Northern blot hybridization analysis of virus RNAs acquired by nymphal <i>B.</i><i>cockerelli</i> after feeding on virus-infected plants.

<p>Approximately 60 nymphal <i>B. cockerelli</i> were allowed to feed for 24 h on leaf discs from tomatillo, tomato or tobacco plants that were infected with TMV-GFP, PVX-GFP or TRV-GFP. The leaf discs were taken from plants that showed GFP expression under UV light illumination at 14 days post rub-inoculation. Total RNAs from leaf tissues (at 14 days post inoculation) and psyllid nymphs (at 24 h post feeding) were separated by denaturing agarose gel electrophoresis, transferred to nylon membranes, and hybridized with ³²P-UTP-labeled GFP-specific probes. Lane 1: tomatillo infected with TMV-GFP. Lane 2: tomato infected with TMV-GFP. Lane 3: tobacco plants infected with TMV-GFP. Lane 4: tobacco infected with PVX-GFP. Lane 5: tobacco infected with TRV-GFP. Lane 6: nymphs fed on tomatillo infected with TMV-GFP. Lane 7: nymphs fed on tomato infected with TMV-GFP. Lane 8: nymphs fed on tobacco infected with PVX-GFP. Lane 9: nymphs fed on tobacco infected with TMV-GFP. Lane 10: nymphs fed on tobacco infected with TRV-GFP. Lane 11: control nymphs fed on uninoculated tobacco leaf discs. The migration of size standards (0.5–10 kb RNA ladder) is indicated to the right. The arrows to the left indicate predicted TMV-GFP<i>-</i>specific genomic and subgenomic RNAs from top to bottom, respectively.</p

RNA Interference towards the Potato Psyllid, <i>Bactericera cockerelli</i>, Is Induced in Plants Infected with Recombinant <i>Tobacco mosaic virus</i> (TMV)

<div><p>The potato/tomato psyllid, <i>Bactericera cockerelli</i> (<i>B. cockerelli</i>), is an important plant pest and the vector of the phloem-limited bacterium <i>Candidatus</i> Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in <i>B. cockerelli</i> via <i>in vitro</i>-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding <i>B. cockerelli</i>, here we developed a plant virus vector-based <i>in planta</i> system for evaluating candidate sequences. We show that recombinant <i>Tobacco mosaic virus</i> (TMV) containing <i>B. cockerelli</i> sequences can efficiently infect and generate small interfering RNAs in tomato (<i>Solanum lycopersicum</i>), tomatillo (<i>Physalis philadelphica</i>) and tobacco (<i>Nicotiana tabacum</i>) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in <i>B. cockerelli</i> feeding on these plants. RNAi effects were primarily detected in the <i>B. cockerelli</i> guts. In contrast to our results with TMV, recombinant <i>Potato virus X</i> (PVX) and <i>Tobacco rattle virus</i> (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in <i>B. cockerelli</i>. The greatest RNA interference effects were observed when <i>B. cockerelli</i> nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing <i>B. cockerelli actin</i> or <i>V-ATPase</i> sequences also showed phenotypic effects resulting in decreased <i>B. cockerelli</i> progeny production as compared to plants infected by recombinant TMV containing <i>GFP</i>. These results showed that RNAi effects can be achieved in plants against the phloem feeder, <i>B. cockerelli</i>, and the TMV-plant system will provide a faster and more convenient method for screening of suitable RNAi target sequences <i>in planta</i>.</p></div

Quantitative real-time PCR analysis of <i>BC-ATPase</i> mRNAs in <i>B.</i><i>cockerelli</i> whole insects and specific tissues.

<p>Teneral adults were allowed to feed for 72 h on tomatillo plants that were infected (14 days post inoculation) with TMV-ATPase or TMV-GFP. Following feeding, total RNAs were isolated from the abdomen or gut tissues of ∼100 insects for each treatment. The experiments were repeated three times. cDNAs were generated using random hexamer primers, and the cDNA was used for quantitative real-time PCR using <i>BC-ATPase</i> specific primers. The qRT-PCR results were normalized against constitutively expressed <i>BC-Actin</i> levels. The level of <i>BC-ATPase</i> mRNA in psyllids that fed on TMV-GFP-infected plants was arbitrarily designated as 1. Differences between <i>BC-ATPase</i> mRNA levels in <i>B. cockerelli</i> fed on TMV-ATPase-infected and control TMV-GFP-infected plants were analyzed using the Bonferroni <i>t-test</i>. The double asterisks indicate a significant difference at p<0.01. Gut GFP (t = 6.831, df = 11), Gut ATPase (t = 15.660, df = 11); Abdomen GFP (t = 5.889, df = 6), Abdomen ATPase (t = 19.265, df = 6).</p

Quantitative real-time PCR detection for <i>B. cockerelli</i> endogenous <i>BC-ATPase</i> mRNAs after feeding on the plants infected with virus expressing <i>BC-ATPase</i> interfering sequence.

1<p>The plants used for virus infection and <i>B. cockerelli</i> feeding.</p>2<p>The virus vector expressing <i>BC-ATPase</i> and control GFP sequences for plant inoculation.</p>3<p>The number of psyllids used for qRT-PCR analysis in one experiment. For each experiment, the same numbers of GFP samples were used as controls for test sample.</p>4<p>Teneral adult <i>B. cockerelli</i> or nymphs for feeding experiments and RNA abundance detection were indicated. All of the samples labeled “Nymph” were done using leaf disc method, and the samples labeled “Adult” were done using whole plant feeding experiment.</p>5<p>The mRNA abundance of <i>BC-ATPase</i> gene after feeding on virus-ATPase –infected plants was shown as test sample, and the average value of the control GFP group was designated as 1. Expression of <i>BC-ATPase</i> was normalized to the level of <i>BC-Actin</i> in the same sample.</p>6<p>Differences between GFP and test group were calculated using the Bonferroni t-test. Single asterisk indicates p<0.05 and double asterisk indicates p<0.01.</p

RNAi effects targeting <i>ATPase</i> and <i>actin</i> mRNAs and survival and fecundity of <i>B.</i><i>cockerelli</i>.

<p>Teneral adults (groups of 20) were allowed to feed for 7 days on tomatillo plants that were inoculated with TMV-GFP, TMV-ATPase and TMV-Actin (10 days post inoculation). After 7 days of feeding on the virus-infected tomatillo, all of the adult psyllids were removed and numbers of survivors was determined. The tomatillo plants (without any adults) were then returned to the growth chamber for another 14 days and the number of progeny nymphs was assessed. The experiments were repeated three times and five biological replicates were included in each experiment. Differences in survival and fecundity between the insects that fed on TMV-ATPase- or TMV-actin-infected tomatillo and control (TMV-GFP-infected) plants were analyzed using the Bonferronit-test. The asterisk indicates a significance at p<0.01. (A), Number of surviving <i>B. cockerelli</i> after feeding for 7 days on virus-infected tomatillo; GFP (T = 14.736, df = 4), ATPase (t = 12.863, df = 4), Actin (T = 14.969, df = 4). (B), number of progeny nymphs at 14 days after removal of the adult psyllids; GFP (T = 7.8, df = 4), ATPase (T = 4.935, df = 4), Actin (T = 3.519, df = 4).</p

Quantitative real-time PCR analysis of the oral acquisition of virus RNAs by <i>B. cockerelli</i>.

<p>Teneral adults and nymphs were allowed to feed on whole plants or leaf discs, respectively, of TMV-GFP-infected tomatillo plants (14 days post inoculation) for 24 h. Following feeding, total RNAs were isolated from whole adults, adult abdomens, adult guts, and whole nymphs. cDNAs were generated using primers that were specific for positive sense (pJL36R) or negative sense (pJL36L) TMV-GFP RNA, or random hexamer primers. Quantification of the amount of TMV-GFP RNA that was acquired by <i>B. cockerelli</i> was performed with <i>GFP</i>-specific qRT-PCR and was normalized against constitutively-expressed psyllid <i>BC-Actin</i> mRNA. BC is the abbreviation for <i>B. cockerelli</i>, and pJL24-indicates the pJL24 (TMV-GFP)-infected tomatillo plants. We did obtain consistent positive signals for GFP RNA in the BC control samples when using pJL36R cDNA. By contrast, both pJL36L and Random Hexamer cDNAs showed essentially zero for the BC controls, thus supporting our interpretation of these data.</p

Infection of four different plant hosts with various virus vectors expressing GFP.

<p>(A), <i>N. benthamiana</i> (3–4 leaf stage), Turkish tobacco (4-leaf stage), tomatillo (cotyledons), and tomato (cotyledons) were inoculated by rub inoculation. Tomato plants infected with TMV-GFP were photographed under UV light to visualize GFP expression at 21 days post inoculation, whereas other plants (<i>N. benthamiana</i>, tomatillo, Turkish tobacco) were examined at 14 days post inoculation. The virus inocula were prepared in <i>N. benthamiana</i> (3–4 leaf stage) that were agro-infiltrated with TMV-GFP or TRV-GFP vectors using <i>Agrobacterium tumefaciens</i> GV3101, or rub-inoculated with <i>in vitro</i>-generated PVX-GFP transcripts in FES buffer. (B), Northern blot analysis showed the accumulation of ∼21 nt siRNAs in virus-infected plants. One µg of total small RNA was separated on a 15% PAGE gel containing 8 M Urea and transferred to a nylon membrane. ³²P-UTP-labeled negative strand <i>GFP</i> RNA was used to probe the blot. MicroRNA markers were electrophoresed on the same gel. The migration of the 21 nt standard is indicated to the right of each blot. Below each blot are shown loading controls, ethidium bromide stained rRNAs.</p

Additional file 2: Figure S2. of Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors

Expression analysis of MseChi2 in non-gut tissues. MseChi2 expression was analyzed in non-gut tissues after five days’ feeding of HT115 cell expressing MseChi2 specific dsRNA. Larvae fed with L4440 transformant were used as control. RO, reproductive organ; FB, fat body; T, thorax; H, head; A, abdomen. (JPG 97 kb