Defence reactions in the apoplastic proteome of oilseed rape (Brassica napus var. napus) attenuate Verticillium longisporum growth but not disease symptoms

Abstract

Background: Verticillium longisporum is one of the most important pathogens of Brassicaceae that remains strictly in the xylem during most stages of its development. It has been suggested that disease symptoms are associated with clogging of xylem vessels. The aim of our study was to investigate extracellular defence reactions induced by V. longisporum in the xylem sap and leaf apoplast of Brassica napus var. napus in relation to the development of disease symptoms, photosynthesis and nutrient status.

Results: V. longisporum (strain VL43) did not overcome the hypocotyl barrier until 3 weeks after infection although the plants showed massive stunting of the stem and mild leaf chlorosis. During this initial infection phase photosynthetic carbon assimilation, transpiration rate and nutrient elements in leaves were not affected in VL43-infected compared to non-infected plants. Proteome analysis of the leaf apoplast revealed 170 spots after 2-D-protein separation, of which 12 were significantly enhanced in response to VL43-infection. LS-MS/MS analysis and data base searches revealed matches of VL43-responsive proteins to an endochitinase, a peroxidase, a PR-4 protein and a beta-1,3-glucanase. In xylem sap three up-regulated proteins were found of which two were identified as PR-4 and beta-1,3-glucanase. Xylem sap of infected plants inhibited the growth of V. longisporum.

Conclusion: V. longisporum infection did not result in drought stress or nutrient limitations. Stunting and mild chlorosis were, therefore, not consequences of insufficient water and nutrient supply due to VL43-caused xylem obstruction. A distinct array of extracellular PR-proteins was activated that might have limited Verticillium spreading above the hypocotyl. In silico analysis suggested that ethylene was involved in up-regulating VL43-responsive proteins.

Figure 1

Stunting symptoms in oilseed rape (Brassica napus var. napus) after infection with Verticillium longisporum (VL43). (A) Time course. White circles = non-infected plants, black circles = VL43-infected plants. Data indicate means (n = 20 ± SE). Occasionally, SE is smaller than the symbols. * indicate significant differences between treated and non-treated plants at P ≤ 0.05. (B) Typical phenotype of controls and infected plants 21 dpi.

Figure 2

Time course of leaf, stem and root biomass production of oilseed rape (Brassica napus var. napus) after infection with Verticillium longisporum VL43. White circles = non-infected plants, black circles = VL43-infected plants. Data indicate means (n = 5 ± SE). * indicate significant differences between treated and non-treated plants at P ≤ 0.05.

Figure 3

Net photosynthesis (A) and transpiration (B) of oilseed rape (Brassica napus var. napus) 15 and 21 dpi after infection with Verticillium longisporum VL43. White bars = non-infected plants, black bars = VL43-infected plants. Data indicate means (n = 8 ± SE). Significant differences were not observed.

Figure 4

Quantum yield of photosynthesis (A) and chlorophyll concentration (B) of oilseed rape (Brassica napus var. napus) after infection with Verticillium longisporum VL43. White symbols = non-infected plants, black symbols = VL43-infected plants. Data indicate means (n = 20 for quantum yield of PSII and n = 9 for chlorophyll, ± SE). * indicate significant differences between treated and non-treated plants at P ≤ 0.05.

Figure 5

Nitrogen, phosphorus and sulphur concentrations in leaves of oilseed rape (Brassica napus var. napus) after infection with Verticillium longisporum VL43. White circles = non-infected plants, black circles = VL43-infected plants. Data indicate means (n = 5 ± SE). Different letters indicate significant differences at P ≤ 0.05.

Figure 6

Protein patterns in leaf apoplastic washing fluids and in the xylem sap of oilseed rape (Brassica napus var. napus) of non-infected plants and 21 dpi after infection with Verticillium longisporum VL43. Each lane was loaded with 100 μg protein. Proteins were separated by gradient SDS-PAGE and stained with Coomassie Blue.

Figure 7

2-D-protein pattern in leaf apoplastic washing fluids of oilseed rape (Brassica napus var. napus) of non-infected plants and at 21 dpi after infection with Verticillium longisporum VL43. Each gel was loaded with 80 μg protein and stained with silver. Circles indicate differentially appearing protein spots. Numbers indicate proteins for which peptides were obtained.

Figure 8

Fungal growth (A) and protein content (B) in xylem sap of non-infected (X-C) and VL43-infected (X-VL43) oilseed rape (Brassica napus var. napus). Data indicate means (n = 13 for non-infected and 15 for infected xylem sap, ± SE). ** indicate significant differences between treated and non-treated plants at P ≤ 0.01, ns = not significant.