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Article

Plasmodiophora brassicae Infection Modulates Expansin Genes of Brassica rapa ssp. pekinensis

by
Muthusamy Muthusamy
1,
Sang Ryeol Park
1,
Jong-In Park
2 and
Soo In Lee
1,*
1
Department of Agricultural Biotechnology, National Institute of Agricultural Sciences (NAS), RDA, Jeonju 54874, Korea
2
Department of Horticulture, Sunchon National University, Sunchon 57922, Korea
*
Author to whom correspondence should be addressed.
Agriculture 2022, 12(9), 1416; https://doi.org/10.3390/agriculture12091416
Submission received: 16 August 2022 / Revised: 30 August 2022 / Accepted: 6 September 2022 / Published: 8 September 2022
(This article belongs to the Special Issue Integrated Pest Management of Field Crops: Series II)
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Abstract

:
Clubroot is a soil-borne disease of cruciferous crops, including Brassica rapa ssp. pekinensis, and causes substantial yield losses. In an attempt to develop clubroot-resistant B. rapa cultivars, we investigated the role of a root-abundant expansin-like B1 (EXLB1) during Plasmodiophora brassicae inoculation. The histochemical analyses of infected transgenic reporter lines showed a role for BrEXLB1 in disease response as early as 3 dpi. The transgenic overexpression of EXLB1 in B. rapa conferred disease-sensitive phenotypes and was comparable to non-transgenic controls at 30 dpi. In contrast, the heterogeneous population of antisense BrEXLB1-overexpressing lines conferred disease resistance against highly pathogenic P. brassicae race 2 inoculations under greenhouse conditions. Additionally, we profiled the relative expression of 32 other BrEXPs in wild-type seedlings, sampled on different days (1–10) after inoculation using qRT-PCR. The results indicate that the expression pattern of most BrEXP genes was significantly altered during different infection times, suggesting their participation in clubroot responses. In particular, the expressions of EXPA20, EXPA21, and EXPA34 were consistently downregulated, while the expression of EXPA5 was upregulated (log2FC ≥ 2) compared to controls. Altogether, our study showed that BrEXPs participate in clubroot disease response, and their genetic manipulation is likely to provide clubroot disease resistance.

1. Introduction

Clubroot is a soil-borne disease of cruciferous crops, including Brassica rapa ssp. pekinensis (popularly known as Chinese cabbage), and one of the characteristic symptoms is the formation of large galls on the host plant’s roots, which leads to significant yield (20–100%) losses by impeding root functions [1]. The clubroot pathogen Plasmodiophora brassicae is an obligate biotrophic protist that can survive as resting spores in the infested soil for long periods. Therefore, the identification and development of clubroot resistance is an ideal choice and is unavoidable, given the global agro-economic losses in Brassica crops. However, decades-long research in clubroot disease resistance has identified several useful major loci, quantitative trait loci (QTL), and genes for developing disease-resistant plants [2,3,4,5,6]. The emergence of new pathotypes in P. brassicae and the report of resistance breakdowns mandates additional sources of resistance [7]. Additionally, different P. brassicae pathotypes exist in different hosts [8].
On the other hand, plants have evolved a plethora of constitutive and pathogen-induced resistance mechanisms to cope with continuous pathogen threats, depending on their genotypes [9]. Plant cell walls are complex, and the dynamic structures act as a physical barrier to pathogen invasion, while the cell’s bioactive constituents play a crucial role in defense responses [10]. Wall structural modifications can influence cell wall integrity and disease-resistance phenotypes [11,12]. The infectivity of the pathogens is partly attributable to their ability to degrade the cell wall of the host plants. In some cases, the pathogen can hijack endogenous host mechanisms in cell wall loosening to create an optimal cellular environment for its intracellular proliferation [12]. In fact, clubroot pathogen infection in Arabidopsis triggered changes in the expression of cell wall metabolic genes [13]. Therefore, engineering plants with altered cell wall structural characteristics that prevent pathogen entry is an exciting strategy for developing clubroot resistance. Approximately 10% of plant genes participate in the biosynthesis, transport, deposition, remodeling, and turnover of cell walls during plant development and biotic/abiotic threats [14]. Additionally, the cell wall’s constant assembly, remodeling, and disassembly are essential for cell growth and stress adaptation [15]. Several structural and functional components, including expansins, are secreted into the cell wall space to regulate cell wall metabolism. Expansins are pH-dependent cell wall loosening proteins that play a crucial role in cell growth and division. Cell wall-localized expansins can disrupt the extracellular matrix for cell wall relaxation and expansion, thereby contributing to plant growth and development. Cell wall relaxation was shown to be useful in conferring tolerance to abiotic stress conditions, and it can pave easy access to plant pathogens [16]. Plant pathogens were sometimes shown to hijack host regulatory pathways associated with cell growth [17] and cell wall loosening proteins such as expansins. The EXPA4 overexpression in tobacco increased its susceptibility against Tobacco mosaic virus and Pseudomonas syringae by reducing the transcript expression levels of defense genes [18], suggesting expansin would also regulate plant defense mechanisms. In another study, suppressing expansin genes (EXPA1, EXPA5, EXPA10, EXPB3, EXPB4, and EXPB7) in rice was attributable to bacterial blight resistance in rice lines [19]. The results indicate that enhanced cell wall loosening/cell wall relaxation activities by expansin genes in hosts might favor pathogen entry/attacks. In addition, it is worth noting that expansin-mediated disease response is specific to phytopathogens [20]. So far, sixteen races of P. brassicae have been known/identified; of these, almost all of the races, except races 10 and 12, are known to be present in Korea, and race 4 was found to have the high pathogenicity that is widely distributed among all races [21].
B. rapa is an economically important vegetable and, more importantly, has expanded the expansin superfamily with 53 members, including several segmental and eight tandem duplicates [22,23]. In some cases, the retention of duplicate gene pairs was known to offer neofunctionalization under certain physiological conditions in plants. In the B. rapa expansin superfamily, only five genes, including BrEXLB1, retained all three duplicates, which draws attention to functional characterization. Additionally, in a previous study dealing with the root-abundant, expansin-like B1 (EXLB1) gene of Brassica rapa ssp. Pekinensis, it was shown to influence primary root growth by altering the root elongation size in Arabidopsis [24,25]. Herein, we investigated the role of BrEXLB1 in clubroot disease response through transgenic approaches. For this purpose, we used BrELXB1promoter::GUS, BrEXLB1 sense (BrEXLB1-S), as well as antisense (BrEXLB1-AS), overexpressing B. rapa transgenic lines developed previously [25,26]. Additionally, we profiled the expression changes of 32 B. rapa expansin genes under P. brassicae race 2 inoculations by qRT-PCR assays. The present study showed that BrEXP genes play a role in clubroot disease responses, and manipulating root-abundant BrEXPs would alter the degree of resistance to P. brassicae race 2 in Chinese cabbage.

2. Materials and Methods

2.1. Plant Material, Treatments, Early Detection, and GUS Assay

Seeds of B. rapa ssp. pekinensis (‘DH03′) and transgenic seedlings overexpressing BrEXLB1promoter::GUS, developed previously [26], were used. The seeds were stratified at 4 °C for a day, and then sown on compost soil (Baroker, Seoulbio, Seoul, Korea) composed of cocopeat (65–70%), peat moss (8–12%), vermiculite (10–14%), zeolite (3–5%), and perlite (5–8%), containing a multi-well plastic tray in greenhouse conditions (23°/20 °C and 14-h photoperiod). One set of control and transgenic seedlings (5 d-old) were inoculated with 4.5 mL of P. brassicae, race 4 (1 × 106 resting spores/mL of H2O) suspension (supplied by the National Institute of Horticultural and Herbal Science, Wanju, Korea), sampled at 1, 3, 5, 7, and 10 days post-inoculation (dpi), and designated as inoculated, while the other set of plants, mock-inoculated with 4.5 mL of sterile H2O, were designated as controls. The inoculated and control seedlings were used for GUS assay, PCR-based early detection, and qRT-PCR-based BrEXP expression profiling. The whole seedlings of treated and controls collected at 1, 3, 5, 7, and 10 dpi were thoroughly rinsed with distilled water and subjected to a GUS staining assay, which was performed using a β-Glucuronidase Reporter Gene Staining Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. For phenotyping, the leaf growth parameters of the fully expanded top leaf (one per plant) were considered, and the mean values from five or more plants were plotted in graphs, while the root growth parameters, such as maximum width and depth, were calculated at 28 dpi for each line by image analysis. The roots of three independent plants were photographed with a scale, and the parameters such as root depth (length/height) and root width (at the widest portion) were calculated using ImageJ software.
To confirm the infection at early time points where no characteristic symptoms are visible and to assess the uniform infection in all the plants, a pair of PCR primers specific to P. brassicae [27] was used. The primers targeting either the 18S ribosomal RNA (18S rRNA) gene (TC1 primer pair), or a fragment of the 18S rRNA and internal transcribed spacer 1 (ITS1) region of the rDNA repeat (TC2 primer pair) of the clubroot pathogen, were used with genomic DNA (10 ng) derived from each treatment and control mentioned above. A positive control was included by performing PCR on the genomic DNA of the pathogen and DNA derived from infected tissues with typical symptoms. The genomic DNA was extracted from frozen gall tissues and young seedlings, using the DNeasy Plant Pro Kit (Qiagen, Hilden, Germany), and the amplification cycle conditions were followed, as reported by Cao et al. [27]. Furthermore, the PCR amplicons at the expected molecular size were gel-purified and subcloned into a pGEM-T-easy vector to facilitate Sanger sequencing with universal T7 and SP6 primers. The homology of the resultant sequences with existing clubroot sequences at NCBI was analyzed using the BLAST tool.

2.2. Screening of BrEXLB1 Transgenic Seedlings for Clubroot Resistance against P. brassicae Race 2

The seeds of BrEXLB1-S- and BrEXLB1-AS-overexpressing lines, non-transgenic controls, and P. brassicae resistant B. rapa cultivars, were sown on compost soil (Baroker, Seoulbio, Seoul, Korea) containing a multi-well plastic tray in greenhouse conditions (23°/20 °C and 14-h photoperiod). At 8 days post-sowing, uniform-sized, visually healthy seedlings were treated with 5 mL of spore (106 spores mL−1 H2O) suspension (Asia Seed company, Seoul, Korea) of clubroot pathogen P. brassicae (race 2, Yeoncheon isolate, Gyeonggi) in the soil at the base of each plant and designated as treated plants. The other plants were mock-inoculated with 5 mL of H2O and were as designated controls (B. rapa (‘DH03’)) for phenotyping. Inoculated seedlings were maintained at a high soil moisture level for one week to facilitate pathogen proliferation. At 30 days post-inoculation, the plants were removed, and the roots were washed thoroughly before assessing the clubroot symptoms. A score was assigned based on the presence or absence of galls on the roots. Plants with clubs on primary or lateral roots were considered susceptible, while plants with no galls were designated as resistant to P. brassicae race 2. Disease incidence (DIC) was recorded as the percentage of diseased plants (with club roots) in the total number of inoculated plants.

2.3. Relative Quantification of BrEXP Genes in P. brassicae Treated Young B. rapa Seedlings at Different Developmental Stages

Total RNA (5 µg) was extracted from control (mock) seedlings and seedlings (three independent biological replicates) inoculated with P. brassicae race 4, collected at 1, 3, 5, 7, and 10 dpi using RNeasy Plant Mini Kits (Qiagen, Germany), and cDNA was prepared in 20 µL reactions with amfiRivert cDNA Synthesis Platinum Master Mix according to the manufacturer’s protocol (GenDEPOT, Baker, TX, USA). For BrEXP gene expression profiling, qRT-PCR was performed using the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with gene-specific primers (Table S1), AccuPower®2X GreenStar Master Mix (Bioneer, Daejeon, Korea), and 1:20 diluted cDNA of each treatment as a template. The PCR conditions were as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s, followed by a melting curve step to confirm the specificity of the amplified products. BrActin2 was used as an internal control for expression normalization. The relative quantity of target gene transcripts was determined by applying the 2−ΔΔCT method [28]. The expression changes of EXP genes (log2 FC) at different sampling intervals post-inoculation were calculated in relation to their expression values observed in equivalent mock samples/controls (1, 3, 5, 7, and 10 dpi) and directly presented as a heatmap. To understand the trend in expression changes among BrEXPs or sampling times, principal component analysis (PCA) was performed using Tbtools [29].

2.4. Promoter Motif Analysis

To identify putative cis-acting elements of downregulated genes (EXPA20/21/34), the promoter/upstream region (−1500 bp to +1 bp) of each gene was manually retrieved from the NCBI gene database (Table S1) and searched against known motifs of the plantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database (accessed on 29 March 2022) [30].

3. Results

3.1. Experimental Design and Molecular Detection of Clubroot Pathogens

In our preliminary studies to decide on the inoculum dosage and pathogen proliferation and to confirm the uniform infection in experimental seedlings, a PCR with primers, which specifically amplifies the 18S rRNA or a fragment of the 18S rRNA and ITS region rDNA repeat of P. brassicae from plant-derived genomic DNA, was performed. As mentioned in the methods section, mock-inoculated and clubroot pathogen-inoculated wild-type seedlings sampled at 1, 3, 5, 7, and 10 dpi were analyzed. As shown in Figure 1, PCR amplicons were present in all treated plants, starting as early as 1 dpi. The amplicon intensity from the positive controls was relatively higher than that of the test samples. In comparison, TC2 performed better in detecting Korean P. brassicae isolates than TC1, as it showed better amplification efficiency on targets. Contrastingly, non-infected plants did not contain any PCR amplicons, thus validating the uniform infection in treated plants. The resolved PCR amplicons were subcloned and sequenced to verify and know their relatedness to other pathotypes based on 18S rRNA sequence similarities. The resultant sequences had 100% sequence similarity (as queried on 21.1.2022, 4.48 pm) with the 18S rRNA and ITS region of P. brassicae isolate Yeoncheon 2. Interestingly, they also matched other Korean isolates, such as Hoengseong 1, Haenam 1, Pyeongchang 2, Seosan 1, and Gangneung 1.

3.2. BrEXLB1 Promoter Is Activated during P. brassicae (Race 4) Inoculations in Young B. rapa Seedlings

A GUS reporter-aided approach was adopted to evaluate the role of BrEXLB1 promoter activity during clubroot infection. The histochemical localization of BrEXLB1 promoter-driven GUS activity was observed prominently in the roots and leaves of P. brassicae-infected plants (Figure 2). The expression was especially strong at 3 and 5 dpi. Interestingly, at 1 dpi, GUS expression was found in root tissues. In contrast, control seedlings showed no notable GUS activity in any part of the seedlings, indicating that the early response is exclusive to the clubroot pathogen. At 3 to 5 dpi, GUS activity was extended to hypocotyls and primary leaves and maintained until 10 dpi. No GUS activity was observed in untreated plants, except for some root-specific activities (at the 3rd and 5th days). The in silico analysis of cis-regulatory elements in BrEXLB1 putative promoter motifs revealed that BrEXLB1p has wounding and pathogen responses, i.e., W box (TTGACC), defense and stress responsiveness motifs (TC-rich repeats; ATTTTCTTCA/GTTTTCTTAC), ABRE4 (CACGTA), and unnamed_4 motifs (Table S2). The potential roles of those cis-elements in clubroot disease responses are not known.

3.3. Enhanced Growth in P. brassicae-Inoculated BrEXLB1-AS Lines

P. brassicae race 4 inoculations in BrEXLB1-S, BrEXLB1-AS, and non-transgenic control plants did not develop typical clubroot symptoms for 1–28 days (Figure 3). The relative expression of BrEXLB1 in test lines prior to infections was measured to confirm the transgenics (Figure 3A). To confirm the biomasses of P. brassicae, a conventional PCR with primers (TC2) specific to P. brassicae was carried out, and the PCR amplicons were seen in most of the inoculated seedlings (Figure 3E), although the band intensity between sense and antisense lines appeared to be different. Interestingly, BrEXLB1-AS lines had notably more enhanced leaf and root growth than wild types and sense lines under similar conditions (Figure 3B–D). While the leaf growth of BrEXLB1-S lines seemed to be comparable with wild lines (Figure 3C), it was significantly different from BrEXLB1-AS lines. In most cases, the enhanced leaf growth of AS lines was restricted to 19 dpi. At 28 dpi, the leaf growth of AS was not significantly different from wild lines (except AS-9), possibly indicating that the leaf growth in AS lines is transient. However, as shown in Figure 3D, the root growth/root biomass in AS lines was visibly more enhanced at 28 dpi than that of wild and S-lines. The root growth of AS was comparable to S-lines under normal/control conditions (data not shown), suggesting that the enhanced root growth of EXLB1-AS lines is attributable to clubroot infection.

3.4. Antisense-Mediated Suppression of BrEXLB1 Likely to Improve P. brassicae Resistance in Transgenic B. rapa Lines

To investigate the BrEXLB1 effect on disease incidence upon P. brassicae race 2 infections, 8-d-old BrEXLB1-S and -AS transgenic seedlings were inoculated, and the experimental plants were evaluated and compared with controls, resistant B. rapa cultivars for clubroot symptoms at 30 dpi (Figure 4). Disease incidence (DIC) was recorded as the percentage of diseased plants (with club roots) in the total number of inoculated plants (10–14 seedlings per treatment). Unlike race 4 infections, race 2 strains developed typical disease symptoms in BrEXLB1-S lines (100 DIC) and non-transgenic control (DB) lines (100 DIC), indicating that the control and BrEXLB1-overexpressing lines were highly susceptible to the clubroot pathogen, and all lines had characteristic gall formation on their roots. However, the antisense-mediated suppression of BrEXLB1 at transcript levels showed no gall formation/disease symptoms in some seedlings derived from the heterogeneous populations (50 DIC), while few others had symptomatic roots, indicating moderate resistance to the pathogen. The resistant cultivar had the lowest disease incidence of 7.69, which indicates their high resistance to race 2 P. brassicae infection.

3.5. Expression Changes of BrEXP Genes in P. brassicae-Inoculated Seedlings

The expression pattern of 32 BrEXPs was investigated at transcript levels in 5-d-old- B. rapa seedlings inoculated with P. brassicae and sampled at different time intervals (1, 3, 5, 7, and 10 dpi). The overall results showed that most BrEXPs responded significantly to the clubroot pathogen by expression changes as early as 1 dpi, suggesting its potential as early response genes for clubroot disease (Figure 5).
Altogether, 28 α-expansin (EXPA), 2 β-expansin (EXPB), 1 expansin-like A (EXLA), and expansion-like B (EXLB) genes showed differential expression in any one of five samples collected during P. brassicae inoculations. Among these, EXPA20/21/34 was consistently downregulated compared to their respective equivalent controls at all sampling intervals, while the EXPA5 expression showed constant upregulation (log2FC ≥ 2) compared to equivalent controls. Additionally, the expression pattern of most genes was relatable to sampling/infection times. The highest number of genes (12) showed the peak of their induced expression at 5 dpi, followed by 1 and 10 dpi. Similarly, the maximum number of genes (nine) showed their peak of reduced expression was also 5 dpi, suggesting that major transcriptional reprogramming of BrEXPs occurs at 5 dpi. EXLB1 and EXPA16 showed some unique trends in expression changes. Their expression was slightly upregulated at 1 dpi; however, a gradual reduction in expression was observed during the disease progression (3, 5, 7, and 10 dpi). Most other genes showed no clear trend in expression changes, and it seems the plant EXP response to pathogens is also governed by the growth stages of host plants or infection times, as indicated by the qRT-PCR results.
To investigate the possible molecular basis of the clubroot pathogen regulating BrEXP expressions (EXPA20/21/34), we analyzed the promoter motifs of those genes. There were nine putative cis-regulatory elements (CREs), such as TCT-motif, ethylene response element (ERE), MyB binding site response element (MRE), unnamed__4, MYC, MYB-like sequence, and core promoter motifs (TATA-box and CAAT-box), that were commonly found in EXPA20/21/34 genes (Table S2).
The gene expression data collected at different intervals were also analyzed by principal component analysis (PCA) to understand the expression/variation trends among BrEXP genes (Figure 6A,B). The PCA score plot shows the overlapping and clustering of 15 genes (EXPA2/6/8/10/12/20/21/23/25/30/32/33/34/35 and EXLB3), mostly belonging to the α-EXPs in the negative axis of PC1, with the total variance accounting 34.1% as the first principal component (PC1) and 23.8% as the second principal component (PC2). The clubroot response expression patterns of EXLA2 and EXPA5/7/13/15/17 are unique and distinct from others. Among sampling times, 5 dpi stand out different from the others, while the expression pattern of most EXP genes at 3 dpi and 10 dpi looks similar. These results also indicate the critical influence of infection times. The correlation analysis between EXLB1 and other expansin genes across different sampling times (1, 3, 5, 7, and 10 dpi) showed that their expression pattern positively correlated with EXLA1 and EXPA7/8/20/25/16, and it negatively correlated with EXLA2, EXLB2/3, and EXPA1/2/4/9/11/12/14/15 genes (Table S3).

4. Discussion

Modifications of cell wall composition and structure occur throughout the plant lifecycle. It directly affects cell wall integrity (CWI), which determines the plant defense responses against pathogen attacks by multiple modes of action [9,11]. The impairment or enhancement of the expression of wall-related genes can alter the CWI. Although the emphasis was given to plant cell walls, cell wall-associated genes, and wall structural modification in pathogen invasion and disease resistance responses [10,11,12], the resources demonstrate that the role of expansin/cell wall loosening proteins in plant disease responses is relatively limited. The current climatic changes are favoring the widespread incidence of clubroot disease in the Brassica species [31]; hence, it is important to develop new resistant resources. Until recently, the role of expansin in clubroot disease responses in B. rapa was not available, although the central mechanism by which EXPs disrupt the extracellular matrix for cell wall relaxation and expansion, and thereby contribute to plant growth and development, was clearly known for a long time in terrestrial plants [15,16,32]. Herein, the histochemical analyses of BrEXLB1 promoter-driven GUS activity during clubroot infection confirm BrEXPs’ participation in clubroot disease. Prominent GUS activity in transgenic reporter lines and qRT-PCR-based expression patterns revealed that BrEXPs respond to the clubroot pathogen as early as 1–3 dpi, suggesting that BrEXPs are crucial in early responses, while the minimal GUS activity found in non-infected control roots at 3, 5 dpi can be attributed to BrEXLB1 in root development, as reported previously [25]. The GUS assay results with BrEXLB1 promoter indirectly imply that BrEXLB1 might play a role, either positively or negatively, in clubroot disease responses. The expression pattern of BrEXLB1 during early clubroot infection (1, 3, 7, and 10 dpi) showed induced expression, which was different from later infections, where a drastic reduction was observed as the disease progressed [25]. Additionally, BrEXLB1 is one of five EXP genes that retained all three gene copies in the Brassica genome after the triploidization event [23]. The retention of gene duplicates could lead to neofunctionalization; hence, it draws attention to crop improvement programs. Some of the EXLB class genes were shown to enhance the abiotic stress tolerance, in particular, drought [25,33] and heat stress conditions [34]. Therefore, the identification of EXLB roles in disease resistance would strengthen both biotic and abiotic stress protection in crop plants. Additionally, the instant availability of BrEXLB1 transgenic lines and the possibility of them being a potential candidate gene for clubroot disease experiments made us investigate the impact of transgenic overexpression and the antisense-mediated suppression of BrEXLB1 on clubroot disease resistance.
Both races (2 and 4) of P. brassicae were previously reported in Korea. In this study, the screening of BrEXLB1-S and -AS lines with P. brassicae race 4 produced some interesting phenotypes, with none of the plants, including wild-type lines, having typical clubroot symptoms. Although the PCR-based early detection and sequencing confirmed the presence of P. brassicae race 4 in all the plants, it is unclear how the plants, including wild-type lines, remained symptomless. The possible reason could be an insufficient proliferation of pathogens under semi-controlled environments. However, we observed differential influences on leaf and root growth parameters of BrEXLB1-AS lines. Considering our previous study, we can rule out the possibility of the transgenic expression of BrEXLB1 affecting leaf or root growth parameters in AS lines [26]. On the other hand, P. brassicae inoculation can transiently enhance plant growth/biomass via enhanced IAA synthesis and XTH action at early infection periods [35]. Nonetheless, in our study, the enhanced growth pattern was observed only in BrEXLB1-AS lines, thus warranting further studies on how BrEXLB1 interaction with pathogens affects growth patterns in B. rapa. Phytohormones could also contribute to BrEXLB1-mediated clubroot disease response, as BrEXLB1 expression was induced by the exogenous application of IAA, ABA, SA, and ethylene in B. rapa [25]. In order to know the performances of transgenic lines under another prevalent, P. brassicae race 2, we conducted similar screening experiments, and the results revealed that BrEXLB1 is a negative regulator of clubroot disease resistance in B. rapa. One of the possibilities is that enhanced cell wall relaxation by ELXB1 overexpression might favor pathogen entry in host root cells. Nevertheless, a recent study that dealt with one of the EXLB class members (AdEXLB8) in wild Arachis showed that overexpression could offer tolerance to both abiotic and biotic stress conditions by activating hormonal and antioxidant-based defense mechanisms [36]. However, unlike BrEXLB1 [25], AdEXLB8 does not significantly contribute to root growth. Although no direct evidence supporting EXP in clubroot resistance is available, their expression pattern in infected or resistant cultivars confirms they could participate in plant disease responses through multiple mechanisms [13,18,20,37]. Previous studies by Park et al. [38] and Otulak-Kozieł et al. [39] demonstrated that the overexpression of NbEXPA1 and potato EXPA3 leads to disease susceptibility in respective host plants against viral pathogens. Additionally, in another recent study, atexp1-1 (loss of function mutant) improved the resistant phenotypes against a necrotrophic fungus, Plectosphaerella cucumerina, compared to the wild type [9]. To progress further or to have concrete evidence of clubroot resistance in AS-lines, it is essential to select homozygous/stable transgenic AS populations. This preliminary screening for resistance showed that the alteration of BrEXLB1 expression is likely to impart P. brassicae disease resistance to B. rapa. The symptomatic AS populations can be attributed to possible unstable transgenic phenotypes/overexpression of antisense BrEXLB1. Therefore, BrEXLB1 knockout lines may be developed by applying the CRISPR/Cas system via Agrobacterium-mediated genetic transformation in B. rapa. In some cases, the potential suppression of EXP expression can lead to undesirable traits, such as retarded plant growth and development (Ding et al., 2008) in transgenic plants, in addition to disease resistance [40]. However, the suppression of BrEXLB1 does not display any visible morphological abnormalities in B. rapa under normal conditions. Previously, it was shown that the exogenous application of phytohormones, such as indole-3-acetic acid and jasmonic acid, as well as other factors, such as white light and drought stress, induce BrEXLB1 promoter activity [26]. Hence, we presume that the inhibition of BrEXLB1 might suppress auxin signaling to impart P. brassicae resistance [40]. A previous study by Gil et al. [16] identified that beet necrotic yellow vein virus, a causative agent of rhizomania in sugar beets, can hijack auxin-regulated pathways and reduce taproot growth by interacting with host AUX/IAA proteins and thereby inducing the expansin activity. This could be explained as a possible reason why BrEXLB1 OX lines show reduced root growth during clubroot infection. However, further study is required for the clear understanding of clubroot pathogen and expansin interaction in B. rapa plants.
In order to understand the role of other BrEXPs in clubroot disease response, we quantified the relative expression changes of 32 BrEXPs during P. brassicae race 4 infections. All the BrEXPs were differentially expressed during clubroot pathogen attacks. The early infection led to a consistent reduction in the expression of some α-EXP family members (EXPA20/21/34). Contrastingly, P. brassicae infection in Arabidopsis induced the expression of α-expansin (EXP) gene family members, albeit in relatively later stages of infection [13]. Another study dealing with comparative transcriptome analyses of symptomless roots and gall roots of B. oleracea revealed that the downregulation of EXPA20 may help maintain roots free of clubs [37]. A recent tissue-specific transcriptome-wide analysis of BrEXPs revealed that BrEXPA20/21 are abundantly expressed in root tissues [24], while BrEXPA34 is not abundant, suggesting that BrEXPs are also regulated by tissue-specificity. Previously, it was shown that the downregulation of cell wall loosening proteins, such as EXP and XTH, is important for disease resistance or symptomless roots [37]. As shown in PCA analysis, the expression pattern of BrEXPs under clubroot infection is likely to be influenced either by infection times or growth stages of plants. Moreover, to understand the clubroot-mediated downregulation of some BrEXPs, we scanned the potential promoter motifs that could perceive the pathogen signal and transcribe target genes as a counter-response. The functional characterization of nine putative cis-regulatory elements (CREs) identified in this study might provide clues about clubroot response gene transcription.

5. Conclusions

We found that BrEXLB1 is response to clubroot disease in B. rapa and its effective suppression likely to enhance disease resistance. Most of the BrEXPs were differentially expressed during clubroot infection suggesting their participation in the clubroot disease response of B. rapa. The future studies dealing genetic manipulation of root-abundant BrEXPs could facilitate clubroot disease resistance in Chinese cabbage.

6. Patents

Transgenic reporter B. rapa lines overexpressing BrEXLB1p:GUS constructs for its response to clubroot pathogen inoculation (Application No. 10-2021-0136083) and BrEXLB1 antisense-overexpressing lines for enhanced protection against clubroot disease in B. rapa (Application No. 10-2021-0136091) were filed for Korean patent.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture12091416/s1, Table S1: List of primers used for qRT-PCR-based relative quantification of Brassica rapa EXP genes; Table S2: List of cis-regulatory elements presented in the putative promoters of EXP genes; Table S3: Pearson correlation statistics for Clubroot response BrEXP genes; Data S1: The putative promoter sequences of BrEXPA34, BrEXPA20 and BrEXPA21 genes.

Author Contributions

Conceptualization, S.I.L.; investigation, M.M., S.R.P., J.-I.P. and S.I.L.; writing—original draft preparation, M.M.; writing—review and editing, M.M. and S.I.L.; supervision, funding acquisition, S.I.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Rural Development Administration (Korea) through the Rural Program for Agricultural Science and Technology Development, grant number PJ01495701, and the New Breeding Technology Center, grant number PJ01653701.

Institutional Review Board Statement

Not applicable.

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References

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Figure 1. PCR-based early detection of P. brassicae in young B. rapa seedlings. N, negative control; P, genomic DNA derived either from the pathogen or infected plants with typical clubroot symptoms as positive control; C, DNA template from control seedlings; T, seedlings treated with P. brassicae race 4; 1, 3, 5, 7, and 10 represent the sampling time (in days) after inoculation; “+” or “−” indicates the presence or absence, respectively, of PCR amplicons in 1.5% agarose gel, representing the 18S ribosomal RNA (TC1) and a fragment of 18S rRNA and internal transcribed spacer 1 (ITS1) region of the rDNA repeat (TC2) of P. brassicae, as mentioned in the methods section.
Figure 1. PCR-based early detection of P. brassicae in young B. rapa seedlings. N, negative control; P, genomic DNA derived either from the pathogen or infected plants with typical clubroot symptoms as positive control; C, DNA template from control seedlings; T, seedlings treated with P. brassicae race 4; 1, 3, 5, 7, and 10 represent the sampling time (in days) after inoculation; “+” or “−” indicates the presence or absence, respectively, of PCR amplicons in 1.5% agarose gel, representing the 18S ribosomal RNA (TC1) and a fragment of 18S rRNA and internal transcribed spacer 1 (ITS1) region of the rDNA repeat (TC2) of P. brassicae, as mentioned in the methods section.
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Figure 2. Histochemical localization of GUS activity in BrEXLB1promoter::GUS-overexpressing transgenic B. rapa lines. (A) represents the simplified vector map comprised BrEXLB1promoter::GUS expression cassette. (B) shows the GUS activity after clubroot pathogen inoculation in young Brassica rapa seedlings. In (B), C denotes controls/mock-inoculated transgenic lines; T stands for treated and denotes P. brassicae race 4-inoculated lines; 1, 3, 5, 7, and 10 represent sampling time points (in days) post-inoculation. Scale bar = 130 mm.
Figure 2. Histochemical localization of GUS activity in BrEXLB1promoter::GUS-overexpressing transgenic B. rapa lines. (A) represents the simplified vector map comprised BrEXLB1promoter::GUS expression cassette. (B) shows the GUS activity after clubroot pathogen inoculation in young Brassica rapa seedlings. In (B), C denotes controls/mock-inoculated transgenic lines; T stands for treated and denotes P. brassicae race 4-inoculated lines; 1, 3, 5, 7, and 10 represent sampling time points (in days) post-inoculation. Scale bar = 130 mm.
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Figure 3. The root and leaf traits in P. brassicae-inoculated BrEXLB1 transgenic and wild-type B. rapa spp. pekinensis lines at greenhouse conditions. The 5-d-old B. rapa transgenic seedlings overexpressing BrEXLB1 sense (S-2, 4, 5, and 6), antisense (AS-2, 5, 9, and 10), and wild type (DB) were inoculated with P. brassicae race 4. The leaf growth was measured randomly at 14, 19, and 28 dpi, while the root growth was photographed at 28 dpi; 3A denotes the three representative lines from treatments. In 3B, the relative quantification (in folds) of BrEXLB1 in transgenic lines is mentioned in 3A over the controls before infection, while 3C indicates the mean leaf growth (mm) parameters measured from P. brassicae race 4-infected plants (n = 5) at different times; 3D shows the mean root growth parameters (maximum root width and depth) measured at 28 dpi; 5E shows the P. brassicae presence in inoculated seedlings (BrEXLB1 sense and antisense OX lines) through PCR amplicons of TC2. * indicates that the mean differences between treatment and controls are statistically significant (statistics by ANOVA test are shown; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).
Figure 3. The root and leaf traits in P. brassicae-inoculated BrEXLB1 transgenic and wild-type B. rapa spp. pekinensis lines at greenhouse conditions. The 5-d-old B. rapa transgenic seedlings overexpressing BrEXLB1 sense (S-2, 4, 5, and 6), antisense (AS-2, 5, 9, and 10), and wild type (DB) were inoculated with P. brassicae race 4. The leaf growth was measured randomly at 14, 19, and 28 dpi, while the root growth was photographed at 28 dpi; 3A denotes the three representative lines from treatments. In 3B, the relative quantification (in folds) of BrEXLB1 in transgenic lines is mentioned in 3A over the controls before infection, while 3C indicates the mean leaf growth (mm) parameters measured from P. brassicae race 4-infected plants (n = 5) at different times; 3D shows the mean root growth parameters (maximum root width and depth) measured at 28 dpi; 5E shows the P. brassicae presence in inoculated seedlings (BrEXLB1 sense and antisense OX lines) through PCR amplicons of TC2. * indicates that the mean differences between treatment and controls are statistically significant (statistics by ANOVA test are shown; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).
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Figure 4. Clubroot pathogen P. brassicae race 2-infected BrEXLB1 transgenic, wild-type, and resistant B. rapa spp. pekinensis cultivars. (A) BrEXLB1-S plants; (B) BrEXLB1-AS; (C) wild type; (D) clubroot-resistant B. rapa. The plus (+) and minus (−) symbols indicate the presence and absence, respectively, of the clubs in roots.
Figure 4. Clubroot pathogen P. brassicae race 2-infected BrEXLB1 transgenic, wild-type, and resistant B. rapa spp. pekinensis cultivars. (A) BrEXLB1-S plants; (B) BrEXLB1-AS; (C) wild type; (D) clubroot-resistant B. rapa. The plus (+) and minus (−) symbols indicate the presence and absence, respectively, of the clubs in roots.
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Figure 5. The expression pattern of B. rapa expansin (BrEXP) genes during P. brassicae race 4 infections. The expression changes in folds (Log2FC) (depicted by color keys) were derived from qRT-PCR-based relative quantification of biological replicates; 1, 3, 5, 7, and 10 dpi represent the sampling times (in days) post-inoculation. The changes in expression (folds) of EXP genes during inoculation (displayed in heatmap blocks) were calculated in relation to their expression values in equivalent mocked samples/controls. BrActin2 was used for expression normalization.
Figure 5. The expression pattern of B. rapa expansin (BrEXP) genes during P. brassicae race 4 infections. The expression changes in folds (Log2FC) (depicted by color keys) were derived from qRT-PCR-based relative quantification of biological replicates; 1, 3, 5, 7, and 10 dpi represent the sampling times (in days) post-inoculation. The changes in expression (folds) of EXP genes during inoculation (displayed in heatmap blocks) were calculated in relation to their expression values in equivalent mocked samples/controls. BrActin2 was used for expression normalization.
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Figure 6. Principal component analysis performed among differentially expressed BrEXP genes (A) with their expression changes in folds and sampling times (dpi) after inoculation of P. brassicae (B).
Figure 6. Principal component analysis performed among differentially expressed BrEXP genes (A) with their expression changes in folds and sampling times (dpi) after inoculation of P. brassicae (B).
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Muthusamy, M.; Park, S.R.; Park, J.-I.; Lee, S.I. Plasmodiophora brassicae Infection Modulates Expansin Genes of Brassica rapa ssp. pekinensis. Agriculture 2022, 12, 1416. https://doi.org/10.3390/agriculture12091416

AMA Style

Muthusamy M, Park SR, Park J-I, Lee SI. Plasmodiophora brassicae Infection Modulates Expansin Genes of Brassica rapa ssp. pekinensis. Agriculture. 2022; 12(9):1416. https://doi.org/10.3390/agriculture12091416

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Muthusamy, Muthusamy, Sang Ryeol Park, Jong-In Park, and Soo In Lee. 2022. "Plasmodiophora brassicae Infection Modulates Expansin Genes of Brassica rapa ssp. pekinensis" Agriculture 12, no. 9: 1416. https://doi.org/10.3390/agriculture12091416

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