Brassica-Specific Orphan Gene CROG1 Confers Clubroot Resistance in Arabidopsis via Phenylpropanoid Pathway Activation

Abstract

Clubroot disease, caused by Plasmodiophora brassicae, poses a serious threat to global Brassica crop production. Orphan genes (OGs), which are species or lineage-specific and lack detectable homologs in other taxa, have been implicated in various biotic stress responses. Here, we identified a novel Brassica rapa-specific orphan gene, designated CROG1, that confers resistance to clubroot. Heterologous overexpression of CROG1 in Arabidopsis thaliana significantly enhanced resistance to P. brassicae. Transcriptomic profiling of CROG1-overexpressing lines highlighted the essential role of the phenylpropanoid biosynthesis pathway, showing upregulation of key lignin synthesis genes (including CCoAMT, CAD6, PER4, and AZI1) and defense-related regulators (RBOHC and WAKs). Weighted co-expression network analysis further corroborated the link between CROG1-mediated resistance and enhanced lignin deposition and cell wall reinforcement. Our findings establish CROG1 as a Brassica-specific orphan gene that enhances clubroot resistance via phenylpropanoid pathway activation. These results highlight the potential of orphan genes as novel genetic resources for breeding clubroot-resistant Brassica varieties, offering a sustainable strategy to mitigate yield losses caused by this devastating disease.

1. Introduction

Clubroot, a soil-borne disease caused by Plasmodiophora brassicae Woronin, is a significant threat to Brassica crop production [1]. The life cycle of this pathogen comprises three stages: (1) survival as dormant spores in the soil; (2) primary infection of root hairs and epidermal cells; and (3) secondary infection of the cortex [2]. This infection process induces abnormal cell proliferation and expansion in infected tissues, leading to gall formation. Plasmodiophora brassicae resting spores can persist in soil for over 15 years, and the frequent mixed occurrence of physiological races in field populations is a challenge for disease control [3]. This complexity has increased the demand for disease resistance gene identification. Clubroot resistance genes and quantitative trait loci have been mapped in Brassica species, originating from A-genome crops, such as turnip (Brassica rapa ssp. rapa) and Chinese cabbage (Brassica rapa ssp. pekinensis). To date, 28 resistance genes/loci have been identified in the A genome, concentrated on chromosomes A01, A02, A03, A05, A06, and A08 [4]. Two major clusters of CR genes are present on A03: one cluster harboring three CR loci (CRd, CRk, and Crr3), and the second cluster harboring seven CR loci (CRa, CRb, CRq, Rcr1, Rcr2, Rcr4, and Rcr5) [5]. Among these genes, CRa [6] and Rcr1 [7] have been successfully cloned.

A critical defense strategy against P. brassicae involves the fortification of the plant cell wall, which serves as a primary physical and chemical barrier against pathogens [8]. Dynamic cell wall remodeling, including lignification and structural modifications, constitutes a fundamental component of the plant immune response [9,10,11]. Notably, genomic analyses reveal that P. brassicae possesses a limited repertoire of cell wall-degrading enzymes [12], suggesting that observed alterations in host cell walls during infection are likely driven primarily by the plant’s own defense mechanisms rather than direct pathogen-mediated degradation. Central to this defense-associated cell wall reinforcement is the phenylpropanoid pathway, a major secondary metabolic route responsible for the biosynthesis of lignin, flavonoid, and phenolic acids [13]. Lignin deposition directly enhances cell wall integrity [14], while flavonoids and phenolic acids function as antimicrobials, antioxidants, and signaling molecules [15]. Accumulating evidence underscores the pivotal role of the phenylpropanoid pathway, particularly lignin biosynthesis, in clubroot resistance across Brassica species. For instance, Tu et al. [16] demonstrated that clubroot resistance conferred by the Rcr1 gene stems from its induction of lignin accumulation, supporting the contribution of basal defense activation and lignin synthesis via the phenylpropanoid pathway for clubroot resistance. Similarly, both flavonoid and lignin biosynthesis pathways are significantly induced in resistant plant material [17]. Irian et al. [18] demonstrated that infection by P. brassicae activates the expression of phenylpropanoid pathway-related genes in oilseed rape (Brassica napus). Disease-resistant lines exhibited upregulated expression of genes involved in lignin and flavonoid biosynthesis. Ergothioneine (EGT) enhances clubroot resistance by inducing the expression of lignin synthesis-related genes in Chinese cabbage [19]. The critical role of cell wall synthesis and phenylpropanoid-associated lignin synthesis has been corroborated by multiple omics studies. Transcriptome studies have consistently reported the altered expression of cell wall-related genes in Brassica crops infected by P. brassicae [20,21,22,23,24]. These findings have been further corroborated by metabolomic analyses across multiple plant species. The differential accumulation of key secondary metabolites, particularly flavonoids, lignans, and phenylpropanoid derivatives, has been documented in A. thaliana, B. napus, and B. rapa after pathogen infection [14,19,25,26]. Collectively, these findings establish the enhancement of the phenylpropanoid pathway—particularly lignin biosynthesis for cell wall reinforcement—as a central mechanism of clubroot resistance. However, the key regulatory genes orchestrating this defense-specific activation in Brassica species remain poorly characterized. This knowledge gap is particularly significant given the potential contribution of lineage-specific genes to host-pathogen coevolution and specialized defense responses.

Orphan genes have arisen through diverse pathways [27] and have been linked to environmental adaptation, growth, and development. These genes exhibit evolutionary lineage specificity [28]. Brassica rapa shows substantial phenotypic variation and diverse resistance responses to P. brassicae. The potential involvement of clubroot-resistant orphan genes in host adaptation to P. brassicae represents a compelling research direction. Observed correlations in abundance shifts suggest that orphan genes play a functional role in mediating the resistance responses of B. rapa to pathogen infection [29]. In our prior study, we conducted a functional screen of a Brassica orphan gene overexpression library to identify novel components of plant immunity [30]. Our systematic analysis revealed that transgenic Arabidopsis plants overexpressing BrOG51 had significantly enhanced resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) infection, as indicated by the reduction in bacterial proliferation and disease symptoms. These findings establish BrOG51 as a previously uncharacterized genetic determinant of pathogen resistance, suggesting that orphan genes are an untapped reservoir for plant defense mechanisms.

Given the well-documented role of phenylpropanoid-mediated cell wall reinforcement in clubroot resistance, coupled with emerging evidence that orphan genes (OGs) may encode novel regulators of plant defense pathways, we hypothesized that Brassica OGs could play significant roles in conferring resistance to P. brassicae. To test this hypothesis, the present study screened a panel of B. rapa orphan gene (BrOG) overexpression lines in Arabidopsis for altered responses to P. brassicae infection; identified and characterized candidate resistance-conferring OGs through phenotypic, molecular, and transcriptomic analyses; and elucidated the molecular mechanisms by which candidate OGs enhance clubroot resistance, with particular emphasis on their potential role in regulating the phenylpropanoid biosynthesis pathway and associated cell wall reinforcement processes. Through this approach, we identified BrOG51 (designated CROG1) as a novel Brassica-specific orphan gene involved in clubroot resistance and further investigated its functional mechanisms using comprehensive transcriptomic and molecular analyses.

2. Results

2.1. Characterization of Clubroot Resistance in BrOGs Overexpression Lines

We screened 20 transgenic A. thaliana lines from our pre-established BrOG overexpression library in order to determine the roles of orphan genes in plant disease resistance, and [30] for resistance to P. brassicae. The disease rate (DR) and disease index (DI) were determined at 3, 4, 5 and 6 weeks post-P. brassicae inoculation (wpi). Three transgenic lines—BrOG36OE, BrOG51OE, and BrOG103OE—exhibited significantly reduced susceptibility compared with wild-type (WT) plants (Figure S1). At 6 wpi, these lines maintained lower disease rates (77.78–91.57%), but all of the WT plants showed 100% disease rates (Figure 1). Further, their disease index values (59.70–80.60) were substantially lower than that of WT (96.4) (Figure 2). BrOG51OE displayed the strongest resistance phenotype, suggesting that its orphan gene plays a critical role in pathogen defense.

2.2. BrOG51OE Showed Significant Resistance to Both Pseudomonas syringae pv. Tomato DC3000 and P. brassicae Infection

Inoculation of the three transgenic lines (BrOG36OE, BrOG51OE, and BrOG103OE) with Pst DC3000 resulted in enhanced disease resistance compared with the WT plants. Leaves of these transgenic lines showed less chlorosis (Figure S2A) and milder disease symptoms (Figure S2B) at 3 days post-inoculation (dpi). As Pst DC3000 triggers the salicylic acid (SA) signaling pathway in A. thaliana, we quantified the transcript levels of the SA-responsive marker gene AtPR1. AtPR1 expression was significantly upregulated in all three transgenic lines (Figure S2C), especially BrOG51OE.

BrOG51OE exhibited significant resistance to clubroot disease caused by P. brassicae. Its resistance persisted even at 6 wpi, contrasting with WT plants (Figure 1 and Figure 2), which were susceptible at this stage (Figure 3A). Consistent with the phenotypic observations, BrOG51OE plants showed a lower P. brassicae DNA content than WT controls (Figure 3B), particularly at 5 and 6 wpi, indicating effective suppression of pathogen proliferation. These findings collectively support the hypothesis that the orphan gene BrOG51 contributes to clubroot resistance. Consequently, we designated this gene CROG1 (Clubroot-Related Orphan Gene 1) to reflect its status as the first reported orphan gene involved in clubroot resistance.

2.3. Analysis of CROG1 Expression in Brassica rapa After Inoculation with P. brassicae

CROG1 (BrOG51, BraA05004304), which was identified in the B. rapa reference genome v2.5 [30], is a Brassica-specific gene with no detectable homologs outside the Brassica genus. The gene spanned 737 bp, contained two exons and one intron, and encoded a predicted 49-amino-acid protein (Figure S3). Subcellular localization assays with a 35S:: CROG1::GFP fusion construct (versus 35S::GFP control) revealed the predominant localization of CROG1-GFP in the nucleus and cell membrane (Figure 4).

As CROG1 is a unique orphan gene in the Brassica A genome, we analyzed its expression dynamics in B. rapa after P. brassicae infection. In the susceptible B. rapa inbred line BJN3-2, mock-inoculated plants showed no symptoms, whereas inoculated plants exhibited 100% disease incidence with severe root galling at 30 dpi (Figure 5A). CROG1 transcript levels were measured at three time points after inoculation, revealing that P. brassicae infection significantly induced CROG1 expression compared with the mock-inoculated control (Figure 5B), establishing that CROG1 expression is responsive to P. brassicae inoculation.

2.4. Transcriptome Analysis of Arabidopsis thaliana CROG1 Transgenic Plants Inoculated with P. brassicae

To analyze the mechanism by which CROG1 regulates clubroot resistance, root samples were collected from WT and CROG1-overexpressing (CROG1OE) plants at 3, 4, and 5 wpi for cDNA library construction and transcriptome sequencing. The sequencing quality was high, with the Q20 and Q30 base percentages of all of the samples exceeding 96.51% and 91.46%, respectively (Table S1). Alignment to the TAIR10 reference genome (http://www.arabidopsis.org) (accessed on 10 September 2024) was effective, yielding uniquely mapped read rates above 97.08% for all of the samples. These results confirm the reliability of the data for subsequent analyses (Table S2).

Transcriptome profiling revealed 3731 differentially expressed genes (DEGs) between WT and CROG1OE plants, with 2244 upregulated and 1487 downregulated DEGs (Figure 6A, Table S3). Gene Ontology (GO) analysis demonstrated significant enrichment of DEGs in key biological processes, including “response to stimulus” (GO:0050896) and “secondary metabolic process” (GO:0019748), and cellular components, including “cell wall” (GO:0005618) and “cell periphery” (GO:0071944) (Figure 6B). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified three significantly enriched metabolic pathways: “phenylpropanoid biosynthesis” (ko00940), “starch and sucrose metabolism” (ko00500), and “plant hormone signal transduction” (ko04075) (Figure 6C). The phenylpropanoid biosynthesis pathway showed consistent enrichment across all three time point comparison groups. These findings suggest that CROG1-mediated clubroot resistance functions through the modulation of the phenylpropanoid biosynthesis pathway, having downstream effects on cell wall reinforcement and lignin deposition.

2.5. CROG1 Is Involved in Clubroot Resistance by Affecting the Phenylpropane Metabolic Pathway

To investigate the molecular mechanisms underlying clubroot resistance, we integrated the DR and DI with the transcriptome data using weighted correlation network analysis (WGCNA). WGCNA identified 18 co-expression modules, among which the brown module showed the strongest positive correlation with disease phenotypes (Figure 7A). Functional enrichment analysis of genes within this module revealed that the phenylalanine, tyrosine, and tryptophan biosynthesis pathway was significantly enriched (Figure S4). These findings indicate that CROG1 participates in clubroot resistance through its involvement in the phenylpropanoid metabolic pathway.

In the brown module, we identified genes involved in lignin synthesis and cell wall formation via the phenylpropanoid pathway, including CCoAMT and CAD6, which are established regulators of lignin biosynthesis [31]. Real-time quantitative polymerase chain reaction (RT-qPCR) validation confirmed that the expression patterns of these genes aligned with the transcriptome data, showing significant induction in CROG1OE plants compared with WT controls. In addition, CHI encodes a chitinase, a protein that plays important roles in plant defense against biotic and abiotic stressors. CHI expression differed significantly between CROG1OE and WT plants, suggesting that its expression may be regulated by CROG1.

Transcriptomic profiling revealed multiple DEGs functionally associated with plant defense responses, cell wall biosynthesis, and structural defense mechanisms. PAL is an entry enzyme for the phenylpropanoid pathway and is activated after sensing H₂O₂ accumulation. Respiratory burst oxidase homolog (RBOHC), a marker gene of the plant defense response level, was significantly upregulated in CROG1OE plants, indicating that CROG1 overexpression induces a more intense defense response in plants and may affect the phenylpropane metabolic pathway. PER4 has been reported to play a role in cell elongation and plant–pathogen interactions. We found that the PER4 transcript levels were affected by CROG1 overexpression. AZI1, which improves plant disease resistance by enhancing lignin synthesis [32], was affected by CROG1 overexpression and showed very high transcript levels at 5 wpi. In addition, cell wall-associated kinases showed a different trend in the CROG1 overexpression lines compared with WT (Figure 7B). These transcriptional changes implicate the role of CROG1 in modulating clubroot resistance through the regulation of the phenylpropanoid metabolic pathway and associated defense mechanisms.

3. Discussion

Orphan genes, which are uniquely present in plant genomes, remain understudied in current research and are largely confined to a limited number of species. Research has indicated their significant role not only in plant growth and development but in plant defense against biotic stress [33,34,35,36,37,38]. In cruciferous crops, the orphan gene AtQQS exemplifies this function and interacts with AtNF-YC4 to enhance antiviral and antimicrobial immunity. AtQQS overexpression in A. thaliana and soybean increases plant resistance to aphids, fungal pathogens, and other microbes [34]. Studies have also shown that the cruciferous-specific orphan gene EWR1 and its kale homolog BoEWR1 confer resistance to Verticillium wilt [39]. Similarly, Jiang et al. [35] demonstrated that 41 of 52 assessed orphan genes (BrOGs) in B. rapa responded transcriptionally to P. brassicae infection. Collectively, these findings establish a strong association between orphan genes and plant biotic stress responses.

Liu et al. [2] demonstrated that P. brassicae infection is initiated in the host cortex and that cell wall modifications occur in both susceptible and resistant plant materials. The phenylpropanoid pathway, a crucial secondary metabolic process responsible for lignin and flavonoid synthesis, plays a significant role in biotic stress responses. Studies on Brassica–P. brassicae interactions have implicated the phenylpropanoid pathway and its branches. As P. brassicae lacks genes for cell wall degradation or modification [15], host-induced cell wall alterations during infection are likely derived from immune responses activated by disease resistance genes, such as Rcr1 [24]. Transcriptomic analysis of Rcr1-mediated resistance revealed its potential influence on the jasmonic acid signaling pathway and the subsequent upregulation of phenylpropanoid pathway genes [20,40]. Tu et al. [16] observed enhanced lignification, particularly in the outer cortex of resistant plants, which impedes pathogen entry. This increase in lignification was attributed to the activation of genes involved in the phenylpropanoid pathway and the S-lignin unit biosynthesis branch. These findings collectively indicate that Rcr1 confers resistance, at least partially, by inducing lignin accumulation. BJN3-2 is susceptible to clubroot, whereas CROG1 remains upregulated following inoculation (Figure 5B). Moreover, overexpressing CROG1 in Arabidopsis reduced the disease rate and disease index (Figure 1 and Figure 2), yet disease development was not completely prevented. Therefore, we propose that CROG1 may contribute to resistance-related responses, but is not the only determining factor of resistance to clubroot. In our study, CROG1 overexpression in Arabidopsis altered the expression of numerous genes, including several involved in phenylpropanoid metabolism, which exhibited differential expression patterns compared with WT plants after inoculation. Cell wall-associated kinases WAK1 and WAK3 have both been associated with plant antimicrobial responses [41,42], and their expression was affected in our experiments (Figure 6B). Therefore, we hypothesize that CROG1 modulates clubroot resistance by influencing lignin or flavonoid biosynthesis via the phenylpropanoid pathway.

There are two theories that explain the origin of orphan genes. The first posits that they arose through rapid sequence evolution and lost detectable homology to other genes over evolutionary time [43]. The second theory suggests that de novo evolution occurred in non-coding genomic regions, offering a framework for understanding their functional emergence [44,45,46]. Compared with evolutionarily conserved genes, orphan genes exhibit accelerated evolutionary rates and high sequence specificity, resulting in strong species specificity. Plasmodiophora brassicae exhibits host specificity for cruciferous crops, and there are several characterized disease resistance loci in the B. rapa genome. Stressors induce genomic changes, including gene birth and death events [47]. It remains unknown whether selective pressure from P. brassicae infection drives the evolution of orphan genes in B. rapa. Supporting this hypothesis, our study showed that overexpression of the orphan gene CROG1 reduced clubroot resistance in A. thaliana (Figure 2). Furthermore, CROG1 expression was significantly induced by P. brassicae in susceptible Chinese cabbage (B. rapa) lines (Figure 3). Collectively, these findings suggest that the selective pressures imposed by P. brassicae during the evolution of B. rapa could have generated orphan genes associated with susceptibility or resistance mechanisms.

Here, we report CROG1 as an orphan gene associated with clubroot resistance. Transcriptome analysis suggests that CROG1 influences resistance, potentially by modulating lignin biosynthesis and/or cell wall integrity. However, this is a preliminary finding, and further molecular studies are needed to determine how CROG1 integrates into the complex signaling networks governing the host–P. brassicae interaction. Our results provide new avenues for enhancing disease resistance in cruciferous crops.

4. Materials and Methods

4.1. Plant Materials

A. thaliana ecotype Col-0 and 20 BrOG transgenic lines [30] were used to screen plant materials responding to Plasmodiophora brassicae. They were grown under long day conditions (16/8 h light/dark photoperiod) at 22 ± 1 °C with a cool-white fluorescent light and a relative humidity of 65–70%. Brassica rapa inbred line ‘BJN3-2’ is susceptible to clubroot disease [21]. It was inoculated with P. brassicae to determine the expression pattern of CROG1 in B. rapa.

4.2. Plasmodiophora brassicae Inoculation

The pathotype used for P. brassicae identification was ‘LAB3’, which was identified as Pb4 by the SCD system [48]. The roots were ground with water, filtered with gauze, and collected in a 50 mL centrifuge tube. The spore count of the filtered bacterial solution was determined using a hemocytometer. The spore concentration was adjusted to 10⁷ spores/mL. We selected 4-week-old A. thaliana plants and injected spore solution into their roots. Brassica rapa inbred line BJN3-2 was inoculated at the 2–3 true leaf stage. Each plant was injected with 2 mL of spore solution into the roots [26,49]. Each treatment was conducted with three biological replicates, each consisting of at least twelve individual plants.

4.3. Clubroot Disease Assessment

The disease rate (DR) and disease index (DI) were determined in A. thaliana at 3, 4, and 5 wpi with P. brassicae. Disease severity was divided into five levels: level 0, asymptomatic; level 1, small galls on lateral roots; level 2, larger galls on lateral roots; level 3, galls on the main root; and level 4, galls on both main and lateral roots. DR was calculated by dividing the number of non-0 level plants by the total number of inoculated plants. DI was calculated according to the following formula:

DI = [(n₁ + 2n₂ + 3n₃ + 4n₄)/N_T× 4] × 100,

where n_i is the number of plants in each disease severity class, and N_T is the total number of plants tested [50]. Total root DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method, and the DNA content of P. brassicae was quantified using specific primers, named Pb-F, Pb-R, Fbox-F, and Fbox-R [26], the sequences of which are shown in Table S4.

4.4. Inoculation with Pseudomonas syringae pv. Tomato DC3000

Four-week-old A. thaliana wild type and transgenic lines BrOG36OE, BrOG51OE, and BrOG103OE were hand-infiltrated with a Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) bacterial suspension (OD₆₀₀ = 0.0002 in 10 mM MgCl₂), and the bacterial load was quantified at 3 dpi. Ps. syringae pv. tomato DC3000 was cultured as previously described 50. There were three biological replicates.

4.5. RNA Extraction and RT-qPCR Analysis

Total RNA was isolated from the root of Col-0 and BrOGOE plants at 3, 4, and 5 wpi, and BJN3-2 at 10, 20, and 30 dpi using the TRNzol universal reagent (4992730; TIANGEN; Beijing, China), each with three biological replicates. Key genes were detected using RT-qPCR. cDNA was synthesized from total RNA using a PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time; RR047A; TaKaRa; China) according to the manufacturer’s instructions. Relative expression was calculated using the 2^−ΔΔCT method. Each assay was repeated three times. The primer sequences for RT-qPCR are listed in Table S4.

4.6. Subcellular Localization Assays

To determine the subcellular localization of CROG1, a fusion construct was generated by cloning the full-length coding sequence of CROG1 (BraA05004304) into the pCAMBIA1302-GFP vector downstream of the Cauliflower Mosaic Virus (CaMV) 35S constitutive promoter, creating a 35S::CROG1::GFP translational fusion. The empty pCAMBIA1302-GFP vector (35S::GFP) served as a control. Agrobacterium tumefaciens strain GV3101 harboring either the fusion construct or the control vector was individually infiltrated into the abaxial air spaces of young leaves of 4–5-week-old Nicotiana benthamiana plants using the transient agroinfiltration method. Infiltrated plants were maintained under standard growth conditions (22 °C, 16 h light/8 h dark cycle) for 48–72 h post-infiltration to allow optimal protein expression. They were examined across three biological replicates.

4.7. Library Construction and RNA-Seq

Library construction and sequencing were performed by Shanghai Personalbio Technology Co., Ltd. (Shanghai, China). Total RNA was isolated from root of Col-0 and CROG1OE plants at 3 wpi, 4 wpi, and 5 wpi after infection, each with three biological replicates. Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. First-strand cDNA was synthesized using random oligonucleotides and Super Script II. Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities, and the enzymes were removed. The library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). The sequencing library was then sequenced on the NovaSeq 6000 platform (Illumina, Shanghai, China).

4.8. RNA-Seq Data Analysis

After sequencing, the reads with low sequencing quality or sequencing connectors in the raw data were filtered. The Arabidopsis genome (TAIR10) was used as the reference genome (http://www.arabidopsis.org) (accessed on 10 September 2024). The filtered reads were mapped to the reference genome using HISAT2 (v2.1.0). For each transcription region, the fragment per kilobase of transcript per million mapped reads (FPKM) value was calculated to quantify its expression abundance and variations using RSEM software (v1.2.31). Then, the difference expression of genes was analyzed by DESeq (v1.38.3) with screened conditions as follows: expression difference multiple |log₂FoldChange| > 1. The DEGs were then subjected to KEGG enrichment analysis and GO enrichment analysis.

4.9. Weighted Gene Co-Expression Network Analysis (WGCNA)

WGCNA was performed to identify functionally coordinated gene modules associated with CROG1-mediated clubroot resistance, using high-quality transcriptome data from root tissues of Col-0 (WT) and CROG1OE Arabidopsis plants at 3, 4, and 5 wpi. Weighted gene co-expression networks were generated in R (version 3.4.2) utilizing the WGCNA package, employing its automated network construction function (blockwiseModules) with default parameters to identify gene modules. Key modules showing strong genotype-dependent enrichment (p < 10⁻⁵) were functionally annotated via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses.

4.10. Statistical Analysis

Data were analyzed with SPSS v19.0 software using Student’s t-test or one-way ANOVA followed by individual comparisons with Tukey’s test, significant p-value < 0.05.

5. Conclusions

Based on the functional screening of a Brassica rapa orphan gene (BrOG) overexpression library in Arabidopsis thaliana, this study identifies BrOG51 (designated CROG1) as a novel B. rapa-specific orphan gene conferring significant resistance to Plasmodiophora brassicae. Overexpression of CROG1 in Arabidopsis substantially reduced disease incidence and severity, suppressed pathogen proliferation, and was transcriptionally induced in susceptible B. rapa upon pathogen challenge. Transcriptome profiling and weighted gene co-expression network analysis (WGCNA) revealed that CROG1 orchestrates clubroot resistance primarily by modulating the phenylpropanoid biosynthesis pathway, leading to the upregulation of key lignin synthesis genes (CCoAMT, CAD6, PER4, AZI1) and defense regulators (RBOHC, WAKs), thereby enhancing cell wall fortification through lignin deposition and structural modifications. These findings provide the first evidence of a Brassica-specific orphan gene directly regulating clubroot resistance via phenylpropanoid-mediated defense mechanisms, offering a novel genetic target and molecular strategy for breeding resistant Brassica crops.