No abstract

In this report, we describe a method for the delivery of small interfering RNAs (siRNAs) into plant cells. In vitro synthesized siRNAs that were designed to target the coding region of a GREEN FLUORESCENT PROTEIN (GFP) transgene were applied by various methods onto GFP-expressing transgenic Nicotiana benthamiana plants to trigger RNA silencing. In contrast to mere siRNA applications, including spraying, syringe injection, and infiltration of siRNAs that all failed to induce RNA silencing, high pressure spraying of siRNAs resulted in efficient local and systemic silencing of the GFP transgene, with comparable efficiency as was achieved with biolistic siRNA introduction. High-pressure spraying of siRNAs with sizes of 21, 22, and 24 nucleotides (nt) led to local GFP silencing. Small RNA deep sequencing revealed that no shearing of siRNAs was detectable by high-pressure spraying. Systemic silencing was basically detected upon spraying of 22 nt siRNAs. Local and systemic silencing developed faster and more extensively upon targeting the apical meristem than spraying of mature leaves.

Since its discovery, RNA interference has been widely used in crop protection. Recently, transgene-free procedures that were based on exogenous application of RNA molecules having the capacity to trigger RNAi in planta have been reported. Yet, efficient delivery of such RNA molecules to plants and particularly to trees poses major technical challenges. Here, we describe simple methods for efficient delivery of hairpin RNAs (hpRNAs) and small interfering RNAs (siRNAs) to Malus domestica, Vitis vinifera, and Nicotiana benthamiana that are based on trunk injection and/or petiole absorption. The applied RNA molecules were efficiently taken up and systemically transported. In apical leaves, the RNA was already detectable 1 day post-application (dpa) and could be detected at least up to 10 dpa, depending on the method of application. Confocal microscopy revealed that the uptaken and systemically transported RNA molecules were strictly restricted to the xylem and apoplast which may illustrate why the applied hpRNAs were not processed into siRNAs by plant DICER-LIKE (DCL) endonucleases. These innovative methods may have great impact in pest management against chewing and/or xylem sap-feeding vectors and eukaryotic pathogens that reside in the xylem.

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.

Crop loss due to viral diseases is still a major problem for agriculture today. We present a strategy to achieve virus resistance based on the expression of single-chain Fv fragments (scFvs) against a conserved domain in a plant viral RNA-dependent RNA polymerase (RdRp), a key enzyme in virus replication. The selected scFvs inhibited complementary RNA synthesis of different plant virus RdRps in vitro and virus replication in planta. Moreover, the scFvs also bound to the RdRp of the distantly related hepatitis C virus. T(1) and T(2) progeny of transgenic lines of Nicotiana benthamiana expressing different scFvs either in the cytosol or in the endoplasmic reticulum showed varying degrees of resistance against four plant viruses from different genera, three of which belong to the Tombusviridae family. Virus resistance based on antibodies to RdRps adds another tool to the repertoire for combating plant viruses.