Bacterial spot, caused by Xanthomonas species, is a devastating disease of tomato (Solanum lycopersicum) and pepper (Capsicum annuum) (Schwartz et al., 2015). The recessively inherited resistance, bacterial spot 5 (bs5), in pepper (hereafter referred to as Cabs5) can confer resistance against different Xanthomonas strains (Jones et al., 2002). The Cabs5 resistance is characterized by the absence of disease symptoms, faint chlorosis at the site of infection, and reduced bacterial growth. Remarkably, commercial pepper varieties containing the bs5 allele show durable resistance, effectively impeding hypervirulent strain emergence in agricultural fields (Vallejos et al., 2010). The CaBs5 gene, together with its paralog CaBs5-like (CaBs5L), has recently been cloned (Sharma et al., 2023; Szabó et al., 2023). CaBs5 encodes a 92 amino acid long protein possessing a cysteine-rich transmembrane (CYSTM) domain, which is implicated in various biotic and abiotic responses. Typically, the CYSTM domain contains conserved residues composed of four consecutive cysteines, followed by two hydrophobic amino acids. A recent study suggested that Cabs5 mediating the resistance against bacterial spot lacks these two conserved leucine residues within the CYSTM domain (Szabó et al., 2023). Tomatoes and peppers are close relatives in the Solanaceae family and commonly susceptible to Xanthomonas infection. Based on the current findings in pepper, we hypothesized that modifying the ortholog of CaBs5 in tomato could confer resistance against Xanthomonas. Consequently, putative Bs5 (SlBs5) and Bs5L (SlBs5L) were identified in tomato based on homology to CaBs5. Both SlBs5 and SlBs5L were located on chromosome 9 with the same head-to-head orientation as their pepper homologues on chromosome 3 (Figure 1a). Despite short and highly similar amino acid sequences of SlBs5 and SlBs5L (Figure 1b), the conserved synteny and gene order in pepper and tomato genomes allowed the assignment of orthology for Bs5 and Bs5L. The mechanism by which the double leucine deletion in Cabs5 leads to resistance against Xanthomonas remains elusive (Figure 1b). Yet, this deletion in the conserved CYSTM domain could potentially impair CaBs5's native functionality (Abell and Mullen, 2011). Following this assumption, we postulated that knocking out SlBs5 would produce similar outcomes to Cabs5. We aimed to disrupt both SlBs5 and SlBs5L to prevent possible functional complementation by SlBs5L, given their greater amino acid sequence similarity compared to CaBs5 and CaBs5L (Figure 1b). We constructed a binary vector for Cas9 and a single-guide RNA (sgRNA) targeting conserved sequences present in both SlBs5 and SlBs5L (Figure 1c). Tomato variety Fla. 8000 was transformed with Agrobacterium. From the progeny of successful transformants, we selected two homozygous lines, Slbs5-1 and Slbs5-2, containing frameshift mutations in both genes (Figure 1c). These mutant lines were self-pollinated or backcrossed to the wild-type parent variety to segregate the T-DNA containing the Cas9-sgRNA cassette. The resistance of the two selected mutant lines was qualitatively evaluated against Xanthomonas perforans GE485 with dip inoculation assays (Figure 1d). At 21 days post-inoculation, the wild-type leaves were covered by black spots indicative of Xanthomonas infection, while both Slbs5-1 and Slbs5-2 retained green leaves with fewer visible symptoms. These phenotypes remained consistent in inoculations of X. perforans 4B and Xanthomonas gardneri 153 (Figure S1). Quantitative evaluation of bacterial growth further supported these findings. At 5 days post-infiltration with a low-density bacterial suspension, Slbs5-1 showed significant decreases in Xanthomonas populations compared to wild-type plants (Figure 1e). Such reductions were consistently observed for Slbs5-2 (Figure S2). However, Slbs5-1 could not significantly hinder Pseudomonas population growth. We additionally examined the growth penalty associated with Slbs5-1 and Slbs5-2 in controlled conditions (Figure 1f). The height of plants was measured at two different time points, but no significant differences were observed between the wild type and the two mutant lines (Figure 1f; Figure S3). This suggested that the resistance to Xanthomonas species comes at no developmental cost in the vegetative stage in the laboratory setting. Although Cabs5-mediated immunity is subtle, it has shown practical value in commercial pepper cultivation. To examine the commercial potential of Slbs5, field trials were conducted with both Slbs5-1 and Slbs5-2 lines at the Gulf Coast Research and Education Center in Florida, a major state for tomato production. Along with naturally occurring Xanthomonas populations, a two-isolate cocktail of X. perforans race T4 was inoculated in the field to heighten disease pressure. Plants were grown with recommended fertilizers and pest management programs, excluding the use of any bactericides or activators of systemic acquired resistance. Despite seasonal variations, Slbs5 mutant lines consistently maintained reduced disease symptoms (Figure 1g). Additionally, no developmental defects, such as stunting, were observed in these mutants (Figure 1h). Quantification of disease severity, based on visible symptoms caused by Xanthomonas infection on plant leaf surfaces, revealed higher percentages of Slbs5-2 leaves with reduced disease symptoms than wild-type leaves in all tested seasons (Figure 1i; Figure S4). Notably, the Slbs5-2 mutants demonstrated effective resistance during three periods of elevated disease pressure, Spring 2018, Fall 2019, and Fall 2023. The marketable yield of fruits is a critical consideration in tomato cultivation. We quantified total marketable yield across five seasonal trials, except for two seasons impacted by a hurricane (Fall 2022) and extremely dry weather (Spring 2023). Throughout all seasons, there was no statistically significant difference in marketable fruit yields between Slbs5-2 and the wild-type plants (Figure 1j; Figure S5). However, during the three periods of increased disease prevalence in Spring 2018, Fall 2019, and Fall 2023 (Figure 1i), the mutants consistently showed a tendency to produce a greater quantity of marketable tomatoes (Figure 1j). This possibly suggests a correlation between Xanthomonas resistance of the mutant lines and improved fruit yields. Overall, this study shows that a knockout of SlbBs5 and SlBs5L in tomatoes represents a promising strategy to achieve broad-spectrum resistance to bacterial spot disease. Compared to stronger sources of resistance, the resistance mediated by Slbs5 and Slbs5L may be considered subtle. However, our mutant lines consistently led to a reduced population of Xanthomonas in laboratory and field conditions. This decrease in pathogen populations could lessen the likelihood of hypervirulent strain emergence. Furthermore, when these mutants are combined with other sources of downstream resistance genes, they may serve as a prior layer of defence. This initial protection has the potential to diminish the probability of pathogen effectors directly interacting with and overcoming the resistance genes, possibly extending the efficacy of durable resistance in the agricultural field. This research was funded by the Innovative Genomics Institute Founders Fund and the 2Blades Foundation. A.O. and B.J.S. conceptualized the project. B.J.S. supervised the project. A.O., D.D. and B.J.S. designed the experiments and helped analyze the data. K.S. and E.S performed bioinformatics analyses. K.S. led statistical analyses and designed the figures. A.S. helped plan the project, designed and tested the guide RNAs and did preliminary genotyping and bacterial disease assays. A.O. did further genotyping, guide RNA testing and conducted disease and phenotype assays of progeny. D.P.T.T., J.V.W., J.B.J, G.M, E.S.O and D.D. performed supplemental bacterial growth assays. E.S. and S.H. conducted field trials. M.J.C. supervised the generation of tomato mutant lines. E.Z. and J.P. conducted tomato transformations. A.O., K.S. and D.P.T.T. analyzed the data and wrote the manuscript. The data that support the findings of this study are available on request from the corresponding author. Appendix S1 Materials and Methods. Figure S1 Qualitative evaluation of disease symptoms with a dip inoculation assay. Figure S2 Quantitative evaluation of bacterial growth after inoculation. Figure S3 Height comparison between wild type and mutant plants. Figure S4 The disease symptoms on wild type and Slbs5-1 plant leaves in the field trials. Figure S5 Fruit yields of wild type and Slbs5-1 plants in the Fall 2023 field trial. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Abstract We examined two variants of turnip mosaic virus (TuMV) KJS 5 and KJS 5 − 1, the polyproteins of which differ by only a single amino acid residue, located in HC-Pro motif CDNQLD. When KJS 5 and KJS 5 − 1 were inoculated to Nicotiana benthamiana and Raphanus sativus differential symptom severity were observed in each host. To complement these results, smGFP and the variant HC-Pros were co-expressed in N. benthamiana leaves by agro-infiltration. Real-time qPCR analysis showed that the relative expression levels of smGFP differed. This shows that a single amino acid substitution affects both symptom severity and RNA silencing suppression efficiency.

Two infectious clones of turnip mosaic virus (TuMV), pKBC-1 and pKBC-8, with differential infectivity in Chinese cabbage (Brassica rapa subsp. pekinensis), were obtained. Both infected Nicotiana benthamiana systemically, inducing similar symptoms, whereas only virus KBC-8 infected Chinese cabbage systemically. To identify the determinants affecting infectivity on Chinese cabbage, chimeric clones were constructed by restriction fragment exchange between the parental clones and tested on several Chinese cabbage cultivars. Chimeric clones p1N8C and p8N1C demonstrated that the C-terminal portion of the polyprotein determines systemic infection of Chinese cabbage despite only three amino acid differences in this region, in the cylindrical inclusion (CI), viral protein genome-linked (VPg), and coat protein (CP). A second pair of hybrid constructs, pHindIII-1N8C and pHindIII-8N1C, failed to infect cultivars CR Victory and Jinseonnorang systemically, yet pHindIII-1N8C caused hypersensitive response-like lesions on inoculated leaves of these cultivars, and could systemically infect cultivars CR Chusarang and Jeongsang; this suggests that R genes effective against TuMV may exist in the first two cultivars but not the latter two. Constructs with single amino acid changes in both VPg (K2045E) and CP (Y3095H) failed to infect Chinese cabbage, implying that at least one of these two amino acid substitutions is essential for successful infection on Chinese cabbage. Successful infection by mutant KBC-8-CP-H and delayed infection with mutant HJY1-VPg-E following mutation or reversion suggested that VPg (2045K) is the residue required for infection of Chinese cabbage and involved in the interaction between VPg and eukaryotic initiation factor eIF(iso)4E, confirmed by yeast two-hybrid assay.

Solanum habrochaites LA1353 is a self-incompatible, highly heterozygous wild tomato that is a useful germplasm resource for the study of metabolism, reproduction and disease resistance. We generated a draft genome assembly with PacBio HiFi reads and genome annotations, which underscored the expansion of gene families associated with metabolite-production, self-incompatibility, DNA regulation and immunity. After manually curating intracellular nucleotide-binding leucine-rich repeat immune receptors (NLRs), we found that S. habrochaites LA1353 has a larger NLR inventory than other wild tomato species. A great number of heterozygous local copy number variations (CNVs) driven by haplotypic structural variations further expands the inventory, both enhancing NLR diversity and providing more opportunities for sequence evolution. The NLRs associated with local CNVs predominantly appear in the helper NLR (NRC)-related phylogenetic clades and are concentrated in a few physical NLR gene clusters. Synteny analysis points out that these genomic regions correspond to the known NLR clusters from which experimentally validated, functional NLRs, such as Hero, Mi-1.2 and Rpi-amr1 , have been identified. Producing and incorporating Resistance Gene Enrichment Sequencing (RenSeq) data across wild tomato species, we reveal that the regions with local CNVs might have been shaped nearly equally by recent NLR gains and losses, along with enhanced sequence diversification that diminishes one-to-one orthology between heterozygous alleles. Our analysis suggests that these genomic regions may have accelerated evolutionary dynamics for NLR diversity generation in S. habrochaites LA1353.

Bacterial canker caused by Clavibacter michiganensis (Cm) is one of the most economically important vascular diseases causing unilateral leaf wilting, stem canker, a bird's-eye lesion on fruit, and whole plant wilting in tomato. There is no commercially available cultivar with bacterial canker resistance, and genomics-assisted breeding can accelerate the development of cultivars with enhanced resistance. Solanum lycopersicum "Hawaii 7998" was found to show bacterial canker resistance. A Quantitative trait loci (QTL)-seq was performed to identify the resistance loci using 909 F-2 individuals derived from a cross between S. lycopersicum "E6203" (susceptible) and "Hawaii 7998," and a genomic region (37.24-41.15 Mb) associated with bacterial canker resistance on chromosome 6 (Rcm6) was found. To dissect the Rcm6 region, 12 markers were developed and several markers were associated with the resistance phenotypes. Among the markers, the Rcm6-9 genotype completely matched with the phenotype in the 47 cultivars. To further validate the Rcm6 as a resistance locus and the Rcm6-9 efficiency, subsequent analysis using F-2 and F-3 progenies was conducted. The progeny individuals with homozygous resistance allele at the Rcm6-9 showed significantly lower disease severity than those possessing homozygous susceptibility alleles. Genomes of five susceptible and two resistant cultivars were analyzed and previously known R-genes were selected to find candidate genes for Rcm6. Nucleotide-binding leucine-rich repeat, receptor-like kinase, and receptor-like protein were identified to have putative functional mutations and show differential expression upon the Cm infection. The DNA markers and candidate genes will facilitate marker-assisted breeding and provide genetic insight of bacterial canker resistance in tomato.

In this work, two new turnip mosaic virus (TuMV) strains (Canola-12 and Canola-14) overcoming resistance in canola ( Brassica napus ) were isolated from a B. napus sample that showed typical TuMV-like symptoms and was collected in the city of Gimcheon, South Korea, in 2020. The complete genome sequence was determined and an infectious clone was made for each isolate. Phylogenetic analysis indicated that the strains isolated from canola belonged to the World-B group. Both infectious clones, which used 35S and T7 promoters to drive expression, induced systemic symptoms in Nicotiana benthamiana and B. napus. To our knowledge, this is the first report of TuMV infecting B. napus in South Korea.

Perilla is an annual herb with a unique aroma and taste that has been cultivated in Korea for hundreds of years. It has been widely cultivated in many Asian and European countries as a food and medicinal crop. Recently, several viruses have been reported to cause diseases in perilla in Korea, including turnip mosaic virus (TuMV), which is known as a brassica pathogen due to its significant damage to brassica crops. In this study, we determined the complete genome sequences of two new TuMV isolates originating from perilla in Korea. Full-length infectious cDNA clones of these two isolates were constructed, and their infectivity was tested by agroinfiltration of Nicotiana benthamiana and sap inoculation of Chinese cabbage and radish plants. In addition, we analyzed the phylogenetic relationship of six new Korean TuMV isolates to members of the four major groups. We also used RDP4 software to conduct recombination analysis of recent isolates from Korea, which provided new insight into the evolutionary relationships of Korean isolates of TuMV.

Three infectious clones of radish mosaic virus (RaMV) were generated from isolates collected in mainland Korea (RaMV-Gg) and Jeju Island (RaMV-Aa and RaMV-Bb). These isolates differed in sequences and pathogenicity. Examination of the wild-type isolates and reassortants between the genomic RNA1 and RNA2 of these three isolates revealed that severe symptoms were associated with RNA1 of isolates Aa or Gg causing systemic necrosis in Nicotiana benthamiana, or with RNA1 of isolate Bb for induction of veinal necrosis and severe mosaic symptoms in radish. Reverse transcription, followed by quantitative real-time PCR (Q-RT-PCR), results from infected N. benthamiana confirmed that viral RNA2 accumulation level was correlated to RaMV necrosis-inducing ability, and that the RNA2 accumulation level was mostly dependent on the origin of RNA1. However, in radish, Q-RT-PCR results showed more similar viral RNA2 accumulation levels regardless of the ability of the isolate to induce necrosis. Phylogenetic analysis of genomic RNAs sequence including previously characterized isolates from North America, Europe, and Asia suggest possible recombination within RNA1, while analysis of concatenated RNA1+RNA2 sequences indicates that reassortment of RNA1 and RNA2 has been more important in the evolution of RaMV isolates than recombination. Korean isolate Aa is a potential reassortant between isolates RaMV-J and RaMV-TW, while isolate Bb might have evolved from reassortment between isolates RaMV-CA and RaMV-J. The Korean isolates were shown to also be able to infect Chinese cabbage, raising concerns that RaMV may spread from radish fields to the Chinese cabbage crop in Korea, causing further economic losses.

Ginseng flower bud (GFB), as an inexpensive part of Panax ginseng, attracted significant attention as a beneficial functional food with medicinal potentials due to its high content of ginsenosides. A few studies focused on the utilization of heat treatment and citric acid treatment to process ginseng flowers, converting its polar ginsenosides into rare ginsenosides to improve its biological activities. Thus, in this study, we compared the changes of ginsenosides in GFB after citric acid and heat treatment by HPLC method. The results revealed that less-polar ginsenoside, Rg(6) and F-4, increased to 1.01 and 0.27% by heat treatment, respectively. Further, ginsenoside F-2 increased to 1.13% with 1 M citric acid treatment. Furthermore, based on the combination of these two processing methods for the first time, the conversion rate of less-polar ginsenosides surged to 80%. The content of ginsenoside Rg3(s) and Rg(5) increased to 1.509 and 1.871%, respectively, by simultaneous heat and citric acid treatment. Therefore, a processing approach that simultaneously performs heat and citric acid treatments has been proposed, and this considerably inexpensive and convenient processing method could be applied to the processing of GFBs and produce less-polar ginsenosides.

Background Nucleotide-binding and leucine-rich repeat immune receptors (NLRs) are an important component of plant immunity that provides resistance against diverse pathogens. NLRs often exist as large gene families, the members of which display diverse multi-domain architectures (MDAs) and evolve through various mechanisms of duplications and selections. Results We conducted resistance gene enrichment sequencing (RenSeq) with single-molecule real time (SMRT) sequencing of PacBio for 18 accessions in Solanaceae including 15 wild tomatoes. We demonstrate what was previously known as Solanaceae Domain (SD) not only is more diverse in structure and function but also far anciently originated from the most recent common ancestor (MRCA) between Asterids and Amaranthaceae. In tomato, NLRs with the extended N-terminus displayed distinct patterns of evolution based on phylogenetic clades by proliferation, continuous elongation and domain losses. Conclusions Our study provides high quality gene models of NLRs that can serve as resources for future studies for crop engineering and elucidates greater evolutionary dynamics of the extended NLRs than previously assumed.

Infectious clones of Korean turnip mosaic virus (TuMV) isolates KIH1 and HJY1 share 88.1% genomic nucleotides and 96.4% polyprotein amino acid identity, and they induce systemic necrosis or mild mosaic, respectively, in Nicotiana benthamiana. Chimeric constructs between these isolates exchanged the 5', central, and 3' domains of KIH1 (K) and HJY1 (H), where the order of the letters indicates the origin of these domains. KIH1 and chimeras KHH and KKH induced systemic necrosis, whereas HJY1 and chimeras HHK, HKK, and HKH induced mild symptoms, indicating the determinant of necrosis to be within the 5' 3.9 kb of KIH1; amino acid identities of the included P1, Helper component protease, P3, 6K1, and cylindrical inclusion N-terminal domain were 90.06, 98.91, 93.80, 100, and 100%, respectively. Expression of P1 or P3 from a potato virus X vector yielded symptom differences only between P3 of KIH1 and HJY1, implicating a role for P3 in necrosis in N. benthamiana. Chimera KKH infected Brassica rapa var. pekinensis 'Norang', which was resistant to both KIH1 and HJY1, indicating that two separate TuMV determinants are required to overcome the resistance. Ability of diverse TuMV isolates, chimeras, and recombinants to overcome resistance in breeding lines may allow identification of novel resistance genes.

Infectious clones were generated from 17 new Korean radish isolates of Turnip mosaic virus (TuMV). Phylogenetic analysis indicated that all new isolates, and three previously characterized Korean radish isolates, belong to the basal-BR group (indicating that the pathotype can infect both Brassica and Raphanus spp.). Pairwise analysis revealed genomic nucleotide and polyprotein amino acid identities of >87.9 and >95.7%, respectively. Five clones (HJY1, HJY2, KIH2, BE, and prior isolate R007) had lower sequence identities than other isolates and produced mild symptoms in Nicotiana benthamiana. These isolates formed three distinct sequence classes (HJY1/HJY2/R007, KIH2, and BE), and several differential amino acid residues (in P1, P3, 6K2, and VPg) were present only in mild isolates HJY1, HJY2, and R007. The remaining isolates all induced systemic necrosis in N. benthamiana. Four mild isolates formed a phylogenetic subclade separate from another subclade including all of the necrosis-inducing isolates plus mild isolate KIH2. Symptom severity in radish and Chinese cabbage genotypes was not correlated with pathogenicity in N. benthamiana; indeed, Chinese cabbage cultivar Norang was not infected by any isolate, whereas Chinese cabbage cultivar Chusarang was uniformly susceptible. Four isolates were unable to infect radish cultivar Iljin, but no specific amino acid residues were correlated with avirulence. These results may lead to the identification of new resistance genes against TuMV.

In 2017, two new tomato mosaic virus (ToMV) isolates were collected from greenhouses in Buyeo, Chungcheongnam-do, South Korea. Full-length cDNAs of the new ToMV isolates were cloned into dual cauliflower mosaic virus 35S and T7 promoter-driven vectors, sequenced and their pathogenicities investigated. The nucleotide sequences of isolates GW1 (MH507165) and GW2 (MH507166) were 99% identical, resulting in only two amino acid differences in nonconserved region II and the helicase domain, Ile668Thr and Val834Ile. The two isolates were most closely related to a ToMV isolate from Taiwan (KJ207374). Isolate GW1 (Ile668, Val834) induced a systemic hypersensitive response in Nicotiana benthamiana compared with the isolate GW2, which a single residue substitution showed was due to Val834.

Rosacea is a chronic inflammatory disorder usually affecting the central face. Chronic use of topical steroids on the face causes lesions similar to rosacea. Synthesis and function of glucocorticoid (GC) have been established in healthy skin and various cutaneous diseases but not in rosacea. In most skin disorders, increased inflammation is usually coupled with a decline in the production of cutaneous GC. We investigated the de novo GC synthesis in the skin and GC receptor expression in the lesional and the nonlesional skin of patients with erythematotelangiectatic rosacea (ETR). Both lesional and non-lesional facial skin samples from the same individuals were provided by 12 ETR patients (mean age, 40.1 years; range, 30 to 55 years). The key steroidogenic enzymes, CYP11A1, 3βHSD2 and CYP21A2, were significantly increased in the lesional skin at the gene and protein levels. CYP17A1 and steroidogenic acute regulatory protein (StAR) tended to be higher in the lesional skin. GC receptor was also significantly increased in the lesional skin. Progesterone was increased significantly in the lesional skin using liquid chromatography-mass spectrometry Simultaneously, p65/p50 subunits of nuclear factor kappa-light-chain enhancer of activated B cells (NFκB) and matrix metalloproteinases (MMP) gene expressions were significantly increased in the lesional skin. Furthermore, p65 and p105 protein levels were significantly increased. These results demonstrate that crosstalk between GC receptor and NFκB may be hampered in the lesional skin of ETR and may contribute to rosacea pathogenesis. We demonstrate that ETR is an inherently inflammatory disorder mediated by NFκB and that GC synthesis and GC receptor expression is increased in the lesional skin of ETR unlike in other cutaneous inflammatory diseases.

For several years, temperatures in the Korean peninsula have gradually increased due to climate change, resulting in a changing environment for growth of crops and vegetables. An associated consequence is that emerging species of insect vector have caused increased viral transmission. In Jeju Island, Korea, occurrences of viral disease have increased. Here, we report characterization of five newly collected turnip mosaic virus (TuMV) isolates named KBJ1, KBJ2, KBJ3, KBJ4 and KBJ5 from a survey on Jeju Island in 2017. Full-length cDNAs of each isolate were cloned into the pJY vector downstream of cauliflower mosaic virus 35S and bacteriophage T7 RNA polymerase promoters. Their full-length sequences share 98.9-99.9% nucleotide sequence identity and were most closely related to previously reported Korean TuMV isolates. All isolates belonged to the BR group and infected both Chinese cabbage and radish. Four isolates induced very mild symptoms in Nicotiana benthamiana but KBJ5 induced a hypersensitive response. Symptom differences may result from three amino acid differences uniquely present in KBJ5; Gly(382)Asp, Ile(891)Val, and Lys(2522)Glu in P1, P3, and NIb, respectively.

Effector-triggered immunity (ETI) in plants involves a large family of nucleotide-binding leucine-rich repeat (NLR) immune receptors, including Toll/IL-1 receptor-NLRs (TNLs) and coiled-coil NLRs (CNLs). Although various NLR immune receptors are known, a mechanistic understanding of NLR function in ETI remains unclear. The TNL Recognition of XopQ 1 (Roq1) recognizes the effectors XopQ and HopQ1 from Xanthomonas and Pseudomonas, respectively, which activates resistance to Xanthomonas euvesicatoria and Xanthomonas gardneri in an Enhanced Disease Susceptibility 1 (EDS1)-dependent way in Nicotiana benthamiana In this study, we found that the N. benthamiana N requirement gene 1 (NRG1), a CNL protein required for the tobacco TNL protein N-mediated resistance to tobacco mosaic virus, is also essential for immune signaling [including hypersensitive response (HR)] triggered by the TNLs Roq1 and Recognition of Peronospora parasitica 1 (RPP1), but not by the CNLs Bs2 and Rps2, suggesting that NRG1 may be a conserved key component in TNL signaling pathways. Besides EDS1, Roq1 and NRG1 are necessary for resistance to Xanthomonas and Pseudomonas in N. benthamiana NRG1 functions downstream of Roq1 and EDS1 and physically associates with EDS1 in mediating XopQ-Roq1-triggered immunity. Moreover, RNA sequencing analysis showed that XopQ-triggered gene-expression profile changes in N. benthamiana were almost entirely mediated by Roq1 and EDS1 and were largely regulated by NRG1. Overall, our study demonstrates that NRG1 is a key component that acts downstream of EDS1 to mediate various TNL signaling pathways, including Roq1 and RPP1-mediated HR, resistance to Xanthomonas and Pseudomonas, and XopQ-regulated transcriptional changes in N. benthamiana.