The plasma membrane H+ -ATPase FgPMA1 regulates the development, pathogenicity, and phenamacril sensitivity of Fusarium graminearum by interacting with FgMyo-5 and FgBmh2

Abstract

Fusarium graminearum, as the causal agent of Fusarium head blight (FHB), not only causes yield loss, but also contaminates the quality of wheat by producing mycotoxins, such as deoxynivalenol (DON). The plasma membrane H⁺ -ATPases play important roles in many growth stages in plants and yeasts, but their functions and regulation in phytopathogenic fungi remain largely unknown. Here we characterized two plasma membrane H⁺ -ATPases: FgPMA1 and FgPMA2 in F. graminearum. The FgPMA1 deletion mutant (∆FgPMA1), but not FgPMA2 deletion mutant (∆FgPMA2), was impaired in vegetative growth, pathogenicity, and sexual and asexual development. FgPMA1 was localized to the plasma membrane, and ∆FgPMA1 displayed reduced integrity of plasma membrane. ∆FgPMA1 not only impaired the formation of the toxisome, which is a compartment where DON is produced, but also suppressed the expression level of DON biosynthetic enzymes, decreased DON production, and decreased the amount of mycelial invasion, leading to impaired pathogenicity by exclusively developing disease on inoculation sites of wheat ears and coleoptiles. ∆FgPMA1 exhibited decreased sensitivity to some osmotic stresses, a cell wall-damaging agent (Congo red), a cell membrane-damaging agent (sodium dodecyl sulphate), and heat shock stress. FgMyo-5 is the target of phenamacril used for controlling FHB. We found FgPMA1 interacted with FgMyo-5, and ∆FgPMA1 showed an increased expression level of FgMyo-5, resulting in increased sensitivity to phenamacril, but not to other fungicides. Furthermore, co-immunoprecipitation confirmed that FgPMA1, FgMyo-5, and FgBmh2 (a 14-3-3 protein) form a complex to regulate the sensitivity to phenamacril and biological functions. Collectively, this study identified a novel regulating mechanism of FgPMA1 in pathogenicity and phenamacril sensitivity of F. graminearum.

Keywords: Fusarium graminearum; DON production; FgPMA1; pathogenicity; phenamacril.

FIGURE 1

Impact of FgPMA1 and FgPMA2 deletion mutants on vegetative growth, and sexual and asexual development. (a) The wild‐type PH‐1, FgPMA1 deletion mutant (∆FgPMA1), FgPMA2 deletion mutant (∆FgPMA2), and complemented strains (∆FgPMA1‐C and ∆FgPMA2‐C) were grown on potato dextrose agar (PDA), complete medium (CM), minimal medium (MM), and V8 at 25°C for 3 days. (b) Hyphal branching pattern of each strain growing on water agar (WA). The branching of ∆FgPMA1 was increased in the extension zone of the colony. Bar = 72 μm. (c) The mutant ∆FgPMA1 was defective in sexual development. Strains grown on carrot agar (CA) were self‐fertilized. Bar = 200 μm. Photographs of perithecia were taken after 10 days of incubation and photographs of ascospores were taken after 2 weeks of incubation. Bar = 250 μm. (d) Conidia of the same set of strains were examined by differential interference contrast (DIC) microscopy. Bar = 20 μm. (e) Conidia of each strain were covered on the WA surface. After 2 and 8 h of incubation, germination of 300 conidia was examined under a microscope. Error bars in each column denote SE of three experiments. Bars with the same letter indicate no significant difference at p = 0.05. (f) The length of germ tube of the strains after incubation for 2 and 8 h (bar = 20 μm)

FIGURE 2

FgPMA1 affects the integrity of the plasma membrane. (a) FgPMA1 was localized to the plasma membrane. (b) FgPMA1 regulated the integrity of the plasma membrane in mycelium. All strains were incubated in yeast exact peptone dextrose (YEPD) for 12 h. Wild type, PH‐1; complemented strains, ‐C. Mycelium was stained with the plasma membrane tracker 3,3′‐dioctadecyloxacarbocyanine perchlorate (DiD). (c) FgPMA1 regulated the integrity of the plasma membrane in conidia. All mutants were incubated in carboxymethy‐cellulose (CMC) for 3 days. All the photographs were taken under a confocal microscope (Leica)

FIGURE 3

FgPMA1 is important for pathogenicity of Fusarium graminearum. (a) Wheat heads infected by various strains were examined at 14 days after inoculation with a conidial suspension. The inoculation sites are indicated by black dots. Wild type, PH‐1; complemented strains, ‐C. (b) The lesion length on wheat coleoptiles infected by various strains. The photographs were taken at 14 days postinoculation (dpi). (c) Confocal images of various strains growing inside wheat leaves at 5 dpi

FIGURE 4

FgPMA1 affects deoxynivalenol (DON) synthesis. (a) ∆FgPMA1 displayed decreased DON content. Wild type, PH‐1; complemented strains, ‐C. All strains were incubated in trichothecene biosynthesis induction (TBI) medium for 7 days. Bars with the same letter indicate no significant difference at p = 0.05. (b) FgPMA1 impaired the formation of DON toxisomes, visualized by by labelling the toxisome with Tri‐GFP. Strains were incubated in TBI for 36 h. (c) ∆FgPMA1 showed a decreased expression level of Tri‐GFP. Strains were incubated in TBI for 36 h and mycelia were harvested for western blot analysis

FIGURE 5

Phenotypes of strains in response to various stresses. (a) ∆FgPMA1 decreased the sensitivity towards a cell membrane‐damaging agent (sodium dodecyl sulphate, SDS) and a cell wall‐damaging agent (Congo red, CR). (b) Statistical analysis of the growth inhibition rate of all strains under CR and SDS stresses. (c) Colony morphology is shown after 3 days of incubation on potato dextrose agar (PDA). Growth phenotype of wild type (PH‐1), mutant, and complementation strains (‐C) on PDA with or without supplementation of KCl, NaCl, CaCl₂, and sorbitol after 3 days of incubation at 25°C. (d) Statistical analysis of the growth inhibition rate of all strains under osmotic stress. (e) ∆FgPMA1 was more tolerant to heat at 30°C and did not show any difference compared with the wild type and complementation strains at 15°C. (f) Statistical analysis of the growth inhibition rate of all strains under heat shock. Bars with the same letter indicate no significant difference at p = 0.05

FIGURE 6

FgPMA1 regulates the sensitivity to phenamacril. (a) The ΔFgPMA1 mutation in sensitive strains increased the sensitivity to phenamacril. All strains were incubated on complete medium (CM) for 3 days. Wild‐type phenamacril‐sensitive strain, PH‐1; wild‐type phenamacril‐resistant strain YP‐1, ‐Y; complemented strains, ‐C; (b) The ΔFgPMA1 mutation in resistant strains decreased the resistance to phenamacril. All strains were incubated on CM for 3 days. (c) Co‐immunoprecipitation confirmed the interaction between FgPMA1 and FgMyo‐5. Strains were cultured in yeast extract peptone dextrose (YEPD) medium for 36 h. (d) The FgPMA1 deletion mutant increased the expression level of FgMyo‐5. Strains were cultured in YEPD for 36 h

FIGURE 7

FgPMA1, FgMyo‐5, and FgBmh2 form a complex to regulate the sensitivity to phenamacril. (a) Yeast two‐hybrid assay showed that FgPMA1 and FgBmh2 interacted directly. (b) Co‐immunoprecipitation (Co‐IP) confirmed the interaction between FgPMA1 and FgBmh2. (c) Co‐IP confirmed the interaction between FgBmh2 and FgMyo‐5. (d) ∆FgBmh2 decreased the sensitivity to phenamacril. All the strains were incubated on potato dextrose agar (PDA) with or without phenamacril for 3 days. Wild type, PH‐1. (e) Statistical analysis of the growth inhibition rate of all strains treated with phenamacril. Bars with the same letter indicate no significant difference at p = 0.05

FIGURE 8

A proposed model showing the role of FgPMA1‐FgMyo‐5‐FgBmh2 complex. When FgPMA1 is deleted, this complex is disrupted, leading to the changed activity or stability of FgMyo‐5 as evidenced by ∆FgPMA1 affecting the sensitivity to phenamacril. This disrupted protein complex also affects the development and pathogenicity of Fusarium graminearum