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.


Introduction
Trehalose is a nonreducing disaccharide with two glucose molecules linked through an α, α-1,1-glucosidic bond, which has been found to exist in bacteria, fungi, algae, invertebrates, and plants [1]. Previous studies have found that trehalose is known as a living substance that exists in all living organisms and is involved in growth and development [2,3]. Trehalose biosynthesis pathways are widely distributed in nature. There are five pathways for trehalose biosynthesis that have been identified according to the different catalytic enzymes [4] ( Figure S1). The first pathway is called the TS pathway; trehalose synthase (TreS) in Pimelobacter sp. catalyzes the conversion of maltose into trehalose by intramolecular transglycosylation [5] ( Figure S1A). The second pathway (TreY/TreZ pathway) has been reported in thermophilic archaea of the genus Sulfolobus, which converts maltodextrin to trehalose via the catalysis of maltooligosyl trehalose synthase (TreY) and by pathogens [4]. Multi-host pathogen Pseudomonas aeruginosa synthesizes trehalose, which is required during infection of Arabidopsis leaves [38]. The inhibition of the biosynthesis of trehalose by the plant pathogen Ralstonia solanacearum contributes to the reduction of its pathogenicity, and it also indicates the important role of trehalose in the interaction between plants and pathogens [39].
Clubroot, a devastating disease affecting Brassica plants, is caused by the obligate biotroph protist Plasmodiophora brassicae and is characterized by the development of large galls on infected roots, inhibiting the uptake of nutrients and water from the soil [40]. Altered carbohydrate metabolism, including that of starch, soluble sugars, and inositol, is an important symptom of clubroot disease. Notably, a previous study found that high trehalose levels accumulated in infected tissues after P. brassicae inoculation [41], and this accumulation pattern was consistent with the expression of PbTPS1, a putative trehalose-6phosphate synthase gene from P. brassicae. Scholars have speculated that a large amount of trehalose is most likely synthesized by P. brassicae rather than by the host. The release of trehalose synthesized by P. brassicae into plants might interfere with the plant's trehalose-sensing system and alter the host's carbohydrates in the pathogen's favor [41]. Plasmodiophora brassicae is an obligate parasite that parasitizes the roots of cruciferous plants, but research on it is limited because it cannot be isolated and cultured in vitro. Therefore, it is unclear whether accumulated trehalose is synthesized by host plants or P. brassicae. To address this, we focused on the role of TPS genes in the trehalose synthesis pathway in the interaction between Brassica rapa and P. brassicae and performed genome-wide identification, characterization, and expression analysis of the TPS genes from P. brassicae and its host plant B. rapa.
We identified the TPS genes in the B. rapa and P. brassicae genomic data and investigated the functional classification, evolutionary characterization, and expression patterns of the TPS gene family. The present study enhances our understanding of the function and evolution of BrTPSs and PbTPSs. The study is also of great significance in clarifying the biosynthesis of trehalose in the interaction between Brassica rapa and P. brassicae.

Trehalose Sugar Content in Cabbage Roots after Infection with P. brassicae
Five weeks after inoculation with P. brassicae, each of the infected Chinese cabbage roots clearly showed typical symptoms of clubroot, and from 4 to 5 weeks post-inoculation (wpi), the clubroot became increasingly obvious ( Figure 1A). To investigate changes during the development of clubroot, the trehalose content of the roots was analyzed at four different stages after infection (from the second week to the fifth week) ( Figure 1B). From 2 to 3 wpi, the trehalose content was extremely low, and there was no difference between the healthy and infected plants. However, trehalose accumulated at 4 wpi when the trehalose content was slightly higher in infected plants than in healthy plants. Most notably at 5 wpi, trehalose increased greatly in infected plants, with a 1000-fold increase relative to healthy plants. This indicates that P. brassicae infection leads to the accumulation of a large amount of trehalose in Chinese cabbage roots.

Identification of TPS Family Members in B. rapa and P. brassicae
To analyze the biosynthesis of trehalose, we identified TPS genes in the B. rapa and P. brassicae genomes. Based on similarities with the 11 Arabidopsis AtTPSs, a total of 14 BrTPSs were identified in the B. rapa genome and named BrTPS1a to BrTPS11 based on their identity with AtTPSs. Among the 14 BrTPSs, 8 BrTPS genes had two copies corresponding to AtTPSs, while the orthologous gene of AtTPS3 was not found in the B. rapa genome. The majority of the BrTPS gene-coding sequences were about 2700 base pairs (bp), and the length of amino acid residues was about 900 aa. The shortest coding sequence of BrTPS5a was only 390 bp, and the length of the amino acids was 129 aa. The isoelectric point (pI) value of BrTPSs ranged from 4.7 to 9.48, and the protein molecular weight ranged The trehalose contents in the roots of Chinese cabbage after P. brassicae inoculation. Th abscissa represents the time post-inoculation, and the ordinate represents the value of sugar content The data represent mean values ± SDs. The asterisks indicate p-values (** p < 0.01) according to Stu dent's t test. Horizontal axis is the time point from 2 weeks to 5 weeks post-inoculation (wpi).

Identification of TPS Family Members in B. rapa and P. brassicae
To analyze the biosynthesis of trehalose, we identified TPS genes in the B. rapa and P. brassicae genomes. Based on similarities with the 11 Arabidopsis AtTPSs, a total of 14 BrTPSs were identified in the B. rapa genome and named BrTPS1a to BrTPS11 based on their identity with AtTPSs. Among the 14 BrTPSs, 8 BrTPS genes had two copies corre sponding to AtTPSs, while the orthologous gene of AtTPS3 was not found in the B. rapa genome. The majority of the BrTPS gene-coding sequences were about 2700 base pair (bp), and the length of amino acid residues was about 900 aa. The shortest coding se quence of BrTPS5a was only 390 bp, and the length of the amino acids was 129 aa. The isoelectric point (pI) value of BrTPSs ranged from 4.7 to 9.48, and the protein molecula weight ranged from 14.7 to 165.8 kDa. Subcellular localization predictions showed tha they were mainly localized in the chloroplast, vacuole, and cytoplasm (Table 1).
A total of three TPS genes were acquired by keyword search against the P. brassica genome in NCBI's nr database, namely PbTPS1, PbTPS2, and PbTPS3. The coding se quence length of the three PbTPS genes was longer than 2500 bp, and the length of the amino acid ranged from 853 to 860 aa. The molecular weight ranged from 95.50 to 96.49 kDa, and the predicted pI value ranged from 6.18 to 6.93. Subcellular localization predic tion indicated that the three PbTPS proteins were located in the cytoplasm (Table 2).  A total of three TPS genes were acquired by keyword search against the P. brassicae genome in NCBI's nr database, namely PbTPS1, PbTPS2, and PbTPS3. The coding sequence length of the three PbTPS genes was longer than 2500 bp, and the length of the amino acid ranged from 853 to 860 aa. The molecular weight ranged from 95.50 to 96.49 kDa, and the predicted pI value ranged from 6.18 to 6.93. Subcellular localization prediction indicated that the three PbTPS proteins were located in the cytoplasm ( Table 2).

Phylogenetic Analysis of BrTPSs and PbTPSs
To analyze the evolutionary relationships of BrTPS and PbTPS genes in B. rapa and P. brassicae, an unrooted phylogenetic tree was constructed using full-length amino acid sequences ( Figure 2). In total, The TPS sequences from 30 species were assessed in the phylogenetic tree (Supplementary Table S2). The TPSs of B. rapa were grouped into two major clades; BrTPS1a to BrTPS4 belonged to Clade I, and BrTPS5a to BrTPS11 belonged to Clade II. The PbTPSs belonged to a separate clade, which indicated that PbTPS is far away from other species in evolution. In addition, PbTPS3 and PbTPS1 were clustered into the same branch; PbTPS2 failed to cluster with them, which indicated that there are sequence differences between PbTPSs, and there may be functional differences.   Table S2). The TPSs of B. rapa were grouped into two major clades; BrTPS1a to BrTPS4 belonged to Clade I, and BrTPS5a to BrTPS11 belonged to Clade II. The PbTPSs belonged to a separate clade, which indicated that PbTPS is far away from other species in evolution. In addition, PbTPS3 and PbTPS1 were clustered into the same branch; PbTPS2 failed to cluster with them, which indicated that there are sequence differences between PbTPSs, and there may be functional differences.  All TPS proteins were divided into three subgroups, represented by three colors. The green color represents Class I proteins, the blue color represents Class II proteins. The yellow circle symbol represents the BrTPS proteins. The unrooted phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 bootstrap replications by MEGA6 software.

Gene Structure and Conserved Domain Analyses of BrTPSs and PbTPSs
Gene structure analysis is an available method for understanding gene evolutions and their potential roles. Thus, the structures of the BrTPS and PbTPS genes were investigated. For 14 BrTPS genes, the number of exons ranged from 2 to 17, and the majority of BrTPS genes (accounting for 42.8%) had three exons. BrTPS4 contained the most exons (17), whereas BrTPS5a harbored the fewest exons (2). Interestingly, BrTPS genes clustered in the same clade generally possessed a similar exon-intron structure, and genes in Clade I had more exons than genes in Clade II ( Figure 3A). Generally, analysis of these TPS gene structures showed that the conserved exon-intron structure within each cluster agreed with the classification of TPS genes in an NJ phylogenetic tree based on TPS sequences ( Figure 2). For PbTPS genes, the number of exons ranged from 7 to 10. Among them, PbTPS1 and PbTPS3 had 10 exons, but PbTPS2 contained 7 exons ( Figure 4A).
Domain analysis of the identified BrTPS and PbTPS protein sequences showed that, except BrTPS5a, the other TPS proteins contained a TPS structure domain (Glyco_transf_20) located at the N-terminal and a TPP domain (Trehalose_PPase) at the C-terminal. However, BrTPS5a contained only the TPP domain ( Figures 3B and 4B). To further elucidate the structural and functional features of BrTPSs and PbTPSs, 10 conserved motifs of the TPS proteins were identified using the MEME program. For BrTPS proteins, these motifs were conserved in most BrTPS proteins, except BrTPS7a and BrTPS5a, which harbored one and three motifs, respectively. The lengths of these motifs ranged from 21 to 50 amino acids. Among them, Motifs 1, 3, 4, 5, 6, 8, and 9 together composed the TPS domain (Glyco_transf_20). Motifs 2, 7, and 10 composed the TPP domain (Trehalose_PPase) ( Figure 3C). For PbTPS proteins, these motifs were almost conserved in PbTPS proteins, except PbTPS2, which lacked Motifs 5 and 8. The lengths of these motifs ranged from 18 to 50 aa. According to Figure

Identification of Cis-Acting Elements in the Promoter Region of BrTPS Genes
To ascertain the potential biological roles of BrTPS genes in B. rapa, 2000 bp sequences upstream of the start site of BrTPS genes were used to identify the potential cis-acting elements in the promoter region. A total of 285 functionally annotated cis-acting elements were predicted in these genes. Many cis-acting elements were involved in light responsiveness, stress responsiveness, hormone responsiveness, site binding, and other functions ( Figure 6A). Generally, the cis-acting elements were roughly classified into three categories of cis-elements linked to growth and development, defense and stress responsiveness, and hormone responsiveness ( Figure 6B). The cis-acting elements in growth and development were the most involved, followed by the cis-acting elements in response to hormones, and the elements in defense and stress were the least involved. Among the 14 BrTPS genes, BrTPS1a had the least cis-acting elements, while BrTPS11 contained the most ( Figure 6C). In the plant growth and development category (159/285), 79 cis-elements (accounting for 50%) were involved in light responsiveness, which accounted for the largest proportion in this category, and this element was contained in all 14 BrTPS genes. Eleven cis-elements were involved in anaerobic induction, 16 in endosperm expression, 8 in meristem expression, 4 in zein metabolism regulation, 10 and 24 as MYB and MYC binding sites, respectively, and 2 in circadian control and regulation. Some BrTPS genes elicit specific cis-acting elements, such as the maximal elicitor-mediated activation element only in BrTPS5b, the cell cycle regulatory element only in BrTPS10b, and the flavonoid biosynthetic element only in BrTPS4, which indicates the specificity of the function of these genes ( Figure 6B,D). In the defense and stress responsiveness category (42/285), eight cis-elements (accounting for 19%) were involved as WUN-motifs, eight (accounting for 19%) in stress responsiveness, six (accounting for 14%) in low-temperature responsiveness, four (accounting for 10%) in salicylic acid responsiveness, four in defense and stress responsiveness, and three (accounting for 7%) in drought inducibility. Additionally, the W-box element (accounting for 17%) was found in seven BrTPS genes. The dehydration responsiveness element (accounting for 2%) was found in BrTPS7b ( Figure 6B,D). In the hormone responsiveness category (84/285), various cis-elements were related to ethylene responsiveness (accounting for 17%), abscisic acid responsiveness (accounting for 43%), MeJA responsiveness (accounting for 19%), auxin responsiveness (accounting for 7%), and gibberellin responsiveness (accounting for 14%). Notably, the largest number of cis-elements was in the abscisic acid-responsive elements. The results suggest that most BrTPS genes might be acid-induced and/or -repressed genes ( Figure 6D).

Identification of Cis-Acting Elements in the Promoter Region of BrTPS Genes
To ascertain the potential biological roles of BrTPS genes in B. rapa, 2000 bp sequen upstream of the start site of BrTPS genes were used to identify the potential cis-ac elements in the promoter region. A total of 285 functionally annotated cis-acting elem were predicted in these genes. Many cis-acting elements were involved in light resp siveness, stress responsiveness, hormone responsiveness, site binding, and other fu tions ( Figure 6A). Generally, the cis-acting elements were roughly classified into three egories of cis-elements linked to growth and development, defense and stress respons

Expression of BrTPS Genes under P. brassicae Infection
To further explore the role of BrTPS genes responsive to P. brassicae infection, the expression patterns of 14 BrTPSs were determined in the roots of Chinese cabbage from 1 to 5 wpi using qRT-PCR (Figure 7). Generally, BrTPS genes exhibited distinct time-specific expression profiles, suggesting the functional divergence of BrTPS genes at different stages during growth and development. We found that most of the genes were highly expressed at 1 to 3 wpi but downregulated at 4 to 5 wpi by investigating the differences in the expression of these 14 BrTPS at five stages after infection with P. brassicae between the Ck and Pb plants. At 1 wpi, the expression levels of four genes (BrTPS1b, BrTPS5a, BrTPS7b, and BrTPS11) in Pb plants were significantly higher than those in Ck plants; at 2 wpi, the expression levels of six genes (BrTPS5a, BrTPS6, BrTPS7a, BrTPs7b, BrTPs10b, and BrTPS11) in Pb plants were significantly higher than those in Ck plants; at 3 wpi, the expression levels of six genes (BrTPS2, BrTPS8, BrTPS9, BrTPs10a, BrTPs10b, and BrTPS11) in Pb plants were upregulated than those in Ck plants. Interestingly, only BrTPS5b and BrTPS5a were upregulated at 4 and 5 wpi, compared to Ck plants. Since trehalose was mainly accumulated at 4 to 5 wpi, it was worth noting that although BrTPS5a was upregulated relative to CK plants at 5 wpi, it has a higher expression at 2 wpi, and trehalose did not accumulate in a large amount at that time; similarly, we also noticed that the expression of BrTPS5b in Ck plants was higher than that in Pb plants at 5 wpi. Therefore, we speculated that BrTPS genes may play little role in trehalose synthesis.

Expression of BrTPS Genes under P. brassicae Infection
To further explore the role of BrTPS genes responsive to P. brassicae infection, the expression patterns of 14 BrTPSs were determined in the roots of Chinese cabbage from 1 to 5 wpi using qRT-PCR (Figure 7). Generally, BrTPS genes exhibited distinct time-specific expression profiles, suggesting the functional divergence of BrTPS genes at different stages during growth and development. We found that most of the genes were highly expressed at 1 to 3 wpi but downregulated at 4 to 5 wpi by investigating the differences in the expression of these 14 BrTPS at five stages after infection with P. brassicae between the Ck and Pb plants. At 1 wpi, the expression levels of four genes (BrTPS1b, BrTPS5a BrTPS7b, and BrTPS11) in Pb plants were significantly higher than those in Ck plants; at 2 wpi, the expression levels of six genes (BrTPS5a, BrTPS6, BrTPS7a, BrTPs7b, BrTPs10b and BrTPS11) in Pb plants were significantly higher than those in Ck plants; at 3 wpi, the expression levels of six genes (BrTPS2, BrTPS8, BrTPS9, BrTPs10a, BrTPs10b, and BrTPS11) in Pb plants were upregulated than those in Ck plants. Interestingly, only BrTPS5b and BrTPS5a were upregulated at 4 and 5 wpi, compared to Ck plants. Since trehalose was mainly accumulated at 4 to 5 wpi, it was worth noting that although BrTPS5a was upregulated relative to CK plants at 5 wpi, it has a higher expression at 2 wpi, and trehalose did not accumulate in a large amount at that time; similarly, we also noticed that the expression of BrTPS5b in Ck plants was higher than that in Pb plants at 5 wpi. Therefore, we speculated that BrTPS genes may play little role in trehalose synthesis

Expression Analyses of PbTPS Genes
To determine the role of PbTPS genes correlated with the accumulation of trehalose, a semi-quantitative PCR analysis was carried out in the root samples of Pb and Ck plants to analyze the expression of the three PbTPS genes. PbTPS genes were amplified only in Pb plants ( Figure 8A). A semi-quantitative PCR analysis of PbTPSs was carried out at the five stages (from 1 to 5 wpi) after inoculation. The transcripts were detected in the fourth and fifth weeks after inoculation, while the PbTPS3 gene was not detected at any stage ( Figure 8B). Quantitative RT-PCR showed that PbTPS1 and PbTPS2 were significantly upregulated from 4 to 5 wpi. Specifically, PbTPS2 had the highest expression level, which was increased by more than 10-fold ( Figure 8C).

Expression Analyses of PbTPS Genes
To determine the role of PbTPS genes correlated with the accumulation of tre a semi-quantitative PCR analysis was carried out in the root samples of Pb and Ck to analyze the expression of the three PbTPS genes. PbTPS genes were amplified Pb plants ( Figure 8A). A semi-quantitative PCR analysis of PbTPSs was carried ou five stages (from 1 to 5 wpi) after inoculation. The transcripts were detected in the and fifth weeks after inoculation, while the PbTPS3 gene was not detected at an ( Figure 8B). Quantitative RT-PCR showed that PbTPS1 and PbTPS2 were significan regulated from 4 to 5 wpi. Specifically, PbTPS2 had the highest expression level, was increased by more than 10-fold ( Figure 8C).

Discussion
Plant pathogens tend to alter carbohydrate transport and distribution in host t a process involving different types of sugars [42,43]. It has been proposed that the gen attempts to manipulate the carbohydrate metabolism of the host in the path favor [44]. Previous studies have shown the accumulation of soluble sugars in C cabbage tissues after P. brassicae infection, suggesting that P. brassicae infection cou ger active sugar translocation between the sugar-producing tissues and the clubbed [45]. In addition, a previous study reported a significant accumulation of trehalose abidopsis roots and hypocotyls after P. brassicae infection. In this study, we observ the contents of trehalose were significantly increased in Chinese cabbage clubroo wpi, a 1000-fold increase compared to control plants. Almost no trehalose was dete infected plants at 2 and 3 wpi, indicating that trehalose was mainly synthesized the later stages of clubroot development. Simultaneously, smaller amounts of tre were also found in healthy plants, indicating that plants are capable of synthesizin amounts of trehalose during growth and development. This result was consisten previous reports that trehalose accumulates in the roots and hypocotyls of infecte bidopsis [41].
TPS is the primary enzyme responsible for catalyzing the trehalose formation fore, elucidating the role of TPS genes in trehalose biosynthesis and their identif and analysis in both host plants and P. brassicae are of interest. Genes encoding TP been identified in many plants in the form of a gene family [46]. The Arabidopsis TP

Discussion
Plant pathogens tend to alter carbohydrate transport and distribution in host tissues, a process involving different types of sugars [42,43]. It has been proposed that the pathogen attempts to manipulate the carbohydrate metabolism of the host in the pathogen's favor [44]. Previous studies have shown the accumulation of soluble sugars in Chinese cabbage tissues after P. brassicae infection, suggesting that P. brassicae infection could trigger active sugar translocation between the sugar-producing tissues and the clubbed tissue [45]. In addition, a previous study reported a significant accumulation of trehalose in Arabidopsis roots and hypocotyls after P. brassicae infection. In this study, we observed that the contents of trehalose were significantly increased in Chinese cabbage clubroots at 5 wpi, a 1000-fold increase compared to control plants. Almost no trehalose was detected in infected plants at 2 and 3 wpi, indicating that trehalose was mainly synthesized during the later stages of clubroot development. Simultaneously, smaller amounts of trehalose were also found in healthy plants, indicating that plants are capable of synthesizing small amounts of trehalose during growth and development. This result was consistent with previous reports that trehalose accumulates in the roots and hypocotyls of infected Arabidopsis [41].
TPS is the primary enzyme responsible for catalyzing the trehalose formation; therefore, elucidating the role of TPS genes in trehalose biosynthesis and their identification and analysis in both host plants and P. brassicae are of interest. Genes encoding TPS have been identified in many plants in the form of a gene family [46]. The Arabidopsis TPS gene family contains 11 members (AtTPS1-11) [16], rice contains 11 members (OsTPS1-11) [26], tomato contains 10 members (SlTPS1-10) [47], and watermelon contains 7 members (ClTPS1-7) [3]. However, the TPS gene family in B. rapa and P. brassicae has not been well studied. Herein, 14 BrTPS genes were identified in the B. rapa genome, and 3 PbTPS genes were identified in the P. brassicae genome. According to their gene structure and enzyme activity, the TPS family of genes in plants are classified into two major clades: Clades I and II. In the Arabidopsis genome, four genes belong to Clade I (AtTPS1-4) and seven to Clade II (AtTPS5-11) [25]. Brassica rapa TPS genes were also divided into two subfamilies: Clades I (BrTPS1a to BrTPS4) and II (BrTPS5a to BrTPS11), which was consistent with the classification in Arabidopsis [25] and rice [26]. There were 14 BrTPS genes, which was greater than the number of genes in A. thaliana and rice. In addition, Arabidopsis, a species closely related to the Brassica genus, contained more TPS genes than B. rapa, suggesting that there may be functional redundancy or divarication between the TPS members. However, the occurrence of gene loss during polyploid speciation was also found in the B. rapa genome corresponding to Arabidopsis TPS genes. For example, the AtTPS3 homolog was absent in the B. rapa genome. Some traits differed between the two BrTPS clades, such as in gene structure and gene length, but especially the gene structure. We observed that the structure of the BrTPS genes in Clades I and II was very different, and the number of introns and exons in Clade I was significantly greater than that in Clade II. Previous studies have suggested three mechanisms (exon/intron gain/loss, exonization/pseudoexonization, and insertion/deletion) that may lead to differences in gene structure [48], and a close relationship between the structure and function of genes has been observed [49]. Moreover, it was stated that the exon/intron number could affect the expression level; genes with fewer introns might be quickly induced [50,51]. Therefore, the BrTPS genes in Clades I and II may have experienced functional differentiation during evolution. Domain analysis showed that most BrTPS proteins had a TPS domain (Glyco_transf_20) at the N-terminus and a TPP domain (Trehalose_PPase) at the C-terminus, which was consistent with the results of other studies [11,16,27,46]. However, BrTPS5a contains only a Trehalose_PPase domain. Similarly, three GhTPS proteins (GhTPS6, GhTPS4, and GhTPS9) in cotton also lack a TPP domain [11], and the loss of the domain may be the result of evolution [46]. Conserved motif analysis demonstrated that the conserved number of BrTPS5a and BrTPS7a was less than that of the other BrTPSs, which may be related to the length of the genes. As for PbTPSs, there was no significant difference in the gene structure and conserved motif of the three PbTPSs, and all possessed a TPS domain at the N-terminal and a TPP domain at the C-terminal. Cis-acting elements were involved in the regulation of gene expression [46]. Previous studies have shown that TPS genes provide stress tolerance in different plant species. For example, plants overexpressing AtTPS1 improved drought resistance in Arabidopsis [52]. Overexpression of OsTPS8 was adequate to confer enhanced salinity tolerance [53], and watermelon ClTPS3 overexpression in Arabidopsis thaliana significantly improved salt tolerance [3]. In this study, a variety of signal response elements were contained in the promoter regions of BrTPS genes. Moreover, many BrTPS genes contain different stress response elements, indicating that BrTPS genes are involved in stress defense processes.
Trehalose is not only used as a stored energy but also serve as a protectant, when encountering drought, cold, osmotic stress, oxidation, and other stress conditions [5]. To determine whether large amounts of trehalose were synthesized by the host or P. brassicae, we examined the expression patterns of BrTPS and PbTPS genes under P. brassicae stress. At 4 and 5 wpi, the expression level of most BrTPS genes in Pb plants was not significantly higher than that in Ck plants, except for BrTPS5a and BrTPS5b. Although the expression level of BrTPS5a in Pb plants was higher than that in Ck at 5 wpi, its expression level was still lower than that at 2 wpi, in which trehalose did not accumulate. Therefore, the substantial accumulation of trehalose is likely not due to synthesis within the host plant. According to a previous study, the resting spores of P. brassicae were detected to contain 14.7 mg/g dry weight of trehalose, which was much higher than that of the infected Arabidopsis tissue, which indicated that P. brassicae is capable of synthesizing a large amount of trehalose. This result correlated with the expression of PbTPS1 in resting spores [41]. Here, we identified other TPS genes in P. brassicae and analyzed PbTPS gene expression patterns, showing that PbTPS3 was not expressed and that PbTPS1 and PbTPS2 were significantly upregulated from 4 to 5 wpi. Our results are consistent with the previous study and further confirm the view that increased trehalose is probably synthesized by pathogens rather than by the host plant. Most fungi and bacteria produce trehalose, and the virulence of some of these plant pathogens is dependent on their trehalose metabolism [10,38,54]. In addition, trehalose plays a key role in protecting bacteria and fungi against a range of stressors [55] and is abundant in the spores of fungi and yeast. For example, more than 7% trehalose and trace amounts of glucose were found on a dry-weight basis in the spores and macrocysts of Dictyostelium mucoroides but not in other lifecycle stages; thus, trehalose restricted in the spores and macrocysts was utilized as energy for germination [56]. Furthermore, trehalose in Neurospora tetrasperma served to activate the ascospores [57]. Similarly, trehalose in P. brassicae may be used as a stress protectant to protect the resting spores of P. brassicae from various stressors. The resting spores of P. brassicae have been reported to survive in the soil for decades [58], perhaps because excess energy allows the germination of resting spores in P. brassicae.
In summary, a large amount of trehalose accumulated in the roots of B. rapa during clubroot development. We identified 14 BrTPS genes from B. rapa and 3 PbTPS genes from P. brassicae. The phylogenetic analysis and gene structure, conserved motif, and cis-acting element analyses of BrTPS genes were performed. Expression analysis showed that most BrTPS genes were not significantly induced by P. brassicae infection, but the expression of the three PbTPS genes of P. brassicae was upregulated. Therefore, much of the accumulated trehalose most likely originated from the pathogen, and the trehalose in the resting spores of P. brassicae may serve as energy for germination.

Plant Material and P. brassicae Inoculation
The Chinese cabbage susceptible variety "BJN3-2" was used as a host and maintained in the culture room under a 16 h light/8 h dark photoperiod at 25 • C. Chinese cabbage plant material was sown in the greenhouse of Shenyang Agricultural University in May under natural conditions. The P. brassicae resting spores of a single-spore isolate (Pb4) were collected from clubbed roots and diluted to a density of 1 × 10 7 spores/mL with sterile distilled water until inoculation. "BJN3-2" and the single-spore isolate (Pb4) of P. brassicae were preserved in the Laboratory of Vegetable Molecular Biology, College of Horticulture, Shenyang Agricultural University. The roots of 2-week-old cabbage seedlings were inoculated with 1 mL of a resting spore suspension, and plants inoculated with an equal volume of distilled water were used as controls. Plants were sampled and analyzed at 5 time points (from the first week to the fifth week) after inoculation. Each treatment was carried out with three biological replicates, and each replicate contained eight plants.

Soluble Sugar Extraction and Trehalose Content Determination
Soluble sugars were extracted from the roots of Chinese cabbage at weeks 2, 3, 4, and 5 after P. brassicae inoculation according to the previous method [45]. The GC-MS QP2010 ultra instrument (Shimadzu, Kyoto, Japan) was used for the gas quality analyses. The detailed setting information is as follows: the inlet temperature was 300 • C, the split ratio was 10:1, the carrier gas was high-purity helium, and the flow rate was 1 mL/min. The heating program was as follows: 120 • C for 3 min, 5 • C/min to 210 • C for 5 min, and 15 • C/min to 300 • C for 10 min. The ion source temperature was 200 • C, and the interface temperature was 280 • C. The solvent removal time was 3 min, and the scanning m/z was 45-500. Soluble sugars were extracted from three biological replicates at each time point. Gas quality analyses were repeated three times for each treatment.

Identification of TPS Genes in B. rapa and P. brassicae
To identify the TPS genes in the B. rapa genome, amino acid sequences of 11 A. thaliana Please use consistent spacing around headers. TPS (AtTPS) genes were selected as bait to search the B. rapa database (http://brassicadb.cn/#/) (accessed on 9 June 2021) by performing a BLASTP analysis. The paralogous genes of BrTPSs were named according to the level of sequence similarity with the corresponding genes in AtTPSs, and a suffix (a, b, c, etc.) was added to the gene according to the E-value from high to low. The TPS genes of P. brassicae were screened using the keywords "Plasmodiophora brassicae trehalose-6phosphate synthase" from the whole genome sequencing data of P. brassicae in the National Center for Biotechnology Information database (NCBI) (https://www.ncbi.nlm.nih.gov/) (accessed on 5 May 2022) [59][60][61][62]. The Conserved Domains database (http://pfam.xfam. org/) (accessed on 5 May 2022) was used to ensure that all candidate TPSs contained the TPS domain. Proteins that contained typical domains (Glyco_transf_20 or Trehalose_PPase) were selected. The physicochemical parameters, including isoelectric points (pI) and molecular weight (kDa), were calculated using the online ExPASy2 database (https://www. expasy.org/resources/compute-pi-mw) (accessed on 10 May 2022). Subcellular localization prediction was performed using Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) (accessed on 10 May 2022).

BrTPS and PbTPS Phylogenetic Evolution Analysis
The TPS protein sequences of Arabidopsis were downloaded from the Arabidopsis genome database (https://www.arabidopsis.org/index.jsp) (accessed on 9 June 2021). Brassica napus and Brassica oleracea TPS protein sequences were downloaded from the BRAD database (http://brassicadb.cn/#/) (accessed on 9 June 2021). The TPS genes in Arabidopsis, B. napus, B. oleracea, B. rapa, and P. brassicae were named AtTPSs, BnTPSs, BoTPSs, BrTPSs, and PbTPSs, respectively. Other organisms' TPS protein sequences were subjected to protein BLAST in NCBI's nr database (Non-Redundant Protein Sequence Database). All TPS protein sequences were used for phylogenetic analysis. Multiple sequence alignment of all TPS protein sequences was conducted with the ClustalW program [63]. An unrooted neighbor-joining (NJ) phylogenetic tree was constructed using MEGA6 [64] software with a bootstrap test of 1000 replicates.

Analysis of BrTPS and PbTPS Gene Structures and Conserved Motifs
The exon-intron structures of the BrTPS and PbTPS genes were identified using the Gene Structure Display Server (GSDS, http://gsds.cbi.-pku.edu.cn/) (accessed on 11 May 2022) [65]. The putative conserved motifs of BrTPS and PbTPS proteins were then identified by online MEME tool (http://meme-suite.org/tools/meme) (accessed on 11 May 2022). Protein sequences were analyzed using the MEME program with any number of repetitions. The maximum retrieval value for the motif was set to 10, and the other parameters were set to default. The conserved domains were visualized using the TBtools software package (http://www.tbtools.com/) (accessed on 11 May 2022) [66].

Chromosomal Location and Cis-Acting Element Analysis of BrTPS Genes
The chromosomal positions of the BrTPSs genes were acquired from the BRAD database (http://brassicadb.cn/#/) (accessed on 11 May 2022). MapChart software [67] was used to map BrTPS genes' chromosomal positions and relative distances; the 2 kb sequences in the BrTPS genes' upstream region were obtained using the BRAD database (http://brassicadb.cn/#/) (accessed on 12 May 2022). Subsequently, the online tool Plant-CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 12 May 2022) was employed to investigate putative cis-regulatory elements in the promoter region of BrTPS genes in B. rapa. TBtools [66] software was used to visualize these cisregulatory elements.

Total RNA Extraction and Quantitative Real-Time PCR
RNA was extracted from Chinese cabbage roots using Trizol reagent (Tiangen, Beijing, China) according to the manufacturer's protocol. First-strand cDNA was obtained using reverse transcription performed with MonScript™ RTIII Super Mix (Monad, China). Quantitative real-time RT-PCR was performed using the SYBR ® Green Premix Pro Taq HS qPCR Kit AG11701 (Accurate Biotechnology, Changsha, China). For reference genes, 18s and Pbactin were used for B. rapa. and P. brassicae, respectively. PCR reactions were carried out in triplicate with three independent RNA samples, and the primers were synthesized by Hongxun Biological Company (Suzhou, China) and are listed in Supplementary Table  S1. The 2 −∆∆Ct [68] method was used to analyze the relative gene expression level.

Statistical Analysis
The data are presented as the means ± SDs (standard deviations). Other statistical evaluations and significance tests were performed via Student's t-tests with the SPSS statistical software (Version 19.0; SPSS, Inc., Chicago, IL, USA). The data were graphically analyzed using GraphPad Prism V8.0.2 (San Diego, CA, USA).