain, to establish an experimental pipeline for identifying PpECs with immune-related functions and establishing their subcellular localizations. Several PpECs were identified that could suppress or activate immune responses in nonhost Nicotiana benthamiana, N. tabacum, Arabidopsis, tomato, or pepper plants.

The Coiled-Coil and Leucine-Rich Repeat Domain of the Potyvirus Resistance Protein Pvr4 Has a Distinct Role in Signaling and Pathogen Recognition.

The pepper Pvr4 protein encoding coiled-coil (CC) nucleotide-binding (NB) leucine-rich repeat (LRR) (NLR) confer hypersensitive response (HR) to potyviruses, including Pepper mottle virus (PepMoV), by recognizing the viral avirulence protein NIb. To figure out the Pvr4-mediated HR mechanism, we analyzed signaling component genes and structure-function relationships of Pvr4, using chimeras and deletion mutants in Nicotiana benthamiana. Molecular chaperone components including HSP90, SGT1, and RAR1 were required, while plant hormones and mitogen-activated protein kinase signaling components had little effect on Pvr4-NIb-mediated HR cell death. Domain swap analyses indicated that the LRR domain of Pvr4 determines recognition of PepMoV-NIb. Our deletion analysis further revealed that the CC domain or CC-NBARC domain alone can trigger autoactive cell death in N. benthamiana. However, the fragments having only an LRR domain could suppress CC-NBARC domain-induced cell death in trans. Further, C-terminal truncation analysis of Pvr4 revealed that a minimum three of five LRR exons showing high similarity was essential for Pvr4 function. The LRR domain may maintain Pvr4 in an inactive state in the absence of NIb. These results provide further insight into the structure and function of NLR protein signaling in plants.

Genome and evolution of the shade-requiring medicinal herb Panax ginseng.

Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane-type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome-scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics-assisted breeding or metabolic engineering.