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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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Expression of Nlsid-1 and Nlaub in N. lugens.(A) Developmental expression of Nlsid-1 and Nlaub in N. lugens from 1st nymph to male adult (Ma) and female adult (Fa). (B) Tissue distribution of Nlsid-1 and Nlaub in N. lugens 3rd instar nymph. qRT-PCR analyses were performed using total RNA from midgut (Mi), salivary gland (Sg), fat body (Fb), cuticle (Cu), leg (Le), and head (He). Data shown are means ± standard errors (N = 3).
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pone-0020504-g002: Expression of Nlsid-1 and Nlaub in N. lugens.(A) Developmental expression of Nlsid-1 and Nlaub in N. lugens from 1st nymph to male adult (Ma) and female adult (Fa). (B) Tissue distribution of Nlsid-1 and Nlaub in N. lugens 3rd instar nymph. qRT-PCR analyses were performed using total RNA from midgut (Mi), salivary gland (Sg), fat body (Fb), cuticle (Cu), leg (Le), and head (He). Data shown are means ± standard errors (N = 3).

Mentions: To probe the functions of the Nlsid-1 and Nlaub genes, their mRNA levels were analyzed, using qRT-PCR, at various developmental stages of N. lugens insects, including nymphs from 1st to 5th instars, female adults and male adults. The developmental expression pattern revealed that Nlsid-1 and Nlaub transcripts were present at all development stages. For both genes, mRNA levels were highest in female adults, but Nlaub mRNA had lower expression compared to that of Nlsid-1 (Figure 2A). In order to further assess the tissue distribution of the transcripts, qRT-PCR was used to amplify Nlsid-1 and Nlaub from cDNA of midgut, salivary gland, fat body, cuticle, leg, and head tissues. The results indicated that Nlsid-1 and Nlaub were expressed in all six tissues tested (Figure 2B).


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)

Expression of Nlsid-1 and Nlaub in N. lugens.(A) Developmental expression of Nlsid-1 and Nlaub in N. lugens from 1st nymph to male adult (Ma) and female adult (Fa). (B) Tissue distribution of Nlsid-1 and Nlaub in N. lugens 3rd instar nymph. qRT-PCR analyses were performed using total RNA from midgut (Mi), salivary gland (Sg), fat body (Fb), cuticle (Cu), leg (Le), and head (He). Data shown are means ± standard errors (N = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020504-g002: Expression of Nlsid-1 and Nlaub in N. lugens.(A) Developmental expression of Nlsid-1 and Nlaub in N. lugens from 1st nymph to male adult (Ma) and female adult (Fa). (B) Tissue distribution of Nlsid-1 and Nlaub in N. lugens 3rd instar nymph. qRT-PCR analyses were performed using total RNA from midgut (Mi), salivary gland (Sg), fat body (Fb), cuticle (Cu), leg (Le), and head (He). Data shown are means ± standard errors (N = 3).
Mentions: To probe the functions of the Nlsid-1 and Nlaub genes, their mRNA levels were analyzed, using qRT-PCR, at various developmental stages of N. lugens insects, including nymphs from 1st to 5th instars, female adults and male adults. The developmental expression pattern revealed that Nlsid-1 and Nlaub transcripts were present at all development stages. For both genes, mRNA levels were highest in female adults, but Nlaub mRNA had lower expression compared to that of Nlsid-1 (Figure 2A). In order to further assess the tissue distribution of the transcripts, qRT-PCR was used to amplify Nlsid-1 and Nlaub from cDNA of midgut, salivary gland, fat body, cuticle, leg, and head tissues. The results indicated that Nlsid-1 and Nlaub were expressed in all six tissues tested (Figure 2B).

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