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MOL ECUL AR P LA NT P ATHOL OG Y D OI: 10. 111 1/mpp. 124 88

1 The resistance of sour orange to Citrus tristeza virus is mediated

2 by both the salicylic acid and RNA silencing defence pathways
AQ11 3 N E U S G OM E Z - M U NO ~ Z 1 , K A R E L I A V E L AZ
 Q U E Z 1 , M A RIA C A R M E N V I V E S 1 , S U S A N A R U I Z - R U I Z 1 ,
4 J O S E A N T O N I O P I N A 1 , R I C A R D O F L O R E S 2 , P E D R O M O R E N O 1 A N D J O S E G U E R R I 1 , *
1
5 Instituto Valenciano de Investigaciones Agrarias (IVIA), Centro de Protecci
o n Vegetal y Biotecnologa, Moncada, Valencia, 46113, Spain
2
6 Instituto de Biologa Molecular y Celular de Plantas (UPV-CSIC), Universidad Polite cnica de Valencia, Avenida de los Naranjos, Valencia, 46022, Spain

7
8 assembly, movement or interactions with the host (Dawson et al., 46
9 SUMMARY 2015; Moreno et al., 2008). The p25, p20 and p23 proteins act as 47

10 Citrus tristeza virus (CTV) induces in the field the decline and RNA silencing suppressors in Nicotiana benthamiana (Lu et al., 48

11 death of citrus varieties grafted on sour orange (SO) rootstock, 2004; Ruiz-Ruiz et al., 2013), and p23 has been mapped as 49

12 which has forced the use of alternative decline-tolerant root- responsible for the seedling yellows (SY) syndrome (Albiach-Mart 50

13 stocks in affected countries, despite the highly desirable agro- et al., 2010). Moreover, transgenic Mexican lime [Citrus aurantifo- 51

14 nomic features of the SO rootstock. Declining citrus plants lia (Christm.) Swing.], expressing p23, displays symptoms closely 52

15 display phloem necrosis below the bud union. In addition, SO is resembling those produced by CTV infection (Soler et al., 2015). 53

16 minimally susceptible to CTV compared with other citrus vari- CTV causes economically important diseases on citrus world- 54

17 eties, suggesting partial resistance of SO to CTV. Here, by silenc- wide, including the decline of trees propagated on sour orange 55

18 ing different citrus genes with a Citrus leaf blotch virus-based (SO) (C. aurantium L.) rootstock, SY syndrome on grapefruit (C. 56

19 vector, we have examined the implication of the RNA silencing paradisi Macf.) and SO, and stem pitting (SP) on different citrus 57

20 and salicylic acid (SA) defence pathways in the resistance of SO varieties (Dawson et al., 2015; Moreno et al., 2008). The decline 58

21 to CTV. Silencing of the genes RDR1, NPR1 and DCL2/DCL4, of sweet orange [C. sinensis (L.) Osb.], mandarin (C. reticulata 59

22 associated with these defence pathways, enhanced virus spread Blanco) and grapefruit varieties grafted on SO led to the loss of 60

23 and accumulation in SO plants in comparison with non-silenced about 100 million trees propagated on this rootstock and forced 61

24 controls, whereas silencing of the genes NPR3/NPR4, associated the development of new citrus industries based on decline- 62

25 with the hypersensitive response, produced a slight decrease in tolerant rootstocks in affected countries (Moreno and Garnsey, 63

26 CTV accumulation and reduced stunting of SO grafted on CTV- 2010), despite the excellent agronomic qualities of SO, which 64

27 infected rough lemon plants. We also found that the CTV RNA include adaptation to most soil types, the ability to bear fruits of 65

28 silencing suppressors p20 and p23 also suppress the SA signal- high quality, and tolerance to many biotic and abiotic factors (Lee 66

29 ling defence, with the suppressor activity being higher in the and Keremane, 2013; Moreno and Garnsey, 2010). Declined field 67

30 most virulent isolates. trees grafted on SO usually show sieve tube necrosis and reduced 68

31 functional phloem below the bud union, whereas no necrosis is 69

32 Keywords: citrus decline, CLBV, reverse genetics, salicylic acid observed in CTV-inoculated SO seedlings. Phloem necrosis in 70

33 signalling defence suppressor, viral vector. grafted plants impairs appropriate carbohydrate supply to the 71

34 roots, leading to rootlet death. This results in the deficient supply 72

35 of water and minerals to the canopy and, ultimately, in decline 73

symptoms (Schneider, 1959). Previous results have indicated that 74

36 INTRODUCTION this bud union disorder, which does not occur with decline- 75
tolerant rootstocks, could be caused by a defence mechanism of 76
37 Citrus tristeza virus (CTV), genus Closterovirus, family Closteroviri-
SO to limit CTV invasion and accumulation. Thus: (i) the maximum 77
38 dae, has a single-stranded (ss) positive-sense genomic RNA
virus load in CTV-susceptible hosts, such as sweet orange, Mexi- 78
39 (gRNA) of 19.3 kb, organized in 12 open reading frames (ORFs)
can lime or C. macrophylla (Wester), is detected in the first flush 79
40 and 50 - and 30 -untranslated terminal regions (UTRs) of 107 and
after inoculation, whereas, in SO, the virus titre increases progres- 80
41 296 nucleotides (nt), respectively. ORFs 1a and 1b, encoding pro-
sively over several months (Comellas, 2009); (ii) CTV distribution 81
42 teins of the replicase complex, are translated from gRNA, whereas
in SO is uneven and its titre is low in comparison with that in sus- 82
43 ORFs 211, encoding proteins p33, p6, p65, p61, p27, p25, p18,
ceptible hosts (Comellas, 2009; Folimonova et al., 2008; Ruiz-Ruiz 83
44 p13, p20 and p23, are expressed via 30 -coterminal subgenomic
et al., 2007); (iii) inoculation of SO with a CTV construct express- 84
45 RNAs (sgRNAs). These latter proteins are involved in virion
ing the green fluorescent protein (GFP) showed that the virus 85
*Correspondence: Email: jguerri@ivia.es barely moves from cell to cell (Folimonova et al., 2008); and (iv) 86

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87 ectopic expression of p23 in transgenic SO increased CTV accumu- technology exploits the RNA silencing mechanism using a virus 137
88 lation (Fagoaga et al., 2011). Moreover, virus accumulation and genome as a vector in which plant genes or gene fragments are 138
89 tropism in SO roots and shoots may differ between CTV isolates inserted. After virus inoculation, processing of the virus-derived 139
90 (Comellas, 2009; Harper et al., 2014). dsRNA by DCLs generates siRNAs that guide the cleavage or 140
91 The replication and movement of viruses within their hosts are translational arrest of mRNAs of the gene or genes homologous 141
92 usually restricted by the triggering of the salicylic acid (SA)-medi- to the sequence inserted in the virus vector. Therefore, the plant 142
93 ated defence and antiviral RNA silencing pathways. SA is a plant displays a loss-of-function phenotype of the gene tested (Burch- 143
94 hormone required for the induction of basal defence and systemic Smith et al., 2004; Senthil-Kumar and Mysore, 2011). Viral vectors 144
95 acquired resistance (SAR) against many pathogens, including offer advantages over mutagenesis or genetic transformation, as 145
96 viruses. Increased SA accumulation has been observed in incom- it is possible to test the function of many genes in a short time. 146
97 patible plantpathogen interactions, leading to cell death (hyper- This point is especially important with citrus varieties, which have 147
98 sensitive reaction, HR) in the infection zone as a result of a long juvenile periods (usually 68 years) and are very difficult to 148
99 resistance gene response, but also in the defence reaction against transform, especially adult plants (Cervera et al., 2008). 149
100 pathogens with a compatible interaction without necrosis (Fu and Previously, we have used Citrus leaf blotch virus (CLBV) for 150
101 Dong, 2013; Mandadi and Scholthof, 2013). Treatments with SA either silencing or protein expression in citrus (Aguero et al., 151
102 or over-expression of the SA biosynthesis genes enhances resist- 2012, 2013, 2014; Velazquez et al., 2016). The CLBV gRNA has 152
103 ance to viruses in N. benthamiana (Lee et al., 2011; White, 1979), 8747 nts organized in three ORFs and 50 - and 30 -UTRs of 73 and 153
104 whereas the expression of the bacterial enzyme salicylate hydrox- 541 nts, respectively. ORF 1 is expressed from the gRNA and enc- 154
105 ylase in transgenic plants compromises plant defence (Baebler odes a polyprotein required for virus replication, and ORFs 2 and 155
106 et al., 2014; Jovel et al., 2011). The Non-expressor of 3, expressed by sgRNAs, encode the movement and coat proteins, 156
107 pathogenesis-related genes 1 (NPR1) is a master regulator of the respectively (Renovell et al., 2012; Vives et al., 2001, 2002). Dif- 157 AQ1
108 SA signalling pathway (Dong, 2004). In npr1 mutant plants, resist- ferent viral vectors, based on a full-genome infectious cDNA clone 158
109 ance to pathogens is compromised, whereas mutants of the NPR1 of CLBV (Vives et al., 2008), have been obtained (Aguero et al., 159
110 paralogues NPR3 and NPR4 (npr3-npr4), two SA adaptor proteins 2012, 2014). 160
111 involved in NPR1 degradation, show enhanced pathogen resist- A knowledge of the molecular mechanism of SO resistance to 161
112 ance (Fu et al., 2012; Zhang et al., 2006). CTV is a necessary step to develop techniques aimed to reduce or 162
113 RNA silencing is an important component of antiviral defence avoid decline/death of CTV-infected citrus varieties grafted on SO. 163
114 in plants. This process is triggered by double-stranded RNA In this work, using VIGS with a CLBV-based vector, we have 164
115 (dsRNA), generated in the replication of ssRNA viruses or present examined the involvement of RNA silencing and SA signalling 165
116 in gRNA and sgRNA regions with high secondary structure, which pathways in SO resistance. 166
117 is cleaved by class III RNases (termed Dicer-like, DCL) into 2124-
118 nt fragments called small interfering RNAs (siRNAs). The mature
RESULTS 167
119 double-stranded siRNA is incorporated into Argonaute (AGO) and,
120 later, is further processed by releasing the passenger strand,
Symptoms and virus distribution in SO seedlings 168
121 whilst keeping bound the guide strand (Meister, 2013). siRNAs
infected with different CTV isolates 169
122 can also function as primers for the synthesis of new dsRNAs, cat-
123 alysed by host RNA-dependent RNA polymerases (RDRs), which To elucidate whether symptom severity in SO is related to CTV 170
124 are cleaved by DCLs into secondary siRNAs, thus amplifying the accumulation in infected tissues, groups of five SO seedlings were 171
125 silencing signal (Baulcombe, 2004). RDR1 is involved in viral graft inoculated with three CTV isolates of different pathogenicity: 172
126 resistance mediated by both the SA signalling and RNA silencing a GFP-tagged version of the Florida isolate T36 (T36-GFP), T318A 173
127 mechanisms, and its expression appears to be up-regulated in cit- and T385. T36-GFP is moderately pathogenic and induces decline 174
128 rus after CTV infection (Alamillo et al., 2006; Ganda et al., 2007; in plants propagated on SO rootstock, SP in sensitive hosts such 175
129 Hunter et al., 2013). DCL2 and DCL4 mediate the genesis of 21- as Mexican lime, and mild to moderate SY syndrome (Satyanar- 176
130 and 22-nt siRNAs, which are the most abundant CTV-derived ayana et al., 2001). T318A is a highly pathogenic isolate of Spain 177
131 sRNAs in infected citrus plants (Ruiz-Ruiz et al., 2011). To over- causing severe decline, intense SY syndrome and SP in different 178
132 come these defence mechanisms, plant viruses encode proteins hosts, including grapefruit and sweet orange (Moreno et al., 179
133 that suppress RNA silencing (Csorba et al., 2015) or SA- 1993; Ruiz-Ruiz et al., 2006; Sambade et al., 2007). T385 is a mild 180
134 responsive defence signalling (Laird et al., 2013). Spanish isolate inducing inconspicuous vein clearing in Mexican 181
135 A very attractive strategy used in recent years to determine lime, but not decline, SY syndrome or SP (Moreno et al., 1991, 182
136 gene function is virus-induced gene silencing (VIGS). This 1993; Vives et al., 1999). 183

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Study of the resistance of sour orange to CTV 3

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Fig. 1 Symptoms induced and virus accumulation on sour orange (SO) seedlings infected with different Citrus tristeza virus (CTV) isolates. (A) Seedling yellows
symptoms following inoculation with the CTV isolate T318A. (B) Weight of shoot bark and roots in the second flush. (C) Roots of plants healthy or inoculated with
different CTV isolates. (D) Comparison of the CTV titre estimated by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) in shoot bark
and rootlets in two consecutive flushes. The average number of CTV target cDNA copies per nanogram (ng) of total RNA (RNAt) 6 standard deviation was estimated
in each assay using five biological replications with two technical repetitions per sample. Bars represent standard deviation.

184 SO seedlings inoculated with the isolate T318A showed severe plants infected with T318A showing the smallest root develop- 193
185 SY syndrome with reduced growth and short internodes and yel- ment, followed by plants infected with T36-GFP, whereas those 194
F1 186 low small-sized leaves (Fig. 1A), whereas those inoculated with infected with T385 displayed the largest root system, similar to 195
187 T36-GFP showed moderate stunting with essentially no leaf yel- that of the non-inoculated controls (Fig. 1C). 196
188 lowing, and those inoculated with T385 remained symptomless, To compare the tropism and viral load of different CTV isolates 197
189 similar to the healthy controls. SO plants infected with these iso- in SO seedlings, the amount of CTV gRNA in shoots and roots 198
190 lates differed with respect to the weight of shoot bark and roots was estimated at the end of the first and second flushes by 199
191 collected at the second flush time (Fig. 1B). The growth of the aer- reverse transcription-quantitative real-time polymerase chain reac- 200
192 ial part was correlated with the size of the root system, with tion (RT-qPCR) (Ruiz-Ruiz et al., 2007) (Fig. 1D). CTV accumulation 201

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202 in plants inoculated with the SY-inducing isolates T318A and T36- the second flush, whereas, in roots of the first flush, differences 251
203 GFP was higher in the shoots than in the roots, whereas, in plants were significant only for plants silenced for DCL2/4. 252
204 inoculated with the asymptomatic isolate T385, CTV accumulation As the isolate T36-GFP was engineered to express GFP, we 253
205 predominated in the roots and reached the highest level of the monitored CTV infection using a fluorescence stereomicroscope 254
206 three isolates. The lowest CTV accumulation in both shoots and with a GFP filter. Stem bark pieces from control plants (healthy or 255
207 roots was found in plants inoculated with T36-GFP, indicating pre-inoculated with WT-CLBV) inoculated with T36-GFP showed a 256
208 that symptom severity is not associated with virus accumulation. red fluorescence background, caused by chlorophyll, and some 257
209 In all CTV-infected plants, the viral load in both shoots and roots isolated green fluorescent spots, caused by CTV infection (Fig. 3). 258 F3
210 increased between the first and the second flushes, indicating The number of GFP spots and their intensity was higher in plants 259
211 that CTV invasion of SO seedlings progressed with time. silenced for the genes RDR1, NPR1 and DCL2/4 than in the non- 260
silenced controls, indicating that silencing of SO defence genes 261
212 VIGS-mediated silencing of genes RDR1, NPR1 and increased the number of infection foci and enhanced virus accu- 262
213 DCL2/DCL4 results in enhanced CTV spread and mulation (Fig. 3). In the silenced plants, a positive correlation was 263
214 accumulation in SO seedlings observed between the number of infection foci and the viral load 264
estimated by RT-qPCR (Figs 2 and 3). The highest number of fluo- 265
215 To assess whether the SA signalling defence and/or RNA silencing
rescent spots was found in plants silenced for gene DCL2/4, fol- 266
216 mechanisms were involved in the resistance of SO to CTV, we
lowed by those silenced for genes RDR1 and NPR1 (Fig. 3). GFP 267
217 silenced the expression of genes RDR1, NPR1, DCL2 and DCL4 by
expression was also observed in roots, where strong green fluo- 268
218 VIGS. For this purpose, cDNA fragments homologous to the Arabi-
rescence was observed in plants silenced for genes RDR1, NPR1 269
219 dopsis thaliana genes RDR1, NPR1 and DCL2/DCL4 were RT-PCR
or DCL2/4, in comparison with the weak yellowish fluorescence 270
220 amplified from Valencia sweet orange and cloned into the clbv30
caused by lignin, generally observed in roots from non-silenced 271
221 vector (see Experimental procedures) to yield vectors clbv30 -RDR1,
plants (Fig. 4). 272 F4
222 clbv30 -NPR1 and clbv30 -DCL2-4, respectively. The vectors were
The effectiveness of VIGS in SO seedlings was evaluated by 273
223 agroinoculated in N. benthamiana leaves and the resulting
comparing the expression of genes RDR1, NPR1, DCL2 and DCL4 274
224 recombinant virions were slash inoculated onto rough lemon (C.
by RT-qPCR in two successive flushes of plants pre-inoculated 275
225 jambhiri Lush) seedlings. Bark pieces of rough lemon infected
with the different constructs and the controls inoculated with WT- 276
226 with virions derived from the three constructs or from the empty
CLBV. Plants pre-inoculated with the clbv30 -RDR1 virions showed 277
227 vector clbv30 (WT-CLBV) were used to graft inoculate 15 SO seed-
a 60%70% reduction in RDR1 mRNA accumulation in shoots 278
228 lings per viral vector. One month after inoculation, groups of five
and roots compared with the non-silenced controls. Plants pre- 279
229 plants of each treatment were graft inoculated with the CTV iso-
inoculated with the clbv30 -NPR1 virions displayed a 70%80% 280
230 lates T36-GFP, T318A and T385. Symptom expression was
reduction in the NPR1 mRNA expressed in the shoots, but no 281
231 observed visually and CTV accumulation in shoots and roots was
NPR1 mRNA was detected in the roots, suggesting that this gene 282
232 estimated by RT-qPCR.
233 The viral titre was higher in RDR1-, NPR1- and DCL2/4-silenced is expressed at a very low level if at all in citrus roots. Reduction 283

234 plants than in the non-silenced controls. These differences were in the DCL2 mRNA level in plants inoculated with clbv30 -DCL2-4 284

235 more evident in plants inoculated with T36-GFP than in those ino- virions was about 90% in the shoots and 65%75% in the roots, 285

236 culated with the T318A or T385 isolates. The highest CTV accumu- whereas reduction in the DCL4 mRNA in the same plants was 286

237 lation was found in DCL2/4-silenced plants, followed by RDR1- 40%50% in both shoots and roots (Fig. S1, see Supporting 287

F2 238 and NPR1-silenced plants (Fig. 2). Tukeys studentized range test, Information). 288

239 used to compare mean pairs, indicated that plants inoculated with
Effect of the silencing of genes RDR1, NPR1, DCL2/4 289
240 T36-GFP showed significant differences when the RDR1, NPR1 or
and NPR3/4 of SO on the growth and virus 290
241 DCL2/4 genes were silenced vs. the non-silenced controls
accumulation of plants propagated on a CTV-infected 291
242 (P < 0.05), but not in plants silenced for RDR1 and NPR1, or RDR1
rough lemon rootstock 292
243 and DCL2/4. Among the plants inoculated with isolate T318A, the
244 only significant differences in gRNA accumulation between In this experiment, we also studied the role in SO defence of 293
245 silenced and non-silenced plants were observed in rootlets of the genes NPR3 and NPR4 in addition to RDR1, NPR1 and DCL2/4. It 294
246 first flush or shoots of the second flush of plants silenced for has been reported previously that the challenge with virulent 295
247 RDR1 or DCL2/4. Finally, among plants inoculated with T385, sig- pathogens of the A. thaliana double mutant npr3-npr4 results in a 296
248 nificant differences in gRNA accumulation between plants with significantly reduced HR (Fu et al., 2012). To examine the role of 297
249 the genes RDR1, NPR1 or DCL2/4 silenced and non-silenced con- these genes in citrus, cDNA fragments of the genes from Valencia 298
250 trols were observed in shoots of the two flushes and in roots of late sweet orange, homologous to the A. thaliana genes NPR3 299

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Study of the resistance of sour orange to CTV 5

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Fig. 2 Virus accumulation in sour
orange (SO) seedlings silenced for
RDR1, NPR1 and DCL2/4, and in
non-silenced control plants [non
pre-inoculated or pre-inoculated
with the wild-type Citrus leaf
blotch virus (WT-CLBV)],
inoculated with Citrus tristeza
virus (CTV) isolates T36-GFP (A),
T318A (B) or T385 (C). The
number of CTV gRNA copies was
estimated by reverse transcription-
quantitative real-time polymerase
chain reaction (RT-qPCR) in shoot
bark and rootlets in the first and
second flushes. Experimental
procedures as in Fig. 1.

300 and NPR4, were cloned in tandem (see Experimental procedures) (Fig. 5), but CTV accumulation was higher in plants propagated 311 F5
301 into the clbv30 vector to yield the construct clbv30 -NPR3-4. on rough lemon rootstock than in the cognate SO seedlings with 312
302 To assess the behaviour of the SO plants silenced for the the same gene silenced (Figs 2 and 5). Plants silenced for NPR3/4 313
303 genes RDR1, NPR1, DCL2/4 and NPR3/4, when subjected to a con- showed decreased CTV titre in comparison with the non-silenced 314
304 tinuous supply of CTV virions, buds of SO were propagated on control (Fig. 5), probably resulting from an enhanced basal resist- 315
305 rough lemon seedlings (in which CTV accumulates to high titres) ance caused by an increased accumulation of the NPR1 protein. 316
306 co-inoculated with virions clbv30 -RDR1, clbv30 -NPR1, clbv30 -DCL2- Bark observation of these plants for GFP expression confirmed the 317
307 4 or clbv30 -NPR3-4 and with the CTV isolate T36-GFP. Plants co- above results (data not shown). No significant growth difference 318
308 inoculated with WT-CLBV and T36-GFP were used as controls. As was observed between silenced and non-silenced plants (healthy 319
309 with SO seedlings, the highest CTV load was found in DCL2/4- or pre-inoculated with WT-CLBV) inoculated with T36-GFP 320
310 silenced plants, followed by those silenced for RDR1 and NPR1 (P > 0.05). 321

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Fig. 3 Detection of green fluorescent protein (GFP) fluorescence in the internal surface of bark from healthy (A) and RDR1- (B), NPR1- (C) or DCL2/4-silenced (D)
sour orange (SO) seedlings inoculated with T36-GFP, using a fluorescence stereomicroscope with a GFP filter. Green fluorescent spots indicate infected foci.

322 In previous work, we have observed that, in comparison with the defence mechanism mounted by their hosts. The different tro- 341
323 mock-inoculated controls, SO propagated on sweet orange seed- pism and accumulation level in SO plants of CTV isolates could 342
324 lings infected with isolate T318A shows a reduced growth that result from the different ability of their p20, p23 and p25 suppres- 343
325 has been associated with abnormal bud union in CTV-infected sors to overcome the SO defence system. These proteins act as 344

326 plants (Pina et al., 2005). To test whether the silencing of genes RNA silencing suppressors in N. benthamiana (Lu et al., 2004; 345

327 NPR3/4 could affect the growth reduction of SO caused by CTV, Ruiz-Ruiz et al., 2013), but their effect on the SA signalling path- 346

328 buds of SO were propagated onto rough lemon seedlings, simulta- way has not been tested. Using the Agrobacterium-mediated tran- 347

329 neously graft inoculated with clbv30 -NPR3-4 and T318A viruses. sient assay in the GFP-expressing N. benthamiana line 16c 348

330 Plants co-inoculated with WT-CLBV and T318A were used as con- (Voinnet et al., 1999), no differences were observed in the RNA 349

331 trols. The NPR3/4-silenced plants grew significantly more strongly silencing suppressor activity of the p20, p23 and p25 proteins 350

F6 332 than their non-silenced counterparts (Fig. 6AC); however, the encoded by CTV isolates T36-GFP, T318A and T385 (Comellas, 351

333 abnormal bud union was not observed in either group of plants 2009; and data not shown). 352

334 (data not shown). The CTV load estimated by RT-qPCR was lower To investigate whether p20, p23 and p25 CTV proteins were 353

335 in NPR3/4-silenced plants than in non-silenced controls (Fig. 6D). able to suppress SA signalling, we used two Agrobacterium-medi- 354
ated transient assays. In the first, we exploited the ability of SA 355
suppressors to delay cell death in response to a gene-for-gene 356
336 The CTV p20, p23 and p25 proteins act as
elicitor. In the second assay, we studied the ability to suppress the 357
337 suppressors of SA signalling defence in N. tabacum
expression of the SA response marker gene PR1a (pathogenesis- 358
338 and N. benthamiana
related 1a) (Laird et al., 2013). 359 AQ3
339 Plant viruses encode proteins that suppress RNA silencing (Voinet, The p19 protein of Tomato bushy stunt virus (TBSV) induces a 360
AQ2 340 2005) or SA-responsive signalling (Laird et al., 2013) to overcome strong HR in N. tabacum L. cv. Xanthi in an SA-dependent manner 361

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Study of the resistance of sour orange to CTV 7

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Fig. 4 Detection of green fluorescent protein (GFP) fluorescence in the external surface of roots from plants in Fig. 3.

362 (Angel and Schoelz, 2013), which reaches its maximum 36 h after expressing p20, p23 or p25 of the different CTV isolates, or p6 of 370
363 agroinoculation. However, co-infiltration of p19 with the p6 pro- CaMV as a positive control. Agroinoculation with empty pCAMBIA 371
364 tein of Cauliflower mosaic virus (CaMV) delays the onset of HR by was used as a negative control. The monitoring of plants at differ- 372
365 approximately 24 h (Laird et al., 2013). To screen the ability of ent post-infiltration times revealed that p20 and p23 of the three 373
366 p20, p23 and p25 from CTV isolates T36-GFP, T318A and T385 CTV isolates suppressed cell death, albeit at lower efficiency than 374
367 for suppression of SA signalling activity, leaves of N. tabacum p6, with p20 being a more efficient HR suppressor than p23. p20 375
368 Xanthi were infiltrated with Agrobacterium cultures transformed and p23 from isolate T318 were slightly more suppressive than 376
369 with pBIN-p19 or co-infiltrated with pBIN-p19 and pCAMBIA those from T36-GFP and T385, the latter being the least 377

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Fig. 5 Virus accumulation in RDR1-, NPR1-, NPR3/4- or DCL2/4-silenced and non-silenced [wild-type Citrus leaf blotch virus (WT-CLBV)] SO plants propagated on
rough lemon rootstock inoculated with T36-GFP. The number of Citrus tristeza virus (CTV) gRNA copies was estimated by reverse transcription-quantitative real-time
polymerase chain reaction (RT-qPCR) in shoot bark in two consecutive flushes. Experimental procedures as in Fig. 1.

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Fig. 6 Growth and Citrus tristeza virus (CTV) accumulation in NPR3/4-silenced and non-silenced [wild-type Citrus leaf blotch virus (WT-CLBV)] SO plants propagated
on rough lemon rootstock inoculated with T-318A. Increased growth of the NPR3/4-silenced plants (A) with respect to the non-silenced control plants (B). Shoot
length of silenced and non-silenced plants in two consecutive flushes (C). CTV accumulation in shoot bark estimated by reverse transcription-quantitative real-time
polymerase chain reaction (RT-qPCR) analysis in two consecutive flushes (D). Experimental procedures as in Fig. 1.

378 suppressive. However, only p25 from T36-GFP afforded a slight expressing p25 from the other CTV isolates. Overall, these results 399
379 delay of cell death. The experiments were repeated three times, suggest that p20 and p23 from CTV show suppressor activity of 400
F7 380 agroinfiltrating four leaves per plant in each assay (Fig. 7). SA signalling defence, and that this activity is stronger for proteins 401
381 To confirm the above results, we triggered the expression of from the more virulent isolates. 402
382 gene PR1a by agroinoculation with a binary plasmid, a response
383 that can be suppressed by the transient expression of p6 (Laird
DISCUSSION 403
384 et al., 2013; Love et al., 2012). We compared, by RT-qPCR, the
385 expression of PR1a mRNA in N. benthamiana infiltrated with The number and size of fluorescent infection foci produced by a 404
386 pCAMBIA or pCAMBIA carrying the p20, p23 or p25 genes from GFP-expressing CTV construct have been shown to be larger in a 405
387 the CTV isolates T36-GFP, T318A or T385. Plants infiltrated with a CTV-susceptible host, such as C. macrophylla, than in SO (Folimo- 406
388 plasmid expressing p6 were used as positive controls and non- nova et al., 2008), indicating that SO deploys an efficient barrier 407
389 infiltrated plants as negative controls. A strong reduction in PR1a against CTV infection. As the SA and RNA silencing pathways 408
390 mRNA accumulation was observed in N. benthamiana agroinfil- have been associated with basal and long-distance plant defence 409
391 trated with plasmids expressing p20 or p23 from the three CTV (Mandadi and Scholthof, 2013), we examined the effect of the 410
392 isolates in comparison with those infiltrated with pCAMBIA, silencing of genes RDR1, NPR1, DCL2/DCL4 and NPR3/4, involved 411
393 although this reduction was smaller than that observed in plants in these pathways, on CTV infection of SO seedlings or SO propa- 412
394 infiltrated with p6. The suppressor activity of p20 and p23 from gated onto a rough lemon rootstock. Our results with VIGS experi- 413
395 isolates T318A and T36-GFP was higher than that observed with ments, followed by CTV inoculation, indicated that RNA silencing 414
396 the homologous proteins from T385. Plants expressing p25 from and SA signalling are key components in SO defence against CTV. 415
397 the T36-GFP isolate showed a slight reduction in the PR1a mRNA RDR1 is an element of the RNA silencing pathway involved in 416
398 level in comparison with the control plants or with plants plant defence against viruses (Garcia-Ruiz et al., 2010), and its 417

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Fig. 7 Suppressive effect of the Citrus tristeza virus (CTV) p20, p23 and p25
proteins on the salicylic acid (SA) signalling pathway indicated by the delay in
cell death induced by the Tomato bushy stunt virus (TBSV) p19 protein in
Nicotiana tabacum Xanthi. Leaves were infiltrated with Agrobacterium
cultures transformed with pCAMBIA empty vector, pBIN-p19 or co-infiltrated
with pBIN-p19 and pCAMBIA expressing the p20, p23 or p25 proteins of the
different CTV isolates or the Cauliflower mosaic virus (CaMV) p6 as a positive
control. Photographs were taken at 3 days post-inoculation. Experiments were
repeated three times, agroinfiltrating four leaves per plant in each assay, with
similar results in all cases.
Fig. 8 Suppressive effect of the Citrus tristeza virus (CTV) p20, p23 and p25
proteins on the salicylic acid (SA) signalling pathway assessed by the relative
accumulation of PR1a mRNA, as estimated by reverse transcription-
quantitative real-time polymerase chain reaction (RT-qPCR), in Nicotiana
benthamiana leaves inoculated with pCAMBIA expressing p20, p23 or p25
from different CTV isolates or Cauliflower mosaic virus (CaMV) p6. Data were
compared with the PR1a mRNA expression induced by the empty pCAMBIA
vector (100%). EF1a was used as internal standard. The average number of AQ9
PR1a cDNA copies per nanogram (ng) of total RNA (RNAt) 6 standard
deviation values were estimated from three different assays using three plants
per assay and agroinfiltrating four leaves per plant. Bars as in Fig. 1. AQ10

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418 expression is induced by SA treatment or virus infection (Xie SY symptoms are determined by the p23 sequence and not by 468
419 et al., 2001; Yu et al., 2003). The rdr1 mutants of A. thaliana p23 accumulation (Albiach-Mart et al., 2010). However, studies 469
420 exhibit marked susceptibility to Tobacco mosaic virus and Tobacco with infectious clones of CMV carrying different versions of the 470
421 rattle virus (Yu et al., 2003), and N. benthamiana transformed gene 2b have shown that symptoms depend on this gene and are 471
422 with gene RDR1 from Medicago truncatula shows a resistance to not associated with virus accumulation (Shi et al., 2002). In SO 472
423 tobamoviruses absent in wild-type N. benthamiana (Yang et al., seedlings inoculated with the SY-inducing CTV isolates T318A and 473
424 2004). In addition, RDR1 expression in citrus is up-regulated after T36-GFP, virus accumulation was higher in the shoots than in the 474
425 CTV infection (Ganda et al., 2007). roots, whereas the opposite situation was observed with the 475
426 The NPR1 protein is a key regulator of the SA signalling path- asymptomatic isolate T385, which reached the highest virus load 476
427 way (Wu et al., 2012). The accumulation of NPR1 is needed for in the roots. These results support previous findings indicating 477
428 the expression of the basal defence genes, whereas its subse- that the roots of citrus genotypes considered to be resistant to 478
429 quent turnover is required for SAR. NPR1 is required in A. thaliana CTV could be infected despite the inability of the virus to infect 479
430 for SA-induced expression of PR genes and pathogen resistance shoots, with this infection capacity being isolate specific (Harper 480
431 (Dong, 2004). Although npr1 mutants show enhanced susceptibil- et al., 2014). Perhaps the antiviral defence in citrus is less effec- 481
432 ity to pathogens (Zhang et al., 2006), blocking NPR1 degradation tive in roots than in shoots, as occurs in N. benthamiana (Andika 482
433 with proteasome inhibitors or by genetic knockdown of Cullin3 in et al., 2005, 2013). Failure to detect NPR1 mRNA in citrus roots 483
434 Arabidopsis (Cul3 is a component of cullin-RING ubiquitin ligases) by RT-qPCR supports this hypothesis. Variable virus accumulation 484
435 increases basal resistance, but blocks SAR induction (Spoel et al., in shoots of SO inoculated with different CTV isolates could be 485
436 2009). Contrasting with the pathogen susceptibility of the npr1 explained, in part, by the different ability of p20 and p23 to sup- 486
437 mutant, the double mutants npr3-npr4 of A. thaliana show press SA signalling defence, with this suppression capacity being 487
438 enhanced disease resistance. However, the npr1-npr3-npr4 triple higher in isolates inducing symptoms in SO. Potential interactions 488
439 mutants display the npr1 phenotype, indicating that the enhanced between viral and host factors required for virus infection and 489
440 resistance of the npr3-npr4 mutants is NPR1 dependent (Zhang movement could also differ depending on the virus isolate and 490
441 et al., 2006). NPR3 and NPR4 interact directly with Cul3 to medi- host variety (Harper et al., 2014; Tatineni et al., 2011). 491
442 ate NPR1 degradation (Fu et al., 2012). Ectopic expression of p23 via transgenesis increased CTV accu- 492
443 Finally, DCL2 and DCL4 are the enzymes responsible for the mulation in SO, but not in Mexican lime or sweet orange, two sus- 493
444 generation of virus-derived siRNAs (Garcia-Ruiz et al., 2010; ceptible host species (Fagoaga et al., 2011). As p23 is a 494
445 Molnar et al., 2005). Their loss of function by mutation of both suppressor of both the SA signalling and RNA silencing defence 495
446 genes DCL2 and DCL4 is sufficient to make plants highly suscepti- responses, these results support the involvement of both path- 496
447 ble to several RNA viruses (Deleris et al., 2006). The most abun- ways in SO resistance to CTV. The RNA silencing suppressors 2b 497
448 dant CTV-derived sRNAs found by deep sequencing in CTV- of CMV and HC-Pro of Tobacco etch virus (TEV) have been found 498
449 infected citrus plants are of 21 and 22 nts in size (Ruiz-Ruiz et al., to interfere with the SA-mediated signalling and RNA silencing 499
450 2011), suggesting the involvement of DCL2 and DCL4 in anti-CTV pathways (Alamillo et al., 2006; Hui and Ding, 2001). 500
451 defence. In A. thaliana plants challenged with virulent pathogens, the 501
452 Evidence of SO genes associated with CTV resistance has been double mutant npr3-npr4 showed a significantly weaker HR (Fu 502 AQ4
453 reported, with a quantitative trait locus (QTL) being associated et al., 2012). When SO plants silenced for the genes NPR3/NPR4 503
454 with significant reduction of CTV accumulation (Asins et al., were propagated onto CTV-susceptible rough lemon rootstock 504
455 2004). In a study on the bark proteome and phosphoproteome of infected with the T318A isolate, thus providing a continuous virus 505
456 CTV-infected Tarocco sweet orange grafted on SO rootstock, it supply, increased growth of SO was observed in comparison with 506
457 was observed that about 52% of the modulated proteins are non-silenced control plants. This difference might be a result of 507
458 related to defence response pathways (Laino et al., 2016). increased NPR1 accumulation that would increase SO resistance. 508
459 The intensity of symptoms induced in SO by different CTV iso- An attractive hypothesis to explain the phloem necrosis usually 509
460 lates did not correlate with virus titre, suggesting that such symp- observed in the field below the bud union of infected citrus vari- 510
461 toms are more dependent on the pathogenicity of each isolate eties propagated on SO rootstock is that CTV induces an HR in the 511
462 than on its accumulation. The 30 -terminal region of the CTV SO rootstock to delay or stop virus invasion. However, in our 512
463 gRNA, encompassing gene p23 and the 30 -UTR, has been mapped experiments, we were unable to observe a necrosis line between 513
464 as responsible for the SY syndrome (Albiach-Mart et al., 2010). In the scion and the rootstock in either the control or the NPR3/ 514
465 this study, no correlation was observed between the amount of NPR4-silenced plants, despite the differences observed in plant 515
466 p23 expressed and the intensity of the SY symptoms induced by growth. This could be the result of: (i) the need for longer infec- 516
467 T36 or by T36/T30 hybrids not inducing SY in SO, suggesting that tion periods to develop bud union necrosis; or (ii) the amount of 517

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Study of the resistance of sour orange to CTV 11

518 virus arriving at SO from a susceptible CTV-infected variety is sinensis genes RDR1, NPR1, NPR3, NPR4, DCL2 and DCL4 were 67%, 566
519 higher when the variety is used as scion than when it is used as 67%, 73%, 72%, 67% and 88%, with query cover values of 93%, 54%, 567

520 rootstock. The first hypothesis is supported, in part, by the obser- 8%, 40%, 89% and 73%, respectively. Different fragments of the citrus 568

521 vation that CTV isolates taken from declining field trees usually do genes RDR1 (GenBank 102608479), NPR1 (GenBank 102617188), NPR3 569
(GenBank 102621158), NPR4 (GenBank 102622086), DCL2 (GenBank 570
522 not induce decline in the sweet orange/SO combination grown in
102617691) and DCL4 (GenBank 102621372) were RT-PCR amplified from 571
523 the glasshouse (Albiach-Mart et al., 2010).
Valencia sweet orange total RNA extracts (RNAt) using selected primers 572
524 In summary, our results suggest that SO offers a resistance to
(Table S1, see Supporting Information). The amplified cDNA fragments of 573
525 CTV invasion that is variable depending on the virus isolate. As the gene pairs NPR3/NPR4 and DCL2/DCL4 were fused and re-amplified, 574
526 CTV accumulation in SO protoplasts is similar to that observed in using the forward primers NPR3-F or DCL2-F and the reverse primers 575
527 protoplasts from susceptible hosts (Albiach-Mart et al., 2004; per- NPR4-R or DCL4-R, respectively, to produce the tandem fragments NPR3-4 576
528 sonal communication), SO resistance must be caused by deficient and DCL2-4, respectively. These fragments were inserted into the Pmll 577
529 cell-to-cell or long-distance movement. Silencing of the genes restriction site of the clbv30 vector (Aguero et al., 2012) to generate the 578
530 associated with the SA and RNA silencing defence pathways in clbv30 -RDR1, clbv30 -NPR1, clbv30 -NPR3-4 and clbv30 -DCL2-4 constructs. 579

531 SO improves both cell-to-cell and long-distance CTV movement. To examine whether the three CTV silencing suppressor genes (p20, 580

532 Comellas (2009) observed that, in CTV-susceptible hosts, the high- p23 and p25) were involved in suppression of the SA signalling pathway 581

533 est virus accumulation was detected in the first flush after inocula- important in SO defence, the coding sequences of these genes from the 582
CTV isolates T36 (GenBank accession AY170468), T318A (GenBank acces- 583
534 tion, whereas, in SO, the viral load increased in successive flushes
sion DQ151548) and T385 (GenBank accession Y18420) were RT-PCR 584
535 over 2 years, depending on the CTV isolate. Moreover, although,
amplified using selected primers (Table S1). The amplified cDNA sequen- 585
536 in susceptible hosts, CTV-derived siRNAs were detected in the first
ces were inserted into the vector pCAMBIA to obtain the plasmids 586
537 flush, detection in SO occurred 1 year after inoculation (Comellas, pCAMBIA-p20T36, pCAMBIA-p20T318A, pCAMBIA-p20T385, pCAMBIA- 587
538 2009), probably because of the need for a threshold viral RNA p23T36, pCAMBIA-p23T318A, pCAMBIA-p23T385, pCAMBIA-p25T36, 588
539 accumulation to trigger the RNA silencing machinery. The finding pCAMBIA-p25T318A and pCAMBIA-p25T385. 589
540 that 53.3% of the sRNAs detected by deep sequencing in CTV-
541 infected Mexican lime were derived from CTV, whereas, in SO, Plant growth and inoculation 590
542 this fraction was only 3.5% (Ruiz-Ruiz et al., 2011), further sup-
Nicotiana benthamiana and N. tabacum Xanthi plants were grown in 591
543 ports delayed CTV accumulation in SO. These results suggest that
small pots with 50% vermiculite and 50% peat moss in a plant growth 592
544 the initial resistance of SO to CTV accumulation probably results chamber at 20/248C (night/day), 60% humidity and a 16 h/8 h (light/dark- 593
545 from triggering of SA-mediated defence, rather than from RNA ness) regime. Citrus plants were grown in a glasshouse at 18/268C (night/ 594
546 silencing, although other antiviral pathways cannot be discarded. day), using 2-L plastic containers filled with 50% sand and 50% peat 595
547 The relative importance of the two defence routes in SO resistance moss and a standard fertilizing procedure (Arregui et al., 1982). SO plants 596
548 to CTV is difficult to assess because there is evidence of the inter- were grown as seedlings or propagated on a rough lemon rootstock. 597
549 play between SA signalling and antiviral RNA silencing pathways. All the recombinant plasmid constructs were transfected to Agrobacte- 598
550 For example, treatment with SA increased RDR1 in N. tabacum rium tumefaciens, strain COR 308, as described previously (Vives et al., 599

551 and A. thaliana (Alamillo et al., 2006; Hunter et al., 2013). In N. 2008). In silencing experiments, recombinant CLBV constructs were agro- 600

552 tabacum expressing salicylate hydrolase (NahG), which catalyses infiltrated on N. benthamiana leaves as described previously (Vives et al., 601
2008). Semipurified virion extracts (Galipienso et al., 2000) from N. ben- 602
553 the degradation of SA, accumulation of Plum pox virus (PPV)-
thamiana infected plants were inoculated to rough lemon plants by stem 603
554 derived sRNAs was reduced (Alamillo et al., 2006). In addition,
slashing (Garnsey et al., 1977). Bark pieces from CLBV-infected rough 604
555 expression of the genes DCL1, DCL2, RDR1 or RDR2 in tomato
lemon were used to graft inoculate SO or rough lemon plants. 605
556 plants was induced after infection with Tomato mosaic virus or by In the experiments to compare the effect of the CTV genes p20, p23 606
557 SA treatment (Campos et al., 2014). Variable accumulation of CTV and p25 on the SA signalling pathways, leaves of N. benthamiana were 607
558 isolates in SO could result from the different capacity of their p20 infiltrated with Ag. tumefaciens cultures carrying the different recombi- 608
559 and p23 proteins to suppress the SA signalling activity of the host. nant pCAMBIA plasmids, whereas N. tabacum Xanthi leaves were co- 609
infiltrated with equal volumes of Ag. tumefaciens cultures carrying the 610
560 EXPERIMENTAL PROCEDURES recombinant pCAMBIA plasmids and the pBIN-19 plasmid. 611

561 Plasmid constructs RNA extraction 612

562 The amino acid sequences of the RDR1, NPR1, NPR3, NPR4, DCL2 and Total RNA preparations (RNAt) from 50 mg of agroinfiltrated leaf areas 613
563 DCL4 proteins from A. thaliana were used to search for their citrus ortho- from N. benthamiana, or from 500 mg of fresh citrus tissues (young bark 614
564 logues in the Phytozome 9.1 database (https://phytozome.jgi.doe.gov/pz/ or rootlets), were obtained with a standard protocol that includes two 615
565 portal.htm) with BLASTX. The nt identities between the A. thaliana and C. phenolchloroformisoamyl alcohol extractions, followed by precipitation 616

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617 with 12 M lithium chloride (Ancillo et al., 2007). RNA was resuspended in ACKNOWLEDGEMENTS 668
618 80 mL of RNase-free water and treated with RNase-free DNase (Turbo
This work was supported by grant AGL2012-32429, co-financed by 669
AQ5 619 DNA-free, Ambion). RNA concentration was measured in a NanoDrop
FEDER (European Fund for Regional Development) and the Spanish Min- 670
620 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and isterio de Economa y Competitividad (MINECO). We thank Dr W. O. 671
621 adjusted to approximately 10 ng/mL. Dawson for providing the GFP-tagged T36 isolate of CTV. Neus Gomez 672
was the recipient of a doctoral fellowship from MINECO. Mara-Carmen 673
622 Estimation of the CTV load and plant gene expression Vives was the recipient of a contract from Instituto Valenciano de Investi- 674
gaciones Agrarias (IVIA). We thank Maria Boil for excellent laboratory 675
623 level by RT-qPCR 676
assistance.
624 To estimate CTV accumulation in citrus shoots and roots, RT-qPCR was
625 performed in a LightCyclerV R platform (Roche Molecular Diagnostics, REFERENCES 67 7
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627 SYBR Green I, 0.3 mM of primers PM197R and PM198R, and 2 mL of RNAt Moreno, P. and Guerri, J. (2012) Development of viral vectors based on Citrus 679
628 (10 ng RNA/mL). The cycling conditions and procedure to estimate the leaf blotch virus to express foreign proteins or analyze gene function in citrus 680
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637 mutant virions or with WT-CLBV was reverse transcribed and then qPCR Albiach-Mart, M.R., Grosser, J.W., Gowda, S., Mawassi, M., Satyanarayana, 692
638 amplified using the primer pairs qRDR1F-qRDR1R, qNPR1F-qNPR1R, T., Garnsey, S.M. and Dawson, W.O. (2004) Citrus tristeza virus replicates and 693
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642 expression level of the gene actin11 from citrus measured in the same 699
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659 BIA. Data were analysed by one-factor ANOVA using the software SPSS sable component of the Ny-1 resistance gene mediated response against Potato 721
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665 Switzerland) using a high-energy light source and a GFP filter. Images
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666 were taken with a Leica DFC490 digital camera using IM50 software genic adult plants from clementine mandarin by enhancing cell competence for 732
667 (Leica Microsystems). transformation and regeneration. Tree Physiol. 28, 5566. 733

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Study of the resistance of sour orange to CTV 13

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888
SUPPORTING INFORMATION 917
nucleotide sequence and genomic organization of citrus leaf blotch virus: candidate
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918
890 Vives, M.C., Galipienso, L., Navarro, L., Moreno, P. and Guerri, J. (2002) Charac- Additional Supporting Information may be found in the online 919
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892
version of this article at the publishers website: 920
Virology, 295, 328336.
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929

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