RNA-Seq Bulked Segregant Analysis of an Exotic B. napus ssp. napobrassica (Rutabaga) F2 Population Reveals Novel QTLs for Breeding Clubroot-Resistant Canola.

In this study, a rutabaga (Brassica napus ssp. napobrassica) donor parent FGRA106, which exhibited broad-spectrum resistance to 17 isolates representing 16 pathotypes of Plasmodiophora brassicae, was used in genetic crosses with the susceptible spring-type canola (B. napus ssp. napus) accession FG769. The F2 plants derived from a clubroot-resistant F1 plant were screened against three P. brassicae isolates representing pathotypes 3A, 3D, and 3H. Chi-square (χ2) goodness-of-fit tests indicated that the F2 plants inherited two major clubroot resistance genes from the CR donor FGRA106. The total RNA from plants resistant (R) and susceptible (S) to each pathotype were pooled and subjected to bulked segregant RNA-sequencing (BSR-Seq). The analysis of gene expression profiles identified 431, 67, and 98 differentially expressed genes (DEGs) between the R and S bulks. The variant calling method indicated a total of 12 (7 major + 5 minor) QTLs across seven chromosomes. The seven major QTLs included: BnaA5P3A.CRX1.1, BnaC1P3H.CRX1.2, and BnaC7P3A.CRX1.1 on chromosomes A05, C01, and C07, respectively; and BnaA8P3D.CRX1.1, BnaA8P3D.RCr91.2/BnaA8P3H.RCr91.2, BnaA8P3H.Crr11.3/BnaA8P3D.Crr11.3, and BnaA8P3D.qBrCR381.4 on chromosome A08. A total of 16 of the DEGs were located in the major QTL regions, 13 of which were on chromosome C07. The molecular data suggested that clubroot resistance in FGRA106 may be controlled by major and minor genes on both the A and C genomes, which are deployed in different combinations to confer resistance to the different isolates. This study provides valuable germplasm for the breeding of clubroot-resistant B. napus cultivars in Western Canada.

Plasmodiophora brassicae affects host gene expression by secreting the transcription factor-type effector PbZFE1.

The protist pathogen Plasmodiophora brassicae hijacks the metabolism and development of host cruciferous plants and induces clubroot formation, but little is known about its regulatory mechanisms. Previously, the Pnit2int2 sequence, a sequence around the second intron of the nitrilase gene (BrNIT2) involved in auxin biosynthesis in Brassica rapa ssp. pekinensis, was identified as a specific promoter activated during clubroot formation. In this study, we hypothesized that analysis of the transcriptional regulation of Pnit2int2 could reveal how P. brassicae affects the host gene regulatory system during clubroot development. By yeast one-hybrid screening, the pathogen zinc finger protein PbZFE1 was identified to specifically bind to Pnit2int2. Specific binding of PbZFE1 to Pnit2int2 was also confirmed by electrophoretic mobility shift assay. The binding site of PbZFE1 is essential for promoter activity of Pnit2int2 in clubbed roots of transgenic Arabidopsis thaliana (Pnit2int2-2::GUS), indicating that PbZFE1 is secreted from P. brassicae and functions within plant cells. Ectopic expression of PbZEF1 in A. thaliana delayed growth and flowering time, suggesting that PbZFE1 has significant impacts on host development and metabolic systems. Thus, P. brassicae appears to secrete PbZFE1 into host cells as a transcription factor-type effector during pathogenesis.

New endophytic strains of Trichoderma promote growth and reduce clubroot severity of rapeseed (Brassica napus).

Rapeseed (Brassica napus L.) is the world's third most important edible oilseed crop after soybean and palm. The clubroot disease caused by Plasmodiophora brassicae poses a significant risk and causes substantial yield losses in rapeseed. In this study, 13 endophytic fungal strains were isolated from the healthy roots of rapeseed (B. napus) grown in a clubroot-infested field and molecularly identified. Based on germination inhibition of resting spores of P. brassicae, two endophytic fungal antagonists, Trichoderma spp. ReTk1 and ReTv2 were selected to evaluate their potential for plant growth promotion and biocontrol of P. brassicae. The Trichoderma isolates were applied as a soil drench (1×107 spore/g soil) to a planting mix and field soil, in which plants were grown under non-infested and P. brassicae-infested (2×106 spore/g soil) conditions. The endophytic fungi were able to promote plant growth, significantly increasing shoot and root length, leaf diameter, and biomass production (shoots and root weight) both in the absence or presence of P. brassicae. The single and dual treatments with the endophytes were equally effective in significantly decreasing the root-hair infection, root index, and clubroot severity index. Both ReTk1 and ReTv2 inhibited the germination of resting spores of P. brassicae in root exudates. Moreover, the endophytic fungi colonized the roots of rapeseed extensively and possibly induced host resistance by up-regulated expression of defense-related genes involved in jasmonate (BnOPR2), ethylene (BnACO and BnSAM3), phenylpropanoid (BnOPCL and BnCCR), auxin (BnAAO1) and salicylic acid (BnPR2) pathways. Based on these findings, it is evident that the rapeseed root endophytes Trichoderma spp. ReTk1 and ReTv2 could suppress the gall formation on rapeseed roots via antibiosis, induced systemic resistance (ISR), and/or systemic acquired resistance (SAR). According to our knowledge, this is the first report of the endophytic Trichoderma spp. isolated from root tissues of healthy rapeseed plants (B. napus.), promoting plant growth and reducing clubroot severity.

bra-miR167a Targets ARF8 and Negatively Regulates Arabidopsis thaliana Immunity against Plasmodiophora brassicae.

Clubroot is a soil-borne disease caused by Plasmodiophora brassicae, which can seriously affect the growth and production of cruciferous crops, especially Chinese cabbage crops, worldwide. At present, few studies have been conducted on the molecular mechanism of this disease's resistance response. In this experiment, we analyzed the bioinformation of bra-miR167a, constructed a silencing vector (STTM167a) and an overexpression vector (OE-miR167a), and transformed them to Arabidopsis to confirm the role of miR167a in the clubroot resistance mechanism of Arabidopsis. Afterwards, phenotype analysis and expression level analysis of key genes were conducted on transgenic plants. From the result, we found that the length and number of lateral roots of silence transgenic Arabidopsis STTM167a was higher than that of WT and OE-miR167a. In addition, the STTM167a transgenic Arabidopsis induced up-regulation of disease resistance-related genes (PR1, PR5, MPK3, and MPK6) at 3 days after inoculation. On the other hand, the auxin pathway genes (TIR1, AFB2, and AFB3), which are involved in maintaining the balance of auxin/IAA and auxin response factor (ARF), were down-regulated. These results indicate that bra-miR167a is negative to the development of lateral roots and auxins, but positive to the expression of resistance-related genes. This also means that the STTM167a can improve the resistance of clubroot by promoting lateral root development and the level of auxin, and can induce resistance-related genes by regulating its target genes. We found a positive correlation between miR167a and clubroot disease, which is a new clue for the prevention and treatment of clubroot disease.

WeiTsing, a pericycle-expressed ion channel, safeguards the stele to confer clubroot resistance.

Plant roots encounter numerous pathogenic microbes that often cause devastating diseases. One such pathogen, Plasmodiophora brassicae (Pb), causes clubroot disease and severe yield losses on cruciferous crops worldwide. Here, we report the isolation and characterization of WeiTsing (WTS), a broad-spectrum clubroot resistance gene from Arabidopsis. WTS is transcriptionally activated in the pericycle upon Pb infection to prevent pathogen colonization in the stele. Brassica napus carrying the WTS transgene displayed strong resistance to Pb. WTS encodes a small protein localized in the endoplasmic reticulum (ER), and its expression in plants induces immune responses. The cryoelectron microscopy (cryo-EM) structure of WTS revealed a previously unknown pentameric architecture with a central pore. Electrophysiology analyses demonstrated that WTS is a calcium-permeable cation-selective channel. Structure-guided mutagenesis indicated that channel activity is strictly required for triggering defenses. The findings uncover an ion channel analogous to resistosomes that triggers immune signaling in the pericycle.

Integrative Transcriptome, miRNAs, Degradome, and Phytohormone Analysis of Brassica rapa L. in Response to Plasmodiophora brassicae.

Clubroot is an infectious root disease caused by Plasmodiophora brassicae in Brassica crops, which can cause immeasurable losses. We analyzed integrative transcriptome, small RNAs, degradome, and phytohormone comprehensively to explore the infection mechanism of P. brassicae. In this study, root samples of Brassica rapa resistant line material BrT24 (R-line) and susceptible line material Y510-9 (S-line) were collected at four different time points for cytological, transcriptome, miRNA, and degradome analyses. We found the critical period of disease resistance and infection were at 0-3 DAI (days after inoculation) and 9-20 DAI, respectively. Based on our finding, we further analyzed the data of 9 DAI vs. 20 DAI of S-line and predicted the key genes ARF8, NAC1, NAC4, TCP10, SPL14, REV, and AtHB, which were related to clubroot disease development and regulating disease resistance mechanisms. These genes are mainly related to auxin, cytokinin, jasmonic acid, and ethylene cycles. We proposed a regulatory model of plant hormones under the mRNA-miRNA regulation in the critical period of P. brassicae infection by using the present data of the integrative transcriptome, small RNAs, degradome, and phytohormone with our previously published results. Our integrative analysis provided new insights into the regulation relationship of miRNAs and plant hormones during the process of disease infection with P. brassicae.

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.

Multi-Omics Approaches to Improve Clubroot Resistance in Brassica with a Special Focus on Brassica oleracea L.

Brassica oleracea is an agronomically important species of the Brassicaceae family, including several nutrient-rich vegetables grown and consumed across the continents. But its sustainability is heavily constrained by a range of destructive pathogens, among which, clubroot disease, caused by a biotrophic protist Plasmodiophora brassicae, has caused significant yield and economic losses worldwide, thereby threatening global food security. To counter the pathogen attack, it demands a better understanding of the complex phenomenon of Brassica-P. brassicae pathosystem at the physiological, biochemical, molecular, and cellular levels. In recent years, multiple omics technologies with high-throughput techniques have emerged as successful in elucidating the responses to biotic and abiotic stresses. In Brassica spp., omics technologies such as genomics, transcriptomics, ncRNAomics, proteomics, and metabolomics are well documented, allowing us to gain insights into the dynamic changes that transpired during host-pathogen interactions at a deeper level. So, it is critical that we must review the recent advances in omics approaches and discuss how the current knowledge in multi-omics technologies has been able to breed high-quality clubroot-resistant B. oleracea. This review highlights the recent advances made in utilizing various omics approaches to understand the host resistance mechanisms adopted by Brassica crops in response to the P. brassicae attack. Finally, we have discussed the bottlenecks and the way forward to overcome the persisting knowledge gaps in delivering solutions to breed clubroot-resistant Brassica crops in a holistic, targeted, and precise way.

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

Plasmodiophora brassicae is a soil-borne pathogen that attacks the roots of cruciferous plants and causes clubroot disease. CircRNAs are noncoding RNAs, widely existing in plant and animal species. Although knowledge of circRNAs has been updated continuously and rapidly, information about circRNAs in the regulation of clubroot disease resistance is extremely limited in Brassica rapa. Here, Chinese cabbage (BJN 222) containing clubroot resistance genes (CRa) against P. brassicae Pb4 was susceptible to PbE. To investigate the mechanism of cicRNAs responsible for clubroot disease resistance in B. rapa, circRNA-seq was performed with roots of 'BJN 222' at 0, 8, and 23 days post-inoculated (dpi) with Pb4 and PbE. A total of 231 differentially expressed circRNAs were identified between the groups. Based on the differentially expressed circRNAs, the circRNA-miRNA-mRNA network was constructed using the target genes directly or indirectly related to plant resistance. Upregulated novel_circ_000495 suppressed the expression of miR5656-y, leading to the upregulation of Bra026508, which might cause plant resistance. Our results provide new insights into clubroot resistance mechanisms and lay a foundation for further studies exploring complex gene regulation networks in B. rapa.

Comparative transcriptome analysis reveals distinct responsive biological processes in radish genotypes contrasting for Plasmodiophora brassicae interaction.

Plasmodiophora brassicae is a protozoan pathogen that causes clubroot disease, which is one of the most destructive diseases for Brassica crops, including radish. However, little is known about the molecular mechanism of clubroot resistance in radish. In this study, we performed a comparative transcriptome analysis between resistant and susceptible radish inoculated with P. brassicae. More differentially expressed genes (DEGs) were identified at 28 days after inoculation (DAI) compared to 7 DAI in both genotypes. Gene ontology (GO) and KEGG enrichment indicated that stress/defense response, secondary metabolic biosynthesis, hormone metabolic process, and cell periphery are directly involved in the defense response process. Further analysis of the transcriptome revealed that effector-triggered immunity (ETI) plays key roles in the defense response. The plant hormones jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA) related genes are activated in clubroot defense in the resistant line. Auxin (AUX) hormone related genes are activated in the developing galls of susceptible radish. Our study provides a global transcriptional overview for clubroot development for insights into the P. brassicae defense mechanisms in radish.

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.

Application of exogenous salicylic acid reduces disease severity of Plasmodiophora brassicae in pakchoi (Brassica campestris ssp. chinensis Makino).

Clubroot is one of the most serious diseases affecting Brassicaceae plants worldwide. However, there is no effective control method for clubroot. Salicylic acid (SA) is a plant hormone that plays a critical role in plant defense. In our study, we found the disease severity of a clubroot-sensitive cultivar of pakchoi, Xinxiaqing, was reduced with 0.6mM exogenous SA after the infection of P. brassicae. To investigate the mechanism of SA-reduced disease severity against clubroot, then we analyzed the plant growth, alteration of antioxidant enzyme system, and related gene expression of Xinxiaqing. Results showed that the clubroot incidence rate and disease index were decreased after being treated with 0.6 mM exogenous SA. Furthermore, plant growth, reactive oxygen species (ROS) contents, and membrane lipid peroxidation were changed. The activities of antioxidant enzymes, including superoxide dismutase (SOD), ascorbic acid-peroxidase (APX), catalase (CAT), and glutathione reductase (GR), were increased. Additionally, the production rates of malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion (O2·-) were also inhibited. The expression levels of genes, encoding SOD, APX, CAT, and GR, were increased. By summering all results, we conclude that 0.6 mM SA contributes to the reduction of disease severity to clubroot by increasing the activities of antioxidant enzymes, abilities of osmotic regulation, and ROS scavenging to reduce the clubroot-induced damage in pakchoi.

Exogenous inoculation of endophytic bacterium Bacillus cereus suppresses clubroot (Plasmodiophora brassicae) occurrence in pak choi (Brassica campestris sp. chinensis L.).

MAIN CONCLUSION: The presence of Bacillus cereus plays a key role in clubroot suppression and improves plant biomass in pak choi. B. cereus is reported for the first time as a novel biocontrol agent against clubroot. Plasmodiophora brassicae Woronin causes a devastating infectious disease known as clubroot that is damaging to cruciferous vegetables. This study aimed to isolate beneficial bacteria from the rhizosphere soil of pak choi (Brassica campestris sp. chinensis) and to evaluate the ability of the isolate to reduce the severity of clubroot. Strains obtained from the rhizosphere of symptomless pak choi were first selected on the basis of their germination inhibition rate and effects on the viability of P. brassicae resting spores. Eight bacterial isolates had inhibitory effects against the resting spores of clubroot causing pathogen. However, MZ-12 showed the highest inhibitory effect at 73.4%. Inoculation with MZ-12 enhanced the plant biomass relative to plants grown without MZ-12 as well as P. brassicae infected plants. Furthermore, enhanced antioxidant enzymatic activities were observed in clubroot-infected plants during bacterial association. Co-inoculation of the plant with both P. brassicae and MZ-12 resulted in a 64% reduction of gall formation in comparison to plants inoculated with P. brassicae only. Three applications of MZ-12 to plants infected with P. brassicae at 7, 14 and 21 days after seeding (DAS) were more effective than one application and repressed root hair infection. According to 16S rDNA sequence analysis, strain MZ-12 was identified as had a 100% sequence similarity with type strain Bacillus cereus. The findings of the present study will facilitate further investigation into biological mechanisms of cruciferous clubroot control.

Physiological change alters endophytic bacterial community in clubroot of tumorous stem mustard infected by Plasmodiophora brassicae.

BACKGROUND: Endophytic bacteria are considered as symbionts living within plants and are influenced by abiotic and biotic environments. Pathogen cause biotic stress, which may change physiology of plants and may affect the endophytic bacterial communiy. Here, we reveal how endophytic bacteria in tumorous stem mustard (Brassica juncea var. tumida) are affected by plant physiological changes caused by Plasmodiophora brassicae using 16S rRNA high-throughput sequencing. RESULTS: The results showed that Proteobacteria was the dominant group in both healthy roots and clubroots, but their abundance differed. At the genus level, Pseudomonas was dominant in clubroots, whereas Rhodanobacter was the dominant in healthy roots. Hierarchical clustering, UniFrac-weighted principal component analysis (PCA), non-metric multidimensional scaling (NMDS) and analysis of similarities (ANOSIM) indicated significant differences between the endophytic bacterial communities in healthy roots and clubroots. The physiological properties including soluble sugar, soluble protein, methanol, peroxidase (POD) and superoxide dismutase (SOD) significantly differed between healthy roots and clubroots. The distance-based redundancy analysis (db-RDA) and two-factor correlation network showed that soluble sugar, soluble protein and methanol were strongly related to the endophytic bacterial community in clubroots, whereas POD and SOD correlated with the endophytic bacterial community in healthy roots. CONCLUSIONS: Our results illustrate that physiologcial changes caused by P. brassicae infection may alter the endophytic bacterial community in clubroots of tumorous stem mustard.

Two Clubroot-Resistance Genes, Rcr3 and Rcr9wa, Mapped in Brassica rapa Using Bulk Segregant RNA Sequencing.

Genetic resistance is widely used to manage clubroot (Plasmodiophora brassicae) in brassica crops, but new pathotypes have recently been identified on canola (Brassica napus) on the Canadian prairies. Resistance effective against both the most prevalent pathotype (3H, based on the Canadian Clubroot Differential system) and the new pathotypes is needed. BC1 plants of Brassica rapa from a cross of line 96-6990-2 (clubroot resistance originating from turnip cultivar 'Waaslander') and a susceptible doubled-haploid line, ACDC, exhibited a 1:1 segregation for resistance against pathotypes 3H and 5X. A resistance gene designated as Rcr3 was mapped initially based on the percentage of polymorphic variants using bulked segregant RNA sequencing (BSR-Seq) and further mapped using Kompetitive Allele Specific PCR. DNA variants were identified by assembling short reads against a reference genome of B. rapa. Rcr3 was mapped into chromosome A08. It was flanked by single nucleotide polymorphisms (SNP) markers (A90_A08_SNP_M12 and M16) between 10.00 and 10.23 Mb, in an interval of 231.6 Kb. There were 32 genes in the Rcr3 interval. Three genes (Bra020951, Bra020974, and Bra020979) were annotated with disease resistance mechanisms, which are potential candidates for Rcr3. Another resistance gene, designated as Rcr9wa, for resistance to pathotype 5X was mapped, with the flanking markers (A90_A08_SNP_M28 and M79) between 10.85 and 11.17 Mb using the SNP sites identified through BSR-Seq for Rcr3. There were 44 genes in the Rcr9wa interval, three of which (Bra020827, Bra020828, Bra020814) were annotated as immune-system-process related genes, which are potential candidates for Rcr9wa.

Metabolomic Fingerprinting of Potato Cultivars Differing in Susceptibility to Spongospora subterranea f. sp. subterranea Root Infection.

Plants defend themselves from pathogens by producing bioactive defense chemicals. The biochemical mechanisms relating to quantitative resistance of potato to root infection by Spongospora subterranea f. sp. subterranea (Sss) are, however, not understood, and are not efficiently utilized in potato breeding programs. Untargeted metabolomics using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to elucidate the biochemical mechanisms of susceptibility to Sss root infection. Potato roots and root exudate metabolic profiles of five tolerant cultivars were compared with those of five susceptible cultivars, following Sss inoculation, to identify tolerance-related metabolites. Comparison of the relative metabolite abundance of tolerant versus susceptible cultivars revealed contrasting responses to Sss infection. Metabolites belonging to amino acids, organic acids, fatty acids, phenolics, and sugars, as well as well-known cell wall thickening compounds were putatively identified and were especially abundant in the tolerant cultivars relative to the susceptible cultivars. Metabolites known to activate plant secondary defense metabolism were significantly increased in the tolerant cultivars compared to susceptible cultivars following Sss inoculation. Root-exuded compounds belonging to the chemical class of phenolics were also found in abundance in the tolerant cultivars compared to susceptible cultivars. This study illustrated that Sss infection of potato roots leads to differential expression of metabolites in tolerant and susceptible potato cultivars.

Integrating long noncoding RNAs and mRNAs expression profiles of response to Plasmodiophora brassicae infection in Pakchoi (Brassica campestris ssp. chinensis Makino).

The biotrophic protist Plasmodiophora brassicae causes serious damage to Brassicaceae crops grown worldwide. However, the molecular mechanism of the Brassica rapa response remains has not been determined. Long noncoding RNA and mRNA expression profiles in response to Plasmodiophora brassicae infection were investigated using RNA-seq on the Chinese cabbage inbred line C22 infected with P. brassicae. Approximately 5,193 mRNAs were significantly differentially expressed, among which 1,345 were upregulated and 3,848 were downregulated. The GO enrichment analysis shows that most of these mRNAs are related to the defense response. Meanwhile, 114 significantly differentially expressed lncRNAs were identified, including 31 upregulated and 83 downregulated. Furthermore, a total of 2,344 interaction relationships were detected between 1,725 mRNAs and 103 lncRNAs with a correlation coefficient greater than 0.8. We also found 15 P. brassicaerelated mRNAs and 16 lncRNA interactions within the correlation network. The functional annotation showed that 15 mRNAs belong to defense response proteins (66.67%), protein phosphorylation (13.33%), root hair cell differentiation (13.33%) and regulation of salicylic acid biosynthetic process (6.67%). KEGG annotation showed that the vast majority of these genes are involved in the biosynthesis of secondary metabolism pathways and plant-pathogen interactions. These results provide a new perspective on lncRNA-mRNA network function and help to elucidate the molecular mechanism of P. brassicae infection.

An Improved Evans Blue Staining Method for Consistent, Accurate Assessment of Plasmodiophora brassicae Resting Spore Viability.

Clubroot caused by Plasmodiophora brassicae is an important disease of brassica crops. The use of vital stains to determine the viability of P. brassicae resting spores can provide useful information regarding spore longevity, inoculum potential, or the efficacy of antimicrobial treatments. Evans blue is one example of a vital stain that has been reported to differentially stain viable and nonviable resting spores. Some previously published protocols using Evans blue to stain P. brassicae resting spores have not provided accurate or consistent results. In this study, we modified the Evans blue method by increasing the staining time to 8 h or more and evaluated P. brassicae resting spores after heat treatment at various combinations of temperature and time. Extending staining times significantly increased the numbers of stained resting spores up to 7 h, after which the numbers of stained spores did not change significantly (R2 = 96.88; P ≤ 0.001). The accuracy of the modified method to discriminate viable and nonviable spores was evaluated in repeated experiments and by comparing the staining data with those derived from inoculation assays and propidium monoazide quantitative PCR (qPCR). The results demonstrated that the modified Evans blue staining method improved the accuracy and consistency of measurement of P. brassicae resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores (R2 = 99.84; P ≤ 0.001) and confirmed in canola infection bioassays.

Transcriptomic response in symptomless roots of clubroot infected kohlrabi (Brassica oleracea var. gongylodes) mirrors resistant plants.

BACKGROUND: Clubroot disease caused by Plasmodiophora brassicae (Phytomyxea, Rhizaria) is one of the economically most important diseases of Brassica crops. The formation of hypertrophied roots accompanied by altered metabolism and hormone homeostasis is typical for infected plants. Not all roots of infected plants show the same phenotypic changes. While some roots remain uninfected, others develop galls of diverse size. The aim of this study was to analyse and compare the intra-plant heterogeneity of P. brassicae root galls and symptomless roots of the same host plants (Brassica oleracea var. gongylodes) collected from a commercial field in Austria using transcriptome analyses. RESULTS: Transcriptomes were markedly different between symptomless roots and gall tissue. Symptomless roots showed transcriptomic traits previously described for resistant plants. Genes involved in host cell wall synthesis and reinforcement were up-regulated in symptomless roots indicating elevated tolerance against P. brassicae. By contrast, genes involved in cell wall degradation and modification processes like expansion were up-regulated in root galls. Hormone metabolism differed between symptomless roots and galls. Brassinosteroid-synthesis was down-regulated in root galls, whereas jasmonic acid synthesis was down-regulated in symptomless roots. Cytokinin metabolism and signalling were up-regulated in symptomless roots with the exception of one CKX6 homolog, which was strongly down-regulated. Salicylic acid (SA) mediated defence response was up-regulated in symptomless roots, compared with root gall tissue. This is probably caused by a secreted benzoic acid/salicylic acid methyl transferase from the pathogen (PbBSMT), which was one of the highest expressed pathogen genes in gall tissue. The PbBSMT derived Methyl-SA potentially leads to increased pathogen tolerance in uninfected roots. CONCLUSIONS: Infected and uninfected roots of clubroot infected plants showed transcriptomic differences similar to those previously described between clubroot resistant and susceptible hosts. The here described intra-plant heterogeneity suggests, that for a better understanding of clubroot disease targeted, spatial analyses of clubroot infected plants will be vital in understanding this economically important disease.

Genius Architect or Clever Thief-How Plasmodiophora brassicae Reprograms Host Development to Establish a Pathogen-Oriented Physiological Sink.

When plants are infected by Plasmodiophora brassicae, their developmental programs are subjected to extensive changes and the resultant clubroot disease is associated with formation of large galls on underground tissue. The pathogen's need to build an efficient feeding site as the disease progresses drives these changes, ensuring successful production of resting spores. This developmental reprogramming is an outcome of interactions between the pathogen and the infected host. During disease progression, we can observe alteration of growth regulator dynamics, patterns of cell proliferation and differentiation, increased cell expansion, and eventual cell wall degradation as well as the redirection of nutrients toward the pathogen. Recently, detailed studies of anatomical changes occurring during infection and studies profiling transcriptional responses have come together to provide a clearer understanding of the sequence of events and processes underlying clubroot disease. Additionally, genome sequencing projects have revealed P. brassicae's potential for the production of signaling molecules and effectors as well as its requirements and capacities with respect to taking up host nutrients. Integration of these new findings together with physiological studies can significantly advance our understanding of how P. brassicae brings about reprogramming of host development. This article summarizes the current state of knowledge on cellular changes induced by P. brassicae infection and aims to explain their impact and importance for both the host and the pathogen.