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Title:   Author  item
                                            SUN, DAOYANG - University Of Californiaitem
                                            NANADETY, RAJA SEKHAR - University Of Californiaitem
                                            ZHANG, YANLONG - Northwest Universityitem
                                            REID, MICHAEL - University Of Californiaitem
                                            NIU, LIXIN - Northwest Universityitem
                                            Jiang, Cai-Zhong
  Submitted to: Journal of Experimental Botany
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 3/24/2016
                                    Publication  Date: 4/19/2016
                                    Citation: Sun, D., Nanadety, R., Zhang, Y., Reid, M.S., Niu, L., Jiang, C. 2016. A petunia ethylene-responsive element binding factor, PhERF2, plays an important role in antiviral RNA silencing. Journal of Experimental Botany. 67(11):3353-3365. doi: 10.1093/jxb/erw155.Interpretive Summary: Virus-induced gene silencing (VIGS) is a useful technique for functional characterization of plant genes. A recombinant plant virus carrying a host-derived sequence fragment initiates RNA-mediated post-transcriptional gene silencing (PTGS), leading to a transient and specific degradation of the corresponding endogenous mRNA. A modified Tobacco rattle virus (TRV) vector has proven to be an excellent tool for VIGS, due to this virus’ wide host range and ability to infect meristematic cells. This system is composed of binary transformation plasmids, TRV1 and TRV2, with a region harboring multiple cloning sites in TRV2. The TRV vector has been successfully used for gene function analysis in a number of eudicots, including the commonly used model plants Arabidopsis, Nicotiana benthamiana, tomato and petunia. In petunia, silencing of phytoene desaturase (PDS) or Chalcone synthase (CHS) provides useful phenotypical markers in VIGS studies for functional characterization of genes in leaf and floral tissues respectively.

The silencing efficiency of the VIGS system is variable, largely depending on growth temperature during the post inoculation and compatibility between the host and the virus. Growth temperature seems to have profound effects on the efficiency of VIGS silencing. Gene silencing efficiency with a TRV system in tomato is enhanced by low temperature and low humidity. Low temperature also enhances gene silencing efficiency throughout the life of cotton plants when Gemini virus-mediated VIGS is employed. But Szittya et al. reported that low temperatures inhibit silencing by preventing siRNA generation. However, the underlying mechanisms of these apparently conflicting findings are not yet well understood. Previously, we tested the effects of silencing CHS on a range of purple-flowered petunia cultivars and found significant variations in the silencing phenotypes. In studies with silencing PDS in tomato we have also observed cultivar-dependent variations in the silencing phenotype.  In four o’clocks (Mirabilis jalapa L.), we found that the Mirabilis Antiviral Protein (MAP) is primarily responsible for poor VIGS efficiency. Simultaneous silencing of the MAP and a gene of interest allowed us to use VIGS to down-regulate a range of genes in this species. Compatibility has limited the range of taxa where TRV-based VIGS has successfully been employed. The genetic basis for the silencing variation and limitation is unknown.

In Petunia (Petunia hybrida), an ethylene-responsive element binding factor, PhERF2, is induced by Tobacco rattle virus (TRV) infection. Inclusion of a PhERF2 fragment in a TRV silencing construct containing reporter’s fragments of Phytoene desaturase (PDS) or Chalcone synthase (CHS) substantially impaired silencing efficiency of both the PDS and CHS reporters. Silencing was also impaired in PhERF2-silenced RNAi transgenic lines, where TRV-PhPDS infection did not show the expected silencing phenotype (photobleaching), but did show more severe symptoms of viral infection. In contrast, photobleaching in response to infiltration with the TRV-PhPDS construct was enhanced in plants overexpressing PhERF2. Transcript abundance of the RNA silencing-related genes RDR2, RDR6, DCL2 and AGO2, was lower in PhERF2-silenced plants but higher in PhERF2-overexpressing plants. Moreover, PhERF2-silenced lines showed higher susceptibility to Cucumber mosaic virus (CMV) than wildtype (WT) plants, while plants overexpressing PhERF2 exhibited increased resistance. Interestingly, growth and development of PhERF2-RNAi transgenic plants were substantially slower, whereas the overexpressing lines were more vigorous than the controls. Taken together, our results indicate that PhERF2 functions as a positive regulator in antiviral RNA silencing.
                                Technical Abstract: Virus-induced gene silencing (VIGS) is a useful technique for functional characterization of plant genes. However, the silencing efficiency of the VIGS system is variable largely depending on compatibility between the host and the virus. Antiviral RNA silencing is involved in plant antiviral defense and requires key enzyme components, RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs) and Argonaute proteins (AGOs). In Petunia (Petunia hybrida), an ethylene-responsive element binding factor, PhERF2, is induced by Tobacco rattle virus (TRV) infection. Inclusion of a PhERF2 fragment in a TRV silencing construct containing reporter’s fragments of Phytoene desaturase (PDS) or Chalcone synthase (CHS) substantially impaired silencing efficiency of both the PDS and CHS reporters. Silencing was also impaired in PhERF2-silenced RNAi transgenic lines, where TRV-PhPDS infection did not show the expected silencing phenotype (photobleaching), but did show more severe symptoms of viral infection. In contrast, photobleaching in response to infiltration with the TRV-PhPDS construct was enhanced in plants overexpressing PhERF2. Transcript abundance of the RNA silencing-related genes RDR2, RDR6, DCL2 and AGO2, was lower in PhERF2-silenced plants but higher in PhERF2-overexpressing plants. Moreover, PhERF2-silenced lines showed higher susceptibility to Cucumber mosaic virus (CMV) than wildtype (WT) plants, while plants overexpressing PhERF2 exhibited increased resistance. Interestingly, growth and development of PhERF2-RNAi transgenic plants were substantially slower, whereas the overexpressing lines were more vigorous than the controls. Taken together, our results indicate that PhERF2 functions as a positive regulator in antiviral RNA silencing.


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