A Genetic Network for Systemic RNA Silencing in …...A Genetic Network for Systemic RNA Silencing...

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Short Title: A Network for Systemic RNA Silencing in plants 1

Corresponding Author: Yiguo Hong 2

Research Centre for Plant RNA Signaling, College of Life and Environmental Sciences, 3

Hangzhou Normal University, Hangzhou 310036, China; Warwick-Hangzhou RNA Signaling 4

Joint Laboratory, School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK 5

and Worcester-Hangzhou Joint Molecular Plant Health Laboratory, Institute of Science and 6

the Environment, University of Worcester, WR2 6AJ, UK. 7

Telephone: +86-571-28866065 8

Fax: +86-571-28866065 9

E-mails: [email protected]; [email protected]; [email protected] 10

Primary Research Area: Systemic Transportation/Cell Biology 11

2nd

Research Area: Small RNAs in Systemic PTGS/Signaling and Response 12

Plant Physiology Preview. Published on February 8, 2018, as DOI:10.1104/pp.17.01828

Copyright 2018 by the American Society of Plant Biologists

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A Genetic Network for Systemic RNA Silencing in Plants1 13

Weiwei Chen2, Xian Zhang

2, Yaya Fan

2, Bin Li

2, Eugene Ryabov

2, Nongnong Shi, Mei Zhao, 14

Zhiming Yu, Cheng Qin, Qianqian Zheng, Pengcheng Zhang, Huizhong Wang, Stephen 15

Jackson, Qi Cheng, Yule Liu, Philippe Gallusci, Yiguo Hong

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Research Centre for Plant RNA Signaling, College of Life and Environmental Sciences, 17

Hangzhou Normal University, Hangzhou 310036, China (W.C., X.Z., Y.F., B.L., E.R., N.S., 18

M.Z., Y.Z., C.Q., Q.Z., P.Z., H.W., Y.H.); Warwick-Hangzhou RNA Signaling Joint 19

Laboratory, School of Life Sciences, University of Warwick, Warwick CV4 7AL, UK (E.R., 20

S.J., Y.H.); Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 21

Beijing 100081, China (Q.C.); Centre for Plant Biology and MOE Key Laboratory of 22

Bioinformatics, School of Life Sciences, Tsinghua University, Beijing 100084, China (Y.L.); 23

UMR EGFV, Bordeaux Sciences Agro, INRA, Université de Bordeaux, 210 Chemin de 24

Leysotte, CS 50008, 33882 Villenave d’Ornon, France (P.G.); Worcester-Hangzhou Joint 25

Molecular Plant Health Laboratory, Institute of Science and the Environment, University of 26

Worcester, WR2 6AJ, UK (Y.H.) 27

1Funding: This work was supported by grants from National Natural Science Foundation of 28

China (NSFC 31370180 to Y.H.); Ministry of Agriculture of the People’s Republic of China 29

(the National Transgenic Program of China 2016ZX08009001-004 to Y.H.); Hangzhou 30

Normal University (Pandeng Program 201108 to Y.H.); the Hangzhou City Government 31

(Innovative Program for Science Excellence 20131028 to Y.H.); NSFC (31601765 to W.C., 32

31201490 to X.Z., 31500251 to C.Q.); Zhejiang Provincial Natural Science Foundation 33

(LY15C140006 to X.Z., LY14C010005 to N.S.) and the UK Biotechnology and Biological 34

Sciences Research Council (UK-China Partnering Award BB/K021079/1 to S.J. and Y.H.). 35

2These authors contributed equally to this work. 36

Author Contributions: W.C., X.Z., Y.F., B.L. and E.R. designed, performed experiments 37

and analysed data; N.S., M.Z., Y.Z., C.Q., Q.Z. and P.Z. performed experiments; H.W., S.J., 38

Q.C., Y.L. and P.G. were involved in the analysis of data and helped writing the paper. Y.H. 39

initiated the project, designed experiments, analysed data and wrote the paper. 40

One-sentence Summary: A DCL2-dependent DCL genetic pathway is crucial for systemic 41

PTGS in plants. 42



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*Address correspondence to [email protected], [email protected] or 43

[email protected]. The author responsible for distribution of materials integral to the 44

findings presented in this article in accordance with the policy described in the instructions 45

for Authors (www.plantphysioll.org) is: Yiguo Hong ([email protected], 46

[email protected] and [email protected]).47









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ABSTRACT 48

Non-cell autonomous RNA silencing can spread from cell to cell and over long-distances in 49

animals and plants. However, the genetic requirements and signals involved in plant mobile 50

gene silencing are poorly understood. Here, we identified a DICER-LIKE2 (DCL2)-51

dependent mechanism for systemic spread of post-transcriptional RNA silencing, also known 52

as post-transcriptional gene silencing (PTGS), in Nicotiana benthamiana. Using a suite of 53

transgenic DCL RNAi lines coupled with a GFP reporter, we demonstrated that N. 54

benthamiana DCL1, DCL2, DCL3, and DCL4 are required to produce microRNAs and 22, 55

24, and 21nt small interfering RNAs (siRNAs), respectively. All investigated siRNAs 56

produced in local incipient cells were present at low levels in distal tissues. Inhibition of 57

DCL2 expression reduced the spread of gene silencing, while suppression of DCL3 or DCL4 58

expression enhanced systemic PTGS. In contrast to DCL4 RNAi lines, DCL2-DCL4 double-59

RNAi lines developed systemic PTGS similar to that observed in DCL2 RNAi. We further 60

showed that the 21 or 24 nt local siRNAs produced by DCL4 or DCL3 were not involved in 61

long-distance gene silencing. Grafting experiments demonstrated that DCL2 was required in 62

the scion to respond to the signal, but not in the rootstock to produce/send the signal. These 63

results suggest a coordinated DCL genetic pathway in which DCL2 plays an essential role in 64

systemic PTGS in N. benthamiana, while both DCL4 and DCL3 attenuate systemic PTGS. 65

We discuss the potential role of 21, 22 and 24 nt siRNAs in systemic PTGS. 66

Keywords: DICER-LIKEs; mobile silencing signal; Nicotiana benthamiana; siRNAs; 67

systemic PTGS 68

INTRODUCTION 69

RNA silencing is a cellular gene regulatory mechanism that is conserved across fungal, plant, 70

and animal kingdoms (Baulcombe, 2004; Sarkies and Miska, 2014). Through sequence-71

specific targeting, RNA silencing can degrade mRNA for post-transcriptional gene silencing 72

(PTGS) or modify related DNA for transcriptional gene silencing (TGS). In plants, primary 73

silencing is triggered by dsRNA molecules, which are processed into 21–24 nucleotide (nt) 74

small-interfering (si) RNA duplexes, known as primary siRNAs, by DICER-LIKE (DCL) 75

ribonuclease III-like enzymes (Baulcombe, 2004; Sarkies and Miska, 2014). For instance, 76

dsRNA can be directly produced from a hairpin transgene in primary silencing (Mlotshwa et 77

al., 2008). Primary silencing differs from transitive silencing, in which the trigger is initially 78



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not itself dsRNA. In contrast to primary silencing, the dsRNA in transitive silencing is 79

produced from a single-stranded (ss) RNA template, and requires the coordinated activity of a 80

set of genes. These genes include DICER-LIKE 2 (DCL2), RNA dependent RNA polymerase 81

6 (RDR6), and SGS3, the latter of which encodes a coiled-coiled domain protein (Mlotshwa et 82

al., 2008; Parent et al., 2015). Transitive silencing cascades and amplifies cell and non-cell 83

autonomous silencing to other parts of the target RNA, leading to generation of secondary 84

siRNAs. It is well known that DCL2 plays an essential role in cell-autonomous silencing 85

transitivity and accumulation of secondary siRNAs in Arabidopsis (Arabidopsis thaliana) 86

(Mlotshwa et al., 2008; Parent et al., 2015). 87

In animals and plants, TGS and PTGS can spread from cell to cell, systemically, or 88

even between different organisms (Weiberg et al., 2013). Intercellular and long-distance 89

movement of non-cell autonomous RNA silencing involves mobile signals and various 90

genetic components (Melnyk et al., 2011b; Molnar et al., 2010). For example, SNF2, a JmjC 91

domain protein JMJ14, the THO/TREX mRNA export complex, and RDR6 are all associated 92

with intercellular RNA silencing (Qin et al., 2012; Searle et al., 2010; Smith et al., 2007; 93

Yelina et al., 2010). Moreover, RDR6 also contributes to initial signal perception for 94

systemic PTGS (Melnyk et al., 2011b). Intriguingly, NRPD1a, encoding RNA polymerase 95

IVa, RDR2, and DCL3, which all function- in a chromatin silencing pathway, are required for 96

the reception of long-distance mRNA PTGS, and Argonaute4 is also partially involved in the 97

reception of such long-distance silencing. DCL4 and DCL2 then act hierarchically, as they do 98

in antiviral resistance, to produce 21 and 22 nt siRNAs, respectively, which guide mRNA 99

degradation in systemic tissues (Brosana et al., 2007). 100

Arabidopsis, and most likely other plant species, possess four DCL RNase III family 101

enzymes for small RNA (sRNA) biogenesis. DCL1 is involved in microRNA (miRNA) 102

production and DCL2, DCL3, and DCL4 are involved in 22, 24, and 21 nt small interfering 103

RNA (siRNA) production, respectively (Henderson et al., 2006; Mukherjee et al., 2013; Xie 104

et al., 2004). DCL1-generated miRNAs can move and act as local and distal signals that play 105

essential roles in plant growth and development, although direct evidence for mobile miRNA 106

signalling is lacking (Carlsbecker et al., 2010; Pant et al., 2008; Skopelitis et al., 2017). The 107

DCL3-processed 24 nt siRNA has been shown to direct systemic TGS that controls genome-108

wide DNA methylation in recipient cells through RNA-directed DNA methylation (RdDM) 109

of homologous genomic DNA sequences (Henderson et al., 2006; Lewsey et al., 2016; 110

Melnyk et al., 2011a; Molnar et al., 2010; Sarkies and Miska, 2014). However, systemic 111

silencing has also been reported to occur in the absence of sRNAs (Mallory et al., 2001) and 112



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no specific siRNAs produced by any of the four DCLs were found to be associated with 113

systemic silencing (Brosnan et al., 2007). Moreover, even in Arabidopsis, different genetic 114

factors required for mobile PTGS have been discovered from mutant screens in very similar 115

experimental systems (Melnyk et al., 2011b). Thus, the precise nature of long-distance 116

mobile signal remains to be elucidated, as this signal is not exclusively produced by any of 117

the four DCLs. Crucial molecular events during systemic RNA silencing reception were 118

elegantly investigated in Arabidopsis (Brosnan et al., 2007). Nevertheless, these findings 119

contrast with systemic silencing, known as RNA interference (RNAi) in animals, where the 120

mobile signal is dsRNA (Jose et al., 2011). In Caenorhabditis elegans, systemic RNAi 121

involves inter-tissue transfer and entry of gene-specific dsRNAs into the cytosol by the 122

dsRNA-selective importer SID-1 (Winston et al., 2002; Feinberg and Hunter, 2003; Shih and 123

Hunter, 2011). Consequently, mobile dsRNAs expressed in a variety of somatic tissues can 124

lead to SID-1-dependent homologous RNAi of target mRNA in other somatic tissues, or even 125

transgenerational silencing (Jose et al., 2009; Devanapally et al., 2015). 126

The intricate functions of DCL2 and DCL4 are partially redundant in trans-acting 127

siRNA biogenesis and in plant antiviral defence. However, DCL2 is responsible for 22 nt 128

siRNA biosynthesis and has different roles to DCL4 in the production of primary and 129

secondary siRNAs (Chen et al., 2010; Henderson et al., 2006; Qu et al., 2008; Wang et al., 130

2011; Xie et al., 2005). DCL4-processed 21nt siRNA is mainly involved in intracellular 131

PTGS as well as represents the signalling molecule that moves from leaf companion cells to 132

adjacent cells to induce limited intercellular PTGS in Arabidopsis (Dunoyer et al., 2005). 133

Interestingly, DCL2 expression in leaf vascular tissues enhances PTGS in surrounding cells 134

in Arabidopsis. DCL2 is thought to promote the production of 22 nt siRNAs that then 135

stimulate 21 nt siRNA biogenesis via RDR6 and DCL4, resulting in increased cell-to-cell 136

spread of PTGS (Parent et al., 2015). By contrast, DCL4 inhibits the cell-to-cell spread of 137

virus-induced gene silencing (VIGS), a form of PTGS, while DCL2, likely along with a 138

DCL2-dependent RNA signal, is required for efficient trafficking of VIGS from epidermal to 139

adjacent cells (Qin et al., 2017). However, the roles of 21 and 24 nt siRNAs and other types 140

of RNA molecules in mobile PTGS and TGS are under debate (Mallory et al., 2001; Brosnan 141

et al., 2007; Melnyk et al., 2011b; Sarkies and Miska, 2014) and remain highly controversial 142

(Berg, 2016). 143

On the other hand, impairment of DCL2 or DCL4 can reduce or promote cell-144

autonomous PTGS, and DCL2 promotes intracellular transitive silencing in Arabidopsis 145

(Mlotshwa et al., 2008; Parent et al., 2015). Moreover, two different strong viral suppressors 146



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of silencing (Turnip crinkle virus P38 and Turnip mosaic virus HC-Pro) block the 147

accumulation of secondary siRNAs produced by transitive silencing (Mlotshwa et al., 2008). 148

Thus, DCL2 may have in a role in viral defense that can be coupled to intercellular and 149

systemic PTGS. This view is indeed supported by the requirement of DCL2 and DCL2-150

dependent mobile signals for intercellular antiviral silencing in N. benthamiana (Qin et al., 151

2017). A genetic screen for impaired systemic RNAi revealed that DCL2 is crucial for RDR6-152

dependent systemic PTGS (Taochy et al., 2017). Nevertheless, it is not known whether, and 153

to what extent, DCL2 and other DCLs regulate systemic silencing. However, DCL2 and its 154

cognate 22 nt siRNA are clearly able to affect secondary siRNA biogenesis, antiviral defence, 155

and plant development (Bouche et al., 2006; Chen 2010; Garcia-Ruiz et al., 2010; Qin et al., 156

2017; Wang et al., 2011). 157

In this article, we report the genetic requirement of a DCL network and the 158

involvement of mobile siRNAs in the systemic spread of PTGS in N. benthamiana. 159

RESULTS 160

Genetic Resources for Examining Mobile RNA Silencing in N. benthamiana 161

To investigate systemic RNA silencing in N. benthamiana, we used gene-specific RNAi to 162

inhibit the four DCLs orthologous to Arabidopsis DCL1, DCL2, DCL3 and DCL4 163

(Supplemental Data Set S1). Specific genes or gene fragments were PCR-amplified and 164

cloned into the overexpression vector pCAMBIA1300 to produce p35S-GFP714 (the GFP 165

coding sequence of this variant is 714 nt long; Ryabov et al., 2004) or the hairpin RNAi 166

vector pRNAi-LIC (Xu et al., 2010) to generate pRNAi-GFP714 and pRNAi-DCLs 167

(Supplemental Fig. S1). These cloned DCL gene fragments shared no detectable nucleotide 168

sequence similarity (Supplemental Table S1). Plants were then transformed with each of the 169

pRNAi-DCL constructs. We selected two single-copy homozygous RNAi lines DCL2Ai and 170

DCL2Bi for DCL2; DCL3Ai and DCL3Bi for DCL3; DCL4Ai and DCL4Bi for DCL4, and 171

produced a double RNAi line DCL24i through crossing DCL2Ai with DCL4Bi (Table 1). We 172

crossed all of the individual DCL RNAi lines with the 16cGFP plants bearing a single copy 173

of a GFP transgene (referred to as GFP792, as the coding region of this gene variant is 792 nt 174

long) under the control of the 35S promoter (Haseloff et al., 1997; Ruiz et al., 1998) to create 175

the hybrid lines Gfp, GfpDCL2Ai, GfpDCL2Bi, GfpDCL3Ai, GfpDCL3Bi, GfpDCL4Ai, 176

GfpDCL4Bi, GfpDCL24Ai, and GfpDCL24Bi (Table 1). Through selfing, we generated 177

homozygous lines homGfpDCL2Ai, homGfpDCL3Bi, and homGfpDCL4Ai. An additional 178



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double DCL2-DCL4 RNAi line GfpDCL24Ci was obtained through crossing homGfpDCL2Ai 179

and homGfpDCL4Ai (Table 1). However, we only obtained one hemizygous DCL1i or 180

GfpDCL1i line by transforming N. benthamiana or 16cGFP with pRNAi-DCL1, as 181

homozygous DCL1 RNAi was lethal (Table 1). Lines RDR6i and GfpRDR6i were included in 182

this work (Schwach et al., 2005). To confirm the inhibition caused by RNAi, we performed 183

RT-qPCR and analyzed DCL mRNA levels in the RNAi lines. Gene-specific RNAi 184

repression of DCL2, DCL3 and DCL4 was between 70–90%; and approximately 45% 185

suppression was achieved for DCL1 (Supplemental Fig. S2), consistent with our previous 186

analyses (Qin et al., 2017). 187

DCLs Function Differentially to Generate Small RNAs in N. benthamiana 188

To determine whether individual DCLs function differently to generate 21, 22, and 24 nt 189

siRNAs in N. benthamiana (Fig. 1), we used a GFP reporter system and performed a local 190

PTGS assay (Ruiz et al., 1998; Ryabov et al., 2004). Leaves of the DCL RNAi plants at the 191

six-leaf stage were co-infiltrated with Agrobacterium harbouring p35-GFP714 and 192

Agrobacterium harbouring pRNAi-GFP714 or an empty pRNAi-LIC vector (Supplemental Fig. 193

S1; Supplemental Fig. S3A). Compared to the non-RNAi wild-type plant (Fig. 1A), RNAi of 194

DCL1 (DCL1i; Fig. 1B), DCL2 (DCL2Ai and DCL2Bi; Fig. 1C and D), and DCL3 (DCL3Ai 195

and DCL3Bi; Fig. 1E and F) did not affect local RNA silencing, but RNAi of DCL4 196

(DCL4Ai, DCL4Bi and DCL24i; Fig. 1G-I) weakened local PTGS. This is evident by almost 197

complete (Fig. 1A-F, left panel) or partial reduction (Fig. 1G-I, left panel) in GFP 198

fluorescence intensity in the co-agroinfiltrated lamina (Supplemental Fig. S4). Consistent 199

with the level of RNAi achieved, only a low level of GFP714 mRNA was detected in wild-200

type, DCL2Ai, DCL2Bi, DCL3Ai and DCL3Bi plants, whereas a relatively high level of 201

GFP714 mRNA was found in DCL4Ai, DCL4Bi and DCL24i plants (Fig. 1J). We also noticed 202

that the GFP714 mRNA levels were slightly higher in DCL1i plants (Fig. 1J). 203

We then used northern hybridizations to characterize siRNAs present in the infiltrated 204

leaf tissues of different DCL RNAi lines (Fig. 1K). These local siRNAs are hereafter 205

designated L-siRNAs. L-siRNAs include primary siRNAs that are directly generated from the 206

hairpin GFP714 dsRNA, and secondary siRNAs that are generated from the p35S-GFP714 207

expressed GFP714 mRNA. There is some variation in the levels of siRNAs detected in the two 208

independent RNAi lines with respect to a given DCL gene. Nevertheless 21, 22, and 24 nt L-209

siRNAs were readily detectable in the control and DCL1i plants. This contrasted with the 210

specific loss of 22 nt L-siRNAs in DCL2Ai and DCL2Bi, 24 nt L-siRNAs in DCL3Ai and 211



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DCL3Bi, and 21 nt L-siRNAs in DCL4Ai and DCL4Bi (Fig. 1K). We also noticed a large 212

amount of 22 and 24 nt L-siRNAs in DCL4Ai and DCL4Ai, and a high level of 24 nt L-213

siRNAs in DCL2Ai and DCL2Bi. However, 21 and 22 nt L-siRNAs were undetectable in the 214

DCL24i plants (Fig. 1K). These data reveal that in N. benthamiana, as in Arabidopsis, DCL4, 215

DCL2, and DCL3 are required to produce 21, 22, and 24 nt siRNAs, respectively. 216

RNAseq analysis confirms DCLs for Biosynthesis of Small RNAs in N. benthamiana 217

To further investigate how DCL RNAi affects sRNA synthesis, we collected agro-infiltrated 218

leaf tissues from N. benthamiana (Nb), DCL1i, DCL2Ai, DCL2Bi, DCL3Ai, DCL3Bi, 219

DCL4Ai, DCL4Bi, and RDR6i plants at 7 days post agro-infiltration (dpa). The leaves were 220

co-infiltrated with agrobacterium harboring p35S-GFP714 and agrobacterium harboring 221

pRNAi-GFP714 (Fig. 2; Supplemental Fig. S3A). We then constructed 9 individual local 222

sRNA libraries for NGS and generated approximately 11 million sRNA reads for each library 223

(Supplemental Table S2). We found that (i) gene-specific RNAi of DCLs or RDR6 had no 224

off-target effect on other silencing-related genes (Supplemental Fig. S5); (ii) correlation 225

analyses of total miRNAs among these sRNA libraries validated the authenticity and direct 226

comparability of the detected sRNA profiles (Supplemental Table S3 and S4); and (iii) DCL1 227

RNAi resulted in a clear reduction of miRNAs (Supplemental Fig. S6; Supplemental Table 228

S2). Compared to Nb, DCL1i and RDR6i (Fig. 2A, B and I, left panel), decreased levels in 21, 229

22, and 24 nt GFP714 L-siRNAs and total sRNAs correlated with RNAi of DCL4, DCL2, and 230

DCL3 (Fig. 2C-H, left panel; Supplemental Fig. S3B-J). These data are consistent with the 231

sRNA northern detection (Fig. 1K) and further show that as in Arabidopsis, DCL1 is also 232

required for miRNA biosynthesis in N. benthamiana. 233

Detection of L-siRNAs in Distal Systemic Leaves 234

To investigate whether GFP714 L-siRNAs generated in local leaves are able to move 235

systemically to distant tissues (Fig. 3), we collected a pool of young systemic leaves from the 236

agro-infiltrated DCL RNAi or control plants at 14 dpa and constructed 9 systemic leaf sRNA 237

libraries for NGS (Supplemental Fig. S3A). Similar to the local sRNA libraries made from 238

the infiltrated leaf tissues, we generated approximately 11 million sRNA reads for each of the 239

systemic libraries and DCL-specific RNAi was again found to have little off-target effect on 240

other silencing-related genes in the systemic leaves (Supplemental Table S2; Supplemental 241

Fig. S5). We then scrutinized the systemic leaf sRNA datasets for reads matching the GFP714 242

mRNA. Different levels of both sense and antisense 21, 22, and 24nt GFP714 L-siRNAs were 243



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detected in systemic leaves of Nb, RDR6i, and DCL RNAi plants (Fig. 2A-I, right panel), 244

although the total number of GFP714 L-siRNA (and total sRNA) reads in the infiltrated leaves 245

was similar among all samples (Fig. 2A-I, left panel; Supplemental Table S2). We detected 246

high levels of 22 nt L-siRNAs, some 21 nt L-siRNAs, and a low level of 24 nt L-siRNAs in 247

systemic Nb leaves (Fig. 2A, right panel). However, in the young leaves of DCL1i, DCL2Ai, 248

DCL2Bi, and RDR6i plants (Fig. 2B-D and I, right panel), the number of 22 or 21 nt L-249

siRNA reads was 5-8 times lower than that in Nb. In systemic leaves of DCL3Ai and DCL3Bi 250

as well as those of DCL4Ai and DCL4Bi, only very low levels of 22 or 21 nt L-siRNAs were 251

identified (Fig. 2E-H, right panel), 45-68 fold lower than in Nb. The distribution across the 252

GFP714 mRNA of both sense and antisense 21-24 nt L-siRNAs detected in local or systemic 253

leaf tissues was almost identical among all samples (Fig. 3, left vs right panels A-I). However, 254

we failed to detect any full-length or partial GFP714 RNA in systemic young leaves in any of 255

the agro-infiltrated plants by RT-PCR using GFP714-specific primers (Supplemental Fig. S7; 256

Supplemental Table S5). 257

DCL2 Facilitates, while DCL4 and DCL3 Attenuate, Systemic PTGS 258

To analyze the impact of DCL RNAi on systemic PTGS (Fig. 4), we infiltrated leaves of Gfp 259

(resulting from a cross between Nb and 16cGFP), GfpDCL1i, GfpDCL2Ai, GfpDCL2Bi, 260

GfpDCL3Ai, GfpDCL3Bi, GfpDCL4Ai or GfpDCL4Bi plants at the six-leaf stage with 261

Agrobacterium harbouring pRNAi-GFP714. It is important to note that local PTGS is induced 262

by the hairpin GFP714 that directly produces long dsRNA in the agro-infiltrated leaves. 263

However, sense transgene GFP792 silencing in local and systemic young leaves requires the 264

activity of multiple genes, including RDR6, to produce dsRNA from GFP792 ssRNA 265

(Mlotshwa et al., 2008). Under long-wavelength UV light, we observed red fluorescence in 266

halos around the infiltrated areas on Gfp and RNAi line leaves at 7 dpa (Fig. 4A; 267

Supplemental Table S6), indicating local silencing. 268

DCL1 RNAi had no obvious effect on systemic silencing in GfpDCL1i; however, 269

DCL2 RNAi resulted in no PTGS in systemic young leaves of GfpDCL2Ai and GfpDCL2Bi 270

(Fig. 4B; Supplemental Fig. S8; Supplemental Table S6). This contrasts with enhancement of 271

systemic PTGS in GfpDCL4Ai and GfpDCL4Bi or GfpDCL3Ai and GfpDCL3Bi (Fig. 4B; 272

Supplemental Fig. S8; Supplemental Table S6). Systemic silencing was further illustrated in 273

single young leaves (Fig. 4C-G). In contrast to the positive control (Fig. 4D), systemic PTGS 274

was completely absent in the young leaves of GfpDCL2Ai and GfpDCL2Bi, as in Gfp (Fig. 275

4C). Only limited silencing was observed in one leaf located immediately above the 276



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infiltrated leaves at 18 dpa or later and such limited systemic PTGS was restricted to minor 277

veins of DCL2 RNAi plants (Fig. 4E). However, strong systemic silencing appeared in young 278

leaves of DCL3 and DCL4 RNAi lines (Fig. 4F and G). Similar results of DCL RNAi on local 279

or systemic silencing were observed in 16cGFP, homGfpDCL2Ai, homGfpDCL3Bi and 280

homGfpDCL4Ai plants as in Gfp and GfpDCL RNAi lines (Supplemental Table S6). 281

We then used northern hybridizations to detect systemic siRNA (dubbed Sy-siRNAs; 282

Fig. 4H). Sy-siRNAs are siRNAs resulting from transitive silencing of the sense transgene 283

GFP792 mRNA in non-agroinfiltrated leaves. Such systemic transitive silencing was triggered 284

by mobile signals that were generated in agro-infiltrated local leaves. Sy-siRNAs also include 285

two types of secondary siRNAs generated either from the GFP792 mRNA sequences that were 286

directly targeted by the mobile signals or from the other parts of the GFP792 mRNA 287

sequences that were not directly targeted by the mobile signals. No 21, 22, and 24 nt Sy-288

siRNAs were detected in the young leaves of GfpDCL2Ai and GfpDCL2Bi by northern blot 289

(Fig. 4H). We were also unable to detect 24 nt Sy-siRNA in GfpDCL3Ai and GfpDCL3Bi, in 290

which systemic silencing was apparently enhanced (Fig. 4B, F and H; Supplemental Fig. S8). 291

However, 21, 22, and 24 nt Sy-siRNAs accumulated to a higher level in systemic leaves of 292

GfpDCL4Ai and GfpDCL4Bi plants compared to Gfp (Fig. 4H). Considering the differential 293

functions of DCL4 and DCL2 in primary and secondary siRNA biosynthesis (Chen et al., 294

2010; Cuperus et al., 2010), and the up-regulated DCL2 expression in the DCL4 or DCL3 295

RNAi lines (Qin et al., 2017), we suspect that these Sy-siRNAs are secondary siRNAs 296

generated from the sense-transgene GFP792 mRNA. Taken together, our data demonstrate that 297

DCL2 facilitates, whilst DCL4 and DCL3 attenuate, systemic PTGS, at least for the 298

transgene-induced PTGS in N. benthamiana. 299

Genetic Interplay between DCL4 and DCL2 to Influence Systemic PTGS 300

To investigate a potential genetic link between DCL2 and DCL4 in systemic PTGS (Fig. 4I 301

and L), we infiltrated leaves of the triple-cross GfpDCL24Ai and GfpDCL24Bi (Table 1) with 302

Agrobacterium harbouring pRNAi-GFP714 at the six-leaf stage. Local RNA silencing was 303

sufficiently induced, as evidenced by the appearance of red halos around the agro-infiltrated 304

lamina (Fig. 4J, i). No long-distance spread of PTGS occurred to reach systemic leaves, 305

which showed uniform green fluorescence under long-wavelength UV light (Fig. 4J, ii and iii; 306

Supplemental Table S6), although some minor vein-restricted silencing was observed in one 307

leaf immediately above the agro-infiltrated leaves (Fig. 4I). Similar systemic PTGS patterns 308

were also found in an additional DCL2-DCL4 double RNAi line GfpDCL24Ci (Table 1; 309



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Supplemental Table S6). These findings show that 16cGFP plants with simultaneous RNAi 310

against DCL2 and DCL4 develop systemic PTGS in a manner similar to GfpDCL2Ai and 311

GfpDCL2Bi lines, but in contrast to GfpDCL4Ai and GfpDCL4Bi lines. 312

Comparison of Hairpin GFP714 and Transgene GFP792 Sequences 313

As indicated in Fig. 5, sequences of transgene 792nt-GFP792 (Haseloff et al., 1997; Ruiz et al., 314

1998) in all transgenic Gfp and GfpDCL RNAi lines and hairpin 714nt-GFP714 (Ryabov et al., 315

2004) in the pRNAi-GFP714 vector share three identical and two less-similar regions, the 316

latter named Region 1 and Region 2, respectively. GFP792 also possesses nucleotides 1-66 317

and 778-792 at both 5’- and 3’-ends that are completely absent from the GFP714 sequence. 318

Within Region 1 or Region 2, the maximum interval between any two consecutive 319

mismatched bases is 14 nt. This information is essential and relevant to our subsequent 320

siRNA profiling and bioinformatic analyses of siRNAs that may or may not be associated 321

with transitive silencing. We only counted 20-25 nt siRNAs that were perfectly (100%) 322

matched to target gene mRNAs. This means that the 20-25 nt siRNAs mapped to Region 323

1/Region 2 of the GFP792 mRNA will not be mapped to the equivalent Region 1/Region 2 of 324

the GFP714 mRNA, or vice versa. Thus, these siRNAs are exclusive to either GFP792 or 325

GFP714, and most importantly, any Sy-siRNA unique to the transgene GFP792 Region 326

1/Region 2 mRNA should be generated by transitive silencing in systemic tissues. 327

Mapping L-siRNAs and Sy-siRNAs to Transgene GFP792 mRNAs and to Hairpin 328

GFP714 dsRNA 329

To investigate whether L-siRNAs are associated with Sy-siRNA biosynthesis and systemic 330

PTGS in distal recipient tissues, we performed systemic transgene GFP792 silencing assays 331

(Supplemental Fig. S9A) and sequenced sRNA libraries generated from the infiltrated or 332

systemic leaves of Gfp, GfpDCL1i, GfpDCL2Ai, GfpDCL3Bi, GfpDCL4Ai, and GfpDCL24Ai 333

plants. Systemic transgene GFP792 silencing was triggered by mobile signals originating from 334

local PTGS that was initiated by the hairpin GFP714 dsRNA (Fig. 4; Supplemental Fig. S9B). 335

Similar to the sRNA results obtained from the non-GFP transformed RNAi lines, we 336

generated approximately 12-million sRNA reads for each library (Supplemental Table S7). 337

Interrogation of these sRNA datasets further confirmed DCL-specific RNAi and their roles in 338

siRNA biogenesis (Supplemental Fig. S9B-P; Supplemental Fig. S10). Moreover, RNAi of 339

DCL1 resulted in a clear reduction of miRNAs, although the total number of miRNAs was 340

generally higher in systemic leaves than in local infiltrated leaves (Supplemental Fig. S11). 341



13

We mapped L- and Sy-siRNAs onto the transgene GFP792 mRNA. The distribution of 342

antisense L- and Sy-siRNAs mapped to nucleotides 217-282 and 625-742 (Fig. 5, ©-labelled) 343

were essentially the same and sense L- and Sy-siRNAs mapped to nucleotides 363-402 and 344

730-773 of the transgene GFP792 mRNA (Fig. 5, ℗-labelled) in both local and systemic 345

leaves of Gfp (Fig. 6A and B), GfpDCL1Ai (Fig. 6C and D), GfpDCL2Ai (Fig. 6E-G), 346

GfpDCL3Bi (Fig. 6H and I), GfpDCL4Ai (Fig. 6J and K), and GfpDCL24Ai (Fig. 6L-N). The 347

L-siRNAs in these four clusters (Fig. 6A, C, E, H, J and L; asterisked) corresponded to the 348

occurrence of systemic GFP792 PTGS (Fig. 4; Supplemental Fig. S9B). We also found 349

relatively high numbers of Sy-siRNA reads in young leaves where strong systemic GFP792 350

PTGS occurred in Gfp, GfpDCL1i, GfpDCL3Bi, and GfpDCL4Ai (Fig. 6B, D, I and K). 351

However, between the Gfp control and each of GfpDCL RNAi lines, we observed distinct 352

profiles of L- and Sy-siRNAs across Region 1/Region 2 of the transgene GFP792 mRNA. Sy-353

siRNAs in young leaves specifically mapped to Region 1/Region 2 (Fig. 6B, D, F, G, I, K, M 354

and N) were absent in the agro-infiltrated leaves where local silencing occurred efficiently in 355

Gfp, GfpDCL1i, GfpDCL2Ai, GfpDCL3Bi, GfpDCL4Ai, and GfpDCL24Ai (Fig. 6A, C, E, H, 356

J and L). 357

We also mapped the L- and Sy-siRNAs across the GFP714 mRNA, which was only 358

expressed from pRNAi-GFP714 and formed hairpin dsRNA in the agro-infiltrated leaf lamina 359

of Gfp (Fig. 7A and B), GfpDCL1i (Fig. 7C and D), GfpDCL2Ai (Fig. 7E-G), GfpDCL3Bi 360

(Fig. 7H and I), GfpDCL4Ai (Fig. 7J and K), and GfpDCL24Ai (Fig. 7L-N). All four clusters 361

of siRNAs mapped to the transgene GFP792 mRNA nucleotides 217-282, 363-402, 625-742 362

and 730-773, which also mapped to the equivalent identical sequences of the GFP714 RNA. 363

However, L-siRNAs originating from the hairpin GFP714 dsRNAs were more abundant than 364

the Sy-siRNAs derived from the transgene GFP792 mRNA. Moreover, we were able to map 365

some L-siRNAs to Region 1 (nucleotide 237-306) and Region 2 (nucleotide 354-540) of the 366

hairpin GFP714 RNA (Fig. 7A, C, E, H, J and L). These two regions differ from equivalent 367

nucleotides 300-369 and 409-603 of the transgene GFP792 mRNA (Fig. 5). Intriguingly, only 368

extremely low reads of such L-siRNAs were found in systemic leaves (Fig. 7B, D, F, G, I, K, 369

M and N). 370

Taken together, these data (Figs. 5-7) suggest that (i) the hairpin GFP714 dsRNA is the 371

main sources for biogenesis of L-siRNA in local PTGS; (ii) GFP714-specific L-siRNAs 372

mapped to Regions 1/2 are present in systemic leaf tissues; (iii) L-siRNAs are associated with 373

the Sy-siRNA biogenesis and the induction of systemic GFP792 silencing; and (iv) Sy-374



14

siRNAs unique to the two specific regions i.e. Regions 1/2 of the transgene GFP792 mRNA, 375

are generated by transitive silencing in systemic young leaves. 376

Influence of DCLs on Biogenesis of 21, 22, and 24 nt L-siRNAs for Systemic Transgene 377

GFP792 Silencing 378

To further examine the association of L-siRNAs with systemic GFP792 PTGS in Gfp (Fig. 8A 379

and B), GfpDCL1i (Fig. 8C and D), GfpDCL2Ai (Fig. 8E-G), GfpDCL3Bi (Fig. 8H and I), 380

GfpDCL4Ai (Fig. 8J and K), and GfpDCL24Ai (Fig. 8L-N), we analyzed the size distribution 381

of sense and antisense L- and Sy-siRNAs (Fig. 8). In local silencing induced by the hairpin 382

GFP714 dsRNA, there were generally more antisense than sense L-siRNAs (Fig. 8A, C, E, H, 383

J and L). However, in systemic GFP792 PTGS, sense- and antisense Sy-siRNAs were 384

approximately equal (Fig. 8B, D, F, G, I, K, M and N). Furthermore, the distribution of 21, 385

22, and 24 nt L-siRNAs differed from that of Sy-siRNAs in infiltrated and systemic leaves 386

for Gfp (Fig. 8A and B), GfpDCL1i (Fig. 8C and D), GfpDCL2Ai (Fig. 8E-G), GfpDCL3Bi 387

(Fig. 8H and I), GfpDCL4Ai (Fig. 8J and K), and GfpDCL24Ai (Fig. 8L-N). 388

In Gfp and GfpDCL1Ai, the siRNA profile was almost identical, but shifted from 22, 389

21 and 24 nt L-siRNAs (approximately “2.5:2:1” ratio for sense L-siRNA and about 1.4:1.2:1 390

for antisense L-siRNA) for local silencing to predominantly 21 nt along with some 22 nt 391

(approximately 6 fold less than 21 nt) and low level of 24 nt (around 300 times less than 21 nt) 392

Sy-siRNAs for systemic PTGS (Fig. 8A-D). However, RNAi of DCL2, DCL3, and DCL4 had 393

different impacts on the accumulation of 21, 22, and 24 nt L- and Sy-siRNAs. In local 394

silencing, high levels of 24 and/or 21nt L-siRNAs were generated in GfpDCL2Ai and 395

GfpDCL24Ai (Fig. 8E and L). Only a very low level of Sy-siRNAs (103-10

5 fold less than L-396

siRNAs) was found in young leaves which showed no systemic silencing (Fig. 8G and N), 397

although 100-200 or less of 21, 22, and 24 nt Sy-siRNAs were detected in leaves with minor 398

vein-restricted systemic PTGS (Fig. 8F and M). The decrease in the 22 nt L-siRNAs was 399

closely correlated with the decrease in the levels of Sy-siRNAs, as well as with the abolition 400

and/or reduction of systemic GFP792 silencing in young leaves of GfpDCL2Ai and 401

GfpDCL24Ai (Fig. 4; Supplemental Fig. S8; Supplemental Fig. S9B). 402

By contrast, the enhanced systemic transgene GFP792 PTGS and the increased Sy-403

siRNA reads in young leaves were positively linked with the elevated levels of 22 nt L-404

siRNAs in local tissues of GfpDCL3Ai (Fig. 8H) and GfpDCL4Ai (Fig. 8J). However, we 405

observed a reduction in the level of 24 nt L-siRNAs (nearly none) in GfpDCL3Bi (Fig. 8H) as 406

well as a decrease in 21 nt L-siRNAs (3-4 fold less than 22 nt L-siRNAs) in GfpDCL4Ai (Fig. 407



15

8J), consistent with sRNA northern detection (Fig. 4H). Further siRNA size profiling 408

confirmed that the levels of 22 nt L-siRNA were linked with efficient systemic transgene 409

GFP792 PTGS, whereas variations in 21 and 24 nt L-siRNAs did not affect Sy-siRNA 410

production and the occurrence of systemic GFP792 silencing (Supplemental Fig. S12). 411

Requirement of DCL2 to Respond to Mobile Systemic PTGS 412

To further investigate the genetic mechanism involved in the control of systemic PTGS, we 413

used 16cGFP, homGfpDCL4Ai, and homGfpDCL2Ai (Table 1) as either rootstocks or scions 414

in reciprocal grafting experiments (Fig. 9). When grafted onto the sense transgene GFP792-415

silenced homGfpDCL4Ai stocks, homGfpDCL2Ai scions developed no PTGS and showed 416

strong GFP green fluorescence (Fig. 9A and B). However, in the reciprocal grafting, strong 417

sense transgene-mediated PTGS occurred in homGfpDCL4Ai scions three weeks after 418

grafting (wag), even if homGfpDCL2Ai stocks had very limited transgene GFP792 silencing 419

(Fig. 9C, inlet). In controls, efficient PTGS took place at 3 wag in homGfpDCL4Ai scions 420

that were grafted onto GFP792-silenced homGfpDCL4Ai stocks (Fig. 9D). In a different 421

experimental setting, homGfpDCL2Ai scions grafted onto pre-silenced 16cGFP stocks 422

remained un-silenced throughout the experiments, whilst homGfpDCL4Ai scions developed 423

systemic PTGS at 3 wag (Fig. 9E and F). These results demonstrate that (i) DCL4 is unlikely 424

required to produce and respond to mobile silencing signals for systemic PTGS, and (ii) 425

DCL2 is required in the scion to respond to the signal, but not in the rootstock to 426

produce/send the signal. 427

DISCUSSION 428

Genetic insights into mobile RNA silencing and involvement of mobile signal(s) in systemic 429

silencing remain two of the least understood, but yet most debated and divisive topics in the 430

field of plant RNA silencing. Using a suite of DCL RNAi lines together with a GFP reporter, 431

grafting, sRNA hybridization and NGS approaches, we have found that gene-specific 432

suppression of DCL2 expression reduces systemic PTGS. DCLs play central roles in the 433

biogenesis of 21, 22, and 24 nt antisense and sense L-siRNAs in incipient cells. DCL2 is 434

required to respond to mobile signals in recipient cells, but to produce such signals in 435

incipient cells. DCL4 or DCL3 inhibit non-cell autonomous PTGS in systemic tissues. We 436

also extensively profiled L- and Sy-siRNAs associated with both local hairpin dsRNA-437

mediated silencing and systemic transitive sense-transgene PTGS, and examined how L-438



16

siRNAs generated by DCLs affect long-distance spread of PTGS. We propose that DCL2 is 439

the key player along with DCL4 and DCL3, as well as mobile siRNAs in systemic PTGS (Fig. 440

10). Thus, we report several novel findings that shed light on how systemic PTGS is 441

genetically and molecularly regulated in N. benthamiana. 442

1) We have generated transgenic DCL RNAi lines for dissecting intra-/intercellular and 443

systemic PTGS (Table 1). DCL1, DCL2, DCL3, and DCL4 are responsible for the biogenesis 444

of miRNAs and 22, 24, and 21 nt siRNAs, respectively, in N. benthamiana. DCL4 and the 445

DCL4-processed 21 nt siRNAs are essential for local hairpin dsRNA-mediated PTGS, whilst 446

DCL2 or DCL3 and their cognate 22 or 24 nt L-siRNAs are not critical for such intracellular 447

silencing (Fig. 1). 448

2) Local PTGS induced by the hairpin GFP714 dsRNA can lead to efficient biogenesis of 449

mobile L-siRNAs in incipient cells and local leaf tissues (Figs. 1-3). Our experimental system 450

(Supplemental Fig. 3A) avoids the amplification and cascading production of siRNAs in 451

remote recipient cells and enables unambiguous detection of different sized L-siRNAs in 452

systemic leaves. However, neither long (full-length or partial) GFP714 RNA (Supplemental 453

Fig. S7) nor agrobacterium-originated siRNA (Supplemental Table S2) was detectable in 454

young leaves, although these results do not rule out the possibility that long RNAs are present 455

and serve as the mobile signal. Moreover, the total number of GFP714 L-siRNA (and total 456

sRNA) reads in local leaf tissues and their distributions across the GFP714 RNA are almost 457

identical among Nb, RDR6i and all DCL RNAi lines (Figs. 2 and 3). The L-siRNAs detected 458

in systemic leaves are mainly 22 and 21 nt, but some are 24 nt (Fig. 2). These mobile L-459

siRNAs were not particularly abundant in systemic young leaf tissues, but consistent with the 460

levels of mobile siRNAs reported in Arabidopsis (Molnar et al., 2010). These data indicate 461

that all sized sense and antisense L-siRNAs are capable of trafficking over long distances 462

(Fig. 10A). Compared to Nb, the reduced levels of L-siRNAs in distal tissues of all RNAi 463

lines suggest that RDR6 and DCLs, particularly DCL3 and DCL4, contribute to the systemic 464

spread of L-siRNAs (Fig. 2). Indeed, genetic analyses have demonstrated that RDR6 is 465

required for perception of long-distance silencing signals (Schwach et al., 2005; Melnyk et al., 466

2011b) and DCL2 responds to mobile silencing for efficient systemic PTGS in plants (Fig. 4; 467

Taochy et al., 2017). However, even if locally produced siRNAs are mobile, this does not 468

prove that siRNAs are the signal. 469

3) Genetic analysis indicates that DCL1 is unlikely to be involved in local hairpin 470

dsRNA-mediated PTGS, although a slightly elevated level of target mRNA was detected in 471

DCL1i plants (Fig. 1) and sense-transgene induced systemic PTGS (Figs. 4). This conclusion 472



17

is supported by the identical profiles of L-siRNAs or Sy-siRNAs in both Gfp and GfpDCL1i 473

plants (Figs. 6-8). Further, the altered size profiles between L- and Sy-siRNAs in Gfp and 474

GfpDCL1i (Fig. 8) implies that the local and systemic PTGS are likely two linked processes 475

with distinct molecular and genetic requirements. Indeed, local PTGS is induced by a hairpin 476

GFP714 RNA that directly produces long dsRNA, which differs from the sense transgene 477

GFP792-mediated PTGS in systemic leaf tissues. In the latter case, dsRNA is not produced 478

directly, but its production from ssRNA requires the activity of several genes, including 479

RDR6 and DCL2 (Mlotshwa et al., 2008). 480

4) DCL2 RNAi reduces systemic PTGS and blocks the exit of the mobile silencing 481

signal from vascular tissues to the surrounding mesophyll cells, indicating that DCL2 is 482

required for long-distance spread of PTGS (Fig. 4), consistent with that DCL2 promotes, 483

whilst DCL4 inhibits, cell-to-cell spread of VIGS (Qin et al., 2017). Moreover, DCL2 is 484

required to respond to mobile signals for systemic PTGS (Fig. 9), supported by a recent 485

report of a critical role of DCL2 in systemic PTGS in Arabidopsis (Taochy et al., 2017). 486

DCL2 is thus involved in promoting the long-distance (leaf-to-leaf) and short-distance 487

movement (vascular cells to neighboring cells) of PTGS in addition to its role in generating 488

the 22 nt siRNAs. On the other hand, our grafting experiments showed that DCL2 is not 489

required in the rootstock for producing/sending the signal (Fig. 9). However, in contrast to 490

complete loss-of-function mutants, RNAi lines are partial loss-of-function. It thus remains 491

possible that in the DCL2i rootstock, residual DCL2 could still produce some transportable 492

molecules and such signals could then be perceived by DCL2 in recipient cells where DCL4 493

is knocked-down by RNAi to trigger systemic transitive PTGS (Fig. 9). Taochy et al. (2017) 494

have elegantly shown that Arabidopsis DCL2 is required in both the source rootstock and 495

recipient shoot tissue for RDR6-dependent systemic PTGS, although this latest discovery is 496

in contrast to their previous finding (Brosnan et al., 2007). 497

5) Contrary to DCL2, DCL4 and DCL3 inhibit sense transgene-mediated systemic PTGS, 498

as RNAi of DCL4 or DCL3 enhances systemic PTGS in distant young leaves (Fig. 4). Our 499

data also reveal that DCL4 has an epistatic effect on DCL2, thereby influencing systemic 500

PTGS in N. benthamiana (Fig. 4I and J). Only DCL2 is required to respond to mobile signals 501

for systemic PTGS in the reciprocal DCL2i-DCL4i grafting experiments (Fig. 9) and RNAi of 502

DCL4 or DCL3 has been found to up-regulate DCL2 expression (Qin et al., 2017). Taken 503

together, our findings suggest that a DCL2-dependent DCL genetic network regulates non-504

cell autonomous systemic silencing (Fig. 10B). A hierarchical interaction between DCL4 and 505



18

DCL2 to affect cell autonomous PTGS was also reported in Arabidopsis (Bouche et al., 2006; 506

Henderson et al., 2006; Xie et al., 2005). 507

6) Our work implicates or excludes the involvement of certain types of RNAs in mobile 508

systemic PTGS (Fig. 10A). First, combined with earlier elegant work showing that DCL2 is 509

required for transitive silencing (Mlotshwa et al., 2008; Parent et al., 2015), our results 510

suggest that the plant’s response to the signal requires a set of genes that are unique to 511

transitive silencing (i.e., genes that play a role in the production of dsRNA). Thus, these 512

results suggest that the signal for systemic silencing in plants is not itself dsRNA, in contrast 513

to dsRNA being required for the systemic spread of RNAi in animals (Jose et al., 2011). 514

Second, detection of L-siRNAs in systemic tissues implies that mobile L-siRNAs 515

might contribute to systemic PTGS. This notion is supported by the L- and Sy-siRNA 516

profiles associated with the hairpin GFP714 dsRNA-mediated local silencing and systemic 517

transgene GFP792 silencing. The identical distributions of the four clusters of L- and Sy-518

siRNAs across nucleotides 625-742 and 217-282, 730-773 and 363-402 of the transgene 519

GFP792 mRNA and the equivalent sequences of the hairpin GFP714 dsRNA (Fig. 5, labelled 520

© or ℗) suggest that L-siRNAs originated from these parts of the hairpin GFP714 dsRNA 521

might represent a component of mobile signals for the biosynthesis of Sy-siRNA and for 522

systemic induction of GFP792 silencing in distal recipient cells (Figs. 6 and 7, asterisk). 523

Consistent with this, the abundance of the four clusters of Sy-siRNAs associated with 524

systemic GFP792 PTGS are much less than the corresponding L-siRNAs associated with local 525

silencing induced by the hairpin GFP714 dsRNA in control and all RNAi lines (Figs. 6 and 7, 526

asterisk). Furthermore, L-siRNAs specific to Regions 1/2 of the hairpin GFP714 dsRNA were 527

found in systemic leaf tissues (Fig. 7). By contrast, Sy-siRNAs unique to Regions 1/2 of the 528

transgene GFP792 mRNA (these two regions have different sequences between GFP714 and 529

GFP792; Fig. 5), were not found in local leaves where primary silencing efficiently occurred 530

(Fig. 6). These results suggest that Sy-siRNAs distinct to Regions 1/2 of the GFP792 mRNA 531

must have been generated from transitive sense-transgene silencing in systemic leaf cells and 532

tissues. Such transitive PTGS was likely induced by secondary siRNAs that were produced 533

from primary silencing of the GFP792 mRNA sequences, directly targeted by the four clusters 534

of mobile L-siRNAs that were generated from the identical GFP714 sequences in local PTGS 535

(Figs. 6-8; Fig. 10A). However, these results do not exclude the possible involvement of 536

other types of RNAs in systemic RNA silencing in plants. 537

Third, the 21 or 24 nt L-siRNAs processed by DCL4 or DCL3 and their precursor 538

RNAs are unlikely to be a major component of the mobile signal for systemic PTGS (Fig. 8), 539



19

in line with our genetic and functional analysis of DCLs in systemic silencing (Fig. 4). That 540

suppression of DCL4 or DCL3 enhanced systemic silencing and the levels of 21 or 24 nt L-541

siRNAs were not correlated with the biogenesis of GFP792 Sy-siRNAs (Fig. 8) and the 542

intensity of systemic PTGS in all GfpDCL RNAi lines (Fig. 4) provides evidence to support 543

such a conclusion. This is also consistent with fact that DCL4 and DCL3 proteins are not 544

required for the production of the sRNA signals in Arabidopsis (Brosnan et al., 2007; Taochy 545

et al., 2017). 546

Fourth, the levels of DCL2-processed 22 nt L-siRNAs were weakly correlated with 547

the induction of systemic PTGS. Mapping L- and Sy-siRNAs onto the transgene GFP792 548

mRNA and hairpin GFP714 dsRNA identified sense and antisense 22 nt L-siRNAs that might 549

be required for systemic GFP792 PTGS in distal recipient cells. It is evident that the varied 550

levels of the 22 nt L and Sy-siRNAs mapped to the transgene GFP792 antisense sequences 551

625-742 and 217-282, and sense strand 730-773 and 363-402 (Supplemental Fig. S12) were 552

linked to the induction of systemic GFP792 silencing in each RNAi lines (Fig. 4). This is also 553

consistent with the high levels of 21, 22, and 24 nt Sy-siRNAs in GfpDCL4Ai, likely 554

resulting from DCL2 and DCL2-processed 22 nt siRNAs that may trigger efficient 555

biosynthesis of secondary Sy-siRNAs in the systemic leaf tissues (Chen et al., 2010; Cuperus 556

et al., 2010; Mlotshwa et al., 2008). However, these findings per se are not direct evidence 557

that DCL2-processed 22 nt L-siRNAs and/or their precursors are the bona fide signals for 558

induction of Sy-siRNA biogenesis and systemic transitive PTGS. 559

Nonetheless, the molecular nature of systemic PTGS signals needs further 560

characterisation. DCL4-processed 21 nt siRNA or DCL3-processed 24 nt siRNA and their 561

precursors may not be involved in long-distance PTGS signalling, even though 21 and 24 nt 562

siRNAs act primarily as intra- and intercellular triggers for RNA-directed degradation of 563

target mRNA or RdDM (Lewsey et al., 2016; Melnyk et al., 2011a; 2011b; Sarkies and 564

Miska, 2014). In Arabidopsis, the DCL3-processed 24 nt siRNAs are thought to be the main 565

signals for systemic TGS (Lewsey et al., 2016; Melnyk et al., 2011a; Molnar et al., 2010). 566

However, we have not examined the roles of DCLs and mobile siRNAs in systemic TGS in N. 567

benthamiana. Furthermore, whether the DCL2-processed/dependent 22 nt siRNAs and/or 568

their precursors could serve as signals for systemic PTGS requires further investigation, even 569

though 22 nt sRNAs can effectively trigger biogenesis of secondary siRNAs of various sizes 570

in plants (Chen et al., 2010; Cuperus et al., 2010; Mlotshwa et al., 2008) and DCL2 and 571

DCL2-processed 22 nt siRNAs are involved in the cell-to-cell spread of VIGS in N. 572

benthamiana (Qin et al., 2017). 573



20

Our findings also differ from early works showing that long-distance movement of 574

transitive silencing occurs in the absence of sRNAs in the rootstock (Mallory et al., 2001), 575

and that no specific sRNAs are associated with systemic silencing in Arabidopsis (Brosnan et 576

al., 2007; Taochy et al., 2017). Considering the transitivity and sequence-specificity of RNA 577

silencing along with the fact that various types of RNA molecules can trigger cell-578

autonomous silencing, we concede that various forms of transportable RNAs, if they can be 579

perceived and processed by DCL2 or a DCL2-dependent pathway in recipient cells, could 580

function as systemic silencing signals (Fig. 10A). On the other hand, DCL4 and DCL3 could 581

affect DCL2-mediated systemic PTGS through their negative regulation of DCL2 expression 582

(Qin et al., 2017), which would indirectly influence DCL2 activity in systemic PTGS. 583

Additionally, DCLs may compete with each other for dsRNA substrates. The loss of DCL4 or 584

DCL3 could enhance the DCL2 output, because the level of dsRNA substrate for DCL2 is 585

increased in both incipient and recipient cells. Cross-regulation among DCLs could also 586

occur at transcriptional, post-transcriptional, and/or translational levels. These mechanisms 587

may act synergistically for DCLs to maximize production of the mobile signals for systemic 588

PTGS (Fig. 10B), in contrast to what has been described in Arabidopsis (Melnyk et al., 2011b; 589

Sarkies and Miska, 2014). Nevertheless, findings from different non-cell autonomous 590

silencing systems may not necessarily be exclusive of each other. There may be various 591

routes to mobile silencing in different plants. N. benthamiana is a natural rdr1 mutant and 592

might behave differently compared to Arabidopsis (Nakasugi et al., 2013). The occurrence of 593

cell-type or tissue dependent functions and specificities of different DCL proteins or different 594

domain arrangements of the DCL proteins has also been reported in N. benthamiana (Andika 595

et al., 2015; Nakasugi et al., 2013). 596

In conclusion, along with our recent work (Qin et al., 2017), we demonstrate that a 597

complex genetic and regulatory network involving DCL2, DCL4, and DCL3 may govern the 598

rate and characteristics of the mobile PTGS (Fig. 10). DCL2 plays a centre role in response to 599

mobile signals for systemic PTGS in distal recipient cells. We have also highlighted the 600

potential (non)-involvement of RNAs, including different sized siRNAs, in systemic PTGS. 601

Arabidopsis DCLs have distinct functions in mobile silencing, but in N. benthamiana their 602

counterparts appear to behave similarly in intracellular silencing to generate miRNAs and 21, 603

22, or 24 nt siRNAs (this study; Katsarou et al., 2016; Qin et al., 2017), but act coordinately 604

to fulfil different roles in systemic PTGS. Thus, our work provides a new framework to test 605

whether the DCL2-centered DCL genetic pathway for systemic PTGS is the representative 606



21

model for the majority of plant species and to unravel mobile signals for systemic PTGS in 607

plants, as well as in (and across) organisms of different kingdoms. 608

MATERIALS AND METHODS 609

Plant Materials and Growth Conditions 610

Non-transformed N. benthamiana and transgenic N. benthamiana plants (Table 1) were 611

grown and maintained in insect-free glasshouses and growth-rooms at 25°C with 612

supplementary light (13,000 Lux intensity/Visible 40W LED Light Bulbs) to give a 16-hour 613

photoperiod. 614

Plasmid Constructs and Plant Transformation 615

The GFP714 coding sequence (Ryabov et al., 2004) was PCR-amplified and cloned between 616

the duplicated 35S CaMV promoter and the NOS terminator of pJG045, a pCAMBIA1300-617

based vector, to produce p35S-GFP714 (Supplemental Fig. S1A). To generate gene-specific 618

hairpin DCL- and GFP714-RNAi constructs (Supplemental Fig. S1B), cDNA fragments 619

corresponding to nucleotides 4835-5075 of DCL1 (Genbank number FM986780), nucleotides 620

3382-3582 of DCL2 (Genbank number FM986781), nucleotides 4167-4347 of DCL3 621

(Genbank number FM986782), nucleotides 3283-3482 of DCL4 (Genbank number 622

FM986783), or the full-length GFP714 (nucleotides 1-714) sequence were cloned into the 623

RNAi vector pRNAi-LIC as described (Xu et al., 2010). There is no sequence similarity 624

among the DCL fragments (Supplemental Table S1; Supplemental Data Set S1). The full-625

length GFP714 sequence (Ryabov et al., 2004) was also cloned into pMD18-T (Takara) to 626

produce pT7.GFP from which GFP714 RNA transcripts were produced by in vitro 627

transcription using the T7 RNA polymerase. Primers used for these constructs are listed in 628

Supplemental Table S5. All constructs were confirmed by DNA sequencing. 629

pRNAi-DCL1, pRNAi-DCL2, pRNAi-DCL3, and pRNAi-DCL4 were transformed 630

into the Agrobacterium tumefaciens LBA4404, and pRNAi-GFP714 and p35S-GFP714 were 631

introduced into A. tumefaciens EHA105. Transgenic N. benthamiana plants were generated 632

by Agrobacterium-mediated leaf disc transformation as described (Hong et al., 1996). Two 633

independent homozygous lines (DCL2Ai and DCL2Bi, DCL3Ai and DCL3Bi, DCL4Ai and 634

DCL4Bi) with a single-copy of each RNAi construct were obtained. Line DCL4Bi was 635

crossed with DCL2Ai to produce DCL24i. DCLi lines were crossed with the GFP transgenic 636

line 16cGFP that constitutively expresses GFP792 (Haseloff et al., 1997; Ruiz et al., 1998) to 637



22

produce GfpDCL2Ai, GfpDCL2Bi, GfpDCL3Ai, GfpDCL3B, GfpDCL4Ai, GfpDCL4Bi, 638

GfpDCL24Ai, and GfpDCL24Bi, respectively. At the later stage of this project, we also 639

generated homozygous homGfpDCL2Ai, homGfpDCL3Bi, homGfpDCL4Ai, and 640

GfpDCL24Ci through selfing. Only one hemizygous DCL1i or GfpDCL1i line was generated 641

through direct transformation of N. benthamiana or 16cGFP with pRNAi-DCL1 642

(Supplemental Fig. S1B). All transgenic lines used in this study are summarized in Table 1. It 643

should be noted that DCL RNAi is gene-specific and has no obvious off-target effect on other 644

DCL and RNA silencing-associated genes (Supplemental Data Set S1; Supplemental Fig. S5 645

and S10). 646

Local RNA Silencing and Small RNA Mobility Assay (SRMA) 647

The experimental design for local RNA silencing and SRMA to detect long-distance 648

movement of siRNAs from local to systemic young leaves is outlined in Supplemental Fig. 649

S3A. Two young leaves per N. benthamiana or DCLi plant (six plants at the six-leaf stage in 650

each experiment) were co-infiltrated with 1 OD600 agrobacterium harbouring p35S-GFP714 651

and agrobacterium harbouring pRNAi-GFP714 in repeated experiments. In co-infiltrated leaf 652

cells, pRNAi-GFP714 is expected to generate hairpin GFP714 dsRNA that are diced into 653

siRNAs. These siRNAs targeted and degraded GFP714 mRNA transcribed from the p35S-654

GFP714 expression cassette, leading to disappearance of GFP fluorescence. Green 655

fluorescence intensity was measured using ImageJ software following the software provider’s 656

guidance (https://imagej.nih.gov/ij/). Infiltrated leaf tissues from 3-4 different plants at 7 days 657

post agro-infiltration (dpa), and four newly developed young leaves of each of 3-4 different 658

plants at 14 dpa were collected and pooled for sRNA extraction and construction of sRNA 659

libraries. 660

Systemic RNA Silencing and Grafting Experiments 661

Systemic RNA silencing was performed for each line as outlined in Supplemental Fig. S9A. 662

Briefly, leaves from six seedlings at the six-leaf stage of transgenic GFP792 line Gfp and 663

GfpDCL RNAi lines were agroinfiltrated with freshly cultured agrobacterium (1 OD600) 664

carrying pRNAi-GFP714 as previously described (Hong et al., 2003). Plant grafting was 665

performed as described by Schwach et al. (2005). Induction and spread of GFP silencing was 666

routinely examined under long-wavelength UV light and recorded photographically using a 667

Nikon Digital Camera D7000. Red halos on the infiltrated leaves and regions of leaf lamina 668

where GFP silencing occurred show red chlorophyll fluorescence, whilst tissues expressing 669

GFP show green fluorescence under long-wavelength UV light. Local and systemic RNA 670


https://imagej.nih.gov/ij/


23

silencing assays were performed for each line in at least two separate experiments. N. 671

benthamiana is an allotetraploid (Nakasugi et al., 2014); however, the specific RNAi 672

constructs described above only knock down specific DCL expression in spite of the 673

homeologous and duplicated gene copies in the genome. This should not interfere with the 674

analysis of the impact of DCL RNAi on cell and non-cell autonomous RNA silencing. 675

Agroinfiltrated leaf tissues were collected from 3-4 plants at 7 dpa and pooled for sRNA 676

extraction and construction of local sRNA libraries. Young leaves with systemic GFP792 677

PTGS from 3-4 plants of each of the Gfp, GfpDCL1i, GfpDCL3Bi and GfpDCL4Ai lines, 678

leaves with weak vein-restricted systemic PTGS from 3-4 plants of each of the GfpDCL2Ai 679

and GfpDCL24Ai lines, or young leaves without systemic PTGS from 3-4 plants of each of 680

the GfpDCL2Ai and GfpDCL24Ai lines were collected at 14 dpa and pooled for sRNA 681

extraction and construction of systemic sRNA libraries. 682

RNA Extraction and Northern Detection of mRNA and siRNA 683

For RT-PCR and quantitative real-time PCR (RT-qPCR), total RNAs were extracted from 684

leaf tissues using the RNAprep Pure Plant Kit as recommended by the manufacturer 685

(Tiangen). For Northern blot, total RNAs were extracted from leaf tissues with Trizol reagent 686

as recommended by the manufacturer (Invitrogen, Carlsbad, CA). RNAs (5 μg) extracted 687

from infiltrated tissues were separated on a 1% formaldehyde agarose gel, transferred to 688

Hybond-N+ membranes (Amersham Biosciences) by upward capillary transfer in 20xSSC 689

buffer, then cross-linked to the membrane with an UVP CX 2000 UV crosslinker four times 690

(upside, downside, upside, downside) at 120 millijoules/cm2, 1 minute each. Membranes 691

were hybridized with digoxigenin (Dig)-labelled GFP DNA probes prepared using the DIG 692

High Prime DNA Labeling Kit (Roche). RNAs were detected using a DIG Nucleic Acid 693

Detection Kit (Roche) as recommended by the manufacturer. Chemilluminescent signals 694

were detected with a ChemiDocTM

XRS+ imaging System (Bio Rad). 695

To analyze siRNAs, low-molecular-mass sRNAs were enriched from total RNA as 696

described (Hamilton and Baulcombe, 1999). The enriched sRNAs (2.5 μg) were fractionated 697

on an 18% denaturing polyacrylamide/7 M urea gel in 1 x Tris–borate–EDTA (TBE) buffer. 698

Small RNAs were transferred to Hybond-N+ membranes (Amersham Biosciences) by 699

upward capillary transfer in 20xSSC buffer, then cross-linked to the membranes with an UVP 700

CX 2000 UV Crosslinker four times (upside, underside, upside, underside) at 120 701

millijoules/cm2, for 1 minute each time. The membranes were hybridized with digoxigenin 702

(Dig)-labelled GFP RNA probes using a DIG RNA Labeling Kit (Roche) as recommended by 703



24

the manufacturer. The hybridization chemiluminescence signals were detected with a 704

ChemiDocTM

XRS+ imaging System (Bio Rad). 705

RT-PCR and RT-qPCR 706

First-strand cDNA was synthesized using 1 μg total RNAs that were pre-treated with 1 unit 707

RNase-free DNase I as templates by the M-MLV Reverse Transcriptase (Promega). RT-PCR 708

analyses of GFP714 RNA were performed using various sets of primers listed in Supplemental 709

Table S5. The RT–qPCR analyses of DCL mRNA levels were performed using DCL-specific 710

primers (Supplemental Table S5) and the SYBR Green Mix. The amplification program for 711

SYBR Green I was performed at 95C for 10 seconds, 58C for 30 seconds and 72C for 20 712

seconds on a CFX96 real-time PCR detection system (Bio-Rad), following the 713

manufacturer’s instructions. Quadruplicate quantitative assays were performed on cDNA of 714

each of the four biological duplicates (leaf tissues from four different treated plants). The 715

relative quantification of DCL mRNA was calculated using the formula 2–ΔΔCt

and normalized 716

to the amount of GAPDH transcripts (GenBank accession number TC17509) as described 717

(Qin et al., 2012). RT-qPCR data between control and various treatments were analysed by 718

Student’s t-test (http://www.physics.csbsju.edu/stats/t-test.html). The statistical significance 719

threshold was P ≤ 0.05. 720

Construction of sRNA Library and sRNA Next Generation Sequencing (NGS) 721

Fragments of 18-30 bases long RNA were isolated from total RNA extracted from pooled 722

samples of 3-4 different plants for each of the biological and technical duplicates after being 723

separated through 15% denaturing PAGE. The pooled samples contained leaf tissues from 3-6 724

leaves per plant. Then sRNAs were excised from the gel and sequentially ligated to a 3’ 725

adapter and a 5’ adapter. After each ligation step, sRNAs were purified after 15% denaturing 726

PAGE. The final purified ligation products were reverse-transcribed into cDNA using reverse 727

transcriptase (Finnzymes Oy). The first strand cDNA was PCR amplified using Phusion* 728

DNA Polymerase (Finnzymes Oy). The purified DNA fragments were used for clustering and 729

sequencing by Illumina Hiseq 2000 (Illumina, San Diego, CA) at the Beijing Genomics 730

Institute, Shenzhen. 731

Bioinformatics Analysis of sRNA Sequences 732

Illumina HighSeq 2000 sequencing produced a similar number of 11-12 million reads per 733

sRNA library. The reads were cropped to remove adapter sequences and were aligned to the 734

reference sequences using Bowtie2 (Langmead and Salzberg, 2012). Reference sequences 735



25

include hairpin GFP714 (Rabyov et al., 2004), GFP792 transgene (Haseloff et al., 1997), 736

DCL1, DCL2, DCL3, and DCL4 gene sequences (Nakasugi et al., 2013) and a set of 737

50 tobacco miRNAs identified in Nicotiana plants (Pandey et al., 2008; Fig. 5; Supplemental 738

Data Set S1). SAMtools pileup was used to produce the siRNA and miRNA coverage profiles 739

(Li et al., 2009). For correlation analyses for sRNA libraries, the number of miRNA hits 740

corresponding to the previously identified set of 50 Nicotiana miRNAs was determined. All 741

analyzed sRNA libraries for local and systemic leaf samples contained similar proportions of 742

host-encoded miRNA reads (Nakasugi et al., 2013; Ryabov et al., 2014), indicating 743

equivalence and direct comparability of the datasets and no variation due to the efficiency of 744

library preparation and sequencing (Supplemental Tables S2 and S7; Supplemental Figs. S5, 745

S6, S10 and S11). Outcomes of comparisons between normalized siRNAs against the total 746

sRNA reads (per 10 million sRNA reads) are consistent with the numbers of siRNA reads 747

directly compared. 748

Accession Numbers 749

DCL1: Genbank number FM986780; DCL2: Genbank number FM986781; DCL3: Genbank 750

number FM986782; DCL4: Genbank number FM986783; and GAPDH: GenBank accession 751

number TC17509. 752

Supplemental Material 753

754

Supplemental Tables 755

Supplemental Table S1. Nucleotide Sequence Similarity (%) between DCL Fragments in the 756

pRNAi-DCLs Constructs. 757

Supplemental Table S2. Summary of sRNAseq datasets of 18 GFP714 sRNA Libraries for 758

Long-distance Spread of siRNAs. 759

Supplemental Table S3. Correlation analyses of miRNA profiles among agro-infiltrated leaf 760

sRNA libraries (Nb background). 761

Supplemental Table S4. Correlation analyses of miRNA profiles among systemic leaf sRNA 762

libraries (Nb background). 763

Supplemental Table S5. Primers Used in This Study. 764

Supplemental Table S6. Impact of DCL RNAi on Systemic PTGS. 765

Supplemental Table S7. Summary of sRNAseq Datasets of 14 sRNA Libraries for Local 766

and Systemic PTGS. 767



26

Supplemental Table S8. Correlation analyses of miRNA profiles among agro-infiltrated leaf 768

sRNA libraries (16cGFP Transgenic Background). 769

Supplemental Table S9. Correlation analyses of miRNA profiles among systemic leaf sRNA 770

libraries (16cGFP Transgenic Background). 771

Supplemental Figures 772

Supplemental Figure S1. Construction of Gene Expression and RNAi Vectors. 773

Supplemental Figure S2. RNAi Effects on DCL Gene Expression. 774

Supplemental Figure S3. NGS detection of total small RNAs in local and systemic leaves. 775

Supplemental Figure S4. Effect of DCL RNAi on Local GFP714 RNA Silencing. 776

Supplemental Figure S5. DCL RNAi Does Not Cause Off-target Silencing of Genes 777

Associated with RNAi Pathways in Nb Backgound Plants –sRNA Reads in 18 sRNA 778

Libraries. 779

Supplemental Figure S6. miRNA Reads in 18 sRNA Libraries (Nb Background). 780

Supplemental Figure S7. No Long-distance Movement of Large GFP714 RNA. 781

Supplemental Figure S8. Impact of DCL RNAi on Systemic PTGS. 782

Supplemental Figure S9. Size Profiles for Total sRNAs in Local and Systemic Leaves. 783

Supplemental Figure S10. DCL RNAi Does Not Cause Off-target Silencing of Genes 784

Associated with RNAi Pathways in 16cGFP Transgenic Background Plants – sRNA Reads 785

in 14 sRNA Libraries. 786

Supplemental Figure S11. miRNA Reads in 14 sRNA Libraries (16cGFP Transgenic 787

Background). 788

Supplemental Figure S12. Specific 21-24nt siRNA Distributions across the Transgene 789

GFP792 mRNA. 790

Supplemental Data 791

Supplemental Data Set S1. Reference Sequences Used in This Study. 792

Supplemental Data Set S2. DCL RNAi Does Not Cause Off-target Silencing of Genes 793

Associated with RNAi Pathways in Nb Backgound Plants – sRNA Reads in 18 sRNA 794

Libraries. 795

Supplemental Data Set S3. miRNA Reads in 18 sRNA Libraries (Nb Background). 796

Supplemental Data Set S4. DCL RNAi Does Not Cause Off-target Silencing of Genes 797

Associated with RNAi Pathways in 16cGFP Transgenic Background Plants – sRNA Reads 798

in 14 sRNA Libraries. 799

Supplemental Data Set S5. miRNA Reads in 14 sRNA Libraries (16cGFP Transgenic 800

Background). 801



27

ACKNOWLEDGEMENTS 802

We are indebted to David Baulcombe for his kind gift of 16cGFP, RDR6i and GfpRDR6i 803

seeds and for his constructive and critical comments on the manuscript. The corresponding 804

author thanks Mahmut Tör for reading the manuscript. No conflict of interest is declared. 805



28

FIGURE LEGENDS 806

Figure 1. Impact of DCL RNAi on Local Hairpin dsRNA-mediated PTGS. (A-I) Local PTGS 807

assay. The left half of leaves of N. benthamiana (A), DCL1i (B), DCL2Ai and DCL2Bi (C, D), 808

DCL3Ai and DCL3Bi (E, F), DCL4Ai and DCL4Bi (G, H) and DCL24i (I) plants were co-809

infiltrated with agrobacterium harbouring p35S-GFP714 and pRNAi-GFP714 and the right half with 810

p35S-GFP714 and the pRNAi-empty vector. Without PTGS, GFP714 expression from p35S-GFP714 811

shows strong green fluorescence. Induction of PTGS by the hairpin GFP714 dsRNA generated 812

from pRNAi-GFP eliminates GFP714 expression and the infiltrated lamina show red auto-813

fluorescence. DCL4 RNAi attenuated intracellular silencing and the level of PTGS decreased in 814

DCL4Ai, DCL4Bi and DCL24i plants. Some green fluorescence is still be visible in the infiltrated 815

lamina of these plants (G-I). Photographs were taken under long-wavelength UV light (upper 816

panel) or through a stereo-fluorescent microscope (lower panel) at 4 days post-agroinfiltration 817

(dpa). Bar=100 μm. The ratio in the bottom right corner of each panel indicates the number of 818

plants out of the number of agro-infiltrated plants that developed local PTGS in two experiments. 819

The green fluorescence intensity was measured using ImageJ software (Supplemental Fig. S4). 820

(J) Analysis of GFP714 mRNA in DCL RNAi lines. Northern detection was performed using 821

GFP714-specific probes (upper panel). Equal loading of total RNAs extracted from leaf tissues at 822

4 dpa is indicated by the equal amount of 18S and 28S rRNAs shown on the gel (lower panel). 823

The position and size of GFP714 mRNA and 18S and 28S rRNA are indicated. (K) Detection of 824

local L-siRNAs. L-siRNAs were analysed by small RNA Northern blots (upper panel). Equal 825

loading of sRNAs extracted from leaf tissues at 4 dpa is indicated by the equal amount of 5S 826

rRNA/tRNAs shown on gel (lower panel). The position of 21, 22 and 24 nt siRNA as well as 5S 827

rRNA/tRNAs are indicated. 828

Figure 2. Detection of L-siRNAs in local and systemic leaves. (A-I) GFP714 L-siRNAs 829

generated from hairpin dsRNA-mediated local PTGS can be detected in distal systemic young 830

leaves. Sense (blue) and antisense (red) 21, 22 and 24 nt L-siRNAs were present in local (left 831

panel) and systemic (right panel) tissues of N. benthamiana (Nb, A) and RNAi lines DCL1i (B), 832

DCL2Ai (C), DCL2Bi (D), DCL3Ai (E), DCL3Bi (F), DCL4Ai (G), DCL4Bi (H) and RDR6i (I). 833

Outline of mobile siRNA assay is indicated in Supplemental Fig. S3A. 834

835 Figure 3. Distribution of 20-25nt L-siRNAs across the GFP714 mRNA. (A-I) Distribution of 836

20-25nt GFP714 L-siRNAs for N. benthamiana (Nb; A); DCL1i (B); DCL2Ai and DCL2Bi (C, D); 837

DCL3Ai and DCL3Bi (E, F); DCL4Ai and DCL4Bi (G, H); and RDR6i (I). Total GFP714 L-838

siRNA reads are from sRNA libraries of agro-infiltrated leaves (7 days post agroinfiltration (dpa); 839



29

left panel) and systemic young leaves (14 dpa; right panel). Outline of mobile siRNA assay is 840

indicated in Supplemental Fig. S3A. 841

Figure 4. Antagonistic Roles of DCL2 Against DCL3 and DCL4 in Systemic PTGS. (A, B) 842

Impact of DCL RNAi on local and systemic PTGS. Local GFP silencing was induced by the 843

hairpin GFP714 dsRNA in all plants by infiltration of leaves with agrobacterium harbouring 844

pRNAi-GFP714, evident by the red halos around the infiltrated lamina. A close-up of the red halo 845

(red arrow) below each image shows clearer silencing effects (A). Sense transgene GFP792-846

mediated systemic PTGS was assessed on young leaves (B). At 14 days post agroinfiltration 847

(dpa), all infiltrated plants of GfpDCL4Ai and GfpDCL4Bi lines developed very strong systemic 848

GFP792 PTGS in young leaves. GfpDCL3Ai and GfpDCL3Ai plants also developed strong 849

systemic PTGS in young leaves, although some relatively weak GFP792 silencing was observed in 850

some of Gfp and GfpDCL1i plants and no systemic silencing appeared on any GfpDCL2Ai and 851

GfpDCL2Bi plants. An enlarged snapshot (lower) of each photograph in its panel shows a clearer 852

image of the silencing phenotypes (B). The ratio in the bottom right corner of each photo in panel 853

B indicates the number of plants out of the number of agro-infiltrated plants that developed 854

systemic PTGS in two separate experiments. Photographs were taken at 7 (A) and 14 dpa (B). (C-855

G) Systemic transgene GFP792 silencing in young leaves. Transgene GFP792 expression was not 856

silenced in the mock-infiltrated Gfp plant and uniform green fluorescence occurred in the whole 857

lamina of the non-infiltrated young leaf (C). Systemic GFP792 silencing in young leaf of Gfp 858

plants infiltrated with agrobacterium harbouring pRNAi-GFP714 was evident by the red 859

fluorescence of chlorophyll across the leaf lamina of a newly-grown young leaf (D). Only very 860

weak GFP792 silencing was observed in the minor veins of a leaf immediately above the 861

infiltrated leaves of GfpDCL2Ai (E), but extremely strong systemic GFP792 silencing appeared in 862

GfpDCL3Bi (F) and GfpDCL4Ai (G). (I) Weak minor vein-restricted systemic silencing in 863

GfpDCL24Ai. Photographs were taken at 18 dpa. (H) Northern blot detection of Sy-siRNAs in 864

the systemic young leaves at 14 dpa. Total sRNA extracted from systemic leaves of GfpRDR6i 865

plants that were agro-infiltrated with pRNAi-GFP714 was included as a negative control. Upper: 866

siRNA blot, lower: 5S rRNA/tRNA control showing equal loading of sRNA samples. The sizes 867

and positions of siRNAs and 5S rRNA/tRNA are indicated. (J) Local (i) and systemic (ii, iii) 868

GFP792 silencing in GfpDCL24Ai. A section of panel J(ii) is enlarged to show a clearer young 869

leaf image [J(iii)]. Photographs were taken at 7 dpa [J(i)] or 14 dpa [J(ii) and (iii)]. Influence of 870

DCL RNAi on the development of local and systemic transgene GFP792 silencing is also 871

summarized in Supplemental Table S6. 872



30

Figure 5. Comparisons of the transgene GFP792 and the hairpin GFP714 sequences. 873

Completely distinct sequences between the GFP792 transgene (16cGFP) present in the Gfp and 874

GfpDCL RNAi lines and the hairpin GFP714 (GFPi) in the pRNAi-GFP vector are outlined in red-875

boxes, identical sequences are in green boxes, and similar sequences are in dotted boxes 876

(highlighted yellow for the actual sequences). Sequence coordinates are indicated. The four 877

clusters of the sense (s) and antisense (as) siRNAs corresponding to the positive (℗) and 878

complementary (©) stands (underlined) of the GFP714 and GFP792 mRNA are also indicated. 879

Figure 6. Distribution of L- and Sy-siRNA across the transgene GFP792 mRNA. (A) and (B) 880

Gfp. (C) and (D) GfpDCL1i. (E-G) GfpDCL2Ai. (H) and (I) GfpDCL3Bi. (J) and (K) 881

GfpDCL4Ai. (L-N) GfpDCL24Ai. Total L- and Sy-siRNAs are from local agro-infiltrated leaves 882

(A, C, E, H, J and L) and systemic young leaves (B, D, F, G, I, K, M and N). Sense- (blue) and 883

antisense (red) siRNAs were mapped to the transgene GFP792 mRNA sequence. The profiles of 884

L-siRNAs generated largely from the hairpin GFP714 dsRNA differ from that of Sy-siRNAs that 885

were derived from the GFP792 mRNA, particularly in the two less similar Region 1/Region 2 (Fig. 886

5). These differences indicate that elevated levels of L-siRNAs (*) together with their long RNA 887

precursor RNAs, despite the latter being unlikely (Supplemental Fig. S7), moved from local to 888

systemic leaves and contributed to silencing signal. Such mobile signals might act as the primary 889

trigger for production of Sy-siRNAs and for systemic transgene GFP792 silencing in distal young 890

leaves. Sense and antisense Sy-siRNAs associated with Region 2 and Region 1 (highlighted) in 891

systemic recipient cells are likely secondary and resulted from transitive silencing. Experimental 892

design for systemic silencing assays is indicated in Supplemental Fig. S9A. Systemic transgene 893

GFP792 PTGS are shown in Fig. 4, Supplemental Fig. S8 and Supplemental Fig. S9B. Compared 894

to Gfp (B) and GfpDCL1i (D), strong systemic PTGS occurred in GfpDCL3Bi (I) and GfpDCL4i 895

(K); no systemic PTGS was observed in young leaves of GfpDCL2Ai (F) and GfpDCL24Ai (M), 896

and only very weak vein-restricted silencing in the leaf located immediately about the agro-897

infiltrated leaves of GfpDCL2Ai (G) and GfpDCL24Ai (N). 898

Figure 7. Distribution of L- and Sy-siRNAs across the hairpin GFP714 RNA. (A) and (B) Gfp. 899

(C) and (D) GfpDCL1i. (E-G) GfpDCL2Ai. (H) and (I) GfpDCL3Bi. (J) and (K) GfpDCL4Ai. (L-900

N) GfpDCL24Ai. Total L- or Sy-siRNAs are from local agro-infiltrated leaves (A, C, E, H, J and 901

L) or systemic young leaves (B, D, F, G, I, K, M and N). Sense (blue) and antisense (red) 902

siRNAs were mapped to the hairpin GFP714 RNA. The L-siRNA profiles differ from Sy-siRNAs 903

in the two less similar Region 1/Region 2 (Fig. 5). Experimental design for systemic silencing 904

assays is indicated in Supplemental Fig. S9A. Systemic transgene GFP792 PTGS are shown in Fig. 905

4, Supplemental Fig. S8 and Supplemental Fig. S9B. 906



31

Figure 8. Size profiles for L- and Sy-siRNAs. (A-N) Fourteen sRNA libraries were generated 907

from sRNA samples extracted from agroinfiltrated leaves at 7 days post agroinfiltration (dpa; A, 908

C, E, H, J and L) and systemic young leaves at 14 dpa (B, D, F, G, I, K, M and N) of Gfp (A, B), 909

GfpDCL1i (C, D), GfpDCL2Ai (E-G), GfpDCL3Bi (H, I), GfpDCL4Ai (J, K) and GfpDCL24Ai 910

(L-N). Plants were infiltrated with agrobacterium harbouring pRNAi-GFP714. Blue and red bars 911

represent L- and Sy-siRNAs aligned to the sense and antisense strand of GFP792 mRNA, 912

respectively. The abundance of the 22-nt L-siRNA is closely correlated with the induction and 913

reduction of systemic transgene GFP792 silencing in systemic young leaves. Experimental design 914

for systemic silencing assays is indicated in Supplemental Fig. S9A. Systemic transgene GFP792 915

PTGS are shown in Fig. 4, Supplemental Fig. S8 and Supplemental Fig. S9B. 916

Figure 9. Systemic Silencing in Grafted homGfpDCL2Ai and homGfpDCL4Ai Plants. (A, B) 917

16cGFP, homGfpDCL2Ai and homGfpDCL4Ai plants were used as silenced stocks to produce or 918

as scions to receive systemic silencing signals in three experiments. No systemic transgene 919

GFP792 silencing was observed in homGfpDCL2Ai scions grafted onto GFP792-silenced 920

homGfpDCL4Ai stocks 3 weeks after grafting (wag, A) and 6-wag (B). (C) Systemic GFP792 921

silencing at 3 wag in a homGfpDCL4Ai scion grafted onto a homGfpDCL2Ai stock. An enlarged 922

section (boxed) of the stock plant showed very limited GFP792 silencing with red fluorescence 923

marked with an asterisk in the inset leaf. (D) Systemic GFP792 silencing in a homGfpDCL4Ai 924

scion grafted onto a GFP792-silenced homGfpDCL4Ai stock at 3 wag as a control. (E) A 925

homGfpDCL2Ai scion grafted onto GFP792-silenced 16c stocks remained unsilenced at 3 wag and 926

afterwards. (F) Systemic transgene GFP792 silencing in a homGfpDCL4Ai scion grafted onto a 927

GFP792-silenced 16cGFP stock at 3 wag. All plants are photographed under long-wavelength UV 928

illumination. Red fluorescence shows transgene GFP792 silencing whilst green fluorescence 929

indicates no silencing. Scions and stocks are indicated by arrows in each panel. 930

Figure 10. Systemic PTGS in N. benthamiana. (A) A regulatory complex among DCL2, DCL3, 931

and DCL4 contributes to biogenesis of mobile silencing signals for systemic PTGS. Regulation of 932

DCL2 expression by DCL4 or DCL3 is demonstrated in our recent work (Qin et al., 2017). DCL1 933

is not included because it seems to have no obvious effect on systemic PTGS. In this model, 934

mobile 21, 22, and 24 nt L-siRNAs are generated from the hairpin GFP714 dsRNA by DCL2, 935

DCL3, or DCL4 in the incipient cells of local leaf tissues. However, only the levels of the DCL2-936

processed 22 nt L-siRNAs, particularly those L-siRNAs matching to the four identical regions 937

indicated by ℗ (positive strand) or © (complementary strand) of the hairpin GFP714 and the 938

transgene GFP792 mRNA (Fig. 5), are somewhat correlated with the DCL2-dependent induction 939

of systemic transgene GFP792 PTGS and Sy-siRNA biogenesis in the recipient cells of systemic 940



32

leaf tissues. Sy-siRNAs unique to Region 1/Region 2 of the transgene GFP792 mRNA are 941

generated by transitive PTGS in recipient cells. Different color “=” signs represent siRNAs 942

matching to each of the four identical sequences between GFP714 and GFP792 (Fig. 5). (B) DCL2-943

dependent DCL genetic network in systemic silencing. Reduction of DCL2 reduces systemic 944

PTGS, suggesting that DCL2 acts an important activator of the long-distance spread of PTGS. 945

DCL2 is also required to perceive incoming signals, likely in the form of sense and antisense 946

siRNAs and their long RNA precursor (although the latter is unlikely to contribute to systemic 947

silencing), for induction of systemic siRNA biogenesis and execution of systemic PTGS in distal 948

recipient cells. DCL4 or DCL3 may contribute to systemic silencing through their positive 949

influences on long-distance movement of siRNAs; however, both are negative regulators (T sign) 950

that down-regulate DCL2 expression (Qin et al., 2017). Thus, DCL2 is a key player in the 951

systemic silencing network. Suppression of either DCL4 or DCL3 alleviates the negative control 952

of DCL2 expression and the elevated DCL2 could then respond more effectively to mobile 953

signals for more efficient systemic PTGS in N. benthamiana. 954

TABLES 955

Table 1. Summary of Transgenic Lines Generated and Used in This Study. Wild-type and

transgenic lines Genetic background and cross Transgene information

Nb N. benthamiana Wild-type, no transgene DCL1i RNAi of DCL1 in Nb pRNAi-DCL1, single copy, hemizygous

DCL2Ai RNAi of DCL2 in Nb pRNAi-DCL2, single copy, homozygous DCL2Bi



RDR6i (Gift from David Baulcombe) RNAi of NbRDR6 in Nb RDR6 hairpin, single copy, homozygous

DCL24i Cross between DCL2Ai and DCL4Bi pRNAi-DCL2, single copy, hemizygous pRNAi-DCL4, single copy, hemizygous

16cGFP GFP transgenic line in Nb 35S-GFP, single copy, homozygous GfpRDR6i (Gift from David Baulcombe) RNAi of RDR6 in 16cGFP

35S-GFP, single copy, homozygous RDR6 hairpin, single copy, homozygous

GfpDCL1i RNAi of DCL1 in 16cGFP 35S-GFP, single copy, homozygous

pRNAi-DCL1, single copy, hemizygous GfpDCL2Ai Cross between 16cGFP and DCL2Ai 35S-GFP, single copy, hemizygous

pRNAi-DCL2, single copy, hemizygous GfpDCL2Bi Cross between 16cGFP and DCL2Bi GfpDCL3Ai Cross between 16cGFP and DCL3Ai 35S-GFP, single copy, hemizygous

pRNAi-DCL3, single copy, hemizygous GfpDCL3Bi Cross between 16cGFP and DCL3Bi



33

GfpDCL4Ai Cross between 16cGFP and DCL4Ai 35S-GFP, single copy, hemizygous pRNAi-DCL4, single copy, hemizygous GfpDCL4Bi Cross between 16cGFP and DCL4Bi

GfpDCL24Ai Triple cross among 16c, DCL2Ai and

DCL4Ai 35S-GFP, single copy, hemizygous

pRNAi-DCL2, single copy, hemizygous pRNAi-DCL4, single copy, hemizygous GfpDCL24Bi

Triple cross among 16c, DCL2Ai and

DCL4Bi homGfpDCL2Ai Self from GfpDCL2Ai

35S-GFP, single copy, homozygous pRNAi-DCL2, single copy, homozygous

homGfpDCL3Ai Self from GfpDCL3Ai 35S-GFP, single copy, homozygous

pRNAi-DCL3, single copy, homozygous homGfpDCL4Ai Self from GfpDCL4Ai

35S-GFP, single copy, homozygous pRNAi-DCL4, single copy, homozygous

GfpDCL24Ci Cross between homGfpDCL2Ai and homGfpDCL4Ai

35S-GFP, single copy, homozygous pRNAi-DCL2, single copy, hemizygous pRNAi-DCL4, single copy, hemizygous

Gfp Cross between Nb and 16cGFP 35S-GFP, single copy, hemizygous 956



34

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Figure 1. Impact of DCL RNAi on Local Hairpin dsRNA-mediated PTGS. (A-I) Local PTGS

assay. The left half leaves of N. benthamiana (A), DCL1i (B), DCL2Ai and DCL2Bi (C, D), DCL3Ai

and DCL3Bi (E, F), DCL4Ai and DCL4Bi (G, H) and DCL24i (I) plants were co-infiltrated with

agrobacterium harbouring p35S-GFP714 and pRNAi-GFP714; and the right half with p35S-GFP714 and

the pRNAi-empty vector. Without PTGS, GFP714 expression from p35S-GFP714 shows strong green

fluorescence. Induction of PTGS by the hairpin GFP714 dsRNA generated from pRNAi-GFP

eliminates GFP714 expression and the infiltrated lamina show red auto-fluorescence. DCL4 RNAi

attenuated intracellular silencing and the level of PTGS decreased in DCL4Ai, DCL4Bi and DCL24i

plants. Some green fluorescence can still be visible in the infiltrated lamina of these plants (G-I).

Photographs were taken under long-wavelength UV light (upper panel) or through a stereo-

fluorescent microscope (lower panel) at 4 days post-agroinfiltration (dpa). Bar=100μm. The ratio in

the bottom right corner of each panel indicates the number of plants out of the number of agro-

infiltrated plants that developed local PTGS in two experiments. The green fluorescence intensity was

measured using the ImageJ software (Supplemental Fig. S4). (J) Analysis of GFP714 mRNA in DCL

RNAi lines. Northern detection was performed using GFP714-specific probes (upper panel). Equal

loading of total RNAs extracted from leaf tissues at 4-dpa is indicated by the equal amount of 18S and

28S rRNAs shown on gel (lower panel). The position and size of GFP714 mRNA and 18S and 28S

rRNA are indicated. (K) Detection of local L-siRNAs. L-siRNAs were analysed by small RNA

Northern blots (upper panel). Equal loading of sRNAs extracted from leaf tissues at 4-dpa is indicated

by the equal amount of 5S rRNA/tRNAs shown on gel (lower panel). The position of 21-, 22- and

24nt siRNA as well as 5S rRNA/tRNAs are indicated.



Figure 2. Detection of L-siRNAs in local and systemic leaves. (A-I) GFP714 L-siRNAs generated

from hairpin dsRNA-mediated local PTGS can be detected in distal systemic young leaves. Sense

(blue) and antisense (red) 21, 22 and 24nt L-siRNAs were present in local (left panel) and systemic

(right panel) tissues of N. benthamiana (Nb, A) and RNAi lines DCL1i (B), DCL2Ai (C), DCL2Bi (D),

DCL3Ai (E), DCL3Bi (F), DCL4Ai (G), DCL4Bi (H) and RDR6i (I). Outline of mobile siRNA assay

is indicated in Supplemental Fig. S3A.



Figure 3. Distribution of 20-25nt L-siRNAs across the GFP714 mRNA. (A-I) Distribution of 20-

25nt GFP714 L-siRNAs for N. benthamiana (Nb; A); DCL1i (B); DCL2Ai and DCL2Bi (C, D);

DCL3Ai and DCL3Bi (E, F); DCL4Ai and DCL4Bi (G, H); and RDR6i (I). Total GFP714 L-siRNA

reads are from sRNA libraries of agro-infiltrated leaves (7 days post agro-infiltration (dpa); left panel)

and systemic young leaves (14dpa; right panel). Outline of mobile siRNA assay is indicated in

Supplemental Fig. S3A.



Figure 4. Antagonistic Roles of DCL2 Against DCL3 or DCL4 in Systemic PTGS. (A, B) Impact

of DCL RNAi on local and systemic PTGS. Local GFP silencing was induced by the hairpin GFP714

dsRNA in all plants by infiltration of leaves with agrobacterium harbouring pRNAi-GFP714,

evidenced by the red halos around the infiltrated lamina. A close-up of the red halo (red arrow) below

each image shows clearer silencing effects (A). Sense transgene GFP792-mediated systemic PTGS was

assessed on young leaves (B). At 14 days post agro-infiltration (dpa), all infiltrated plants of

GfpDCL4Ai and GfpDCL4Bi lines developed very strong systemic GFP792 PTGS on young leaves.

GfpDCL3Ai and GfpDCL3Ai plants also developed strong systemic PTGS in young leaves, although

some relatively weak GFP792 silencing was observed in some of Gfp and GfpDCL1i plants and no

systemic silencing appeared on any GfpDCL2Ai and GfpDCL2Bi plants. An enlarged snapshot (lower)

of each photograph in panel shows a clearer image of the silencing phenotypes (B). The ratio in the

bottom right corner of each photo in panel B indicates the number of plants out of the number of agro-

infiltrated plants that developed systemic PTGS in two separate experiments. Photographs were taken

at 7 (A) and 14dpa (B). (C-G) Systemic transgene GFP792 silencing in young leaves. Transgene

GFP792 expression was not silenced in the mock-infiltrated Gfp plant and uniform green fluorescence

occurred in the whole lamina of the non-infiltrated young leaf (C). Systemic GFP792 silencing in

young leaf of Gfp plants infiltrated with agrobacterium harbouring pRNAi-GFP714 was evidenced by

the red fluorescence of chlorophyll across the leaf lamina of a newly-grown young leaf (D). Only very

weak GFP792 silencing was observed in minor veins of a leaf immediately above the infiltrated leaves

of GfpDCL2Ai (E), but extremely strong systemic GFP792 silencing appeared in GfpDCL3Bi (F) and

GfpDCL4Ai (G). (I) Weak minor vein-restricted systemic silencing in GfpDCL24Ai. Photographs

were taken at 18dpa. (H) Northern blot detection of Sy-siRNAs in the systemic young leaves at 14dpa.

Total sRNA extracted from systemic leaves of GfpRDR6i plants that were agro-infiltrated with

pRNAi-GFP714 was included as a negative control. Upper: siRNA blot, lower: 5S rRNA/tRNAs

control showing equal loading of sRNA samples. The sizes and positions of siRNAs and 5S

rRNA/tRNAs are indicated. (J) Local (i) and systemic (ii, iii) GFP792 silencing in GfpDCL24Ai. A

section of panel J(ii) is enlarged to show a clearer young leaf image [J(iii)]. Photographs were taken

at 7dpa [J(i)], or 14dpa [J(ii) and (iii)]. Influence of DCL RNAi on development of local and systemic

transgene GFP792 silencing was also summarised in Supplemental Table S6.



Figure 5. Comparisons between the transgene GFP792 and the hairpin GFP714 sequences.

Completely distinct sequences between the GFP792 transgene (16cGFP) presented in Gfp and

GfpDCL RNAi lines and the hairpin GFP714 (GFPi) in the pRNAi-GFP vector are outlined in red-

boxes, identical sequences in green boxes, similar sequences in dotted boxes (highlighted yellow for

the actual sequences). Sequence coordinates are indicated. The four clusters of the sense (s) and

antisense (as) siRNAs corresponding to the positive (℗) and complementary (©) stands (underlined)

of the GFP714 and GFP792 mRNA are also indicated.



Figure 6. Distribution of L- and Sy-siRNA across the transgene GFP792 mRNA. (A) and (B) Gfp. (C)

and (D) GfpDCL1i. (E-G) GfpDCL2Ai. (H) and (I) GfpDCL3Bi. (J) and (K) GfpDCL4Ai. (L-N)

GfpDCL24Ai. Total L- and Sy-siRNAs are from local agro-infiltrated leaves (A, C, E, H, J and L) and

systemic young leaves (B, D, F, G, I, K, M and N). Sense- (blue) and antisense (red) siRNAs were

mapped to the transgene GFP792 mRNA sequence. The profiles of L-siRNAs generated largely from the

hairpin GFP714 dsRNA differ from that of Sy-siRNAs that were derived from the GFP792 mRNA,

particularly in the two less similar Region 1/Region 2 (Fig. 5). These differences indicate that the high

abundance of L-siRNAs (asterisked) together with their long RNA precursor RNAs, despite the latter is

unlikely (Supplemental Fig. S7) moved from local to systemic leaves. Such mobile signals might act as the

primary trigger for production of Sy-siRNAs and for systemic transgene GFP792 silencing in distal young

leaves. Sense and antisense Sy-siRNAs associated with Region 2 and Region 1 (highlighted) in systemic

recipient cells are likely secondary and resulted from transitive silencing. Experimental design for systemic

silencing assays is indicated in Supplemental Fig. S9A. Systemic transgene GFP792 PTGS are shown in Fig.

4, Supplemental Fig. S8 and Supplemental Fig. S9B. Compared to Gfp (B) and GfpDCL1i (D), strong

systemic PTGS occurred in GfpDCL3Bi (I) and GfpDCL4i (K); no systemic PTGS was observed in young

leaves of GfpDCL2Ai (F) and GfpDCL24Ai (M), and only very weak vein-restricted silencing in the leaf

located immediately about the agroinfiltrated leaves of GfpDCL2Ai (G) and GfpDCL24Ai (N).



Figure 7. Distribution of L- and Sy-siRNAs across the hairpin GFP714 RNA. (A) and (B) Gfp. (C)

and (D) GfpDCL1i. (E-G) GfpDCL2Ai. (H) and (I) GfpDCL3Bi. (J) and (K) GfpDCL4Ai. (L-N)

GfpDCL24Ai. Total L- or Sy-siRNAs are from local agro-infiltrated leaves (A, C, E, H, J and L) or

systemic young leaves (B, D, F, G, I, K, M and N). Sense (blue) and antisense (red) siRNAs were

mapped to the hairpin GFP714 RNA. The L-siRNA profiles differ from Sy-siRNAs, in the two less

similar Region 1/Region 2 (Fig. 5). Experimental design for systemic silencing assays is indicated in

Supplemental Fig. S9A. Systemic transgene GFP792 PTGS are shown in Fig. 4, Supplemental Fig. S8

and Supplemental Fig. S9B.



Figure 8. Size profiles for L- and Sy-siRNAs. (A-N) Fourteen sRNA libraries were generated from

sRNA samples extracted from agro-infiltrated leaves at 7 days post agro-infiltration (dpa; A, C, E, H,

J and L) and systemic young leaves at 14dpa (B, D, F, G, I, K, M and N) of Gfp (A, B), GfpDCL1i

(C, D), GfpDCL2Ai (E-G), GfpDCL3Bi (H, I), GfpDCL4Ai (J, K) and GfpDCL24Ai (L-N). Plants

were infiltrated with agrobacterium harbouring pRNAi-GFP714. Blue and red bars represent L- and

Sy-siRNAs aligned to the sense and antisense strand of GFP792 mRNA, respectively. The abundance

of the 22-nt L-siRNA is closely correlated with the induction and reduction of systemic transgene

GFP792 silencing in systemic young leaves. Experimental design for systemic silencing assays is

indicated in Supplemental Fig. S9A. Systemic transgene GFP792 PTGS are shown in Fig. 4,

Supplemental Fig. S8 and Supplemental Fig. S9B.



Figure 9. Systemic Silencing in Grafted homGfpDCL2Ai and homGfpDCL4Ai Plants. (A, B)

16cGFP, homGfpDCL2Ai and homGfpDCL4Ai plants were used as silenced stocks to produce or as

scions to receive systemic silencing signals in three experiments. No systemic transgene GFP792

silencing was observed in homGfpDCL2Ai scions grafted onto GFP792-silenced homGfpDCL4Ai

stocks 3 weeks after grafting (wag, A) and 6-wag (B). (C) Systemic GFP792 silencing at 3-wag in a

homGfpDCL4Ai scion grafted onto a homGfpDCL2Ai stock. An enlarged section of the stock plant

showed very limited GFP792 silencing with red fluorescence asterisked in the inset leaf. (D) Systemic

GFP792 silencing in a homGfpDCL4Ai scion grafted onto a GFP792-silenced homGfpDCL4Ai stock at

3-wag as a control. (E) A homGfpDCL2Ai scion grafted onto GFP792-silenced 16c stocks remained

unsilenced at 3-wag and afterwards. (F) Systemic transgene GFP792 silencing in a homGfpDCL4Ai

scion grafted onto a GFP792-silenced 16cGFP stock at 3-wag. All plants are photographed under long-

wavelength UV illumination. Red fluorescence shows transgene GFP792 silencing whilst green

fluorescence indicates no silencing. Scions and stocks are indicated by arrows in each panel.



Figure 10. Systemic PTGS in N. benethamiana. (A) A regulatory complex among DCL2, DCL3 and

DCL4 contributes to biogenesis of mobile silencing signals for systemic PTGS. Regulation of DCL2

expression by DCL4 or DCL3 is demonstrated in our recent work (Qin et al., 2017). DCL1 is not

included because it seems to have no obvious effect on systemic PTGS. In this model, mobile 21, 22

and 24nt L-siRNAs are generated from the hairpin GFP714 dsRNA by DCL2, DCL3 or DCL4 in the

incipient cells of local leaf tissues. However, only the levels of the DCL2-processed 22nt L-siRNAs,

particularly these L-siRNAs matching to the four identical regions indicated by ℗ (positive strand) or

© (complementary strand) of the hairpin GFP714 and the transgene GFP792 mRNA (Fig. 5), are

somewhat correlated with the DCL2-dependent induction of systemic transgene GFP792 PTGS and

Sy-siRNA biogenesis in the recipient cells of systemic leaf tissues. Sy-siRNAs unique to Region

1/Region 2 of the transgene GFP792 mRNA are generated by transitive PTGS in recipient cells.

Different colour “=” signs represent siRNAs matching to each of the four identical sequences between

GFP714 and GFP792 (Fig. 5). (B) DCL2-dependent DCL genetic network in systemic silencing.

Reduction of DCL2 reduces systemic PTGS, suggesting DCL2 acts an important activator for long-

distance spread of PTGS. DCL2 is also required to perceive incoming signals, likely in the form of

sense and antisense siRNAs and their long RNA precursor (although the latter is unlikely to contribute

systemic silencing), for induction of systemic siRNA biogenesis and execution of systemic PTGS in

distal recipient cells. DCL4 or DCL3 may contribute to systemic silencing through their positive

influences on long-distance movement of siRNAs, however both are negative regulators (T sign) that

down-regulate DCL2 expression (Qin et al., 2017). Thus, DCL2 is a key player in the systemic

silencing network. Suppression of either DCL4 or DCL3 alleviates the negative control of DCL2

expression and the elevated DCL2 could then respond more effectively to mobile signals for more

efficient systemic PTGS in N. benthamiana.



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http://www.ncbi.nlm.nih.gov/pubmed?term=Andika IB%2C Maruyama K%2C Sun L%2C Kondo H%2C Tamada T%2C Suzuki N %282015%29 Differential contributions of plant Dicer%2Dlike proteins to antiviral defences against potato virus X in leaves and roots%2E Plant J 81%3A 781%2D793%2E&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Andika IB, Maruyama K, Sun L, Kondo H, Tamada T, Suzuki N (2015) Differential contributions of plant Dicer-like proteins to antiviral defences against potato virus X in leaves and roots. Plant J 81: 781-793.

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http://search.crossref.org/?page=1&rows=2&q=Baulcombe D (2004) RNA silencing in plants. Nature 431: 356-363.

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Melnyk CW, Molnar A, Baulcombe DC (2011b) Intercellular and systemic movement of RNA silencing. EMBO J 30: 3553-3563.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Mlotshwa S, Pruss GJ, Peragine A, Endres MW, Li J, Chen X, Poethig RS, Bowman LH, Vance V (2008) DICER-LIKE2 plays a primaryrole in transitive silencing of transgenes in Arabidopsis. PLoS One 3: e1755.


Molnar A, Melnyk CW, Bassett A, Hardcastle TJ, Dunn R, Baulcombe DC (2010) Small silencing RNAs in plants are mobile and directepigenetic modification in recipient cells. Science 328: 872-875.


Mukherjee K, Campos H, Kolaczkowski B (2013) Evolution of animal and plant dicers: early parallel duplications and recurrentadaptation of antiviral RNA binding in plants. Mol Biol Evol 30: 627-641.


Nakasugi K, Crowhurst R, Bally J, Waterhouse P (2014) Combining transcriptome assembly from multiple De novo assemblers in theallo-tetraploid plant Nicotiana benthamiana. PLoS One 9: e91776.


Nakasugi K, Crowhurst RN, Bally J, Wood CC, Hellens RP, Waterhouse PM (2013) De novo transcriptome sequence assembly andanalysis of RNA silencing genes of Nicotiana benthamiana. PLoS One 8: e59534.


Pandey SP, Shahi P, Gase K, Baldwin IT (2008). Herbivory-induced changes in the small-RNA transcriptome and phytohormonesignaling in Nicotiana attenuate. Proc Natl Acad Sci USA 105: 4559-4564.


Pant BD, Buhtz A, Kehr J, Scheible W-R (2008) MicroRNA399 is a long-distance signal for the regulation of plant phosphatehomeostasis. Plant J 53: 731-738.


Parent J-S, Bouteiller N, Elmayan T, Vaucheret H (2015) Respective contribution of Arabidopsis DCL2 and DCL4 to RNA silencing.Plant J 81: 223-232.


Qin C, Shi N, Gu M, Zhang H, Li B, Shen J, Mohammed A, Ryabov E, Li C, Wang H, Liu Y, Osman T, Vatish M, Hong Y (2012) Involvementof RDR6 in short-range intercellular RNA silencing in Nicotiana benthimiana. Sci Rep 2: 467.


Qin C, Li B, Fan Y, Zhang X, Yu Z, Ryabov E, Zhao M, Wang H, Shi N, Zhang P, Jackson S, Tör M, Cheng Q, Liu Y, Gallusci P, Hong Y(2017) Roles of DCL2 and DCL4 in Intra- and Intercellular Antiviral Silencing in Nicotiana benthmiana. Plant Physiol 174: 1067-1081.


Qu F, Ye X, Morris TJ (2008) Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathwaynegatively regulated by DCL1. Proc Natl Acad Sci USA 105: 14732-14737.


Ruiz MT, Voinnet O, Baulcombe DC (1998) Initiation and maintenance of virus-induced gene silencing. Plant Cell 10: 937-946.Pubmed: Author and Title www.plantphysiol.orgon April 11, 2020 - Published by Downloaded from

Copyright © 2018 American Society of Plant Biologists. All rights reserved.

http://search.crossref.org/?page=1&rows=2&q=Melnyk CW, Molnar A, Bassett A, Baulcombe DC (2011a) Mobile 24 nt small RNAs direct transcriptional gene silencing in the root meristems of Arabidopsis thaliana. Curr Biol 21: 1678-1683.

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http://search.crossref.org/?page=1&rows=2&q=Melnyk CW, Molnar A, Baulcombe DC (2011b) Intercellular and systemic movement of RNA silencing. EMBO J 30: 3553-3563.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Melnyk&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Intercellular and systemic movement of RNA silencing&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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http://search.crossref.org/?page=1&rows=2&q=Mlotshwa S, Pruss GJ, Peragine A, Endres MW, Li J, Chen X, Poethig RS, Bowman LH, Vance V (2008) DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis. PLoS One 3: e1755.

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Mlotshwa&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Mlotshwa&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

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http://search.crossref.org/?page=1&rows=2&q=Molnar A, Melnyk CW, Bassett A, Hardcastle TJ, Dunn R, Baulcombe DC (2010) Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 328: 872-875.

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http://scholar.google.com/scholar?as_q=Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=Mukherjee K%2C Campos H%2C Kolaczkowski B %282013%29 Evolution of animal and plant dicers%3A early parallel duplications and recurrent adaptation of antiviral RNA binding in plants%2E Mol Biol Evol 30%3A 627%2D641%2E&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Mukherjee K, Campos H, Kolaczkowski B (2013) Evolution of animal and plant dicers: early parallel duplications and recurrent adaptation of antiviral RNA binding in plants. Mol Biol Evol 30: 627-641.

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http://search.crossref.org/?page=1&rows=2&q=Nakasugi K, Crowhurst R, Bally J, Waterhouse P (2014) Combining transcriptome assembly from multiple De novo assemblers in the allo-tetraploid plant Nicotiana benthamiana. PLoS One 9: e91776.

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http://search.crossref.org/?page=1&rows=2&q=Nakasugi K, Crowhurst RN, Bally J, Wood CC, Hellens RP, Waterhouse PM (2013) De novo transcriptome sequence assembly and analysis of RNA silencing genes of Nicotiana benthamiana. PLoS One 8: e59534.

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http://search.crossref.org/?page=1&rows=2&q=Pandey SP, Shahi P, Gase K, Baldwin IT (2008). Herbivory-induced changes in the small-RNA transcriptome and phytohormone signaling in Nicotiana attenuate. Proc Natl Acad Sci USA 105: 4559-4564.

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http://search.crossref.org/?page=1&rows=2&q=Pant BD, Buhtz A, Kehr J, Scheible W-R (2008) MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis. Plant J 53: 731-738.

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http://scholar.google.com/scholar?as_q=MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pant&as_ylo=2008&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Parent J%2DS%2C Bouteiller N%2C Elmayan T%2C Vaucheret H %282015%29 Respective contribution of Arabidopsis DCL2 and DCL4 to RNA silencing%2E Plant J 81%3A 223%2D232%2E&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Parent J-S, Bouteiller N, Elmayan T, Vaucheret H (2015) Respective contribution of Arabidopsis DCL2 and DCL4 to RNA silencing. Plant J 81: 223-232.

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http://scholar.google.com/scholar?as_q=Respective contribution of Arabidopsis DCL2 and DCL4 to RNA silencing&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Parent&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Qin C%2C Shi N%2C Gu M%2C Zhang H%2C Li B%2C Shen J%2C Mohammed A%2C Ryabov E%2C Li C%2C Wang H%2C Liu Y%2C Osman T%2C Vatish M%2C Hong Y %282012%29 Involvement of RDR6 in short%2Drange intercellular RNA silencing in Nicotiana benthimiana%2E Sci Rep 2%3A 467%2E&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Qin C, Shi N, Gu M, Zhang H, Li B, Shen J, Mohammed A, Ryabov E, Li C, Wang H, Liu Y, Osman T, Vatish M, Hong Y (2012) Involvement of RDR6 in short-range intercellular RNA silencing in Nicotiana benthimiana. Sci Rep 2: 467.

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http://scholar.google.com/scholar?as_q=Involvement of RDR6 in short-range intercellular RNA silencing in Nicotiana benthimiana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Involvement of RDR6 in short-range intercellular RNA silencing in Nicotiana benthimiana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Qin&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Qin C%2C Li B%2C Fan Y%2C Zhang X%2C Yu Z%2C Ryabov E%2C Zhao M%2C Wang H%2C Shi N%2C Zhang P%2C Jackson S%2C T�r M%2C Cheng Q%2C Liu Y%2C Gallusci P%2C Hong Y %282017%29 Roles of DCL2 and DCL4 in Intra%2D and Intercellular Antiviral Silencing in Nicotiana benthmiana%2E Plant Physiol 174%3A 1067%2D1081%2E&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Qin C, Li B, Fan Y, Zhang X, Yu Z, Ryabov E, Zhao M, Wang H, Shi N, Zhang P, Jackson S, T�r M, Cheng Q, Liu Y, Gallusci P, Hong Y (2017) Roles of DCL2 and DCL4 in Intra- and Intercellular Antiviral Silencing in Nicotiana benthmiana. Plant Physiol 174: 1067-1081.

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http://scholar.google.com/scholar?as_q=Roles of DCL2 and DCL4 in Intra- and Intercellular Antiviral Silencing in Nicotiana benthmiana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Qin&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

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Ryabov EV, Wood GR, Fannon JM, Moore JD, Bull JC, Chandler D, Mead A, Burroughs N, Evans DJ (2014) A virulent strain ofDeformed Wing Virus (DWV) of Honeybees (Apis mellifera) prevails after Varroa destructor-mediated, or in vitro, transmission. PLoSPathog 10: e1004230.


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Schwach F, Vaistij FE, Jones L, Baulcombe DC (2005) An RNA-dependent RNA polymerase prevents meristem invasion by potato virusX and is required for the activity but not the production of a systemic silencing signal. Plant Physiol 138: 1842-1852.


Shih JD, Hunter CP (2011) SID-1 is a dsRNA-selective dsRNA-gated channel. RNA 17: 1057–1065.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Skopelitis DS, Benkovics AH, Husbands AY, Timmermans MCP (2017) Boundary formation through a direct threshold-based readout ofmobile small RNA gradients. Dev Cell 43: 265-273.


Smith LM, Pontes O, Searle I, Yelina N, Yousafzai FK, Herr AJ, Pikaard CS, Baulcombe DC (2007) An SNF2 protein assoicated withnuclear RNA silencing and the spread of a silencing signal between cells in Arabidopsis. Plant Cell 19: 1507-1521.


Taochy C, Gursanscky NR, Cao J, Fletcher SJ, Dressel U, Mitter N, Tucker MR, Koltunow AMG, Bowman JL, Vaucheret H, Carroll BJ(2017) A Genetic Screen for Impaired Systemic RNAi Highlights the Crucial Role of DICER-LIKE 2. Plant Physiol 175: 1424-1437.


Wang XB, Jovel J, Udomporn P, Wang Y, Wu Q, Li WX, Gasciolli V, Vaucheret H, Ding SW (2011) The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defence by two cooperative argonautes in Arabidopsisthaliana. Plant Cell 23: 1625-1638.


Weiberg A, Wang M, Lin FM, Zhao H, Zhang Z, Kaloshian I, Huang HD, Jin H (2013). Fungal small RNAs suppress plant immunity byhijacking host RNA interference pathways. Science 342: 118-123.


Winston WM, Molodowitch C, Hunter CP (2002) Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1.Science 295: 2456–2459.


Xie Z, Allen E, Wilken A, Carrington JC (2005) DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegative www.plantphysiol.orgon April 11, 2020 - Published by Downloaded from Copyright © 2018 American Society of Plant Biologists. All rights reserved.

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http://www.ncbi.nlm.nih.gov/pubmed?term=Searle IR%2C Pontes O%2C Melnyk CW%2C Smith LM%2C Baulcombe DC %282010%29 JMJ14%2C a JmjC domain protein%2C is requried for RNA silencing and cell%2Dto%2Dcell movement of an RNA silencing signal in Arabidopsis%2E Genes %26 Dev 24%3A 986%2D991%2E&dopt=abstract

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http://scholar.google.com/scholar?as_q=Putative Arabidopsis THO/TREX mRNA export complex is involved in transgene and endogenous siRNA biosynthesis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yelina&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1


A Genetic Network for Systemic RNA Silencing in …...A Genetic Network for Systemic RNA Silencing...

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