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Functional characterization of the principal sigma factor RpoD of phytoplasmas via an in vitro transcription assay.

Miura C, Komatsu K, Maejima K, Nijo T, Kitazawa Y, Tomomitsu T, Yusa A, Himeno M, Oshima K, Namba S - Sci Rep (2015)

Bottom Line: In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools.The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns.These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.

ABSTRACT
Phytoplasmas (class, Mollicutes) are insect-transmissible and plant-pathogenic bacteria that multiply intracellularly in both plants and insects through host switching. Our previous study revealed that phytoplasmal sigma factor rpoD of OY-M strain (rpoDOY) could be a key regulator of host switching, because the expression level of rpoDOY was higher in insect hosts than in plant hosts. In this study, we developed an in vitro transcription assay system to identify RpoDOY-dependent genes and the consensus promoter elements. The assay revealed that RpoDOY regulated some housekeeping, virulence, and host-phytoplasma interaction genes of OY-M strain. The upstream region of the transcription start sites of these genes contained conserved -35 and -10 promoter sequences, which were similar to the typical bacterial RpoD-dependent promoter elements, while the -35 promoter elements were variable. In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools. The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns. These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.

No MeSH data available.


Related in: MedlinePlus

Identification of rrnB TSSs and analysis of rrnB promoter activity based on the in vitro transcription assay.(a) Schematic representation of the upstream promoter region and TSSs of the rrnB gene. The rrnB TSSs identified by 5′ RACE analysis, designated as P1, P2, and P3 (91, 254, 444 nt upstream of rrnB, respectively), are represented by arrows. Putative –35 and –10 promoter elements of each of the three TSSs are underlined with dotted and continuous lines, respectively. (b) In vitro transcription assays using the RNAP holoenzyme with RpoDOY. RNAPEc and a DNA template were incubated with NTP, including [γ-32P]CTP in the absence (–) or presence (+) of RpoDOY. A 784-bp DNA fragment named PrrnB covering the region from –500 to +284 of rrnB was used as a template. White arrowheads indicate the positions of the transcripts that are possibly transcribed from P2 and P3.
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f1: Identification of rrnB TSSs and analysis of rrnB promoter activity based on the in vitro transcription assay.(a) Schematic representation of the upstream promoter region and TSSs of the rrnB gene. The rrnB TSSs identified by 5′ RACE analysis, designated as P1, P2, and P3 (91, 254, 444 nt upstream of rrnB, respectively), are represented by arrows. Putative –35 and –10 promoter elements of each of the three TSSs are underlined with dotted and continuous lines, respectively. (b) In vitro transcription assays using the RNAP holoenzyme with RpoDOY. RNAPEc and a DNA template were incubated with NTP, including [γ-32P]CTP in the absence (–) or presence (+) of RpoDOY. A 784-bp DNA fragment named PrrnB covering the region from –500 to +284 of rrnB was used as a template. White arrowheads indicate the positions of the transcripts that are possibly transcribed from P2 and P3.

Mentions: RpoD recognizes specific promoter elements located at positions 35 and 10 bp upstream of TSSs1, and hence, an experimental determination of TSSs provides important clues in estimating their upstream promoter elements. For the purpose of identifying promoter elements recognized by RpoDOY, we investigated the TSSs of the 16S ribosomal RNA (rrn) B gene of OY-M, as rrn is often transcribed from RpoD-dependent promoter elements in many bacteria21222324. A 5′ rapid amplification of cDNA ends (5′ RACE) analysis of rrnB was performed using total RNA from phytoplasma-infected plants, resulting in the detection of three TSSs located 91, 254, and 444 bp upstream of rrnB (Fig. 1a). The same results were obtained from total RNA extracted from phytoplasma-infected insects. We designated these TSSs as P1, P2, and P3, respectively, and estimated their putative promoter elements as follows: P1 promoter elements, –35 5′-TTCACA-3′ and –10 5′-TAATCT-3′; P2 promoter elements, –35 5′-TTGCTA-3′ and –10 5′-TATAAT-3′; and P3 promoter elements, –35 5′-TTGCCA-3′ and –10 5′-TATAAT-3′. Among these promoter elements, putative P2 and P3 promoter elements were highly similar to each other.


Functional characterization of the principal sigma factor RpoD of phytoplasmas via an in vitro transcription assay.

Miura C, Komatsu K, Maejima K, Nijo T, Kitazawa Y, Tomomitsu T, Yusa A, Himeno M, Oshima K, Namba S - Sci Rep (2015)

Identification of rrnB TSSs and analysis of rrnB promoter activity based on the in vitro transcription assay.(a) Schematic representation of the upstream promoter region and TSSs of the rrnB gene. The rrnB TSSs identified by 5′ RACE analysis, designated as P1, P2, and P3 (91, 254, 444 nt upstream of rrnB, respectively), are represented by arrows. Putative –35 and –10 promoter elements of each of the three TSSs are underlined with dotted and continuous lines, respectively. (b) In vitro transcription assays using the RNAP holoenzyme with RpoDOY. RNAPEc and a DNA template were incubated with NTP, including [γ-32P]CTP in the absence (–) or presence (+) of RpoDOY. A 784-bp DNA fragment named PrrnB covering the region from –500 to +284 of rrnB was used as a template. White arrowheads indicate the positions of the transcripts that are possibly transcribed from P2 and P3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493692&req=5

f1: Identification of rrnB TSSs and analysis of rrnB promoter activity based on the in vitro transcription assay.(a) Schematic representation of the upstream promoter region and TSSs of the rrnB gene. The rrnB TSSs identified by 5′ RACE analysis, designated as P1, P2, and P3 (91, 254, 444 nt upstream of rrnB, respectively), are represented by arrows. Putative –35 and –10 promoter elements of each of the three TSSs are underlined with dotted and continuous lines, respectively. (b) In vitro transcription assays using the RNAP holoenzyme with RpoDOY. RNAPEc and a DNA template were incubated with NTP, including [γ-32P]CTP in the absence (–) or presence (+) of RpoDOY. A 784-bp DNA fragment named PrrnB covering the region from –500 to +284 of rrnB was used as a template. White arrowheads indicate the positions of the transcripts that are possibly transcribed from P2 and P3.
Mentions: RpoD recognizes specific promoter elements located at positions 35 and 10 bp upstream of TSSs1, and hence, an experimental determination of TSSs provides important clues in estimating their upstream promoter elements. For the purpose of identifying promoter elements recognized by RpoDOY, we investigated the TSSs of the 16S ribosomal RNA (rrn) B gene of OY-M, as rrn is often transcribed from RpoD-dependent promoter elements in many bacteria21222324. A 5′ rapid amplification of cDNA ends (5′ RACE) analysis of rrnB was performed using total RNA from phytoplasma-infected plants, resulting in the detection of three TSSs located 91, 254, and 444 bp upstream of rrnB (Fig. 1a). The same results were obtained from total RNA extracted from phytoplasma-infected insects. We designated these TSSs as P1, P2, and P3, respectively, and estimated their putative promoter elements as follows: P1 promoter elements, –35 5′-TTCACA-3′ and –10 5′-TAATCT-3′; P2 promoter elements, –35 5′-TTGCTA-3′ and –10 5′-TATAAT-3′; and P3 promoter elements, –35 5′-TTGCCA-3′ and –10 5′-TATAAT-3′. Among these promoter elements, putative P2 and P3 promoter elements were highly similar to each other.

Bottom Line: In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools.The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns.These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.

ABSTRACT
Phytoplasmas (class, Mollicutes) are insect-transmissible and plant-pathogenic bacteria that multiply intracellularly in both plants and insects through host switching. Our previous study revealed that phytoplasmal sigma factor rpoD of OY-M strain (rpoDOY) could be a key regulator of host switching, because the expression level of rpoDOY was higher in insect hosts than in plant hosts. In this study, we developed an in vitro transcription assay system to identify RpoDOY-dependent genes and the consensus promoter elements. The assay revealed that RpoDOY regulated some housekeeping, virulence, and host-phytoplasma interaction genes of OY-M strain. The upstream region of the transcription start sites of these genes contained conserved -35 and -10 promoter sequences, which were similar to the typical bacterial RpoD-dependent promoter elements, while the -35 promoter elements were variable. In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools. The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns. These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.

No MeSH data available.


Related in: MedlinePlus