Primer sequences.

<p>Primer sequences.</p

Nucleases as a barrier to gene silencing in the cotton boll weevil, <i>Anthonomus grandis</i>

<div><p>RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (<i>Anthonomus grandis</i>) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted <i>AgraNuc1</i>, <i>AgraNuc2</i>, and <i>AgraNuc3</i>, and the influences of these nucleases on the gene silencing of <i>A</i>. <i>grandis</i> chitin synthase II (<i>AgraChSII</i>) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the <i>AgraChSII</i> gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that <i>A</i>. <i>grandis</i> midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.</p></div

Analysis of CBW nucleases two days after gene silencing by RT-qPCR and dsRNA digestion assay.

<p>(A) Insect microinjection was performed with 500 ng of dsRNA against each nuclease and a mixture of all three dsRNAs (in a total of 1500 ng of dsRNA) and the analysis was performed two days after the microinjection. dsRNA against <i>gus</i> was used as a negative control, and <i>Agra-β-actin</i> and <i>Agra-β-tubulin</i> were used as reference genes. The relative expression (UA) was calculated based on the lowest expression value that was obtained. Statistical analyses of the average transcripts expression levels were performed using Tukey’s test with a 0.05% significance level for comparisons between treatments. The bar chart shows that the expression of the nucleases, including each individual nuclease and all three nucleases together, was silenced. (B) dsRNA (~ 200 bp) was incubated with CBW gut juice (GJ) for 30 minutes at 37°C. GJ was collected two days after RNAi nuclease gene silencing, and 1% agarose gel electrophoresis was performed to analyze dsRNA digestion. GJ was collected from uninjected insects and from injected insects with all three nucleases silenced at once. GJ: Gut Juice, KD: knocked down, WT: wild type, CBW: cotton boll weevil.</p

Biochemical characterization of CBW gut juice.

<p>(A) CBW gut juice (GJ), which is able to degrade both dsRNA, ~ 200bp, and dsDNA, > 5000 bp (as observed), has non-specific nuclease activity. MM: Molecular Marker 1-Kb Plus DNA ladder (Invitrogen); GJ: Gut Juice. Samples were incubated with GJ for 30 minutes at 37°C. (B) The optimal pH for nuclease activity ranges from 5.5 to 6.5, indicating that the nucleases function best at acidic pH.</p

Analysis of CBW <i>ChSII</i> gene expression after nuclease gene silencing.

<p>Two days after microinjection of the nuclease dsRNA into the CBW body cavity, which silenced the <i>AgraNuc</i> genes, the insect was starved for two days, and 500 ng of <i>AgraChSII</i> dsRNA was orally administered. The insects with silenced nucleases (fourth bar) showed a decrease in <i>AgraChSII</i> transcript expression compared with the control insects (first, second and third bars). RNA extraction, cDNA synthesis and RT-qPCR were performed with the whole insect. dsRNA against <i>gus</i> was used as a negative control, and <i>Agra-β-actin</i> and <i>Agra-β-tubulin</i> were used as reference genes. The relative expression (UA) was calculated based on the lowest expression value that was obtained the average transcripts expression levels were performed using Tukey’s test with a 0.05% significance level for comparisons between treatments.</p

RT-qPCR analysis of CBW nuclease expression at different developmental stages.

<p>(A and B) CBW was dissected to obtain the gut and carcass, and nuclease expression was then measured in these samples. The bar chart shows that <i>AgraNuc1</i> expression is similar in the gut and carcass of the adult (A) and larvae (B), whereas <i>AgraNuc2</i> and <i>AgraNuc3</i> are highly expressed in the gut only. (C and D) The insect gut was sectioned into the anterior midgut (AMG), posterior midgut (PMG) and posterior gut (PG), and the expression levels of the nucleases in these sections were evaluated. Higher expression of <i>AgraNuc2</i> and <i>AgraNuc3</i> was observed in the PMG of both adults (C) and larvae (D), whereas <i>AgraNuc1</i> expression was similar in all gut sections. <i>Agra-β-actin</i> and <i>Agra-β-tubulin</i> were used as reference genes. The relative expression (UA) was calculated based on the lowest expression value that was obtained. Statistical analyses of the average transcripts expression levels were performed using Tukey’s test with a 0.05% significance level for comparisons between treatments.</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

<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

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

Transcriptome Analysis in Cotton Boll Weevil (<i>Anthonomus grandis</i>) and RNA Interference in Insect Pests

<div><p>Cotton plants are subjected to the attack of several insect pests. In Brazil, the cotton boll weevil, <i>Anthonomus grandis</i>, is the most important cotton pest. The use of insecticidal proteins and gene silencing by interference RNA (RNAi) as techniques for insect control are promising strategies, which has been applied in the last few years. For this insect, there are not much available molecular information on databases. Using 454-pyrosequencing methodology, the transcriptome of all developmental stages of the insect pest, <i>A. grandis</i>, was analyzed. The <i>A. grandis</i> transcriptome analysis resulted in more than 500.000 reads and a data set of high quality 20,841 contigs. After sequence assembly and annotation, around 10,600 contigs had at least one BLAST hit against NCBI non-redundant protein database and 65.7% was similar to <i>Tribolium castaneum</i> sequences. A comparison of <i>A. grandis</i>, <i>Drosophila melanogaster</i> and <i>Bombyx mori</i> protein families’ data showed higher similarity to dipteran than to lepidopteran sequences. Several contigs of genes encoding proteins involved in RNAi mechanism were found. PAZ Domains sequences extracted from the transcriptome showed high similarity and conservation for the most important functional and structural motifs when compared to PAZ Domains from 5 species. Two SID-like contigs were phylogenetically analyzed and grouped with <i>T. castaneum</i> SID-like proteins. No RdRP gene was found. A contig matching chitin synthase 1 was mined from the transcriptome. dsRNA microinjection of a chitin synthase gene to <i>A. grandis</i> female adults resulted in normal oviposition of unviable eggs and malformed alive larvae that were unable to develop in artificial diet. This is the first study that characterizes the transcriptome of the coleopteran, <i>A. grandis</i>. A new and representative transcriptome database for this insect pest is now available. All data support the state of the art of RNAi mechanism in insects.</p> </div