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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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Related in: MedlinePlus

Northern blot analysis of the expression pattern of NlHT1, Nlcar and Nltry dsRNAs in different transgenic rice lines.RNA blots for the expression of (A) NlHT1, (B) Nlcar and (C) Nltry dsRNA in T1 lines homozygous for the dsRNA transgenes (H2 and H4, C8 and C9, T3 and T18 respectively) and in the absence of dsRNA in the T1 line homozygous for the empty transformation vector homozygous T1 line (V): L, leaf; P, phloem sap. (D) NlHT1, (E) Nlcar and (F) Nltry siRNAs were detected in the three dsRNA transgenic lines, respectively. Loading of equal amounts of RNA was confirmed by ethidium bromide staining.
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pone-0020504-g006: Northern blot analysis of the expression pattern of NlHT1, Nlcar and Nltry dsRNAs in different transgenic rice lines.RNA blots for the expression of (A) NlHT1, (B) Nlcar and (C) Nltry dsRNA in T1 lines homozygous for the dsRNA transgenes (H2 and H4, C8 and C9, T3 and T18 respectively) and in the absence of dsRNA in the T1 line homozygous for the empty transformation vector homozygous T1 line (V): L, leaf; P, phloem sap. (D) NlHT1, (E) Nlcar and (F) Nltry siRNAs were detected in the three dsRNA transgenic lines, respectively. Loading of equal amounts of RNA was confirmed by ethidium bromide staining.

Mentions: RNA blot analysis showed that the NlHT1, Nlcar and Nltry dsRNAs were transcribed in the transgenic lines and some of them were processed to siRNAs (Figure 6). The expression and processing of dsRNAs for the target N. lugens genes in the transgenic rice lines provided dsRNA and siRNA molecules for potential ingestion by the insects.


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)

Northern blot analysis of the expression pattern of NlHT1, Nlcar and Nltry dsRNAs in different transgenic rice lines.RNA blots for the expression of (A) NlHT1, (B) Nlcar and (C) Nltry dsRNA in T1 lines homozygous for the dsRNA transgenes (H2 and H4, C8 and C9, T3 and T18 respectively) and in the absence of dsRNA in the T1 line homozygous for the empty transformation vector homozygous T1 line (V): L, leaf; P, phloem sap. (D) NlHT1, (E) Nlcar and (F) Nltry siRNAs were detected in the three dsRNA transgenic lines, respectively. Loading of equal amounts of RNA was confirmed by ethidium bromide staining.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020504-g006: Northern blot analysis of the expression pattern of NlHT1, Nlcar and Nltry dsRNAs in different transgenic rice lines.RNA blots for the expression of (A) NlHT1, (B) Nlcar and (C) Nltry dsRNA in T1 lines homozygous for the dsRNA transgenes (H2 and H4, C8 and C9, T3 and T18 respectively) and in the absence of dsRNA in the T1 line homozygous for the empty transformation vector homozygous T1 line (V): L, leaf; P, phloem sap. (D) NlHT1, (E) Nlcar and (F) Nltry siRNAs were detected in the three dsRNA transgenic lines, respectively. Loading of equal amounts of RNA was confirmed by ethidium bromide staining.
Mentions: RNA blot analysis showed that the NlHT1, Nlcar and Nltry dsRNAs were transcribed in the transgenic lines and some of them were processed to siRNAs (Figure 6). The expression and processing of dsRNAs for the target N. lugens genes in the transgenic rice lines provided dsRNA and siRNA molecules for potential ingestion by the insects.

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