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Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target.

Ulrich J, Dao VA, Majumdar U, Schmitt-Engel C, Schwirz J, Schultheis D, Ströhlein N, Troelenberg N, Grossmann D, Richter T, Dönitz J, Gerischer L, Leboulle G, Vilcinskas A, Stanke M, Bucher G - BMC Genomics (2015)

Bottom Line: Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death.Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites.Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes.

View Article: PubMed Central - PubMed

Affiliation: Johann-Friedrich-Blumenbach-Institut, GZMB, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany. julia.ulrich@biologie.uni-goettingen.de.

ABSTRACT

Background: Insect pest control is challenged by insecticide resistance and negative impact on ecology and health. One promising pest specific alternative is the generation of transgenic plants, which express double stranded RNAs targeting essential genes of a pest species. Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death. However, the identification of efficient RNAi target genes remains a major challenge as genomic tools and breeding capacity is limited in most pest insects impeding whole-animal-high-throughput-screening.

Results: We use the red flour beetle Tribolium castaneum as a screening platform in order to identify the most efficient RNAi target genes. From about 5,000 randomly screened genes of the iBeetle RNAi screen we identify 11 novel and highly efficient RNAi targets. Our data allowed us to determine GO term combinations that are predictive for efficient RNAi target genes with proteasomal genes being most predictive. Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites.

Conclusions: Our results will aid the identification of RNAi target genes in many pest species by providing a manageable number of excellent candidate genes to be tested and the proteasome as prime target. Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes. Our off target analysis is relevant for the sequence selection used in transgenic plants.

No MeSH data available.


Related in: MedlinePlus

GO term clustering reveals the proteasome as efficient target. a All 11 top RNAi target genes have Drosophila orthologs and overall, their protein sequence identity with other species is high. Only Cact, Inr-a and Gw show a low degree of sequence conservation. Dm Drosophila melanogaster, Am Apis mellifera, Aa Aedes aegypti, Ap Acyrthosiphon pisum. b GO term clustering of the top 40 RNAi target genes revealed ten clusters with enriched biological processes. Searching for genes that share the respective GO term combinations identified additional RNAi target genes. For cluster 1, seven novel genes with the respective combination were found and six of them (86 %) turned out to be highly lethal at day 11 (d11) after injection. Cluster 1, 2 and 7 showed the highest predictive power. c The GO terms of the most predictive clusters are shown. Count is the number of genes annotated with a given term. GOTERM_BP_FAT are annotations with respect to biological processes (see Additional file 2: Figure S4 for all clusters)
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Fig3: GO term clustering reveals the proteasome as efficient target. a All 11 top RNAi target genes have Drosophila orthologs and overall, their protein sequence identity with other species is high. Only Cact, Inr-a and Gw show a low degree of sequence conservation. Dm Drosophila melanogaster, Am Apis mellifera, Aa Aedes aegypti, Ap Acyrthosiphon pisum. b GO term clustering of the top 40 RNAi target genes revealed ten clusters with enriched biological processes. Searching for genes that share the respective GO term combinations identified additional RNAi target genes. For cluster 1, seven novel genes with the respective combination were found and six of them (86 %) turned out to be highly lethal at day 11 (d11) after injection. Cluster 1, 2 and 7 showed the highest predictive power. c The GO terms of the most predictive clusters are shown. Count is the number of genes annotated with a given term. GOTERM_BP_FAT are annotations with respect to biological processes (see Additional file 2: Figure S4 for all clusters)

Mentions: In order to protect non-target organisms it would be desirable to use dsRNA fragments that are specific to the pest species and do not contain sequences targeting genes in non-target organisms (off targets). Therefore, we asked whether protein sequence conservation of our RNAi target genes correlated with the number of potential off target sites in other species. On the protein sequence level most of our RNAi target genes showed a strong conservation between some well sequenced species covering insect diversity (Drosophila melanogaster, Aedes aegypti (Diptera), Apis mellifera (Hymenoptera), Acyrthosiphon pisum (Hemiptera). L10, L67 and L82 were the least conserved (Fig. 3a). For protein L76 no ortholog could be identified in Aedes aegypti (Fig. 3a; see Additional file 2: Figure S3 H for phylogenetic analysis).Fig. 3


Large scale RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as prime target.

Ulrich J, Dao VA, Majumdar U, Schmitt-Engel C, Schwirz J, Schultheis D, Ströhlein N, Troelenberg N, Grossmann D, Richter T, Dönitz J, Gerischer L, Leboulle G, Vilcinskas A, Stanke M, Bucher G - BMC Genomics (2015)

GO term clustering reveals the proteasome as efficient target. a All 11 top RNAi target genes have Drosophila orthologs and overall, their protein sequence identity with other species is high. Only Cact, Inr-a and Gw show a low degree of sequence conservation. Dm Drosophila melanogaster, Am Apis mellifera, Aa Aedes aegypti, Ap Acyrthosiphon pisum. b GO term clustering of the top 40 RNAi target genes revealed ten clusters with enriched biological processes. Searching for genes that share the respective GO term combinations identified additional RNAi target genes. For cluster 1, seven novel genes with the respective combination were found and six of them (86 %) turned out to be highly lethal at day 11 (d11) after injection. Cluster 1, 2 and 7 showed the highest predictive power. c The GO terms of the most predictive clusters are shown. Count is the number of genes annotated with a given term. GOTERM_BP_FAT are annotations with respect to biological processes (see Additional file 2: Figure S4 for all clusters)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4559001&req=5

Fig3: GO term clustering reveals the proteasome as efficient target. a All 11 top RNAi target genes have Drosophila orthologs and overall, their protein sequence identity with other species is high. Only Cact, Inr-a and Gw show a low degree of sequence conservation. Dm Drosophila melanogaster, Am Apis mellifera, Aa Aedes aegypti, Ap Acyrthosiphon pisum. b GO term clustering of the top 40 RNAi target genes revealed ten clusters with enriched biological processes. Searching for genes that share the respective GO term combinations identified additional RNAi target genes. For cluster 1, seven novel genes with the respective combination were found and six of them (86 %) turned out to be highly lethal at day 11 (d11) after injection. Cluster 1, 2 and 7 showed the highest predictive power. c The GO terms of the most predictive clusters are shown. Count is the number of genes annotated with a given term. GOTERM_BP_FAT are annotations with respect to biological processes (see Additional file 2: Figure S4 for all clusters)
Mentions: In order to protect non-target organisms it would be desirable to use dsRNA fragments that are specific to the pest species and do not contain sequences targeting genes in non-target organisms (off targets). Therefore, we asked whether protein sequence conservation of our RNAi target genes correlated with the number of potential off target sites in other species. On the protein sequence level most of our RNAi target genes showed a strong conservation between some well sequenced species covering insect diversity (Drosophila melanogaster, Aedes aegypti (Diptera), Apis mellifera (Hymenoptera), Acyrthosiphon pisum (Hemiptera). L10, L67 and L82 were the least conserved (Fig. 3a). For protein L76 no ortholog could be identified in Aedes aegypti (Fig. 3a; see Additional file 2: Figure S3 H for phylogenetic analysis).Fig. 3

Bottom Line: Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death.Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites.Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes.

View Article: PubMed Central - PubMed

Affiliation: Johann-Friedrich-Blumenbach-Institut, GZMB, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany. julia.ulrich@biologie.uni-goettingen.de.

ABSTRACT

Background: Insect pest control is challenged by insecticide resistance and negative impact on ecology and health. One promising pest specific alternative is the generation of transgenic plants, which express double stranded RNAs targeting essential genes of a pest species. Upon feeding, the dsRNA induces gene silencing in the pest resulting in its death. However, the identification of efficient RNAi target genes remains a major challenge as genomic tools and breeding capacity is limited in most pest insects impeding whole-animal-high-throughput-screening.

Results: We use the red flour beetle Tribolium castaneum as a screening platform in order to identify the most efficient RNAi target genes. From about 5,000 randomly screened genes of the iBeetle RNAi screen we identify 11 novel and highly efficient RNAi targets. Our data allowed us to determine GO term combinations that are predictive for efficient RNAi target genes with proteasomal genes being most predictive. Finally, we show that RNAi target genes do not appear to act synergistically and that protein sequence conservation does not correlate with the number of potential off target sites.

Conclusions: Our results will aid the identification of RNAi target genes in many pest species by providing a manageable number of excellent candidate genes to be tested and the proteasome as prime target. Further, the identified GO term combinations will help to identify efficient target genes from organ specific transcriptomes. Our off target analysis is relevant for the sequence selection used in transgenic plants.

No MeSH data available.


Related in: MedlinePlus