A Proteome-Level Investigation Into Plasmodiophora brassicae Resistance in Brassica napus Canola

Abstract

Clubroot of Brassicaceae, an economically important soil borne disease, is caused by Plasmodiophora brassicae Woronin, an obligate, biotrophic protist. This disease poses a serious threat to canola and related crops in Canada and around the globe causing significant losses. The pathogen is continuously evolving and new pathotypes are emerging, which necessitates the development of novel resistant canola cultivars to manage the disease. Proteins play a crucial role in many biological functions and the identification of differentially abundant proteins (DAP) using proteomics is a suitable approach to understand plant-pathogen interactions to assist in the development of gene specific markers for developing clubroot resistant (CR) cultivars. In this study, P. brassicae pathotype 3 (P3H) was used to challenge CR and clubroot susceptible (CS) canola lines. Root samples were collected at three distinct stages of pathogenesis, 7-, 14-, and 21-days post inoculation (DPI), protein samples were isolated, digested with trypsin and subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. A total of 937 proteins demonstrated a significant (q-value < 0.05) change in abundance in at least in one of the time points when compared between control and inoculated CR-parent, CR-progeny, CS-parent, CS-progeny and 784 proteins were significantly (q < 0.05) changed in abundance in at least in one of the time points when compared between the inoculated- CR and CS root proteomes of parent and progeny across the three time points tested. Functional annotation of differentially abundant proteins (DAPs) revealed several proteins related to calcium dependent signaling pathways. In addition, proteins related to reactive oxygen species (ROS) biochemistry, dehydrins, lignin, thaumatin, and phytohormones were identified. Among the DAPs, 73 putative proteins orthologous to CR proteins and quantitative trait loci (QTL) associated with eight CR loci in different chromosomes including chromosomes A3 and A8 were identified. Proteins including BnaA02T0335400WE, BnaA03T0374600WE, BnaA03T0262200WE, and BnaA03T0464700WE are orthologous to identified CR loci with possible roles in mediating clubroot responses. In conclusion, these results have contributed to an improved understanding of the mechanisms involved in mediating response to P. brassicae in canola at the protein level.

Keywords: Brassica napus; calcium binding; clubroot; plant–pathogen interaction; proteomics.

FIGURE 1

Clubroot gall development following inoculation with P. brassicae pathotype 3. Control at 7-, 14-, and 21-DPI, Clubroot resistant (CR) inoculated line at 7-, 14-, and 21-DPI Clubroot susceptible (CS) inoculated 7-, 14-, and 21-DPI.

FIGURE 2

Histology images of root cross sections after P. brassicae infection at 7-, 14-, and 21-days post inoculation (DPI). Root tissues were stained with eosin and hematoxylin. Each column indicates DPI of the pathogen and the rows show the control and inoculated genotypes [clubroot- susceptible (CS) and resistant (CR) lines]. At 7 DPI, infected cells showed primary plasmodia with dark purple mass within cells indicated by the solid yellow arrow. At 14 DPI, CS inoculated line showed the presence of secondary plasmodia; however, the pathogen development on the CR inoculated line was not progressed to secondary plasmodia phase. At 21 DPI, pathogen clearly progressed to secondary plasmodia phase, maturing into developing resting spores in the CS line. However, the infection development was not progressed further at the same time point on the CR line.

FIGURE 3

Scanning electron micrograph (SEM) of root cross sections after P. brassicae infection at 7-, 14-, and 21-DPI. Each column indicates days after inoculation of the pathogen and the rows show the control and inoculated genotypes (CR and CS). At 7 DPI, infected cells showed primary plasmodia within cells indicated by the solid yellow arrow. At 14 DPI, CS inoculated line showed the presence of secondary plasmodia; however, the pathogen development on the CR inoculated line was not progressed to secondary plasmodia phase. At 21 DPI, pathogen clearly progressed to secondary plasmodia phase, maturing into resting spores. However, the infection development was not progressed in the CR line at the timepoint.

FIGURE 4

Differentially accumulated proteins (DAP) between control (CC) and inoculated (In), In/CC CR and In/CC CS in the progeny lines. The numeric values indicate the number of proteins that were significantly changing in abundance (q-value < 0.05) in protein abundance. (A) 7 DPI, (B) 14 DPI, and (C) 21 DPI. CR, clubroot resistant; CS, clubroot susceptible; Inc and Dec represent Increase and Decrease; respectively.

FIGURE 5

Differentially accumulated proteins (DAP) between control (CC) and inoculated (In), In/CC CR and In/CC CS in the parent lines. The numeric values indicate the number of proteins that were significantly changing in abundance (q-value < 0.05) in protein abundance. (A) 7 DPI, (B) 14 DPI, (C) 21 DPI. CR, clubroot resistant; CS, clubroot susceptible; Inc and Dec represent Increase and Decrease; respectively.

FIGURE 6

Differentially accumulated proteins (DAP) between clubroot resistant (CR) and susceptible (CS) inoculated, CR/CS progeny and CR/CS parent lines in the progeny and parental lines. The numeric values indicate the number of proteins that were significantly changing in abundance (q-value < 0.05) in protein abundance. (A) 7 DPI, (B) 14 DPI, (C) 21 DPI. Inc and Dec represent Increase and Decrease; respectively.

FIGURE 7

Schematic diagram of plant-pathogen interaction upon P. brassicae inoculation in B. napus. Heatmaps were developed using log₂ fold change values at 7-, 14-, and 21-DPI and were shown besides each major metabolic processes in the plant-pathogen interaction pathway indicated by a dark solid arrow. Dark Green indicates increase in abundance and light color indicates decrease in abundance. Predicted proteins that were included in the heatmaps were significantly (q < 0.05) differentially abundant at least in one time point across all genotypes investigated. During P. brassicae infection, pathogen releases signal metabolites such as PAMPs and effectors and these elicitors were recognized by host cells. Plant receptors such as receptor like -kinases and -proteins (RLKs/RLPs) and resistance (R) proteins interact with the pathogen signal compounds, thereby activates the MAP kinases (signal transduction). Transcription factors are regulated and successively plant defense related metabolic pathways are activated, proteins related to phytohormone biosynthesis pathway, especially SA, JA, and ET biosynthetic pathway and other secondary metabolism were activated. Pathogen defense related secondary metabolites such as glucosinolates, small molecules thaumatin, polyssacharides (lignin), glycosyl hydrolases (chitinases), and a spectrum of antimicrobial and defense related proteins including dehydrin proteins were differentially accumulated in response to the pathogen. Growth regulators, auxin (AU) and cytokinin (CK) were manipulated by the pathogen, the hormones are regulated abnormally, thereby leading to the hypertrophy in the root tissues. There is an interplay between ROS and calcium signaling compounds during plant and pathogen (P. brassicae) interaction. Calcium acts as a second messenger and with the involvement of Ca²⁺ -pumps, -buffers, and –exchangers, they orchestrates and regulates cellular processes and participates in an interplay with other signaling transduction processes such as ROS. Detail annotation of protein ids shown in the heatmaps are provided in the supplementary tables: TS7 – TS13 (Supplementary Tables 7–13). Avr, avirulence factor; RLP, receptor like proteins; RLK, receptor like kinase; SA, salicylic acid; JA, jasmonic acid; ET, ethylene; CY, cytokinin; AU, auxin; PRR, pathogen recognition receptor; PAMP, pathogen associated molecular pattern; MAPK, mitogen-activated protein kinase; TIR, toll/interleukin-1 receptor; NBS, nucleotide binding site; LRR, leucine rich repeat; APR, adult plant resistance. PgR-7d, CR progeny 7 DPI; PgR-14d, CR progeny 14 DPI; PgR-21d, CR progeny 21 DPI; PgS, CS progeny; PrR, CR parent; PrS, CS parent.

FIGURE 8

Differentially accumulated proteins (DAP) at all three time points between control (CC) and inoculated (In) lines, In/CC CS and In/CC CR lines across parent and progeny, these proteins are from (A) chromosome A3 and (B) A8. Heatmaps were developed using log₂ fold change values at 7-, 14-, and 21-DPI. Dark Green indicates increase in abundance and light color indicates decrease in abundance. Proteins included in the heatmaps were significantly (q-value < 0.05) differentially abundant at least in one time point across all genotypes investigated.

FIGURE 9

Differentially accumulated proteins (DAP) between inoculated- CR/CS progeny and CR/CS parent, these proteins are orthologous to clubroot resistance related proteins. QTL information (Rcr1, Rcr4, Rcr8, Crr3, CRs, SingleGene-Brookfield, and qBrCR38-2) was obtained from published articles (Hirai et al., 2004; Saito et al., 2006; Chu et al., 2014; Hasan and Rahman, 2016; Yu et al., 2017; Laila et al., 2019; Zhu et al., 2019). The physical location in each chromosome and reference sequence was based on Brassica rapa ‘Chifu-401’ whole genome sequence assembly V3.0 (http://brassicadb.cn/#/syntenic-gene/). (A) Heatmap shows the protein abundance of each CR related proteins at each time point across all genotypes investigated. (B) Dendrogram shows the distribution of putative proteins and the known clubroot resistance related proteins. The red solid arrows indicate the clubroot resistance loci. PgR-7d, CR progeny 7 DPI; PgR-14d, CR progeny 14 DPI; PgR-21d, CR progeny 21 DPI; PgS, CS progeny; PrR, CR parent; PrS, CS parent.