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

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

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

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

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

Prognostic Value of Focal Adhesion Kinase (FAK) in Human Solid Carcinomas: A Meta-Analysis

<div><p>Background</p><p>Recently, the number of reports on focal adhesion kinase (FAK) as a vital therapeutic target in solid carcinomas has increased; however, the prognostic role of FAK status remains poorly understood. This study aims to evaluate the prognostic effect of FAK by means of a meta-analysis.</p><p>Methods</p><p>We performed a systematic literature search in order to examine the correlation between expression of FAK and overall survival(OS). The hazard ratio (HR) of OS was used to measure survival. A random-effects model was used to pool study statistics. Sensitivity and publication bias analyses were also conducted.</p><p>Results</p><p>Thirty eligible studies involving 4702 patients were included. The median expression rate of FAK was 54%. Meta-analysis of the HRs demonstrated that high FAK expression was associated with worse OS (average HR = 2.073, 95%confidence interval[CI]:1.712–2.510, p = 0.000). Regarding cancer type, FAK was associated with worse OS in gastric cancer (HR = 2.646,95% CI:1.743–4.017, p = 0.000), hepatocellular carcinoma (HR = 1.788,95% CI:1.228–2.602, p = 0.002), ovarian cancer (HR = 1.815, 95% CI: 1.193–2.762, p = 0.005), endometrial cancer (HR = 4.149, 95% CI:2.832–6.079, p = 0.000), gliomas (HR = 2.650, 95% CI: 1.205–5.829, p = 0.015), and squamous cell carcinoma (HR = 1,696, 95% CI: 1.030–2.793, p = 0.038). No association was found between HR and disease staging according to our meta-regression analysis.</p><p>Conclusions</p><p>Our study shows that high expression of FAK is associated with a worse OS in patients with carcinomas, but the association between FAK and prognosis varies according to cancer type. The value of FAK status in clinical prognosis in cancer needs further research.</p></div

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