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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

Sequence conservation in the RpoDOY-dependent promoter region.(a) Putative promoter sequences deduced from the alignment of upstream sequences of TSSs identified by 5′ RACE analysis. Bold letters indicate the putative –35 and –10 promoter elements. ‘+1’ represents the position of the TSS. Numbers of the right side show the distance to the 5´-end of mature rRNA or ATG. (b) Consensus sequences of RpoDOY-dependent promoters. The consensus sequences of –35 (left) and –10 (right) promoter elements were determined using the BioProspector program63 and illustrated with the WebLogo tool64.
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f4: Sequence conservation in the RpoDOY-dependent promoter region.(a) Putative promoter sequences deduced from the alignment of upstream sequences of TSSs identified by 5′ RACE analysis. Bold letters indicate the putative –35 and –10 promoter elements. ‘+1’ represents the position of the TSS. Numbers of the right side show the distance to the 5´-end of mature rRNA or ATG. (b) Consensus sequences of RpoDOY-dependent promoters. The consensus sequences of –35 (left) and –10 (right) promoter elements were determined using the BioProspector program63 and illustrated with the WebLogo tool64.

Mentions: To more closely define the RpoDOY-dependent promoter elements, we performed a 5′ RACE analysis of nine genes shown in this study to be transcribed by RNAPEc–RpoDOY (Fig. 3; ibpA, infC, rplM, rpsD, PAM157, PAM289, PAM486, tengu, and amp) using total RNA from phytoplasma-infected plants or insects. As a result, we mapped the 5′-ends of ibpA, infC, rplM, rpsD, PAM157, PAM289, PAM486, tengu, and amp transcripts at 170, 415, 166, 301, 177, 222, 52, 153, and 81 nt upstream, respectively, of their start codons (Fig. 4a). In agreement with a previous study on the detection and identification of mycoplasma promoter sequences35, the 5′-end of these transcripts was either adenine or guanine. The predicted size from these identified TSSs to the 3′-ends of the above-mentioned in vitro transcription templates roughly corresponded to the length of transcripts produced by the in vitro transcription assays (Fig. 3). Next, to further characterize the RpoDOY-dependent promoter elements, we searched for consensus promoter elements using a motif finding tool, BioProspector, and found conserved –35 and –10 hexamers located at appropriate positions upstream of the TSSs (Fig. 4a). The consensus –10 promoter element (5′-TAtAAT-3′) was found in all sequences examined (Fig. 4b). The consensus –35 promoter element (5′-TTgaca-3′) was also found, even though this element was less conserved compared with the –10 promoter element (Fig. 4b). The spacing between the –35 and –10 promoter elements could vary from 17 to 19 nt. We also found two relatively conserved regions, an ‘extended –10 motif’ (5′-TnTG-3′) positioned around –17 to –14 and an A-rich region positioned around –42 to –39 (see Supplementary Fig. S5 online), which are common features of the promoter regions in other bacteria3637.


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)

Sequence conservation in the RpoDOY-dependent promoter region.(a) Putative promoter sequences deduced from the alignment of upstream sequences of TSSs identified by 5′ RACE analysis. Bold letters indicate the putative –35 and –10 promoter elements. ‘+1’ represents the position of the TSS. Numbers of the right side show the distance to the 5´-end of mature rRNA or ATG. (b) Consensus sequences of RpoDOY-dependent promoters. The consensus sequences of –35 (left) and –10 (right) promoter elements were determined using the BioProspector program63 and illustrated with the WebLogo tool64.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Sequence conservation in the RpoDOY-dependent promoter region.(a) Putative promoter sequences deduced from the alignment of upstream sequences of TSSs identified by 5′ RACE analysis. Bold letters indicate the putative –35 and –10 promoter elements. ‘+1’ represents the position of the TSS. Numbers of the right side show the distance to the 5´-end of mature rRNA or ATG. (b) Consensus sequences of RpoDOY-dependent promoters. The consensus sequences of –35 (left) and –10 (right) promoter elements were determined using the BioProspector program63 and illustrated with the WebLogo tool64.
Mentions: To more closely define the RpoDOY-dependent promoter elements, we performed a 5′ RACE analysis of nine genes shown in this study to be transcribed by RNAPEc–RpoDOY (Fig. 3; ibpA, infC, rplM, rpsD, PAM157, PAM289, PAM486, tengu, and amp) using total RNA from phytoplasma-infected plants or insects. As a result, we mapped the 5′-ends of ibpA, infC, rplM, rpsD, PAM157, PAM289, PAM486, tengu, and amp transcripts at 170, 415, 166, 301, 177, 222, 52, 153, and 81 nt upstream, respectively, of their start codons (Fig. 4a). In agreement with a previous study on the detection and identification of mycoplasma promoter sequences35, the 5′-end of these transcripts was either adenine or guanine. The predicted size from these identified TSSs to the 3′-ends of the above-mentioned in vitro transcription templates roughly corresponded to the length of transcripts produced by the in vitro transcription assays (Fig. 3). Next, to further characterize the RpoDOY-dependent promoter elements, we searched for consensus promoter elements using a motif finding tool, BioProspector, and found conserved –35 and –10 hexamers located at appropriate positions upstream of the TSSs (Fig. 4a). The consensus –10 promoter element (5′-TAtAAT-3′) was found in all sequences examined (Fig. 4b). The consensus –35 promoter element (5′-TTgaca-3′) was also found, even though this element was less conserved compared with the –10 promoter element (Fig. 4b). The spacing between the –35 and –10 promoter elements could vary from 17 to 19 nt. We also found two relatively conserved regions, an ‘extended –10 motif’ (5′-TnTG-3′) positioned around –17 to –14 and an A-rich region positioned around –42 to –39 (see Supplementary Fig. S5 online), which are common features of the promoter regions in other bacteria3637.

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