Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

Sebastian Kurscheid; Ala E. Lew-Tabor; Manuel Rodriguez Valle; Anthea G. Bruyeres; Vivienne J. Doogan; Ulrike G. Munderloh; Felix D. Guerrero; Roberto A. Barrero; Matthew I. Bellgard

doi:10.1186/1471-2199-10-26

Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

Sebastian Kurscheid, Ala E. Lew-Tabor, Manuel Rodriguez Valle, Anthea G. Bruyeres, Vivienne J. Doogan, Ulrike G. Munderloh, Felix D. Guerrero, Roberto A. Barrero, Matthew I. Bellgard

Entomology

Research output: Contribution to journal › Article › peer-review

58 Scopus citations

Abstract

Background: The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. Results: We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. Conclusion: We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.

Original language	English (US)
Article number	26
Journal	BMC Molecular Biology
Volume	10
DOIs	https://doi.org/10.1186/1471-2199-10-26
State	Published - Mar 26 2009

Bibliographical note

Funding Information:
The authors acknowledge Dr Bing Zhang for his assistance with culture qRT-PCR analysis and Ms Catherine Minchin for maintenance of the BME26 cell lines and for undertaking the culture knockdown experiments. The authors also wish to acknowledge the expertise and diligence provided by Mr Daniel Jarrett in the preparation of molecules for the RNAi diagram (Figure 5) and Dr Leo Salividar (USDA) for assistance with identifying Gen-Bank Accession numbers for all relevant R. microplus consensus and clone sequences. We would like to thank Dr Wayne Jorgensen and Prof Rudi Appels for a critical review of the manuscript. This research was funded by the Cooperative Research Centre for Beef Genetic Technologies, Armi-dale, NSW, Australia.

Publisher link

10.1186/1471-2199-10-26

Find It

Find It at U of M Libraries

Cite this

Kurscheid, S., Lew-Tabor, A. E., Rodriguez Valle, M., Bruyeres, A. G., Doogan, V. J., Munderloh, U. G., Guerrero, F. D., Barrero, R. A., & Bellgard, M. I. (2009). Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila. BMC Molecular Biology, 10, Article 26. https://doi.org/10.1186/1471-2199-10-26

@article{c0f51757809043dba2b1552d5b81f4e4,

title = "Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila",

abstract = "Background: The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. Results: We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. Conclusion: We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.",

author = "Sebastian Kurscheid and Lew-Tabor, {Ala E.} and {Rodriguez Valle}, Manuel and Bruyeres, {Anthea G.} and Doogan, {Vivienne J.} and Munderloh, {Ulrike G.} and Guerrero, {Felix D.} and Barrero, {Roberto A.} and Bellgard, {Matthew I.}",

note = "Funding Information: The authors acknowledge Dr Bing Zhang for his assistance with culture qRT-PCR analysis and Ms Catherine Minchin for maintenance of the BME26 cell lines and for undertaking the culture knockdown experiments. The authors also wish to acknowledge the expertise and diligence provided by Mr Daniel Jarrett in the preparation of molecules for the RNAi diagram (Figure 5) and Dr Leo Salividar (USDA) for assistance with identifying Gen-Bank Accession numbers for all relevant R. microplus consensus and clone sequences. We would like to thank Dr Wayne Jorgensen and Prof Rudi Appels for a critical review of the manuscript. This research was funded by the Cooperative Research Centre for Beef Genetic Technologies, Armi-dale, NSW, Australia.",

year = "2009",

month = mar,

day = "26",

doi = "10.1186/1471-2199-10-26",

language = "English (US)",

volume = "10",

journal = "BMC Molecular Biology",

issn = "1471-2199",

publisher = "BioMed Central",

}

TY - JOUR

T1 - Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

AU - Kurscheid, Sebastian

AU - Lew-Tabor, Ala E.

AU - Rodriguez Valle, Manuel

AU - Bruyeres, Anthea G.

AU - Doogan, Vivienne J.

AU - Munderloh, Ulrike G.

AU - Guerrero, Felix D.

AU - Barrero, Roberto A.

AU - Bellgard, Matthew I.

N1 - Funding Information: The authors acknowledge Dr Bing Zhang for his assistance with culture qRT-PCR analysis and Ms Catherine Minchin for maintenance of the BME26 cell lines and for undertaking the culture knockdown experiments. The authors also wish to acknowledge the expertise and diligence provided by Mr Daniel Jarrett in the preparation of molecules for the RNAi diagram (Figure 5) and Dr Leo Salividar (USDA) for assistance with identifying Gen-Bank Accession numbers for all relevant R. microplus consensus and clone sequences. We would like to thank Dr Wayne Jorgensen and Prof Rudi Appels for a critical review of the manuscript. This research was funded by the Cooperative Research Centre for Beef Genetic Technologies, Armi-dale, NSW, Australia.

PY - 2009/3/26

Y1 - 2009/3/26

N2 - Background: The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. Results: We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. Conclusion: We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.

AB - Background: The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. Results: We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. Conclusion: We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.

UR - http://www.scopus.com/inward/record.url?scp=65649094845&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=65649094845&partnerID=8YFLogxK

U2 - 10.1186/1471-2199-10-26

DO - 10.1186/1471-2199-10-26

M3 - Article

C2 - 19323841

AN - SCOPUS:65649094845

SN - 1471-2199

VL - 10

JO - BMC Molecular Biology

JF - BMC Molecular Biology

M1 - 26

ER -

Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

Abstract

Bibliographical note

Publisher link

Find It

Other files and links

Fingerprint

Cite this