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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

Phylogenetic relationships of the SID and Argonaute proteins from N. lugens and other species, and the structure of the N. lugens Aubergine protein.Phylogenetic analysis of (A) SID protein sequences, and (B) Argonaute protein sequences from N. lugens and other animals were conducted using MEGA 4.0; nodes with a bootstrap similarity of at least 50% are shown. (C) Conserved domains of Aubergine in N. lugens. It has the PAZ and Piwi domains that are typical motifs of Argonaute family proteins. The catalytic triad DDH is also shown. Sequence motifs were predicted by the NCBI Conserved Domains Server.
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pone-0020504-g001: Phylogenetic relationships of the SID and Argonaute proteins from N. lugens and other species, and the structure of the N. lugens Aubergine protein.Phylogenetic analysis of (A) SID protein sequences, and (B) Argonaute protein sequences from N. lugens and other animals were conducted using MEGA 4.0; nodes with a bootstrap similarity of at least 50% are shown. (C) Conserved domains of Aubergine in N. lugens. It has the PAZ and Piwi domains that are typical motifs of Argonaute family proteins. The catalytic triad DDH is also shown. Sequence motifs were predicted by the NCBI Conserved Domains Server.

Mentions: Because of the probable role of the SID protein in the uptake of dsRNA from the environment in C. elegans, we cloned the sid-1 gene from N. lugens. The full-length cDNA of the sid-1 gene is 2,119 bp long and contains an open reading frame (ORF) of 1,875 bp (GenBank accession no. JF915743), encoding a protein of 624 amino acids with a calculated molecular mass of 70.8 kDa and an isolectric point (pI) of 6.67 (Figure S1). Multiple alignment and phylogenetic analysis of the deduced amino acid sequences confirmed that this gene is a sid-1 like gene, hence we named it Nlsid-1 (Nilaparvata lugens sid-1). The phylogenetic tree of deduced amino acid sequences showed that the N. lugens SID-1 protein is most closely related to the A. mellifera SID-1 like protein (40% identity, Figure 1A).


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)

Phylogenetic relationships of the SID and Argonaute proteins from N. lugens and other species, and the structure of the N. lugens Aubergine protein.Phylogenetic analysis of (A) SID protein sequences, and (B) Argonaute protein sequences from N. lugens and other animals were conducted using MEGA 4.0; nodes with a bootstrap similarity of at least 50% are shown. (C) Conserved domains of Aubergine in N. lugens. It has the PAZ and Piwi domains that are typical motifs of Argonaute family proteins. The catalytic triad DDH is also shown. Sequence motifs were predicted by the NCBI Conserved Domains Server.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020504-g001: Phylogenetic relationships of the SID and Argonaute proteins from N. lugens and other species, and the structure of the N. lugens Aubergine protein.Phylogenetic analysis of (A) SID protein sequences, and (B) Argonaute protein sequences from N. lugens and other animals were conducted using MEGA 4.0; nodes with a bootstrap similarity of at least 50% are shown. (C) Conserved domains of Aubergine in N. lugens. It has the PAZ and Piwi domains that are typical motifs of Argonaute family proteins. The catalytic triad DDH is also shown. Sequence motifs were predicted by the NCBI Conserved Domains Server.
Mentions: Because of the probable role of the SID protein in the uptake of dsRNA from the environment in C. elegans, we cloned the sid-1 gene from N. lugens. The full-length cDNA of the sid-1 gene is 2,119 bp long and contains an open reading frame (ORF) of 1,875 bp (GenBank accession no. JF915743), encoding a protein of 624 amino acids with a calculated molecular mass of 70.8 kDa and an isolectric point (pI) of 6.67 (Figure S1). Multiple alignment and phylogenetic analysis of the deduced amino acid sequences confirmed that this gene is a sid-1 like gene, hence we named it Nlsid-1 (Nilaparvata lugens sid-1). The phylogenetic tree of deduced amino acid sequences showed that the N. lugens SID-1 protein is most closely related to the A. mellifera SID-1 like protein (40% identity, Figure 1A).

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