Fusarium graminearum ATP-Binding Cassette Transporter Gene FgABCC9 Is Required for Its Transportation of Salicylic Acid, Fungicide Resistance, Mycelial Growth and Pathogenicity towards Wheat

Abstract

ATP-binding cassette (ABC) transporters hydrolyze ATP to transport a wide range of substrates. Fusarium graminearum is a major causal agent of Fusarium head blight, which is a severe disease in wheat worldwide. FgABCC9 (FG05_07325) encodes an ABC-C (ABC transporter family C) transporter in F. graminearum, which was highly expressed during the infection in wheat and was up-regulated by the plant defense hormone salicylic acid (SA) and the fungicide tebuconazole. The predicted tertiary structure of the FgABCC9 protein was consistent with the schematic of the ABC exporter. Deletion of FgABCC9 resulted in decreased mycelial growth, increased sensitivity to SA and tebuconazole, reduced accumulation of deoxynivalenol (DON), and less pathogenicity towards wheat. Re-introduction of a functional FgABCC9 gene into ΔFgABCC9 recovered the phenotypes of the wild type strain. Transgenic expression of FgABCC9 in Arabidopsis thaliana increased the accumulation of SA in its leaves without activating SA signaling, which suggests that FgABCC9 functions as an SA exporter. Taken together, FgABCC9 encodes an ABC exporter, which is critical for fungal exportation of SA, response to tebuconazole, mycelial growth, and pathogenicity towards wheat.

Keywords: ABC transporter; Fusarium head blight; SA; mycotoxin.

Figure A1

Structure of FgABCC9. (a) Distribution of conserved protein domain in the amino acid sequence of FgABCC9. (b) Predicted tertiary structure of FgABCC9 protein. Dark blue, purple, cyan, and brown colors indicate the “ATTR1” (ABC transporter transmembrane region 1; pfam00116; 190-503 aa), “AMD1” (ABC transporter C family MRP (multidrug resistance-associated protein) domain 1; cd03250; 546-783 aa), “ATTR2” (ABC transporter transmembrane region 2; pfam00664; 873-1138 aa), and “AMD2” (ABC transporter C family MRP domain 2; cd03250; 1180-1411 aa), respectively. (c) Schematic of ABC exporters [26].

Figure 1

Construction of deletion and complementation mutants for FgABCC9. (a) The left border (LB) and the right border (RB) were amplified from the wild type (WT) strain to construct the recombinant plasmid. ΔFgABCC9 mutants were then created by homologous recombination between the plasmid and FgABCC9. (b) PCR verification of ΔFgABCC9. (c) Gene sequence of FgABCC9 was amplified from the genomic DNA of WT and ligated into the complementation vector. The T-DNA region of the complementation vector was inserted into the genome of ΔFgABCC9 to create C-FgABCC9. SacI, ApaI, SpeI, HindIII, NcoI, and SpeI show the restriction enzymes used. (d) RT (reverse transcription)-PCR of FgABCC9 in C-FgABCC9 using RJ-ABCC9-F + RJ-ABCC9-R. FgGAPDH was used as reference. The black lines under the elements in (a) and (c) represent the targeted location of primers listed in Table 1. LB-F + LB-R, RB-F + RB-R, C-FgABCC9-F + C-FgABCC9-R were located in the genomic sequence of FgABCC9 of the WT strain. P1-F + P2-R and P3-F + P4-R were mapped in the T-DNA fragment of the pRF-HU2 vector. ΔJ-U-F, ΔJ-U-R, ΔJ-D-F, and ΔJ-D-R were, respectively, positioned into the upstream and downstream of inserted T-DNA sequence of ΔFgABCC9. All the PCR products were verified by sequencing in the commercial company (Qingke, Chengdu, China).

Figure 2

Effect of FgABCC9 on mycelial growth. (a) Mycelial growth on mSNA (modified Synthetischer Nährstoffarmer Agar) plates with SA and tebuconazole. CK, control treatment. Plates were photographed on the fourth day post inoculation. (b) Percentage of mycelial growth inhibited by SA ([A1-B1]/A1 for WT, [A2-B2]/A2 for ΔFgABCC9). (c) Percentage of mycelial growth inhibited by tebuconazole ([A1-C1]/A1 for WT, [A2-C2]/A2 for ΔFgABCC9). Asterisks represent significance at p < 0.05. A1, B1, and C1 indicate mycelial areas of WT strain under the CK, SA, and tebuconazole treatments, respectively. A2, B2, and C2 indicate mycelial areas of ΔFgABCC9 under the CK, SA, and tebuconazole treatments, respectively. The experiments were repeated three times with 10 plates for each treatment. (d) Expression changes of FgABCC9 under SA and tebuconazole treatments in the WT strain. Different letters above each column indicate significance at p < 0.05. (e) Subcellular localization of the FgABCC9 protein. The fluorescent signal is marked with red arrows. E, Optical microscope. F, fluorescence microscope. Scale bar, 10 µm.

Figure 3

FgABCC9 affected pathogenesis of F. graminearium in spikes. (a) FHB disease in wheat heads photographed on the eighth day after inoculation. Arrows indicate the inoculated spikelets. (b) the numbers of infected and bleached spikelets on the fourth, sixth, eighth, and twelfth day after inoculation. (c) Relative expression level of FgGAPDH (glyceraldehyde 3-phosphate dehydrogenase gene of F. graminearium, FG05_06257) in wheat spikes inoculated with WT, ΔFgABCC9, and C-FgABCC9, respectively. (d) DON contents in wheat spikes. (e) Concentration of DON in liquid medium. (f) Comparison of the accumulation of SA in wheat spikes after inoculation. Values are the mean ± standard deviation. Different letters above each column indicate a significant difference (p < 0.05). FW, fresh weight.

Figure 4

Evaluation of the function of FgABCC9 in A. thaliana. (a) The WT and AtFgABCC9 plants on the 30th day under SD condition. (b) Comparison of leaf numbers of WT and AtFgABCC9 plants as used in (a). (c) Comparison of visual disease symptoms in leaves of WT and AtFgABCC9 plants infected by F. graminearium at the 48th hour after inoculation. Scale bar, 1 cm (d) Comparison of infected areas in leaves at the 20th and 40th hour after inoculation. (e) Comparison of the contents of SA in leaves (without inoculation) of WT and AtFgABCC9 plants on the 30th day post germination under the SD (short day) condition. (f) Comparison of gene expression in leaves (without inoculation) of WT and AtFgABCC9 plants by qPCR. The cross indicates the absence of FgABCC9 from Col-0. Different letters above each column indicate significance at p < 0.05.