Silencing RNA-directed RNA polymerase 2 increases the susceptibility of Nicotiana attenuata to UV in the field and in the glasshouse

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Abstract

Summary

RNA-directed RNA-polymerases (RdRs) are essential in small interfering RNA (siRNA) biogenesis and appear to be functionally specialized. We examined the consequences of silencing RdR2 in Nicotiana attenuata with a field release, and transcriptional, two-dimensional proteomic and metabolite analyses. NaRdR2-silenced plants (irRdR2) had large reductions (46% of wild type) in 22–24-nt small RNAs (smRNAs), and smaller reductions (35, 23 and 26% of wild type) in the 19–21, 25–27 and 28–30-nt smRNAs, respectively. When planted into their native habitats in the Great Basin Desert, irRdR2 plants had impaired growth and reproductive output, which were associated with reduced levels of leaf phenolics (rutin and 4′-chlorogenic acid) and MYB and PAL transcripts, but were unaffected in their herbivore resistance. These phenotypes were confirmed in glasshouse experiments, but only when irRdR2 plants were grown with UV-B radiation. irRdR2 plants had wild-type levels of elicited phytohormones and resistance to Manduca sexta attack, but when exposed to UV-B, had reduced growth, fitness, levels of MYB and PAL transcripts, and phenolics. Proteins related to protection against oxidative and physiological stresses, chromatin remodeling and transcription were also downregulated. Silencing the MYB gene by virus-induced gene silencing (VIGS) in wild-type plants reduced levels of PAL transcripts and phenolics, as it did in UV-exposed irRdR2 plants. Bioinformatic analysis revealed that genes involved in phenylpropanoid biosynthesis contained a large number of smRNA binding motives, suggesting that these genes are targets of smRNAs. We conclude that although NaRdR2 transcripts are upregulated in response to both UV-B and herbivore elicitation, the responses they regulate have been tailored to provide protection from UV-B radiation.

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