Abstract
RNA silencing is a powerful tool deployed by plants against viral infection and abnormal gene expression. Plant viruses have evolved a suite of silencing suppressors for counter-defense, which are also widely used to boost transcript and protein accumulation in transient assays. However, only wild type silencing suppressor proteins have been reported to date. Here we demonstrate that P0 of Potato leafroll virus (PLRV), PLP0, can be split into two proteins that only show silencing suppression activity upon co-expression. We cloned each of these proteins in two different constructs and transiently co-infiltrated them in N. benthamiana leaves. We expressed a fluorescent protein from one of the vectors and observed that cells expressing both halves of PLP0 suppressed gene silencing. Further, we showed that Q system of Neurospora crassa, based on co-expression of a transcription activator and inhibitor, is functional in agroinfiltrated leaves of N. benthamiana. Q system combined with the split PLP0 system showed very tight co-expression of Q system’s transcriptional activator and inhibitor. Altogether, our experiments demonstrate a functioning conditional silencing suppressor system and its potential as a powerful tool for transient expression in N. benthamiana leaves, as well as the application of the Q system in plants.
Highlights
Most plant viruses have RNA genomes and, as they replicate, long double-stranded RNAs are produced and generically recognized by the host’s endonucleases, Dicer-like (DCL) enzymes
We further show that the binary system of Neurospora crassa, Q system, is functional in N. benthamiana agroinfiltrated leaves and, when combined with the conditional silencing suppressor PLP0 system, results in very tight co-expression of each of its binary components for transcriptional regulation
Consistent with a previous report showing that the F-box domain of P0 from Cucurbit aphid-borne yellows virus is located within its N-terminus[5], the PLP0-A sequence was found to be highly conserved among Potato leafroll virus (PLRV) isolates (Fig. 1A and Table S1)
Summary
Most plant viruses have RNA genomes and, as they replicate, long double-stranded RNAs (dsRNA) are produced and generically recognized by the host’s endonucleases, Dicer-like (DCL) enzymes. DCLs catalyze the formation of siRNAs that, upon loading into Argonaute (AGO) proteins, promote sequence-specific antiviral defense through RNA silencing[1]. P19 suppresses RNA silencing, thereby aiding higher levels of transgenic-encoded proteins, by sequestering siRNAs in a manner that is neither sequence-specific nor organism-dependent[2] This property of effectively preventing siRNAs loading into AGO proteins has made P19 an essential component in most transient assays, such as those using Nicotiana benthamiana leaves for Agrobacterium infiltration[3]. We observe that PLP0 can be split into two functional proteins that are ineffective as silencing suppressors on their own, but upon co-expression show activity similar to that of PLP0 full-length protein We applied this system to co-infiltration assays in N. benthamiana leaves by inserting each half of PLP0 into two separate vectors. This work presents a conditional silencing suppressor system as a powerful tool for transient co-expression assays in N. benthamiana leaves, and demonstrates the feasibility of the Q system in plants
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