The M35 Metalloprotease Effector FocM35_1 Is Required for Full Virulence of Fusarium oxysporum f. sp. cubense Tropical Race 4

Abstract

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes Fusarium wilt of banana, the most devastating disease on a banana plant. The genome of Foc TR4 encodes many candidate effector proteins. However, little is known about the functions of these effector proteins on their contributions to disease development and Foc TR4 virulence. Here, we discovered a secreted metalloprotease, FocM35_1, which is an essential virulence effector of Foc TR4. FocM35_1 was highly upregulated during the early stages of Foc TR4 infection progress in bananas. The FocM35_1 knockout mutant compromised the virulence of Foc TR4. FocM35_1 could interact with the banana chitinase MaChiA, and it decreased banana chitinase activity. FocM35_1 induced cell death in Nicotiana benthamiana while suppressing the INF1-induced hypersensitive response (HR), and its predicted enzymatic site was required for lesion formation and the suppression to INF1-induced HR on N. benthamiana leaves. Importantly, treatment of banana leaves with recombinant FocM35_1 accelerates Foc TR4 infection. Collectively, our study provides evidence that metalloprotease effector FocM35 seems to contribute to pathogen virulence by inhibiting the host immunity.

Keywords: Fusarium wilt; banana; effector; metalloprotease; virulence.

Figure 1

Sequence analysis of FocM35_1. (a) Schematic structure of the FocM35_1 protein, a M35 metalloprotease. (b) Maximum likelihood tree based on P46076 (M35 protein of Aspergillus oryzae) and Avr-Pita and their orthologous proteins from Fusarium spp. genomes. Red dot indicates FocM35_1.

Figure 2

FocM35_1 expression at different development stages. The relative expression levels were compared to that of the conidia (set as 1). The fungal constitutive gene FocEF1α was used as internal reference. Data are the means of tree independent experiments. The letters above the bars indicate statistically significant differences at p < 0.05 (Student’s t-test).

Figure 3

FocM35_1 contributes to the virulence of Foc TR4. (a) Colony morphologies of wild type (WT), ∆FocM35_1 and ∆FocM35_1-C strains cultured on potato dextrose agar (PDA) plates. Photos were taken 5 days after incubation at 28 °C. (b) Colony diameter of the WT, ∆FocM35_1 and ∆FocM35_1-C strains shown in a. (c) Disease phenotype and (d) disease index distribution in banana plantlets inoculated with the WT, ∆FocM35_1 and ∆FocM35_1-C strains at 30 days post inoculation (dpi). (e) Fungal growth in banana roots, determined by quantitative polymerase chain reaction (qPCR). Data presented in (b) and (e) are means ± SDs from three independent experiments. The letters above the bars indicate statistically significant differences at p < 0.05 (Student’s t-test).

Figure 4

Metalloprotease activity of FocM35_1. (a) Treatment with His-FocM35_1 protein decreased chitinase activity. Banana embryonic suspension cells were incubated with elusion buffer (EB), different concentrations of BSA or His-FocM35_1 protein individually. Chitinase activity was assayed. All data are means ± SDs from three independent experiments. Different letters above the histograms indicate statistically significant differences at p < 0.05 (Student’s t-test). (b) FocM35_1 and MaChiA interaction detected in rice membrane by bimolecular fluorescence complementation (BiFC) assay. The mVenus channel exhibit Venus fluorescence reflecting the direct interaction of FocM35_1 and MaChiA. Bars = 10 μm.

Figure 5

The signal peptide and enzymatic activity of FocM35_1 is required for its cell death-promoting effects. (a) Schematic illustration of FocM35_1 deletion and point mutation mutants used in this study. (b) Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens carrying vector, INF1, FocM35_1 and its mutants. Photographs were taken at 5 dpi. Similar results were obtained in two additional experiments. (c) Western blotting detection of control, INF1, FocM35_1 and its mutants using anti-GFP. (d) Quantification of cell death by electrolyte leakage measurement. Different letters above the histograms indicate statistically significant differences at p < 0.05 (Student’s t-test). (e) Subcellular localization of FocM35_1 by transient expression of green fluorescent protein (GFP)-fused FocM35_1 in N. benthamiana leaves. Photographs were taken at 2 dpi. The vector was used as control.

Figure 6

FocM35_1 suppress INF1-induced cell death and its enzymatic activity is required for immunosuppressive ability. (a) 1 day after infiltration with A. tumefaciens carrying vector, FocM35_1 and FocM35_1-nsp (without signal peptide), N. benthamiana leaves were further challenged with A. tumefaciens carrying INF1 at the same sites. The photographs were taken at 3 dpi. Similar results were obtained in two additional experiments. (b) Western blotting detection of FocM35_1 and INF1 using anti-GFP. (c) Quantification of cell death by electrolyte leakage measurement. Different letters above the histograms indicate statistically significant differences at p < 0.05 (Student’s t-test).

Figure 7

FocM35_1 promoted lesion formation of Foc TR4 in banana leaves. Banana leaves were infiltrated with elution buffer, recombinant FocM35_1, Foc TR4 or recombinant FocM35_1 together with Foc TR4. Photographs were taken 4 dpi. The areas within the red squares are enlarged into pictures at the right side. Bar = 1 cm.