Interaction Between Glycoside Hydrolase FsGH28c from Fusarium solani and PnPUB35 Confers Resistance in Piper nigrum

Abstract

Pathogens deploy various molecular mechanisms to overcome host defenses, among which glycoside hydrolases (GHs) play a critical role as virulence factors. Understanding the functional roles of these enzymes is essential for uncovering pathogen-host interactions and developing strategies for disease management. Fusarium wilt has occurred in the main Piper nigrum cultivation regions, which seriously affects the yield and quality of P. nigrum. Here, we identified and characterized FsGH28c, a GH28 family member in Fusarium solani. Its expression was significantly upregulated during the infection of black pepper (Piper nigrum) roots by F. solani cv. WN-1, indicating its potential role in pathogenicity. FsGH28c elicited cell death in Nicotiana benthamiana and modulated the expression of genes related to pathogenesis. FsGH28c exerts a positive influence on the pathogenicity of F. solani. The knockout of FsGH28c mutant strains markedly attenuated F. solani 's virulence in black pepper plants. The knockout mutant strains decrease the ability of F. solani to utilize carbon sources. The FsGH28c deletion did not affect mycelial growth on PDA but did impact spore development. We identified a U-box protein, PnPUB35, interacting with FsGH28c using yeast two-hybrid and bimolecular fluorescence complementation assays. PnPUB35 conferred enhanced resistance to F. solani in black pepper through positive regulation. These findings suggest that FsGH28c may function as a virulence factor by modulating host immune responses through its interaction with PnPUB35.

Keywords: Fusarium solani; cell death; glycoside hydrolase.

Figure 1

Identification of the cell death induced by FsGH28c in Fusarium solani: (a) Cell death assays for 9 F. solani genes in 6-week-old N. benthamiana leaves were performed by Agrobacterium-mediated transient expression. Leaves were imaged 6 days after infiltration with Agrobacterium carrying the FsGH28 genes. BAX and GFP were used as the positive and negative controls, respectively. Scale bar = 0.5 cm. (b) Semi-quantitative reverse transcription PCR analysis of transiently expressed FsGH28 genes in N. benthamiana leaves 48 h after infiltration. NbActin was used as the control. (c) Detection of the cell death activity of FsGH28c. The FsGH28c was transiently expressed in 6-week-old N. benthamiana leaves. Sq RT-PCR analysis showed that the FsGH28c was expressed in N. benthamiana leaves. The number 1 represents the sample that was only infiltrated with BAX, number 2 represents the sample that was only infiltrated with GFP, number 3 represents the sample that was infiltrated with truncation mutation FsGH28c^-C, number 4 represents the sample that was only infiltrated with truncation mutation FsGH28c^-N, number 5 represents the sample that was only infiltrated with FsGH28c^-SP, and number 6 represents the sample that was only infiltrated with FsGH28c. NbActin was used as the control. Scale bar = 0.5 cm. (d) Confirmation of the function of the signal peptide of FsGH28c by yeast signal trap assay. Fusion of the functionality of the signal peptide of FsGH28c can grow on YPRAA medium. The functionality of the signal peptide of Avr1b was used as the positive control. (e) The qRT-PCR analysis of the pathogenesis-related genes. The blue, yellow, and grey columns represent the samples that were infiltrated by BAX, FsGH28c, and GFP at 00 h, 06 h, 12 h, and 24 h, respectively. NbActin was used as a control. Values represent means ± standard deviation of three replicates. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

FsGH28c plays an important role in utilizing carbon source: (a) Phenotype analysis of the wild-type, ∆FsGH28c, and C-∆FsGH28c strains grown on PDA and Cazpek Dox medium with sucrose, galactose, pectin, raffinose, or cellulose as the sole carbon source for 7 days. Scale bar = 3 cm. (b) The spore concentration of the wild-type, ∆FsGH28c, and C-∆FsGH28c strains grown in liquid Czapek Dox medium for 3 days. Values represent means ± standard deviation of three replicates. The asterisks represent statistical differences performed by a t-test (* p < 0.05) in comparison with the wild-type strains. (c) The mycelial growth of the wild-type, ∆FsGH28c, and C-∆FsGH28c strains on Cazpek Dox medium at 7 days. Values represent means ± standard deviation of three replicates. The measurement unit represents centimeters. The asterisks represent statistical differences performed by a t-test (* p < 0.05, ** p < 0.01, *** p < 0.001) in comparison with the wild-type strains.

Figure 3

Observation of the mycelium and conidia on a scanning electron microscope: Mycelium of the wild-type, ∆FsGH28c, and C-∆FsGH28c strains grown on PDA medium for 5 days, and microconidia of the wild-type, ∆FsGH28c, and C-∆FsGH28c strains incubated in Cazpek Dox for 3 days were observed via scanning electron microscope, respectively. The red arrows point to the mycelium and microconidia of each strain. Scale bar = 20 μm.

Figure 4

FsGH28c plays a positive role in the virulence of Fusarium solani: (a) Disease symptoms of black pepper after the wild-type, ∆FsGH28c, and C-∆FsGH28c strains infection. Photographs were taken at 8 weeks after fungal inoculation. Each treatment has more than 30 black peppers. Each treatment has three replicates. Scale bar = 3 cm. (b) Re-isolation of F. solani WN-1 strains from the stem of inoculated black pepper plants at 26 °C for 3 days. Scale bar = 1.5 cm. (c) Disease index of black pepper plants at 4 weeks, 6 weeks, and 8 weeks after the wild-type, ∆FsGH28c, and C-∆FsGH28c strains infection. (d) Relative fungal biomass in stems of black pepper after the wild-type, ∆FsGH28c, and C-∆FsGH28c strains infection at 8 weeks. Values represent means ± standard deviation of three replicates. The asterisks represent statistical differences performed by a t-test (* p < 0.05, ** p < 0.01) in comparison with the wild-type strains.

Figure 4

FsGH28c plays a positive role in the virulence of Fusarium solani: (a) Disease symptoms of black pepper after the wild-type, ∆FsGH28c, and C-∆FsGH28c strains infection. Photographs were taken at 8 weeks after fungal inoculation. Each treatment has more than 30 black peppers. Each treatment has three replicates. Scale bar = 3 cm. (b) Re-isolation of F. solani WN-1 strains from the stem of inoculated black pepper plants at 26 °C for 3 days. Scale bar = 1.5 cm. (c) Disease index of black pepper plants at 4 weeks, 6 weeks, and 8 weeks after the wild-type, ∆FsGH28c, and C-∆FsGH28c strains infection. (d) Relative fungal biomass in stems of black pepper after the wild-type, ∆FsGH28c, and C-∆FsGH28c strains infection at 8 weeks. Values represent means ± standard deviation of three replicates. The asterisks represent statistical differences performed by a t-test (* p < 0.05, ** p < 0.01) in comparison with the wild-type strains.

Figure 5

FsGH28c interacts with the PnPUB35: (a) The interaction between FsGH28c and PnPUB35 was confirmed by Yeast two-hybrid assays. FsGH28c^-SP was an SP truncation mutation, FsGH28c^-C was an N-terminal truncation mutation, and FsGH28c^-N was a C-terminal truncation mutation. Scale bar = 0.3 cm. (b) BiFC assay showing that the interaction between PnPUB35-nYFP and FsGH28c^-SP-cYFP formed a functional YFP in the plasma membrane. Scale bar = 100 µm.

Figure 6

PnPUB35 positively regulates black pepper resistance against Fusarium solani: (a) Disease symptoms of the black pepper plants after WN-1 infection. Photographs were taken at 8 weeks after inoculation. Scale bar = 2 cm. (b) The expression level of PnPUB35 in the treatment plants. Total RNA was isolated from roots at 21 days post-agroinfiltration. PnMLF1 was used as the reference. Each experiment was performed using three independent replicates (** p < 0.01). (c) Disease index of the treatment plants at 4 weeks, 6 weeks, and 8 weeks after inoculation with WN-1. Each experiment was performed using three replicates (* p < 0.05). (d) qPCR analysis of the relative fungal biomass in stems of the treatment plants at 8 weeks after WN-1 inoculation. Each experiment was performed using three replicates. Differences between groups were compared using the t-test (** p < 0.01). (e) Re-isolation of F. solani from the stem of inoculated black pepper plants at 26 °C for 3 days. Scale bar = 1 cm. (f) Callose deposition in leaves of the treatment plants at 6 weeks after WN-1 inoculation. Leaves were imaged on fluorescence microscopy. Scale bar = 200 μm.

Figure 6

PnPUB35 positively regulates black pepper resistance against Fusarium solani: (a) Disease symptoms of the black pepper plants after WN-1 infection. Photographs were taken at 8 weeks after inoculation. Scale bar = 2 cm. (b) The expression level of PnPUB35 in the treatment plants. Total RNA was isolated from roots at 21 days post-agroinfiltration. PnMLF1 was used as the reference. Each experiment was performed using three independent replicates (** p < 0.01). (c) Disease index of the treatment plants at 4 weeks, 6 weeks, and 8 weeks after inoculation with WN-1. Each experiment was performed using three replicates (* p < 0.05). (d) qPCR analysis of the relative fungal biomass in stems of the treatment plants at 8 weeks after WN-1 inoculation. Each experiment was performed using three replicates. Differences between groups were compared using the t-test (** p < 0.01). (e) Re-isolation of F. solani from the stem of inoculated black pepper plants at 26 °C for 3 days. Scale bar = 1 cm. (f) Callose deposition in leaves of the treatment plants at 6 weeks after WN-1 inoculation. Leaves were imaged on fluorescence microscopy. Scale bar = 200 μm.

Figure 7

Overexpression of PnPUB35 enhances Arabidopsis thaliana resistance to Fusarium solani: (a) Disease symptoms of the A. thaliana plants after WN-1 infection. WT represents wild-type A. thaliana plants, and OE^PnPUB35 represents PnPUB35 overexpression transgenic A. thaliana line plants. Photograph was taken 14 days after inoculation with WN-1. Scale bar = 1 cm. (b) The necrotic leaves rate of A. thaliana plants at 14 days after WN-1 infection. Differences between groups were compared using the t-test (* p < 0.05). (c) qPCR analysis of the relative fungal biomass in leaves of the treatment plants at 14 days after WN-1 inoculation. Each experiment was performed using three replicates. Differences between groups were compared using the t-test (* p < 0.05). (d) Western blot analysis of PnPUB35 expression in A. thaliana plants. CBB was used as the control.

Figure 8

Schematic model of the interaction between FsGH28c and PnPUB35. When Fusarium solani infects plants, F. solani secretes a virulence factor FsGH28c. FsGH28c can induce immune responses. It is a positive regulator of F. solani virulence. The PnPUB35 transfers localization from the nucleus to the plant cell membrane in the presence of F. solani, and interacts with FsGH28c to protect the plant against F. solani. The red X represents the F. solani strain without FsGH28c.