**9. Understanding the mechanism of clubroot disease resistance**

The formation of galls on primary and secondary roots is typically characteristic of clubroot disease. The modification of root structure and decaying of root galls eventually damages plant root systems so the plants may completely die or dramatically reduce productivity. Arabi‐ dopsis is a model plant and relative to the Brassica species, thus it has been successfully used in clubroot research. Malinowski et al. [96] investigated the relationship of cell division, gall formation, and clubroot disease development in Arabidopsis. Using those genes involved in cell division as molecular markers, their data suggested that reducing gall formation by inhibiting cell division would not prevent pathogen from finishing the life cycle while large galls may help pathogens produce more resting spores.

been fine mapped and some clubroot resistance genes have been eventually cloned (Table 1). The *Crr3* locating on chromosome R3 was first mapped to a small genetic region between 0.35 cM genetic distance using 888 F2 individual plants [85]. In another report, the clubroot resistance locus *CRa* has been further analyzed to identify the candidate gene [93]. Over 1,600 F2 individual plants were used to select 80 recombinants using two closely linked molecular markers. Further analysis of those recombinants allowed identifying one open reading frame located on chromosome R3, which belongs to a typical resistance gene family and encodes a TIR-NBS-LRR protein [93]. More recently, there are two other independent reports that focused on fine mapping of clubroot resistance loci on chromosome R3. The *CRb* clubroot resistance locus which was described to be effective to *P. brassicae* isolates No. 14, a very aggressive isolate in Japan, has been fine mapped [94]. Using over 2,000 F2 individual plants and F3 progeny testing, 92 F2 recombinants between two closely linked molecular markers were identified. The analysis of these 92 F2 recombinants suggested that the *CRb* clubroot resistance locus might be the same as the *CRa* locus and the *CRa* and *CRb* clubroot resistance loci are different from the clubroot resistance locus *Crr3* [94]. Similarly, gene mapping of five Chinese cabbage cultivars was performed and all these hybrid cultivars were found to contain the same clubroot resistance locus on chromosome R3 [95]. They further fine mapped the clubroot resistance locus in Chinese cabbage to a 187 kilo-base pair (kb) chromosomal region using a large segregating population with over 8,000 individual plants. Molecular markers which are closely linked to the mapped clubroot resistance locus have been developed and those molecular markers can be used in marker-assisted selection to breed Chinese cabbage with clubroot

Characterization of clubroot resistance genes offers opportunities for further understanding clubroot resistance and interactions of resistance genes and pathogens. Hatakeyama et al. [86] cloned one clubroot resistance gene *Crr1a* on chromosome R8 and confirmed the resistance through plant transformation. Some transgenic *B. rapa* plants are resistant while others are susceptible, suggesting that the *Crr1a* gene might not explain the whole clubroot resistance in the original locus. They also found that *Crr1a* and *Crr1b* were tandem repeats in the same locus and both genes encode typical resistance gene proteins with TIR-NBS-LRR structures.

Based on the previous reports and whole genome sequencing data, clubroot resistance loci on chromosome 3 in *B. rapa* also contain multiple genes that encode TIR-NBS-LRR proteins. The complexity of those clubroot resistance loci needs to be investigated further. When a clubroot resistance locus contains multiple genes encoding the similar proteins, it becomes challenging to know how each individual gene plays a role in the clubroot resistance and how they contribute to the differences of alleles from various resistant sources. It is necessary to further dissect those complex clubroot resistance loci and investigate each individual gene to under‐ stand the functional properties of those loci. Therefore, gene functional analysis for clubroot

The formation of galls on primary and secondary roots is typically characteristic of clubroot disease. The modification of root structure and decaying of root galls eventually damages plant

resistance is still an important research focus in Brassica species.

**9. Understanding the mechanism of clubroot disease resistance**

resistance.

12 Plants for the Future

The expression of genes involved in the progression of clubroot disease may change so transcriptome analysis can be used to pinpoint the dynamic changing of gene expression in metabolic pathways for clubroot disease development. Schuller et al. [97] used laser micro‐ dissection and microarray analysis to check the changes of gene expression and found that the genes involved in the metabolism of plant hormones, especially auxin, cytokinin, and brassi‐ nosteriod, and plant defense-related hormones such as jasmonate and ethylene were differ‐ entially regulated. In another microarray analysis in Arabidopsis, Jubault et al. [98] observed that the major differences of gene expression in partial resistance interaction and susceptible interaction of the same Arabidopsis accession inoculated with two different clubroot isolates. The results showed that reduced or delayed metabolomic changes by pathogen and early induced classical defense responses were the major scenarios leading to partial clubroot phenotype instead of full susceptibility. More recently, Chu et al. [99] used RNA sequencing technology to identify over 2,000 genes that were expressed differentially in clubroot-resistant and susceptible plants. They found that those genes involved in defense responses such as jasmonic acid, ethylene, callose deposition, and indole glucosinolates were upregulated, and the expression of some genes in the pathway of salicylic acid did not show changes while the genes in the auxin biosynthesis and cell growth and development showed reduced expression in clubroot-resistant plants. By inducing clubroot resistance with an endophytic fungus, *Heteroconium chaetospira*, Lahlali et al. [100] detected the upregulation of genes involved in plant defense interaction such as PR-2 and genes in phenylpropanoid biosynthesis, and in the metabolism of plant hormones such as jasmonic acid, auxin, and ethylene using qPCR. Moreover, Verma et al. [101] performed miRNA analysis using miRNA-based microarray to detect differentially expressed miRNA during clubroot development. They further predicted the targets of those differentially expressed miRNA which belong to transcription factors, plant hormone-related and stress-related genes. In general, the data collected in those reports are quite preliminary and more research are required to know how each individual dominant clubroot resistance gene interacts with some avirulence genes in pathogen and eventually the interaction changes the expression of downstream genes which leads to clubroot resistance.
