# Revisiting the roles of tobamovirus replicase complex proteins in viral replication and silencing suppression

Nachelli Malpica-Lopez, Rajendran Rajeswaran, Daria Beknazariants, Jonathan Seguin, Victor Golyaev, Laurent Farinelli, Mikhail Pooggin

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Nachelli Malpica-Lopez, Rajendran Rajeswaran, Daria Beknazariants, Jonathan Seguin, Victor Golyaev, et al.. Revisiting the roles of tobamovirus replicase complex proteins in viral replication and silencing suppression. Molecular Plant-Microbe Interactions, 2017, in press (1), pp.1-97.
10.1094/mpmi-07-17-0164-r hal-01607395

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1 Revisiting the roles of tobamovirus replicase complex proteins in viral replication

2 and silencing suppression

3

4 Nachelli Malpica-López,¹ Rajendran Rajeswaran,¹ Daria Beknazariants,¹ Jonathan

5 Seguin,¹ Victor Golyaev,¹ Laurent Farinelli,² Mikhail M. Pooggin¹,³

6

7 ¹University of Basel, Department of Environmental Sciences, Basel, Switzerland;

8 ²Fasteris SA, Plan-les-Ouates, Switzerland;

9 ³INRA, UMR BGPI, Montpellier, France.

10

11

12 N. Malpica-López. and R. Rajeswaran are joint first authors.

13

14 Corresponding author: Mikhail M. Pooggin; E-mail: mikhail-pooggin@inra.fr.

15
16 **ABSTRACT**

17 Tobamoviral replicase possesses an RNA-dependent RNA polymerase (RDR) domain
18 and is translated from genomic (g)RNA via a stop codon readthrough mechanism at a
19 one-to-ten ratio relative to a shorter protein lacking the RDR domain. The two proteins
20 share methyltransferase and helicase domains and form a heterodimer implicated in
21 gRNA replication. The shorter protein is also implicated in suppressing RNA silencing-
22 based antiviral defenses. Using a stop codon mutant of Oilseed rape mosaic tobamovirus
23 (ORMV), we demonstrate that the readthrough replicase (p182) is sufficient for gRNA
24 replication and for subgenomic RNA transcription during systemic infection in *Nicotiana
25 benthamiana* and *Arabidopsis thaliana*. However, the mutant virus displays milder
26 symptoms and does not interfere with HEN1-mediated methylation of viral short
27 interfering (si)RNAs or plant small (s)RNAs. The mutant virus tends to revert the stop
28 codon, thereby restoring expression of the shorter protein (p125), even in the absence of
29 plant Dicer-like activities that generate viral siRNAs. Plant RDR activities that generate
30 endogenous siRNA precursors do not prevent replication or movement of the mutant
31 virus, and double-stranded precursors of viral siRNAs representing the entire virus
32 genome are likely synthesized by p182. Transgenic expression of p125 partially
33 recapitulates the ORMV disease symptoms associated with overaccumulation of plant
34 sRNAs. Taken together, the readthrough replicase p182 is sufficient for viral replication
35 and transcription but not for silencing suppression. By contrast, the shorter p125 protein
36 suppresses silencing, provokes severe disease symptoms, causes overaccumulation of
37 unmethylated viral and plant sRNAs but it is not an essential component of the viral
38 replicase complex.

39
40 INTRODUCTION

41 Tobamoviruses are positive-sense RNA viruses replicating via a negative-sense RNA
42 intermediate that serves as a template for the synthesis of their genomic RNA (gRNA)
43 and the transcription of two subgenomic RNAs (sgRNAs) (Buck et al. 1999; Ishikawa
44 and Okada 2004). The viral coat protein (CP) and the movement protein (MP) are both
45 translated from these sgRNAs (sgRNA-CP and sgRNA-MP), while viral replication-
46 associated proteins are synthesized directly from gRNA. The tobamoviral replicase
47 polypeptide (178-183 kDa) is translated from an ORF coding for a shorter 122-130 kDa
48 protein, via a stop codon readthrough mechanism that engages ca. 10% of arriving
49 ribosomes and adds a C-terminal RNA-dependent RNA polymerase (RdRP, designated
50 here as viral RDR) domain. The shorter protein, which is present at an approximately 10-
51 fold molar excess over the readthrough replicase (Watanabe et al. 1999), shares with it
52 methyl transferase/guanylyl transferase and helicase domains that play a role in
53 replication and capping of gRNA and sgRNAs. Both proteins associate into membrane-
54 bound heterodimers that participate in viral RNA replication (Osman and Buck 1996;
55 Watanabe et al. 1999; Hagiwara et al. 2003; Komoda et al. 2007; Nishikiori et al. 2012).
56 The excessive shorter protein also forms soluble oligomers (Watanabe et al. 1999).
57 Notably, non-membrane-bound (but not membrane-bound) fraction of these proteins has
58 been implicated in the suppression of RNA silencing-based plant antiviral defenses
59 (Hagiwara-Komoda et al. 2008).

60 RNA silencing is a sequence-specific mechanism that regulates gene expression and
61 defends against invasive nucleic acids such as transposons, transgenes and viruses in
62 most eukaryotes (Ghildiyal and Zamore 2009). In plants, RNA silencing is directed by
63 21-24 nucleotide (nt) small RNAs (sRNA), classified into short interfering RNAs
64 (siRNA) and microRNAs (miRNA), which are produced as duplexes with 2-nt 3'-
65 overhangs from longer double-stranded RNA (dsRNA) precursors or hairpin-like
66 secondary structures, respectively, by Dicer-like (DCL) enzymes. Both strands of each
67 sRNA duplex are methylated at the 2'-hydroxyl of their 3'-terminal nucleotide by
68 methyltransferase HEN1 (Yu et al. 2005; Yang et al. 2006) and then sorted, based mostly
69 on size and the identity of their 5'-terminal nucleotide, by Argonaute (AGO) family
70 proteins to form RNA-induced silencing complexes (RISCs) (Mi et al. 2008; Havecker et
71 al. 2010). Mature RISCs contain a single-stranded sRNA that guides AGO towards
72 complementary RNA targets to silence gene expression post-transcriptionally through
73 AGO-sRNA-directed cleavage or translational repression of the target mRNA, or
74 transcriptionally through AGO-sRNA-directed DNA methylation and chromatin
75 modification (Havecker et al. 2010). Gene silencing directed by both miRNAs and
76 siRNAs can, in some cases, be reinforced by plant RNA-dependent RNA polymerase
77 (RDR) enzymes that generate dsRNA precursors for secondary siRNAs (Wassenegger
78 and Krczal 2006; Howell et al. 2007; Rajeswaran and Pooggin 2012; Rajeswaran et al.
79 2012).

80 In plants, RNA silencing pathways are extremely diverse, involving multiple
81 combinations of RDRs, AGOs and DCLs of differing specificities. For instance, the
82 genome of *Arabidopsis thaliana* encodes six RDRs, ten AGOs and four DCLs. Although
83 the latter can all process RDR-independent dsRNA (Fusaro et al. 2006; Blevins et al.
84 2011), they do exhibit differences regarding substrate preference (DCL1 processes
85 preferentially Pol II-transcribed *MIR* gene transcripts, while DCL2, 3 and 4 prefer RDR6-
86 or RDR2-dependent dsRNA precursors) and product size (21-22 nt miRNA duplexes for
87 DCL1, 22-nt siRNA for DCL2, 24-nt siRNA for DCL3 and 21-nt siRNA for DCL4)
88 (Bouché et al. 2006; Howell et al. 2007; Mlotshwa et al. 2008; Rajeswaran et al. 2012;
89 Parent et al. 2015; Blevins et al. 2015; Zhai et al. 2015). It is therefore unsurprising that,
90 although all four *A. thaliana* DCLs are involved in the generation of viral siRNAs
91 (Fusaro et al. 2006; Bouché et al. 2006; Blevins et al. 2006; Diaz-Pendon et al. 2007),
92 their impact on antiviral defense depends on the identity of the infecting virus. RNA
93 viruses with cytoplasmic replication/transcription (such as tobamoviruses) are targeted
94 mainly by DCL4 and, in some cases, DCL2 (Xie et al. 2004; Fusaro et al. 2006; Bouché
95 et al. 2006; Blevins et al. 2006; Andika et al. 2015), while DNA viruses with nuclear
96 replication and/or transcription are targeted by DCL3 and DCL1, in addition to DCL4
97 and DCL2 (Akbergenov et al. 2006; Blevins et al. 2006; Blevins et al. 2011; Aregger et
98 al. 2012).

99 A similar picture has emerged regarding other components of the RNA silencing
100 machinery: RDR1, RDR2 and RDR6 have been implicated in defense against RNA
101 viruses, albeit the relative contribution (if any) of these RDRs to the biogenesis of viral
102 secondary siRNAs appears to depend on viral genus, strain, and/or mutant variant (Yu et
103 al. 2003; Yang et al. 2004; Qu et al. 2005; Schwach et al. 2005; Qi et al. 2009; Vaistij and
104 Jones 2009; Donaire et al. 2008; Wang et al. 2010; Garcia-Ruiz et al. 2010; Wang et al.
105 2011; Lee et al. 2016), while neither RDR1, RDR2 nor RDR6 appear to play a significant
106 role in the biogenesis of DNA virus-derived siRNAs in *A. thaliana* (Blevins et al. 2011;
107 Aregger et al. 2012). Likewise, among multiple AGOs implicated in plant antiviral
108 defense mechanisms (Carbonell and Carrington 2015), AGO2 seems to play a major role
109 (Harvey et al. 2011; Scholthof et al. 2011; Ghoshal and Sanfaçon 2014; Ma et al. 2015;
110 Odokonyero et al. 2015), owing to its ability to bind viral siRNAs (Harvey et al. 2011;
111 Wang et al. 2011) and form antiviral RISCs with cleavage/slicer activity (Garcia-Ruiz et
112 al. 2015), while AGO1 may play both direct and indirect roles via its association with
113 viral siRNAs (Wang et al. 2011; Garcia-Ruiz et al. 2015) or plant miRNAs, respectively:
114 in the latter case, AGO1-miR403-directed repression of *AGO2* mRNA controls the
115 accumulation of AGO2 protein and hence its antiviral activity (Harvey et al. 2011).

116 In order to counteract these defense mechanisms, most plant viruses have evolved anti-
117 silencing strategies targeting different steps of the biogenesis and/or action of viral
118 siRNAs as well as plant miRNAs (Csorba et al. 2015). One such strategy involves the
119 induction of miR168 (Várallyay et al. 2010; Várallyay and Havelda 2013), which
120 participates in *AGO1* mRNA repression via a feedback loop involving AGO1 itself
121 (Vaucheret et al. 2006), while another, very common strategy employed by RNA viruses
122 involves the binding of sRNA duplexes by a viral suppressor protein, preventing their
123 methylation by HEN1 and/or RISC assembly, as shown in *A. thaliana* and *N.
124 benthamiana* (Lakatos et al. 2006; Mérai et al. 2006; Lózsa et al. 2008). The shorter
125 tobamoviral replication protein appears to be one such RNA silencing suppressor, as
126 demonstrated by transient assays in *N. benthamiana* with 130 kDa protein (p130) of
127 *Tomato mosaic virus* (ToMV) (Kubota et al. 2003), p126 of *Tobacco mosaic virus*
128 (TMV) (Ding et al. 2004; Vogler et al. 2007) and TMV-Cg (Kurihara et al. 2007), and
129 p122 of crucifer-infecting TMV (Csorba et al. 2007). p122 and p126 from two different
130 crucifer-infecting tobamoviruses were shown to bind sRNA duplexes (Kurihara et al.
131 2007; Csorba et al. 2007), which might inhibit their methylation and subsequent RISC
132 assembly (Csorba et al. 2007). Interestingly, sRNA duplex-binding activity was exhibited
133 only by a non-membrane-bound form of the ToMV replicase complex (Hagiwara-
134 Komoda et al. 2008). In any case, since antisilencing activities of the shorter replication
135 protein have so far been demonstrated in transient assays or *in vitro*, it remains unclear
136 whether during tobamovirus infection this protein alone is sufficient for suppressing
137 HEN1-mediated methylation and antiviral silencing, or if it needs to form a heterodimer
138 with the longer readthrough replicase. Likewise, it remains unknown whether the longer
139 protein can also suppress silencing on its own.

140 *Oilseed rape mosaic virus* (ORMV) (Aguilar et al. 1996), also known as *Youcai mosaic virus*, *Ribgrass mosaic virus* or *Chinese rape mosaic virus*, is a tobamovirus that can establish systemic infection and cause severe disease in both *A. thaliana* and *N. benthamiana* (Mansilla et al. 2009; Seguin et al. 2014). In *A. thaliana*, ORMV interferes with HEN1-mediated methylation of viral siRNAs (Akbergenov et al. 2006; Blevins et al. 2006), leading to the accumulation of non-methylated viral siRNAs and plant sRNAs including miRNAs and transacting siRNAs (tasiRNAs), as well as their passenger strands (miRNA* and tasiRNA*), which are normally degraded (Akbergenov et al. 2006; Blevins et al. 2006; Vogler et al. 2007; Hu et al. 2011). However, the precise roles played by the two ORMV replicase components (the shorter protein p125 and the readthrough replicase p182) in this phenomenon have not been determined. Also, although it has been shown that ORMV siRNA biogenesis in *A. thaliana* (Columbia-0 [Col-0]) plants depends largely on DCL4 and is independent of RDR2 and RDR6 (Blevins et al. 2006), further detail on the mechanisms of antiviral silencing and its suppression during ORMV infections is lacking. Therefore, we have constructed a p125-deficient mutant of ORMV and used it to dissect the function of both the p125 and p182 proteins in *A. thaliana* and its siRNA biogenesis-deficient mutant lines as well as in *N. benthamiana*, employing blot hybridization and deep sequencing of sRNAs to characterize their impact on the RNA silencing machinery.

159 RESULTS

160 To better understand the induction and suppression of antiviral silencing during ORMV
161 infection we performed an in-depth molecular characterization of wild-type *A. thaliana*
162 (Col-0) and of the siRNA biogenesis-deficient triple mutant lines *dcl234* (*dcl2-5 dcl3-1 dcl4-2*) (Blevins et al. 2006) and *rdr126* (*rdr1-1 rdr2-1 rdr6-15*) (Blevins et al. 2011) at 163 5, 14 and 22 days post inoculation (dpi) with wild-type ORMV or a p125 stop codon 164 mutant (UAG-to-UAC) derivative expressing p182 but not p125 (Fig. 1E). The plants 165 were inoculated either with ORMV sap or with *in vitro* transcripts of viral gRNA from 166 wild-type and mutant virus clones, designated W41 and M41, respectively (Materials and 167 Methods; Fig. 1E; Supplementary Fig. S1A). Viral RNAs and proteins were analyzed by 168 Northern (Fig. 1A and 1C) and Western (Fig. 1F) blotting respectively, while viral and 169 endogenous sRNAs were analyzed by blot hybridization (Fig. 1B and 1D) and then by 170 Illumina sequencing. 171

172 **Double-stranded intermediates of viral replication appear to be major substrates for**
173 **DCLs.**

174 At all three time points, viral gRNA and sgRNAs accumulated to higher levels in *dcl234* 175 than in Col-0 plants (Fig. 1A and 1C; Supplementary Fig. S1G), coinciding with much 176 lower levels of viral siRNAs (Fig. 1B and 1D; Supplementary Fig. S1H; see below). This 177 finding is consistent with previous results implicating DCL4 along with DCL2 and DCL3 178 in ORMV siRNA biogenesis and antiviral defense (Blevins et al. 2006). In the *rdr126* 179 host, viral gRNA and sgRNAs also accumulated to higher levels than in Col-0 at 14 and 180 22 dpi (Fig. 1A; Supplementary Fig. S1G), and to comparable levels at 5 dpi (Fig. 1C; 181 Supplementary Fig. S1I). However, despite the absence of three RDRs known to generate 182 plant siRNA precursors, viral siRNA production was not abolished in this background 183 (Fig. 1B and 1D; Supplementary Fig. S1H and S1I; see below). *A. thaliana* has three 184 additional *RDR* genes of unknown function (*RDR3a*, *RDR3b* and *RDR3c*) (Wassenegger 185 and Krczal 2006) whose potential contribution to viral siRNA biogenesis in *rdr126* plants 186 cannot be formally ruled out. However, it would be plausible for DCLs to directly 187 generate ORMV siRNAs from intermediate dsRNA forms arising during gRNA 188 replication and sgRNA transcription. 189

189 A genome-sized long antisense RNA (designated gRNA(-)) likely functioning as a
190 template for gRNA replication and sgRNA transcription, as well as a relatively more
191 abundant short antisense RNA, comparable in size to sgRNA-CP and hence designated
192 CP(-), were detected by means of strand-specific ORMV probes (Fig. 1A and 1C).

193 Previously, dsRNA species with sizes corresponding to viral gRNA and sgRNAs were
194 isolated from TMV-infected plants (Palukaitis et al. 1983). At 5 dpi, gRNA(-) was
195 detectable only in *dcl234* (Fig. 1C), which might suggest that DCLs are targeting dsRNA
196 viral replication intermediates since the earliest stages of viral infection. However, at the
197 later time points (14 and 22 dpi), gRNA(-) accumulation in *dcl234* was not higher than in
198 Col-0 (Fig. 1A).

199 **Small RNA sequencing uncovers mechanisms of viral siRNA biogenesis.**

200 Illumina sequencing of the 19-30 nt fraction from total RNA samples of mock-inoculated
201 and ORMV sap-infected *A. thaliana* Col-0, *rdr126* and *dcl234* plants at 14 and 22 dpi
202 (used for Northern analysis in Fig. 1A) yielded 12 libraries, each containing 3.1 to 7.8
203 million reads (Supplementary Dataset S1). The majority of these reads fell into a size
204 range of 20 to 25 nts, known to be populated by endogenous plant miRNAs and siRNAs
205 as well as viral siRNAs. This size range was selected for further bioinformatics analysis
206 (see Materials and Methods).

207 Zero-mismatch mapping of the sRNA reads to the 120 Mb *A. thaliana* TAIR10 reference
208 genome and the 6.3 Kb ORMV genome revealed that viral sRNAs represented a large
209 fraction (27% at 14 dpi and 23% at 22 dpi) (Fig. 2A; Supplementary Dataset S1) of the
210 20-25 nt sRNA population in ORMV-infected Col-0 plants. This proportion was even
211 larger in ORMV-infected *rdr126* plants (35% at 14 dpi and 31% at 22 dpi) (Fig. 2A).
212 However, since endogenous siRNA biogenesis is diminished in *rdr126*, thereby
213 increasing the proportion of 21-nt reads (mostly representing miRNAs) within the 20-25
214 nt *Arabidopsis* reads (Fig. 2B), we decided to normalize viral reads to the number of 21-
215 nt endogenous *Arabidopsis* reads. The resulting figures were roughly equivalent between
216 *rdr126* and Col-0 at 14 dpi (894,214 vs 908,107) and only slightly higher in *rdr126* at 22
217 dpi (898,249 vs 792,706) (Supplementary Dataset S1). Moreover, the size profiles of
218 viral reads did not differ significantly between *rdr126* and Col-0; in both cases, 21-nt
219 reads predominated (67-70% of 20-25 nt viral reads), followed by 20-nt (13-14%) and
220 22-nt (7-9%) reads (Fig. 2B). Likewise, no substantial differences in the 5'-nucleotide
221 profile (Supplementary Dataset S1) or viral genome distribution (Fig. 3; see below) of
222 viral reads were observed between Col-0 and *rdr126* at any time point. Taken together, 223 our findings indicate that RDR1, RDR2 and RDR6 are not essential for viral siRNA 224 biogenesis.

225 The size profile of endogenous sRNAs changed significantly upon ORMV infection: at 226 both 14 dpi and 22 dpi, 21-nt reads became the most abundant, surpassing the 24-nt reads 227 that dominated in mock-inoculated Col-0 plants (Fig. 2B, Supplementary Dataset S1). A 228 similar shift in sRNA size profiles was previously reported for ORMV-infected *A*. 229 *thaliana* at 7 dpi, where it was found to correlate with overaccumulation of many 230 miRNAs as well as miRNA* and siRNA* passenger strands (Hu et al. 2011), and at 22 231 dpi in an earlier study (Blevins et al. 2006). We validated these findings for selected 232 miRNAs and tasiRNAs by blot hybridization (Fig. 1B and 1D; Supplementary Fig. S1H). 233 Indeed, the accumulation of miR168, miR472, miR472*, and siR255* was much higher 234 in ORMV-infected than mock-inoculated plants at both 14 and 22 dpi, while extra bands 235 of longer and/or shorter size appeared for miR403 and miR173 in ORMV-infected plants 236 (Fig. 1B). Similar (although less pronounced) effects were observed for miR168, 237 miR472*, and siR255* at 5 dpi (Fig. 1D; Supplementary Fig. S1I). Notably, the levels of 238 both miRNA and miRNA* were comparable between Col-0, *dcl234* and *rdr126* plants 239 (Fig. 1B and 1D; Supplementary Fig. S1H and S1I). The latter finding indicates that 240 endogenous RDRs are not involved in the upregulation of miRNA and miRNA* species 241 triggered by ORMV infection, and validates the normalization procedure followed here 242 for comparing viral siRNA levels between siRNA-deficient mutant lines and Col-0.

243 The number of viral sRNA reads was substantially lower in ORMV-infected *dcl234*
244 plants compared to Col-0 or *rdr126* individuals. In this genetic background, viral reads
245 represented only 11% and 12% of 20-25 nt reads at 14 and 22 dpi respectively which,
246 when normalized per million *Arabidopsis* 21-nt reads, represents frequencies 4- and 3-
247 fold lower than those of the corresponding time points in *rdr126* or Col-0 plants (Fig. 2B;
248 Supplementary Dataset S1). The size profile of viral reads in *dcl234* plants was different
249 as well at the two time points, with 21-nt reads constituting only 23% of 20-25 nt viral
250 reads, i.e., 3 times lower than in *rdr126* or Col-0 plants (Fig. 2B). This is consistent with
251 previous results implicating DCL4 in the biogenesis of 21-nt viral siRNAs in ORMV-
252 infected *Arabidopsis* (Blevins et al. 2006). However, our finding that viral siRNA
253 production was not abolished in *dcl234* plants indicates that in this species the viral
254 siRNA precursors can also be processed, although in a less efficient and precise manner,
255 by DCL1 or some other RNase III-like endonuclease(s) that produces an sRNA
256 population of broader and more evenly distributed size classes, exhibiting comparable
257 numbers of 20-, 22-, 23-, 24- and 25-nt reads in addition to the slightly more abundant
258 21-nt reads (Fig. 2B). Note that the Illumina protocol used in our study only sequences
259 RNA molecules bearing 5'-monophosphate and 3'-hydroxyl groups, which are typical of
260 DCL and RNase III cleavage products.

261 An inspection of single-nucleotide resolution maps of ORMV-derived 20-25 nt reads
262 revealed that viral siRNAs span the entire virus genome in both sense and antisense
263 orientations without gaps in any strand (Supplementary Dataset S3; Fig. 3). No strong
264 strand bias of total 20-25 nt reads was observed in Col-0 or *rdr126* plants (Fig. 3).
265 Furthermore, no substantial difference in the sRNA maps for individual size classes was
266 observed between the wild-type Col-0 and the *rdr126* mutant lacking RDR1, RDR2, and
267 RDR6 at both 14 and 22 dpi (Fig. 3; Supplementary Dataset S3), supporting our
268 hypothesis that viral siRNAs are processed from RDR-independent dsRNA precursors. In
269 contrast, a strong bias towards gRNA(+) polarity was observed in *dcl234* plants, where
270 sense reads exceeded antisense reads ca. 9-fold at both 14 dpi (324,360 vs 34,565) and 22
271 dpi (420,344 vs 46,262). Interestingly, most antisense reads belonged to the 21-nt size
272 class (Fig. 3) or the less abundant 20-nt size class, while sense reads were more evenly
273 distributed across other size classes (Supplementary Dataset S2), pointing at possible
274 involvement of DCL1 in production of viral sRNAs in the absence of other DCLs (see
275 Discussion). Interestingly, in Col-0 and *rdr126* plants (possessing all four DCLs) a strong
276 sense strand bias was only detected among the low abundance 23-nt, 24-nt and 25-nt
277 reads (Supplementary Datasets S1 and S2; Fig. 3).

278 **p125 is not essential for viral replication or movement but has a strong impact on**
279 **silencing suppression and disease severity.**

280 Viral silencing suppressors can interfere with the antiviral activities of specific
281 components of the RNA silencing machinery, which explains why the susceptibility of
282 wild-type plants to certain viruses is often undistinguishable from that of mutant plants
283 lacking the targeted components. For instance, it was only by using suppressor-deficient
284 mutants of *Cucumber mosaic virus* (CMV) and *Turnip mosaic virus* (TuMV) that the
285 involvement of RDR6 and RDR1 in the biogenesis of secondary viral siRNAs and
286 antiviral defense in *Arabidopsis* was demonstrated (Wang et al. 2010; Garcia-Ruiz et al.
287 2010; Wang et al. 2011). Therefore, in an effort to dissect the involvement of the short
288 and long (p125 and p182, respectively) components of the ORMV replicase complex in
289 silencing suppression, we generated a mutant virus that can synthesize p182 but not p125
290 by changing the stop codon of the latter from TAG to TAC (which codes for tyrosine, the
291 amino acid normally incorporated at this position via translational readthrough in other
292 tobamoviruses) (Fig. 1E). The mutation was introduced into ORMV infectious clone
293 W41, where the 630 nt consensus viral sequence (Seguin et al. 2014) is placed under
294 control of a T7 RNA polymerase promoter, as well as into its W4 derivative, which
295 carries a non-consensus T-to-G substitution at position 5612 (Supplementary Fig. S1A),
296 obtaining the mutant clones M41 and M4. The parental clones W41 and W4 produce,
297 upon delivery into *A. thaliana* Col-0 or *N. benthamiana* plants via inoculation of *in vitro*
298 transcripts, a disease whose symptoms and severity (Supplementary Fig. S1A-S1C) are
299 similar to those appearing after inoculation with wild-type ORMV virions (Hu et al.
300 2011; Seguin et al. 2014; and data not shown).

301 Following inoculation into *A. thaliana* Col-0, *rdr126* and *dcl234* plants, clones M41 and
302 M4 exhibited lower virulence than their parental constructs, producing less severe
303 symptoms by 22 dpi (Supplementary Fig. S1C), which correlated with a reduced
304 accumulation of viral gRNA(+) (Supplementary Fig. S1G). RT-PCR/sequencing analysis
305 revealed that in all the M41- and M4-infected Col-0, *rdr126* and *dcl234* plants of that
306 time point, a p125 stop codon had reappeared in most viral gRNA molecules via an
307 UAC-to-UAA reversion, and only a small fraction of viral RNA still carried the mutant
308 UAC codon at this position (Supplementary Fig. S1D-S1F). These results confirm earlier
309 findings in TMV, where a similar UAG to UAU mutation at the stop codon was reverted
310 *in vivo* to UAA (Ishikawa et al. 1986). The stop codon reversions restored p125 protein
311 expression, which accumulated by 22 dpi at the levels comparable to those from the wild
312 type virus (W41) in all the plants (Fig. 1F; lower blot).
313 It is notable that even the impairment in siRNA biogenesis in *rdr126* and *dcl234* plants
314 (see e.g. the tasiRNA siR255 in Fig. 1B and 1D and the repeat-associated siRNA siR1003
315 in Supplementary Fig. S1G) did not compensate for the strong selective disadvantage
316 suffered by p125-deficient viruses, as evidenced by their displacement by stop codon
317 revertants along the course of the infection. However, the p125-deficient mutants could
318 replicate and move systemically during the early stages of infection, because no p125
319 expression was detected in systemic leaves of M41-infected Col-0, *rdr126* or *dcl234*
320 plants at 5 dpi (Fig. 1F; upper blot, lanes 2, 5, 8; Supplementary Fig. S1I). Consistently,
321 no reversion of the stop codon was detected by deep sequencing and bioinformatic
322 analysis of M41 gRNA-derived small RNAs at 5 dpi (see below). The gRNA and
323 sgRNAs of M41 accumulated in all these plants, albeit at lower levels than in W41-
324 infected plants (Fig. 1C, compare lanes 2, 5, 8 with lanes 3, 6, 9). Notably, mutant virus
325 RNAs did not accumulate to higher levels in *rdr126* than in Col-0 (Fig. 1C;
326 Supplementary Fig. S1I), indicating that replication and/or systemic movement of the
327 p125-deficient virus are not promoted in the absence of RDR1, RDR2 and RDR6. On the
328 other hand, they accumulated to higher levels in *dcl234* than in Col-0 (Fig. 1C),
329 indicating that the three DCLs substantially inhibit replication and/or systemic movement
330 of the p125-deficient virus. Viral gRNA accumulated to comparable levels in M41- and
331 W41-infected *dcl234* plants (Fig. 1C, lane 8 vs 9), as was the p182 protein (Fig. 1F,
332 upper blot, lane 8 vs 9), which, by contrast, was barely detectable in M41-infected Col-0
333 and *rdr126* plants at 5 dpi (Fig. 1F, upper blot, lanes 2, 5; Supplementary Fig. S1I).
334 Hence, p125-deficient viruses replicated gRNA and translated p182 more efficiently
335 when DCL-mediated viral siRNA biogenesis was impaired.

336 To further examine whether p125 might affect viral siRNA biogenesis by inhibiting the
337 activity of plant RDR1, RDR2 or RDR6, and whether p182 alone has any influence on
338 the plant sRNA-generating machinery, we deep-sequenced sRNAs from Col-0 and
339 *rdr126* plants systemically infected with M41 or W41 at 5 dpi. Bioinformatic analysis of
340 viral sRNA reads revealed no stop codon reversion, confirming that the mutant virus
341 population had not yet been overtaken by p125 stop codon revertants at this time point.
342 W41-derived sRNAs comprised a large fraction of the 20-25 nt reads in both Col-0 and
343 *rdr126*, even larger than that of endogenous *Arabidopsis* sRNAs reads (Fig. 4A). When
344 normalized to 21-nt endogenous *Arabidopsis* reads, the accumulation of viral siRNAs
345 was similar in Col-0 (2,800,511) and *rdr126* plants (2,852,744) (Supplementary Dataset
346 S1). Moreover, an analysis of viral sRNA size classes (Fig. 4B), single nucleotide
347 resolution maps (Fig. 5; Supplementary Dataset S3) and 5'-nucleotide profiles
348 (Supplementary Dataset S1) did not find any significant difference between the ORMV-
349 derived siRNAs of Col-0 and *rdr126* plants, indicating that RDR1, RDR2 and RDR6 do
350 not play a significant role in the generation of siRNAs from wild-type virus at 5 dpi and
351 thus extending the scope of our above-described findings for 14 and 22 dpi. Mutant
352 (M41) virus-derived sRNAs accumulated to lower levels than W41-derived sRNAs in
353 both Col-0 and *rdr126* (Fig. 4A; Fig. 1D; Supplementary Fig. S1I), which correlated with
354 the lower amount of viral long RNAs in the corresponding samples (Fig. 1C). Although
355 the normalized numbers of viral 20-25 nt reads for M41 in *rdr126* (181,155) were 2.5-
356 fold lower than in Col-0 (442,742), which might be interpreted as a sign of RDR
357 involvement in the production of secondary viral siRNAs in wild-type plants, as argued
358 in earlier studies (Wang et al. 2010; Garcia-Ruiz et al. 2010; Wang et al. 2011), it should
359 be pointed out that the amount of M41 long RNAs from which siRNAs are derived is also
360 approximately 3-fold lower in *rdr126* than in Col-0 (Fig. 1C, lanes 5 vs 2;
361 Supplementary Fig. S1I). Furthermore, there are no significant differences between the
362 size profiles (Fig. 4B), single-nucleotide resolution maps (Fig. 5) and 5'-teminal
363 nucleotide identities (Supplementary Dataset S1) of mutant virus-derived sRNAs
364 between Col-0 and *rdr126*. Taken together, our data indicate that at the early stages of
365 infection, host RDR activity does not appear to restrict replication of the p125-deficient
366 mutant virus. Also, viral p125 does not appear to inhibit the activities of any of the three
367 host RDRs during wild-type virus infections, as no differences are apparent between the
368 studied RDR-dependent endogenous siRNAs in wild-type or mutant virus infections (Fig.
369 1D; Supplementary Fig. S1H and S1I).

370 In order to verify and extend the above findings to another host plant species, we tested if
371 p125 deficiency also affects ORMV infection in the solanaceous plant *N. benthamiana*.
372 Inoculation of *N. benthamiana* seedlings with wild-type ORMV virions or with *in vitro*
373 transcribed RNA from W41 or W4 resulted in severe disease symptoms, including stem
374 necrosis and wilting at 5 dpi, followed by rapid plant death within a few days (Seguin et
al. 2014) (Supplementary Fig. S1B). In contrast, inoculation with *in vitro*-transcribed RNA from p125-deficient viruses (M41 or M4) did not result in plant death, although disease symptoms were observed in upper (systemic) leaves of M41- and M4-infected plants at 26 dpi (Supplementary Fig. S1B). The accumulation of viral gRNA and sgRNAs at 5 dpi for M41 and M4 was about 2-fold lower than for W41 and W4, and the accumulation of the mutant virus RNAs were significantly lower at 26 dpi than at 5 dpi, suggesting that plant defense mechanisms dampen virus replication in the absence of p125 (Supplementary Fig. S1J).

Importantly, in systemic leaves of the mutant virus-infected plants no reversion of the mutated stop codon was observed at 5 dpi, and only a small fraction of viral RNA contained a reverted stop codon at 26 dpi (Supplementary Fig. S1E). Thus, the p125-deficient virus is able to replicate and spread systemically in *N. benthamiana* plants. Nonetheless, in one of the three repeated experiments there was detectable reversion of the stop codon and restoration of p125 expression in a single M41-inoculated plant (Supplementary Fig. S1L), and when the sap of M41- or M4-infected plants was used to inoculate new individuals, p125 expression was quickly restored, leading to rapid death of the hosts. Thus, even though p125 is not essential for ORMV infection in *N. benthamiana*, p125-deficient viruses are eventually overtaken by stop codon revertants, although the process takes place at a much slower rate than in *A. thaliana*.

Deep sequencing of sRNAs confirmed that the mutant viruses M41 and M4 did not undergo any substantial reversion of the stop codon at 26 dpi in *N. benthamiana* plants. In wild-type virus (W41)-infected *N. benthamiana* at 5 dpi (before plant death), viral siRNAs were found to be highly abundant, since viral sequences comprise 65% of total 20-25 nt reads (Fig. 4A). In contrast, p125-deficient virus-derived siRNAs accumulated to much lower levels at 26 dpi (9% and 20% of total 20-25 nt reads for M41 and M4, respectively) (Fig. 4A; Supplementary Dataset S1), which correlates with lower levels of gRNA and sgRNAs. An analysis of single-base resolution maps of viral 20-25 nt reads revealed that the two most abundant viral siRNA size classes (21-nt and 22-nt) were equally distributed between both strands alongside the entire virus genome, and that siRNA hotspot patterns were similar between M41 and W41 (Fig. 5; Supplementary
405 Dataset S3). Furthermore, 5'-terminal nucleotide frequency in viral reads was also
406 comparable between M41 and W41 (Supplementary Dataset S1). Thus, despite
407 substantial differences in overall accumulation levels, DCL-mediated processing of viral
408 siRNAs from dsRNA precursors does not appear to be influenced by p125 in N.
409 *benthamiana*, in agreement with our findings for *A. thaliana*.

410 p125 interferes with the methylation of viral and endogenous sRNAs during ORMV
411 infection.

412 It has been shown that ORMV interferes with HEN1-mediated methylation of viral
413 siRNAs as well as of endogenous siRNAs and miRNAs in *A. thaliana* (Akbergenov et al.
414 2006; Blevins et al. 2006). To test whether p125 is responsible for this effect, we assessed
415 the methylation status of sRNAs from W41- and M41-infected plants by a classical β-
416 elimination method. If RNA is not modified at the 3'-terminal nucleotide, β-elimination
417 removes it, leaving a truncated RNA with a 3'-phosphate whose mobility in denaturing
418 15% PAGE is shifted by two nucleotides (Akbergenov et al. 2006) (see non-methylated
419 22-nt RNA (+) control, Fig. 6). HEN1 methylates plant miRNAs and siRNAs at the 2'-
420 hydroxyl of their 3'-terminal nucleotide, which blocks β-elimination. As can be observed
421 (Fig. 6), *A. thaliana* miR168 and miR173, *A. thaliana* siR255 and *N. benthamiana*
422 miR168 and miR482 are methylated in non-infected plants and therefore fully resistant to
423 β-elimination. In contrast, a significant fraction of viral siRNAs and endogenous sRNAs
424 in wild-type virus (W41)-infected *A. thaliana* and *N. benthamiana* was sensitive to β-
425 elimination (Fig. 6, lanes 4, 10; Supplementary Fig. S1I), while both viral and
426 endogenous (miR168 and miR482) sRNAs from *N. benthamiana* plants infected with the
427 mutant virus (M41) were resistant to β-elimination (Fig. 6, lane 6). This indicates that in
428 the absence of p125, the virus is not able to block the methylation of viral siRNAs. Taken
429 together, our data indicate that p182, when expressed in the absence of p125, does not
430 interfere with HEN1-mediated methylation of viral and endogenous sRNAs. Note that in
431 the case of *N. benthamiana*, p182 accumulation in the mutant virus-infected plants was
432 comparable to p125 accumulation in the wild-type virus-infected plants (Supplementary
433 Fig. S1L).
Although it has been shown that in *A. thaliana* HEN1 deficient mutant lines sRNA 434 accumulation is impaired (Yu et al. 2005; Yang et al. 2006), such a phenomenon was not 435 observed in ORMV-infected plants, where HEN1-mediated methylation is partially 436 inhibited. On the contrary, viral siRNAs and many endogenous sRNAs accumulated to 437 much higher levels in W41-infected plants compared to M41-infected plants, and plants 438 infected with wild-type ORMV produced higher-than-normal levels of non-methylated 439 passenger strands from endogenous miRNA and siRNA duplexes (Blevins et al. 2006) 440 (Fig. 6). 441

In order to ascertain whether sRNA-mediated cleavage and degradation of target mRNAs 442 is affected in ORMV-infected plants, we examined the relative accumulation of *AGO1* 443 and *AGO2* mRNAs, known to be targeted by miR168 and miR403, respectively. 444 Northern blot hybridization analysis revealed that the levels of these mRNAs were higher 445 in 14 and 22 dpi ORMV-infected plants than in mock-inoculated controls (Fig. 1A). This 446 finding is consistent with a previous transcriptome profiling study, where ORMV 447 infection of Col-0 plants was found to gradually elevate the levels of *AGO1* and *AGO2* 448 mRNAs at 7, 14 and 21 dpi (Hu et al. 2011). However, a corresponding increase in the 449 levels of the proteins encoded by these mRNAs was not detected by Western blot 450 analysis; rather, the levels of *AGO1* had decreased drastically by 15 dpi (Fig. 1G). Hence, 451 despite the apparent compromise of miRNA-directed mRNA cleavage, the translation of 452 target mRNAs appears to be repressed by the overaccumulation of their miRNAs. Indeed, 453 the degree of miR168 overaccumulation upon ORMV infection is much higher than that 454 of miR403 (Fig. 1B), fitting with the much more pronounced drop in the levels of *AGO1*, 455 compared to *AGO2* protein (Fig. 1G). It is worth noting that ORMV-mediated up- 456 regulation of miR168 depends on p125, as the p125-deficient virus did not up-regulate 457 this miRNA in *A. thaliana* (Fig. 1D; Supplementary Fig. S1I) or *N. benthamiana* (Fig. 6). 458

**Transgenic expression of p125 partially recapitulates viral disease symptoms and impact on plant sRNAs.**

In order to verify that p125 is solely responsible for the enhancement of disease 461 symptoms and the impact on sRNA biogenesis in the context of ORMV infections, we 462 engineered transgenic plants expressing p125 and p182 either individually or in 463
464 combination. To this end, the corresponding coding sequences were subcloned from a
465 full-length ORMV clone or its derivative with the p125 stop codon mutation into the
466 pEarleygate binary vectors 100 (no tag), 201 (HA-tag) and 202 (FLAG-tag) under control
467 of the CaMV 35S promoter (Supplementary Fig. S2C). The resulting constructs were first
468 tested in a classical transient expression assay in leaves of the *N. benthamiana* green
469 fluorescent protein (GFP) transgenic line 16c for their ability to suppress GFP transgene-
470 induced silencing (Supplementary Fig. S2A-C), and then used for stable transformation
471 of *A. thaliana* Col-0 plants (Supplementary Fig. S2D-G).

472 Transient expression of p125 or its derivative with an N-terminal FLAG-tag (p125-FG)
473 suppressed GFP silencing as strongly as potyviral HC-Pro (pHC-Pro) or tombusviral p19,
474 ranked among the strongest silencing suppressors in this system (Hamilton et al. 2002)
475 (Fig. 7B; Supplementary Fig. S2A and S2B). Blot hybridization analysis revealed that the
476 strong suppression of GFP silencing by p125 and pHC-Pro was not associated with a
477 reduced accumulation of GFP transgene-derived siRNAs of sense or antisense polarities
478 (Fig. 7B; Supplementary Fig. S2C), which is consistent with previous research
479 implicating the duplex sRNA-binding activity of both HC-Pro (Garcia-Ruiz et al. 2015)
480 and p125 homologs (Kurihara et al. 2007; Csorba et al. 2007) in silencing suppression.
481 Further supporting this hypothesis, we found that transient expression of either p125,
482 p125-FG or pHC-Pro increased the accumulation of miR482*, which is normally
483 degraded (Fig. 7B; Supplementary Fig. S2C).

484 Phenotyping of *Arabidopsis* transgenic lines carrying each of the nine ORMV replicase
485 constructs revealed that the expression of p125 without tags produced strong
486 developmental abnormalities, resulting in adult plants exhibiting serration of the rosette
487 leaf margins and curling of the stem (Fig. 7C). This phenotype, being reproducible in T2
488 and T3 progenies of two independent transgenic lines (Supplementary Fig. S2D), is
489 remarkably similar to that produced by ORMV infection, the only difference being that
490 the latter produces chlorosis in older leaves (Fig. 7C). The severity of the developmental
491 defects exhibited by p125-transgenic lines was correlated with the accumulation levels of
492 some plant miRNAs (miR168 and miR472) and of the passenger strands of some miRNA
493 (miR472*) and tasiRNA (siR255*) duplexes (Fig. 7E, lanes 2-3). Remarkably, these
494 plant sRNAs also accumulated to high levels in ORMV-infected *A. thaliana* plants (Fig. 1B and 1D). Taken together these findings indicate that p125 is a strong silencing suppressor that interferes with sRNA biogenesis and plant development.

497 In contrast to p125, expression of p182 alone did not suppress GFP silencing or alter plant phenotype and sRNA accumulation, while expression of p182 together with p125 weakened the ability of the latter to suppress GFP silencing and compromise plant development and sRNA biogenesis (Supplementary Fig. S2A-G). However, accumulation of p182 protein was below detection in these assay systems (Supplementary Fig. S2G; and data not shown), which does not allow for definitive conclusions on the role of p182.

## 504 DISCUSSION

505 In this work we investigated the roles of proteins p125 and p182 from the tobamovirus ORMV in viral gRNA replication, sgRNA transcription and suppression of sRNA-directed antiviral silencing during systemic infection in *A. thaliana* and *N. benthamiana*. 506 507 To this end we constructed a mutant virus expressing exclusively p182 but not p125 by eliminating the stop codon preceding the RDR domain of the former, and tested it for infectivity in both wild-type plants and silencing-mutant plants deficient in DCL- and RDR-dependent siRNA biogenesis. Furthermore, we examined the effects of both viral proteins, alone or in combination, on the plant sRNA-generating silencing machinery during their transient expression in *N. benthamiana* leaves and upon stable constitutive expression in *Arabidopsis* transgenic lines. 508 509 510 511 512 513 514

515 Functions of p125 and p182 in viral replication vs silencing suppression.

516 Our analysis of the p125-deficient mutant virus in systemic leaf tissues of *A. thaliana* and *N. benthamiana* revealed that p125 is not essential for gRNA replication or sgRNA transcription. However, gRNA and sgRNAs of the p125-deficient mutant accumulated to levels lower than those of the wild type virus (Fig. 1C, Supplementary Fig. S1I), and stop codon revertants often accumulated and overtook the mutant population, restoring p125 expression (Fig. 1F, Supplementary Fig. S1L). These findings are consistent with previous observations in other tobamoviruses, where mutants expressing exclusively the
523 readthrough replicase do replicate in tobacco protoplasts and plant tissues albeit with a
524 reduced efficiency, and stop codon revertants restoring expression of the shorter protein
525 appear at a high frequency (Ishikawa et al. 1986; Lewandowski and Dawson 2000;
526 Knapp et al. 2001).

527 Theoretically, p125 can facilitate viral replication and/or transcription either indirectly
528 through its silencing suppressor activity, or directly through the formation of a putative
529 heterodimer with p182. In other tobamoviruses, the replicase and guanylyltransferase
530 enzyme activities localize to membrane-bound heterodimers of the corresponding
531 p125/p182 homologs (Osman and Buck 1996; Watanabe et al. 1999; Hagiwara et al.
532 2003; Komoda et al. 2007; Nishikiori et al. 2012), and leftover p125, which is present at
533 molar excess, forms oligomers (Watanabe et al. 1999), which might assume additional
534 roles unrelated to viral gRNA replication and sgRNA transcription. Indeed, non-
535 membrane-bound fraction of the tobamoviral replication proteins have been implicated in
536 silencing suppression via their sRNA binding activities (Hagiwara-Komoda et al. 2008).
537 We demonstrate here that p125 is a strong silencing suppressor in the classical transient
538 expression assay in N. benthamiana 16c plants (Fig. 7B) and that p125 is required for the
539 inhibition of HEN1 activity during ORMV infection (Fig. 6). We favor a scenario in
540 which p125 facilitates viral replication and transcription only indirectly, through its
541 antisilencing activity. In support of this hypothesis, the replication of evolutionarily
542 related potexviruses in the alpha-like virus supergroup, such as Potato virus X, requires
543 only one virus-encoded protein (P1) that is equivalent to the tobamoviral readthrough
544 replicase, and the silencing suppressor activity resides in a separate viral gene (p25)
545 (Bayne et al. 2005). Our data do not support an essential direct role of p125 in ORMV
546 replication, as the p125-deficient virus was clearly able to infect systemically the entire
547 plant without reverting the stop codon at 5 dpi in A. thaliana (Fig. 1C and 1F; and the
548 sRNA sequencing analysis showing no reversion) and up to 26 dpi in N. benthamiana
549 (Supplementary Fig. S1D and S1E; and the sRNA sequencing analysis showing no
550 reversion). Hence, p125 is not essential for viral replication, cell-to-cell movement
551 through plasmodesmata or systemic movement via vascular tissues. It should be noted,
552 however, that our results do not rule out a possibility that p125 may facilitate viral
553 replication by forming a heterodimer with p182.
554 Unlike p125, the readthrough replicase p182 does not appear to possess any silencing suppressor activity. The most compelling evidence for the inability of p182 to suppress antiviral silencing comes from the molecular analysis of virus-derived sRNAs: the p125-deficient mutant virus was not able to interfere with HEN1-mediated methylation of viral siRNAs and plant sRNAs in systemically-infected *N. benthamiana* plants (Fig. 6), where p182 accumulation was detectable and comparable to p125 accumulation in wild type virus-infected plants (Supplementary Fig. S1K). Additionally, we did not observe silencing of GFP or impacts on plant small RNAs when we attempted to express the p182 protein transiently or stably, although we did not observe expression of the protein by Western blotting with anti-p125 antibody (Supplementary Fig. S2G; and data not shown).

564 Why is it that p182, containing the entire p125 polypeptide, is not able to suppress silencing? The C-terminal RdRP/RDR domain of p182, which is missing in p125, may plausibly inhibit a potential anti-silencing activity residing elsewhere on the protein. Moreover, p125-mediated suppression of antiviral silencing likely takes place in the cytoplasm, where soluble p125 may bind sRNA duplexes and interfere with the activity of HEN1, preventing RISC assembly. Being localized in a membrane-bound replication complex, p182 may not be available for other functions, unless it is overexpressed. During viral infection, p125 is produced in a ca. ten-to-one molar excess over p182 (Fig. 1F; Supplementary Fig. S1L), which would ensure that sufficient p125 is available in the cytoplasm to exert its antisilencing activity. p125 is likely the first viral protein to be translated from gRNA in a virus-infected cell, followed by p182 via p125 stop codon readthrough and then by replication of the gRNA from which both p125 and 182 were translated. It is tempting to speculate that the overexpression of p125 at the earliest stage of the viral replication cycle has the primary objective of ensuring that any primary viral siRNAs arising from the processing of gRNA replication and sgRNA transcription dsRNA intermediates by DCL activities are promptly bound and sequestered.

580 The biogenesis of viral siRNAs in ORMV-infected plants.

581 Our data from deep sequencing of viral siRNAs (Figs. 2-5) and blot hybridization (Fig. 1) support the hypothesis that viral dsRNA replication and transcription intermediates, which span the entire ORMV genome, are already targeted by DCLs at the early stages of
584 viral infection. First, both gRNA and gRNA(-) as well as sgRNA-CP and CP(-)
585 accumulate to higher than wild-type levels at 5 dpi in *A. thaliana* dcl234 (Fig. 1C), where
586 viral siRNA generation is impaired. Second, the population of virus-derived siRNAs in
587 wild-type *A. thaliana* or *N. benthamiana* does not exhibit any site preference or strand
588 bias throughout the ORMV genome, and this viral siRNA profile remains unaltered in
589 *Arabidopsis rdr126* plants lacking functional RDR activities (Figs. 2-5). Thus, DCLs
590 appear to use directly dsRNA intermediates of viral gRNA replication (and possibly
591 sgRNA transcription) for their processing into siRNAs. A similar mechanism whereby
592 DCL directly process viral dsRNA replication intermediates has previously been
593 proposed not only for other positive sense plant RNA viruses, such as the potyvirus
594 TuMV (Garcia-Ruiz et al. 2010) and the cucumovirus CMV (Wang et al. 2010; Wang et
595 al. 2011), but even for a positive-sense RNA virus in *Drosophila melanogaster*, where
596 RDR genes do not exist (Flynt et al. 2009).

597 The analysis of the genetic requirements for viral siRNA biogenesis in ORMV-infected
598 *A. thaliana* [(Blevins et al. 2006) and this study], coupled with viral sRNA profiling by
599 deep sequencing in this study, confirms the notion that DCL4 is the primary antiviral
dicer producing 21-nt viral siRNAs in this host, while DCL2 and DCL3 assume the role
600 of secondary dicers producing 22-nt and 24-nt viral siRNAs respectively in the absence
601 of DCL4 activity (Xie et al. 2004; Fusaro et al. 2006; Bouché et al. 2006; Blevins et al.
602 2006). Surprisingly, the comparative analysis performed here between ORMV-infected
603 Col-0 and *dcl234* plants revealed a strong positive-strand bias in *dcl234* for all major
604 sRNA size-classes except for 21-nt class where a much weaker bias was
605 observed(Supplementary Datasets S1 and S2; Fig. 3). A strong positive strand bias was
606 observed in Col-0 only for minor size classes such as 24-nt class (Fig. 3; Supplementary
607 Datasets S1 and S2). This finding implies that in *dcl234*, viral dsRNAs are processed into
608 21-nt siRNA duplexes, presumably by DCL1, whereas viral single-stranded RNAs of
609 positive sense polarity, i.e., gRNA(+) and sgRNAs, give rise to single-stranded sRNAs of
610 all sizes, presumably through random cleavage by DCL1 or other RNase III-like
611 endonuclease(s). The processing of ORMV siRNA precursors in *dcl234* plants, whether
612 by DCL1 or another RNase III-like activity, appears to be less efficient and precise,
613 giving rise to sRNAs of a broader size range with a strong positive-strand bias, in
614
addition to 21-nt siRNA duplexes (Fig. 2B; Fig. 3). DCL1 has previously been shown to be involved in viral siRNA biogenesis for the DNA viruses *Cabbage leaf curl geminivirus* (Blevins et al. 2006; Aregger et al. 2012) and *Cauliflower mosaic pararetrovirus* (Blevins et al. 2006; Blevins et al. 2011), using *A. thaliana* triple and quadruple *dcl* mutants. Notably, in the latter case, DCL1 produced a major fraction of 21-nt siRNAs from a viral dsRNA decoy (Blevins et al. 2011). A candidate RNase III-like activity that might also process ORMV siRNA precursors would be RTL1 and/or other members of the recently characterized *A. thaliana* RTL1 gene family (Shamandi et al. 2015; Elvira-Matelot et al. 2016). Interestingly, RTL1 can be induced by viral infection and inactivated by viral silencing suppressors, and its transgenic overexpression in *A. thaliana* interferes with the activity of DCL2, DCL3 and DCL4 by targeting their endogenous dsRNA substrates (Shamandi et al. 2015).

A strong sense strand bias has been previously reported for viral siRNAs produced in wild-type plants infected with some positive sense RNA viruses, leading to the hypothesis that the main substrates for DCL4 and other DCLs are secondary structures formed by viral RNAs [see e.g., (Molnár et al. 2005)]. Our findings in ORMV-infected *Arabidopsis* wild type and *rdr126* plants at 5, 14 or 22 dpi and *N. benthamiana* plants at 5 or 26 dpi, along with previous findings for ORMV- and TMV-infected *Arabidopsis* plants at 2 and 4 dpi (Zavallo et al. 2015), do not support this hypothesis. However, we found that ORMV-infected *Arabidopsis dcl234* plants do exhibit a strong positive-strand bias. Perhaps the inactivation of the siRNA-generating DCLs by viral suppressors may unmask DCL1 or another RNase III-like enzyme that randomly cleaves abundant viral single-stranded RNAs of positive-sense polarity, although such bias might also arise from technical issues with certain sRNA cloning, sequencing and detection protocols (Smith et al. 2010; Harris et al. 2015).

Our analysis of wild-type and p125-deficient viruses in Col-0 and *rdr126* plants suggests that neither RDR1, RDR2 nor RDR6 are directly involved in the process whereby viral dsRNA gets processed into siRNAs, implying that most ORMV-derived siRNAs are primary, RDR-independent species. It should be noted, though, that despite the similar viral siRNA profiles of Col-0 and *rdr126* plants (Figs. 2-5), the latter exhibited higher
645 levels of wild-type ORMV gRNA by the late stages of infection (Fig. 1A; Supplementary
646 Fig. S1G), suggesting that RDR-dependent secondary siRNAs may contribute to defense
647 against the wild-type virus. Earlier studies of suppressor-deficient mutants of RNA
648 viruses CMV and TuMV in Col-0 vs *rdr*-mutant plants revealed RDR1- and/or RDR6-
649 dependent secondary viral siRNAs restricting replication and systemic infection of the
650 mutant viruses (Wang et al. 2010; Garcia-Ruiz et al. 2010; Wang et al. 2011). In the
651 present case, however, the lack of substantial differences between Col-0 and *rdr126*
652 plants regarding global and local profiles of viral siRNA sizes, 5'-nucleotide identities
653 and hot-spots along the wild type or p125-mutant ORMV genome indicates that RDR1
654 and RDR6 may not contribute to viral siRNA biogenesis, although the quantitative
655 differences in viral siRNA accumulation were observed. Presumably the absence of RDR
656 activities facilitates ORMV replication only indirectly, through the loss of endogenous
657 plant siRNAs such as tasiRNAs and heterochromatic siRNAs that may normally
658 downregulate the expression of gene products that facilitate viral replication. Still, it
659 should be noted that replication of the p125-deficient virus was not facilitated in *rdr126*
660 compared to Col-0 plants (Fig. 1C; Supplementary Fig. S1I). An interesting possibility
661 would be that, in the absence of functional RDRs, p125-deficient virus might be
662 preferentially targeted by an RNA decay mechanism known to compete with RDRs for
663 aberrant RNA substrates (Christie et al. 2011). In line with this hypothesis, both TMV
664 infection and transgenic expression of TMV MP or CP genes induce the plant RNA
665 decay genes (Conti et al. 2017).

666 **Effect of p125 on the production, modification and activity of viral siRNAs and**
667 **plant siRNAs and miRNAs.**

668 By comparing wild-type and p125-deficient mutant viruses, we demonstrated that p125
669 does not block the production of viral siRNAs; rather, viral siRNAs are produced at much
670 higher levels in the presence of p125, concomitant with higher levels of viral gRNA
671 replication, sgRNA transcription and increased disease severity. This suggests that virus-
672 derived sRNAs are failing to restrict viral replication and transcription. We found that in
673 the presence of p125, viral siRNAs become sensitive to β-elimination, although this does
674 not lead to their destabilization, as expected based on the fact that endogenous siRNAs
675 become unstable in *Arabidopsis* mutant lines lacking HEN1 (Yu et al. 2005; Yang et al. 2006). The observed stability of β-elimination-sensitive viral siRNAs during infection 676 with wild-type ORMV may arise from the binding of siRNA duplexes by p125, which 677 would in turn prevent HEN1-mediated methylation, prevent the formation of RISCs and 678 thus interfere with siRNA-directed cleavage and/or translational repression of viral 679 RNAs, as has been proposed for p122, a p125 homolog from crucifer-infecting TMV 680 (Csorba et al. 2007). Still, we do not have any direct evidence supporting this hypothesis. 681 On the contrary, what indirect evidence we do have, based on the analysis of *AGO1* and 682 *AGO2* gene expression in ORMV-infected plants, indicates that an increased 683 accumulation of miR168 and miR403 actually leads to translational repression of their 684 target mRNAs (*AGO1* and *AGO2*, respectively) despite higher-than-normal levels of the 685 latter (Fig. 1A and 1G). Concomitant increases in the levels of miR168 and *AGO1* 686 mRNA accompanied by a drop in the levels of AGO1 protein have previously been 687 observed during infections of *A. thaliana* with different RNA viruses, including the 688 cruciferous strain of TMV (Várallyay et al. 2010; Várallyay and Havelda 2013), so our 689 data extend these findings to ORMV and demonstrate that p125 is responsible for the 690 perturbation of miR168 and AGO1 expression during ORMV infection. The induction of 691 miR168 and miR403 mediated by p125 can be part of a viral counter-defense strategy to 692 repress AGO1 and AGO2 translation and thus prevent the formation of viral siRNA- 693 AGO1 and viral siRNA-AGO2 complexes, whose involvement in antiviral defense is 694 well documented (Wang et al. 2011; Carbonell et al. 2012; Garcia-Ruiz et al. 2015). The 695 increased accumulation of *AGO1* and *AGO2* mRNAs observed at later stages of ORMV- 696 infection (Fig. 1A) is likely part of a plant counter-counter-defense strategy involving a 697 feedback regulation loop to restore and maintain normal levels of AGO1 and AGO2 698 proteins. 699

700 Transcriptome profiling of ORMV-infected Col-0 plants at 7, 14 and 21 dpi during a 701 previous study revealed a general up-regulation of cellular mRNAs targeted by miRNAs 702 and/or tasiRNAs, concomitant with an increased accumulation of their respective 703 miRNAs and tasiRNAs (Hu et al. 2011). This major perturbation of the host miRNA and 704 siRNA pathways, triggered by p125 as we established here, may constitute a contributing 705 factor for the development of the severe disease symptoms that characterize the later
706 stages of ORMV infection. Indeed, our analysis of *Arabidopsis* transgenic lines carrying
707 p125, p182 or p125/182 constructs revealed that p125 expression partially recapitulates
708 viral disease symptoms, such as serration of the rosette leaves and curling of the stem
709 (Fig. 7C; Supplementary Fig. S2D). This is consistent with previous studies
710 demonstrating that the transgenic expression of viral suppressors from different viruses
711 causes developmental abnormalities in *Arabidopsis*, accompanied by major perturbations
712 of its miRNA and siRNA pathways [e.g., (Chapman et al. 2004; Shivaprasad et al.
713 2008)].

714 The analysis of p125-transgenic lines and plants infected with either wild-type or p125-deficient viruses indicates that in addition to the abovementioned miR168 and miR403, 715 other miRNA and miRNA* species are induced in a p125-dependent manner. One 716 particularly important example is miR472 (Fig. 1B and 1D) which has been shown to 717 regulate plant innate immunity against non-viral pathogens (Boccara et al. 2015). 718 Because pattern-triggered immunity (PTI) has been implicated in antiviral defense 719 (Kørner et al. 2013; Niehl et al. 2016; Nicaise and Candresse 2016) and a viral silencing 720 suppressor protein has been shown to suppress PTI and other immune responses in virus-infected *A. thaliana* (Zvereva et al. 2016), it is conceivable that p125 may also suppress 721 immune responses. As proposed earlier (Zvereva and Pooggin 2012), such an activity 722 might trigger plant counter-counter-defense responses resembling those of effector-triggered immunity (ETI), typically aimed at non-viral pathogens. p125-mediated 723 induction of miR472 would thus downregulate ETI-related genes, contributing to 724 suppress antiviral ETI. Whether p125-mediated HEN1 suppression and p125-triggered 725 perturbations of the plant sRNA-directed silencing pathways (including those that 726 regulate immune responses) stem from a significant contribution of PTI to plant defenses 727 against ORMV, and whether p125 is directly involved in PTI suppression, constitute 728 therefore valuable avenues of research. It is worth pointing out that even in *dcl234* and 729 *rdr126* plants, where major components of the RNA silencing machinery are missing, 730 stop codon revertants where p125 expression is restored are quickly selected during 731 infections with p125-deficient ORMV mutants, indicating the presence of a selective 732 pressure for this effector imposed by other defense pathways such as those of plant innate 733 immunity. On the other hand, no differences regarding susceptibility to infection with 734
737 either wild-type or p125-deficient ORMV mutants were noticeable in our preliminary experiments with *A. thaliana* mutants lacking BAK1, a major PTI response co-receptor, 738 thus contradicting a previous study (Kørner et al. 2013) while supporting a more recent 739 observation (Nicaise and Candresse 2016). In either case, RNA silencing and innate 740 immunity work in concert to restrict viral replication and systemic movement (Zvereva 741 and Pooggin 2012), so that relieving the selection pressure for p125 expression may 742 require suppressing the activities of both PTI and the siRNA silencing pathways. 743

744 MATERIAL AND METHODS

745 Construction of an ORMV wild-type infectious clone and its derivatives.

746 A plasmid named pORMV (designated here as W), containing a full-length cDNA of the 747 ORMV genome (Aguilar et al. 1996) downstream a T7 promoter (kindly provided by Dr. 748 Fernando Ponz) was used as a template for site-directed mutagenesis to replace the p125 749 amber stop codon TAG with a tyrosine codon TAC as follows. Two overlapping PCR 750 products (662 bp and 248 bp), each containing the TAC codon, were separately amplified 751 using the primers pairs p180_ClaI_s and p126_stop_sup_as, and p126_stop_sup_s and 752 p180_XmaIII_as (see Supplementary Table S1 for primer sequences). The resulting PCR 753 products were used for "PCR ligation" with the flanking primers (p180_ClaI_s and 754 p180_Xma III_as). The resulting product (881 bp) containing the TAC mutation was gel 755 purified, trimmed with *Cla* I and *Xma* III restriction enzymes (New England Biolabs) at 756 the sites located in the flanking primers, and cloned between the *Cla* I and *Xma* III sites 757 of pORMV, replacing the wild type ORMV sequence. The resulting mutant clone 758 (designated as M) was selected following transformation of *E.coli* DH5α cells and 759 sequenced to confirm the presence of the introduced mutation.

760 As the available pORMV clone W did not display wild-type disease symptoms, we 761 sequenced sRNAs from *A. thaliana* Col-0 plants infected with ORMV virions and 762 reconstructed *de novo* a 6,303 nt consensus master sequence of the ORMV genome as a 763 single sRNA contig from two independent sRNA libraries (Seguin et al. 2014). The 764 reconstructed sequence differed from the ORMV cDNA sequence at three positions (G- 765 to-A at position 12, A-to-T at position 231, and G-to-T at position 5612). To correct these
766 errors, two synthetic DNA fragments (Eurofins; Supplementary Table S1) with suitable
767 restriction sites were used to obtain a new wild type ORMV genome clone, designated
768 W41 (deposited into NCBI Genbank as KF137561). Briefly, the original clone W was
digested with *EcoN* I and *Nar* I in order to replace a 418 bp fragment comprising the T7
769 promoter and the adjacent ORMV sequence by a synthetic fragment containing an A at
770 position 12 and a T at position 231. This partially repaired clone, named W4, was then
digested with *Nco* I and *Pst* I to replace a 1 Kb 3'-portion of the ORMV cDNA by a
771 synthetic fragment containing a T at position 5612 and a new *Nsi* I restriction site
772 adjacent to the ORMV sequence and followed by the *Pst* I site. This fully repaired clone,
773 named W41, was tested for infectivity in *N. benthamiana* and found to be biologically
774 active, causing disease symptoms indistinguishable from those of wild-type ORMV
775 (Seguin et al. 2014) (Supplementary Fig. S1). A third partially repaired clone was also
776 obtained, designated W1, after digestion of the original clone W with *Nco* I and *Pst* I to
777 replace exclusively the 1Kb fragment as described above. The same procedure was
778 followed in parallel to repair the p125 stop codon mutant clone M, yielding the constructs
779 M4, M41 and M1, respectively. All the constructs were sequenced to confirm the
780 introduced mutations and corrections and then tested for infectivity in *N. benthamiana*.
781 The results revealed that the corrections in the 5'-portion of the ORMV genome present
782 in clones W4 and W41 had fully restored infectivity and disease severity, while the
783 correction in the 3'-portion of the genome had only a minor effect. Interestingly, the latter
784 correction (position 5612) mutated the MP ORF stop codon, thereby elongating viral MP
785 by an additional 19 amino acids.
786
787

788 Plant growth and virus inoculation.

789 Plants of *A. thaliana* genotype Col-0 and its mutant or transgenic derivatives were grown
790 in light chambers (Sanyo) at 20°C under a 12/12 hours light/dark cycle. *N. benthamiana*
791 plants were grown in soil at 20-25°C in an open greenhouse or in Sanyo chambers at
792 25°C, also under a 12/12 hours light/dark cycle.

793 Five weeks post-germination *A. thaliana* seedlings (three per genotype) were inoculated
794 mechanically with sap from wild-type ORMV-infected *N. benthamiana* (Akbergenov et
795 al. 2006), or with *in vitro* RNA transcripts from the ORMV clones described above.
Briefly, the sap was distributed onto two leaves per plant (10 µl per leaf) and mixed with a pinch of Celite® 545 (Merck). After rubbing the mix softly with a finger against the upper surface of the leaves, the plants were returned to the light chamber under controlled conditions. The *in vitro* transcripts were obtained from plasmid DNA of the ORMV constructs (W41, W4, M41 and M4) using the T7 Megascript Kit (Ambion) and the cap analog m7G(5')ppp(5')G (Ambion), following the manufacturer's instructions. Briefly, for a 20 µl reaction, 2 µl of ATP, CTP and UTP (75 mM each) were added to 1 µg of plasmid DNA resuspended in 4 µl of DEPC-treated water. Then 3 µl of the cap analogue (6 mM) and 2 µl of GTP (15 mM) were added to promote capping of the transcripts, as well as 2 µl of 10X T7 reaction buffer, 2 µl of T7 Enzyme Mix and 1 µl of RNase inhibitor RNasin (40 units/µl) (Promega). After a 2 hour incubation at 37°C, 1 µl GTP (75 mM) was added to the mix and left at 37°C for 2 hrs. Before inoculation, the reaction was diluted 1:4 with DEPC-treated, a 20 µl aliquot was used as inoculum and another 20 µl aliquot was analyzed by agarose gel electrophoresis. Interestingly, the use of linearized (at the introduced Nsi I or Pst I site) or non-linearized ORMV plasmid templates for *in vitro* transcription had no detectable effects on the relative infectivity of the resulting transcripts *in planta*. We speculate that the artificial tails added *in vitro* to the 3'-end of the tobamoviral gRNA transcript might be removed by a plant 3'-5' exonucleolytic activity that is stalled by the secondary structure of the tRNA-like sequence located at the 3' terminus of the tobamoviral genome.

## Western blotting.

Systemic (non-inoculated) leaves from three plants of *A. thaliana* or two plants of *N. benthamiana* were pooled and ground in liquid nitrogen (in some cases with *N. benthamiana* and the mutant virus, the leaves were ground individually). Thirty milligrams of ground frozen material were resuspended in 150 µl of 6X Laemmli SDS-PAGE Sample Loading Buffer (0.35 M Tris-HCl pH 6.8, 22.4% glycerol, 10% SDS, 0.6 M DTT, bromophenol), vortexed briefly and heated for 3 minutes at 95°C. The samples were vortexed again briefly and centrifuged at 12,000 g for 2 minutes. Twelve-microliter aliquots of the supernatants were loaded in a 9% SDS-PAGE gel with a 4% stacking gel in a Mini-PROTEAN Tetra Cell (BioRad). Five microliters of PageRuler Plus pre-stained
826 protein ladder (Thermo Fisher Scientific) were loaded as a molecular weight reference.

827 After gel electrophoresis for 1 hour at 100 V in Tris-glycine buffer pH 8.3 (25 mM Tris-HCl, 250 mM glycine, 0.1% SDS), the proteins were transferred to an Amersham Hybond-P PVDF membrane 0.45 µm (GE Healthcare) for two hours in the presence of transfer buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, 0.025% SDS, 20% ethanol).

831 The membrane was blocked at room temperature with 1% BSA (Sigma) in TBS-Tween buffer (25 mM Tris-HCl pH 7.4, 3 mM KCl, 140 mM NaCl, 0.1% Tween 20) for 1 hour.

833 After 3 washes with TBS-Tween buffer, the following primary antibodies were used according to the different assays in TBS-Tween-1% BSA 1% buffer at the indicated dilutions and incubated with the membrane at 4°C overnight: rabbit anti-p125/p182 (1:4000) (custom antibody from Eurogentec SA; see below), rabbit anti-AGO1 from *Arabidopsis* (1:8000) (Agrisera), or rabbit anti-AGO2 from *Arabidopsis* (1:4000) [kindly provided by D. Baulcombe; (Harvey et al. 2011)]. After washing the membrane 3 times with TBS-Tween buffer, a secondary goat anti-rabbit antibody (1:10000) (Agrisera) was added in TBS-Tween-BSA 1% at room temperature for 1 hour. After 3 three additional washes with TBS-Tween buffer, the signals were developed by incubation at room temperature with Amersham's ECL Prime Western Blotting Detection Reagent (1 ml of solution A + 1 ml of solution B) for 3 minutes, acquiring with an Azure c600 Imager. The membrane was then stained with amidoblack to control for amount of protein loaded.

845 In order to detect ORMV p125 and p182, a primary anti-peptide serum (Eurogentec SA) was raised in rabbits by immunization with a synthetic peptide (GITRADKDNVRTVDS) shared by both proteins. After purification, the antibodies were used at the suggested dilution (1:4000).

849 **Total RNA preparation.**

850 For both long and small RNA blot hybridization analyses as well as Illumina deep-sequencing of sRNAs, systemic (non-inoculated) leaf tissue samples from ORMV-infected and mock-inoculated *A. thaliana* plants were harvested at days 5, 14 and 22 post-inoculation and frozen in liquid nitrogen. Total RNA was isolated from 0.5 g of ground frozen tissue from a pool of three plants, using TRI Reagent (Sigma) according to the manufacturer's protocol. In the case of *N. benthamiana*, pools of leaves from two plants
856 (infected with each ORMV construct or mock-inoculated) were harvested at day 5 and 26
857 post-inoculation and frozen in liquid nitrogen. Total RNA was isolated by resuspending
858 0.5 g of frozen ground tissue into 5 ml of GHCL extraction buffer (6.5 M guanidinium
859 hydrochloride, 100 mM Tris-HCI pH 8.0, 0.1 M sodium acetate pH 5.5, 0.1 M β-
860 mercaptoethanol), centrifuging the resulting homogenate at 10,000 g for 10 minutes at
861 4°C and treating the supernatant according to the manufacturer's standard protocol with
862 TRI Reagent (Sigma). In all cases, RNA was quantified with a ND-1000 UV-Vis
863 Spectrophotometer (NanoDrop Technologies).

864 RT-PCR analysis of ORMV RNA.

865 Two micrograms of total RNA dissolved in DEPC-treated water were mixed with 1 µl of
866 ORMV CP_qPCR_as primer (10 µM), 1 µl of 10 mM dNTP mix (Sigma-Aldrich), 4 µl
867 of 5X First-strand buffer [250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl₂] and
868 2 µl of 0.1 M DTT and adjusted to a final volume of 18 µl. The mix was heated at 65°C
869 for 2 min and then left on ice to cool down. One microliter of RNAsin (40 units/µl,
870 Promega) and 1 µl of Superscript III Reverse Transcriptase (200 units/µl; Invitrogen)
871 were added, and the mix was left for 2 minutes at RT, incubated at 50°C for 1 hour and
872 then left at 85°C for 5 minutes to stop the reaction. Each reaction was diluted 1:100 with
873 DEPC-treated water before further use.

874 To verify the p125 stop codon mutation/reversion, 4 µl of the diluted cDNA were mixed
875 with 2 µl of oligonucleotide p180_Cla_s (10 µM), 2 µl of oligonucleotide
876 p180_XmaIII_as (10 µM), 1 µl of 10mM dNTP mix (Sigma-Aldrich), 5 µl of 10X PCR
877 Buffer (Sigma-Aldrich) and 1 µl of Vent DNA polymerase (2000 units/ml; NEB),
878 adjusting the resulting reaction to a final volume of 50 µl. The thermal cycling conditions
879 were 95°C for 2 min, 34 cycles of 95°C (30 sec), 62°C (40 sec), and 72°C (1 min),
880 followed by a final extension at 72°C (10 min). The reaction was purified with a
881 GenElute™ PCR Clean-Up Kit (Sigma) following manufacturer's instructions, and an
882 aliquot from the resulting DNA was analyzed by agarose gel electrophoresis to check the
883 size of the PCR products. The remaining DNA was sequenced using the p180_Cla_s and
884 p180_XmaIII_as primers. All primer sequences are provided in Supplementary Table S1.
885 **Long RNA blot hybridization analysis.**

886 Twenty micrograms of total RNA were dried in a SpeedVac and resuspended in 6 µl of
887 DEPC-treated water, mixed with 6 µl of 2X RNA Gel Loading Dye (Thermo Fisher
888 Scientific), heated at 65°C for 3 minutes and loaded in a 1.2% denaturing agarose gel
889 [1.2% agarose, 3% formaldehyde in MOPS buffer (0.02 M MOPS pH 7.0, 1 mM EDTA,
890 5mM NaOAc)]. A RiboRuler High Range RNA Ladder from 200 to 6000 bases (Thermo
891 Fisher Scientific) was also loaded as size marker. The gel was run in a Sub-Cell GT Cell
892 (BioRad) nucleotide electrophoresis device at 100 V with MOPS buffer for 4 hours, and
893 the RNA was transferred to a Hybond N+ membrane (Amersham) by capillary transfer
894 for 24 h using 50 mM NaH₂PO₄, 5 mM EDTA, pH 6.5 as transfer buffer. After cross-
895 linking twice with 1200 µjoules x100 UV light in a Stratalinker 1800 (Stratagene), the
896 hybridization was performed as described below for sRNA blot hybridization analysis.

897 **Small RNA blot hybridization analysis.**

898 High-resolution sRNA blot hybridization was performed as described previously
899 (Akbergenov et al. 2006) with some modifications. Ten micrograms or, in some cases, 30
900 µg of total RNA were dried in a SpeedVac and resuspended into 10 µl of loading buffer
901 (95% formamide, 20 mM EDTA pH 8.0, 0.05% bromophenol blue, 0.05% xylene
902 cyanol), heated at 95°C for 2 min and loaded in a 15% polyacrylamide-urea gel (19:1
903 acrylamide:bis-acrylamide and 8 M urea). The gel was run using an SE 600
904 electrophoresis device (Hoefer, San Francisco, CA) at 300 V for 4 hours and the RNA
905 was then transferred to a Hybond N+ membrane (Amersham) by electroblotting in TBE
906 buffer at 10 V overnight at 4°C. The RNA was cross-linked to the membrane twice with
907 1200 µjoules x100 UV light in a Stratalinker 1800 (Stratagene). Blot hybridization was
908 performed at 35°C overnight in UltraHyb-oligo buffer (Ambion) using as probes one or
909 more short DNA oligonucleotides end-labeled with P³² gamma ATP (Hartmann Analytic,
910 Germany) by T4 polynucleotide kinase (Roche, Switzerland) and purified through
911 MicroSpin G-25 columns (Amersham) following manufacturer's recommendations. The
912 membrane was washed 3 times with 2X SCC, 0.5% SDS for 30 min at 35°C, and the data
913 was acquired by exposure for 1 to 6 days to a phosphor screen in a GE Typhoon 8600
914 imager (GE Healthcare Life Sciences). For repeated hybridizations the membrane was
915 stripped with 0.5X SSC, 0.5% SDS for 30 min at 80°C and then with 0.1X SSC, 0.5%
916 SDS for 30 min at 80°C. The DNA oligonucleotides were designed and synthesized by
917 Eurofins S.A. (see Supplementary Table S1 for all probes and PCR primers).

918 β-elimination assay.

919 The β-elimination experiment was performed as described previously (Akbergenov et al.
920 2006). Briefly, 20 µg of total RNA were dissolved in 17.5 µl borax buffer, pH 8.6 [4.375
921 mM borax (Fluka), 50 mM boric acid (Fluka)] and 2.5 µl of freshly prepared 0.2 M
922 sodium periodate (Fluka). As an internal control of the reaction, 250 pg of 22-nt non-
923 methylated RNA (5'-UUCACCUUGAUGCCGUUCUUCU-3') were spiked into each
924 sample. The reaction mixture was incubated for 10 min at room temperature in the dark
925 and, after addition of 2 µl of glycerol, the incubation was repeated. The mixture was
926 dried under vacuum in a SpeedVac, dissolved in 50 µl borax buffer, pH 9.5 (33.75 mM
927 borax, 50 mM boric acid, pH adjusted by NaOH) and incubated for 90 min at 45°C. After
928 treatment, the RNA samples were purified with Illustra MicroSpin G-25 Columns (GE
929 Healthcare Life Sciences) and used for sRNA blot hybridization as described above.

930 Illumina sequencing and bioinformatics analysis of viral and plant sRNAs.

931 Ten-microgram aliquots from the total RNA samples analyzed by sRNA blot
932 hybridization were used to prepare sRNA libraries following Illumina's modified protocol
933 for the sRNA library construction kit. Briefly, the 19-30 nt RNA fraction from total RNA
934 was purified on a 15% TBE-Urea acrylamide gel. A 5'-adenylated single-stranded
935 adapter was first ligated to the 3'-end of the RNA using T4 RNA ligase without ATP,
936 followed by a second single-stranded adapter ligated to the 5'-end of the RNA using T4
937 RNA ligase in the presence of ATP. The ligation products were purified on a 10% TBE-
938 Urea acrylamide gel before cDNA synthesis and PCR amplification, after which the
939 resulting libraries were sequenced on an Illumina Genome Analyzer HiSeq2000 (BPO-
940 36-44) or HiSeq2500 (BPO-137-146) following the manufacturer's protocols.

941 After trimming adaptor sequences, the datasets of all (redundant) reads were mapped to
942 the reference genomes of A. thaliana Col-0 (TAIR10), N. benthamiana
943 (Niben.genome.v0.4.4; http://solgenomics.net/organism/Nicotiana_benthamiana/genome;
944 ftp://ftp.solgenomics.net/genomes/Nicotiana_benthamiana/assemblies/Niben.genome.v0.
945 4.4.scaffolds.nrcontigs.fasta.gz), or ORMV (W41, KF137561; or its mutant derivative
946 M41) using the Burrows-Wheeler Aligner (BWA) v0.5.9 (Li and Durbin 2009). Then,
947 20-25 nt redundant reads were taken for further bioinformatics analysis as described
948 previously (Rajeswaran et al. 2014). The results are summarized in Supplementary
949 Datasets S1-S3. The analysis of virus-derived sRNAs and the reconstruction of a
950 consensus master sequence and its quasispecies variants (single nucleotide
951 polymorphisms) were done using MISIS-2 (www.fasteris.com/apps) (Seguin et al. 2016).
952 For each reference genome and sRNA size class, MISIS-2 counted total reads as well as
953 reads in each orientation, generating single-base resolution maps (Supplementary
954 Datasets S2 and S3) that displayed, for each position starting from the 5' end of the
955 reference genome, the number of matches starting at that position in forward (first base of
956 the read) and reverse (last base of the read) orientation per size class.

957 Construction of expression vectors for ORMV p125, p125/182 and p182 proteins.

958 Gateway technology was used to construct vectors for expressing the ORMV proteins
959 transiently in N. benthamiana or stably in transgenic A. thaliana. The coding sequences
960 for p125 and p125/p182 were amplified from pORMV clone W as attB-PCR products
961 using Vent DNA Polymerase (NEB) and primers AttB1_Rep_s and 5AttB2_p125_as for
962 p125 or AttB1_Rep_s and AttB2_p182_as for p125/p182 (see Supplementary Table S1
963 for primer sequences). The latter pair of primers was also used to amplify the p182
964 coding sequence from the p125 stop codon mutant clone M. The resulting attB-PCR
965 products were separately cloned into a Gateway pDONR/Zeo vector through a BP
966 Clonase reaction (Gateway BP Clonase Enzyme Mix; Invitrogen) to yield three different
967 entry clones. An LR reaction (Gateway LR Clonase Enzyme mix; Invitrogen) using three
968 different destination vectors [pEarleyGate binary constructs 100 (no tag), 201 (HA-tag),
969 and 202 (FLAG-tag), (Earley et al. 2006)] -- per entry clone was then used to obtain the
970 ORMV protein expression constructs (p125, p125-HA, p125-FG, p182, p182-HA, p182-
971 FG, p125/182, 201-p125/182-HA and p125/182-FG). All the constructs contained the
972 ORMV coding sequence between a CaMV 35S promoter and an octopine synthase (OCS)
973 terminator, as well as kanamycin- and phosphinotricine (or herbicide Basta)-resistance
974 markers genes for bacterial and plant selection, respectively. The pEarleyGate 100
975 constructs contained the wild type ORMV protein sequences, while the pEarleyGate 201
976 and 202 constructs contained an HA-tag or a FLAG (FG)-tag fused to the N-terminus of
977 the ORMV proteins, respectively. All final pEarleyGate constructs were amplified in
978 *E.coli* DH5a in the presence of kanamycin, sequenced downstream the 35S promoter and
979 upstream the OCS terminator to verify fragment junctions, and then mobilized to
980 *Agrobacterium tumefaciens* strain GV3101 (rifampicin-resistant) by electroporation. The
981 *Agrobacterium* colonies were selected on rifampicin and kanamycin, and the presence of
982 each construct was confirmed by PCR using primers CaMV35Spro\_#1 and Ormv323\_as
983 (see Supplementary Table S1).

984 Generation of transgenic *A. thaliana* plants.

985 Transgenic plants were obtained by floral dipping transformation (Zhang et al. 2006)
986 with *Agrobacterium* strains carrying the ORMV expression constructs. Briefly, *A.
987 *thaliana* Col-0 plants (3 plants per construct) at an early bolting stage (two months old)
988 were cleaned from flowers and left only with closed flower buds, and then dipped into an
989 *Agrobacterium* suspension supplemented with 5% sucrose and 0.02% Silwet L-77 for
990 transformation. After removing excess liquid, the plants were kept in the dark at 20°C
991 overnight and then placed in a light chamber at 20°C until siliques with mature seeds
992 were produced. The harvested seeds were germinated and the seedlings were sprayed
993 with a 250 mg/L Basta (Bayer CropScience) solution two times per week. Basta-resistant
994 seedlings (generation T1) were transferred to new pots and grown until seeds were
995 produced. These seeds were then germinated on soil and the seedlings were sprayed with
996 the same Basta solution two times per week. Afterwards, T2 Basta-resistant seedlings
997 were transferred to new pots, and the same procedure was repeated to obtain generations
998 T3 and T4 of Basta-resistant plants. The presence of ORMV expression constructs in the
999 transgenic lines was confirmed by PCR genotyping using primers CaMV35Spro\_#1 and
1000 Ormv141\_as (see Supplementary Table S1). For each transgenic line, seedlings of T2 or
1001 T3 generations were phenotyped (Supplementary Fig. S2B-S2C) and analyzed for
1002 ORMV protein expression and endogenous sRNAs by Western and sRNA blot
1003 hybridization, respectively, as described above.
Transient expression in *N. benthamiana*.

Transient expression experiments using the *N. benthamiana* 16c line were performed as described previously (Hamilton et al. 2002; Himber et al. 2003). *Agrobacterium tumefaciens* strains carrying a binary plasmid with a green fluorescent protein (GFP) coding sequence under control of the CaMV 35S promoter and nopaline synthase terminator (35S:GFP), its derivatives with a potyviral pHC-Pro (35S:HcPro) or tombusviral p19 (35S:P19) coding sequence [all kindly donated by D. Baulcombe (Hamilton et al. 2002)], and the *Agrobacterium* strains harboring the ORMV constructs were grown in liquid LB medium with 100 mg/L kanamycin overnight at 28°C at 180 rpm. After centrifugation, the bacterial pellets were resuspended in 40 ml of an infiltration buffer [10 mM 2-(N-morpholino)ethanesulphonic acid (MES), 10 mM MgCl₂, pH 5.6], induced with 150 µM acetosyringone for 3 hours at room temperature and adjusted to OD600 = 0.5 with the infiltration buffer before agroinfiltration. The cell suspension carrying the GFP construct was mixed with an equal volume of cell suspensions carrying each viral construct or an empty agro suspension, infiltrating the resulting mixtures on the abaxial surface of leaves of the *N. benthamiana* 16C line with a 1 ml syringe (without needle) after gently scratching it with a needle. Leaf patches infiltrated with the suspension carrying exclusively the GFP construct were used as a positive silencing control, whereas leaf patches infiltrated with the suspension carrying the GFP construct plus the pHC-Pro or p19 construct served as a strong silencing suppression control. Images of the agroinfiltrated leaves under UV light were taken at days 5, 7 and 10 days post infiltration (dpi) in order to monitor transient GFP expression in the presence or absence of the putative viral silencing suppressors. Representative images are shown in Supplementary Fig. S1A and S1B. The plants were grown in soil under open greenhouse conditions at 20-25°C and kept under the same conditions after agroinfiltration. Total RNA was extracted at 10 days post-agroinfiltration from the infiltrated leaf tissue patches ground in liquid nitrogen, using TRIzol reagent (Sigma), following the manufacturer's instructions but including an additional first step with GHCL extraction buffer as described above for total RNA extraction. For sRNA analysis, 10 µg samples of total RNA were separated on 15% polyacrylamide gel, images of the EtBr-stained gels were acquired to be used as loading controls, and the RNA species
1035 were transferred to a Hybond N+ membrane (Amersham). The blot was hybridized with
1036 ³²P-ATP-labeled, GFP 3'UTR-specific probe mGFP5_Nosterm_as, then stripped and
1037 hybridized with probe mGFP5_Nosterm_s, and then stripped again and hybridized with a
1038 miR482-specific probe (see Supplementary Table S1 for probe details).

1039 ACKNOWLEDGEMENTS

1040 This work was supported by the Swiss National Science Foundation (grant
1041 31003A_143882/1 to M.M.P.) and European Cooperation in Science and Technology
1042 (COST; grant SERI No. C09.0176 to L.F. and M.M.P.). We thank Fernando Ponz for the
1043 pORMV plasmid, David Baulcombe for the AGO2 antibody, Todd Blevins for critical
1044 reading of the manuscript, and Alejandro Martin for English editing.

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1345 **FIGURES LEGENDS**

1346 **Fig. 1.** Blot hybridization analyses of viral and plant long RNAs, small RNAs and
1347 proteins in systemic (non-inoculated) leaves of *A. thaliana* (Col-0, *dcl234*, or *rdr126*)
1348 plants infected with wild-type virus (ORMV, W41) or its p125 stop codon mutant
1349 (M41). Panels (A) and (C) show long RNA analysis (1% agarose denaturing gel) of
1350 ORMV positive-sense RNAs (gRNA, sgRNA-MP, sgRNA-CP) and negative-sense
1351 RNAs [gRNA(-) and CP(-)], and of *A. thaliana* *AGO1* and *AGO2* mRNAs in mock-
1352 inoculated (-) and virus-infected (+) Col-0, *rdr126* and *dcl234* plants at 14 and 22 days
1353 post-inoculation (dpi) with ORMV sap (A), and at 5 dpi with W41 and M41 *in vitro*
1354 transcripts (C). In each experiment, total RNA was extracted from systemic leaf pools of
1355 three plants for each condition. The cropped scans represent the blots successively
1356 hybridized with DNA oligonucleotide probes specific for the viral CP sense and antisense
1357 strands and the *AGO1* and *AGO2* coding strands (sequences for these and all other probes
1358 are given in Supplementary Table S1). The positions of RNA size markers are indicated
1359 on each scan. An abundant plant rRNA species cross-hybridized with the viral CP
1360 antisense-specific oligonucleotide probe is indicated by an asterisk. The question mark
1361 (?) indicates a presumptive viral MP(-) RNA species, the reliable detection of which is
1362 prevented by probe cross-hybridization to a co-migrating plant RNA species. EtBr-
1363 stained rRNA is shown as a loading control. Panels (B) and (D) show high-resolution
1364 sRNA analysis (15% PAGE) of the same total RNA samples from panels (A) and (C).
The blots were successively hybridized with DNA oligonucleotide probes specific for viral antisense siRNAs derived from ORMV p125 and CP, as well as for plant miRNA (miR168, miR403, miR472, miR173), miRNA* (miR472*), siRNA (siR255) and siRNA* (siR255*) species. sRNA sizes are indicated. The EtBr-stained gel is shown as loading control. Panel (E) shows the genome organization of ORMV wild-type (W41) and mutant (M41) viruses. The ORFs are boxed and the encoded proteins (p125, p182, MP, and CP) are indicated above them. Positions of the p125 stop codon (UAG) in W41 and its mutation (UAG to UAc) in M41 are indicated. Viral genomic (gRNA) and subgenomic (sgRNA-CP and sgRNA-MP) RNAs are depicted by lines. Panels (F) and (G) show Western blots of ORMV p125 and p182 and *A. thaliana* AGO1 and AGO2 in Col-0, *rdr126*, *dcl234* and *ago2* plants infected with M41 or W41 at 5, 15 and 22 dpi. In each case, a systemic leaf pool of three plants was used for protein extraction. The membranes were incubated with anti-p125, anti-AGO1, or anti-AGO2 antibodies, and were stained with Amido Black afterwards to use the Rubisco bands as loading controls. The positions of p125 and p182 are indicated with arrows. Note that, in this particular experiment, the ORMV p125/p182-specific antibody cross-hybridized to a plant protein migrating just above p182 ("Col-0 mock" lane). Note that the experiments were replicated at least two times with similar results, and long and small RNA analyses for replicated experiments at 5 dpi and 22 dpi are shown in Supplementary Fig. S1G-S11.

**Fig. 2. Illumina sequencing counts of endogenous and viral sRNAs in mock-inoculated and ORMV-infected *A. thaliana* (Col-0, rdr126, or dcl234) at 14 and 22 dpi.** (A) The graph shows the percentages of virus- and plant-derived sRNAs that mapped to ORMV or *Arabidopsis* genome reference sequences with zero mismatches in the pool of total 20-25 nt reads. (B) The graphs show the percentages of each size class for 20-25 nt virus- or *Arabidopsis*-derived sRNA reads that mapped to the corresponding reference sequence with zero mismatches. Note that aliquots of the same total RNA samples (extracted from systemic leaf pools of three plants per each condition) was used for both Illumina sequencing and blot hybridization analyses in Fig. 1A and 1B. The details of all sRNA libraries with absolute and normalized counts of plant and viral sRNA reads are given in Supplementary Datasets S1.
Fig. 3. Single-nucleotide resolution maps of 20-25 nt viral siRNAs from ORMV-infected *A. thaliana* (Col-0, *rdr126*, or *dcl234*) at 14 and 22 dpi. For each plant genotype and time point, the histograms plot the numbers of total 20-25-nt, 21-nt or 24-nt viral sRNA reads at each nucleotide position of the 6303 bp ORMV reference genome sequence (mapped with zero mismatches). The bars above the axis represent sense reads starting at each position and those below represent antisense reads ending at the respective position. A scaled ORMV genome diagram is shown above the histograms, with the ORFs boxed and their nucleotide positions indicated. The details of sRNA counts and single-nucleotide resolution maps are given in Supplementary Datasets S1 and S2. Note that Supplementary Dataset S1 contains both absolute and normalized counts of viral siRNA reads for each sRNA library. As explained in the Results section the normalization per million of host 21-nt sRNA reads is the most appropriate method for comparison of viral siRNA accumulation between wild type (Col-0) and siRNA-deficient (*rdr126* and *dcl234*) Arabidopsis plants.

Fig. 4. Illumina sequencing counts of endogenous and viral sRNAs in mock-inoculated and virus (W41, or M41)-infected *A. thaliana* (Col-0 or *rdr126*) at 5 dpi and *N. benthamiana* (N.b.) at 5 and 26 dpi. (A) Percentages of virus- and plant-derived sRNAs (mapped to ORMV and plant genome reference sequences with zero mismatches) in the pool of total 20-25 nt reads. (B) Size class percentages for 20-25 nt virus- and plant-derived sRNA reads (mapped with zero mismatches to the corresponding reference sequence). Note that aliquots of the same total RNA samples (extracted from systemic leaf pools of three plants per each condition) was used for both Illumina sequencing and blot hybridization analysis in Fig. 1C and 1D. In the case of *N. benthamiana*, total RNA was extracted from systemic leaf pools of two plants per condition and analyzed by blot hybridization (Supplementary Fig. S1J-S1K, experiment 2). The details of all sRNA libraries with absolute and normalized counts of plant and viral sRNA reads are given in Supplementary Datasets S1.

Fig. 5. Single-nucleotide resolution maps of 20-25 nt viral siRNAs from virus (W41, or M41)-infected *A. thaliana* (Col-0 or *rdr126*) at 5 dpi and *N. benthamiana* (N.b.) at 5 and 26 dpi. For each plant genotype and time point, the histograms plot the numbers of
1425 total 20-25 nt, 21-nt and 24-nt viral sRNA reads at each nucleotide position of the 6303
1426 bp W41 or M41 reference genome (mapped with zero mismatches); the bars above the
1427 axis represent sense reads starting at each respective position and those below represent
1428 antisense reads ending at the respective position. A scaled ORMV genome diagram is
1429 shown above the histograms, with the ORFs boxed and their nucleotide positions
1430 indicated. The details of sRNA counts and single-nucleotide resolution maps are given in
1431 Supplementary Datasets S1 and S3. Note that Supplementary Dataset S1 contains both
1432 absolute and normalized counts of viral siRNA reads for each sRNA library. As
1433 explained in the Results section the normalization per million of host 21-nt sRNA reads is
1434 the most appropriate for comparison of viral siRNA accumulation between wild type
1435 (Col-0) and siRNA-deficient (rdr126 and dcl234) *Arabidopsis* plants.

1436 **Fig. 6. β-elimination analysis of viral and endogenous sRNAs in plants infected with wild-type (W41) or p125-deficient (M41) virus.** Two aliquots were taken from each
1437 total RNA sample from *N. benthamiana* (N.b.) systemically infected with W41 (5 dpi;
1438 systemic leaf pool of two plants) or M41 (26 dpi; systemic leaf pool of two plants), or *A.
1439 thaliana* (Col-0) infected with W41 (22 dpi; systemic leaf pool of three plants). One was
1440 treated with periodate (+) and the other with buffer (-) and both were analyzed by 15%
1441 PAGE and blotted. The blot was successively hybridized with DNA oligonucleotide
1442 probes specific for viral antisense siRNAs derived from ORMV p125 or CP genes, for
1443 plant miRNAs (miR168, miR482), miRNA* (miR482*), siRNA (siR255) and siRNA*
1444 (siR255*) species and for a synthetic non-methylated 22-nt RNA (spiked into each total
1445 RNA sample as an internal control). sRNA sizes are indicated on each scan. The EtBr-
1446 stained gel is shown as loading control. An additional β-elimination analysis of viral and
1447 endogenous sRNAs in *A. thaliana* plants infected with W41 or M41 at 5 dpi is shown in
1448 Supplementary Fig. S1I.
1449

1450 **Fig. 7. Transgenic expression of p125 alone partially recapitulates viral disease symptoms and the impact of viral infection on plant sRNAs.** (A) Schematic
1451 representation of a pEarleygate expression construct carrying ORMV p125 coding
1452 sequence under control of the CaMV 35S promoter and OSC terminator. (B) Effects of
1453 transient expression of the ORMV p125, pHC-Pro or p19 constructs on transgene-
1455 induced silencing (measured as relative GFP fluorescence; see Supplementary S2A and S2B) and the accumulation of GFP siRNAs and plant miRNAs (examined by sRNA blot hybridization at 10 dpi) in *N. benthamiana* GFP transgenic 16c plants. The sRNA blot was successively hybridized with DNA oligonucleotide probes specific for GFP sense and antisense siRNAs and plant miR482, miR160 and miR482*. sRNA sizes are indicated.
1456
1457
1458
1459
1460 The EtBr-stained gel is shown as a loading control. (C) Representative phenotypes of the p125-transgenic plants (T2-plants of line 32) before and after bolting (see Supplementary Fig. S2D-S2F for other transgenic lines and controls), compared to ORMV-infected *A. thaliana* Col-0 plants (the rosette leaves following early inoculation and the stem following late inoculation). (D) Western blotting analysis of viral p125 and p182 proteins in *Arabidopsis* Col-0 plants infected with ORMV (W41) at 5 and 15 dpi, and in *Arabidopsis* transgenic plants carrying the p125 (T3-plant of line 33) construct. The membrane was incubated with an anti-p125 antibody or stained with AmidoBlack, shown here as loading control (Rubisco). The expected positions of p125 and p182 are indicated with arrowheads. (E) sRNA blot hybridization analysis (15% PAGE) of total RNA samples from two individual plants of the *Arabidopsis* transgenic line carrying the p125 expression construct (two T3-plants of line 32, lanes 2-3), or control wild-type plant (Col-0; lane 1). The blot was successively hybridized with DNA oligonucleotide probes specific for plant miR168, miR403, miR173, miR472*, siR255 and siR255* and for Met-tRNA. sRNA sizes are indicated on each scan. The EtBr-stained gel is shown as loading control. See Supplementary Fig. S2 for analysis of other ORMV constructs in transient assay and transgenic plants.
1461
1462
1463
1464
1465
1466
1467
1468
1469
1470
1471
1472
1473
1474
1475
1476
<figure><img src="image_2.png" /><figcaption>Fig. 1. Blot hybridization analyses of viral and plant long RNAs, small RNAs and proteins in systemic (non-inoculated) leaves of A. thaliana (Col-0, dcl234, or rdr126) plants infected with wild-type virus (ORMV, W41) or its p125 stop codon mutant (M41). Panels (A) and (C) show long RNA analysis (1% agarose denaturing gel) of ORMV positive-sense RNAs (gRNA, sgRNA-MP, sgRNA-CP) and negative-sense RNAs [gRNA(-) and CP(-)], and of A. thaliana AGO1 and AGO2 mRNAs in mock-inoculated (-) and virus-infected (+) Col-0, rdr126 and dcl234 plants at 14 and 22 days post-inoculation (dpi) with ORMV sap (A), and at 5 dpi with W41 and M41 in vitro transcripts (C). In each experiment, total RNA was extracted from systemic leaf pools of three plants for each condition. The cropped scans represent the blots successively hybridized with DNA oligonucleotide probes specific for the viral CP sense and antisense strands and the AGO1 and AGO2 coding strands (sequences for these and all other probes are given in Supplementary Table S1). The positions of RNA size markers are indicated on each scan. An abundant plant rRNA species cross-hybridized with the viral CP antisense-specific oligonucleotide probe is indicated by an asterisk. The question mark (?) indicates a presumptive viral MP(-) RNA species, the reliable detection of which is prevented by probe cross-hybridization to a co-migrating plant RNA species. EtBr-stained rRNA is shown as a loading control. Panels (B) and (D) show high-resolution sRNA analysis (15% PAGE) of the same total RNA samples from panels (A)</figcaption></figure>
and (C). The blots were successively hybridized with DNA oligonucleotide probes specific for viral antisense siRNAs derived from ORMV p125 and CP, as well as for plant miRNA (miR168, miR403, miR472, miR173), miRNA* (miR472*), siRNA (siR255) and siRNA* (siR255*) species. sRNA sizes are indicated. The EtBr-stained gel is shown as loading control. Panel (E) shows the genome organization of ORMV wild-type (W41) and mutant (M41) viruses. The ORFs are boxed and the encoded proteins (p125, p182, MP, and CP) are indicated above them. Positions of the p125 stop codon (UAG) in W41 and its mutation (UAG to UAc) in M41 are indicated. Viral genomic (gRNA) and subgenomic (sgRNA-CP and sgRNA-MP) RNAs are depicted by lines. Panels (F) and (G) show Western blots of ORMV p125 and p182 and A. thaliana AGO1 and AGO2 in Col-0, rdr126, dcl234 and ago2 plants infected with M41 or W41 at 5, 15 and 22 dpi. In each case, a systemic leaf pool of three plants was used for protein extraction. The membranes were incubated with anti-p125, anti-AGO1, or anti-AGO2 antibodies, and were stained with Amido Black afterwards to use the Rubisco bands as loading controls. The positions of p125 and p182 are indicated with arrows. Note that the experiments were replicated at least two times with similar results, and long and small RNA analyses for replicated experiments at 5 dpi and 22 dpi are shown in Supplementary Fig. S1G-S1I.
A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /></figure>

Fig. 2. Illumina sequencing counts of endogenous and viral sRNAs in mock-inoculated and ORMV-infected A. thaliana (Col-0, rdr126, or dcl234) at 14 and 22 dpi. (A) The graph shows the percentages of virus- and plant-derived sRNAs that mapped to ORMV or Arabidopsis genome reference sequences with zero mismatches in the pool of total 20-25 nt reads. (B) The graphs show the percentages of each size class for 20-25 nt virus- or Arabidopsis-derived sRNA reads that mapped to the corresponding reference sequence with zero mismatches. Note that aliquots of the same total RNA samples (extracted from systemic leaf pools of three plants per each condition) was used for both Illumina sequencing and blot hybridization analyses in Fig. 1A and 1B. The details of all sRNA libraries with absolute and normalized counts of plant and viral sRNA reads are given in Supplementary Datasets S1.
<figure><img src="image_5.png" /><figcaption>ORMV sRNAs in Col-0 at 14 dpi</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>ORMV sRNAs in rdr126 at 14 dpi</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>ORMV sRNAs in dcl234 at 14 dpi</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>ORMV genomic RNA</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>ORMV sRNAs in Col-0 at 22 dpi</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>ORMV sRNAs in rdr126 at 22 dpi</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>ORMV sRNAs in dcl234 at 22 dpi</figcaption></figure>

<figure><img src="image_12.png" /></figure>

Fig. 3. Single-nucleotide resolution maps of 20-25 nt viral siRNAs from ORMV-infected A. thaliana (Col-0, rdr126, or dcl234) at 14 and 22 dpi. For each plant genotype and time point, the histograms plot the numbers of total 20-25-nt, 21-nt or 24-nt viral sRNA reads at each nucleotide position of the 6303 bp ORMV reference genome sequence (mapped with zero mismatches). The bars above the axis represent sense reads starting at each position and those below represent antisense reads ending at the respective position. A scaled ORMV genome diagram is shown above the histograms, with the ORFs boxed and their nucleotide positions indicated. The details of sRNA counts and single-nucleotide resolution maps are given in Supplementary Datasets S1 and S2. Note that Supplementary Dataset S1 contains both absolute and normalized counts of viral siRNA reads for each sRNA library. As explained in the Results section the normalization per million of host 21-nt sRNA reads is the most appropriate method for comparison of viral siRNA accumulation between wild type (Col-0) and siRNA-deficient (rdr126 and dcl234) Arabidopsis plants.
A

Percentage of plant genome- and virus-derived 20-25 nt sRNAs
in a total 20-25 nt sRNA population

<figure><img src="image_13.png" /></figure>

B

Percentage of 20-nt, 21-nt, 22-nt, 23-nt, 24-nt and 25-nt sRNAs in total 20-25 nt sRNA populations of the plant endogenous and viral sRNAs

<figure><img src="image_14.png" /></figure>

Fig. 4. Illumina sequencing counts of endogenous and viral sRNAs in mock-inoculated and virus (W41, or M41)-infected *A. thaliana* (Col-0 or rdr126) at 5 dpi and *N. benthamiana* (N.b.) at 5 and 26 dpi. (A)

Percentages of virus- and plant-derived sRNAs (mapped to ORMV and plant genome reference sequences with zero mismatches) in the pool of total 20-25 nt reads. (B) Size class percentages for 20-25 nt virus- and plant-derived sRNA reads (mapped with zero mismatches to the corresponding reference sequence). Note that aliquots of the same total RNA samples (extracted from systemic leaf pools of three plants per each condition) was used for both Illumina sequencing and blot hybridization analysis in Fig. 1C and 1D. In the case of *N. benthamiana*, total RNA was extracted from systemic leaf pools of two plants per condition and analyzed by blot hybridization (Supplementary Fig. S1J-S1K, experiment 2). The details of all sRNA libraries with absolute and normalized counts of plant and viral sRNA reads are given in Supplementary Datasets S1.
<figure><img src="image_15.png" /><figcaption>ORMV genomic RNA: mutant M41</figcaption></figure>

<figure><img src="image_16.png" /></figure>

Fig. 5. Single-nucleotide resolution maps of 20-25 nt viral siRNAs from virus (W41, or M41)-infected A. thaliana (Col-0 or rdr126) at 5 dpi and N. benthamiana (N.b.) at 5 and 26 dpi. For each plant genotype and time point, the histograms plot the numbers of total 20-25 nt, 21-nt and 24-nt viral sRNA reads at each nucleotide position of the 6303 bp W41 or M41 reference genome (mapped with zero mismatches); the bars above the axis represent sense reads starting at each respective position and those below represent antisense reads ending at the respective position. A scaled ORMV genome diagram is shown above the histograms, with the ORFs boxed and their nucleotide positions indicated. The details of sRNA counts and single-nucleotide resolution maps are given in Supplementary Datasets S1 and S3. Note that Supplementary Dataset S1 contains both absolute and normalized counts of viral siRNA reads for each sRNA library. As explained in the Results section the normalization per million of host 21-nt sRNA reads is the most appropriate for comparison of viral siRNA accumulation between wild type (Col-0) and siRNA-deficient (rdr126 and dcl234) Arabidopsis plants.
<figure><img src="image_17.png" /><figcaption>Fig. 6. β-elimination analysis of viral and endogenous sRNAs in plants infected with wild-type (W41) or p125-deficient (M41) virus. Two aliquots were taken from each total RNA sample from N. benthamiana (N.b.) systemically infected with W41 (5 dpi; systemic leaf pool of two plants) or M41 (26 dpi; systemic leaf pool of two plants), or A. thaliana (Col-0) infected with W41 (22 dpi; systemic leaf pool of three plants). One was treated with periodate (+) and the other with buffer (-) and both were analyzed by 15% PAGE and blotted. The blot was successively hybridized with DNA oligonucleotide probes specific for viral antisense siRNAs derived from ORMV p125 or CP genes, for plant miRNAs (miR168, miR482), miRNA* (miR482*), siRNA (siR255) and siRNA* (siR255*) species and for a synthetic non-methylated 22-nt RNA (spiked into each total RNA sample as an internal control). sRNA sizes are indicated on each scan. The EtBr-stained gel is shown as loading control. An additional β-elimination analysis of viral and endogenous sRNAs in A. thaliana plants infected with W41 or M41 at 5 dpi is shown in Supplementary Fig. S1I.</figcaption></figure>
A ORMV p125 construct

<figure><img src="image_18.png" /></figure>

B Transient expression in *N.benthamiana* leaves

Relative GFP fluorescence

<table><tr><td>Empty</td><td>-</td></tr><tr><td>p125</td><td>++++</td></tr><tr><td>pHC-Pro / p19</td><td>++++</td></tr></table>

Small RNA blot hybridization

<figure><img src="image_19.png" /></figure>

C Phenotypes of p125-trasgenic versus ORMV-infected *A. thaliana* plants

p125-transgenic

<figure><img src="image_20.png" /><figcaption>Before bolting</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>After bolting</figcaption></figure>

ORMV-infected

<figure><img src="image_22.png" /><figcaption>Inoculated early</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>Inoculated late</figcaption></figure>

D Western blot analysis

<figure><img src="image_24.png" /></figure>

E Small RNA analysis of *A. thaliana* transgenics

<figure><img src="image_25.png" /></figure>

Fig. 7. Transgenic expression of p125 alone partially recapitulates viral disease symptoms and the impact of viral infection on plant sRNAs. (A) Schematic representation of a pEarleygate expression construct carrying ORMV p125 coding sequence under control of the CaMV 35S promoter and OSC terminator. (B) Effects of transient expression of the ORMV p125, pHC-Pro or p19 constructs on transgene-induced silencing (measured as relative GFP fluorescence; see Supplementary S2A and S2B) and the accumulation of GFP siRNAs and plant miRNAs (examined by sRNA blot hybridization at 10 dpi) in N. benthamiana GFP transgenic 16c plants. The sRNA blot was successively hybridized with DNA oligonucleotide probes specific for GFP sense and antisense siRNAs and plant miR482, miR160 and miR482*. sRNA sizes are indicated. The EtBr-stained gel is shown as a loading control. (C) Representative phenotypes of the p125-transgenic plants (T2-plants of line 32) before and after bolting (see Supplementary Fig. S2D-S2F for other transgenic lines and controls), compared to ORMV-infected A. thaliana Col-0 plants (the rosette leaves following early inoculation and the stem following late inoculation). (D) Western blotting analysis of viral p125 and p182 proteins in Arabidopsis Col-0 plants infected with ORMV (W41) at 5 and 15 dpi, and in Arabidopsis transgenic plants carrying the p125 (T3-plant of line 33) construct. The membrane was incubated with an anti-p125 antibody or stained with AmidoBlack, shown here as loading control (Rubisco). The expected positions of p125 and p182 are indicated with arrowheads. (E) sRNA blot hybridization analysis (15% PAGE) of total RNA samples from two individual plants of the Arabidopsis transgenic line carrying the p125 expression construct (two T3-plants of line 32, lanes 2-3), or control wild-type plant (Col-0; lane 1). The blot was successively hybridized with DNA oligonucleotide probes specific for plant miR168, miR403, miR173, miR472*, siR255 and siR255* and for Met-tRNA. sRNA sizes are indicated on each scan. The EtBr-stained gel is shown as loading control. See Supplementary Fig. S2 for analysis of other ORMV constructs in transient assay and transgenic plants.
**Supplementary Figure S1. Infectivity tests and molecular analysis of the p125-deficient virus in *N. benthamiana*, *A. thaliana* and its mutant lines with impaired siRNA biogenesis.**

(S1A) In vitro transcription of ORMV genome constructs. The top panel contains linear diagrams of the genome organization of each ORMV construct, depicting the p125, p182, movement protein (MP) and coat protein (CP) ORFs, and a diagram of a prototypical ORMV genome construct in the context of a circular plasmid (pORMV). The leaky stop codon of p125 is shown as a vertical red line in the two p125-expressing constructs (W41 and W4) and the mutated p125 stop codon is shown as a red circle in constructs M41 and M4. The T-to-G substitution at position 5612 (which eliminates the original MP stop codon, elongating the protein by an additional 19 amino acids) is shown as a blue circle in constructs W4 and M4. The location of the T7 promoter for in vitro transcription and of the restriction sites (Nsi I and Pst I) used to linearize the constructs before in vitro transcription are also shown. The bottom panel shows an analysis by non-denaturing agarose gel electrophoresis of two in vitro transcription experiments (N1 and N2) using the linearized constructs, indicating the positions of the linear template DNA and viral RNA transcripts with arrows. The positive control was an in vitro transcription reaction from the linear plasmid pTRI-Xef, provided by the kit supplier (Ambion).
<figure><img src="image_26.png" /><figcaption>Fig. S1A. *In vitro* transcription of ORMV genome constructs</figcaption></figure>
This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.

(S1B) Infection of N. benthamiana with in vitro transcripts. The figure shows the disease symptoms caused by infection with in vitro transcripts from two different experiments (N1 and N2). N. benthamiana plants (two per construct) were inoculated with in vitro transcripts from wild-type (W41 or W4) or mutant (M41 or M4) ORMV constructs. The symptoms of viral infection at 5, 10 and 26 dpi are shown, indicating their relative severity. Note that after stem necrosis and wilting at 5 dpi, systemic tissue death had occurred in all plants infected with W41 or W4 by 26 dpi (not shown). In experiment N2, one of the two plants inoculated with M41 (plant 1) remained asymptomatic up to 26 dpi (not shown), even though plant 2 and both plants inoculated with M4 had developed disease symptoms by that date. In both cases, the figures shows only plant 2 of both conditions at 26 dpi.
<figure><img src="image_27.png" /><figcaption>Fig. S1B. *N. benthamiana* infected with the *in vitro* transcripts</figcaption></figure>

Experiment N1

<figure><img src="image_28.png" /></figure>

Experiment N2

<figure><img src="image_29.png" /></figure>

Symptom severity at 5 dpi: **W4 ≥ W41 >>> M4 ≥ M41**
(S1C) Infection of A. thaliana with in vitro transcripts. Disease symptoms caused in Col-0, rdr126 and dcl234 plants by infection with in vitro transcripts. Each A. thaliana line (two plants per construct) was inoculated with in vitro transcripts from wild-type (W41 or W4) or mutant (M41 or M4) ORMV constructs. The figure shows the symptoms of viral infection at 22 dpi for one representative plant per construct, together with the relative severity of disease symptoms. The experiment was repeated two times with similar results.
<figure><img src="image_30.png" /><figcaption>Fig. S1C. *A. thaliana* plants infected with the *in vitro* transcripts</figcaption></figure>
(S1D) RT-PCR amplification and sequencing of viral RNA to examine the stability of the mutated stop codon of p125 in planta. RT-PCR analysis of viral RNA prepared from A. thaliana and N. benthamiana plants inoculated with wild-type or mutant viral constructs (leaf tissues were harvested from plants shown in Figures S1B and S1C).

The top panel shows a diagram of the ORMV genome, the positive sense genomic RNA transcribed from the ORMV constructs and replicated in infected plants (red line), the location of the primer (short green arrow) used to generate the cDNA (black line) and the location of the primers (orange and red arrows) used to generate a PCR amplicon containing the p125 stop codon (original, mutated, or reverted). The table below summarizes the RT-PCR sequencing results (shown in Supplementary Figures S1E and S1F). For each construct (W41, W4, M41, M41) the columns show the sequence context of the p125 stop codon (underlined) in the constructs used for in vitro transcription and the actual sequence of the codon obtained by sequencing of the viral RNA from the infected plants at 22 dpi for A. thaliana (Col-0) and its mutant lines (rdr126 and dcl234) and at 5 and 26 dpi for N. benthamiana (N.b.). Major and minor reversions of the p125 stop codon mutation are marked in red and orange.
<figure><img src="image_31.png" /><figcaption>Fig. S1D. RT-PCR amplification and sequencing of the viral RNA</figcaption></figure>

<table><thead><tr><th></th><th>Type</th><th>Transcript</th><th>Col-0<br>22 dpi</th><th>rdr126<br>22 dpi</th><th>dcl234<br>22 dpi</th><th>N. b.<br>5 dpi</th><th>N. b.<br>26 dpi</th></tr></thead><tbody><tr><td>W41</td><td>Original</td><td>TAG CAA</td><td>TAG CAA</td><td>TAG CAA</td><td>TAG CAA</td><td>TAG CAA</td><td>-</td></tr><tr><td>W4</td><td>Original</td><td>TAG CAA</td><td>TAG CAA</td><td>TAG CAA</td><td>TAG CAA</td><td>TAG CAA</td><td>-</td></tr><tr><td>M41</td><td>Mutated</td><td>TAC CAA</td><td>TAA CAA<br>(Major)</td><td>TAA CAA<br>(Major)</td><td>TAA CAA<br>(Major)</td><td>TAC CAA</td><td>TAC CAA<br>(Minor rev)</td></tr><tr><td>M4</td><td>Mutated</td><td>TAC CAA</td><td>TAA CAA<br>(Major)</td><td>TAA CAA<br>(Major)</td><td>TAA CAA<br>(Major)</td><td>TAC CAA</td><td>TAC CAA<br>(Minor rev)</td></tr></tbody></table>
(S1E) No (or minor) reversion of the mutated stop codon in N. benthamiana.

Partial sequencing electrophoregrams of cDNA-PCR amplicons from viral RNA
of N. benthamiana plants inoculated with the mutant ORMV constructs (M41
and M4) at 5 dpi and 26 dpi. The mutated stop codon of p125 is boxed and the
peak showing a minor reversion to the stop codon is labeled with an asterisk.
<figure><img src="image_32.png" /><figcaption>Fig. S1E. No (or minor) reversion of the mutated stop codon
in *N. benthamiana*</figcaption></figure>
(S1F) Major reversions recreating the mutated stop codon of p125 in A. thaliana. Partial sequencing electrophoregrams of cDNA-PCR amplicons from viral RNA of A. thaliana plants (Col-0, rdr126 and dcl234) inoculated with the mutant ORMV constructs (M41 and M4) at 22 dpi. The mutated stop codon of p125 is boxed and the peak showing a major reversion to the stop codon is labeled with an asterisk.
<caption>Fig. S1F. Major reversions that recreate the p125 stop codon in *A. thaliana*</caption>

This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.

<figure><img src="image_33.png" /></figure>
(S1G) Blot hybridization analysis of viral gRNAs and sgRNAs in A. thaliana plants (Col-0, rdr126 and dcl234) inoculated with in vitro transcripts from ORMV constructs (W41, W4, M41, M4). Total RNA was extracted from leaf tissues harvested at 22 dpi in pools (two plants per condition). The top panels show long RNA analysis (in denaturing 1% agarose gels) of total RNA samples from a pool of two plants per construct. The blots show viral positive-sense RNAs (gRNA, sgRNA-MP, sgRNA-CP) detected by the probe specific for the CP region of the ORMV genome and gRNA(-) and CP(-) detected by the reverse-complementary probe. The EtBr-stained gels used for the blots are shown below as loading controls. The positions of the gRNA, sgRNA-MP and sgRNA-CP are marked with arrows. Note that by 22 dpi, stop codon revertants had overtaken the initial population of mutant viruses (M41 and M4) in all the lines (Col-0, rdr126 and dcl234, see Fig. S1F), thereby restoring p125 expression (see Fig. 1F). Note that aliquots of the same total RNA samples were taken for small RNA analysis shown in Fig. S1H.
<figure><img src="image_34.png" /><figcaption>Fig. S1G. Blot hybridization analysis of viral gRNA and sgRNAs in *A. thaliana* wild type (Col-0) and mutant (rdr126 and dcl234) plants at 22 dpi</figcaption></figure>

Molecular Plant-Microbe Interactions "First Look" paper • http://dx.doi.org/10.1094/MPMI-07-17-0164-R • posted 09/19/2017
This paper has been peer reviewed and accepted for publication but has not yet been copyedited or proofread. The final published version may differ.
(S1H) Blot hybridization analysis of viral and endogenous sRNAs from A. thaliana (Col-0, rdr126 and dcl234 inoculated with in vitro transcripts from ORMV constructs (W41, W4, M41, M4). Total RNA was extracted from leaf tissues harvested at 22 dpi in pools (two plants per condition). The panels show high-resolution sRNA analysis (15% PAGE) of total RNA samples from a pool of two plants per construct. The blots were successively hybridized with DNA oligonucleotide probes specific for viral antisense siRNAs derived from the ORMV p125 region as well as for plant miRNA (miR160), rasiRNA (siR1003), miRNA* (miR173*, miR472*) and tasiRNA * (siR255*) species. Note that by 22 dpi the stop codon mutation of viruses M41 and M4 has been fully reverted (see Fig. S1F), thereby restoring p125 expression (see Fig 1F). Note that aliquots of the same total RNA samples were taken for the small RNA analysis (shown in this Figure) and long RNA analysis shown in Fig. S1G.
<figure><img src="image_35.png" /><figcaption>Fig. S1H. Blot hybridization analysis of viral and endogenous sRNAs in *A. thaliana* wild type (Col-0) and mutant (rdr126 and dcl234) plants at 22 dpi</figcaption></figure>
(S1) Analysis of viral long and small RNAs from *A. thaliana* plants (Col-0, rdr126) inoculated with in vitro transcripts from ORMV constructs W41 and M41 at 5 dpi.

(a) RNA blot hybridization analysis (in denaturing 1% agarose gels) of total RNA samples from a pool of two plants per construct per condition. The blot shows viral positive-sense RNAs (gRNA, sgRNA-MP, sgRNA-CP, marked with arrows) detected by the probe specific for the CP region of ORMV genome. The EtBr-stained gel is shown below as loading controls. The relative accumulation of combined viral (g+sg) RNAs was calculated as total viral RNA signal intensities normalized by EtBr staining intensities, with W41-infected Col-0 set to 1.0.

(b) Small RNA blot hybridization analysis (15% PAGE) of total RNA samples from panel (a). The blot was successively hybridized with DNA oligonucleotide probes specific for viral antisense siRNAs derived from ORMV p125 and CP regions as well as for plant miR168, miR168*, miR472, miR472*, siR225) and siR255* species. sRNA sizes are indicated on each scan. EtBr staining of the gel is shown as loading control.

(c) β-elimination analysis of viral and endogenous sRNAs in total RNAs from panel (a). Two aliquots were taken from each total RNA sample. One was treated with periodate (+) and the other with buffer (-) and both were analyzed by 15% PAGE and blotted. The blot was successively hybridized with DNA oligonucleotide probes specific for viral antisense siRNAs derived from ORMV p125 or CP genes and for plant miRNA* species (miR472*). sRNA sizes are indicated on each scan. The EtBr-stained gel is shown as loading control.

(d) Western blot analysis of ORMV p125 and p182 protein accumulation in *A. thaliana* Col-0 and rdr126 plants infected with M41 or W41 at 5 dpi. The membrane was incubated with anti-p125 antibodies, scanned and then stained with Amido Black for loading control. The positions of p125 and p182 are indicated with arrows.
<caption>Fig. S1I. Blot hybridization analysis of **M41** and **W41** gRNA, sgRNAs and sRNAs in *A. thaliana* wild type (**Col-0**) and mutant (**rdr126**) plants at **5 dpi**</caption>

Molecular Plant-Microbe Interactions "First Look" paper • http://dx.doi.org/10.1094/MPMI-07-17-0164-R • posted 09/19/2017
This paper has been peer reviewed and accepted for publication; it has not yet been copyedited or proofread. The final published version may differ.

<figure><img src="image_36.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_37.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>EtBr</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>(d)</figcaption></figure>
(S1J) Blot hybridization analysis of 5 and 26 dpi viral long RNAs from N. benthamiana plants inoculated with in vitro transcripts from ORMV constructs (W41, W4, M41, M4). The top panel shows a long RNA blot (1% agarose denaturing gel electrophoresis) of total RNA samples from two experiments: in experiment N2, the RNA was extracted from a systemic leaf pool of two plants, while in experiment N3 it was extracted from systemic leaves of individual plants selected on the basis of the Western blot data shown in Fig. S1K (plants indicated with black asterisks). The blots show ORMV positive-sense RNAs (gRNA, sgRNA-MP, sgRNA-CP) detected by a probe specific for the CP region of the ORMV genome. The EtBr-stained gel used for the blot is shown below as a loading control. The position of the gRNA, sgRNA-MP and sgRNA-CP species are marked with arrows. Note that in experiment N2 one of the two plants of pool M41 was asymptomatic (Fig. S1B, plant 1) and had not developed disease symptoms at 26 dpi. The relative accumulation of combined viral genomic and subgenomic RNAs was calculated as total viral RNA signal intensities normalized by EtBr staining intensities, with W41-infected plants set to 1.0.
<figure><img src="image_40.png" /><figcaption>Fig. S1J. Northern blot hybridization analysis of viral long RNAs
in *N. benthamiana* plants at **5 dpi** and **26 dpi**</figcaption></figure>

* Pool of two plants: one symptomatic + one non-symptomatic (see Fig. S1B)

** Experiment N2 pool of two plants per construct

*** Experiment N3
Individual plants
no reversion of p125
in selected M41 and M4
plants, see Fig. S1L
(S1K) Blot hybridization analysis of 5 dpi viral and endogenous sRNAs from N. benthamiana plants inoculated with in vitro transcripts from ORMV constructs (W41, W4, M41, M4). The panels show high-resolution sRNA blots (15% PAGE) of total RNA samples from experiment N2 (pool of two plants per construct), hybridized successively with DNA oligonucleotide probes specific for the viral antisense siRNAs derived from ORMV p125 region, plant miRNAs (miR160 and miR482) and the * species of miR482 (miR482*). Note that in experiment N2 one of the two plants of pool M41 was asymptomatic (Fig. S1B, plant 1) and had not developed disease symptoms at 26 dpi.
<figure><img src="image_41.png" /><figcaption>Fig. S1K.Blot hybridization analysis of ORMV and endogenous sRNAs in *N. benthamiana* plants at 5 dpi (Experiment N2)</figcaption></figure>

* Pool of two plants: one symptomatic + one not symptomatic see Fig. S1B
(S1L) Western blot analysis of ORMV p125 and p182 proteins. Total protein samples were obtained from upper (systemic) leaf tissues of individual N. benthamiana plants at 5 dpi following inoculation with in vitro transcripts of the viral constructs M4, M41, W4 or W41 (3 plants per construct) and separated in a 9% SDS-PAGE gel. ORMV p182 and p125 were detected with a primary anti-p125 antibody (see Material and Methods). The AmidoBlack-stained membrane is shown below as a loading control. Note that in plants inoculated with W4- and W41-derived transcripts, both proteins were present (although p125 constituted the most abundant species of the two), while in plants inoculated with M4- and M41-derived transcripts only p182 was present, with the exception of plant M41-3 (indicated with red asterisks), where stop codon revertants overtook the mutant population and restored p125 expression. The black asterisks indicate the individual plants taken for the RNA blot hybridization shown in Fig. S1J (Experiment N3).
<caption>Fig. S1L. Western blot analysis of ORMV p125 and p182 proteins</caption>

in *N. benthamiana* plants at 5 dpi

(Experiment N2, individual plants)

<figure><img src="image_42.png" /></figure>
Supplementary Fig. S2. Transient and stable expression of ORMV p125 and/or p182 proteins from pEarleygate constructs in *N. benthamiana* and *A. thaliana*.

**(S2A)** Effects of the transient expression of p125, p125/182 and p182 constructs on GFP transgene-induced silencing in N. benthamiana 16c leaves. In this experiment each construct was expressed on a separate leaf. GFP fluorescence images of the leaves under UV light were taken at 6 and 8 days dpi, following infiltration with an agro strain carrying a GFP expression cassette in combination with buffer (no suppressor control), an agro strain carrying the potyviral pHC-Pro (strong silencing suppressor control), or each ORMV protein expression cassette. The ORMV constructs without tag (p125, p125/182 and p182) contain the wild type ORMV protein sequences, while the constructs p125-HA, p125/182-HA and p182-HA have an HA-tag and the constructs p125-FG, p125/182-FG and p182-FG have a FLAG-tag fused to the N-terminus of the ORMV proteins.
<figure><img src="image_43.png" /><figcaption>Fig. S2A. Effects of transient expression of *p125*, *p125/182* and *p182* constructs on GFP transgene-induced silencing in *N. benthamiana* 16c leaves</figcaption></figure>
(S2B) Effects of the transient expression of p125, p125/182 and p182 constructs on GFP transgene-induced silencing in N. benthamiana 16c leaves. In this experiment each set of three ORMV constructs either without tag (p125, p125/182 and p182), with an HA-tag (p125-HA, p125/182-HA and p182-HA), or with a FLAG-tag (p125-FG, p125/182-FG and p182-FG) were expressed in separate patches of the same leaf. GFP fluorescence images of the leaves under UV light were taken at 7 and 10 days after infiltration with an agro strain carrying a GFP expression cassette in combination with either an empty agro strain (no suppressor control, patch N2 on each leaf), an agro strain carrying tombusviral p19 (strong silencing suppressor control; patch N1 on each leaf), an agro strain carrying p125 (patch N3 on each leaf), p125/182 (patch N4 on each leaf), or p182 (batch N5 on each leaf) without or with tag as indicated above the images. Note that a so-called "red ring", evidence of cell-to-cell movement of the GFP silencing signal (Himber et al. 2003) is clearly visible at 10 dpi around control patch N2 (no suppressor) as well as around patch N5 for all three p182 constructs and around patch N3 for the p125-HA construct. No red ring is apparent for the p125 or p125-FG constructs, indicating, similarly to tombusviral p19, that the wild-type p125 protein and its N-terminal FLAG-tagged version strongly suppress Both cell-autonomous silencing and cell-to-cell movement of the silencing signal (see Himber et al. 2003).
<figure><img src="image_44.png" /><figcaption>Fig. S2B. Effects of transient expression of *p125*, *p125/182* and *p182* constructs on GFP transgene-induced silencing in *N. benthamiana* 16c leaves</figcaption></figure>
**Fig. S2C. Effect on transiently expressed p125 and/or p182 on the GFP transgenic and plant small RNAs in *N. benthamiana* 16c leaves.**

(a) Schematic representation of pEarleygate expression constructs carrying ORMV p125, p125/182 or p182 protein coding sequences (with or without N-terminal HA- or FLAG-tags) under control of the CaMV 35S promoter. (b) Effects of transient expression of the ORMV p125, p125/182 and p182 constructs on transgene-induced silencing (measured as relative GFP fluorescence; see Fig. S2A and S2B) and the accumulation of GFP siRNAs and plant miRNAs (examined by sRNA blot hybridization at 10 dpi) in *N. benthamiana* GFP transgenic 16c plants. The sRNA blot was successively hybridized with DNA oligonucleotide probes specific for GFP sense and antisense siRNAs and plant miR482, mi160 and miR482*. sRNA sizes are indicated. The EtBr-stained gel is shown as a loading control.

<figure><img src="image_45.png" /><figcaption>(b) Transient expression in *N.benthamiana* 16c leaves</figcaption></figure>

<table><thead><tr><th colspan="2">Relative GFP fluorescence</th></tr></thead><tbody><tr><td>Empty</td><td>-</td></tr><tr><td>p125</td><td>++++</td></tr><tr><td>p125-HA</td><td>++</td></tr><tr><td>p125-FG</td><td>++++</td></tr><tr><td>p182</td><td>-</td></tr><tr><td>p182-HA</td><td>-</td></tr><tr><td>p182-FG</td><td>-</td></tr><tr><td>p125/182</td><td>+</td></tr><tr><td>p125/182-HA</td><td>+/-</td></tr><tr><td>p125/182-FG</td><td>+</td></tr><tr><td>pHC-Pro / p19</td><td>++++</td></tr></tbody></table>

<figure><img src="image_46.png" /></figure>
(S2D) Phenotypes of Arabidopsis transgenic lines carrying the wild-type p125 expression construct. At maturity, seeds from each dipped T0 plant were collected and placed on soil to germinate. Transgenic seedlings were identified by their resistance to the herbicide BASTA (see Materials and Methods). The typical serrated morphology of the rosette leaves is shown for T2 and T3 plants obtained from 2 different T1 events (lines 32 and 33). A "zig-zag" stem curling phenotype can also be observed in T2 plants from both lines at the bolting stage.
<caption>Fig. S2D. Phenotypes of *Arabidopsis* transgenic lines carrying wild-type p125</caption>

<figure><img src="image_47.png" /><figcaption>Representative T2 plant of p125 line 32 (after bolting)</figcaption></figure>

<figure><img src="image_48.png" /><figcaption>Representative T3-plants of p125 line 32</figcaption></figure>

<figure><img src="image_49.png" /><figcaption>T2-plants of p125 line 33</figcaption></figure>

<figure><img src="image_50.png" /><figcaption>Representative T2 plant p125 line 33 (after bolting)</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>Representative T3-plants of p125 line 33</figcaption></figure>

<figure><img src="image_52.png" /></figure>
(S2E) Phenotypes of Arabidopsis transgenic lines carrying p125 constructs with HA- or FLAG (FG)-tags. Images of the pools and of representative individuals of Basta-resistant T2-plants from two independent T1 transgenic events for p125-HA (lines 40 and 45) and p125-FG (lines 50 and 49). Note that p125-HA transgenic lines 40 and 45 exhibit almost no serration of the rosette leaves, and line 45 has a normal stem. p125-FG transgenic line 50 exhibits a strong serrated leaf phenotype and an aberrant silique arrangement at the bolting stage, while line 49 has two different rosette leaf phenotypes.
Fig. S2E. Phenotypes of *Arabidopsis* transgenic lines carrying p125 with N-terminal HA or FLAG (FG) tags

Pool of p125-HA line 40 T2-plants and individual representative T2-plants from pools of p125-HA line40 and p125-HA line45

<figure><img src="image_53.png" /></figure>

Pool of p125-FG line 50 T2-plants and individual representative T2-plants from pools of p125-FG line 50 and p125-FG line49

<figure><img src="image_54.png" /></figure>
(S2F) Phenotypes of A. thaliana transgenic lines carrying p182 or p125/182 transgenes without tag or with HA or FG tags. The top panels contain images of the pool and of representative individuals of Basta-resistant T2-plants from two T1 transgenic events for each construct: p182 (lines 3 and 2), p182-HA (lines 10 and 5) and p125-FG (lines 3 and 4). Note that the transgenic T2 plants carrying the p182 constructs, regardless of tag status, exhibit normal rosette leaf phenotypes without serration. No stem curling or other inflorescence abnormalities were observed after bolting (not shown). The bottom panels show pictures of representative Basta-resistant T2-, T3- and T4-Plants from independent T1 transgenic events for p125/182 constructs with and without tag: p125/182 (line 0), p125/182-HA (lines 5 and 3) and p125/182-FG (line 1). Note that some of the T2 plants exhibit slightly abnormal leaf phenotypes but no obvious serration or stem curling (not shown).
<caption>**Fig. S2F.** Phenotypes of *A. thaliana* transgenic lines carrying p182 and p125/182 constructs</caption>

Pools and representative T2-plants of transgenic lines carrying p182, p182-HA, and p182-FG constructs

<figure><img src="image_55.png" /></figure>

Representative T2- or T3-plants of transgenic lines carrying p125/182, p125/182-HA, and p125/182-FG constructs

<figure><img src="image_56.png" /></figure>
**Fig. S2G. Transgenic expression of p125 alone partially recapitulates the impact of viral infection on plant sRNAs.**

(a) sRNA blot hybridization analysis (15% PAGE) of total RNA samples from individual plants of *Arabidopsis* transgenic lines carrying p125 (T3-plants of line 32, lanes 2-3), p125-HA (T3-plants of line 40, lanes 4-5), p125-FG (T3-plants of line 49, lanes 6-7), p182 (T3-plant of line 1, lane 8), p182-HA (T3-plant of line 2, lane 9), p182-FG (T3-plant of line 1, lane 10), p125/182 (T2-plants of line 0, lanes 13-14; T2-plant of line 3, lane 15), p125/182-HA (T2-plants of line 0, lanes 13-14; T2-plant of line 3, lane 15), and p125/182-FG (T3-plants of line 1, lanes 16-18) expression constructs, or control wild-type plants (Col-0; lanes 1,11-12). The blot was successively hybridized with DNA oligonucleotide probes specific for plant miR168, miR403, miR173, miR472*, siR255 and siR255* and for Met-tRNA. sRNA sizes are indicated on each scan. The EtBr-stained gel is shown as loading control.

(b) Western blotting analysis of viral p125 and p182 proteins in *Arabidopsis* Col-0 plants infected with ORMV (W41) at 5 and 15 dpi, and in *Arabidopsis* transgenic plants carrying the p125 (T3-plant of line 33) and p182 (T3-plant of line 3) constructs. The membrane was incubated with an anti-p125 antibody or stained with AmidoBlack, shown here as loading control (Rubisco). The expected positions of p125 and p182 are indicated with arrowheads.

(c) Quantitative (q)RT-PCR analysis of relative accumulation levels of transgenic transcripts in individual plants from Arabidopsis transgenic lines carrying p125-HA (A-C), p125/182-HA (D,E) and p182-HA (F,G). Col-0 plant was used as a negative control. The Arabidopsis Actin2 gene was used for normalization of transgene transcript expression levels.
Fig. S2G. Transgenic expression of p125 alone partially recapitulates the impact of viral infection on plant sRNAs.

<figure><img src="image_57.png" /><figcaption>(b) Western blot analysis</figcaption></figure>

<figure><img src="image_58.png" /><figcaption>(c) qRT-PCR analysis of transcript levels in Arabidopsis lines carrying p125, p125/182 or p182 constructs</figcaption></figure>

<figure><img src="image_59.png" /></figure>
<table><caption>Supplementary Table S1. DNA oligonucleotide probes, PCR primers and synthetic gene fragments</caption><thead><tr><th>Name original</th><th>Detects</th><th>(Gene/region)</th><th>Sequence (5' to 3')</th></tr></thead><tbody><tr><td colspan="4"><strong>A. thaliana genome sRNA specific probes:</strong></td></tr><tr><td>miR160a_as</td><td>miR160</td><td>miRNA</td><td>TGGCATACAGGGAGCCAGGCA</td></tr><tr><td>miR168a_as</td><td>miR168</td><td>miRNA</td><td>TTCCCGACCTGCACCAAGCGA</td></tr><tr><td>miR173_as</td><td>miR173</td><td>miRNA</td><td>GTGATTTCTCTCTGCAAGCGAA</td></tr><tr><td>miR173<sup>*</sup>_as</td><td>miR173*</td><td>miRNA*</td><td>CTTTCGCTTACACAGAGAATC</td></tr><tr><td>miR403a_as</td><td>miR403</td><td>miRNA</td><td>CGAGTTTGTGCGTGAATCTAA</td></tr><tr><td>miR472_as</td><td>miR472</td><td>miRNA</td><td>GGTATGGGCGGAGTAGGAAAAA</td></tr><tr><td>miR472<sup>*</sup>_as</td><td>miR472*</td><td>miRNA*</td><td>GATTTTGCCTACTTCGACCAT</td></tr><tr><td>siR255_as</td><td>siR255</td><td>TAS1a/b/c siRNA</td><td>TACGCTATGTTGGACTTAGAA</td></tr><tr><td>siR255<sup>*</sup>_as</td><td>siR255*</td><td>TAS1a/b/c siRNA*</td><td>TATTCTAAGTCCAACATAGCG</td></tr><tr><td>siR1003_as</td><td>siR1003</td><td>5S rRNA siRNA</td><td>ATGCCTATGTTGGCCTCACGGTCT</td></tr><tr><td>Met_tRNA_as</td><td>Met-tRNA</td><td>tRNA</td><td>TGGTATCAGAGCCAGGTTTCGATCC</td></tr><tr><td colspan="4"><strong>N. benthamiana genome sRNA specific probes:</strong></td></tr><tr><td>miR160a_as</td><td>miR160</td><td>miRNA</td><td>TGGCATACAGGGAGCCAGGCA</td></tr><tr><td>nbmiR482a_as</td><td>miR482</td><td>miRNA</td><td>TAGGAATGGGTGGAATTGGAAA</td></tr><tr><td>nbmiR482a_star_as</td><td>miR482*</td><td>miRNA*</td><td>GCTTTCCAACCCACCAATCCC</td></tr><tr><td colspan="4"><strong>N. benthamiana GFP transgene-specific probes</strong></td></tr><tr><td>NOSterm_s</td><td>3'UTR a/sense siRNAs</td><td></td><td>CAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCG</td></tr><tr><td>NOSterm_as</td><td>3'UTR a/sense siRNAs</td><td></td><td>CGCAAGACCGGCAACAGGATTCAATCTTAAGAAACTTTATTG</td></tr><tr><td colspan="4"><strong>β-elimination internal control RNA-specific probe:</strong></td></tr><tr><td>GFP_22ntRNA_as</td><td>non-methylated GFP RNA</td><td></td><td>AGAAGAACGGCATCAAGGTGAA</td></tr><tr><td colspan="4"><strong>ORMV genome-specific probes for long RNA blot hybridization:</strong></td></tr><tr><td>OrmvCP_qPCR_s</td><td>ORMV CP antisense</td><td></td><td>GTACCAGTATTTCGCAGCGATGTG</td></tr><tr><td>OrmvCP_qPCR_as</td><td>ORMV CP sense</td><td></td><td>GTATCTCTTGCCGCTTGAGTTTGG</td></tr><tr><td colspan="4"><strong>Arabidopsis genome-specific probes for long RNA blot hybridization:</strong></td></tr><tr><td>AGO1_as</td><td>AGO1 mRNA</td><td></td><td>CCACTGTCTGATGTCTCTGGCTCCATGTAGAATC</td></tr><tr><td>AGO2_as</td><td>AGO2 mRNA</td><td></td><td>GAGATTACAAATTCGTTTCAACACACCAAAACC</td></tr><tr><td colspan="4"><strong>ORMV genome specific probes for small RNA blot hybridization:</strong></td></tr><tr><td>OrmvCP_qPCR_s</td><td>ORMV CP antisense siRNAs</td><td></td><td>GTACCAGTATTTCGCAGCGATGTG</td></tr><tr><td>Mixture of 3 oligos</td><td>ORMV p125 antisense siRNAs</td><td></td><td>GCACGCTCGAGACGCCCTAAGGTCCAT</td></tr><tr><td>ormv5_1s</td><td></td><td></td><td>AGGCCGTACACTCCCTAGCGGGTGGTC</td></tr><tr><td>ormv5_2s</td><td></td><td></td><td>CGCTGCGCACCTTTTCAAGGGACGCGA</td></tr><tr><td>ormv5_3s</td><td></td><td></td><td></td></tr><tr><td colspan="4"><strong>ORMV qPCR primers</strong></td></tr><tr><td>OrmvRep_qPCR_s</td><td>ORMV p125 sense</td><td></td><td>TGCCTCATTGTCAGCTACAGTCTC</td></tr><tr><td>OrmvRep_qPCR_as</td><td>ORMV p125 antisense</td><td></td><td>TAACCAGCGTCCTCTTCTCACATC</td></tr><tr><td>OrmvMP_qPCR_s</td><td>ORMV MP sense</td><td></td><td>CTGGCTCTGGAGGTGGTTTCTG</td></tr><tr><td>OrmvMP_qPCR_as</td><td>ORMV MP antisense</td><td></td><td>CTTCGACGTTCGGATCATTCACAG</td></tr><tr><td>OrmvCP_qPCR_s</td><td>ORMV CP sense</td><td></td><td>GTACCAGTATTTCGCAGCGATGTG</td></tr><tr><td>OrmvCP_qPCR_as</td><td>ORMV CP antisense</td><td></td><td>GTATCTCTTGCCGCTTGAGTTTGG</td></tr><tr><td colspan="4"><strong>PCR primers to introduce the ORMV p125 stop codon mutation:</strong></td></tr><tr><td>p126_stopsup_s</td><td>ORMV p125 stop sense</td><td></td><td>TCGGGTACCCAATAcCAATTACAGATCGACACAGTG</td></tr></tbody></table>
p126_stopsup_as ORMV p125 stop antisense GTCGATCTGTAATTTGgTATTGGGTACCCGACTCTAC
p180_ClaI_s p125 stop sense upstream AGGTTGTTTATCGATGAGGGATTAATGCTG
p180_XmaIII_as p125 stop a/sense downstream GAAAACCGGCCGTTCTTTCGGCACTTCAC

Gateway primers to generate vectors expressing ORMV p125 and/or p182 proteins:

AttB1_Rep_s ORMV p125 start sense ggggacaagtttgtacaaaaaagcaggctACAcCATGGCACAATTTCAACAAACAG
AttB2_p126_as ORMV p125 stop antisense ggggaccactttgtacaagaaagctgggtCCTATTGGGTACCCGACTCTACCT
AttB2_p180_as ORMV p182 stop antisense ggggaccactttgtacaagaaagctgggtcTCAAACAAAAAACAATCTTTAACAACC

PCR primers for genotyping transgenic lines:

CaMV35Spro_#1 CaMV 35S promoter CGCGTCGAGATGCTAAAT
Ormv141_as ORMV p125 ORF CGCGTCGAGATGCTAAATC

Synthetic fragments to repair the pORMV infectious clone:

>Ormv_NarI EcoNI
ggcgccTTAATACGACTCACTATAGTTTTATATTTTATTGCAACAACAACAACAATTACAATAACAACAAAACAATAACACAACAACAACATGGCACAATTTCAACAAACAGTAAAC
ATGCAAACATTGCAGGCTGCCGCGGGCGCAACAGCCTGGTGAATGATTTAGCATCTCGACGCGTTTATGATAATGCTGTCGAGGAGCTAAATGCACGCTCGAGACGCCCTAAGGTCCA
TTTCTCCAAATCAGTGTCTACGGAACAGACGCTGTTGGCTTCAAACGCTTATCCGGAGTTTGAGATTTCCTTTACTCATACCCAACAGGCCGTACACTCCTAGCGGGTGGTCTAAGGA
CTCTTGAGTTGGAGTATCTCATGCAAGTTCCGTTCCGGTTCTCTAACGTACGATATCGGTGGTAACTTCGCTGCGCAccttttcaagg

>Ormv_NcoI_PstI
ccatggTTACTAATAACGTGGTTGTAAAGGTTTGAGGGAAAAGTCATCGCTGTGAATGATCCGAACGTCGAAGGTTTCGAAGGTGTGGTTGACGATTTCGTCGATTCGGTTGCTGCA
TTCAAGGCGATTGACAGTTTCAGGAAGAAAAGAAAAGGATTGGAGGAAGGGATGTAATAGTAATAAGTATAGATATAGACCGGAGATACGCCGGTCCTGATTTCGTTACAATATAA
AGAAGAAAATGGTTTACAACATCACTAGCTCGAATCAGTACCAGTATTTTCGACGCGATGTGGGCAGAGCCCACAGCGATGCTTAACCAGTGCGTGTCTGCGTTGTCGCAGTCGTACCAA
ACTCAAGCGGCAAGAGATACTGTTAGACAGCAGTTCTCTAACCTTCTGAGTGCGATTGTGACACCGAACCCAGCGGTTTCCAGAAGCAGGATACCGGGTGTATATTAATTCAGCAGTTCT
AAAACCGTTGTACGAGTCTCTCATGAAGTCCTTTGATACTAGGAATAGGATCATTGAAACTGAAGAAGAGTCGCGTCCATCGGCTTCCGAAGTAGCTAATGCAACACAACGTGTTGATG
ATGCGACCGTGGCCATCAGGAGTCAAATTCAGCTTTTCTGCTGAACGAGCTCTCCAACGGACATGGTCTGATGAACAGGGCAGAGTTCGAGGTTTTATTACCTTGGGCTACTGCGCCAGCT
ACATAGGCGTGGTCACACGATAGTGCACAGTGTTTTTCTCTCCACTTAAATCGAAGAGATATACTTACGGTGAATTCCGTAAGGGTGGCGTAAACCAAATTACGCAATGTTTTAGGT
TCCATTTAATCGAAACCTGTTATTTCCTGGATCACCTGTTAACGTACGCGTGGCGTATATTACAGTGGGAATAACTAAAAGTGAGAGGTTCGAATCCTCCCTAACCCCGGGTAGGGGC
CCATGCATctgcag