Plasmodiophora Brassicae Research Articles

Transcriptome Analysis of Chinese Cabbage Infected with Plasmodiophora Brassicae in the Primary Stage

Clubroot disease caused by the infection of Plasmodiophora brassicae is widespread in China, and significantly reduces the yield of Chinese cabbage (Brassica rapa L. ssp. pekinensis). However, the resistance mechanism of Chinese cabbage against clubroot disease is still unclear. It is important to exploit the key genes that response to early infection of P. brassicae. In this study, it was found that zoospores were firstly invaded hair roots on the 8th day after inoculating with 1 × 107 spores/mL P. brassicae. Transcriptome analysis found that the early interaction between Chinese cabbage and P. brassicae caused the significant expression change of some defense genes, such as NBS-LRRs and pathogenesis-related genes, etc. The above results were verified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Otherwise, peroxidase (POD) salicylic acid (SA) and jasmonic acid (JA) were also found to be important signal molecules in the resistance to clubroot disease in Chinese cabbage. This study provides important clues for understanding the resistance mechanism of Chinese cabbage against clubroot disease.

Integrated pest management farmer field school of Chinese cabbage for young farmers in Batu City

The Abinaya Milenial Youth Farmers Group in Sumber Brantas Village, Bumiaji District, Batu City, faces significant challenges in cultivating Chinese cabbage due to a high incidence of clubroot disease caused by Plasmodiophora brassicae, with infection rates reaching 50–80 percent, leading to crop failure. The overuse of synthetic pesticides has resulted in pathogen resistance. Therefore, we adopted an integrated pest management (IPM) approach focusing on long-term prevention by integrating ecologically-based control techniques. The principles of IPM include managing healthy plants, preserving natural enemies, monitoring, and enhancing farmers' ecological understanding to become IPM experts. Through the IPM Farmer Field School (IPM-FFS), we aimed to implement ecological IPM, improve agroecosystem health, and support sustainable agriculture through participatory extension methods. Activities were conducted from June to November 2023 in Sumber Brantas Village, Bumiaji District, Batu City. The activities included surveys, planning, socialization, teaching, training, and follow-up. The IPM-FFS successfully developed standard operating procedures for Chinese cabbage management, increasing farmers' knowledge of IPM by 85 percent. The adoption of IPM reduced the incidence of clubroot disease to 20 percent, increased production by 420 kg, and reduced production costs by IDR 403,000 on an area of 400 m2. Farmers independently produce compost, compost tea, botanical pesticides, mycorrhizae, and fungicides through the IPM-FFS, empowering them in sustainable agriculture.

Genetic Mapping and Characterization of the Clubroot Resistance Gene BraPb8.3 in Brassica rapa.

Clubroot, a significant soil-borne disease, severely impacts the productivity of cruciferous crops. The identification and development of clubroot resistance (CR) genes are crucial for mitigating this disease. This study investigated the genetic inheritance of clubroot resistance within an F2 progeny derived from the cross of a resistant parent, designated "377", and a susceptible parent, designated "12A". Notably, "377" exhibited robust resistance to the "KEL-23" strain of Plasmodiophora brassicae, the causative agent of clubroot. Genetic analyses suggested that the observed resistance is controlled by a single dominant gene. Through Bulked Segregant Analysis sequencing (BSA-seq) and preliminary gene mapping, we localized the CR gene locus, designated as BraPb8.3, to a 1.30 Mb genomic segment on chromosome A08, flanked by the markers "333" and "sau332-1". Further fine mapping precisely narrowed down the position of BraPb8.3 to a 173.8 kb region between the markers "srt8-65" and "srt8-25", where we identified 22 genes, including Bra020861 with a TIR-NBS-LRR domain and Bra020876 with an LRR domain. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses confirmed that both Bra020861 and Bra020876 exhibit increased expression levels in the resistant parent "377" following inoculation with P. brassicae, thereby underscoring their potential as key genes implicated in BraPb8.3-mediated clubroot resistance. This study not only identifies molecular markers associated with BraPb8.3 but also enriches the genetic resources available for breeding programs aimed at enhancing resistance to clubroot.

Comparative Metabolome and Transcriptome Analysis Reveals the Defense Mechanism of Chinese Cabbage (Brassica rapa L. ssp. pekinensis) against Plasmodiophora brassicae Infection.

Chinese cabbage (Brassica rapa L. ssp. pekinensis) ranks among the most cultivated and consumed vegetables in China. A major threat to its production is Plasmodiophora brassicae, which causes large root tumors, obstructing nutrient and water absorption and resulting in plant withering. This study used a widely targeted metabolome technique to identify resistance-related metabolites in resistant (DH40R) and susceptible (DH199S) Chinese cabbage varieties after inoculation with P. brassicae. This study analyzed disease-related metabolites during different periods, identifying 257 metabolites linked to resistance, enriched in the phenylpropanoid biosynthesis pathway, and 248 metabolites linked to susceptibility, enriched in the arachidonic acid metabolism pathway. Key metabolites and genes in the phenylpropanoid pathway were upregulated at 5 days post-inoculation (DPI), suggesting their role in disease resistance. In the arachidonic acid pathway, linoleic acid and gamma-linolenic acid were upregulated at 5 and 22 DPI in resistant plants, while arachidonic acid was upregulated at 22 DPI in susceptible plants, leading to the conclusion that arachidonic acid may be a response substance in susceptible plants after inoculation. Many genes enriched in these pathways were differentially expressed in DH40R and DH199S. The research provided insights into the defense mechanisms of Chinese cabbage against P. brassicae through combined metabolome and transcriptome analysis.

A Combined mRNA and microRNA Transcriptome Analysis of B. oleracea Response to Plasmodiophora brassicae Infection

Clubroot disease, caused by the pathogen Plasmodiophora brassicae, is a serious disease that poses a critical threat to cabbage production. However, the molecular mechanism of the microRNAs (miRNAs) involved in the cabbage’s response to P. brassicae infection remains to be elucidated. Here, the mRNA and miRNA expression profiles of cabbage in response to a P. brassicae infection were analyzed. In the transcriptome analysis, 2217 and 5552 differentially expressed genes (DEGs) were identified at 7d and 21d after inoculation, which were enriched in MAPK signaling, plant–pathogen interaction, plant hormone signal transduction, and phenylpropanoid biosynthesis pathways. BolC02g057640.2J, BolC09g006890.2J, BolC02g013230.2J, BolC06g006490.2J, BolC03g052660.2J, BolC07g052580.2J, and BolC04g044910.2J were predicted to be significantly involved in the defense response or plant–pathogen interaction through co-expression network analysis. Small RNA data analysis identified 164 miRNAs belonging to 51 families. miR1515, miR166, miR159, and miR9563 had the greatest number of members among the miRNA families. Integrated analysis revealed 23 miRNA–mRNA interactions related to a P. brassicae infection. The target genes of differentially expressed miRNAs (DEMs) revealed the NAC, ARF, TCP, and SPL transcription factor members that probably participate in the defense response. This study provided new insights into the miRNA-involved regulatory system during the process of disease infection with P. brassicae in cabbage.

Plasmodiophora brassicae effector PbPE23 induces necrotic responses in both host and non-host plants.

Plasmodiophora brassicae is an obligate biotroph that causes clubroot disease in cruciferous plants, including canola and Arabidopsis. In contrast to most known bacterial, oomycete and fungal pathogens that colonize at the host apoplastic space, the protist P. brassicae establishes an intracellular colonization within various types of root cells and secretes a plethora of effector proteins to distinct cellular compartments favourable for survival and growth of the pathogen during pathogenesis. Identification and functional characterization of P. brassicae effectors has been hampered by the limited understanding of this unique pathosystem. Here, we report a P. brassicae effector, PbPE23, containing a Ser/Thr kinase domain, that induces necrosis after heterologous expression by leaf infiltration in both host and non-host plants. While PbPE23 is an active kinase, the kinase activity itself is not required for triggering the necrosis in plants. PbPE23 shows a nucleocytoplasmic localization in Nicotiana benthamiana and its N-terminal 25TPdPAQKQ32 sequence, resembling the contiguous hydrophilic TPAP motif and Q-rich region in many Nep1-like proteins (NLPs) from plant-associated microbes, is required for the induction of necrosis. Further, transcript profiling of PbPE23 reveals its high expression at the transition stages from primary to secondary infection, suggesting its potential involvement in the development of clubroot disease.

Susceptibility of Oilseed Radish (Raphanus sativus subsp. oleiferus) Cultivars and Various Brassica Crops to Plasmodiophora brassicae.

Oilseed radish (OR; Raphanus sativus var. oleiferus) is grown as a cover crop and develops a unique taproot, absorbing nitrogen left by the previous crop. The aim of this project was to investigate the resistance of OR cultivars (cvs.) to Plasmodiophora brassicae, the causal agent of clubroot disease. Twelve market cvs. were compared with cvs. of clubroot-resistant (CR) winter oilseed rape (OSR; Brassica napus) and other selected species of the Brassicaceae family. The study was performed as a replicated bioassay in a growth chamber using a specially composed mixture of field soils holding the natural inoculum of P. brassicae. The results show that the OR cultivars were infected, which implies that OR multiplies the pathogen. The susceptibility of the OR cultivars was not significantly different from that of the CR OSR cultivars Alister and Archimedes, but it was significantly different from that of the OSR cv. Mendel. The disease severity index (DSI) for OR cultivars ranged from 2.3 to 9.3, and disease incidence was 3-17%. The best performance was shown by black radish (Raphanus sativus var. niger) with a DSI of 0.3. For sustainable brassica crop production, we suggest avoiding OR as a cover crop in crop rotations, including OSR or other brassica crops, since there is a risk of increasing inoculum in the soil.

Comparative Analysis of Transcriptomes Reveals Pathways and Verifies Candidate Genes for Clubroot Resistance in Brassica oleracea.

Clubroot, a soil-borne disease caused by Plasmodiophora brassicae, is one of the most destructive diseases of Brassica oleracea all over the world. However, the mechanism of clubroot resistance remains unclear. In this research, transcriptome sequencing was conducted on root samples from both resistant (R) and susceptible (S) B. oleracea plants infected by P. brassicae. Then the comparative analysis was carried out between the R and S samples at different time points during the infection stages to reveal clubroot resistance related pathways and candidate genes. Compared with 0 days after inoculation, a total of 4991 differential expressed genes were detected from the S pool, while only 2133 were found from the R pool. Gene function enrichment analysis found that the effector-triggered immunity played a major role in the R pool, while the pathogen-associated molecular pattern triggered immune response was stronger in the S pool. Simultaneously, candidate genes were identified through weighted gene co-expression network analysis, with Bol010786 (CNGC13) and Bol017921 (SD2-5) showing potential for conferring resistance to clubroot. The findings of this research provide valuable insights into the molecular mechanisms underlying clubroot resistance and present new avenues for further research aimed at enhancing the clubroot resistance of B. oleracea through breeding.

Effects of Streptomyces melanosporofaciens X216 on microbial diversity in oilseed rape soil.

Clubroot disease is a devastating soil borne disease caused by infection with Plasmodiophora brassicae, which primarily affects cruciferous plants. The microbial diversity of the soil is an essential indicator of its quality. This study measured the physicochemical properties of the soil to study the effect of its microbial diversity on the infection of oilseed rape with P. brassicae. High-throughput sequences of the soil bacteria and fungi in the inter-root soils of P. brassicae were analyzed under different treatment conditions. In the study, it was found that the efficiency of strain X216 in preventing and controlling the root disease of rapeseed was positively correlated with the amount of solution used to irrigate the root system. The results of the greenhouse and field trials showed that the efficiency of strain X216 against the root disease of rapeseed was 43.16% in the field and 62.14% in the greenhouse. Proteobacteria, Chloroflexi, Rozellomycota, and Basidiomycota are critical phylum in the development of clubroot disease. The application of biocontrol increased the relative abundance of Actinobacteria, Bacillus, Mesorhizobium, Mycobacterium, Streptomyces and Filobasidium, which affected the structure and abundance of microbial communities. A principal coordinate analysis showed that the microbial structure in the soil varied substantially in the bacterial community, and there was no significant difference in soil structure in the fungal community. The occurrence of clubroot disease affected the structure of inter-root microbial community composition in the soil, which resulted in a decrease in its community diversity. The application of the biocontrol bacterium X216 increased the soil microbial diversity. It effectively reduced the occurrence of P. brassicae, and this study provides a basis to study the microbial diversity in cruciferous crops.

Comparison of Root Transcriptomes against Clubroot Disease Pathogens in a Resistant Chinese Cabbage Cultivar (Brassica rapa cv. 'Akimeki').

Clubroot, caused by Plasmodiophora brassicae, is one of the diseases that causes major economic losses in cruciferous crops worldwide. Although prevention strategies, including soil pH adjustment and crop rotation, have been used, the disease's long persistence and devastating impact continuously remain in the soil. CR varieties were developed for clubroot-resistant (CR) Chinese cabbage, and 'Akimeki' is one of the clubroot disease-resistant cultivars. However, recent studies have reported susceptibility to several Korean pathotypes in Akimeki and the destruction of the resistance to P. brassicae in many Brassica species against CR varieties, requiring the understanding of more fine-tuned plant signaling by fungal pathogens. In this study, we focused on the early molecular responses of Akimeki during infection with two P. brassicae strains, Seosan (SS) and Hoengseong2 (HS2), using RNA sequencing (RNA-seq). Among a total of 2358 DEGs, 2037 DEGs were differentially expressed following SS and HS2 infection. Gene ontology (GO) showed that 1524 and 513 genes were up-regulated following SS and HS2 inoculations, respectively. Notably, the genes of defense response and jasmonic acid regulations were enriched in the SS inoculation condition, and the genes of water transport and light intensity response were enriched in the HS2 inoculation condition. Moreover, KEGG pathways revealed that the gene expression set were related to pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) mechanisms. The results will provide valuable information for developing CR cultivars in Brassica plants.

Pathotype Identification and Host Resistance Evaluation of Clubroot in Zhejiang Province, China.

Clubroot, caused by Plasmodiophora brassicae, is a globally destructive soil-borne disease affecting cruciferous plants. Here, the predominant pathotypes of P. brassicae in six cities within Zhejiang Province were identified using the Williams and European Clubroot Differential (ECD) systems. A phylogenetic analysis of P. brassicae isolates infecting cruciferous crops worldwide was conducted using MEGA, and their ITS2 secondary structures were predicted through the ITS2 database. Accessions of B. rapa, B. oleracea, B. juncea, and Eruca sativa Mill. were employed to assess clubroot resistance. The results revealed that the prevalent pathotypes in Zhejiang Province were pathotype 1, ECD20/31/12 and ECD24/16/30; pathotype 3, ECD20/15/4; pathotype 8, ECD16/0/0 and ECD24/0/0; and pathotype 2, ECD16/15/15. Isolates from distinct genera of Brassicaceae formed separate branches in the evolutionary tree. Moreover, isolates of Brassica crops from Zhejiang Province exhibited homology with those from other global regions, a finding corroborated by their ITS2 secondary structure. Approximately 80% and 95% of B. rapa and B. juncea crops displayed susceptible phenotypes for pathotype 8, ECD16/0/0, whereas approximately 60% of B. oleracea crops exhibited resistance. Furthermore, three Brassica crop accessions showed significant variation in resistance to the pathogen, both among morphological and geographical origin groups. This study contributes to understanding the distribution of diverse P. brassicae pathotypes in different regions of Zhejiang Province and facilitates the identification of Brassica crops with potential disease resistance suitable for cultivation in the province.

Optimizing Clubroot Management and the Role of Canola Cultivar Mixtures.

The sustainable cultivation of canola is under threat from clubroot disease (Plasmodiophora brassicae). The pathogen's resting spores can survive in the soil for extended periods, complicating disease management. Therefore, effective clubroot control requires a combination of tactics that provide multiple layers of protection. Management strategies have focused on pathogen avoidance and reducing disease levels in infested fields. The sanitation of machinery and field equipment remains the most effective method for preventing the pathogen's introduction into non-infested fields. For disease reduction, crop rotation, liming, chemical control, and host resistance are commonly employed, with the use of clubroot-resistant cultivars being the most effective to date. However, resistance breakdown has been observed within four years of the introduction of new cultivars, jeopardizing the long-term effectiveness of this approach. A promising yet underexplored strategy is the use of cultivar mixtures. This approach leverages mechanisms such as the dilution effect, the barrier effect, induced resistance, disruptive selection, and the compensatory effect to control the disease. Cultivar mixtures have the potential to reduce the impact of clubroot on canola production while preserving pathogen population structure, thereby minimizing the likelihood of resistance breakdown. Given its potential, further research into cultivar mixtures as a management strategy for clubroot disease is warranted.

Lignin accumulation in cell wall plays a role in clubroot resistance.

Clubroot, caused by Plasmodiophora brassicae, is a significant disease affecting brassica crops worldwide and poses a threat to canola (Brassica napus) production in western Canada. Management of this disease heavily relies on the use of resistant cultivars, but resistance erosion is a serious concern due to the highly diverse pathogen populations. Understanding resistance mechanisms may aid in better deployment/rotation of clubroot resistance (CR) genes and improve resistance resilience. In this study, we conducted a comparative analysis using resistant canola varieties carrying either a single (Rcr1) or double CR genes (Rcr1+Crr1rutb ) to decipher the resistance modes associated with these genes. Cell wall (CW) biopolymeric compounds in different root layers were mapped and quantified using Fourier-transform mid-infrared microspectroscopy for changes in CW elements associated with clubroot resistance. Transmission electron and confocal microscopy were used to assess root infection details and relative transcript abundance was analyzed to determine the activation of the lignin-related pathway in relation to resistance. Neither resistant variety affected the primary infection of root hairs/epidermal cells compared to the susceptible "Westar", but both exhibited strong inhibition of cortical infection, effectively 'trapping' the pathogen in the exodermis. The most prominent change observed was increased lignin accumulation associated with resistance. In Westar, the pathogen was able to degrade CW lignin, facilitating access to the root cortex by secondary plasmodia of P. brassicae. In contrast, resistant varieties showed clear lignin accumulation around the penetration site on the exodermis, accompanied by elevated expression of genes involved in the phenylpropanoid pathway. These results suggest that induced lignin accumulation plays a role in clubroot resistance mediated by the CR genes Rcr1 and Crr1rutb in canola, providing cellular and structural evidence that supports the data from earlier transcriptomic studies.

Biological Control of Rapeseed Clubroot (Plasmodiophora brassicae) Using the Endophytic Fungus Didymella macrostoma P2.

Didymella macrostoma P2 was isolated from rapeseed (Brassica napus), and it is an endophyte of rapeseed and an antagonist of three rapeseed pathogens, Botrytis cinerea, Leptosphaeria biglobosa, and Sclerotinia sclerotiorum. However, whether P2 has a suppressive effect on infection of rapeseed by the clubroot pathogen Plasmodiophora brassicae remains unknown. This study was conducted to detect production of antimicrobials by P2 and to determine the efficacy of the antimicrobials and P2 pycnidiospores in suppression of rapeseed clubroot. The results showed that cultural filtrates (CFs) of P2 in potato dextrose broth and the substances in pycnidiospore mucilages exuded from P2 pycnidia were inhibitory to P. brassicae. In the indoor experiment, seeds of the susceptible rapeseed cultivar Zhongshuang No. 9 treated with P2 CF and the P2 pycnidiospore suspension (P2 SS, 1 × 107 spores/ml) reduced clubroot severity by 31 to 70% on the 30-day-old seedlings compared with the control (seeds treated with water). P2 was reisolated from the roots of the seedlings in the treatment of P2 SS; the average isolation frequency in the healthy roots (26%) was much higher than that (5%) in the diseased roots. In the field experiment, seeds of another susceptible rapeseed cultivar, Huayouza 50 (HYZ50), treated with P2 CF, P2 CE (chloroform extract of P2 CF, 30 μg/ml), and P2 SS reduced clubroot severity by 29 to 48% on 60-day-old seedlings and by 28 to 59% on adult plants (220 days old) compared with the control treatment. The three P2 treatments on HYZ50 produced significantly (P < 0.05) higher seed yield than the control treatment on this rapeseed cultivar, and they even generated seed yield similar to that produced by the resistant rapeseed cultivar Shengguang 165R in one of the two seasons. These results suggest that D. macrostoma P2 is an effective biocontrol agent against rapeseed clubroot.

The lambs of sheepfoot lane: Oldham Athletic AFC 1960-2023; football, politics and identity.

The article focuses on an overview of the history, politics and cultural identity of Oldham Athletic AFC between 1960 and 2023. During this period the club, a founding member of the Football League, played across six divisions of the game's pyramid. My hometown club, the Latics faced an existential threat to its survival following relegation to the National League in 2022, before being rescued and brought back into local ownership on the long road back to its recapturing its former status. The successes of the Frizzell and Royle eras (1969-1994) contrast with the decline of the club in the 1960s, and its gradual meltdown since the mid-1990s. These are interwoven with the economic, geopolitical and cultural dynamics of the game. The study locates the history of a small club geographically positioned within the vortex of the of the two Manchester giants and Liverpool, in English football's traditional hinterland of the industrial north west. The article has four main themes. Firstly, it examines Oldham Athletic as custodian of local identity, fusing folkloric rituals within the club's historic and contemporary nexus. Secondly, it contextualises the interface between global, glocal and grobal forces impacting on tradition, modernity and post- modernity in the club's lexicon. It then considers the themes of ownership, commodification and globalisation, debating whether Oldham Athletic stands as a bastion of anti- globalisation, or reconfirms existing global patterns at the micro level, incorporating salient theoretical perspectives in this analysis. Finally, the review evaluates whether the political and social class cleavages of the club's roots in Oldham's late Victorian industrial landscape still resonate, or if they have been marginalised by socio- economic changes since 1960.

Exploring the influence of rapeseed cultivar and pathogen isolate on Acremonium alternatum's efficacy in clubroot disease control

Clubroot disease, caused by Plasmodiophora brassicae, ranks among the most significant diseases affecting rapeseed cultivars, leading to substantial annual yield losses. Current control methods are limited to a small selection of chemical or biological treatments. Using biocontrol organisms presents a promising strategy for reducing disease severity and promoting plant vigour. However, their efficacy is strongly dependent on biotic and abiotic factors during the growing season, as well as the specific application conditions. In the present study, we evaluated the efficacy of the biocontrol fungus Acremonium alternatum in reducing clubroot disease symptoms across different susceptible and resistant rapeseed cultivars (Brassica napus) under various experimental greenhouse settings employing different types of P. brassicae inoculum: a uniform single spore isolate e3 and two German field isolates P1 and P1 ( +). We found that A. alternatum can reduce clubroot disease symptoms in susceptible rapeseed cultivars Visby, Ability and Jenifer, but not cv. Jumbo, when inoculated with the aggressive single spore isolate P. brassicae e3 at moderate (106 spores mL−1) and high (107 spores mL−1) densities. A. alternatum enhanced plant vitality and shoot biomass in cv. Visby inoculated with field isolates P1 or P1 ( +) but did not considerably reduce clubroot severity there. The clubroot-resistant cv. Mentor displayed a reduction in clubroot symptoms after A. alternatum treatment. In conclusion, A. alternatum holds some promise in managing moderate P. brassicae levels in the soil and could serve as an option in integrated pest management of clubroot disease when combined with resistant cultivars.

Clubroot-Induced Changes in the Root and Rhizosphere Microbiome of Susceptible and Resistant Canola.

Clubroot is a soilborne disease of canola (Brassica napus) and other crucifers caused by the obligate parasite Plasmodiophora brassicae. In western Canada, clubroot is usually managed by planting-resistant cultivars, but the emergence of resistance-breaking pathotypes of P. brassicae represents a major threat to sustainable canola production. The rhizosphere and root contain beneficial microorganisms that can improve plant health. In this study, we evaluated the effect of two P. brassicae isolates (termed A and B) with different levels of virulence on the root and rhizosphere microbiomes of clubroot-resistant and clubroot-susceptible canola. Additionally, potential biocontrol microorganisms were identified based on taxa antagonistic to clubroot. Although both P. brassicae isolates were classified as pathotype 3A, isolate A caused a higher disease severity index in the resistant canola genotype compared with isolate B. Metabarcoding analysis indicated a shift in the bacterial and fungal communities in response to inoculation with either field isolate. Root endophytic bacterial and fungal communities responded to changes in inoculation, isolate type, sampling time, and canola genotype. In contrast, fungal communities associated with the rhizosphere exhibited significant differences between sampling times, while bacterial communities associated with the rhizosphere exhibited low variability.

Bioinformatics and functional analysis of EDS1 genes in Brassica napus in response to Plasmodiophora brassicae infection

Enhanced Disease Susceptibility 1 (EDS1) is a key regulator of plant-pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) responses. In the Brassica napus genome, we identified six novel EDS1 genes, among which four were responsive to clubroot infection, a major rapeseed disease resistant to chemical control. Developing resistant cultivars is a potent and economically viable strategy to control clubroot infection. Bioinformatics analysis revealed conserved domains and structural uniformity in Bna-EDS1 homologs. Bna-EDS1 promoters harbored elements associated with diverse phytohormones and stress responses, highlighting their crucial roles in plant defense. A functional analysis was performed with Bna-EDS1 overexpression and RNAi transgenic lines. Bna-EDS1 overexpression boosted resistance to clubroot and upregulated defense-associated genes (PR1, PR2, ICS1, and CBP60), while Bna-EDS1 RNAi increased plant susceptibility, indicating suppression of the defense signaling pathway downstream of NBS-LRRs. RNA-Seq analysis identified key transcripts associated with clubroot resistance, including phenylpropanoid biosynthesis. Activation of SA regulator NPR1, defense signaling markers PR1 and PR2, and upregulation of MYC-TFs suggested that EDS1-mediated clubroot resistance potentially involves the SA pathway. Our findings underscore the pivotal role of Bna-EDS1-dependent mechanisms in resistance of B. napus to clubroot disease, and provide valuable insights for fortifying resistance against Plasmodiophora brassicae infection in rapeseed.

Pyramiding of triple Clubroot resistance loci conferred superior resistance without negative effects on agronomic traits in Brassica napus.

Clubroot disease caused by Plasmodiophora brassicae is becoming a serious threat to rapeseed (Brassica napus) production worldwide. Breeding resistant varieties using CR (clubroot resistance) loci is the most promising solution. Using marker-assisted selection and speed-breeding technologies, we generated Brassica napus materials in homozygous or heterozygous states using CRA3.7, CRA08.1, and CRA3.2 loci in the elite parental line of the Zhongshuang11 background. We developed three elite lines with two CR loci in different combinations and one line with three CR loci at the homozygous state. In our study, we used six different clubroot strains (Xinmin, Lincang, Yuxi, Chengdu, Chongqing, and Jixi) which are categorized into three groups based on our screening results. The newly pyramided lines with two or more CR loci displayed better disease resistance than the parental lines carrying single CR loci. There is an obvious gene dosage effect between CR loci and disease resistance levels. For example, pyramided lines with triple CR loci in the homozygous state showed superior resistance for all pathogens tested. Moreover, CR loci in the homozygous state are better on disease resistance than the heterozygous state. More importantly, no negative effect was observed on agronomic traits for the presence of multiple CR loci in the same background. Overall, these data suggest that the pyramiding of triple clubroot resistance loci conferred superior resistance with no negative effects on agronomic traits in Brassica napus.

Identification and Characterization of High-Molecular-Weight Proteins Secreted by Plasmodiophora brassicae That Suppress Plant Immunity.

Plasmodiophora brassicae is an obligate intracellular parasitic protist that causes clubroot disease on cruciferous plants. So far, some low-molecular-weight secreted proteins from P. brassicae have been reported to play an important role in plant immunity regulation, but there are few reports on its high-molecular-weight secreted proteins. In this study, 35 putative high-molecular-weight secreted proteins (>300 amino acids) of P. brassicae (PbHMWSP) genes that are highly expressed during the infection stage were identified using transcriptome analysis and bioinformatics prediction. Then, the secretory activity of 30 putative PbHMWSPs was confirmed using the yeast signal sequence trap system. Furthermore, the genes encoding 24 PbHMWSPs were successfully cloned and their functions in plant immunity were studied. The results showed that ten PbHMWSPs could inhibit flg22-induced reactive oxygen burst, and ten PbHMWSPs significantly inhibited the expression of the SA signaling pathway marker gene PR1a. In addition, nine PbHMWSPs could inhibit the expression of a marker gene of the JA signaling pathway. Therefore, a total of 19 of the 24 tested PbHMWSPs played roles in suppressing the immune response of plants. Of these, it is worth noting that PbHMWSP34 can inhibit the expression of JA, ET, and several SA signaling pathway marker genes. The present study is the first to report the function of the high-molecular-weight secreted proteins of P. brassicae in plant immunity, which will enrich the theory of interaction mechanisms between the pathogens and plants.