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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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NlHT1, Nlcar and Nltry suppression in nymphs fed on transgenic plants.(A) NlHT1 mRNA levels in the empty transformation vector and NlHT1-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on H2 and H4 phloem sap for 2 d and 4 d. (B) Nlcar mRNA levels in the empty transformation vector and Nlcar-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on C8 and C9 phloem sap for 2 d and 4 d. (C) Nltry mRNA levels in the empty transformation vector and Nltry-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on T3 and T18 phloem sap for 2 d and 4 d. Statistical analysis of mRNA levels was performed with student t-test.(*, P<0.05; **, P <0.01.) (D) Northern blot of NlHT1 transcripts of 3rd instar nymphs fed on the empty transformation vector and H2 transgenic plants for 4 d. (E) Northern blot of Nlcar transcripts of 3rd instar nymphs fed on the empty transformation vector and C8 transgenic line for 4 d. (F) Northern blot of Nltry transcripts of 3rd instar nymphs fed on the empty transformation vector and T3 transgenic line for 4 d.
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pone-0020504-g007: NlHT1, Nlcar and Nltry suppression in nymphs fed on transgenic plants.(A) NlHT1 mRNA levels in the empty transformation vector and NlHT1-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on H2 and H4 phloem sap for 2 d and 4 d. (B) Nlcar mRNA levels in the empty transformation vector and Nlcar-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on C8 and C9 phloem sap for 2 d and 4 d. (C) Nltry mRNA levels in the empty transformation vector and Nltry-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on T3 and T18 phloem sap for 2 d and 4 d. Statistical analysis of mRNA levels was performed with student t-test.(*, P<0.05; **, P <0.01.) (D) Northern blot of NlHT1 transcripts of 3rd instar nymphs fed on the empty transformation vector and H2 transgenic plants for 4 d. (E) Northern blot of Nlcar transcripts of 3rd instar nymphs fed on the empty transformation vector and C8 transgenic line for 4 d. (F) Northern blot of Nltry transcripts of 3rd instar nymphs fed on the empty transformation vector and T3 transgenic line for 4 d.

Mentions: Third instar nymphs, previously reared on susceptible rice (cultivar Taichung Native 1, TN1) plants, were transferred onto the dsRNA-transgenic plants and allowed to feed for 2 to 4 days. The transcript levels of the target genes in N. lugens were then detected using qRT-PCR. In nymphs fed on the H2 and H4 lines, NlHT1 transcript levels began to decrease on day 2 and were reduced by 59.3% in H2 and 42.7% in H4 on day 4 (Figure 7A). A similar experiment was performed on the Nlcar and Nltry dsRNA-transgenic plants. The qRT-PCR results revealed that the mRNA transcripts of Nlcar and Nltry after ingestion of the respective dsRNAs were reduced on days 2 and 4. The maximum reductions in the Nlcar transcript level, of 42.1% for the C8 line and 43.3% for the C9 line, occurred on day 2 (Figure 7B). The maximum reductions in the Nltry transcript levels, of 61% for the T3 line and 73.3% for the T18 line, occurred on day 4 (Figure 7C). The northern blotting results confirm that the brown planthopper nymphs fed on H2 plants had less NlHT1 transcript compared to nymphs on the plants transformed with an empty transformation vector (Figure 7D). Similarly, the nymphs from fed on C8 and T3 plants had lower Nlcar and Nltry expression levels than those on the plants transformed with an empty transformation vector (Figure 7E–7F). Figure 7A–7C also shows that the RNAi efficiency for Nltry was significantly higher (p<0.01, t-test) than for NlHT1 and Nlcar on day 4. The results show that ingestion of dsRNA-transgenic rice plants expressing dsRNA and siRNA is an effective way to trigger RNA interference in the Hemipteran insect Nilaparvata lugens.


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)

NlHT1, Nlcar and Nltry suppression in nymphs fed on transgenic plants.(A) NlHT1 mRNA levels in the empty transformation vector and NlHT1-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on H2 and H4 phloem sap for 2 d and 4 d. (B) Nlcar mRNA levels in the empty transformation vector and Nlcar-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on C8 and C9 phloem sap for 2 d and 4 d. (C) Nltry mRNA levels in the empty transformation vector and Nltry-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on T3 and T18 phloem sap for 2 d and 4 d. Statistical analysis of mRNA levels was performed with student t-test.(*, P<0.05; **, P <0.01.) (D) Northern blot of NlHT1 transcripts of 3rd instar nymphs fed on the empty transformation vector and H2 transgenic plants for 4 d. (E) Northern blot of Nlcar transcripts of 3rd instar nymphs fed on the empty transformation vector and C8 transgenic line for 4 d. (F) Northern blot of Nltry transcripts of 3rd instar nymphs fed on the empty transformation vector and T3 transgenic line for 4 d.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105074&req=5

pone-0020504-g007: NlHT1, Nlcar and Nltry suppression in nymphs fed on transgenic plants.(A) NlHT1 mRNA levels in the empty transformation vector and NlHT1-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on H2 and H4 phloem sap for 2 d and 4 d. (B) Nlcar mRNA levels in the empty transformation vector and Nlcar-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on C8 and C9 phloem sap for 2 d and 4 d. (C) Nltry mRNA levels in the empty transformation vector and Nltry-RNAi transgenic lines after feeding; nymphs were monitored by qRT-PCR after feeding on T3 and T18 phloem sap for 2 d and 4 d. Statistical analysis of mRNA levels was performed with student t-test.(*, P<0.05; **, P <0.01.) (D) Northern blot of NlHT1 transcripts of 3rd instar nymphs fed on the empty transformation vector and H2 transgenic plants for 4 d. (E) Northern blot of Nlcar transcripts of 3rd instar nymphs fed on the empty transformation vector and C8 transgenic line for 4 d. (F) Northern blot of Nltry transcripts of 3rd instar nymphs fed on the empty transformation vector and T3 transgenic line for 4 d.
Mentions: Third instar nymphs, previously reared on susceptible rice (cultivar Taichung Native 1, TN1) plants, were transferred onto the dsRNA-transgenic plants and allowed to feed for 2 to 4 days. The transcript levels of the target genes in N. lugens were then detected using qRT-PCR. In nymphs fed on the H2 and H4 lines, NlHT1 transcript levels began to decrease on day 2 and were reduced by 59.3% in H2 and 42.7% in H4 on day 4 (Figure 7A). A similar experiment was performed on the Nlcar and Nltry dsRNA-transgenic plants. The qRT-PCR results revealed that the mRNA transcripts of Nlcar and Nltry after ingestion of the respective dsRNAs were reduced on days 2 and 4. The maximum reductions in the Nlcar transcript level, of 42.1% for the C8 line and 43.3% for the C9 line, occurred on day 2 (Figure 7B). The maximum reductions in the Nltry transcript levels, of 61% for the T3 line and 73.3% for the T18 line, occurred on day 4 (Figure 7C). The northern blotting results confirm that the brown planthopper nymphs fed on H2 plants had less NlHT1 transcript compared to nymphs on the plants transformed with an empty transformation vector (Figure 7D). Similarly, the nymphs from fed on C8 and T3 plants had lower Nlcar and Nltry expression levels than those on the plants transformed with an empty transformation vector (Figure 7E–7F). Figure 7A–7C also shows that the RNAi efficiency for Nltry was significantly higher (p<0.01, t-test) than for NlHT1 and Nlcar on day 4. The results show that ingestion of dsRNA-transgenic rice plants expressing dsRNA and siRNA is an effective way to trigger RNA interference in the Hemipteran insect Nilaparvata lugens.

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