Differences in cell death induction by Phytophthora Elicitins are determined by signal components downstream of MAP kinase kinase in different species of Nicotiana and cultivars of Brassica rapa and Raphanus sativus

Abstract

Elicitins are small, secreted proteins produced by species of the plant-pathogenic oomycete Phytophthora. They induce hypersensitive cell death in most Nicotiana species and in some cultivars of Brassica rapa and Raphanus sativus. In this study, two true-breeding Fast Cycling B. rapa lines were established that showed severe necrosis (line 7-R) or no visible response (line 18-NR) after treatment with elicitin. Unexpectedly, microscopic examination revealed localized cell death in line 18-NR plants, and expression levels of various defense-marker genes were comparable in both lines. These results suggested that both "responsive" and "nonresponsive" plants responded to elicitin but differed in the extent of the cell death response. Expression of a constitutively active form of Arabidopsis (Arabidopsis thaliana) MAP kinase kinase 4 (AtMEK4(DD)) also induced rapid development of confluent cell death in line 7-R, whereas line 18-NR showed no visible cell death. Similarly, elicitin-responsive Nicotiana species and R. sativus cultivars showed significantly stronger cell death responses following expression of AtMEK4(DD) compared with nonresponsive species/cultivars. Line 7-R also showed higher sensitivity to toxin-containing culture filtrates produced by Alternaria brassicicola, and toxin sensitivity cosegregated with elicitin responsiveness, suggesting that the downstream responses induced by elicitin and Alternaria toxin share factors that control the extent of cell death. Interestingly, elicitin responsiveness was shown to correlate with greater susceptibility to A. brassicicola (a necrotroph) in B. rapa but less susceptibility to Phytophthora nicotianae (a hemibiotroph) in Nicotiana, suggesting a more extensive cell death response could cause opposite effects on the outcomes of biotrophic versus necrotrophic plant-pathogen interactions.

Figure 1.

Cell death in Fast Cycling B. rapa plants induced by elicitin solution prepared from P. cinnamomi culture filtrate or by transient expression of a synthetic elicitin gene. A, Original seed stock of Fast Cycling B. rapa contained individuals with different elicitin responsiveness. Cotyledons of 80 Fast Cycling B. rapa individuals were treated with elicitin solution. Differential responses to elicitin were categorized into six classes according to the severity of their visible cell death responses. Numbers of plants in each class are given under each section. B, Cotyledons of responsive (line 7-R) and nonresponsive (line 18-NR) lines treated with elicitin solution. Treated cotyledons are indicated by arrowheads. Photographs in A and B were taken 2 d after treatment. C, Cotyledons of responsive (line 7-R) and nonresponsive (line 18-NR) lines infiltrated with A. tumefaciens carrying pSLJ7292 (vector) or pCBJ-sp-capsicein (synthetic elicitin gene). The pCBJ-sp-capsicein construct contains a synthetic α-capsicein gene, which is fused in frame with the coding sequence of the PR1 signal peptide of N. tabacum and expressed under the control of the CaMV 35S promoter. Photographs were taken 3 d after agroinfiltration.

Figure 2.

A, Microscopic examination of cell death responses in cotyledons of B. rapa lines 7-R and 18-NR. Dead cells were visualized by lactophenol trypan blue staining 0, 6, and 24 h after elicitin treatment. Bar = 100 μm. B, Cell death observed in petiole vascular tissues of cotyledons treated with elicitin. Arrows indicate the direction from the base of the cotyledon toward the hypocotyl. Bar = 200 μm.

Figure 3.

Hypersensitive cell death induced by transient expression of elicitin or AtMEK4^DD genes in B. rapa cultivars, R. sativus cultivars, and Nicotiana species. A. tumefaciens cultures carrying pCBJ-sp-capsicein or pCBJ-AtMEK4^DD were infiltrated into intercellular space of plant leaves. Photographs were taken 3 d after agroinfiltration. Results shown are representative of three separate experiments.

Figure 4.

Expression profiles of marker genes for defense responses in B. rapa line 7-R and line 18-NR at various times after treatment with elicitin solution. Accumulation of mRNA for PR-1, PDF1.2, CSD1, LSD1, and BI-1 was determined by RT-PCR with specific primers. RT-PCR analysis of actin gene expression is shown as an internal control. Results shown are representative of three separate experiments.

Figure 5.

Cell death in B. rapa induced by Alternaria toxin and elicitin solution. A, B. rapa leaves were treated with potato dextrose broth media (Control) or culture filtrate of A. brassicicola isolate MBH3-1 (Ab-cf). B, Leaves of F3 plants from the B. rapa cross line 7-R × line 18-NR were treated with elicitin solution (Elicitin) or culture filtrate of A. brassicicola (Ab-cf) and photographed 2 d after treatment.

Figure 6.

A, Disease symptoms on B. rapa lines 7-R and 18-NR after inoculation with A. brassicicola. Line 7-R showed more severe symptoms compared to line 18-NR at the same time point (top). Extensive fungal growth and sporulation (inset) of A. brassicicola were observed in both lines 7-R and 18-NR (bottom). All photographs were taken 60 h after inoculation. Bars = 50 μm. B, Infection of N. tabacum and N. amplexicaulis with P. nicotianae 24 (top) and 48 (bottom) h after inoculation. Incomplete containment of pathogen growth by dead plant cells was often observed in N. tabacum 24 h after inoculation (indicated by arrowheads) in contrast with extensive growth of pathogen in N. amplexicaulis (top). Extensive growth of P. nicotianae was observed in both N. tabacum and N. amplexicaulis 48 h after inoculation, but production of numerous sporangia (inset) is obvious only in N. amplexicaulis (bottom). Bars = 100 μm (main panels) and 50 μm (inset).

Figure 7.

Schematic diagram summarizing a model for plant signal transduction leading to the induction of hypersensitive cell death in response to elicitin. Both “responsive” and “nonresponsive” plants possesses an elicitin receptor and can initiate signal transduction leading to hypersensitive cell death and expression of defense genes. “Responsive” plants can show visible cell death due to the presence of an enhancer or the absence of a suppressor of cell death progression, while nonresponsive plants only induce localized cell death, normally invisible to the naked eye. The enhancer or suppressor of cell death acts downstream of the MAPK cascade. Severity of cell death induced by Alternaria toxin is also determined by the same enhancer/suppressor of cell death.