RDR6 Has a Broad-Spectrum but Temperature-Dependent Antiviral Defense Role in <em>Nicotiana benthamiana</em>

Feng Qu

Xiaohong Ye

Guichuan Hou

Shirley Sato

Thomas Clemente

T Morris

School of Biological Sciences,^{Center for Biotechnology, University of Nebraska—Lincoln, Lincoln, Nebraska 68588-0666}²

^{Corresponding author. Mailing address: E 229 Beadle Center, University of Nebraska—Lincoln, Lincoln, NE 68588-0666. Phone: (402) 472-8889. Fax: (402) 472-8722. E-mail:}ude.lnu@1sirromj.

Received 2005 Jun 29; Accepted 2005 Sep 27.

Abstract

SDE1/SGS2/RDR6, a putative RNA-dependent RNA polymerase (RdRP) from Arabidopsis thaliana, has previously been found to be indispensable for maintaining the posttranscriptional silencing of transgenes, but it is seemingly redundant for antiviral defense. To elucidate the antiviral role of this RdRP in a different host plant and to evaluate whether plant growth conditions affect its role, we down-regulated expression of the Nicotiana benthamiana homolog, NbRDR6, and examined the plants for altered susceptibility to various viruses at different growth temperatures. The results we describe here clearly show that plants with reduced expression of NbRDR6 were more susceptible to all viruses tested and that this effect was more pronounced at higher growth temperatures. Diminished expression of NbRDR6 also permitted efficient multiplication of tobacco mosaic virus in the shoot apices, leading to serious disruption with microRNA-mediated developmental regulation. Based on these results, we propose that NbRDR6 participates in the antiviral RNA silencing pathway that is stimulated by rising temperatures but suppressed by virus-encoded silencing suppressors. The relative strengths of these two factors, along with other plant defense components, critically influence the outcome of virus infections.

Abstract

RNA silencing is a surveillance system in eukaryotic organisms triggered by double-stranded RNA (dsRNA) that is subsequently digested by a dsRNA-specific RNase (Dicer or Dicer-like) into a small RNA species of 21 to 25 nucleotides (nt) long, called small interfering RNA (siRNA). The resultant siRNAs are then recruited into the RNA-induced silencing complex to direct the degradation of other RNAs with sequence complementarity to siRNAs (13). RNA silencing is thought to function primarily in defending eukaryotic cells against RNA molecular parasites, such as RNA viruses and transposon RNAs. Plant viruses, as well as some animal viruses, counteract this host defense mechanism by encoding suppressors of RNA silencing, which act at different steps of the pathway and with various strengths to ensure their successful systemic invasion of specific hosts (26, 31).

In addition to guarding the host against parasitic RNAs, recent studies have shown that processes highly related to RNA silencing are also involved in developmental regulation (22, 28), methylation of chromosomal DNA and histones, and chromatin maintenance (20, 43). miRNA-mediated regulation of gene expression in both animal and plant systems is a particularly interesting discovery. Unlike siRNAs, miRNAs are encoded by genomes of eukaryotes in the form of partially double-stranded precursor molecules, which are processed by Dicer-like RNase(s) to release mature miRNAs. The miRNAs then mediate degradation or translational repression of the target RNAs (15). One well-studied example in plants is miR165/166. This miRNA targets the mRNA of three class III homeodomain leucine zipper (HD-ZIP III) transcription factors, PHABULOSA (PHB), PHAVOLUTA (PHV), and REVOLUTA (REV), for cleavage (10, 29, 30). Restricted expression of these genes in the shoot apex plays a critical role in patterning adaxial-abaxial polarity in lateral organs. Gain-of-function mutations of PHB, PHV, and REV, which cause adaxialization of leaves and vascular systems, have been mapped mostly to the target sites of miR165/166 and have been found to prevent the miRNA-mediated degradation of their mRNAs (10, 21, 23, 30). Notably, similar phenotypes have also been frequently observed with transgenic plants expressing virus-encoded silencing suppressors and occasionally with virus-infected plants (2, 3, 9, 11, 22), supporting the argument that siRNA- and miRNA-mediated pathways are closely related.

The plant RNA silencing pathway can be mechanistically divided into two stages, initiation and maintenance (5, 36). The initiation stage is characterized by its dependence on the trigger dsRNA and siRNAs directly derived from the trigger. The maintenance stage is independent of the trigger and is responsible for the persistent silencing, even after the inducer dsRNA is cleared from the cells. At this stage, RNA silencing is maintained through secondary synthesis of dsRNA by a cellular RNA-dependent RNA polymerase (RdRP), using the siRNA-complementary target RNA as a template.

The Arabidopsis spp. SDE1/SGS2/RDR6 (RDR6 hereafter) RdRP is a putative RdRP originally identified as required for RNA silencing of transgenes (6, 32). Additional evidence since then has established that it is essential for the maintenance of RNA silencing. RDR6 has been shown to be necessary for the continued silencing of a transgene after the complete elimination of inducer RNA, the cell-to-cell movement of the RNA silencing signal, and the spread of silencing along the target RNA to sequences beyond the region that is homologous to the trigger molecule (7, 17, 24, 40).

Virus-induced silencing differs from transgene-mediated silencing at the initiation stage because the primary dsRNA inducer is generally thought to be the viral double-stranded replication intermediates, hence circumventing the requirement of a host RdRP. However, virus-induced silencing is similar to transgene-mediated silencing at the maintenance stage in that both require siRNA amplification and intercellular silencing signaling. Indeed, several studies have shown that plant viral silencing suppressors function primarily by preventing the movement of silencing signals out of the initially infected cells (14, 34, 35, 42). Therefore, plants encoding defective RDR6 would be expected to become more susceptible to virus infections. It was hence unexpected that mutant Arabidopsis plants lacking a functional RDR6 gene (sde1 and sgs2 plants) failed to show increased susceptibility to most of the RNA plant viruses tested (6, 32).

This inconsistency between the role of RDR6 in RNA silencing maintenance and apparent lack of heightened viral susceptibility in its absence led us to speculate that the effect of RDR6 could be masked by specific experimental conditions that affect the efficacy of RNA silencing. Consistent with this speculation, higher plant growth temperature has recently been found to enhance RNA silencing (39). In this report, we have evaluated the role of a Nicotiana benthamiana homolog of RDR6, NbRDR6, in plant antiviral defense under different temperature conditions. Our results demonstrate that NbRDR6 is actively involved in defending both differentiated and apical plant tissues from invasion by several different RNA plant viruses, including members of the genera Potexvirus, Carmovirus, and Tobamovirus. We show that the consequence of NbRDR6 down-regulation depends on both the plant growth temperature and the nature of the invading virus, reflecting the delicate balance between the efficacy of the host RNA silencing and the ability of the invading virus to counteract this process.

Acknowledgments

We thank Vicki Vance and colleagues for providing us with the modified PVX vector.

This work is supported in part by grants from the U.S. Department of Energy (DE-FG03-98ER20315) and NIH (grant number P20 RR16469) as a part of the INBRE Program of the National Center for Research Resources.

Acknowledgments

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