Comparative Transcriptome Analysis of Rutabaga (Brassica napus) Cultivars Indicates Activation of Salicylic Acid and Ethylene-Mediated Defenses in Response to Plasmodiophora brassicae

Abstract

Clubroot, caused by Plasmodiophora brassicae Woronin, is an important soilborne disease of Brassica napus L. and other crucifers. To improve understanding of the mechanisms of resistance and pathogenesis in the clubroot pathosystem, the rutabaga (B. napus subsp. rapifera Metzg) cultivars 'Wilhelmsburger' (resistant) and 'Laurentian' (susceptible) were inoculated with P. brassicae pathotype 3A and their transcriptomes were analyzed at 7, 14, and 21 days after inoculation (dai) by RNA sequencing (RNA-seq). Thousands of transcripts with significant changes in expression were identified in each host at each time-point in inoculated vs. non-inoculated plants. Molecular responses at 7 and 14 dai supported clear differences in the clubroot response mechanisms of the two genotypes. Both the resistant and the susceptible cultivars activated receptor-like protein (RLP) genes, resistance (R) genes, and genes involved in salicylic acid (SA) signaling as clubroot defense mechanisms. In addition, genes related to calcium signaling and genes encoding leucine-rich repeat (LRR) receptor kinases, the respiratory burst oxidase homolog (RBOH) protein, and transcription factors such as WRKYs, ethylene responsive factors, and basic leucine zippers (bZIPs), appeared to be upregulated in 'Wilhelmsburger' to restrict P. brassicae development. Some of these genes are essential components of molecular defenses, including ethylene (ET) signaling and the oxidative burst. Our study highlights the importance of activation of genes associated with SA- and ET-mediated responses in the resistant cultivar. A set of candidate genes showing contrasting patterns of expression between the resistant and susceptible cultivars was identified and includes potential targets for further study and validation through approaches such as gene editing.

Keywords: Brassica napus; Plasmodiophora brassicae; RNA sequencing (RNA-seq); clubroot; defense mechanisms; ethylene; salicylic acid.

Figure 1

Phenotypes of Plasmodiophora brassicae-inoculated roots of the rutabagas ‘Wilhelmsburger’ (R) and ‘Laurentian’ (S) at 7, 14, and 21 days after inoculation (dai). A red arrow indicates the presence of galls.

Figure 2

Number of differentially expressed transcripts in each rutabaga cultivar and time-point. (A) Number of total differentially expressed transcripts in each cultivar and time-point. (B–D) Venn diagrams showing the number of transcripts with common and unique expression patterns in the two cultivars at 7 (B), 14 (C) and 21 (D) days after inoculation (dai). Up, upregulation; down, downregulation; R, the resistant cultivar ‘Wilhelmsburger’; S, the susceptible cultivar ‘Laurentian’.

Figure 3

Correlation of log₂ (fold-change) values of 10 selected genes based on RNA-seq and qRT-PCR analyses (inoculated versus non-inoculated). The R values indicate the correlation coefficient between the two methods in each host and time-point, and the p-values indicate the significance level of the t-test. (A) ‘Laurentian,’ 7 dai; (B) ‘Laurentian,’ 14 dai; (C) ‘Laurentian,’ 21 dai; (D) ‘Wilhelmsburger,’ 7 dai; (E) ‘Wilhelmsburger,’ 14 dai; (F) ‘Wilhelmsburger,’ 21 dai.

Figure 4

Distribution of differentially expressed genes involved in the biotic stress response in two rutabaga hosts at 7 days after inoculation with Plasmodiophora brassicae. (A) ‘Wilhemsburger’ and (B) ‘Laurentian.’ The log₂ fold-changes are presented on a scale where red represents upregulation and blue represents downregulation. ABA, abscisic acid; JA, jasmonic acid; SA, salicylic acid; bZIP, basic region-leucine zipper; ERF, APETALA2/Ethylene-responsive element binding protein family; WRKY, WRKY transcription factor; MYB, MYB transcription factor; DOF, DNA-binding one zinc finger transcription factor; MAPK, mitogen-activated protein kinase; PR-protein, pathogenesis-related protein; R genes, resistance genes.

Figure 5

Distribution of differentially expressed genes involved in the biotic stress response in two rutabaga hosts at 14 days after inoculation with Plasmodiophora brassicae. (A) ‘Wilhemsburger’ and (B) ‘Laurentian.’ The log₂ fold-changes are presented on a scale where red represents upregulation and blue represents downregulation. ABA, abscisic acid; JA, jasmonic acid; SA, salicylic acid; bZIP, basic region-leucine zipper; ERF, APETALA2/Ethylene-responsive element binding protein family; WRKY, WRKY transcription factor; MYB, MYB transcription factor; DOF, DNA-binding one zinc finger transcription factor; MAPK, mitogen-activated protein kinase; PR-protein, pathogenesis-related protein; R genes, resistance genes.

Figure 6

Heatmaps of differentially expressed genes in the resistant (R) rutabaga ‘Wilhelmsburger’ and the susceptible (S) ‘Laurentian’ in response to Plasmodiophora brassicae through the time course. Only genes that showed significant differential expression in each host at 7 or 14 dai were selected. In the heatmap scale used in this diagram, red indicates upregulation, green indicates downregulation, and gray indicates no significant regulation. ERF, ethylene response factor; JA, jasmonic acid; OPR, oxophytodienoate reductase; AOC, allene oxide cyclase; AOS, allene oxide synthase; SA, salicylic acid; SABATH, SABATH methyltransferase gene family; CBP, calcium binding protein; CaMBP, calmodulin-binding protein; LRR, leucine-rich repeat; RLP, receptor like protein. The number of transcripts for each term are indicated in parentheses.

Figure 7

Distribution of the number of transcripts showing opposite regulation patterns in the resistant (R) rutabaga ‘Wilhelmsburger’ and the susceptible (S) ‘Laurentian’ at 7 days after inoculation with Plasmodiophora brassicae, using Mapman annotation. (A) Pie chart generated from transcripts upregulated in ‘Wilhlemsburger’ and downregulated in ‘Laurentian.’ (B) Pie chart generated from transcripts downregulated in ‘Wilhelmsburger’ and upregulated in ‘Laurentian.’ (C) Number of transcripts related to RNA regulation.

Figure 8

Model illustrating the major networks of the resistance response in the rutabaga ‘Wilhelmsburger’ to Plasmodiophora brassicae pathotype 3A. Important genes in each functional category are indicated in parentheses. Genes in red are upregulated, while those in green are downregulated in the resistant cultivar ‘Wilhelmsburger.’ Genes marked with an asterisk (*) were inversely regulated in the susceptible cultivar ‘Laurentian.’ Lightning bolt symbols indicate defense responses.