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

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

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

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