Plasmodiophora Brassicae Research Articles

Genome-Wide Identification and Characterization of the Trehalose-6-Phosphate Synthetase Gene Family in Chinese Cabbage (Brassica rapa) and Plasmodiophora brassicae during Their Interaction.

Trehalose is a nonreducing disaccharide that is widely distributed in various organisms. Trehalose-6-phosphate synthase (TPS) is a critical enzyme responsible for the biosynthesis of trehalose, which serves important functions in growth and development, defense, and stress resistance. Although previous studies have found that the clubroot pathogen Plasmodiophora brassicae can lead to the accumulation of trehalose in infected Arabidopsis organs, it has been proposed that much of the accumulated trehalose is derived from the pathogen. At present, there is very little evidence to verify this view. In this study, a comprehensive analysis of the TPS gene family was conducted in Brassica rapa and Plasmodiophora brassicae. A total of 14 Brassica rapa TPS genes (BrTPSs) and 3 P. brassicae TPS genes (PbTPSs) were identified, and the evolutionary characteristics, functional classification, and expression patterns were analyzed. Fourteen BrTPS genes were classified into two distinct classes according to phylogeny and gene structure. Three PbTPSs showed no significant differences in gene structure and protein conserved motifs. However, evolutionary analysis showed that the PbTPS2 gene failed to cluster with PbTPS1 and PbTPS3. Furthermore, cis-acting elements related to growth and development, defense and stress responsiveness, and hormone responsiveness were predicted in the promoter region of the BrTPS genes. Expression analysis of most BrTPS genes at five stages after P. brassicae interaction found no significant induction. Instead, the expression of the PbTPS genes of P. brassicae was upregulated, which was consistent with the period of trehalose accumulation. This study deepens our understanding of the function and evolution of BrTPSs and PbTPSs. Simultaneously, clarifying the biosynthesis of trehalose in the interaction between Brassica rapa and P. brassicae is also of great significance.

Advances in Biological Control and Resistance Genes of Brassicaceae Clubroot Disease-The Study Case of China

Clubroot disease is a soil-borne disease caused by Plasmodiophora brassicae. It occurs in cruciferous crops exclusively, and causes serious damage to the economic value of cruciferous crops worldwide. Although different measures have been taken to prevent the spread of clubroot disease, the most fundamental and effective way is to explore and use disease-resistance genes to breed resistant varieties. However, the resistance level of plant hosts is influenced both by environment and pathogen race. In this work, we described clubroot disease in terms of discovery and current distribution, life cycle, and race identification systems; in particular, we summarized recent progress on clubroot control methods and breeding practices for resistant cultivars. With the knowledge of these identified resistance loci and R genes, we discussed feasible strategies for disease-resistance breeding in the future.

Evaluation of cabbage varieties for resistance to clubroot pathogens, Plasmodiophora brassicae woronin in Dhankuta, Nepal

A field experiment was conducted to evaluate the varietal resistance of cabbage against club root disease, Plasmodiophora brassicae Woronin, in a naturally infested field in Sindhuwa, Dhankuta. It was conducted in a randomized complete block design (RCBD) of three replications and seven treatments, viz., T1: Green Coronet, T2: Nepa Green 777, T3: Big Sun 171, T4: Yr Honam, T5: Big Sun 111, T6: Super Green, and T7: Nepa Star. The plant height was comparatively greater in Green Coronet and Super Green in 60 DAT. In contrast, there was no significant difference in the number of leaves. Big Sun 111 and Yr Honam did not show any signs of galling or clubroot, indicating a significantly higher resistance. Despite not being afflicted by clubroot, Nepa star produced less than satisfying results due to black rot and early head decay. The maximum average head weight and marketable cabbage yield were recorded as being highest in Big Sun 111 (2.56 kg) and Yr Honam (2.24 kg). Furthermore, the yield from Big Sun 171 (1.93 kg) and Nepa Green 777 (1.84 kg) was satisfactory, despite the higher PDI (percentage disease index) (40.33%) and (61.04%), respectively. Disease incidence was zero in Big Sun 111, Yr Honam, and Nepa Star. In terms of productivity and disease resistance, Big Sun 111 (160.13 Mt/ha) and Yr Honam (140.51 Mt/ha) provide the best results, which may be beneficial to farmers who face costly and lengthy disease control practices.

Identification of Sources of Resistance to Clubroot (Plasmodiophora brassicae) in a Collection of Brassica napus Plant Introductions

Clubroot is a soilborne disease caused by Plasmodiophora brassicae that is emerging as a production problem in North Dakota, which contributes approximately 90% of the total U.S. canola production. P. brassicae’s resiliency in the soil and its ability to overcome the genetic resistance available in commercial hybrids make this a significant threat to canola production in the state and highlight the need to identify additional sources of resistance. To this effect, 115 Brassica napus plant introduction accessions were evaluated for their reaction to clubroot in field trials conducted between 2019 and 2021 in a naturally infested field. Seven accessions with high levels of clubroot resistance were identified. These resistant materials may contribute to widening the genetic resistance base of modern canola cultivars.

The root meristem growth factor BrRGF6 positively regulates Chinese cabbage to infection of clubroot disease caused by Plasmodiophora Brassicae.

Chinese cabbage has a high annual demand in China. However, clubroot disease caused by the infection of Plasmodiophora brassicae seriously affects its yield. Transcriptome analysis identified a root meristem growth factor 6 (BrRGF6) as significantly up-regulated in Chinese cabbage roots infected with Plasmodiophora brassicae. Quantitative reverse-transcription polymerase chain reaction and in situ hybridization analysis showed higher BrRGF6 expression in susceptible materials than in resistant materials. After Plasmodiophora brassicae infection, BrRGF6 expression was significantly up-regulated, especially in susceptible materials. Gene function analysis showed that the roots of Arabidopsis mutant rgf6 grew faster than the wild-type, and delayed the infection progress of Plasmodiophora brassicae. A Protein, nuclear transcription factor Y subunit C (BrNF-YC), was screened from yeast two-hybrid library of Chinese cabbage induced by Plasmodiophora brassicae, and verified to interact with BrRGF6 by yeast two-hybrid co-transfer. Yeast one-hybrid and β-Glucuronidase activity analysis showed that BrNF-YC could directly bind to and strongly activate the promoter of BrRGF6. Transgenic verification showed that BrRGF6 or BrNF-YC silenced Chinese cabbage significantly decreased the expression of BrRGF6, accelerated root development, and reduced incidence of clubroot disease. However, after overexpression of BrRGF6 or BrNF-YC, the phenotype showed a reverse trend. Therefore, BrRGF6 silencing accelerated root growth and enhanced resistance to clubroot disease, which was regulated by BrNF-YC.

Exploring a rhizobium to fix nitrogen in non-leguminous plants by using a tumor-formation root pathogen

Over 110 million tons of nitrogen fertilizer every year is used for crop production. Scientists have dreamed of enabling rhizobial nitrogen fixation in non-leguminous crops to mitigate the increasing demand for nitrogen fertilizer. However, despite decades of research, rhizobial nitrogen fixation in non-host plants has not been demonstrated. Here, we reported that an N-fixing rhizobium and a clubroot pathogen Plasmodiophora brassicae exhibited a synergistic effect on fixing nitrogen in cruciferous plants. Rhizobia were found to invade P. brassicae-infected rapeseed (Brassica napus) roots in the field. The colonization of rhizobium on rapeseed roots was confirmed by co-inoculating Mesorhizobium huakuii with P. brassicae under controlled laboratory conditions. M. huakuii infection could alleviate clubroot symptoms and promote the growth of diseased rapeseeds. M. huakuii could fix nitrogen in P. brassicae-infected plants based on the results of 15N isotope dilution tests. The expression of homologs of legume genes required for symbiosis and early-nodulin genes was significantly upregulated in Arabidopsis during early infection by P. brassicae. More importantly, M. huakuii could even fix nitrogen in P. brassicae-resistant rapeseed cultivar and promote plant growth when co-inoculated with P. brassicae. Our findings provide a new avenue to understand the interaction of rhizobia with non-host plants, stimulate the exploration of fixing nitrogen in non-leguminous plants by nitrogen-fixing rhizobia, and develop a strategy for both disease control and nitrogen fixation on non-host crops.

Marker-Assisted Pyramiding of CRa and CRd Genes to Improve the Clubroot Resistance of Brassica rapa.

Clubroot, caused by Plasmodiophora brassicae, is an economically important soil-borne disease that threatens Brassicaceae crops worldwide. In recent years, the incidence area of Chinese cabbage (Brassica rapa ssp. pekinensis) clubroot disease has increased, which severely affects the yield and quality of Chinese cabbage. The resistance of varieties harboring the single clubroot-resistance (CR) gene is easily broken through by P. brassicae pathotypes. CRa and CRd, genetically identified in B. rapa, are CR genes known to be highly resistant to different P. brassicaea pathotypes. In our study, we perform the gene pyramiding of CRa and CRd in Chinese cabbages through marker-assisted selection (MAS), and develop homozygous pyramided lines. The newly generated pyramided lines exhibit greater resistance to six different pathotypes than that of two parental lines carrying a single CR gene. This study provides new CR-gene-pyramided lines for the development of clubroot-resistant Brassica varieties for future breeding programs.

High-throughput sequencing reveals tuber mustard genes responsive to Plasmodiophora brassicae in the early stage of infection

Tuber mustard is a kind of Brassica crop, swollen stem of this crop is the material of mustard pickle, which has high economic value. Clubroot is a serious disease of brassicaceae plants, causing huge losses every year. Although some clubroot resistant cultivars of tuber mustard were bred, the mechanism by which tuber mustard responds to the pathogen Plasmodiophora brassicae needs to be elucidated. Morphologic and cytological changes of tuber mustard roots were observed during the pathogenetic process, and 72 h after inoculation was found to be the key stage of the interaction between the root of tuber mustard and the pathogen. A transcriptome sequencing was performed using the roots inoculated and mock-inoculated by P. brassicae at 72 h. A total of 2294 up-regulated and 6533 down-regulated genes were identified, and the expression levels of 11 differentially expressed genes were verified by qPCR. Genes related to MAPK signaling pathway-plant, plant hormone signal transduction, phenylpropanoid biosynthesis, nitrogen metabolism, ABC transporters, plant-pathogen interaction were involved in tuber mustard response to infection by P. brassicae. After the pathogen inoculation, among the differentially expressed genes, most of the PR1 like genes were upregulated, while most of BAK1 like genes, MAPK like genes, SUMM2 like genes, JAZ like genes, and NPR1 like genes were downregulated. The miRNA sequencing results showed that miR160a-5p, miR319a, miR395i, miR400 and their relative targets were negatively co-expressed, which might be involved in the regulation of tuber mustard response to the pathogen. This study is helpful for further studying the functions of tuber mustard genes during P. brassicae infection.

Metabonomic profiling of clubroot-susceptible and clubroot-resistant radish and the assessment of disease-resistant metabolites.

Plasmodiophora brassicae causes a serious threat to cruciferous plants including radish (Raphanus sativus L.). Knowledge on the pathogenic regularity and molecular mechanism of P. brassicae and radish is limited, especially on the metabolism level. In the present study, clubroot-susceptible and clubroot-resistant cultivars were inoculated with P. brassicae Race 4, root hairs initial infection of resting spores (107 CFU/mL) at 24 h post-inoculation and root galls symptom arising at cortex splitting stage were identified on both cultivars. Root samples of cortex splitting stage of two cultivars were collected and used for untargeted metabonomic analysis. We demonstrated changes in metabolite regulation and pathways during the cortex splitting stage of diseased roots between clubroot-susceptible and clubroot-resistant cultivars using untargeted metabonomic analysis. We identified a larger number of differentially regulated metabolites and heavier metabolite profile changes in the susceptible cultivar than in the resistant counterpart. The metabolites that were differentially regulated in both cultivars were mostly lipids and lipid-like molecules. Significantly regulated metabolites and pathways according to the P value and variable important in projection score were identified. Moreover, four compounds, including ethyl α-D-thioglucopyranoside, imipenem, ginsenoside Rg1, and 6-gingerol, were selected, and their anti-P. brassicae ability and effects on seedling growth were verified on the susceptible cultivar. Except for ethyl α-D-thioglucopyranoside, the remaining could inhibit clubroot development of varing degree. The use of 5 mg/L ginsenoside Rg1 + 5 mg/L 6-gingerol resulted in the lowest disease incidence and disease index among all treatments and enhanced seedling growth. The regulation of pathways or metabolites of carbapenem and ginsenoside was further explored. The results provide a preliminary understanding of the interaction between radish and P. brassicae at the metabolism level, as well as the development of measures for preventing clubroot.

Application of the NanoString nCounter System as an Alternative Method to Investigate Molecular Mechanisms Involved in Host Plant Responses to Plasmodiophora brassicae.

Clubroot, caused by the soilborne pathogen Plasmodiophora brassicae, is an important disease of canola (Brassica napus) and other crucifers. The recent application of RNA sequencing (RNA-seq) technologies to study P. brassicae−host interactions has generated large amounts of gene expression data, improving knowledge of the molecular mechanisms of pathogenesis and host resistance. Quantitative PCR (qPCR) analysis has been widely applied to examine the expression of a limited number of genes and to validate the results of RNA-seq studies, but may not be ideal for analyzing larger suites of target genes or increased sample numbers. Moreover, the need for intermediate steps such as cDNA synthesis may introduce variability that could affect the accuracy of the data generated by qPCR. Here, we report the validation of gene expression data from a previous RNA-seq study of clubroot using the NanoString nCounter System, which achieves efficient gene expression quantification in a fast and simple manner. We first confirm the robustness of the NanoString system by comparing the results with those generated by qPCR and RNA-seq and then discuss the importance of some candidate genes for resistance or susceptibility to P. brassicae in the host. The results show that the expression of genes measured using NanoString have a high correlation with the values obtained using the other two technologies, with R > 0.90 and p < 0.01, and the same expression patterns for most genes. The three methods (qPCR, RNA-seq, and NanoString) were also compared in terms of laboratory procedures, time, and cost. We propose that the NanoString nCounter System is a robust, sensitive, highly reproducible, and simple technology for gene expression analysis. NanoString could become a common alternative to qPCR to validate RNA-seq data or to create panels of genes for use as markers of resistance/susceptibility when plants are challenged with different P. brassicae pathotypes.

Comparing the infection biology and gene expression differences of Plasmodiophora brassicae primary and secondary zoospores.

Plasmodiophora brassicae (Wor.) is an obligate plant pathogen affecting Brassicae worldwide. To date, there is very little information available on the biology and molecular basis of P. brassicae primary and secondary zoospore infections. To examine their roles, we used microscope to systematically investigate the infection differences of P. brassicae between samples inoculated separately with resting spores and secondary zoospores. The obvious development of P. brassicae asynchrony that is characterized by secondary plasmodium, resting sporangial plasmodium, and resting spores was observed at 12 days in Brassica rapa inoculated with resting spores but not when inoculated with secondary zoospores at the same time. Inoculation with resting spores resulted in much more development of zoosporangia clusters than inoculation with secondary zoospores in non-host Spinacia oleracea. The results indicated that primary zoospore infection played an important role in the subsequent development. To improve our understanding of the infection mechanisms, RNA-seq analysis was performed. Among 18 effectors identified in P. brassicae, 13 effectors were induced in B. rapa seedlings inoculated with resting spores, which suggested that the pathogen and host first contacted, and more effectors were needed. Corresponding to those in B. rapa, the expression levels of most genes involved in the calcium-mediated signaling pathway and PTI pathway were higher in plants inoculated with resting spores than in those inoculated with secondary zoospores. The ETI pathway was suppressed after inoculation with secondary zoospores. The genes induced after inoculation with resting spores were suppressed in B. rapa seedlings inoculated with secondary zoospores, which might be important to allow a fully compatible interaction and contribute to a susceptible reaction in the host at the subsequent infection stage. The primary zoospores undertook an more important interaction with plants.

The clubroot pathogen Plasmodiophora brassicae: A profile update.

Plasmodiophora brassicae is the causal agent of clubroot disease of cruciferous plants and one of the biggest threats to the rapeseed (Brassica napus) and brassica vegetable industry worldwide. In the advanced stages of clubroot disease wilting, stunting, yellowing, and redness are visible in the shoots. However, the typical symptoms of the disease are the presence of club-shaped galls in the roots of susceptible hosts that block the absorption of water and nutrients. Members of the family Brassicaceae are the primary host of the pathogen, although some members of the family, such as Bunias orientalis, Coronopus squamatus, and Raphanus sativus, have been identified as being consistently resistant to P. brassicae isolates with variable virulence profile. Class: Phytomyxea; Order: Plasmodiophorales; Family: Plasmodiophoraceae; Genus: Plasmodiophora; Species: Plasmodiophora brassicae (Woronin, 1877). Clubroot disease is spread worldwide, with reports from all continents except Antarctica. To date, clubroot disease has been reported in more than 80 countries. Based on its virulence on different hosts, P.brassicae is classified into pathotypes or races. Five main pathotyping systems have been developed to understand the relationship between P.brassicae and its hosts. Nowadays, the Canadian clubroot differential is extensively used in Canada and has so far identified 36 different pathotypes based on the response of a set of 13 hosts. After the identification and characterization of the clubroot pathogen SABATH-type methyltransferase PbBSMT, several other effectors have been characterized. However, no avirulence gene is known, hindering the functional characterization of the five intercellular nucleotide-binding (NB) site leucine-rich-repeat (LRR) receptors (NLRs) clubroot resistance genes validated to date. Canola Council of Canada is constantly updating information about clubroot and P.brassicae as part of their Canola Encyclopedia: https://www.canolacouncil.org/canola-encyclopedia/diseases/clubroot/. PLADBR: EPPO A2 list; Annex designation 9E.

Pathotype Characterization of Plasmodiophora brassicae, the Cause of Clubroot in Central Europe and Sweden (2016-2020).

Clubroot, caused by Plasmodiophora brassicae, is a crucial oilseed rape disease worldwide. Information on the virulence of P. brassicae populations is essential to apply disease control with proper clubroot-resistant cultivars. In 2016-2020, 84 isolates of P. brassicae were collected in the Czech Republic (CZ), Germany (DE), Poland (PL), and Sweden (SW). Pathotypes were designated using 17 Brassica hosts, including the European Clubroot Differentials (ECD), Somé set, and clubroot-resistant oilseed rape cv. Mendel. According to the ECD set, virulence analyses differentiated the isolates into 42 pathotypes. The most common pathotypes were 16/31/31 (in DE, PL, and SW) and 16/06/12 (in CZ, DE, and PL). Six pathotypes were found according to the Somé set, including 1-4 pathotypes per country. P1 was most prevalent in DE, PL, and SW, while P3 was abundant in CZ, DE, and PL. The current study provides clear evidence for a shift towards increased virulence in P. brassicae populations compared to previous studies. Several isolates overcame the resistance of cv. Mendel and of Brassica rapa genotypes ECD 01 to ECD 04. Considering all investigated samples, significant negative correlations were found between clubroot incidence and the frequency of oilseed rape in crop rotation, as for clubroot incidence and soil pH.

Genome-wide survey of Calcium-Dependent Protein Kinases (CPKs) in five Brassica species and identification of CPKs induced by Plasmodiophora brassicae in B. rapa, B. oleracea, and B. napus.

Calcium-dependent protein kinase (CPK) is a class of Ser/Thr protein kinase that exists in plants and some protozoa, possessing Ca2+ sensing functions and kinase activity. To better reveal the roles that Brassica CPKs played during plant response to stresses, five Brassica species, namely Brassica rapa (B. rapa), Brassica nigra (B. nigra), Brassica oleracea (B. oleracea), Brassica juncea (B. juncea), and Brassica napus (B. napus) were selected and analyzed. In total, 51 BraCPK, 56 BniCPK, 56 BolCPK, 88 BjuCPK, and 107 BnaCPK genes were identified genome wide and phylogenetics, chromosomal mapping, collinearity, promoter analysis, and biological stress analysis were conducted. The results showed that a typical CPK gene was constituted by a long exon and tandem short exons. They were unevenly distributed on most chromosomes except chromosome A08 in B. napus and B. rapa, and almost all CPK genes were located on regions of high gene density as non-tandem form. The promoter regions of BraCPKs, BolCPKs, and BnaCPKs possessed at least three types of cis-elements, among which the abscisic acid responsive-related accounted for the largest proportion. In the phylogenetic tree, CPKs were clustered into four primary groups, among which group I contained the most CPK genes while group IV contained the fewest. Some clades, like AT5G23580.1(CPK12) and AT2G31500.1 (CPK24) contained much more gene members than others, indicating a possibility that gene expansion occurred during evolution. Furthermore, 4 BraCPKs, 14 BolCPKs, and 31 BnaCPKs involved in the Plasmodiophora brassicae (P. brassicae) defense response in resistant (R) or susceptible (S) materials were derived from online databases, leading to the discovery that some R-specific induced CPKs, such as BnaC02g08720D, BnaA03g03800D, and BolC04g018270.2J.m1 might be ideal candidate genes for P. brassicae resistant research. Overall, these results provide valuable information for research on the function and evolution of CDK genes.

Genotypic characterization of Plasmodiophora brassicae in the paddy-field weed Cardamine occulta and symptomology reveal a distinct pathogen population in Japan

Genotypic characterization of Plasmodiophora brassicae in the paddy-field weed Cardamine occulta and symptomology reveal a distinct pathogen population in Japan

Genome-Wide Identification and Variation Analysis of JAZ Family Reveals BnaJAZ8.C03 Involved in the Resistance to Plasmodiophora brassicae in Brassica napus.

Clubroot caused by Plasmodiophora brassicae led to a significant decrease in the yield and quality of Brassica napus, one of the most important oil crops in the world. JAZ proteins are an essential repressor of jasmonates (JAs) signaling cascades, which have been reported to regulate the resistance to P. brassicae in B. napus. In this study, we identified 51, 25 and 26 JAZ proteins in B. napus, B. rapa and B. oleracea, respectively. Phylogenetic analysis displayed that the notedJAZ proteins were divided into six groups. The JAZ proteins clustered in the same group shared a similar motif composition and distribution order. The 51 BnaJAZs were not evenly assigned on seventeen chromosomes in B. napus, except for A04 and C07. The BnaJAZs of the AtJAZ7/AtJAZ8 group presented themselves to be significantly up-regulated after inoculation by P. brassicae. Variation analysis in a population with a specific resistance performance in P. brassicae displayed a 64 bp translocation in BnaC03T0663300ZS (BnaJAZ8.C03, homologous to AtJAZ8) with an 8% reduction in the disease index on average. Through protein-protein interaction analysis, 65 genes were identified that might be involved in JAZ8 regulation of resistance to P. brassicae in B. napus, which provided new clues for understanding the resistance mechanism to P. brassicae.

PM 3/66 (3) Guidelines for the management of plant health risks of biowaste of plant origin

<scp>PM</scp> 3/66 (3) Guidelines for the management of plant health risks of biowaste of plant origin

Fine mapping and candidate gene analysis of CRA3.7 conferring clubroot resistance in Brassica rapa.

In this study, we fine-mapped a clubroot resistance gene CRA3.7 in Chinese cabbage and developed its closely linked marker syau-InDel3008 for marker-assisted selection in CR cultivars breeding. Chinese cabbage is an important leafy vegetable rich in many nutrients widely grown in China. Clubroot disease caused by an obligate biotrophic pathogen Plasmodiophora brassicae was rapidly spread and challenged to Chinese cabbage production. A clubroot resistance (CR) gene, CRA3.7, was mapped on chromosome A03 of Brassica rapa. A Chinese cabbage line 'CR510', which harbor homozygous resistance locus CRA3.7 was selected from a BC4F3 family. 'CR510' was crossed with a clubroot susceptible Chinese cabbage inbred line '59-1'. Total 51 recombinant plants were identified from an F2 population including 3000 individuals. These recombinants were selfed and the clubroot resistance of F2/3 families was evaluated. Finally, a clubroot resistance gene CRA3.7 was fine-mapped to an interval of approximately 386 kb between marker syau-InDel3024 and syau-InDel3008. According to the reference genome, total 54 genes including five encoding the TIR-NBS-LRR proteins was annotated in the fine-mapped region. Further, nine candidate's gene expression in parental lines at 7, 14 and 21 days after inoculation of P. brassicae were evaluated. Bra019376, Bra019401, Bra019403 and Bra019410 are highly expressed in 'CR510' than '59-1'. Gene sequence of Bra019410 from 'CR510' was cloned and identified different from CRa. Therefore, Bra019376, Bra019401, Bra019403 and Bra019410 are the most likely candidates for CRA3.7. Our research provides a valuable germplasm resource against P. brassicae Pb3 and CRA3.7 closely linked marker for marker-assisted selection in CR cultivars breeding.

Mapping of a novel clubroot disease resistance locus in Brassica napus and related functional identification.

Clubroot disease, caused by Plasmodiophora brassicae, is a devastating disease that results in substantial yield loss in Brassicaceae crops worldwide. In this study, we identified a clubroot disease resistance (CR) Brassica napus, “Kc84R,” which was obtained by mutation breeding. Genetic analysis revealed that the CR trait of “Kc84R” was controlled by a single dominant locus. We used the bulked segregant analysis sequencing (BSA-seq) approach, combined with genetic mapping based on single nucleotide polymorphism (SNP) markers to identify CR loci from the F2 population derived from crossing CR “Kc84R” and clubroot susceptible “855S.” The CR locus was mapped to a region between markers BnSNP14198336 and BnSNP14462201 on the A03 chromosome, and this fragment of 267 kb contained 68 annotated candidate genes. Furthermore, we performed the CR relation screening of candidate genes with the model species Arabidopsis. An ERF family transcriptional activator, BnERF034, was identified to be associated with the CR, and the corresponding Arabidopsis homozygous knockout mutants exhibited more pronounced resistance compared with the wild-type Col-0 and the transgenic lines of BnERF034 in response to P. brassicae infection. Additionally, the expression analysis between resistant and susceptible materials indicated that BnERF034 was identified to be the most likely CR candidate for the resistance in Kc84R. To conclude, this study reveals a novel gene responsible for CR. Further analysis of BnERF034 may reveal the molecular mechanisms underlying the CR of plants and provide a theoretical basis for Brassicaceae resistance breeding.

Identification and characterization of putative effectors from Plasmodiophora brassicae that suppress or induce cell death in Nicotiana benthamiana.

Clubroot, caused by Plasmodiophora brassicae, is a major disease of crucifers. Effector proteins are important virulence factors in host recognition of pathogens and the interactions between pathogens and hosts. Secretory proteins, as effector candidates, have been studied in the interaction between Plasmodiophora brassicae and its hosts. In this study, 518 secretary proteins were screened from the Plasmodiophora brassicae genome. A total of 63 candidate effectors that induce or suppress cell death were identified using agroinfiltration-mediated transient expression in Nicothiana benthamiana. The candidate effectors, Pb4_102097 and Pb4_108104 showed high expressing level in the stage of rest spore maturity, could induce cell death and were associated with H2O2 accumulation in N. benthamiana leaves. In addition, 55 candidate effectors that could suppress BAX (Bcl-2-associated X protein) induced cell death, and 21 out of which could suppress the immunity caused by bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 in Arabidopsis. Based on the expression pattern in different stages, 28 candidate effectors showed high expression levels during the primary and secondary infection stage. Five candidate effectors containing the RXLR motif functioned in the cytoplasm and cell membrane.