Immunocapture of dsRNA-bound proteins provides insight into tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector

Abstract

27 Plant RNA viruses form highly organized membrane-bound virus replication complexes 28 (VRCs) to replicate their genome and multiply. VRCs contain both virus-and host-encoded 29 proteins, and have been investigated through a number of experimental approaches, including 30 genetic screens on surrogate model experimental hosts and pull-down of viral replicase 31 proteins. Double-stranded RNA (dsRNA) is an obligate intermediate of the replication 32 process of all RNA viruses and is the trigger of potent antiviral defenses in all eukaryotes. In 33 this work, we describe the use of a GFP-tagged dsRNA-binding protein (B2:GFP) to pull 34 down viral replicating RNA and associated proteins in planta. Following infection of A.35 thaliana constitutively expressing B2:GFP with tobacco rattle virus (TRV), we were able 36 through GFP immunoprecipitation to pull down dsRNA. Mass spectrometry analysis of the 37 dsRNA-B2:GFP-bound proteins from TRV-infected plants revealed the presence of (i) viral 38 proteins such as the replicase, which attested to the successful isolation of VRCs, and (ii) a 39 number of host proteins, some of which have previously been involved in virus infection. To 40 verify whether the identified host proteins localized at TRV replication complexes, we 41 selected nine candidates, generated genetic fusions with RFP, and transiently expressed them 42 in healthy and TRV-infected N. benthamiana stably expressing B2:GFP. Confocal 43 microscopy analysis revealed that eight out of nine candidates showed dramatic re-44 localization upon infection, and seven of these co-localized with B2-labeled TRV replication 45 complexes, providing ample validation for the immunoprecipitation results. We therefore 46 propose B2:GFP-mediated pull down of dsRNA to be a novel and robust method to explore 47 the proteome of VRCs in planta. 48 49 50 51 AUTHOR SUMMARY 52 Viruses are an important class of pathogens that represent a major problem for human, animal 53 and plant health. They hijack the molecular machinery of host cells to complete their 54 replication cycle, a process frequently associated with the production of double-stranded 55 RNA (dsRNA) that is regarded as a universal hallmark of infection by RNA viruses. Here we 56 exploited the capacity of a GFP-tagged dsRNA-binding protein stably expressed in transgenic 57 Arabidopsis to pull down dsRNA and associated proteins upon virus infection. In this manner 58 we specifically captured short and long dsRNA from tobacco rattle virus (TRV) infected 59 plants, and successfully isolated viral proteins such as the replicase, which attested to the 60 successful isolation of virus replication complexes (VRCs). More excitingly, a number of 61 host proteins, some of which have previously been involved in virus infection, were also 62 captured. Remarkably, among a set of nine host candidates that were analyzed, eight showed 63 dramatic re-localization to viral factories upon infection, and seven of these co-localized 64 dsRNA-labeled VRCs. Being compatible with any plant virus as ...

Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana

“…To test if TBSV-BS3Ng replication is also associated to the production of spherules, we first infected N. benthamiana constitutively expressing the dsRNA-binding sensor protein B2:GFP [ 47 ]. Confocal and electron microscopy analyses of these plants confirmed that TBSV-BS3Ng replicates in association with severely modified peroxisomes containing numerous spherules ( Supplementary Figure S3 and [ 46 ]). Confocal and electron microscopy analyses of constitutively-expressing B2:GFP A. thaliana Col-0 plants infected with TBSV-BS3Ng ( Figure 2 ) revealed similar cytopathic structures than those observed in N. benthamiana ( Supplementary Figure S3 ).…”

“…All A. thaliana lines used were previously described: drb2-1 , drb4-1 and drb2-1/drb4-1 [ 42 ]; dcl2-1 , dcl4-2 and dcl2-1/dcl4-2 [ 43 , 44 ]; ago2-1 [ 45 ] and ago1-27 [ 45 ]. The 35S: B2:GFP /Col-0 line was also previously described [ 46 ], as was 35S: B2:GFP / N. benthamiana [ 47 ]. A. thaliana were grown in a neon-lit chamber at 22–18 °C, 12 h/12 h light/dark photoperiod throughout the infection experiments, while N. benthamiana plants were grown in a greenhouse at 22–18 °C, 16 h/8 h light/dark photoperiod.…”

“…After a few minutes, leaves were rinsed with water. The pEAQ-ΔP19 plasmids expressing 35S:tRFP , 35S:DRB2:tRFP , 35S:DRB4:tRFP and 35S:tRFP:NFD2 were previously described [ 46 ]. For overexpression and TBSV infection experiments, leaves of 5–6 week-old 35S: B2:GFP/N.…”

“…Immunoprecipitation procedures were carried out as in [ 46 , 54 ]. Rosette leaves (0.15 g) were ground in liquid nitrogen, homogenized in a mortar with 1 mL lysis buffer (50 mM Tris–HCl, pH 8, 50 mM NaCl and 1% Triton X-100) containing a protease inhibitor (Roche, Basel, Switzerland), transferred to a tube, incubated for 15 min at 4 °C on a wheel with slow rotation.…”

“…Mass spectrometry procedures were carried out as in [ 46 ]. Proteins were digested with sequencing-grade trypsin (Promega) and analyzed by nanoLC-MS/MS on a TripleTOF 5600 mass spectrometer (Sciex, Framingham, MA, USA), as previously described [ 55 ].…”