Getting to the root of a club - Understanding developmental manipulation by the clubroot pathogen.

Plasmodiophora brassicae Wor., the clubroot pathogen, is the perfect example of an "atypical" plant pathogen. This soil-borne protist and obligate biotrophic parasite infects the roots of cruciferous crops, inducing galls or clubs that lead to wilting, loss of productivity, and plant death. Unlike many other agriculturally relevant pathosystems, research into the molecular mechanisms that underlie clubroot disease and Plasmodiophora-host interactions is limited. After release of the first P. brassicae genome sequence and subsequent availability of transcriptomic data, the clubroot research community have implicated the involvement of phytohormones during the clubroot pathogen's manipulation of host development. Herein we review the main events leading to the formation of root galls and describe how modulation of select phytohormones may be key to modulating development of the plant host to the benefit of the pathogen. Effector-host interactions are at the base of different strategies employed by pathogens to hijack plant cellular processes. This is how we suspect the clubroot pathogen hijacks host plant metabolism and development to induce nutrient-sink roots galls, emphasizing a need to deepen our understanding of this master manipulator.

Natural variation in Arabidopsis responses to Plasmodiophora brassicae reveals an essential role for Resistance to Plasmodiophora brasssicae 1 (RPB1).

Despite the identification of clubroot resistance genes in various Brassica crops our understanding of the genetic basis of immunity to Plasmodiophora brassicae infection in the model plant Arabidopsis thaliana remains limited. To address this issue, we performed a screen of 142 natural accessions and identified 11 clubroot-resistant Arabidopsis lines. Genome-wide association analysis identified several genetic loci significantly linked with resistance. Three genes from two of these loci were targeted for deletion by CRISPR/Cas9 mutation in resistant accessions Est-1 and Uod-1. Deletion of Resistance to Plasmodiophora brassicae 1 (RPB1) rendered both lines susceptible to the P. brassicae pathotype P1+. Further analysis of rpb1 knock-out Est-1 and Uod-1 lines showed that the RPB1 protein is required for activation of downstream defence responses, such as the expression of phytoalexin biosynthesis gene CYP71A13. RPB1 has recently been shown to encode a cation channel localised in the endoplasmic reticulum. The clubroot susceptible Arabidopsis accession Col-0 lacks a functional RPB1 gene; when Col-0 is transformed with RPB1 expression driven by its native promoter it is capable of activating RPB1 transcription in response to infection, but this is not sufficient to confer resistance. Transient expression of RPB1 in Nicotiana tabacum induced programmed cell death in leaves. We conclude that RPB1 is a critical component of the defence response to P. brassicae infection in Arabidopsis, acting downstream of pathogen recognition but required for the elaboration of effective resistance.

Systematic analysis of Heat Shock Protein 70 (HSP70) gene family in radish and potential roles in stress tolerance.

The 70 kD heat shock proteins (HSP70s) represent a class of molecular chaperones that are widely distributed in all kingdoms of life, which play important biological roles in plant growth, development, and stress resistance. However, this family has not been systematically characterized in radish (Raphanus sativus L.). In this study, we identified 34 RsHSP70 genes unevenly distributed within nine chromosomes of R. sativus. Phylogenetic and multiple sequence alignment analyses classified the RsHSP70 proteins into six distinct groups (Group A-F). The characteristics of gene structures, motif distributions, and corresponding cellular compartments were more similar in closely linked groups. Duplication analysis revealed that segmental duplication was the major driving force for the expansion of RsHSP70s in radish, particularly in Group C. Synteny analysis identified eight paralogs (Rs-Rs) in the radish genome and 19 orthologs (Rs-At) between radish and Arabidopsis, and 23 orthologs (Rs-Br) between radish and Chinese cabbage. RNA-seq analysis showed that the expression change of some RsHSP70s were related to responses to heat, drought, cadmium, chilling, and salt stresses and Plasmodiophora brassicae infection, and the expression patterns of these RsHSP70s were significantly different among 14 tissues. Furthermore, we targeted a candidate gene, RsHSP70-23, the product of which is localized in the cytoplasm and involved in the responses to certain abiotic stresses and P. brassicae infection. These findings provide a reference for further molecular studies to improve yield and stress tolerance of radish.

A CRISPR/Cas9-based vector system enables the fast breeding of selection-marker-free canola with Rcr1-rendered clubroot resistance.

Breeding for disease resistance in major crops is of crucial importance for global food security and sustainability. However, common biotechnologies such as traditional transgenesis or genome editing do not provide an ideal solution, whereas transgenic crops free of selection markers such as cisgenic/intragenic crops might be suitable. In this study, after cloning and functional verification of the Rcr1 gene for resistance to clubroot (Plasmodiophora brassicae), we confirmed that the genes Rcr1, Rcr2, Rcr4, and CRa from Brassica rapa crops and the resistance gene from B. napus oilseed rape cv. 'Mendel' on chromosome A03 were identical in their coding regions. We also determined that Rcr1 has a wide distribution in Brassica breeding materials and renders potent resistance against multiple representative clubroot strains in Canada. We then modified a CRISPR/Cas9-based cisgenic vector system and found that it enabled the fast breeding of selection-marker-free transgenic crops with add-on traits, with selection-marker-free canola (B. napus) germplasms with Rcr1-rendered stable resistance to clubroot disease being successfully developed within 2 years. In the B. napus background, the intragenic vector system was able to remove unwanted residue sequences from the final product with high editing efficiency, and off-target mutations were not detected. Our study demonstrates the potential of applying this breeding strategy to other crops that can be transformed by Agrobacterium. Following the streamlined working procedure, intragenic germplasms can be developed within two generations, which could significantly reduce the breeding time and labor compared to traditional introgression whilst still achieving comparable or even better breeding results.

A Hydroponic-Based Bioassay to Facilitate Plasmodiophora brassicae Phenotyping.

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most devastating diseases affecting the canola/oilseed rape (Brassica napus) industry worldwide. Currently, the planting of clubroot-resistant (CR) cultivars is the most effective strategy used to restrict the spread and the economic losses linked to the disease. However, virulent P. brassicae isolates have been able to infect many of the currently available CR cultivars, and the options to manage the disease are becoming limited. Another challenge has been achieving consistency in evaluating host reactions to P. brassicae infection, with most bioassays conducted in soil and/or potting medium, which requires significant space and can be labor intensive. Visual scoring of clubroot symptom development can also be influenced by user bias. Here, we have developed a hydroponic bioassay using well-characterized P. brassicae single-spore isolates representative of clubroot virulence in Canada, as well as field isolates from three Canadian provinces in combination with canola inbred homozygous lines carrying resistance genetics representative of CR cultivars available to growers in Canada. To improve the efficiency and consistency of disease assessment, symptom severity scores were compared with clubroot evaluations based on the scanned root area. According to the results, this bioassay offers a reliable, less expensive, and reproducible option to evaluate P. brassicae virulence, as well as to identify which canola resistance profile(s) may be effective against particular isolates. This bioassay will contribute to the breeding of new CR canola cultivars and the identification of virulence genes in P. brassicae that could trigger resistance and that have been very elusive to this day.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Fengycin produced by Bacillus subtilis XF-1 plays a major role in the biocontrol of Chinese cabbage clubroot via direct effect and defense stimulation.

Bacillus subtilis XF-1 is a well-investigated biocontrol agent against the biotrophic Plasmodiophora brassicae Woron., the causal agent of clubroot disease of cruciferous crops. The present study demonstrates that XF-1 could efficiently control clubroot disease via leaf spraying and provides an understanding of the biocontrol mechanisms. High-performance thin-layer chromatography (HTPLC) analysis indicated the presence of fengycin-type cyclopeptides in the supernatant. A ppsB deletion mutant of XF-1 resulted in no fengycin production, significantly reduced the lysis rate of testing spores in vitro and the primary infection rate of root hair in vivo, and decreased the protection value against clubroot disease under the greenhouse conditions. Confocal laser scanning microscopy proved that fengycin was not required for leaf internalization and root colonization. Moreover, the expression level of the ppsB gene in XF-1 was regulated by its cell density in root during interaction with P. brassicae. In addition, the ΔppsB mutant of XF-1 could not efficiently control disease because it led to a lower activation level of the jasmonic acid and salicylic acid signaling pathways in roots, which are necessary for the plant defense reaction upon pathogen invasion. Altogether, the present study provides a new understanding of specific cues in the interaction between B. subtilis and P. brassicae as well as insights into the application of B. subtilis in agriculture.

Phagocytosis underpins the biotrophic lifestyle of intracellular parasites in the class Phytomyxea (Rhizaria).

Phytomyxea are intracellular biotrophic parasites infecting plants and stramenopiles, including the agriculturally impactful Plasmodiophora brassicae and the brown seaweed pathogen Maullinia ectocarpii. They belong to the clade Rhizaria, where phagotrophy is the main mode of nutrition. Phagocytosis is a complex trait of eukaryotes, well documented for free-living unicellular eukaryotes and specific cellular types of animals. Data on phagocytosis in intracellular, biotrophic parasites are scant. Phagocytosis, where parts of the host cell are consumed at once, is seemingly at odds with intracellular biotrophy. Here we provide evidence that phagotrophy is part of the nutritional strategy of Phytomyxea, using morphological and genetic data (including a novel transcriptome of M. ectocarpii). We document intracellular phagocytosis in P. brassicae and M. ectocarpii by transmission electron microscopy and fluorescent in situ hybridization. Our investigations confirm molecular signatures of phagocytosis in Phytomyxea and hint at a small specialized subset of genes used for intracellular phagocytosis. Microscopic evidence confirms the existence of intracellular phagocytosis, which in Phytomyxea targets primarily host organelles. Phagocytosis seems to coexist with the manipulation of host physiology typical of biotrophic interactions. Our findings resolve long debated questions on the feeding behaviour of Phytomyxea, suggesting an unrecognized role for phagocytosis in biotrophic interactions.

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.

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.

Effects of Exogenous Ergothioneine on Brassica rapa Clubroot Development Revealed by Transcriptomic Analysis.

Clubroot disease is a soil-borne disease caused by Plasmodiophora brassicae that leads to a serious yield reduction in cruciferous plants. In this study, ergothioneine (EGT) was used to culture P. brassicae resting spores, the germination of which was significantly inhibited. Further exogenous application of EGT and P. brassicae inoculation in Chinese cabbage showed that EGT promoted root growth and significantly reduced the incidence rate and disease index. To further explore the mechanism by which EGT improves the resistance of Chinese cabbage to clubroot, a Chinese cabbage inbred line BJN3-2 susceptible to clubroot treated with EGT was inoculated, and a transcriptome analysis was conducted. The transcriptome sequencing analysis showed that the differentially expressed genes induced by EGT were significantly enriched in the phenylpropanoid biosynthetic pathway, and the genes encoding related enzymes involved in lignin synthesis were upregulated. qRT-PCR, peroxidase activity, lignin and flavonoid content determination showed that EGT promoted the lignin and flavonoid synthesis of Chinese cabbage and improved its resistance to clubroot. This study provides a new insight for the comprehensive prevention and control of cruciferous clubroot and for further study of the effects of EGT on clubroot disease.

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 of biotin carboxyl carrier subunits of acetyl-CoA carboxylase in Brassica and their role in stress tolerance in oilseed Brassica napus.

BACKGROUND: Biotin carboxyl carrier protein (BCCP) is a subunit of Acetyl CoA-carboxylase (ACCase) which catalyzes the conversion of acetyl-CoA to malonyl-CoA in a committed step during the de novo biosynthesis of fatty acids. Lipids, lipid metabolites, lipid-metabolizing and -modifying enzymes are known to play a role in biotic and abiotic stress tolerance in plants. In this regard, an understanding of the Brassica napus BCCP genes will aid in the improvement of biotic and abiotic stress tolerance in canola. RESULTS: In this study, we identified 43 BCCP genes in five Brassica species based on published genome data. Among them, Brassica rapa, Brassica oleracea, Brassica nigra, Brassica napus and Brassica juncea had six, seven, seven, 10 and 13 BCCP homologs, respectively. Phylogenetic analysis categorized them into five classes, each with unique conserved domains. The promoter regions of all BCCP genes contained stress-related cis-acting elements as determined by cis-element analysis. We identified four and three duplicated gene pairs (segmental) in B. napus and B. juncea respectively, indicating the role of segmental duplication in the expansion of this gene family. The Ka/Ks ratios of orthologous gene pairs between Arabidopsis thaliana and five Brassica species were mostly less than 1.0, implying that purifying selection, i.e., selective removal of deleterious alleles, played a role during the evolution of Brassica genomes. Analysis of 10 BnaBCCP genes using qRT-PCR showed a different pattern of expression because of exposure of the plants to biotic stresses, such as clubroot and sclerotinia diseases, and abiotic stresses such as drought, low temperature and salinity stresses. CONCLUSIONS: The identification and functional analysis of the Brassica BCCPs demonstrated that some of these genes might play important roles in biotic and abiotic stress responses. Results from this study could lay the foundation for a better understanding of these genes for the improvement of Brassica crops for stress tolerance.

Allelic variation of a clubroot resistance gene (Crr1a) in Japanese cultivars of Chinese cabbage (Brassica rapa L.).

Clubroot resistance (CR) is an important trait in Chinese cabbage breeding worldwide. Although Crr1a, the gene responsible for clubroot-resistance, has been cloned and shown to encode the NLR protein, its allelic variation and molecular function remain unknown. Here, we investigated the sequence variation and function of three Crr1a alleles cloned from six CR F1 cultivars of Chinese cabbage. Gain-of-function analysis revealed that Crr1aKinami90_a isolated from the cv. 'Kinami 90' conferred clubroot resistance as observed for Crr1aG004 . Because two susceptible alleles commonly lacked 172 amino acids in the C-terminal region, we investigated clubroot resistance in transgenic Arabidopsis harboring the chimeric Crr1a, in which 172 amino acids of the functional alleles were fused to the susceptible alleles. The fusion of the C-terminal region to the susceptible alleles restored resistance, indicating that their susceptibility was caused by the lack of the C-terminus. We developed DNA markers to detect the two functional Crr1a alleles, and demonstrated that the functional Crr1a alleles were frequently found in European fodder turnips, whereas they were rarely introduced into Japanese CR cultivars of Chinese cabbage. These results would contribute to CR breeding via marker-assisted selection and help our understanding of the molecular mechanisms underlying clubroot resistance.

A Loop-Mediated Isothermal DNA Amplification (LAMP) Assay for Detection of the Clubroot Pathogen Plasmodiophora brassicae.

Clubroot caused by Plasmodiophora brassicae is a serious threat to cruciferous crops around the world. The resting spores of P. brassicae are a primary source of infection and can survive in soil for many years. Detection of resting spores in soil is essential for forecasting clubroot prevalence. Detection of P. brassicae has been relying on plant bioassays or PCR-based methods. The loop-mediated isothermal DNA amplification (LAMP) is a promising approach for microorganism detection with the advantage of high sensitivity, accuracy, and convenience in viewing. In this study, we developed a LAMP assay for detection of P. brassicae in soil, roots, and seeds. This method can detect P. brassicae at a minimal amount of 1 fg of plasmid DNA or 10 resting spores in the soil. Compared with conventional PCR, the LAMP was more sensitive in detection of P. brassicae at the lower levels in soil samples. In conclusion, we elaborated a sensitive, accurate, and easy-to-use LAMP assay to detect P. brassicae, which will facilitate sustainable clubroot management and planning.

First Report of Plasmodiophora brassicae causing clubroot on Thlaspi arvense L. in Sichuan Province of China.

Pennycress (Thlaspi arvense L.), an annual herb of the mustard family Brassicacae, is native to Eurasia and now widely distributed throughout temperate North America. This species is currently being developed as a medicinal herb used to treat nephritis in China and an oilseed crop for biofuel production (Roque et al.2012). In November 2020, stunt and wilt symptoms were observed on above ground parts and swollen club-shaped galls were observed on the roots of T. arvense in most of the Chinese cabbage growing area in Kangding (30°03'"N,102°02'"E), Sichuan Province of China. The average disease incidence of swollen roots on T. arvense was 91.2% ( n=80). To identify the causal agent of this disease, the swollen roots of T. arvense were collected, crushed and observed under microscope (Fei et al.2017). Abundant resting spores were found in the root galls, which were spherical and 2.0 to 3.1 µm in diameter with an average length of 2.7 nm (n=100). The healthy roots and the root galls of T. arvense plants were further evaluated by PCR with P. brassicae-specific primers TC2F/TC2R (Cao et al. 2007). The results showed that a DNA fragment with an estimated size of 520 bp, as expected for that of P. brassicae, was consistently amplified in diseased roots, No PCR amplification occurred in the healthy roots with the TC2F/TC2R primers. Blast analysis of the 520 bp segment (GenBankMZ040496) showed the highest identity with the sequence of small subunit ribosomal RNA gene of P. brassicae (GenBankMH762161, 97.7%, E value=0). These results confirmed that the pathogen in the galled roots of T. arvense was P. brassicae. The pathogenicity of isolated P. brassicae was tested on both T. arvense and Chinese cabbage (B. campestris ssp. pekinensis). Resting spores were isolated from the diseased roots (Castlkbury et al. 1994) of T. arvense and suspended in Hoagland's solution to the final concentration of 1 × 107 spores per milliliter. Fifteen plastic pots (10 cm bottom diameter, 16 cm upper diameter, 13 cm high) were filled with soil (1 kg per pot) that was sterilized twice with high-temperature (121℃), high pressure (19 PSI) for 1.5 hours with a time interval of 2 days between. Inoculated pots received 100 mL spore suspension each. Fifteen control pots with sterilized field soil were treated with 100 mL Hoagland's solution each. Seeds of T. arvense and B. campestris were pre-germinated at 20°C on moist filter paper for 7 days and transplanted into the pots, five seedlings each and five pots per treatment. The pots were maintained in a greenhouse with 16 hours photoperiod at 24°C/16°C day/night temperature. After 7 weeks, plants in each pot were uprooted and the roots cleaned in running water and inspected for clubroot symptoms. Plants of T. arvense and Chinese cabbage in pots inoculated with resting spores showed clubroot symptoms while no disease symptoms were observed on any control plants. The disease incidence rate was 95.4% on T. arvense and 81.2% on B. campestris. Therefore, it was confirmed that P. brassicae could cause clubroot disease on T. arvense. To our knowledge, this is the first published report of clubroot disease on T. arvense in China. This finding is helpful for the management of clubroot on herbs and plants of biological origin in the cruciferous family.

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.

Sugar Transporters in Plasmodiophora brassicae: Genome-Wide Identification and Functional Verification.

Plasmodiophora brassicae, an obligate intracellular pathogen, can hijack the host's carbohydrates for survival. When the host plant is infected by P. brassicae, a large amount of soluble sugar accumulates in the roots, especially glucose, which probably facilitates the development of this pathogen. Although a complete glycolytic and tricarboxylic acid cycle (TCA) cycle existed in P. brassicae, very little information about the hexose transport system has been reported. In this study, we screened 17 putative sugar transporters based on information about their typical domains. The structure of these transporters showed a lot of variation compared with that of other organisms, especially the number of transmembrane helices (TMHs). Phylogenetic analysis indicated that these sugar transporters were far from the evolutionary relationship of other organisms and were unique in P. brassicae. The hexose transport activity assay indicated that eight transporters transported glucose or fructose and could restore the growth of yeast strain EBY.VW4000, which was deficient in hexose transport. The expression level of these glucose transporters was significantly upregulated at the late inoculation time when resting spores and galls were developing and a large amount of energy was needed. Our study provides new insights into the mechanism of P. brassicae survival in host cells by hijacking and utilizing the carbohydrates of the host.

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.

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.