iTRAQ-based quantitative analysis reveals proteomic changes in Chinese cabbage (Brassica rapa L.) in response to Plasmodiophora brassicae infection

Abstract

Clubroot disease is one of the major diseases affecting Brassica crops, especially Chinese cabbage (Brassica rapa L. ssp. pekinensis), which is known to be highly susceptible to the disease. In this study, the obligate biotrophic protist Plasmodiophora brassicae Woronin was used to infect the roots of Chinese cabbage seedlings. The disease symptoms were noticeable at 28 and 35 days after inoculation (DAI) in the susceptible (CM) line. Using isobaric tags for relative and absolute quantitation (iTRAQ) analysis, a total of 5,003 proteins of differential abundance were identified in the resistant/susceptible lines, which could be quantitated by dipeptide or polypeptide segments. Gene ontology (GO) analysis indicated that the differentially expressed proteins (DEPs) between the susceptible (CM) and resistant (CCR) lines were associated with the glutathione transferase activity pathway, which could catalyze the combination of glutathione and other electrophilic compounds to protect plants from disease. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEPs may be significantly enriched cytokinin signaling or arginine biosynthesis pathways, both of which are responses to stimuli and are plant defense reactions. The cytokinins may facilitate cell division in the shoot, resulting in the hypertrophy and formation of galls and the presentation of typical clubroot symptoms. In this study, the proteomic results provide a new perspective for creating germplasm resistance to P. brassicae, as well as a genetic basis for breeding to improve Chinese cabbage.

Figure 1

Analysis of a swelled root in the Brassica rapa lines CCR and CM after inoculation with Plasmodiophora brassicae. (A) 14, 21, 28, 35, and 42 days after inoculation with P. brassicae. CCR-ck and CM-ck were two controls, CCR-I denotes CCR-inoculation, and CM-I denotes CM-inoculation. (B) The root-shoot ratios of CCR-ck, CCR-I, CM-ck, and CM-I at five periods in time.

Figure 2

Overview of the unique proteins identified using iTRAQ data. PCA was used to analyze the main proteins in (A) CCR-28/CM-28 and (B) CCR-35/CM-35. PLS-DA was used to analyze the main proteins in (C) CCR-28/CM-28 and (D) CCR-35/CM-35. (E) Analysis of the DEPs between CCR-28 and CM-28 using volcano figures. (F) Analysis of the DEPs between CCR-35 and CM-35 using volcano figures. (G) Signal normalization of the signal boxes for each channel. The figure can be used to understand the normalization effect through the median of each channel. (H) Quality control of quality precision. The abscissa is the mass accuracy distribution of the mass spectrometry detection, and the ordinate is the corresponding distribution of the number of matching results.

Figure 3

Identification of analysis of the DEPs in the nine sample combinations: CM-28-ck/CM-28, CM-28-ck/CM-35, CM-28/CM-35, CCR-28-ck/CCR-28, CCR-28-ck/CCR-35, CCR-28/CCR-35, CM-28-ck/CCR-28-ck, CM-28/CCR-28, and CM-35/CCR-35.

Figure 4

The hierarchical clustering and metabolic pathway analysis of the DEPs in the comparisons of CM-28-ck/CM-28 and CM-28/CCR-28.

Figure 5

The DEP classification analysis from three GO categories in the comparison of (A) CM-28-ck/CM-28 and (B) CM-28/CCR-28, including: biological processes, cellular components, and molecular functions. Black bars represent the biological process, green bars represent the molecular function, and blue bars represent the cellular component.

Figure 6

The DEP analysis from the glutathione transferase activity pathway in the comparison of (A) CM-28-ck/CM-28 and (B) CM-28/CCR-28. Green represents the number of DEPs in the comparison, and “is a” represents a containment relationship.

Figure 7

The number of DEPs from the nine comparisons obtained by KEGG analysis.

Figure 8

Analyses of the arginine metabolism pathway for the purpose of explaining the DEPs in the arginine biosynthesis process.