Root Transcriptome and Metabolome Profiling Reveal Key Phytohormone-Related Genes and Pathways Involved Clubroot Resistance in Brassica rapa L.

Plasmodiophora brassicae, an obligate biotrophic pathogen-causing clubroot disease, can seriously affect Brassica crops worldwide, especially Chinese cabbage. Understanding the transcriptome and metabolome profiling changes during the infection of P. brassicae will provide key insights in understanding the defense mechanism in Brassica crops. In this study, we estimated the phytohormones using targeted metabolome assays and transcriptomic changes using RNA sequencing (RNA-seq) in the roots of resistant (BrT24) and susceptible (Y510-9) plants at 0, 3, 9, and 20 days after inoculation (DAI) with P. brassicae. Differentially expressed genes (DEGs) in resistant vs. susceptible lines across different time points were identified. The weighted gene co-expression network analysis of the DEGs revealed six pathways including “Plant–pathogen interaction” and “Plant hormone signal transduction” and 15 hub genes including pathogenic type III effector avirulence factor gene (RIN4) and auxin-responsive protein (IAA16) to be involved in plants immune response. Inhibition of Indoleacetic acid, cytokinin, jasmonate acid, and salicylic acid contents and changes in related gene expression in R-line may play important roles in regulation of clubroot resistance (CR). Based on the combined metabolome profiling and hormone-related transcriptomic responses, we propose a general model of hormone-mediated defense mechanism. This study definitely enhances our current understanding and paves the way for improving CR in Brassica rapa.

Identification of Candidate Genes for Clubroot-Resistance in Brassica oleracea Using Quantitative Trait Loci-Sequencing

Clubroot caused by Plasmodiophora brassicae is a devastating disease of cabbage (Brassica oleracea). To identify quantitative trait loci (QTLs) for clubroot resistance (CR) in B. oleracea, genomic resequencing was carried out in two sets of extreme pools, group I and group II, which were constructed separately from 110 and 74 F2 cloned lines derived from the cross between clubroot-resistant (R) cabbage “GZ87” (against race 4) and susceptible (S) cabbage “263.” Based on the QTL-sequencing (QTL-Seq) analysis of group I and group II, three QTLs (i.e., qCRc7-2, qCRc7-3, and qCRc7-4) were determined on the C07 chromosome. RNA-Seq and qRT-PCR were conducted in the extreme pools of group II before and after inoculation, and two potential candidate genes (i.e., Bol037115 and Bol042270), which exhibiting upregulation after inoculation in the R pool but downregulation in the S pool, were identified from the three QTLs on C07. A functional marker “SWU-OA” was developed from qCRc7-4 on C07, exhibiting ∼95% accuracy in identifying CR in 56 F2 lines. Our study will provide valuable information on resistance genes against P. brassicae and may accelerate the breeding process of B. oleracea with CR.

MiR1885 Regulates Disease Tolerance Genes in Brassica rapa during Early Infection with Plasmodiophora brassicae

Clubroot caused by Plasmodiophora brassicae is a severe disease of cruciferous crops that decreases crop quality and productivity. Several clubroot resistance-related quantitative trait loci and candidate genes have been identified. However, the underlying regulatory mechanism, the interrelationships among genes, and how genes are regulated remain unexplored. MicroRNAs (miRNAs) are attracting attention as regulators of gene expression, including during biotic stress responses. The main objective of this study was to understand how miRNAs regulate clubroot resistance-related genes in P. brassicae-infected Brassica rapa. Two Brassica miRNAs, Bra-miR1885a and Bra-miR1885b, were revealed to target TIR-NBS genes. In non-infected plants, both miRNAs were expressed at low levels to maintain the balance between plant development and basal immunity. However, their expression levels increased in P. brassicae-infected plants. Both miRNAs down-regulated the expression of the TIR-NBS genes Bra019412 and Bra019410, which are located at a clubroot resistance-related quantitative trait locus. The Bra-miR1885-mediated down-regulation of both genes was detected for up to 15 days post-inoculation in the clubroot-resistant line CR Shinki and in the clubroot-susceptible line 94SK. A qRT-PCR analysis revealed Bra019412 expression was negatively regulated by miR1885. Both Bra019412 and Bra019410 were more highly expressed in CR Shinki than in 94SK; the same expression pattern was detected in multiple clubroot-resistant and clubroot-susceptible inbred lines. A 5′ rapid amplification of cDNA ends analysis confirmed the cleavage of Bra019412 by Bra-miR1885b. Thus, miR1885s potentially regulate TIR-NBS gene expression during P. brassicae infections of B. rapa.

Identification and Characterization of Circular RNAs in Brassica rapa in Response to Plasmodiophora brassicae

BackgroundPlasmodiophora brassicae is a soil-borne pathogen that attacks the roots of cruciferous plants, causing clubroot disease. CircRNAs are non-coding RNAs widely exist in plant and animal species which can acting as “microRNA (miRNA) sponges” and “competing endogenous RNAs (ceRNAs)”. Knowledge of circRNAs has been updated continuously and rapidly. However, the information about circRNAs in the regulation of clubroot-disease resistance is limited in Brassica rapa. ResultsHere, the Chinese cabbage (BJN 222) containing clubroot resistance gene (CRa) resistant to the Pb4 was susceptible to the PbE of P. brassicae. To investigate the mechanism of cicRNAs responsible for clubroot-disease resistance in Brassica rapa, the circRNA-seq was performed roots of BJN 222 at 0 d, 8 d, and 23 d after inoculated with Pb4 and PbE. A total of 1636 circRNAs were detected distributed on 10 chromosomes. Furthermore, total 231 differentially expressed circRNAs between groups were screened. Parental genes of circRNAs functions analysis results indicated that the expression of circRNAs was affected not only by inoculation time but also by the pathogenicity of P. brassicae. However, the “Phenylalanine, tyrosine, and tryptophan biosynthesis” pathway was significant enriched between the two pathotypes at different inoculation times. All the expression of target genes annotated with “receptor-like protein kinase,” “zinc finger protein,” “LRR-repeat protein,” and “hormone-related” identified from the circRNA-miRNA-mRNA network were analyzed. 5 target genes were consistent with the expression pattern of novel_circ_000495 at 8 dpi, but only Bra026508 was significantly up-regulated. ConclusionThe up-regulated novel_circ_000495 might suppressed the expression of miR5656-y, leading to the up-regulation of Bra026508. Our results provided new insights to clubroot resistance mechanisms of B.rapa and laid a foundation for further research on the function of circRNAs responsible for the pathogen infection.

Transcriptome and Coexpression Network Analyses Reveal Hub Genes in Chinese Cabbage (Brassica rapa L. ssp. pekinensis) During Different Stages of Plasmodiophora brassicae Infection

Clubroot, caused by the soil-borne protist Plasmodiophora brassicae, is one of the most destructive diseases of Chinese cabbage worldwide. However, the clubroot resistance mechanisms remain unclear. In this study, in both clubroot-resistant (DH40R) and clubroot-susceptible (DH199S) Chinese cabbage lines, the primary (root hair infection) and secondary (cortical infection) infection stages started 2 and 5 days after inoculation (dai), respectively. With the extension of the infection time, cortical infection was blocked and complete P. brassica resistance was observed in DH40R, while disease scales of 1, 2, and 3 were observed at 8, 13, and 22 dai in DH199S. Transcriptome analysis at 0, 2, 5, 8, 13, and 22 dai identified 5,750 relative DEGs (rDEGs) between DH40R and DH199S. The results indicated that genes associated with auxin, PR, disease resistance proteins, oxidative stress, and WRKY and MYB transcription factors were involved in clubroot resistance regulation. In addition, weighted gene coexpression network analysis (WGCNA) identified three of the modules whose functions were highly associated with clubroot-resistant, including ten hub genes related to clubroot resistance (ARF2, EDR1, LOX4, NHL3, NHL13, NAC29, two AOP1, EARLI 1, and POD56). These results provide valuable information for better understanding the molecular regulatory mechanism of Chinese cabbage clubroot resistance.

Clubroot resistance derived from the European Brassica napus cv. ‘Tosca’ is not effective against virulent Plasmodiophora brassicae isolates from Alberta, Canada

In this study, clubroot resistance in the resynthesized European winter Brassica napus cv. ‘Tosca’ was introgressed into a Canadian spring canola line ‘11SR0099’, which was then crossed with the clubroot susceptible spring line ‘12DH0001’ to produce F1 seeds. The F1 plants were used to develop a doubled haploid (DH) mapping population. The parents and the DH lines were screened against ‘old’ pathotypes 2F, 3H, 5I, 6M and 8N of the clubroot pathogen, Plasmodiophora brassicae, as well as against the ‘new’ pathotypes 5X, 5L, 2B, 3A, 3D, 5G, 8E, 5C, 8J, 5K, 3O and 8P. Genotyping was conducted using a Brassica 15K SNP array. The clubroot screening showed that ‘Tosca, ‘11SR0099’ and the resistant DH lines were resistant to three (2F, 3H and 5I) of the five ‘old’ pathotypes and four (2B, 3O, 8E and 8P) of the 12 ‘new’ pathotypes, while being moderately resistant to the ‘old’ pathotype 8N and the ‘new’ pathotypes 3D and 5G. ‘Tosca’ was susceptible to isolates representing pathotype 3A (the most common among the ‘new’ pathotypes) as well as pathotypes 6M, 5X, 5L, 5K and 8J. Linkage analysis and QTL mapping identified a ca. 0.88–0.95 Mb genomic region on the A03 chromosome of ‘Tosca’ as conferring resistance to pathotypes 2F, 3H, 5I, 2B, 3D, 5G, 8E, 3O and 8P. The identified QTL genomic region housed the CRk, Crr3 and CRd gene(s). However, the susceptibility of ‘Tosca’ to most of the common virulent pathotypes makes it unattractive as a sole CR donor in the breeding of commercial canola varieties in western Canada.

Local Duplication of TIR-NBS-LRR Gene Marks Clubroot Resistance in Brassica napus cv. Tosca

Clubroot, caused by Plasmodiophora brassicae infection, is a disease of growing importance in cruciferous crops, including oilseed rape (Brassica napus). The affected plants exhibit prominent galling of the roots that impairs their capacity for water and nutrient uptake, which leads to growth retardation, wilting, premature ripening, or death. Due to the scarcity of effective means of protection against the pathogen, breeding of resistant varieties remains a crucial component of disease control measures. The key aspect of the breeding process is the identification of genetic factors associated with variable response to the pathogen exposure. Although numerous clubroot resistance loci have been described in Brassica crops, continuous updates on the sources of resistance are necessary. Many of the resistance genes are pathotype-specific, moreover, resistance breakdowns have been reported. In this study, we characterize the clubroot resistance locus in the winter oilseed rape cultivar “Tosca.” In a series of greenhouse experiments, we evaluate the disease severity of P. brassicae-challenged “Tosca”-derived population of doubled haploids, which we genotype with Brassica 60 K array and a selection of SSR/SCAR markers. We then construct a genetic map and narrow down the resistance locus to the 0.4 cM fragment on the A03 chromosome, corresponding to the region previously described as Crr3. Using Oxford Nanopore long-read genome resequencing and RNA-seq we review the composition of the locus and describe a duplication of TIR-NBS-LRR gene. Further, we explore the transcriptomic differences of the local genes between the clubroot resistant and susceptible, inoculated and control DH lines. We conclude that the duplicated TNL gene is a promising candidate for the resistance factor. This study provides valuable resources for clubroot resistance breeding programs and lays a foundation for further functional studies on clubroot resistance.

Clubroot resistance in canola and brassica vegetable cultivars in Ontario, Canada

Clubroot, caused by the obligate pathogen <i>Plasmodiophora brassicae</i> Woronin, has been present on brassica vegetables in Ontario for decades, but was only recently identified on canola (<i>Brassica napus</i> L.). Once <i>P. brassicae</i> is present in a field, eradication is difficult, but resistant cultivars can provide effective management. Pathotype 6 has been the predominant pathotype on vegetable crops for decades, but pathotype 2 is predominant in canola fields in Ontario. Field trials were used to assess the reaction of selected canola and vegetable Brassica cultivars to pathotype 2, and controlled environment studies were conducted to evaluate the reaction of canola the same cultivars to pathotypes 2 and 6. Four canola cultivars with putative clubroot resistance were compared to two cultivars that were expected to be susceptible and three susceptible control cultivars. Several brassica vegetables were assessed: cabbage, cauliflower, broccoli, napa cabbage, rutabaga, and Shanghai pak choi (a susceptible control). The canola cultivars marketed as resistant were highly resistant in both the field and growth room trials. The canola cultivars not marketed as resistant were susceptible to pathotype 2, as expected. All of the canola cultivars were resistant to pathotype 6. The vegetable cultivars marketed as resistant or tolerant were resistant to pathotype 6 and most were resistant to pathotype 2. A putative resistant cultivar of cabbage and one of broccoli were resistant to pathotype 6 but susceptible to pathotype 2. Clubroot consistently reduced fresh shoot weight in susceptible cultivars of canola and brassica vegetables relative to resistant cultivars.