Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling-5

<p><b>Copyright information:</b></p><p>Taken from "Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling"</p><p>http://www.biomedcentral.com/1471-2202/8/65</p><p>BMC Neuroscience 2007;8():65-65.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1963332.</p><p></p>hybrid GFP (first chromosome) and homozygous for mutations were analyzed for the fluorescence in the wing. The rescue in background was analyzed as double heterozygous (). Flies were heat-shocked at day 3 and analyzed two days later. Positive control: MARCM system (chromosome recombination based on flipase under heat-shock promotor induces axonal marker -)

Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling-1

<p><b>Copyright information:</b></p><p>Taken from "Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling"</p><p>http://www.biomedcentral.com/1471-2202/8/65</p><p>BMC Neuroscience 2007;8():65-65.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1963332.</p><p></p>re. In tube (C), homozygous mutants their genetic background give: T value 3.87, P = 0.001, degrees of freedom: 18 ; T value 7.6, P = 0.0001, degrees of freedom: 18 (); T value 4.36, P = 0.0004, degrees of freedom 18 (). The heterozygous mutants show a similar trend (n = 10). Statistics for mutants genetic background in tube C: T value 4, P = 0.001, degrees of freedom: 16 (and ); T value 1.98, P = 0.06, degrees of freedom: 16 (). (2) The experiment was also performed using a derivative protocol: one single pierced transparent tube was used with 1 ml grape juice in a saturated atmosphere. We observed progression of the accumulation from 2 to 24 hours. Values are mean ± SEM (n = 10). For at 24 hours: T value 1.8, P value: 0.085, degrees of freedom: 18 (3) Analysis of the mutant and the rescue (protocol as in (1)). The rescue (, along with , exposed to single acute heat shock at late third instar larva 3 day old adult stage, showed reverse phenotype (similar to control genetic background). Statistics for the mutant the rescue in tube C: T value 3.42, P = 0.003, degrees of freedom: 18 (n = 10)

Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling-0

<p><b>Copyright information:</b></p><p>Taken from "Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling"</p><p>http://www.biomedcentral.com/1471-2202/8/65</p><p>BMC Neuroscience 2007;8():65-65.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1963332.</p><p></p>�l) was placed inside the three systems. The protocols are described in the Methods section. Briefly, tubes are painted black to eliminate any visual interference with exploration. One set of experiments is carried out in an aerated chamber (odour gradients are maintained), the other in grape juice-saturated atmosphere (see figure 4 and Methods section). One hundred flies were placed in the chamber and the flies inside the 3 tubes were counted 10 hours later. (A) tube with 50 μl, (B) 100 μl and (C) 300 μl. The mutants , and were analyzed along with genetic background and . Values are mean ± SEM (n = 10)

Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling-3

<p><b>Copyright information:</b></p><p>Taken from "Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling"</p><p>http://www.biomedcentral.com/1471-2202/8/65</p><p>BMC Neuroscience 2007;8():65-65.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1963332.</p><p></p>nts. Mutants (20 flies, 5 days old, red eyed, females to avoid pheromone interference) were released in the chamber without disturbing the aggregated spot (aggregates stable, probably because of altered vision). Simultaneously, a second fresh spot was placed inside. Here, the graph represents the time course of the ratio of aggregated flies (or ) on the pre-aggregated spot the fresh one. Values are mean ± SEM (n = 10). The mutant against control gave T value: 3.38, P = 0.003 and degrees of freedom: 18. Heat shock rescue was carried out as in figure 1. () The control of flies aggregated the total in the chamber is represented for the strains tested above. We saw no significant differences between the strains. The apparatus was the same than in the figure 5 and 6

Knock-down of <i>Meloidogyne incognita</i> proteases affects nematode reproduction and egg viability (after 45 DAI).

<p>(A) Number of eggs per gram of root. (B) Total egg hatching ratio. Experiments were repeated twice. Different letters mean statistical significance through one-way ANOVA and Tukey test (<i>p</i>≤0.05).</p

<i>In</i><i>silico</i> analyses of all <i>Meloidogyne incognita</i> aspartic, serine and cysteine proteases ESTs present in EST data bank dbEST.

<p>Representation of <i>M. incognita</i> proteases expressed sequence tags (ESTs) in databanks. Bars show the percentage of proteases EST number relative to the total number of EST available for each developmental stage. ESTs from proteases were retrieved from NCBI-dbEST (<a href="http://www.ncbi.nlm.nih.gov/dbest/index.html" target="_blank">http://www.ncbi.nlm.nih.gov/dbEST/index.html</a>) and their representation was assessed by the number of ESTs relative to the total number of ESTs available for the developmental stage considered. The developmental stages considered were; eggs (14,671 ESTs), freshly hatched J2s (33,835 ESTs), mixed parasitic stages (3,133 ESTs) and females (4,427 ESTs). The distribution of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ESTs is indicated for comparison. </p

Relative abundance of specific protease gene transcripts in <i>Meloidogyne incognita</i>.

<p>Real-time qRT-PCR analysis of <i>M. incognita</i> proteases transcript levels at different stages of the nematode life cycle. (A) Cathepsin D-like aspartic proteinase (<i>Mi-asp-1</i>, Accession: DQ360827). (B) Chymotrypsin-like serine proteinase (<i>Mi-ser-1</i>, AY714229). (C) Cathepsin L cystein proteinase (<i>Mi-cpl-1</i>, AJ557572). Each bar represents the mean of duplicate assays repeated twice. Standard errors are shown. Different letters mean statistical difference (<i>p</i>≤0.05) according to the iteration test (Rest 2009 Software). The results are presented as fold change in comparison to the stage that had the smaller relative expression value that was arbitrarily designed as 1.</p

Effect of protease knock-down on progeny virulence of <i>M. incognita</i>.

<p>Hatched J2 from eggs that were laid by females that feed on transgenic and control plants were inoculated in wild-type tobacco. (A) Relative number of galls per plant at 45 DAI; (B) Relative number of egg masses per plant at 45 DAI; Experiments were repeated twice. Different letters mean statistical significance through one-way ANOVA and Tukey test (P≤0.05).</p

Gene cloning and transgenic tobacco plant generation for host-derived RNA-interference of <i>Meloidogyne incognita</i> proteases.

<p>(A) Regions of proteinases genes used in RNAi experiments. Numbers indicate nucleotide positions. (B) Schematic representation of the pK7GWIWG2(I) (Karimi et al. 2002) hairpin double-stranded RNA (dsRNA) constructs containing the sense and antisense coding regions fragments of <i>Mi-asp-1</i>, <i>Mi-ser-1</i>, <i>Mi-cpl-1</i> separately and together. (C) Characterization of RNAi lines for silencing of <i>Mi-ser-1</i>, <i>Mi-cpl-1</i> and the fragments in tandem of <i>Mi-asp-1</i>, <i>Mi-ser-1</i> and <i>Mi-cpl-1</i> (Fusion), by PCR. Attempts for generate ds-<i>Mi-asp-1</i> lines were not successful. Sense (S) fragment, anti-sense (AS) fragment, undistinguishable fragment (Sense or Anti-sense) (F). (D) RT-PCR of the single-stranded pK7GWIWG2(I) intron (spacer) of the hairpin dsRNA was used to confirm the expression of <i>Mi-ser-1</i>, <i>Mi-cpl-1</i> and fusion dsRNAs in seedlings of independent transgenic tobacco lines at 15 d post-germination.</p

Quantitative RT-PCR showing the transcript levels of proteases in eggs exposed to dsRNAs.

<p>Eggs were collected from <i>M. incognita</i> females that infected transgenic tobacco lines expressing dsRNA for Mi-SER, Mi-CPL and Mi-ASP, Mi-SER and Mi-CPL fused (fusion). (A) Analysis of <i>Mi-asp-1</i>; in <i>M. incognita</i> (Mi) eggs of 35S-dsCPL and 35S-dsSER plants <i>Mi-asp-1</i> was not exposed to a specific dsRNA. (B) Analysis of <i>Mi-cpl-1</i> gene; in Mi eggs of 35S-dsSER plants <i>Mi-cpl-1</i> was not exposed to a specific dsRNA. (C) Analysis of <i>Mi-ser-1</i> gene; in Mi eggs from 35S-dsCPL plants <i>Mi-ser-1</i> was not exposed to a specific dsRNA. Significant differences were assessed by Iteration test (REST Software) where proteases gene expression in nematodes eggs from different plants lines were compared to control plants (*, **, *** = P ≤ 0.05, 0.01 and 0.001, respectively). </p