Relative mRNA levels of <i>CHS</i>1 (A) and <i>V-ATPase</i> (B) following microinjection of specific dsRNAs into third instar <i>P. citri</i>.

<p>qRT-PCR results of <i>CHS</i>1 (A) and <i>V-ATPase</i> (B) mRNAs from <i>P. citri</i> following microinjection of elution buffer (EB), green fluorescent protein (GFP), <i>CHS</i>1 or <i>V-ATPase</i> dsRNAs. Total RNA was extracted 4 days post injection and used for cDNA synthesis. qRT-PCR was performed using primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073657#pone-0073657-t003" target="_blank">Table 3</a>. The <i>P. citri</i> 18S ribosomal RNA was used as an endogenous control in all experiments. Calculation of mRNA levels between control and treated groups was done by the comparative CT or 2^–ΔΔCt method and analyzed by ANOVA using statistix 8.1 software. Numbers followed by the same letter are not different at p<0.01 (LSD 0.08). All experiments were performed twice and data were used collectively.</p

<i>P. citri</i> feeding on <i>N. benthamiana</i> plants inoculated with TMV-Actin.

<p>Mealybug crawlers feeding on: A) <i>N. benthamiana</i> plants inoculated with TMV only (pJL36) showing healthy crawlers emerging while B, C and D show <i>N. benthamiana</i> plants inoculated with recombinant TMV (TMV-Actin) and show a very high mortality in crawlers and adults. Arrows in A indicate healthy crawlers; in B & C arrows indicate dead crawlers and D the arrows indicate dead adults.</p

Primers used for <i>in vitro</i> dsRNA synthesis.

a<p>Underlined nucleotide sequence is the T7 RNA polymerase promoter sequence. A pair of gene specific primers with the T7 promoter sequence at the 5′ ends of both primers was used to amplify the desired templates. Amplified PCR products were used as templates for <i>in vitro</i> dsRNA synthesis.</p>b<p>Italic nucleotide sequence is the <i>Not</i> 1 restriction enzyme recognition sequence.</p

Primers used for qRT-PCR.

<p>Primers for qRT-PCR studies were designed from target sequences using Primer Express 3.0 software and the best primers that did not produce secondary structures or primer dimers were selected. These primers targeted sequences flanking the input dsRNA sequences.</p

<i>P. citri</i> feeding on <i>N. benthamiana</i> plants inoculated with TMV-CHS1 and TMV-V-ATPase.

<p>Mealybug crawlers feeding on: A) <i>N. benthamiana</i> plants infected with TMV (pJL36) show healthy crawlers emerging while B and C show <i>N. benthamiana</i> plants infected with TMV-CHS1-S where B shows abnormal ovisac formation and C shows a high mortality of crawlers. D shows an <i>N. benthamiana</i> plant infected with TMV-V-ATPase-S showing a high mortality in crawlers. Arrows in A indicate healthy crawlers, in B an abnormal ovisac and in C & D indicate dead crawlers.</p

Detection of <i>P. citri CHS</i>1 and <i>V-ATPase</i> mRNAs (A) and accumulation of RNAi-induced siRNAs (B) in <i>N. benthamiana</i> plants after TMV-CHS1 inoculation.

<p>Plants were inoculated with recombinant TMV containing <i>P. citri CHS</i>1 (TMV-CHS1-S) and <i>V-ATPase</i> (TMV-V-ATPase-S). At 7 days post inoculation, total and small RNAs were isolated from infected plants and analyzed by RT-PCR and siRNA northern blot hybridization, respectively. A) One step RT-PCR was performed using total RNA as a template and <i>CHS</i>1 and <i>V-ATPase</i> primers and products analyzed on the gel. Lane L: 1Kb plus DNA ladder, Lane 1: <i>CHS</i>1 primers and RNA of TMV-CHS1-S inoculated <i>N. benthamiana</i> plants; Lane 2: <i>CHS</i>1 primers and RNA of TMV inoculated plant; Lane 3: <i>V-ATPase</i> primers and RNA of TMV-V-ATPase-S inoculated plant and Lane 4: <i>V-ATPase</i> primers and RNA of TMV inoculated plant. B) Small RNAs were separated by PAGE in 15% acrylamide, 8 M urea gels and processed for northern blot hybridization using ³²P-UTP-labeled negative strand <i>P.citri CHS</i>1 transcript as a probe. Lane 1: TMV-CHS1-S inoculated plant; Lane 2: TMV-CHS1-AS iinoculated plant; Lane 3: TMV-inoculated plant.</p

Mortality in <i>P. citri</i> adults and crawlers on plants inoculated with different recombinant TMV constructs.

<p>Comparison of corrected mortality in adult <i>P. citri</i> and crawlers following 18 days after release of insects onto plants inoculated with the following viral constructs: HC: healthy control <i>N. benthamiana</i> plant; TMV-GFP  =  TMV carrying GFP (TMV JL24); TMV  =  TMV with no insert (pJL36); TMV-Actin  =  part of the <i>actin</i> RNA inserted into TMV; TMV-CHS1-S and TMV-CHS1-AS  =  the <i>chitin synthase</i> 1 (<i>CHS</i>1) RNA fragment inserted into TMV in sense and antisense orientations, respectively; TMV-V-ATPase-S and TMV-V-ATPase-AS  =  the <i>V-ATPase</i> RNA fragment inserted into TMV in sense and antisense orientations, respectively. Numbers indicated with the same letter are homogenous groups at p<0.05.</p

Relative levels of <i>CHS</i>1 (A) and <i>V-ATPase</i> (B) mRNAs in <i>P. citri</i> after feeding on <i>N. benthamiana</i> plants inoculated with recombinant TMV expressing <i>P. citri CHS</i>1 and <i>V-ATPase</i> fragments.

<p>qRT-PCR results of <i>CHS</i>1 (A) and <i>V-ATPase</i> (B) mRNA levels from <i>P. citri</i> after feeding on <i>N. benthamiana</i> plants infected with recombinant TMVs. TMV-GFP (pJL24) was used as a control and sense and antisense inserts of <i>P. citri CHS</i>1<i>/V-ATPase</i> sequences were compared. Total RNA was extracted after 12 days feeding on plants and used for cDNA synthesis. qRT-PCR was performed using primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073657#pone-0073657-t003" target="_blank">Table 3</a>. The 18S ribosomal RNA was used as an endogenous control in all experiments. Calculation of mRNA levels between control and treated groups was carried out by the comparative CT or 2^–ΔΔCt method and analyzed by ANOVA using statistix 8.1 software. Numbers with the same letter indicate homogenous groups at p<0.01 (LSD 0.04 and 0.21) for <i>CHS</i>1 and <i>V-ATPase</i> respectively. All experiments were performed twice and data were used collectively.</p

Primers used for RT-PCR of RNAi targets.

a<p>Underlined nucleotide sequence is the <i>Sal</i> 1 restriction enzyme recognition sequence, added to facilitate cloning.</p>b<p>Underlined sequence is the <i>Sma</i> 1 restriction enzyme recognition sequence, added to facilitate cloning.</p>c<p>Underlined nucleotide sequence is the <i>Pac</i> 1 restriction enzyme recognition sequence, added to facilitate cloning.</p>d<p>Underlined nucleotide sequence is the <i>Not</i> 1 restriction enzyme recognition sequence, added to facilitate cloning.</p

Emergence of <i>P. citri</i> crawlers and their survival on plants inoculated with different recombinant TMV constructs.

<p>Comparison of alive and total crawlers that emerged 18<i>P. citri</i> onto plants inoculated with the following viral constructs: HC  =  healthy control <i>N. benthamiana</i> plants; TMV-GFP  =  TMV carrying GFP (TMV JL24); TMV  =  TMV with no insert (pJL36); TMV-Actin  =  part of the <i>actin</i> RNA inserted into TMV; TMV-CHS1-S and TMV-CHS1-AS  =  the <i>chitin synthase</i> 1 (<i>CHS</i>1) RNA fragment inserted into TMV in sense and antisense orientations, respectively; TMV-V-ATPase-S and TMV-V-ATPase-AS  =  the <i>V-ATPase</i> RNA fragment inserted into TMV in sense and antisense orientations, respectively. Numbers indicated with the same letter are homogenous groups at p<0.05.</p