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, 86 (12), 6847-54

Temperature-dependent Survival of Turnip Crinkle Virus-Infected Arabidopsis Plants Relies on an RNA Silencing-Based Defense That Requires dcl2, AGO2, and HEN1

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Temperature-dependent Survival of Turnip Crinkle Virus-Infected Arabidopsis Plants Relies on an RNA Silencing-Based Defense That Requires dcl2, AGO2, and HEN1

Xiuchun Zhang et al. J Virol.

Abstract

While RNA silencing is a potent antiviral defense in plants, well-adapted plant viruses are known to encode suppressors of RNA silencing (VSR) that can neutralize the effectiveness of RNA silencing. As a result, most plant genes involved in antiviral silencing were identified by using debilitated viruses lacking silencing suppression capabilities. Therefore, it remains to be resolved whether RNA silencing plays a significant part in defending plants against wild-type viruses. We report here that, at a higher plant growth temperature (26°C) that permits rigorous replication of Turnip crinkle virus (TCV) in Arabidopsis, plants containing loss-of-function mutations within the Dicer-like 2 (DCL2), Argonaute 2 (AGO2), and HEN1 RNA methyltransferase genes died of TCV infection, whereas the wild-type Col-0 plants survived to produce viable seeds. To account for the critical role of DCL2 in ensuring the survival of wild-type plants, we established that higher temperature upregulates the activity of DCL2 to produce viral 22-nucleotide (nt) small interfering RNAs (vsRNAs). We further demonstrated that DCL2-produced 22-nt vsRNAs were fully capable of silencing target genes, but that this activity was suppressed by the TCV VSR. Finally, we provide additional evidence supporting the notion that TCV VSR suppresses RNA silencing through directly interacting with AGO2. Together, these results have revealed a specialized RNA silencing pathway involving DCL2, AGO2, and HEN1 that provides the host plants with a competitive edge against adapted viruses under environmental conditions that facilitates robust virus reproduction.

Figures

Fig 1
Fig 1
DCL2 targets wild-type TCV in a temperature-dependent manner. (A) Detection of TCV gRNA and sgRNAs, as well as vsRNAs, by Northern blot hybridizations in samples of Col-0, dcl2, dcl3, dcl2 dcl3, and dcl4 plants kept at two different temperatures as indicated above the panels. The relative accumulation levels of TCV gRNA and 22-nt vsRNA were estimated by averaging the relative signal strength of each treatment in three independent blots, with the value of TCV-infected Col-0 plants at 18°C set as 1. (B) Detection of CPC gRNA, sgRNAs, and vsRNAs by Northern blot hybridizations in the inoculated leaves of the same set of plants kept at two different temperatures as indicated above the panels. (C) Detection of CPC gRNA, sgRNAs, and vsRNAs by Northern blot hybridizations in the inoculated leaves of Col-0, dcl4, dcl2 dcl4, dcl3 dcl4, and dcl2 dcl3 dcl4 plants kept at two different temperatures as indicated above the panels. (D) RNA samples shown in panel A were enriched for dsRNA and run on a native agarose gel to reveal the accumulation levels of TCV-specific dsRNA. The size marker (M) on the left is a DNA ladder.
Fig 2
Fig 2
Temperature-dependent survival of TCV-infected dcl2, ago2, and hen1 plants. (A). Col-0, dcl2, dcl3, dcl2 dcl3, and dcl4 plants were infected with TCV and placed in two growth chambers set at different temperatures (18°C versus 26°C), and the infected plants were photographed at 28 dpi. (B) Col-0, ago1 (1–27 allele), ago2, and ago1 ago2 plants were likewise infected with TCV and subjected to different temperature conditions, except that the images were taken 1 week earlier, at 21 dpi. (C) Ler-0 (the parental ecotype of hen1-1) and hen1-1 plants were subjected to the treatments described in panels A and B and photographed at 28 dpi. (D) Accumulation levels of viral RNAs and vsRNAs in Ler-0 and hen1-1 plants at 7 dpi as detected with Northern blot hybridizations.
Fig 3
Fig 3
DCL2-produced 22-nt vsRNAs are effective inducers of target gene silencing. (A) Diagrams of TCV-CC, CPB-CC, TCV-CC-PDS, and CPB-CC-PDS constructs, with the R130T mutation of CPB denoted by a red star. The TCV-CC and CPB-CC constructs were produced by changing the AT dinucleotides to CC at nt 3807 to 3808, within the 3′UTR of TCV, resulting in a new KpnI site (GGTACC). A 90-nt PDS fragment was then inserted into the KpnI site to create TCV-CC-PDS and CPB-CC-PDS. (B) The viral RNA and vsRNA accumulation levels of CPB-CC-PDS and TCV-CC-PDS in Col-0, dcl2, dcl4, and dcl2 dcl4 plants kept at two different temperatures as detected by Northern blot hybridization. (C) PDS silencing induced by CPB-CC-PDS in Col-0, dcl2, dcl4, and dcl2 dcl4 plants. Images were recorded at 18 dpi. (D) Downregulation of PDS mRNA levels by CPB-CC-PDS in dcl2 and dcl4 plants as determined with semiquantitative RT-PCR. The samples were collected at 7 dpi, before photobleaching becoming apparent. Actin 3 mRNA was used as a control to ensure that similar amounts of RNA were used in all reactions. Omission of reverse transcriptase led to the failure to obtain the PDS PCR fragment (bottom panel), proving that the PCR products in top panel were dependent on the presence of PDS mRNA.
Fig 4
Fig 4
TCV CP and AGO2 interact to restore YFP fluorescence in a BIFC assay. (A) Illustrations of the BIFC constructs used in these experiments. (B) Laser scanning confocal images of N. benthamiana leaves transiently expressing the indicated constructs. Bar, 50 μm.

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