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Mayuko Shimokawa,Sayaka Hirai,Hiroaki Kodama 한국식물생명공학회 2019 Plant biotechnology reports Vol.13 No.2
The 2b protein of Cucumber mosaic virus (CMV) subgroup IB strain IA (IA2b) suppresses the sense transgene-induced posttranscriptional gene silencing (S-PTGS) but not the inverted repeat-induced post-transcriptional gene silencing (IR-PTGS) of a tobacco microsome-localized α-linolenate synthase gene (NtFAD3). In contrast, the 2b protein of CMV subgroup IB strain SD (SD2b) has been reported to suppress IR-PTGS. We overexpressed the SD2b gene in tobacco, and this transgenic line was crossed with the transgenic plants showing S-PTGS and IR-PTGS of the NtFAD3 gene. The phenotype of offspring showed that SD2b inhibited S-PTGS but not IR-PTGS. Next, we determined the suppressor activity of IA2b and SD2b proteins in a transient IR-PTGS assay. The transient expression of firefly luciferase (LUC) gene was efficiently decreased by IR-PTGS. Co-infiltration of SD2b and IA2b gene partially suppressed IR-PTGS of the LUC gene in the wild-type plants; however, these 2b proteins did not suppress IR-PTGS in the RNA-dependent RNA polymerase6 (RDR6) knockdown plants. It has been reported that RDR6-dependent secondary small interfering RNA synthesis does not occur in the IR-PTGS, when targeting endogenous genes such as NtFAD3, but is efficiently induced in the IR-PTGS of the reporter transgenes. These results indicate that the SD2b and IA2b preferentially suppress the RDR6-dependent silencing pathway but do not suppress the RDR6-independent IR-PTGS pathway.
Tatsuya Mikami,Yuta Saeki,Sayaka Hirai,Mayuko Shimokawa,Yukiko Umeyama,Yusaku Kuroda,Hiroaki Kodama 한국식물생명공학회 2018 Plant biotechnology reports Vol.12 No.6
RNA silencing is a sequence-specific form of epigenetic regulation that targets invasive nucleic acids. RNA-dependent RNA polymerase6 (RDR6) converts target RNA molecules, such as transgene transcripts, into double-stranded RNAs (dsRNAs) during posttranscriptional gene silencing (PTGS). Then, these dsRNAs are processed into small RNAs that guide sequencespecific RNA degradation. T-DNA-derived small RNAs are generated during the transfer of T-DNA from Agrobacterium to plant cells and compromise the function of the genes in the T-DNA. In the present study, we produced selection-markerfree transgenic tobacco plants using the MAT vector system, and expression of the tobacco RDR6 gene (NtRDR6) was suppressed using inverted-repeat-induced PTGS. Reduced expression of the NtRDR6 gene improved the transient expression of the transgene in the agroinfiltrated leaves and enhanced the production of hairy roots after infection with Agrobacterium containing a root-inducing T-DNA. The expression level of the sense transgene was determined in individual hairy roots, and knockdown of the NtRDR6 gene did not affect the distribution of the expression levels in individual transformants. These results indicate that NtRDR6 partially inhibited T-DNA function during T-DNA transfer but did not affect the expression of the transgene in stable transformants, except in transformants showing sense-transgene-induced PTGS.
Sayuri Ohta,Sakie Nakagawara,Sayaka Hirai,Kumi Miyagishima,Gorou Horiguchi,Hiroaki Kodama 한국식물생명공학회 2018 Plant biotechnology reports Vol.12 No.2
Several plant genes have their first intron in the 5′ untranslated region (5′ UTR), and such 5′ UTR introns often show several biological functions, including the intron-mediated enhancement of protein expression through an increase of mRNA level (IME), intron-dependent spatial expression, and intron-mediated enhancement of translation. Here, we show another function of the 5′ UTR intron, i.e., the 5′ UTR intron-mediated enhancement of constitutive splicing. The NtFAD3 gene, which encodes a tobacco microsome ω-3 fatty acid desaturase, has a 552 nucleotide-long 5′ UTR intron (intron 1), and the other seven introns are located in the coding sequence. The splicing of the 5′ half region of the NtFAD3 was studied through an in vivo splicing assay using Arabidopsis leaf explants. The low splicing efficiency of intron 2 was much improved when the assay construct harbored intron 1. Deletion of intron 1 and the replacement of intron 1 to the NtFAD3 intron 8 decreased the splicing efficiency of intron 2. The splicing enhancers were redundant and dispersed in the 5′ splice site-proximal, 284-nucleotides region of intron 1. In addition, the interaction among the cis-elements, i.e., the splicing enhancers in the intron 1 and exon 2, were necessary for the efficient splicing of intron 2. The 5′ UTR intron-mediated constitutive splicing was partially inhibited when an SR-like protein, SR45, was deficient. These results indicated a novel function of the 5′ UTR intron, namely an enhancement of the constitutive splicing.