SUMMARYIn plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference ... more SUMMARYIn plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)‐inducing molecules produced by the endonucleolytic cleavage of double‐stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA‐DIRECTED RNA POLYMERASE 6 (RDR6)‐mediated dsRNA synthesis using siRNA‐targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER‐LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA‐induced silencing complex reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the green fluorescent protein (GFP)‐expressing Nicotiana benthamiana 16C line, high‐pressure spraying protocol, and synthetic 22‐nucleotide (nt) long siRNAs. We found that the 22‐nt siRNA targeting the 3ʹ of the GFP transgene was less efficient in inducing silencing when compared with the siRNAs targeting the 5ʹ and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6‐mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3ʹ‐end of the target RNA.
Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistan... more Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (-)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.
In plants, transgenes frequently become spontaneously silenced for unknown reasons. Typically, tr... more In plants, transgenes frequently become spontaneously silenced for unknown reasons. Typically, transgene silencing involves the generation of small interfering RNAs (siRNAs) that directly or indirectly target cognate DNA and mRNA sequences for methylation and degradation, respectively. In this report, we compared spontaneous silencing of a transgene in Nicotiana benthamiana and Nicotiana tabacum. In both species, abundant siRNAs were produced. In N. benthamiana, the self-silencing process involved mRNA degradation and dense DNA methylation of the homologous coding region. In N. tabacum, self-silencing occurred without complete mRNA degradation and with low methylation of the cognate coding region. Our data indicated that in plants, siRNA-mediated spontaneous silencing is, in addition to mRNA degradation, based on translational inhibition. Differences in the initiation and establishment of self-silencing together with marked differences in the degree of de novo DNA methylation showed that the mechanistic details of RNA silencing, although largely conserved, may vary also in genetically close plant species.
SUMMARYIn plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference ... more SUMMARYIn plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)‐inducing molecules produced by the endonucleolytic cleavage of double‐stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA‐DIRECTED RNA POLYMERASE 6 (RDR6)‐mediated dsRNA synthesis using siRNA‐targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER‐LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA‐induced silencing complex reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the green fluorescent protein (GFP)‐expressing Nicotiana benthamiana 16C line, high‐pressure spraying protocol, and synthetic 22‐nucleotide (nt) long siRNAs. We found that the 22‐nt siRNA targeting the 3ʹ of the GFP transgene was less efficient in inducing silencing when compared with the siRNAs targeting the 5ʹ and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6‐mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3ʹ‐end of the target RNA.
Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistan... more Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (-)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.
In plants, transgenes frequently become spontaneously silenced for unknown reasons. Typically, tr... more In plants, transgenes frequently become spontaneously silenced for unknown reasons. Typically, transgene silencing involves the generation of small interfering RNAs (siRNAs) that directly or indirectly target cognate DNA and mRNA sequences for methylation and degradation, respectively. In this report, we compared spontaneous silencing of a transgene in Nicotiana benthamiana and Nicotiana tabacum. In both species, abundant siRNAs were produced. In N. benthamiana, the self-silencing process involved mRNA degradation and dense DNA methylation of the homologous coding region. In N. tabacum, self-silencing occurred without complete mRNA degradation and with low methylation of the cognate coding region. Our data indicated that in plants, siRNA-mediated spontaneous silencing is, in addition to mRNA degradation, based on translational inhibition. Differences in the initiation and establishment of self-silencing together with marked differences in the degree of de novo DNA methylation showed that the mechanistic details of RNA silencing, although largely conserved, may vary also in genetically close plant species.
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Papers by Gabi Krczal