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Knockdown of midgut genes by dsRNA-transgenic plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens.

Zha W, Peng X, Chen R, Du B, Zhu L, He G - PLoS ONE (2011)

Bottom Line: To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens.When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed.The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.

ABSTRACT

Background: RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders.

Methodology/principal findings: The Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed.

Conclusions: Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants.

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Growth phenotypes of wild type (WT) plants, empty transformation vector plants and transgenic lines.(A) Two-week-old seedlings. (B) Plants at the four leaf stage. (C) Mature plants.
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pone-0020504-g005: Growth phenotypes of wild type (WT) plants, empty transformation vector plants and transgenic lines.(A) Two-week-old seedlings. (B) Plants at the four leaf stage. (C) Mature plants.

Mentions: Double strand RNA interference (dsRNAi) constructs NlHT1-RNAi, Nlcar-RNAi and Nltry-RNAi were developed for the three N. lugens genes NlHT1, Nlcar and Nltry, under the control of the Ubi1 promoter and the nopaline synthase (nos) terminator cassette [38], [39]. The genes were cloned at the BamHI site of the hygromycin gene expression cassette in the pCU vector of Agrobacterium (Figure 4A). Transgenic plants were generated by introducing the constructs into the japonica rice variety Hejiang 19 by Agrobacterium tumefaciens–mediated transformation, and genomic DNA was isolated from the hygromycin-tolerant transgenic rice plants. Positive transformants were detected by PCR of the hygromycin resistance gene, while wild type (WT) plants failed to show such amplification (data not shown). No significant morphological differences were found in these transgenic lines compared with WT plants (Figure 5). Southern blot analysis showed that the PCR-positive plants had 1–3 copies of the target coding sequences (Figure 4B–4D). Conversely, genomic DNA from transgenic plants carrying an empty vector failed to show any hybridization with the probes. Two independently transformed lines (H2 and H4, C8 and C9, T3 and T18) with a single-copy-insertion were chosen for NlHT1-RNAi, Nlcar-RNAi and Nltry-RNAi,respectively, for further analyses. Resistance and non-resistance to hygromycin were observed in progenies of these plants in the ratio 3∶1 (Table S2), confirming integration of the respective transgenes at a single locus in each case.


Knockdown of midgut genes by dsRNA-transgenic plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens.

Zha W, Peng X, Chen R, Du B, Zhu L, He G - PLoS ONE (2011)

Growth phenotypes of wild type (WT) plants, empty transformation vector plants and transgenic lines.(A) Two-week-old seedlings. (B) Plants at the four leaf stage. (C) Mature plants.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105074&req=5

pone-0020504-g005: Growth phenotypes of wild type (WT) plants, empty transformation vector plants and transgenic lines.(A) Two-week-old seedlings. (B) Plants at the four leaf stage. (C) Mature plants.
Mentions: Double strand RNA interference (dsRNAi) constructs NlHT1-RNAi, Nlcar-RNAi and Nltry-RNAi were developed for the three N. lugens genes NlHT1, Nlcar and Nltry, under the control of the Ubi1 promoter and the nopaline synthase (nos) terminator cassette [38], [39]. The genes were cloned at the BamHI site of the hygromycin gene expression cassette in the pCU vector of Agrobacterium (Figure 4A). Transgenic plants were generated by introducing the constructs into the japonica rice variety Hejiang 19 by Agrobacterium tumefaciens–mediated transformation, and genomic DNA was isolated from the hygromycin-tolerant transgenic rice plants. Positive transformants were detected by PCR of the hygromycin resistance gene, while wild type (WT) plants failed to show such amplification (data not shown). No significant morphological differences were found in these transgenic lines compared with WT plants (Figure 5). Southern blot analysis showed that the PCR-positive plants had 1–3 copies of the target coding sequences (Figure 4B–4D). Conversely, genomic DNA from transgenic plants carrying an empty vector failed to show any hybridization with the probes. Two independently transformed lines (H2 and H4, C8 and C9, T3 and T18) with a single-copy-insertion were chosen for NlHT1-RNAi, Nlcar-RNAi and Nltry-RNAi,respectively, for further analyses. Resistance and non-resistance to hygromycin were observed in progenies of these plants in the ratio 3∶1 (Table S2), confirming integration of the respective transgenes at a single locus in each case.

Bottom Line: To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens.When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed.The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.

ABSTRACT

Background: RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders.

Methodology/principal findings: The Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed.

Conclusions: Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants.

Show MeSH
Related in: MedlinePlus