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. 2026 Jan 24;26:328. doi: 10.1186/s12870-026-08210-9

Genome-wide analysis of leucine-rich repeat-only proteins gene family revealed FaLRRop26 responding Colletotrichum Siamense infection by regulated lignin synthesis in cultivated strawberry (Fragaria × ananassa)

Xu Li 2, Lili Liu 2, Chun Luo 1,2, Zhe Chen 3, Bo Shu 1,2,✉
PMCID: PMC12911200  PMID: 41580606

Abstract

Background

Strawberry (Fragaria × ananassa Duch.) is a highly valued crop, susceptible to various diseases, with Colletotrichum siamense-induced crown rot being a major concern. Emerging evidence indicates that leucine-rich repeat only protein (LRRop) participates in plant responses to both biotic and abiotic stresses.

Results

The LRRop gene family was investigated in strawberry. 140 FaLRRop gene family members were identified in the strawberry genome, and 121 orthologous gene pairs with F. vesca and 117 with F. iinumae. C. siamense infection and exogenous MeJA pretreatment inhibited the expression of FaLRRop26 and its alleles, while SA pretreatment upregulated their expressions. The FaLRRop26 and its alleles mutations of octoploid strawberry were achieved by utilizing CRISPR/Cas9 gene editing. Mutants exhibited significantly larger lesions compared to wild-type. Compared with wild-type, the key structural genes for lignin monomer synthesis, including 4-coumarate: CoA ligase, caffeic acid-O-methyltransferase, ferulate-5-hydroxylase, cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase of mutants were significantly inhibited. Furthermore, mutants had 17% lower lignin content than wild-type on 0 days post-inoculation (0 dpi). On 4 dpi, wild-type accumulated approximately 87% lignin, whereas mutants maintained only about 30%.

Conclusions

Our findings suggest that FaLRRop26 and its alleles enhanced strawberry resistance for C. siamense by positive regulating lignin biosynthesis, and which provides new insights into the role of LRRop gene family in strawberry immunity and offers a potential target for strawberry resistance breeding.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08210-9.

Keywords: Leucine-rich repeat only protein, Multiple alleles, Gene editing, Lignin, Resistance

Background

Strawberry (Fragaria × ananassa Duch.) is an economically important crop valued for both edible and ornamental purposes, cultivated worldwide [1]. Strawberries are susceptible to various diseases during cultivation, with crown rot caused by Colletotrichum spp. being particularly damaging [2]. In recent years, C. siamense-induced crown rot has frequently occurred in major strawberry production areas of China, causing significant economic losses [3]. Currently, chemical control remains the primary management approach. However, under the high temperature and humidity conditions of summer and autumn, disease progression accelerates can shorten to about one week, necessitating frequent pesticide applications [4]. This approach shows limited efficacy, high cost and serious environmental pollution.

Developing resistant cultivars represents the most effective strategy for controlling strawberry crown rot. However, due to the scarcity of resistant germplasm, conventional hybridization breeding faces significant challenges [5]. Advances in molecular biology have enabled the screening of pathogenesis and plant–pathogen interactions genes, following transgenesis and genome editing, thereby providing novel approaches to develop C. siamense-resistant strawberry germplasm. Recent multi-omics studies have revealed the molecular mechanisms underlying the response of moderately resistant strawberry cultivars to hemibiotrophic Colletotrichum infection [6]. The resistance response involves recognition of pathogen-associated molecular patterns and effectors by leucine-rich repeat (LRR)-type immune receptors, followed by signal transduction through hormone pathways including salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA), ultimately activating defense processes such as sesquiterpenoid biosynthesis, flavonoid metabolism, and lignin deposition [7–9].

LRR proteins belong to a highly conserved superfamily in eukaryotes. Their characteristic tandem LRR domains mediate critical biological processes such as protein-protein interactions and signal transduction, playing pivotal roles in plant growth, development, and immunity [10]. Based on domain architecture, LRR proteins are classified into composite proteins containing LRR domains along with other functional domains or motifs (LRR receptor-like kinases (LRR-RLKs), LRR receptor-like proteins (LRR-RLPs), nucleotide-binding site LRR (NBS-LRR) proteins, LRR-extensin (LRX) proteins, and polygalacturonase-inhibiting proteins (PGIPs)) and proteins containing only LRR domains (LRR-only proteins, LRRops) [11–14]. Significant progress has been made in plant LRR protein research, with well-characterized functions of plasma membrane-localized pattern recognition receptors (LRR-RLKs and LRR-RLPs), intracellular immune receptors (NBS-LRR), and cell wall-associated proteins (PGIPs and LRX) in pathogen defense [13, 15–17]. However, the LRRop subfamily remains understudied. Emerging evidence indicates that LRRop participates in plant responses to both biotic and abiotic stresses. In Arabidopsis thaliana, 45 LRRop members have been identified. Among these, the lrrop-1 mutant exhibits enhanced abiotic stress tolerance, while AtLRRop2 has been reported as a positive regulator of cold stress tolerance [18, 19]. A study in cucumber (Cucumis sativus) demonstrates that knockout of CsLRR1 increases susceptibility to powdery mildew, whereas its overexpression significantly enhances resistance [20]. Studies on strawberry LRR proteins reveal differential expression patterns during Colletotrichum infection. Both C. gloeosporioides and C. acutatum infections activate the expression of various LRR-encoding genes [5, 8]. Our preliminary research demonstrated that C. siamense infection downregulates multiple LRRop genes, which also respond to exogenous methyl JA (MeJA) and SA pretreatments, suggesting strawberry LRRops in defense against C. siamense.

To address this scientific question systematically, we propose the following research objectives: 1) Comprehensive identification of LRRop gene family members in cultivated strawberry and expression profiling of LRRop genes during C. siamense infection; 2) Generation of key LRRop mutants using CRISPR/Cas9 gene editing to evaluate disease resistance phenotypes; and 3) Elucidation of molecular mechanisms underlying LRRop-mediated resistance through integrated phenotypic and transcriptomic analyses. This study will provide theoretical insights into strawberry-Colletotrichum interactions and identify novel targets for resistant strawberry breeding.

Methods

Identification of the FaLRRop gene family and its expression pattern responding to C. siamense infection, SA and MeJA pretreatment

The protein sequences of 45 AtLRRop members and genomic information of Arabidopsis were obtained from TAIR database (https://www.arabidopsis.org/), while the ‘Benihoppe’ strawberry genome sequence and annotation files were downloaded from GDR database (https://www.rosaceae.org/). Preliminary screening was performed using Diamond tool (https://github.com/bbuchfink/diamond) to align AtLRRop sequences against the strawberry protein database with an e-value < 10− 5. A three-step stringent screening process was implemented: (1) NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd/) was used to predict domain architecture, excluding proteins containing other domains; (2) TMHMM 2.0 server (https://services.healthtech.dtu.dk/services/TMHMM-2.0/) predicted transmembrane helices to exclude membrane-associated proteins; (3) Manual verification ensured all candidates contained only LRR domains. Physicochemical properties (molecular weight, theoretical pI, etc.) were analyzed using ExPASy tool (https://web.expasy.org/). Subcellular localization was predicted by WoLF PSORT (https://wolfpsort.hgc.jp/), and chromosomal distribution was visualized using MG2C online tool (http://mg2c.iask.in/). Conserved motifs of were identified using MEME Suite (https://meme-suite.org/). Based on NCBI-CDD predictions, TBtools software was employed to visualize gene structures (exon-intron organization) and LRR domain distributions [21]. The 2000 bp upstream sequences from transcription start sites were extracted for cis-acting element analysis using PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/), with results visualized by TBtools. Multiple sequence alignment was performed using MUSCLE (http://www.drive5.com/muscle/) and trimAl (https://vicfero.github.io/trimal/). The maximum likelihood phylogenetic tree of Arabidopsis and strawberry LRRop families was constructed using IQ-TREE (https://iqtree.github.io/) with 1000 replicate bootstraps. Genome and annotation files of diploid strawberries (F. vesca and F. iinumae) were obtained from the GDR database. MCScanX (http://chibba.pgml.uga.edu/mcscan2) was employed to analyze intra-species synteny in cultivated strawberry and inter-species synteny with Arabidopsis and diploid ancestors, followed by visualization in TBtools.

Transcriptome data of ‘Benihoppe’ strawberry infected with C. siamense and pretreated with exogenous MeJA/SA (0 d, 2 d, 4 d) were previously generated [22, 23]. The Transcripts per million (TPM) values of 120 homologous FaLRRop genes were visualized as a heatmap using TBtools to analyze their expression patterns.

Vector construction and genetic transformation

After verifying the CDS sequence of FaLRRop26 by TA-cloning, two sgRNA sites targeting conserved regions of FaLRRop26 and its alleles were designed using the CRISPOR (https://crispor.gi.ucsc.edu/), located 79 bp (sgRNA1: TCAGACATCTCAGCTCTCA) and 897 bp (sgRNA2: GCTGTCGGTTCGTTACAAC) downstream of the start codon (Fig. 5A). The dual-sgRNA expression cassette was cloned into the pCBC-T1T2 vector, and subsequently assembled into the pKSE401G binary vector via Golden Gate Assembly method. Following sequence verification, the recombinant vector was transformed into Agrobacterium tumefaciens strain GV3101 competent cells. For genetic transformation, an optimized leaf disc protocol was employed where young leaves of tissue cultured seedlings derived from the same ‘Benihoppe’ strawberry stem tip (Supplied by Hubei Academy of Agricultural Sciences), were cut into 5 × 5 mm discs and pre-cultured for 20 days until calluses occurred. The calluses were infected with Agrobacterium suspension (OD600 = 0.6) for 20 min, with the bacterial culture pre-induced by co-buffer containing 1% acetosyringone (50 mg/mL) for 3 h. After 3 days of co-culture, explants were transferred to antibiotic medium for 1 week, followed by GFP-positive calluses screening under blue light excitation. The positive calluses were transferred to selection medium and the medium was refreshed every 2–3 weeks until shoot regeneration occurred [24]. Shoots derived from different calluses were separately excised for subculture, and each independent shoot line was verified for editing efficiency by PCR sequencing. Positive lines were propagated to sufficient numbers before fully expanded shoots were excised and transferred to rooting medium for root induction. All the component of medium and primer information involved in this study were listed in Table S4 and S6. Genomic DNA was extracted from transgenic lines using the Plant Genomic DNA Kit (Solarbio, Beijing). Target-specific primers flanking 200 bp regions of each allele were designed for PCR amplification. The amplicons were subjected to TA cloning and transformed, with five randomly selected positive clones per allele sequenced. Potential off-target sites predicted by CRISPOR were examined by PCR amplification of 200–300 bp flanking regions, followed by TA cloning and sequencing to assess off-target effects.

Fig. 5.

Fig. 5

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Gene editing of FaLRRop26 and its alleles based on CRISPR/Cas9. A Schematic diagram of dual sgRNA targets location. B Mutation situation of sgRNA1 target in transgenic lines. The blue and green letters represent the sgRNA sequence and PAM site, respectively. The deletion and insertion events are characterized with red letters. C Sequencing maps of the mutation events. D Detection of potential off-target site sequences. E Length and width of lesion of gene-edited (ED) and wild-type (WT) at 4 dpi

Plant materials and experiment design

Gene-edited (ED) and wild-type (WT) ‘Benihoppe’ strawberry plantlets were transplanted into pots containing Pinstrup nursery substrate (5–20 mm) and grown in controlled chambers (25℃/16 h light, 15℃/8 h dark). Plants were watered three times weekly and supplemented with 30 mL Hoagland’s nutrient solution. After 3 months cultivation, disease resistance evaluation against C. siamense SCR-7 was according to description of Peng et al. with modification [22]. Before inoculation, crown regions were surface-sterilized with 70% ethanol for 10 s, rinsed thrice with sterile water, then wounded to 1 mm depth using sterile needles and inoculated with 10 µL spore suspension (1 × 106 spores/mL). Lesion diameters were measured at 4 days post-inoculation (dpi). Four treatment groups were established: ED0D (gene-edited lines at 0 dpi), WT0D (wild-type at 0 dpi), ED4D (gene-edited lines at 4 dpi), and WT4D (wild-type at 4 dpi), with three biological replicates per group (4 plants per replicate). Crown tissues were excised, rinsed with distilled water, blotted dry, and immediately frozen in liquid nitrogen for subsequent analysis.

Transcriptome analysis and qRT-PCR

Total RNA was extracted from each treatment using a commercial kit (Sangon, Shanghai), and its purity was assessed via microspectrophotometry and agarose gel electrophoresis. Sequencing was performed on an Illumina HiSeq 2500 platform, with the strawberry genome (F. × ananassa Benihoppe Genome v1.0, https://www.rosaceae.org/Analysis/18085091) as the reference. Quality-filtered clean reads were aligned and assembled against the reference genome using HISAT2 software. TPM values were calculated using StringTie (https://github.com/gpertea/stringtie) to estimate gene expression levels. Differentially expressed genes (DEGs) were identified with DESeq2 (https://bioconductor.org/packages/DESeq2) under the thresholds of qValue ≤ 0.05 and |log2FoldChange| ≥ 2. Functional annotation of DEGs was performed via Gene Ontology Resource (https://www.geneontology.org/), and their metabolic pathways were analyzed using the KEGG database (https://www.genome.jp/kegg/) as described in Shu et al. [3]. The transcriptome data information was available in Table S5. The relative expression levels of key enzyme-encoding genes in the lignin biosynthesis pathway were calculated by the 2−ΔΔCt method, with the strawberry actin gene (FaActin) as the internal reference [25].

Lignin determination

Lignin contents were determined using a test kit as described by the manufacturer (Solarbio, Beijing, China). Dried samples (~ 5 g) were acetylated, and then absorbance was measured at 280 nm [26].

Statistical analysis

Statistical analysis was performed using SAS 8.1 software (SAS Inc., Cary, N.C., USA). Differences among treatments were evaluated by Duncan’s multiple range test at a significance level of p < 0.05, with different lowercase letters indicating statistically significant differences.

Results

Genome-wide identification and characterization of FaLRRop gene family

Through Diamond alignment and domain prediction, 140 FaLRRop gene family members were identified in the strawberry genome. The FaLRRop proteins ranged from 175-1169 aa in length with molecular weights of 19.17-129.42 kDa, displaying diverse physicochemical properties (Table S1). Subcellular localization prediction revealed 57 members localized to chloroplasts and 30 to nuclei (Table S1). Chromosomal mapping showed uneven distribution: chr_2B contained the highest density (9 members), while chr_5D and chr_6D had the lowest (1 member each), with other chromosomes containing 2–8 members (Fig. 1). Ten characteristic motifs (10–50 aa) were identified in the FaLRRop family, and the motif quantity of each FaLRRop ranged from three to thirty-four, with motif 1, 5, 6, 7, and 8 rich in leucine residues (Fig. 2, S1). Gene structure analysis showed 1–21 exons and 0–20 introns contained in FaLRRop family. 48 genes (34%) had the canonical exon-intron-UTR structure, 61 (44%) were single-exon genes, and the remaining 31 (22%) had exon-intron structures (Fig. 2). The sub-tree of FaLRRop showed that members within the same clade shared conserved motif patterns and gene structures. For example, group Ⅱ contained single-exon genes, while group Ⅶ had multiple exons. Group Ⅲ showed the highest motif diversity despite simpler gene structures (Fig. 2).

Fig. 1.

Fig. 1

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Chromosome location of FaLRRop gene family members. The genes highlighted in red represent tandem duplication gene pairs

Fig. 2.

Fig. 2

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Gene structure and motif analysis of FaLRRop gene family. A Phylogenetic tree of FaLRRop proteins. B Motifs analysis of FaLRRop proteins. C LRR domains of FaLRRop proteins. D Gene structure of FaLRRop genes

Conserved evolution and purifying selection of FaLRRop gene family

Phylogenetic analysis clustered FaLRRop and AtLRRop into 9 clades. The quantity of each clade is significantly different, and clade of Ⅸ had twenty-four FaLRRops while clade of Ⅰ had three FaLRRops (Fig. 3A). Intra-species synteny analysis revealed extensive segmental duplication events among FaLRRop family members, with 188 segmental duplication and 4 tandem duplication gene pairs identified (Fig. 3B). These duplications predominantly occurred between subgenomes within chromosomes, reflecting polyploidization history of strawberry. Inter-species synteny analysis detected 68 orthologous gene pairs between FaLRRop and Arabidopsis, and showed high conservation with diploid ancestors (121 orthologous gene pairs with F. vesca and 117 with F. iinumae), suggesting their genetic conservation (Fig. 3C). Ka/Ks analysis indicated that 403 gene pairs had ratios < 1, demonstrating predominant purifying selection, while 18 pairs showed Ka/Ks > 1, suggesting potential positive selection (Table S2).

Fig. 3.

Fig. 3

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Phylogenetic and evolution analysis of FaLRRop gene family. A Phylogenetic tree of strawberry and Arabidopsis LRRop proteins via Neighbour-Joining method based on 1000 replicate bootstraps. Different colour modules represent the subfamily groups, while the red triangles represent branches with the bootstrap greater than 50. B Inter-species synteny analysis of cultivated strawberry. Red lines represent the segmental duplication events of FaLRRop gene between subgenomes within each homoeologous group, while other colored lines represent segmental duplication events between subgenomes of different homoeologous groups. The orange bar chart represents the chromosome gene density, while the green bar chart represents the GC ratio. C Intra-species synteny analysis of octoploid strawberry with Arabidopsis and diploid strawberries. The red lines represent the collinear relationship of FaLRRop gene family among species

Multiple cis-acting elements distributed in the promoters of FaLRRop gene family

Cis-acting element analysis revealed that FaLRRop gene promoters contain abundant regulatory elements beyond core promoter elements (TATA-box/CAAT-box). Hormone-responsive elements were most prevalent, with MeJA-related elements (CGTCA-motif/TGACG-motif, 614 hits) being the most abundant, followed by ABA-responsive ABRE (375), GA-related elements (GARE-motif/P-box/TATC-box, 123), auxin-responsive elements (TGA-element/AuxRR-core/AuxRE/TGA-box, 112), and SA-responsive elements (TCA-element/SARE, 69) (Table S3). Stress-responsive elements included hypoxia-responsive elements (ARE/GC-motif, 328), low-temperature-responsive elements (LTR, 81), defense and stress-responsive elements (TC-rich repeats, 75), and wound-responsive elements (WUN-motif, 11) (Table S3). All identified transcription factor binding sites were MYB-type (CCAAT-box/MBS/MBSI/MRE, 255). Notably, FaLRRop26 possessed significantly more cis-acting elements (38) than its alleles, encompassing ABA/MeJA response and stress defense elements.

Differential expression in response to C. siamense infection following exogenous MeJA and SA pretreatment of FaLRRop gene family

Transcriptomic profiling uncovered dynamic expression patterns of FaLRRop genes under pathogen challenge and hormone treatments. The expression of FaLRRops in strawberry crown showed diverse patterns in response to C. siamense infection following exogenous MeJA and SA pretreatment. Most of the members (104) were induced during early C. siamense infection, with upregulated genes progressively increasing and downregulated genes decreasing from 0 to 4 dpi (Fig. 4). In addition, exogenous hormone pretreatments exhibited temporal-specific regulation of FaLRRops. MeJA initially activated (0 dpi) then suppressed (2 dpi) before reactivating (4 dpi) gene expression (S), while SA caused sustained suppression after initial activation (Fig. 4). Strikingly, FaLRRop26 and its alleles exhibited coordinated expression patterns under both pathogen and hormone treatments. Infection with C. siamense and exogenous MeJA pretreatment inhibited the expression of FaLRRop26/20/43, while SA pretreatment specifically upregulated their expression levels (FaLRRop37 was newly annotated in the ‘Benihoppe’ genome).

Fig. 4.

Fig. 4

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Expression pattern analysis of FaLRRop gene family in response to C. siamense infection, exogenous MeJA and SA pretreatment. TPM value has been assigned a background value of 0.01 and underwent log2 conversion following by standardization processing. Cs means inoculating with C. siamense SCR-7

Gene-editing of FaLRRpo26 and its alleles increased strawberry susceptibility to C. siamense

Sequencing analysis confirmed effective editing at the sgRNA1 target site but not at sgRNA2. In line #4, all four alleles (FaLRRop20/26/37/43) showed heterozygous mutations (Fig. 5B, C). The copies of allele1 exhibited 25-bp deletion and wild-type. The copies of allele2 and allele3 both displayed 1-bp deletions and wild-type. While, the copies of allele4 showed 1-bp insertion and wild-type. These results demonstrated significantly higher editing efficiency at sgRNA1 than sgRNA2, achieving simultaneous multiplex editing of FaLRRop26 and its alleles. The detection results of all the positive lines obtained were available in Fig. S2 and S3. Furthermore, CRISPOR prediction identified the potential off-target for sgRNA1 (exon of Fxa6Dg04403 with 3 mismatches) and one for sgRNA2 (intergenic region between Fxa3Cg02687 and Fxa3Cg02688 with 4 mismatches) (Fig. 5D). Off-target sequencing verified the absence of nonspecific edits, confirming high editing specificity.

At 4 dpi with C. siamense, mutants exhibited significantly larger lesions (0.51 cm × 0.30 cm) compared to wild-type (0.42 cm × 0.26 cm) (p < 0.05) (Fig. 5E). This phenotype demonstrated that loss-of-function mutations in FaLRRop26 and its alleles increased strawberry susceptibility to C. siamense, indicating their positive regulatory roles in innate immunity.

Transcriptional regulation differences of gene-edited lines compare to wide-type

Transcriptome analysis comparing ED0D vs. WT0D (0 dpi) and ED4D vs. WT4D (4 dpi) revealed that the mutation of FaLRRop26 and its alleles significantly altered gene regulatory networks in strawberry crowns. At baseline (ED0D vs. WT0D), we identified 1,432 differentially expressed genes (DEGs), with 1,091 upregulated and 341 downregulated genes (Fig. 6A). At 4 dpi (ED4D vs. WT4D), DEGs increased dramatically to 3,333, with 456 upregulated and 2,877 downregulated genes (Fig. 6B). Following inoculation with C. siamense SCR-7, the number of DEGs increased dramatically, with downregulated genes substantially outnumbering upregulated ones, indicating that FaLRRop26 and its allele mutants primarily respond to pathogen infection through negative regulation of gene expression.

Fig. 6.

Fig. 6

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Transcriptome analysis of gene-edited (ED) and wild-type (WT) lines in response to C. siamense infection. A The volcano maps of ED0D vs. WT0D and ED4D vs. WT4D comparisons, respectively. B GO enrichment maps of ED0D vs. WT0D and ED4D vs. WT4D comparisons, respectively. C KEGG pathways enrichment scatter plots of ED0D vs. WT0D and ED4D vs. WT4D comparisons, respectively

GO enrichment scatterplot revealed that ED0D vs. WT0D comparison significantly enriched DEGs in molecular functions including ‘oxidoreductase activity’ (GO:0016491), ‘peroxidase activity’ (GO:0004601), and ‘glucosyltransferase activity’ (GO:0046527) (Fig. 6C). ED4D vs. WT4D comparison showed prominent enrichment in biological processes like ‘lignin biosynthesis process’ (GO:0009809), ‘phenylpropanoid metabolism process’ (GO:0009698), and ‘chlorophyll binding’ (GO:0016168) (Fig. 6D). Notably, cell wall components (GO:0005618) were consistently enriched in both comparisons. KEGG enrichment results demonstrated shared significant enrichment in ‘phenylpropanoid biosynthesis’ (ko00940), ‘α-linolenic acid metabolism’ (ko00592), and ‘plant hormone signal transduction’ (ko04075). ED0D vs. WT0D comparison specifically enriched ‘linoleic acid metabolism’ (ko00591), ‘valine, leucine and isoleucine biosynthesis’ (ko00290), and ‘glucosinolate biosynthesis’ (ko00966) (Fig. 6E). Unique to ED4D vs. WT4D comparison were ‘glycolysis/gluconeogenesis’ (ko00010), ‘ubiquinone and other terpenoid-quinone biosynthesis’ (ko00130), and ‘photosynthesis’ (ko00195) pathways (Fig. 6F).

Gene-editing of FaLRRop26 and its alleles suppressed lignin biosynthesis

KEGG pathway analysis of ED4D vs. WT4D after C. siamense infection revealed significant enrichment of the phenylpropanoid biosynthesis pathway toward lignin synthesis branch. This metabolic flux initiates with phenylalanine ammonia-lyase (PAL) and cinnamate-4-hydroxylase (C4H) to form p-coumaric acid, ultimately yielding lignin monomers through subsequent reactions. Notably, both PAL (1 gene) and C4H (2 genes) were significantly downregulated (Fig. 7A). Furthermore, the key structural genes for lignin monomer synthesis also showed significant downregulation, including 4-coumarate: CoA ligase (4CL) (6 genes), caffeic acid-O-methyltransferase (COMT) (4 genes), ferulate-5-hydroxylase (F5H) (3 genes), cinnamoyl-CoA reductase (CCR) (4 genes) and cinnamyl-alcohol dehydrogenase (CAD) (5 genes). Among them, the genes with the most significant downregulation were selected for qRT-PCR analysis (Fig. 7A).

Fig. 7.

Fig. 7

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Analysis of lignin biosynthesis pathway. A Lignin biosynthesis pathway map of ED4D vs. WT4D comparison. B Relative expression levels of the structural genes of lignin biosynthesis at 0 and 4 dpi. C Percentage contents of lignin at 0 and 4 dpi

qRT-PCR analysis of the lignin metabolism pathway revealed significant differences among four treatment groups (Fig. 7B-H). At 0 dpi, most genes in mutants showed suppressed expression, with FaPAL, FaCAD, FaCOMT, and FaF5H significantly downregulated while FaCCR upregulated. At 4 dpi, key lignin biosynthesis genes (FaC4H, FaCCR, FaCAD, FaCOMT, and FaF5H) in wild-type were significantly induced, particularly FaC4H showing about 3-fold increase. Conversely, all seven lignin biosynthesis genes in mutants were downregulated, with FaC4H, Fa4CL, and FaCCR significantly decreased compared to uninfected mutants. These results strongly correlated with transcriptome data. Lignin quantification showed congruent patterns with gene expression. At 0 dpi, mutants had 17% lower lignin content than wild-type (Fig. 7I). By 4 dpi, wild-type accumulated approximately 87% lignin (37% increase from 0 dpi), whereas mutants maintained only about 30% (57% lower than wild-type, 3% decrease from their own 0 dpi level) (Fig. 7I).

Discussion

Structural and functional diversity of FaLRRop gene family

A total of 140 FaLRRop genes were identified in cultivated strawberry through genome-wide analysis. These proteins exhibited substantial length variation (175–1169 aa) and diverse molecular weights (19.17-129.42 kDa). Subcellular localization predictions revealed multi-compartment distribution, with significant enrichment in chloroplast (41%), followed by nucleus (21%) and extracellular space (16%) (Table S1). The chloroplast-localized members were predominant, suggesting potential roles in stress resistance through chloroplast-nuclear retrograde signaling, while the localization of nucleus consistent with established stress response mechanisms [27]. FaLRRop genes showed uneven chromosomal distribution with significant gene number variation among homeologous chromosomes. This heterogeneity likely reflects differential gene retention following whole-genome duplication (WGD) events in octoploid strawberry [28]. Gene structure analysis identified 44% members as single-exon genes, while subgroups like group Ⅶ contained up to 8 exons. This structural diversity mirrors the evolutionary pattern similar to CKX family in wheat (Triticum aestivum), suggesting functional divergence during FaLRRop evolution [29].

The expansion and contraction mechanisms of FaLRRop gene family

Cross-species comparisons identified multiple orthologous gene pairs between strawberry and Arabidopsis, confirming the evolutionary conservation of FaLRRop in dicots. Notably, the octoploid strawberry harbored significantly more LRRop genes (140) than diploid Arabidopsis (44), indicating substantial family expansion during strawberry evolution. Synteny analysis with diploid progenitors (F. vesca and F. iinumae) revealed high conservation between cultivated strawberry LRRop genes and ancestral genomic regions, underscoring their functional stability during evolution [30]. Intraspecific synteny analysis revealed FaLRRop expansion was primarily driven by WGD/segmental duplication, while local tandem duplication also contributed to family expansion. Analogous to GST and LEA families in cultivated strawberry, this pattern reflects subgenome dominance after polyploidization [31, 32]. However, only ~ 80% of expected homoeologs were retained, indicating ~ 20% ancestral genes were lost during octoploidization due to functional redundancy or subgenome rearrangement [30]. This “expansion-contraction” balance may represent a key genomic strategy for environmental adaptation. Selection pressure analysis showed 403 gene pairs exhibited Ka/Ks < 1, under strong purifying selection to conserve core LRR domain functions, while 18 pairs showed Ka/Ks > 1, potentially driving functional divergence with positive selection (Table S2). This heterogeneous selection pattern strongly correlates with Rosaceae MLO family, may contributing to the prevalent molecular mechanism for adapting evolution in Rosaceae species [33].

FaLRRop26 and its alleles regulated basic synthesis and pathogen-induced deposition of lignin

As a critical physical barrier against pathogens, lignin enhances cell wall rigidity and hydrophobicity to restrict pathogen effector diffusion and nutrient acquisition [34, 35]. This study successfully achieved mutations in four FaLRRop alleles (FaLRRop20/26/37/43) in cultivated strawberry. We found that FaLRRop26 and its alleles mutations only significantly caused systemic dysregulation of lignin biosynthesis (inhibition the expression of 4-coumarate: CoA ligase, caffeic acid-O-methyltransferase, ferulate-5-hydroxylase, cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase encoding genes) and which might enhance C. siamense susceptibility of FaLRRop alleles mutation. The gene which located in lignin biosynthesis pathway or its regulator directly influenced plant resistance even in polyploid plant. This suppression is similar to the increased powdery mildew susceptibility in TmPAL/TmCOMT/TmFAH/TmCCoAMT/TmCAD-silenced diploid wheat (T. monococcum) but contradicts the enhanced Sclerotinia sclerotiorum resistance in BnF5H-knockout rape, reflecting the infection strategies of different pathogens and the priority of host defense [36, 37]. Cross-species comparisons further confirm the species-specific lignin defense. Cotton (Gossypium hirsutum) employed MYB transcription factor GhODO1 (combine the promoters of Gh4CL1 and GhCAD3) and GhWRKY1-like (bind the promoters and activate the expression of GhPAL6 and GhCOMT1) to regulate Verticillium dahliae resistance positively [38], while wheat activates lignin deposition via TaRac1-TaCCR module against leaf rust [39]. In this study, 87% lignin accumulation of wild-type strawberry post-infection aligns with conserved defense system, whereas mutants lost this capacity under sustained suppression of structural genes, proving the hub role of FaLRRop26 and its alleles for lignin defense. Notably, different from the sustained activation of PAL1 in wheat against fungal pathogens, the suppression of FaPAL in mutants may create a “metabolic bottleneck” blocking the entire phenylpropanoid pathway (Fig. 7B) [40]. Previous research has shown that disruption of lignin synthesis usually leads to a redistribution of metabolic flux [41]. PAL2 and 4CL2 has been reported to compensatorily upregulated in the ccr1 mutants of Arabidopsis [42]. Similarly, we observed the specific upregulation of FaCCR in uninfected mutants under the suppression of FaPAL, FaCAD, FaCOMT, and FaF5H, suggesting this compensatory regulation may be involved in maintaining normal lignin flux in the mutants (Fig. 7B, E). However, the upregulation of FaCCR failed to completely reverse the overall lignin deficit. This regulatory defect, combined with further suppression under pathogen stress, led to the collapse of defense. This dual defect explained high susceptivity of mutants to C. siamense, offering new target for strawberry resistance breeding.

Conclusion

In summary, this study conducted a genome-wide identification and characterization of the LRRop family in cultivated strawberries, elucidating their distribution and evolutionary relationships. Whole-genome duplication events drove the significant expansion of the FaLRRop gene family, leading to functional divergence during evolution. Furthermore, this study utilized CRISPR/Cas9 gene editing technology to create multi-gene knockout mutants of the key differentially expressed gene FaLRRop26 and its alleles. Pathogenicity tests revealed that the knockout of FaLRRop26 and its alleles reduced the resistance of cultivated strawberry to C. siamense, with significant suppression of lignin biosynthesis. These findings highlight the importance of the FaLRRop gene family in strawberry immunity and provide new insights into the molecular mechanisms underlying disease resistance in octoploid strawberry.

Supplementary Information

12870_2026_8210_MOESM1_ESM.jpg (1.8MB, jpg)

Supplementary Material 1: Fig. S1 The sequence logo maps of motifs contained in the FaLRRop family proteins.

12870_2026_8210_MOESM2_ESM.jpg (355KB, jpg)

Supplementary Material 2: Fig. S2 Electrophoresis detection images of Cas9 (left) and GFP (right) in the transgenic lines. The differential positive lines showed as Line 1 (L2), Line 2 (L2), Line 3 (L3) and Line 4 (L4). M: DL5000 DNA Maker; +: Positive control, using pKSE401G vector as the template; −: Negative control, using ddH2O as the template.

12870_2026_8210_MOESM3_ESM.jpg (3.3MB, jpg)

Supplementary Material 3: Fig. S3 PCR detection results of sgRNA1 (A) and sgRNA2 (B) targets in Line 1 (L1), Line 2 (L2), Line 3 (L3) and Line 4 (L4). The blue and green line segments represent the sgRNA sequence and PAM site, respectively. The red stubs and letters represent mutation sites.

12870_2026_8210_MOESM4_ESM.xlsx (69.4KB, xlsx)

Supplementary Material 4: Table S1 Physicochemical properties of FaLRRop proteins. Table S2 Ka, Ks, and Ka/Ks values of gene pairs of FaLRRop gene family. Table S3 Cis-acting element numbers of promoters of FaLRRop gene family. Table S4 Component of medium. Table S5 Transcriptome data information of gene-editing (ED) and wild-type (WT) strawberry. Table S6 Primer information.

Authors’ contributions

Xu Li, Lili Liu and Bo Shu: carried out the experiments and analyzed the data. Bo Shu: conceived the experiments and wrote the manuscript. Chun Luo and Zhe Chen: critically reviewed the manuscript.

Funding

This work was supported by Project of Guizhou Provincial Department of Science and Technology (No. Qian Ke he Ji Chu QN [2025] 226), Youth Fund of Guizhou Academy of Agricultural Sciences (No. Qian nong ke yuan qing nian ji jin [2026] 03) and Open Fund of Ministry of Agriculture and Rural Affairs/Key Laboratory of Tropical Fruit Tree Biology of Hainan Province (No. ITFT2024PT0204).

Data availability

The data that support the findings of this study are available from the corresponding author. Transcriptomic data were uploaded to the NCBI Sequence Read Archive as PRJNA1354640.

Declarations

Ethics approval and consent to participate

This study did not include human or animal subjects.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12870_2026_8210_MOESM1_ESM.jpg (1.8MB, jpg)

Supplementary Material 1: Fig. S1 The sequence logo maps of motifs contained in the FaLRRop family proteins.

12870_2026_8210_MOESM2_ESM.jpg (355KB, jpg)

Supplementary Material 2: Fig. S2 Electrophoresis detection images of Cas9 (left) and GFP (right) in the transgenic lines. The differential positive lines showed as Line 1 (L2), Line 2 (L2), Line 3 (L3) and Line 4 (L4). M: DL5000 DNA Maker; +: Positive control, using pKSE401G vector as the template; −: Negative control, using ddH2O as the template.

12870_2026_8210_MOESM3_ESM.jpg (3.3MB, jpg)

Supplementary Material 3: Fig. S3 PCR detection results of sgRNA1 (A) and sgRNA2 (B) targets in Line 1 (L1), Line 2 (L2), Line 3 (L3) and Line 4 (L4). The blue and green line segments represent the sgRNA sequence and PAM site, respectively. The red stubs and letters represent mutation sites.

12870_2026_8210_MOESM4_ESM.xlsx (69.4KB, xlsx)

Supplementary Material 4: Table S1 Physicochemical properties of FaLRRop proteins. Table S2 Ka, Ks, and Ka/Ks values of gene pairs of FaLRRop gene family. Table S3 Cis-acting element numbers of promoters of FaLRRop gene family. Table S4 Component of medium. Table S5 Transcriptome data information of gene-editing (ED) and wild-type (WT) strawberry. Table S6 Primer information.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author. Transcriptomic data were uploaded to the NCBI Sequence Read Archive as PRJNA1354640.

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