Fig 1.
M. oryzae ACB1 is required for lipid homeostasis, pathogenicity on rice, and BIC organization.
A. Fatty acid methyl ester (FAME) analysis by GC-MS showing the total (left) and relative (right) abundances of the five most abundant fatty acids in each strain. Samples were extracted at 40 h post inoculation (hpi) from vegetative mycelia incubated for 24 h in liquid complete media followed by 16 h in liquid minimal media at 26°C. Bars depicts the means of three biological replicates (S1 Table). Error bars are SD. Different lowercase letters indicate statistical differences as determined by one-way ANOVA followed by an unpaired t test with Bonferroni correction, P < 0.05. B. Pathogenicity assay of the Guy11 wild-type (WT) strain, the ACB1 gene deletion strain Δacb1, and the Δacb1 ACB1 complementation strain. Images are representative of at least 5 leaves from 3 independent biological replicates and were taken of 3-weeks-old CO-39 rice seedlings at 120 hpi. Spores of the indicated strains were inoculated at a rate of 1×105 spores ml-1.
Fig 2.
ACB1 prevents stochastic loss of BIC integrity.
A,B. Live-cell imaging at 36 hpi of detached rice leaf sheath epidermal cells infected with the indicated strains producing BIC-localized Pwl2-mCherry:NLS and apoplast-localized Bas4-GFP. C. Percentage of IH of each strain displaying the indicated number of BICs/ BIC fragments in the first infected rice cell at 36 hpi. Detached rice leaf sheaths were inoculated with 1x105 spores ml-1 and incubated at 26°C. Bars depict means ± SD and were calculated from observing 50 infected rice cells per treatment, repeated in triplicate. Different lowercase letters denote statistical differences (one-way ANOVA, Welch’s t test, P < 0.05). D. Live-cell imaging at 44 hpi of detached rice leaf sheath epidermal cells infected with the indicated strains producing BIC-localized Pwl2-mCherry:NLS and apoplast-localized Bas4-GFP. A,B, D. Leaf sheaths were inoculated with 3×104 spores ml-1 and incubated at 26°C. White arrowheads indicate sites of appressorial penetration, white arrows show the location of the BICs, thin red arrows indicate the retention of ER-Golgi secreted Bas4-GFP in intracellular compartments, and asterisks indicate where IH have moved to neighboring cells; scale bars, 10 μm. Images are representative of 50 infected rice cells per leaf sheath per strain, repeated in triplicate.
Fig 3.
ACB1 disruption leads to temperature-dependent changes in growth, sporulation and membrane trafficking.
A. Radial growth of the indicated strains on complete media at the given temperatures after 10 days post inoculation (dpi). Images are representative of three biological replications. Δacb1 pBV591 is the mutant strain carrying the pBV591 vector to express PWL2-mCherry:NLS and BAS4-GFP. B. Mean counts ± SD (n = 3) of spores harvested at 10 dpi from CM at the indicated temperatures. Significant differences are denoted by lowercase letters (one-way ANOVA followed by Tukey HSD, P < 0.05). C. Micrographs of endocytosis by vegetative hyphae of the indicated stains following exposure to the amphiphilic styryl dye FM4-64. Cultures were grown for 24 h in CM then switched to minimal media for 20 h at the indicated temperatures, washed and stained with 1.3 mg/ml FM4-64 for 2 min at room temperature. Images are representative of three independent biological replicates. Scale bars, 10μm. D. Rates of hyphal tip staining by FM4-64. Cultures were grown in liquid CM at 26°C for 24 h and then switched to liquid MM at the indicated temperatures for 20 h. Washed cells were stained with 1.3 mg/ml FM4-64 for 2 min at room temperature. Bars show means with error bars representing standard deviation. Different lowercase letters indicate significant differences (one-way ANOVA followed by Tukey HSD, P < 0.05). Values were obtained from observing 50 hyphal tips in each of three biological replicates at each temperature.
Fig 4.
ACB1 is critical for rice blast disease at 22°C and 26°C but not at 29°C.
A. Symptoms of rice blast disease on leaves of 3-weeks-old CO-39 rice seedlings after spraying with spores of the indicated strains at a rate of 1×105 spores ml-1. Inoculated plants were incubated for 120 hpi at the designated temperatures (ie. under constant temperature conditions). B. Lesion coverage rates on leaves of 3-week old rice seedlings infected with the indicated strains at 120 hpi at the indicated temperatures. Bars depict means ± SD. Different lowercase letters indicate significant differences (one-way ANOVA followed by Tukey HSD, P < 0.05). The experiment was performed twice, and calculations were determined from three biological replicates with three technical replicates each. C. Appressorial penetration rates for the indicated strains after incubation of inoculated detached rice leaf sheaths under constant temperatures for 36 hpi (left), or (right) after 24 h incubation at 26°C followed by a temperature switch to the indicated temperatures for an additional 12 h. Spores of the indicated strains were applied at a rate of 3×104 spores ml-1. Bars depict means ± SD and were calculated from observing 50 infected rice cells per treatment, repeated in triplicate. Significant differences were determined by Student’s t-test as indicated with asterisks: *P < 0.05, **P < 0.01; n.s. = not significant. D. Symptoms of rice blast disease on leaves of 3-weeks-old CO-39 rice seedlings after spraying with spores of the indicated strains at a rate of 1×105 spores ml-1. Plants were kept at 26°C for 24 h before switching to the indicated temperatures for an additional 96 h. E. Lesion coverage rates on leaves of 3-week-old rice seedlings infected with the indicated strains. All samples were left at 26°C for 24 h following spore inoculation before being switched to the indicated temperatures for 96 h. Bars depict means ± SD. Different lowercase letters indicate significant differences (one-way ANOVA followed by Tukey HSD, P < 0.05). Values are from three biological replicates with three technical replicates each. F. Symptoms of rice blast disease on leaves of 3-weeks-old CO-39 rice seedlings after spraying with spores of the indicated strains at a rate of 1×105 spores ml-1. Plants were kept at 26°C for 24 h before switching to the indicated temperatures for an additional 96 h. G. Lesion coverage rates on leaves of 3-week-old rice seedlings infected with the indicated strains. All samples were left at 26°C for 24 h following spore inoculation before being switched to the indicated temperatures for 96 h. Bars depict means ± SD. Different lowercase letters indicate significant differences (one-way ANOVA followed by Tukey HSD, P < 0.05). Values are from three biological replicates with three technical replicates each.
Fig 5.
ACB1 is critical for BIC integrity at 22°C and 26°C but not at 29°C.
A. Live-cell imaging of detached rice leaf sheaths inoculated at a rate of 3×104 spores ml-1 with the indicated strains expressing PWL2-mCherry:NLS and BAS4-GFP and incubated under temperature-shift conditions of 24 h at 26°C followed by a switch to the designated temperatures for an additional 12 h. Appressorial penetration sites are indicated with white arrowheads and white arrows show BIC localization; scale bars, 10μm. Images are representative of 50 infected rice cells per leaf sheath per strain, repeated in triplicate. B. Rate of occurrence of abnormal BICs in the indicated strains, including dual- and multi-BIC phenotypes, after the incubation of inoculated detached rice leaf sheaths at 26°C for 24 h followed by a temperature shift to the designated temperatures for an additional 12 h. Bars depict means ± SD and were calculated from observing 50 infected rice cells per treatment, repeated in triplicate. Significant differences were determined by Student’s t-test as indicated with asterisks: *P < 0.05, **P < 0.01; n.s. = not significant.
Fig 6.
Membrane fluidity and membrane integrity are dependent on ACB1 at optimal and suboptimal temperatures.
A. Anisotropy was calculated from single-wavelength measurements of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) using polarized excitation and emission filters and is inversely proportional to membrane fluidity. Cultures were grown in CM at 26°C for 24 h before switching to the indicated temperatures for an additional 20 h. After washing, hyphae were stained with 5 μM DPH for 40 min. Box plots display interquartile range (IQR), with boxes representing the 25th to 75th percentiles; the bold line inside the box is the mean value and whiskers extend to 1.5 times the IQR. Different lowercase letters indicate significance differences (one-way ANOVA followed by Tukey HSD, P < 0.05). The experiment was performed twice with each comprising three biological replicates with four technical replicates each. B. Fractional cellular electrolyte leakage measured in fungal hyphae of the indicated strains across a temperature gradient (22°C to 32°C). Fungal hyphae were grown at 26°C in liquid CM for 24 h and then transferred to liquid MM for 16 h. Thermal stress was simulated by gradually heating samples in a circulator. Samples were removed at 2°C intervals (from 22°C to 32°C) and analyzed for ion leakage, which is an indicator of membrane integrity. Box plots display interquartile range (IQR), with boxes representing the 25th to 75th percentiles; the bold line inside the box is the mean value and whiskers extend to 1.5 times the IQR. Different lowercase letters indicate significant differences (one-way ANOVA followed by Tukey HSD, P < 0.05). The experiment was performed twice with each comprising three biological replicates with one technical replicate each.