Methodic of basic punches and defences in box.

Title: Methodic ofbasic punches and defences in box. The aim of diploma theses: Create a manual ofmethodic ofbasic punches and defences in box. Method: In making my theses I used usual methods like dokument analysis, interrogation and observation. I replenished this acquired information with study of Czech and foreign literature, reading magazines and searching on internet. Also I completed this infonnation with my own experience. Results: In this diploma theses I described all of basic punches and defences in box. Process is systematic from basic pose and simply punches till offensive combinations, active and passive defence. Keywords: Box, punch, defence, offensive.

Wild-type and transformants of <i>Fusarium graminearum</i> strains used in this study.

<p>Wild-type and transformants of <i>Fusarium graminearum</i> strains used in this study.</p

Schematic draw of the pre-mRNA splicing processes and components of tri-snRNP related to this study.

<p>Exons and one intron are represented by boxes and solid line, respectively. Base pairing of U1 to 5’ss and recognition of BP by U2 (formation of complex A) are followed by the integration of preformed U4/U6-U5 tri-snRNP to form complex B. Whereas Prp4 and Prp31 are components of U4/U6 snRNP, Brr2, Prp8, and Prp6 are components of U5 snRNP. Phosphorylation of Prp6 and Prp31 by Prp4 is associated with the activation of B-complex (complex B^act). U1, U4, Prp4, Prp6, and Prp31 are released from the activated spliceosome that catalyzes two sequential transesterifications reactions for intron splicing.</p

Assays for the function of the N-terminal 310 aa of FgPrp4.

<p>(<b>A).</b> Conidia, 12 h germlings, and hyphae of the <i>Fgprp4/FgPRP4-</i>GFP transformant (FPN1) were examined by DIC and epifluorescence microscopy. Bar = 20 μm. (<b>B).</b> The expression level of <i>FgPRP4</i> was assayed by qRT-PCR with RNA isolated from conidia, 12 h germlings, perithecia at 10 days post-fertilization, and infected wheat heads at 7 days post-inoculation (dpi). Mean and standard deviation were calculated with data from three independent biological replicates. The β-tubulin gene FGSG_06611 of <i>F</i>. <i>graminearum</i> was used as the internal control. (<b>C).</b> Three-day old PDA cultures of the <i>Fgprp4</i> mutant (FP1), <i>Fgprp4/FgPRP4</i>-GFP transformant (FPN1), and <i>Fgprp4/FgPRP4</i>^Δ1-310-GFP transformant (FPN310). (<b>D).</b> 12 h germlings of transformant FPN310 were examined by DIC and epifluorescence microscopy. Bar = 20 μm.</p

Effects of <i>FgPRP4</i> deletion on intron splicing.

<p>(<b>A).</b> Box-plot comparison of intron retention levels between the wild type (PH-1) and <i>Fgprp4</i> mutant (FP1) in replica experiments. The statistical significance for each comparison is analyzed by <i>t</i>-test (****, <i>P</i><0.0001). (<b>B).</b> The percentage of introns and genes with the three marked intron retention levels in <i>Fgprp4</i> compared to the wild type. (<b>C)</b>. Introns that were increased in intron retention over 2-fold in <i>Fgprp4</i> in both replica experiments. (<b>D).</b> Intron splicing defects in the labelled genes were verified by RT-PCR with primers flanking the introns with reduced splicing efficiency (marked with *) in the <i>Fgprp4</i> mutant. Lanes 1–3 were PCR results with the genomic DNA, cDNA of PH-1, and cDNA of <i>Fgprp4</i>, respectively. The sizes of amplified bands are labelled on the side.</p

The S289 mutation affected the function but not localization of FgPrp4.

<p>(<b>A).</b> Western blots of total proteins isolated from the wild type (PH-1) and the <i>FgPRP4</i>-3xFLAG transformant were detected with an anti-3xFLAG antibody. (<b>B).</b> 12 h germlings of the <i>Fgprp4/FgPRP4</i>^S289A transformant FPA2 were examined by DIC and epifluorescence microscopy. Bar = 20 μm. <b>(C).</b> Three-day old PDA cultures of the <i>Fgprp4</i> mutant (FP1), complemented transformant (FPN1), and <i>Fgprp4/FgPRP4</i>^S289A transformant (FPA2).</p

Candidate Prp4-target genes sequenced in the selected suppressor strains.

<p>Candidate Prp4-target genes sequenced in the selected suppressor strains.</p

Suppressor mutations in <i>FgBRR2</i> and <i>FgPRP8</i>.

<p>(<b>A)</b>. Schematic drawing of the FgBrr2 protein and alignment of the marked region with its orthologs from <i>M</i>. <i>oryzae</i> (Mo), <i>N</i>. <i>crassa</i> (Nc), <i>Sclerotina sclerotiorum</i> (Ss), <i>S</i>. <i>cerevisiae</i> (Sc), <i>S</i>. <i>pombe</i> (Sp), and human (Hs). G308 of FgBrr2 and A311 of Brr2 (Sp) were boxed with red line. Conserved domains of the N- and C-terminal helicase cassettes, G308E mutation in FgBrr2, and A311E in SpSpp41 were labelled. (<b>B)</b>. The predicted domain structure of FgPrp8 and sequence alignment of its marked regions with its orthologs from other fungi and humans. (<b>C)</b>. 3-D modeling of FgPrp8^846-2370. The regions with suppression mutations (marked with boxes) were magnified on the right to show the differences between D and G at 1153 or E and K at 1429 in the side chains. E1412 (purple) is in the same cleft with E1429.</p

Spontaneous suppressors of the <i>Fgprp4</i> mutant.

<p>(<b>A)</b>. PDA cultures of the <i>Fgprp4</i> mutant (FP1) strains after incubation for two weeks. Fast growing sectors were marked with arrows. (<b>B).</b> CM cultures of representative type I and type II suppressor mutants grew in race tubes for 14 days. (<b>C)</b>. Flowering wheat heads were drop inoculated with conidia of the wild type PH-1 and marked <i>Fgprp4</i> suppressor strains. Typical wheat heads were examined 14 dpi. (<b>D).</b> Perithecia and asci produced by suppressor strains S3, S18, S38, and S30. Lower panels show asci and ascospores released from cracked perithecia. Bar = 50 μm.</p