The defense response in Arabidopsis thaliana against Fusarium sporotrichioides

Abstract

Background: Certain graminaceous plants such as Zea mays and Triticum aestivum serve as hosts for Fusarium sporotrichioides; however, molecular interactions between the host plants and F. sporotrichioides remain unknown. It is also not known whether any interaction between Arabidopsis thaliana and F. sporotrichioides can occur. To understand these interactions, we performed proteomic analysis.

Results: Arabidopsis leaves and flowers were inoculated with F. sporotrichioides. Accumulation of PLANT DEFENSIN1.2 (PDF1.2) and PATHOGENESIS RELATED1 (PR1) mRNA in Arabidopsis were increased by inoculation of F. sporotrichioides. Furthermore, mitogen-activated protein kinase 3 (MPK3) and mitogen-activated protein kinase 6 (MPK6), which represent MAP kinases in Arabidopsis, were activated by inoculation of F. sporotrichioides. Proteomic analysis revealed that some defense-related proteins were upregulated, while the expression of photosynthesis- and metabolism-related proteins was down regulated, by inoculation with F. sporotrichioides. We carried out the proteomic analysis about upregulated proteins by inoculation with Fusarium graminearum. The glutathione S-transferases (GSTs), such as GSTF4 and GSTF7 were upregulated, by inoculation with F. graminearum-infected Arabidopsis leaves. On the other hand, GSTF3 and GSTF9 were uniquely upregulated, by inoculation with F. sporotrichioides.

Conclusions: These results indicate that Arabidopsis is a host plant for F. sporotrichioides. We revealed that defense response of Arabidopsis is initiated by infection with F. sporotrichioides.

Figure 1

Inoculation of Arabidopsis leaves and flower buds with Fusarium sporotrichioides. Photographs of mock-treated (A, E, I) and conidial suspension–treated Arabidopsis leaves (B–D, F–H, J–L) at 2 dpi. (E–H) represents magnification of the inner box of panels A–D, respectively. Trypan blue staining of mock-treated leaves (I) and leaves inoculated with F. sporotrichioides (J–L). (M, N) Cross-sections of Trypan blue staining of leaves inoculated with F. sporotrichioides. Arrowheads show the hyphae. Photographs of mock-treated (O) and conidial suspension–treated flower buds (P) at 2 dpi. The scale bars indicate 1 cm (A–D), 1 mm (E–H, O, P), and 100 μm (I–N).

Figure 2

The expression pattern of PDF1.2a and PR1 mRNA in Arabidopsis by inoculation of F. sporotrichioides. (A) The expression analysis of PR1 and PDF1.2a mRNA in Arabidopsis after inoculation. ACT2 and ACT8 were used as reference genes. These experiments were repeated 3 times. (B) Activity of MPK3 and MPK6 in response to inoculation with F. sporotrichioides. Upper panel: results of an in-gel kinase assay. Lower panel: CBB staining of the loading control. The amplification efficiency for qRT-PCR of PR-1, PDF1.2 and ACT2, 8 were 91.6, 86.7 and 98.2%, respectively. These experiments were repeated 3 times.

Figure 3

Proteomic analysis of Arabidopsis leaves inoculated with a conidial suspension of F. sporotrichioides. (A) Protein spots of mock-treated samples (Cy3-labeled total protein). (B) Protein spots of F. sporotrichioides–inoculated leaves (Cy5-labeled total protein). (C) Merge of (A) and (B) images. Spots of downregulated (D) and upregulated (E) proteins in F. sporotrichioides–inoculated leaves are shown. The spot numbers correspond to the results of protein identification (Table 1). This experiment was repeated 3 times. Functional classification of the identified proteins is shown. (F) The proteins increased and (G) decreased in response to inoculation with F. sporotrichioides.

Figure 4

The expression pattern of GSTs, APX and SOD in Arabidopsis leaves inoculated with a conidial suspension of F. graminearum H3 and ZEA-1. (A, D) Protein spots of mock-treated samples (Cy2-labeled total protein). (B, E) Protein spots of F. graminearum H3-inoculated leaves (Cy3-labeled total protein). (C, F) Protein spots of F. graminearum ZEA-1-inoculated leaves (Cy5-labeled total protein). This experiment was repeated 3 times. (G) Venn diagram showing of expression pattern of GSTs, APX and SOD proteins in Arabidopsis leaves inoculated with a conidial suspension of F. sporotrichioides, F. graminearum H3 and ZEA-1.