Effects of SNF1 pathway mutations on GFP-PTS1 distribution.

<p>(<b>A</b>) Confocal microscopic observation of mutant strains expressing GFP-PTS1. Images shown were representative of the majority of vegetative hyphae. Enlarged peroxisomes were more frequently observed in <i>ΔMosnf1</i> and <i>ΔMosak1ΔMotos3</i> than WT. Arrows point to peroxisomes. Bar  = 5 µm. (<b>B</b>) Colocalization of GFP-PST1-positive peroxisomes and CMAC-stained vacuoles. The amount of cytoplasmic peroxisomes was decreased dramatically in the conidia of <i>ΔMosnf1</i>, <i>ΔMosak1</i>, and <i>ΔMosak1ΔMotos3</i>, while in WT and <i>ΔMotos3</i>, numerous peroxisomal puncta were observed with the absence of vacuolar GFP fluorescence. The localization patterns of GFP-PTS1 in <i>ΔMosip2</i> and <i>ΔMosnf4</i> conidia were indistinguishable from that in <i>ΔMosnf1</i> conidia. Bars  = 5 µm.</p

Mutations in SNF1 pathway affected the utilization of non-fermentable carbons.

<p>Strains were cultured on MM plates supplemented with 1% Glucose, 1% Tween 80, 1% Olive oil, 1% Triolein, or 50 mM Sodium acetate as sole carbon source for 10 d at 25°C.</p

Pathogenicity assay on detached barley leaves.

<p>Intact and abraded barley leaves were inoculated with 20 µl conidial suspensions (1×10⁵ conidia/ml) of the tested strains for 4 days before photography. <i>ΔMosnf1</i>, <i>ΔMosip2</i>, <i>ΔMosnf4</i>, <i>ΔMosak1</i>, and <i>ΔMosak1ΔMotos3</i> were deficient in appressorium-mediated infection of intact barley leaves. Elevated virulence was observed in <i>ΔMosip2</i>, <i>ΔMosnf4</i>, and <i>ΔMosak1</i> when tested on abraded barley leaves, while <i>ΔMosnf1</i> and <i>ΔMosak1ΔMoto3</i> were still non-pathogenic. Addition of 2.5% glucose to conidial suspensions could evidently, yet partially, restored the virulence of the defective mutants.</p

Comparison of the SNF1 pathway mutants with regard to colony morphology and conidial development.

<p>(<b>A</b>) Strains were cultured on CM plates at 25°C for 10 days. <i>ΔMosak1ΔMotos3</i> exhibited a decreased mycelial growth rate, while no significant difference in the colony size was observed between other mutants and Guy11. (<b>B</b>) Microscopic observation of conidial development. Significant reduction in conidial production was observed in <i>ΔMosnf1</i>, <i>ΔMosip2</i>, <i>ΔMosnf4</i>, <i>ΔMosak1</i>, and <i>ΔMosak1ΔMoto3</i> at 24 hpi. However, <i>ΔMotos3</i> developed short, yet dense conidiophores with plenty of spores arrayed thereon. Bars  = 50 µm. (<b>C</b>) Conidia of WT and the mutants were harvested and observed under the light microscope. Conidial shape of <i>ΔMosip2</i>, <i>ΔMosnf4</i>, <i>ΔMosak1</i>, and <i>ΔMosak1ΔMoto3</i> was identical to that of <i>ΔMosnf1</i> (<i>ΔMosnf1</i>-pattern), whereas there was no measurable difference between <i>ΔMotos3</i> and Guy11 (Normal). Bars  = 5 µm.</p

Spray inoculation assay with rice seedlings.

<p>Conidial suspensions (1×10⁵ conidia/ml) with (+) or without (-) 2.5% glucose were evenly sprayed onto rice leaves for 7 days before photographing the typical infected leaves (<b>A</b>) and calculating the percentage of diseased leaf area (<b>B</b>).</p

Indirect assessment of turgor pressure and appressorial porosity.

<p>(<b>A</b>) To measure appressorial turgor pressure, incipient cytorrhysis assay was performed on induced appressoria at 48 hpi with glycerol solutions of varying concentrations (1–4 M). (<b>B</b>) Plasmolysis/cytorrhysis assay with osmotic solutions of different average molecular weights. The solutions were adjusted to the denoted concentrations to exert 4 MPa osmotic pressure on appressoria at 48 hpi. (<b>C</b>) Plasmolyzed appressoria at 48 hpi were photographed after soaked in 1.7 M glycerol solution for 10 min. Bars  = 5 µm.</p

SNF1 pathway mutants exhibited dramatic defects in sporulation, appressorial size, conidial germination, and appressorium formation.

<p>(<b>A</b>) Conidial production of <i>ΔMosnf1</i>, <i>ΔMosip2</i>, <i>ΔMosnf4</i>, <i>ΔMosak1</i>, and <i>ΔMosak1ΔMoto3</i> was severely impaired, while <i>ΔMotos3</i> showed an elevated conidiation in comparison to Guy11. The complemented strains restored the sporulation defects of the corresponding mutants. Conidia were collected and counted from 10-day-old cultures grown on CM plates. The means and standard deviations of three independent experiments are presented as columns with error bars. (<b>B</b>) Appressorial diameters were measured and statistically analyzed after 48 h incubation on the artificial surfaces. Addition of 2.5% glucose to conidial suspensions partially restored the mutant defect in appressorial size. (<b>C</b>) Disruption of SNF1 pathway genes impaired conidial germination and appressorium formation in <i>M. oryzae</i>. Conidial suspensions harvested from 8-day-old CM cultures were incubated on hydrophobic surfaces and observed under a light microscope at indicated time points.</p

Protein interaction and gene expression analyses of SNF1 kinase complex components and its activating kinases in <i>M. oryzae</i>.

<p>(<b>A</b>) Different composition of the heterotrimeric SNF1 kinase complex and upstream kinases between <i>S. cerevisiae</i> and <i>M. oryzae</i>. (<b>B</b>) MoSnf1, MoSip2, and MoSnf4 interacted with each other, while no interaction was observed between MoSnf1 and its activating kinases in yeast two-hybrid assay. Yeast transformants expressing MoSnf1 plus MoSip2, MoSnf1 plus MoSnf4, MoSip2 plus MoSnf4, MoSnf1 plus MoSak1, or MoSnf1 plus MoTos3 were 10-fold serially diluted with a starter culture of 10⁶ cells/ml and then spotted (5 µl) onto SD-Trp-Leu-His-Ade medium. (<b>C</b>) Gene expression profiles of <i>MoSNF1</i>, <i>MoSIP2</i>, <i>MoSNF4</i>, <i>MoSAK1</i>, and <i>MoTOS3</i> among different developmental stages. Tested fungal tissues included vegetative hyphae (VH), conidia (CO), appressoria 8 hpi (AP), and invasive hyphae (72 hpi), which were within infected plant leaves (IP). Gene expression data, obtained from quantitative RT-PCR analysis, were normalized by using β-tubulin as an internal control and calibrated against the transcript abundances of VH stage.</p

Growth rate of the SNF1 pathway mutants on non-fermentable carbon media.

<p>Vegetative growth rate (%)  =  (the diameter of strains on MM with various carbon sources/the diameter of cultures on regular MM plates) ×100; colony diameters on regular MM plates are set as 100% control. Data were collected from 10 days postincubation and presented as means±SD from three independent experiments. Duncan's multiple range tests were used to determine significance at the 0.05 level of probability. The same letters in a column mean no significant difference.</p

Inter-individual differences and intra-individual similarities in the castrated rats.

<p>Inter-individual variability in serum OS and target tissue 8-OHdG staining in rats with similar (castrated) levels of testosterone.</p