A malectin‐like receptor kinase regulates cell death and pattern‐triggered immunity in soybean
2020; Springer Nature; Volume: 21; Issue: 11 Linguagem: Inglês
10.15252/embr.202050442
ISSN1469-3178
AutoresDongmei Wang, Xiangxiu Liang, Yazhou Bao, Suxin Yang, Xiong Zhang, Hui Yu, Qian Zhang, Guangyuan Xu, Xianzhong Feng, Daolong Dou,
Tópico(s)Plant pathogens and resistance mechanisms
ResumoArticle14 September 2020Open Access Source DataTransparent process A malectin-like receptor kinase regulates cell death and pattern-triggered immunity in soybean Dongmei Wang Dongmei Wang Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Changchun, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiangxiu Liang Xiangxiu Liang Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Yazhou Bao Yazhou Bao Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Suxin Yang Suxin Yang Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Changchun, China Search for more papers by this author Xiong Zhang Xiong Zhang orcid.org/0000-0002-2044-6929 Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Hui Yu Hui Yu Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Changchun, China Search for more papers by this author Qian Zhang Qian Zhang Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Guangyuan Xu Guangyuan Xu Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Xianzhong Feng Corresponding Author Xianzhong Feng [email protected] orcid.org/0000-0002-7129-3731 Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Changchun, China Search for more papers by this author Daolong Dou Corresponding Author Daolong Dou [email protected] orcid.org/0000-0001-5226-6642 Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China College of Plant Protection, Nanjing Agricultural University, Nanjing, China Search for more papers by this author Dongmei Wang Dongmei Wang Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Changchun, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiangxiu Liang Xiangxiu Liang Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Yazhou Bao Yazhou Bao Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Suxin Yang Suxin Yang Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Changchun, China Search for more papers by this author Xiong Zhang Xiong Zhang orcid.org/0000-0002-2044-6929 Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Hui Yu Hui Yu Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Changchun, China Search for more papers by this author Qian Zhang Qian Zhang Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Guangyuan Xu Guangyuan Xu Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China Search for more papers by this author Xianzhong Feng Corresponding Author Xianzhong Feng [email protected] orcid.org/0000-0002-7129-3731 Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Changchun, China Search for more papers by this author Daolong Dou Corresponding Author Daolong Dou [email protected] orcid.org/0000-0001-5226-6642 Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China College of Plant Protection, Nanjing Agricultural University, Nanjing, China Search for more papers by this author Author Information Dongmei Wang1,2,‡, Xiangxiu Liang3,‡, Yazhou Bao3, Suxin Yang1, Xiong Zhang3, Hui Yu1, Qian Zhang3, Guangyuan Xu3, Xianzhong Feng *,1 and Daolong Dou *,3,4 1Key Laboratory of Soybean Molecular Design Breeding, Northeast Institute of Geography and Agroecology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Changchun, China 2University of Chinese Academy of Sciences, Beijing, China 3Key Laboratory of Pest Monitoring and Green Management, MOA and College of Plant Protection, China Agricultural University, Beijing, China 4College of Plant Protection, Nanjing Agricultural University, Nanjing, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 0431 85655051; E-mail: [email protected] *Corresponding author. Tel: +86 025 84396973; E-mail: [email protected] EMBO Reports (2020)21:e50442https://doi.org/10.15252/embr.202050442 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Plant cells can sense conserved molecular patterns through pattern recognition receptors (PRRs) and initiate pattern-triggered immunity (PTI). Details of the PTI signaling network are starting to be uncovered in Arabidopsis, but are still poorly understood in other species, including soybean (Glycine max). In this study, we perform a forward genetic screen for autoimmunity-related lesion mimic mutants (lmms) in soybean and identify two allelic mutants, which carry mutations in Glyma.13G054400, encoding a malectin-like receptor kinase (RK). The mutants exhibit enhanced resistance to both bacterial and oomycete pathogens, as well as elevated ROS production upon treatment with the bacterial pattern flg22. Overexpression of GmLMM1 gene in Nicotiana benthamiana severely suppresses flg22-triggered ROS production and oomycete pattern XEG1-induced cell death. We further show that GmLMM1 interacts with the flg22 receptor FLS2 and its co-receptor BAK1 to negatively regulate flg22-induced complex formation between them. Our study identifies an important component in PTI regulation and reveals that GmLMM1 acts as a molecular switch to control an appropriate immune activation, which may also be adapted to other PRR-mediated immune signaling in soybean. Synopsis Plants can sense conserved molecular patterns through pattern-recognition receptors and initiate pattern-triggered immunity. The soybean malectin-like receptor kinase GmLMM1 interacts with these PRR complexes to negatively regulate PTI responses. The malectin-like receptor kinase GmLMM1 is identified in a genetic screen for autoimmunity-related mutants. GmLMM1 is involved in the PRR complex and works as a negative regulator of plant immunity. GMLMM1 regulates the interaction between the flg22 receptor FLS2 and its co-receptor BAK1. Introduction Soybean is one of the major sources of oil and plant proteins worldwide. Its demand in food, feed, and industrial production has increased stably along with the rapid expansion of the world's population. Soybean diseases, including bacterial blight, phytophthora root rot, and soybean rust, continuously cause great losses to soybean yield and quality worldwide. Traditional disease control mainly relies on chemical and breeding methods, which are sometimes outpaced by the evolution of pathogens. Thus, it is important to study the soybean immune system and to understand how soybean defends itself against pathogens (Whitham et al, 2016). Plants are equipped with two layers of immune perception systems: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI; Jones & Dangl, 2006). Plant plasma membrane-localized pattern recognition receptors (PRRs) can sense the presence of pathogens through recognition of microbe-associated molecule, such as bacterial flg22 and Phytophthora sojae XEG1, to activate downstream PTI signaling (Chinchilla et al, 2006; Wang et al, 2018). Successful pathogens can evade plant resistance by secreting effector proteins to suppress plant PTI signaling. Plant intracellular nucleotide-binding and leucine-rich repeat receptors (NLRs) can recognize the presence of microbial effectors to trigger a much stronger ETI response, which is usually accompanied by hypersensitive response (HR; Dou & Zhou, 2012; Jones et al, 2016). Both layers of the plant immune system have been extensively studied in Arabidopsis and some crops, such as rice. However, the limited information on soybean immunity focuses mainly on ETI, few on PTI. For example, several NLRs have been cloned and characterized during the last decade (Whitham et al, 2016), including Rpg1b/Rpg1r that recognizes AvrB/AvrRpm1 from Pseudomonas syringae pv. glycinea (Psg) (Ashfield et al, 2014). Reports of immune responses mediated by PRRs in soybean are scarce (Whitham et al, 2016). Although PTI is not as strong as ETI, it confers a much broader and moderate resistance to most microbes, with potentially lower growth and yield penalty. In plants, PRRs consist of receptor kinase (RK) and receptor protein (RP). RK contains a variable ectodomain potentially involved in ligand perception, a single transmembrane domain, and a cytoplasmic kinase domain for signal transduction. RP contains an ectodomain and a transmembrane domain, but lacks the cytoplasmic kinase domain. Plant RKs and RPs are divided into many subgroups according to the nature of their extracellular domain, such as the leucine-rich repeat (LRR) type, lysine motif (LysM) type, and malectin-like domain type (Tang et al, 2017). The Arabidopsis LRR-RK FLS2 and EFR can recognize bacterial flagellin (or the epitope flg22) and ET-Tu (or the epitope elf18), respectively (Chinchilla et al, 2006; Zipfel et al, 2006), and form a complex with the co-receptor BAK1/SERK3, which acts as a co-receptor for several LRR-type PRRs (Chinchilla et al, 2007; Heese et al, 2007). Fungal cell wall-derived chitin can be recognized by the LysM-RK protein LYK5, which is in complex with CERK1 (Cao et al, 2014). The Nicotiana benthamiana receptor protein RXEG1 recognizes the P. sojae-derived pattern XEG1 and interacts with the co-receptor NbBAK1 and the adapter NbSOBIR1 (Wang et al, 2018). Plant PRRs interact with a subset of proteins, including BIK1 (and its homologs), the NADPH oxidase RbohD, heterotrimeric G proteins (XLG2, AGB1, and AGG1/2), and other regulatory components to constitute a large and dynamic immune receptor complex to activate downstream immune responses (Couto & Zipfel, 2016; Yu et al, 2017). PTI includes a series of immune responses, including production of reactive oxygen species (ROS), transient influx of calcium, activation of MAP kinase and calcium-dependent protein kinase cascades, and transcriptional reprogramming (Couto & Zipfel, 2016). Malectin-like RKs contain two tandem malectin-like domains in their extracellular region to specifically recognize different signal molecules, including rapid alkalinization factors (RALFs; Franck et al, 2018). Since the first member was identified in Catharanthus roseus (CrRLK1; Schulze-Muth et al, 1996), malectin-like RKs have received increasing attention because of their versatile roles in hormone signaling, growth, morphogenesis, reproduction, and stress responses (Franck et al, 2018). Arabidopsis thaliana BUPS1 and BUPS2 recognize the preferentially pollen-expressed RALF4 and RALF19 peptides to regulate pollen tube rupture and cell wall integrity by forming a receptor complex with ANX1/2, suggesting dynamic interactions among this protein family (Boisson-Dernier et al, 2009; Miyazaki et al, 2009; Ge et al, 2017). FERONIA (FER) recognizes RALF1 to regulate pollen tube reception and cell elongation (Escobar-Restrepo et al, 2007; Guo et al, 2009; Kessler et al, 2010; Haruta et al, 2014). Some members also participate in plant immunity. For example, FER positively regulates flg22- or elf18-mediated PTI and facilitates the complex formation between FLS2 and EFR with BAK1 (Stegmann et al, 2017). In contrast, ANX1 negatively regulates PTI by inhibiting the flg22-induced FLS2-BAK1 association (Mang et al, 2017). Interestingly, a fungal pathogen, Fusarium oxysporum, deploys a RALF-like peptide as a virulence effector to hijack plant immunity signaling by targeting FER (Mang et al, 2017). Investigation of plant lesion mimic mutants (lmms) has been a powerful forward genetic approach to unravel plant immunity responses and cell death pathways because they display visible spontaneous cell death phenotypes on leaves, that are often related to autoimmunity (Bruggeman et al, 2015; Chakraborty et al, 2018; Radojicic et al, 2018). Here, we report the isolation and functional characterization of a soybean malectin-like RK, GmLMM1, which negatively regulates resistance to bacterial and oomycete pathogens. GmLMM1 directly couples to the PRR complexes, including PRRs, co-receptors, RbohD, and G proteins from soybean, leading to attenuation of PTI activation. We took advantage of N. benthamiana, a model plant for plant immunology, and revealed that GmLMM1 could inhibit the FLS2-BAK1 association under treatment with flg22 and act as a molecular switch to regulate FLS2-BAK1 interaction and immune activation. Thus, we present a novel soybean PTI regulator and reveal a mechanism by which GmLMM1 maintains disease resistance responses at proper levels. Results Positional cloning of the GmLMM1 gene We generated a mutant population derived from ethyl methane sulfonate (EMS)-treated soybean cultivar "Williams 82" and obtained 16 independent lesion mimic mutants (Fig 1A). We noticed that one mutant exhibited constitutively enhanced resistance to Pseudomonas syringae pv. glycinea (Psg), a causal pathogen of bacterial leaf spot on soybean (Fig 1B), suggesting it is an autoimmune-related mutant. Thus, we selected this for further characterization and named it Gmlmm1-1 (G. max lesion mimic mutant 1-1). The mutant had overall shorter plant height, smaller leaf size, fewer branches, and shorter petioles in comparison to the wild type (WT) (Fig 1A). Spontaneous lesions began to appear on the extended leaves and increased following the growth of each leaf, resulting in the phenotypes developing through the entire growth cycle, and only 3–4 new emerging leaves were eventually normal (Fig 1A). Figure 1. Identification and characterization of the GmLMM1 gene Phenotype appearance of the Gmlmm1-1 mutant. Typical leaves (left panel; scale bar, 1 cm) and whole plants (right panel; scale bar, 10 cm.) were photographed 60 and 118 days after seeding, respectively. Enhanced resistance of Gmlmm1-1 and Gmlmm1-2 to Psg infection. Leaves from 2-week-old soybean plants were infected with Psg, and the bacteria number was determined at 0 and 4 dpi (Mean ± SD, n = 8, n represents sample number, ***P < 0.001, Student's t-test). Physical locations of markers defining the GmLMM1 region. The chromosomal positions (Pd, physical distance in Mb) of each marker are indicated on chromosome 13 (Chr13) according to the soybean reference genome. Recombination ratio (Rr, %) at the corresponding physical location was calculated using a formula (2 × plant number of genotype HD 12 + plant number of heterozygote genotype)/(2 × total number of plants) for rough mapping. Numbers of recombination (Nr) refers to the number of heterozygous plants at a physical location in the 92 plants for gene mapping. The candidate genes were harbored in a 131 kb region spanned by MOL3754 and MOL3780, in which six homologous genes were tandemly repeated and belonged to the malectin-like gene family in the shadows. The direction of the arrow represents the direction of the gene on the chromosome. Genes with high similarity are indicated by arrows of the same color. The number under the genes corresponds to the gene locus on chromosome 13. The Glyma.13G054400 (44) gene harbors a mutation (C to T) according to re-sequencing. Comparison of Arabidopsis FERONIA, ANXUR1, and GmLMM1. SP and TM indicate the predicted signal peptide and transmembrane domain, respectively. Two corresponding mutated sites in Gmlmm1-1 and Gmlmm1-2 are indicated by arrows. Subcellular localization of GmLMM1-GFP and GmLMM1L407H-GFP in N. benthamiana. PI4P indicates the membrane localization. Scale bar, 10 μm. GFP, green fluorescent protein. Autophosphorylation activity of the GmLMM1 intracellular domain in vitro. The proteins purified from E. coli were subjected to pMIAGO phosphorylation detection assay (upper panel) and were detected using Western blot (lower panel) with BIK1-HIS as a control. GmLMM1K564E contains a mutation at the predicted ATP binding site. Data information: The experiments were performed three times (A, B, E) or two times (F), as biological replicates, with similar results. Source data are available online for this figure. Source Data for Figure 1 [embr202050442-sup-0005-SDataFig1.zip] Download figure Download PowerPoint Next, we performed map-based cloning to identify the GmLMM1 gene. The Gmlmm1-1 mutant was back-crossed to the wild type (Williams 82) for four generations to purify the genetic background, and then, the progeny was crossed with another soybean cultivar, "Hedou 12". Two F1 seeds were obtained, and their seedlings showed similar phenotypes to that of the WT. In their F2 progenies, 372 individuals were very similar to that of the WT, and 92 exhibited phenotypes of the mutant. The F2 segregation rate fit the expected 3:1 ratio (x2 = 3.28, df = 1, P = 0.07; Appendix Table S1), indicating the mutation was mono-recessive inheritance. Furthermore, 170 available INDEL molecular markers covering 20 chromosomes were used for rough mapping (Song et al, 2015a). We successfully narrowed down the candidate gene to a 2.59 Mb region on chromosome 13 between MOL3684 (15.06 Mb) and MOL0631 (17.65 Mb). In order to further reduce the scope of the candidates, we designed a set of molecular markers (Appendix Table S2). Eventually, the target gene was positioned in a 131 kb region between markers MOL3754 and MOL3780 (Fig 1C). In this region of the soybean cultivar "Williams 82" (Data ref: Schmutz et al, 2010), we found 13 genes (Appendix Table S3), including six tandemly repeated homologous genes that share high sequence similarity with Arabidopsis FER and ANX1 (Fig 1D and Appendix Table S4). Next, we performed whole genome sequencing of the mutant Gmlmm1-1 and found that only one gene, Glyma.13G054400, was mutated in this region. There is a C to T substitution on its second exon, resulting in an early stop at its 636th amino acid (Figs 1C and D, and EV1A). To confirm that the lesion mimic phenotype was indeed caused by the mutation of Glyma.13G054400, we aimed to find an independent mutant allele for Gmlmm1 from the above 16 Gmlmms using genomic PCR and sequencing. Fortunately, a second mutant allele was successfully identified and named Gmlmm1-2, which harbors a single base (T to A) substitution on its first exon resulting in a leucine to histidine substitution at its 407th amino acid (Figs 1D and EV1A). The Gmlmm1-2 mutant plants also showed enhanced resistance to Psg infection (Fig 1B) and reduced plant height. In addition, the seed number per pod of Gmlmm1 mutants was significantly reduced. For example, the number of three-seed pods of Gmlmm1-2 was reduced to half of that of the WT, while the one-seed pod number doubled (Appendix Table S5), indicating that the fertility of sexual organs or early embryo development were also affected in the mutant plants. Click here to expand this figure. Figure EV1. Identification of the Gmlmm1 mutants and bioinformatics analysis of the GmLMM1 gene Verification of the mutation site of Gmlmm1-1 and Gmlmm1-2 by PCR and sequencing. Typical phenotypes of the plants. The indicated plants were grown for 18 days in a climate chamber. Scale bar, 1 cm. Identification of the F1 plant (Gmlmm1-1 × Gmlmm1-2) genotype by PCR and restriction enzyme digestion. Fragments covering the Gmlmm1-1 and Gmlmm1-2 mutation sites were amplified by PCR, digested by the indicated enzymes, and separated on agarose gel. The corresponding fragment sizes after cleavage are marked with arrows. Identification of the F2 (Gmlmm1-1 × Gmlmm1-2) genotype. Total DNA was extracted from 33 individual F2 plants, and fragments covering the Gmlmm1-1 and Gmlmm1-2 mutation sites were amplified. Phylogenetic analysis of the malectin-like RK family in Arabidopsis and Glycine max. Nucleotide diversity (π) of Glycine max (red line) and Glycine soja (green line) across a ˜150 kb genomic region that harbored the 13 mapped genes. The blue dotted line indicates the location of the GmLMM1 gene. The abscissa represents the physical location (Mb) of differential nucleotides. The ordinate represents nucleotide diversity (π). Data information: The experiments were performed three times (A, B) or two times (C, D), as biological replicates, with similar results. Source data are available online for this figure. Download figure Download PowerPoint We next crossed Gmlmm1-1 with Gmlmm1-2 and observed a similar lesion mimic phenotype in the F1 plants (Fig EV1B). Sequencing and restriction enzyme digestion revealed that both mutation sites of the parents were heterozygous in F1 progenies (Fig EV1C). We also analyzed the phenotypes and genotypes of their F2 progenies. All 33 independent F2 plants exhibited similar phenotypes as their parents and contained three different genotypes, a1a1 (11 lines), a1a2 (16), and a2a2 (6), which is consistent with the Mendelian ratio expectation (a1a1:a1a2:a2a2 = 1:2:1. Square test: x2 = 0.76, df = 2, P = 0.69; Fig EV1D and Appendix Table S6). Taken together, we concluded that Gmlmm1-1 and Gmlmm1-2 are allelic mutants and therefore named the corresponding gene GmLMM1. GmLMM1 is a soybean malectin-like RK Bioinformatic analysis revealed that the GmLMM1 gene encodes a malectin-like RK, which was first identified as the Catharanthus roseus CrRLK1L (Schulze-Muth et al, 1996; Franck et al, 2018). The closest homologs of GmLMM1 in Arabidopsis are the malectin-like RKs, FER (identity = 50.5%), and ANX1 (identity = 43.8%) (Figs 1D and EV1E). We analyzed the nucleic acid polymorphism in a ~150 kb region from 15.07 Mb to 15.22 Mb on chromosome 13 using 62 varieties of Glycine soja and 130 varieties of G. max. In the investigated region, the nucleotide diversity (π) of G. max and G. soja showed no obvious difference, which indicated that GmLMM1 is highly conserved in both cultivated and wild soybeans (Fig EV1F). We examined the subcellular localization of GmLMM1 by transiently expressing GFP-tagged GmLMM1 in N. benthamiana and observed that GmLMM1-GFP was localized to the plasma membrane (Fig 1E). As the Gmlmm1-2 mutant harbors a single-site mutation (L407H) in the extracellular region, the localization assay showed that the GmLMM1L407H mutation abnormally aggregates in the cytosol and membrane (Fig 1E). This abnormal subcellular location of GmLMM1L407H may account for its loss-of-function phenotype of Gmlmm1-2. The malectin-like RK family proteins contain a C-terminal intracellular kinase domain, which is important for the activation of downstream signaling. An in vitro kinase reaction assay revealed that GmLMM1 showed strong autophosphorylation activity. In contrast, a site mutation of GmLMM1K564E, which is predicted as the ATP binding site, resulted in lost autophosphorylation activity (Fig 1F). Collectively, we suggest that GmLMM1 encodes a malectin-like RK with membrane localization and kinase activity. Mutations in GmLMM1 cause cell death mimic phenotypes To further verify that the mutant phenotype was controlled by a loss-of-function mutation of GmLMM1, we designed three CRISPR vectors (C1/2/9) to knock out GmLMM1 and its neighboring genes with high sequence similarity, Gm13G053600 and Gm13G053800 (Fig EV1E and Appendix Table S4). The recombinant plasmids were introduced into wild-type Chinese soybean cultivar, "Dongnong 50 (DN50)", to generate GmLMM1 mutants. Only one T0 (C1-16) line generated from the C1 vector was obtained. In the T1 lines, C1-16-1 exhibited a lesion mimic phenotype, whereas C1-16-5 was similar to the WT (Fig 2A). Consistent with this, the C1-16-1 line harbored substantial mutations causing a frame shift in the GmLMM1 gene and a 3-bp deletion in Gm13G053800. In contrast, C1-16-5 showed a 3-bp deletion in GmLMM1 and multiple mutated sites in Gm13G053800 (Fig 2A). Similarly, the C2 vector targeting GmLMM1, Gm13G053600, and Gm13G053800 was introduced into DN50. In the two T1 lines generated, the gene editing events caused a frame shift in all three target genes in the two T1 lines. Consistent with this, both T1 lines exhibited a lesion mimic phenotype (Fig 2B). The C9 vector was designed to simultaneously target GmLMM1 and Gm13G053600 and was introduced into DN50 (Fig 2C). The C9-109-2 line harbored a 7-bp deletion in Gm13G053600, but GmLMM1 was not edited. Leaves of the C9-109-2 lines did not show any visible lesion mimic spots (Fig 2C). The C9-24-13 line contained the frame shift mutant of GmLMM1, but Gm13G053600 was not edited. Consistent with this, the C9-24-13 lines showed a visible lesion mimic phenotype (Fig 2C). It is noteworthy that the lesion mimic phenotype in C9-24-13 plants was not as strong as that in the C1 and C2 CRISPR lines, which could be due to the fact that the C9 vector targets the C terminus of GmLMM1 (Fig 2C). To further confirm that the lesion mimic phenotype of Gmlmm1 mutants is caused by autoimmunity-related cell death, we examined cell death in Gmlmm1-1, Gmlmm1-2, and the CRISPR line C9-24-13. Trypan blue staining showed that all three mutant lines exhibited a stronger cell death phenotype than the corresponding controls (Fig EV2A and B). We further checked the expression of the immune marker genes GmPR1 and GmPR2 in the Gmlmm1 mutants. Both marker genes were highly expressed in Gmlmm1-1, Gmlmm1-2, and the CRISPR lines (Fig EV2C and D). Taken together, we suggest that the GmLMM1 gene, but not its two homologous genes, is responsible for the autoimmune-related cell death mimic phenotypes. Figure 2. Multiallelic editing of the GmLMM1 gene causes the lesion mimic phenotypeThree constructs were designed to target GmLMM1 and its close homologous genes. The CRISPR target sequence is shown in orange, and the corresponding protospacer adjacent motif (PAM) site is shown in blue. The box indicates the putative sites shear cut by cas9. Two representative T1 transgenic lines (aged 1 month) are shown for each construct. The editing results were examined by PCR and sequencing; – means deletion of the corresponding nucleotide. The red or green face-labeled letters indicate the mutated or added nucleotides, respectively. Editing of GmLMM1 and Gm13G053800 by construct 1 (C1). The alignment of the sequence and phenotypes of two T1 lines (C1-16-1 and C1-16-5) generated from the same T0 line (C1-16) are shown. Simultaneous edits of GmLMM1, Gm13G053600, and Gm13G053800 by the C2 vector. Editing of GmLMM1 and Gm13G053600 by C9 vector. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Gmlmm1 mutants are autoimmune-related lesion mimic mutants Cell death examination in Gmlmm1-1, Gmlmm1-2, and Williams 82. Leaves of the indicated plants (aged 2 weeks) were subjected to trypan blue staining. Scale bar, 1 cm. Cell death examination in DN50 (Dongnong 50) and the CRISPR line C9-24-13. Scale bar, 1 cm. Expression of the defense marker genes in Gmlmm1 mutants. GmPR1 and GmPR2 were examined by qPCR analysis in the indicated plants (Mean ± SD, **P < 0.01, ***P < 0.001, Student's t-test). Expression of GmPR1 and GmPR2 in DN50 and the CRISPR lines. (Mean ± SD, ***P < 0.001, Student's t-test). Data information: The experiments were performed three times (A, B) or two times (C, D), as biological replicates, with similar results. Download figure Download PowerPoint The Gmlmm1 mutant confers enhanced resistance to bacterial and oomycete pathogens As the activation of plant immune responses is often accompanied by accumulation of ROS, we investigated ROS accumulation in the Gmlmm1 mutant lines using DAB staining. Compared to Williams 82, the Gmlmm1-1 and Gmlmm1-2 mutants showed significantly enhanced ROS accumulation (Fig 3A). We examined ROS a
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