The Ras GTPase-activating protein UvGap1 orchestrates conidiogenesis and pathogenesis in the rice false smut fungus Ustilaginoidea virens

Abstract

Ras GTPase-activating proteins (Ras GAPs) act as negative regulators for Ras proteins and are involved in various signalling processes that influence cellular functions. Here, the function of four Ras GAPs, UvGap1 to UvGap4, was identified and analysed in Ustilaginoidea virens, the causal agent of rice false smut disease. Disruption of UvGAP1 or UvGAP2 resulted in reduced mycelial growth and an increased percentage of larger or dumbbell-shaped conidia. Notably, the mutant ΔUvgap1 completely lost its pathogenicity. Compared to the wild-type strain, the mutants ΔUvgap1, ΔUvgap2 and ΔUvgap3 exhibited reduced tolerance to H₂ O₂ oxidative stress. In particular, the ΔUvgap1 mutant was barely able to grow on the H₂ O₂ plate, and UvGAP1 was found to influence the expression level of genes involved in reactive oxygen species synthesis and scavenging. The intracellular cAMP level in the ΔUvgap1 mutant was elevated, as UvGap1 plays an important role in maintaining the intracellular cAMP level by affecting the expression of phosphodiesterases, which are linked to cAMP degradation in U. virens. In a yeast two-hybrid assay, UvRas1 and UvRasGef (Ras guanyl nucleotide exchange factor) physically interacted with UvGap1. UvRas2 was identified as an interacting partner of UvGap1 through a bimolecular fluorescence complementation assay and affinity capture-mass spectrometry analysis. Taken together, these findings suggest that the UvGAP1-mediated Ras pathway is essential for the development and pathogenicity of U. virens.

Keywords: Ustilaginoidea virens; Ras GTPase-activating protein; conidiation; pathogenicity.

FIGURE 1

Identification of the T‐DNA‐tagged gene UvGAP1 and expression patterns of UvGAPs in Ustilaginoidea virens. (a) Virulence of wild type (P‐1) and T‐DNA insertional mutant B‐2510 on rice cultivar Liangyoupeijiu. (b) Average number of false smut balls per panicle. (c) Localization of the T‐DNA insertion sites in the B‐2510 mutant. (d) Domain architecture of four UvGaps in U. virens. (e–h) The relative expression of UvGAP1 (e), UvGAP2 (f), UvGAP3 (g) and UvGAP4 (h) at various developmental and infection stages on rice spikelets ranging from 6 h to 30 days. The expression levels of UvGAPs were normalized to β‐TUBULIN expression. Hy, hyphae on the potato sucrose agar plate for 7 days; IS, initial stage of sporulation; LS, later stage of sporulation; Co, conidia. Asterisks indicate significant differences compared to Hy, as determined by Duncan's new multiple range test (p < 0.05).

FIGURE 2

Role of UvGAPs in mycelial growth and conidial morphology of Ustilaginoidea virens. (a) Colony morphology of wild‐type strain P‐1, mutant strains ΔUvgap1, ΔUvgap2 and ΔUvgap3 and complemented strains Uvgap1‐c and Uvgap2‐c on potato sucrose agar (PSA) plates or in potato sucrose broth (PSB). The strains were incubated on PSA plates for 20 days or cultured in PSB with shaking for 6 days. Scale bar, 5 mm. (b) Conidial morphology and germination of U. virens strains. Conidia were harvested from 6‐day‐old PSB cultures, and conidial germination was observed after 20 h of incubation. Scale bar, 20 μm. (c) Calcofluor white staining of hyphae from U. virens strains. Arrows indicate hyphal septa. Scale bar, 20 μm. (d) Cell length of U. virens hyphae. Asterisks indicate significant differences compared to wild‐type P‐1, as determined by Duncan's new multiple range test (p < 0.05).

FIGURE 3

Role of UvGAP1 in the virulence of Ustilaginoidea virens. (a) Rice false smut balls produced by the wild type (P‐1), ΔUvgap1 and Uvgap1‐c on rice at 30 days post‐inoculation (dpi). (b) Observation of infected rice spikelets at 2, 4, 8 and 14 dpi. Scale bar, 10 μm.

FIGURE 4

Toxicity assessment of Ustilaginoidea virens culture filtrates on rice seeds. (a) Rice seeds were soaked with potato sucrose broth control or filtrates from cultures of the wild type (P‐1), ΔUvgap1, and Uvgap1‐c. Shoot and root growth were evaluated after incubation at 25°C for 5 days. (b) Histogram of shoot and root length in (a). Data are represented as means ± SD from more than 50 rice seeds. Asterisks indicate significant differences compared to P‐1 as estimated by Duncan's new multiple range test (p < 0.05).

FIGURE 5

UvGAP1 modulates stress responses in Ustilaginoidea virens. (a) Mycelial growth of wild type (P‐1), ΔUvgap1, and Uvgap1‐c on potato sucrose agar (PSA) or PSA supplemented with various stress agents including 0.5 M NaCl, 0.6 M sorbitol, 500 μg/mL calcofluor white (CFW), 100 μg/mL Congo red (CR), 0.05% sodium dodecyl sulphate (SDS) or 0.05% H₂O₂. Photographs were obtained after incubation at 28°C for 20 days in the dark. (b) Expression profiles of five peroxidase genes, two catalase genes, two NADPH oxidase genes, and one NADPH oxidase regulator. RNA samples were isolated from vegetative hyphae of P‐1 and ΔUvgap1 cultured in potato sucrose broth (PSB) or PSB with 0.05% H₂O₂. The expression level of each gene in the wild‐type strain P‐1 cultured in PSB was arbitrarily set to 1.0. Different letters represent significant differences as estimated by Duncan's new multiple range test (p < 0.05).

FIGURE 6

UvGAP1 modulates the intracellular cAMP content in Ustilaginoidea virens. (a) Intracellular cAMP levels in the wild type (P‐1), ΔUvgap1, and Uvgap1‐c. (b) Expression levels of UvAC1 and UvPDEH. Asterisks indicate significant differences between the mutant and the wild‐type strain as determined by Duncan's new multiple range test (p < 0.05).

FIGURE 7

Protein interactions between UvGaps, UvRas and UvRasGef. (a) A yeast two‐hybrid assay was conducted to assess the interactions between four UvGaps, two Ras proteins (UvRas1 and UvRas2) and UvRasGef. Yeast cells (10⁴–10⁶ cells/mL) harbouring prey and bait vectors were examined for growth on SD/−Leu/−Trp (SD−2) and SD/−Ade/−His/−Leu/−Trp (SD−4) media. The positive control involved the interaction between pGADT7 (AD‐T) and pGBKT7‐53 (BD‐53), while the negative control examined the lack of growth in the interaction between pGADT7 (AD‐T) and pGBKT7‐Lam (BD‐Lam). (b) The expression of bait and prey proteins were detected using anti‐HA antibody and anti‐Myc antibody, respectively. (c) Bimolecular fluorescence complementation assay to detect the interaction between UvGap1 and UvRas1 or UvRas2. Yellow fluorescent protein signals were examined with a LSM710 confocal microscope (Zeiss). Scale bar, 20 μm.

FIGURE 8

Investigating protein interactions involving UvGap1 in Ustilaginoidea virens. (a) Yeast two‐hybrid assay to assess the interactions between UvGap1 and UvGap1‐interacting partners from the affinity capture‐mass spectrometry assay. SD−2: SD/−Leu/−Trp medium, SD−4: SD/−Ade/−His/−Leu/−Trp medium. Positive control: interaction between pGADT7 (AD‐T) and pGBKT7‐53 (BD‐53). Negative control: interaction between pGADT7 (AD‐T) and pGBKT7‐Lam (BD‐Lam). (b) The expression of bait and prey proteins were detected with anti‐HA antibody and anti‐Myc antibody, respectively.