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Characterization of a Novel Bipartite Double-Stranded RNA Mycovirus 1
Conferring Hypovirulence in the Phytopathogenic Fungus Botrytis porri 2
Mingde Wu, Fengyin Jin, Jing Zhang, Long Yang, Daohong Jiang, and Guoqing Li* 3
The State Key Laboratory of Agricultural Microbiology and the Key Laboratory of Plant 4
Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, Hubei, China. 5
* Corresponding author: Dr. Guoqing Li, 86-27-87286880, E-mail: guoqingli@mail.hzau.edu.cn 6
Running title: A novel bipartite dsRNA mycovirus in Botrytis porri 7
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00292-12 JVI Accepts, published online ahead of print on 11 April 2012
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The ascomycete Botrytis porri causes clove rot and leaf blight of garlic worldwide. Here we 21
report the biological and molecular features of a novel bipartite double-stranded RNA (dsRNA) 22
mycovirus named Botrytis porri RNA virus 1 (BpRV1) from the hypovirulent strain GarlicBc-72 23
of B. porri. The BpRV1 genome comprises two dsRNAs, dsRNA-1 (6,215 bp) and dsRNA-2 24
(5,879 bp), which share sequence identity of 62 and 95% at the 3’- and 5’-terminual regions, 25
respectively. Two open reading frames (ORFs), ORF I (dsRNA-1) and ORF II (dsRNA-2), were 26
detected. The protein encoded by the 3’-proximal coding region of ORF I shows sequence 27
identities of 19–23% with RNA-dependent RNA polymerases encoded by viruses in the families 28
Totiviridae, Chrysoviridae and Megabirnaviridae. However, the proteins encoded by the 29
5’-proximal coding region of ORF I and by the entire ORF II lack sequence similarities to any 30
reported virus proteins. Phylogenetic analysis showed that BpRV1 belongs to a separate clade 31
distinct from those of other known RNA mycoviruses. Purified virions of ~35 nm in diameter 32
encompass dsRNA-1 and dsRNA-2, and three structural proteins (SPs) of 70, 80 and 85 kDa, 33
respectively. Peptide mass fingerprinting analysis revealed that the 80- and 85-kDa SPs are 34
encoded by ORF I, while the 70-kDa SP is encoded by ORF II. Introducing BpRV1 purified 35
virions into the virulent strain GarlicBc-38 of B. porri caused derivative 38T reduced mycelial 36
growth and hypovirulence. These combined results suggest that BpRV1 is a novel bipartite 37
dsRNA virus that possibly belongs to a new virus family. 38
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Mycoviruses (fungal viruses) have been recorded to be widespread in the major taxonomic 44
groups of filamentous fungi, yeasts and oomycetes (16, 39). Most mycoviruses have RNA 45
genomes, which are either single-stranded (ss) or double-stranded (ds), and are now classified 46
into twelve families (16). Members of four families (Chysoviridae, Partitiviridae, Reoviride and 47
Totiviridae) accommodate undivided (e.g. Totiviridae) or divided (e.g. Chysoviridae, 48
Partitiviridae and Reoviride) dsRNA genomes, which are encapsidated within capsid proteins 49
with formation of rigid virus particles. Members of six families (Alphaflexiviridae, Barnaviride, 50
Endornaviridae, Gammaflexiviridae, Hypoviridae and Narnaviridae) accommodate ssRNA 51
genomes, of which only two families (Alphaflexiviridae and Gammaflexiviridae) form virus 52
particles, whereas members of the remaining four virus families are unencapsidated and do not 53
form typical virions. Two families (Metaviridae and Pseudoviridae) accommodate RNA 54
reverse-transcribing genomes. Meanwhile, an increasing number of novel mycoviruses have been 55
reported recently (2, 9, 29, 53, 59). Chiba et al. (9) proposed to establish a new bipartite dsRNA 56
virus family, Megabirnaviridae, to accommodate Rosellinia necatrix megabirnavirus 1 57
(RnMBV1) based on characterization of the dsRNA genome of RnMBV1. Besides RNA 58
mycoviruses, Yu et al. (55) reported a geminivirus-related ssDNA mycovirus infecting 59
Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of stem rot of oilseed rape (Brassica 60
napus). 61
In many cases, mycovirus infection does not cause any visible abnormal symptoms for host 62
fungi (cryptic infections). However, infection by some mycoviruses can cause abnormal 63
symptoms in their host fungi, including reduced mycelial growth and debilitated virulence (e.g. 64
hypovirulence) (37). Successful control of chestnut blight caused by virulent strains of 65
Cryphonectria (= Endothia) parasitica with hypovirulent strains of this pathogen in Europe (1, 66
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19, 30) inspired numerous researchers to study the phenomenon of mycovirus-mediated 67
hypovirulence in plant pathogenic fungi. So far, many mycoviruses belonging to Hypoviridae, 68
Narnaviridae, Reoviridae, Megabirnaviridae and some unassigned virus taxa have been 69
identified to cause (or to be associated with) fungal hypovirulence (16, 38, 39). 70
Allium crops, including garlic (Allium sativum), garlic chives (A. tuberosum), bulb onions 71
(A. cepa) and green onions (A. fistulosum), are important vegetables grown in China, as well as in 72
many other countries. Gray mold disease caused by Botrytis spp., including B. porri, is one of the 73
most important ecological factors limiting both the yield and the quality of these Allium crops 74
(33, 57, 58). Previous studies indicated that B. porri can cause garlic clove rot (12), garlic leaf 75
blight (57) and leek leaf rot (3). Several RNA mycoviruses have been reported in B. cinerea (5, 6, 76
21, 22, 23, 50, 52), a close relative of B. porri and the causal agent of gray mold disease on 77
numerous plants. The genomes of some mycoviruses infecting B. cinerea have been 78
characterized (22, 23, 52). However, no mycoviruses infecting other species of Botrytis, 79
including B. porri, have been reported so far. 80
A strain of B. porri, designated as GarlicBc-72, was isolated from a diseased leaf of garlic 81
grown in Zhushan County, Hubei Province, China (57). We found that it grew slowly on culture 82
media and was hypovirulent to garlic leaves compared to other strains of B. porri. Two dsRNA 83
segments of approximately 6.2 and 6.5 kb in size, respectively, were detected in mycelia of strain 84
GarlicBc-72, but were not detected in virulent strains of B. porri. These results suggested that 85
strain GarlicBc-72 might be infected with an RNA mycovirus. Therefore, a study was conducted 86
to fulfill the following three objectives: (i) to establish the cause-effect relationship between 87
presence of the two dsRNA segments and hypovirulence in strain GarlicBc-72; (ii) to obtain and 88
analyze the full-length cDNA sequences generated from the two dsRNA segments; and (iii) to 89
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purify the virus particles from strain GarlicBc-72 and to characterize their nucleic acids and the 90
structural proteins at the molecular level. 91
MATERIALS AND METHODS 92
Fungal strains/isolates and biological characterization. Fifteen strains or isolates of 93
Botrytis porri were used in this study (Table 1). For simplicity, the names of strains GarlicBc-16, 94
GarlicBc-38, GarlicBc-72, OnionBc-95 and GP72SC35 of B. porri were shortened herein as 95
Bc-16, Bc-38, Bc-72, Bc-95 and SC35, respectively. Maintenance of the stock cultures and 96
establishment of working cultures of these strains of B. porri were carried out as described in our 97
previous study (51). Mycelial agar plugs (6 mm diameter) removed from the colony margin of a 98
3- to 5-day-old culture of each strain/isolate were placed on potato dextrose agar (PDA) in Petri 99
dishes (9 cm diameter), one plug per dish. The dishes were incubated at 20 ºC for determination 100
of the mycelial growth rate and for observation of the colony morphology. Pathogenicity of each 101
strain was determined by inoculating detached leaves of garlic (A. sativum cultivar Feng Suan 102
No. 1) with agar plugs of actively-growing mycelia using the procedures of Zhang et al. (57, 58). 103
Vertical transmission of hypovirulence-associated dsRNAs. Vertical transmission refers 104
to transmission of hypovirulence-associated dsRNAs from mycelia to asexual or sexual spores 105
(38). This experiment was done by determining the pathogenicity and the presence of dsRNAs in 106
single-conidium (SC) isolates from the dsRNA-containing hypovirulent strain Bc-72. For 107
obtaining conidia, strain Bc-72 was incubated at 20 ºC on PDA in Petri dishes (9 cm diameter) 108
for 17 days. Colonies showing conidial production were selected. Conidia in one of the dishes 109
were washed off by flooding the colony with ~3 ml sterile distilled water (SDW), followed by 110
scraping the surface of the colony with a sterilized glass rod. The conidial mixtures from the 111
selected dishes were pooled and filtered through four-layer cheesecloth to remove mycelial 112
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fragments. The resulting conidial suspension was diluted to 1 × 104 conidia/mL with SDW. An 113
aliquot of 100 µL conidial suspension was pipetted onto a PDA dish and the conidial suspension 114
drop was carefully spread on the PDA plate using a sterilized glass rod. Individual conidia on the 115
PDA surface were picked up using a sterilized fine glass needle under a light microscope and 116
transferred to fresh PDA plates, one conidium per plate. The plates were incubated at 20 ºC for 5 117
days and the resulting cultures were identified and those belonging to B. porri were individually 118
tested for the mycelial growth rates on PDA (20 ºC), pathogenicity on detached garlic leaves (20 119
ºC, 72 h) and the presence of the two dsRNA segments in mycelia using previously described 120
methods (51, 57, 58). The hypovirulent strain Bc-72 and the virulent strain Bc-16 of B. porri 121
were used as controls in this experiment. 122
Horizontal transmission of hypovirulence-associated dsRNAs. Horizontal transmission 123
refers to transmission hypovirulence-associated dsRNAs from hypovirulent to virulent fungal 124
strains through hyphal anastomosis (38). The experiment was done on PDA in Petri dishes using 125
the pairing culture technique (51, 52, 56). In the contact culture in each dish (9 cm diameter), the 126
dsRNA-containing hypovirulent strain Bc-72 served as the donor, whereas strains Bc-38, SC35 127
or Bc-95 served as recipients. After incubation of the contact cultures at 20 °C for 8 days, three 128
mycelial derivative isolates were obtained from three colonies of each recipient strain in the three 129
contact cultures of Bc-72/Bc-38, Bc-72/SC35 or Bc-72/Bc-95 using the method described by Wu 130
et al. (51). The isolates were designated as 72-38-1, 72-38-2 and 72-38-3 for derivatives from 131
strain Bc38; 72-35-1, 72-35-2 and 72-35-3 for derivatives from strain SC35; and 72-95-1, 132
72-95-2 and 72-95-3 for derivatives from strain Bc-95. All these derivative isolates were 133
individually tested for the mycelial growth rates on PDA (20 ºC), pathogenicity on detached 134
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garlic leaves (20 ºC, 72 h) and the presence of dsRNA in mycelia (51, 57, 58). The parental 135
strains Bc-72, Bc-38, Bc-95 and SC35 were included as controls in this experiment. 136
Transmission electron microscopy (TEM). Sub-cellular characteristics of hyphal cells of 137
strains Bc-16 (virulent) and Bc-72 (hypovirulent) were examined using TEM. A mycelial agar 138
plug of each strain was inoculated in the center of a piece of sterilized cellophane film placed on 139
PDA in a Petri dish. The culture was incubated at 20 ºC for 3 days for strain Bc-16 and for 5 days 140
for strain Bc-72. Small pieces (2 × 2 mm, length × width) of the cellophane films colonized with 141
mycelia of strains Bc-16 or Bc-72 were carefully cut off from the colony margin (~3 mm in 142
width) and the central area (~10 mm from the margin area) using a razor blade. The mycelial 143
specimens were fixed and dehydrated using conventional procedures. Ultra-thin sections (50–60 144
nm in thickness) for the mycelial specimens were cut using a diamond knife, mounted on slotted 145
and formvar-coated grids, stained with 5% aqueous lead citrate and 5% uranyl acetate, and 146
examined under TEM (Model Tecnai G2 20, FEI CompanyTM, Hillsboro, OR, USA). 147
Extraction and purification of dsRNA from mycelia. Mycelial agar plugs of each 148
strain/isolate of B. porri were inoculated in potato dextrose broth in Petri dishes (9 cm diameter), 149
1 agar plug per dish and 20 dishes per strain. The dishes were incubated at 20 ºC in the dark for 3 150
to 6 days. Mycelial mats in each dish were harvested and stored at at -20 ºC until use. Extraction 151
and purification of the dsRNAs from mycelia of each strain were performed using the procedures 152
of Morris and Dodds (34). Analysis of the dsRNA extracts and identification of the double- 153
strandedness of RNAs were done with the methods described in our previous studies (51). 154
cDNA cloning, sequencing and sequence analysis. The dsRNAs extracted from strain 155
Bc-72 of B. porri were fractioned by agarose gel electrophoresis and the individual dsRNA bands 156
(6.5 kb for dsRNA-1 and 6.2 kb for dsRNA-2) were cut off for further analysis. Cloning of the 157
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cDNAs for the dsRNAs from strain Bc-72 by random primers-mediated PCR, sequencing of the 158
cDNAs and analysis of the sequences were done using the procedures described in our previous 159
studies (51, 52). To clone the terminal sequences of each dsRNA, the 3’-terminus of each strand 160
of dsRNA-1 or dsRNA-2 was ligated to the 5’-terminus of the BPA adaptor (Fig. S1 and Table 161
S1 in Supplemental material) using T4 RNA ligase (TAKARA Biotechnology Co., Ltd., Dalian, 162
China) at 4–8 ºC for 18 h. The primer RCBPA was used for reverse transcription of dsRNA-1 or 163
dsRNA-2 to cDNA. The primer pairs RCBPA/BP5-4 and RCBPA/BP3 were used for 164
PCR-amplification of the 5’-terminal sequence and the 3’-terminal sequence of dsRNA-1, 165
respectively. Similarly, the primer pairs RCBPA/BG1 and RCBPA/BG2 were used for 166
PCR-amplifying the 5’-terminal sequence and the 3’-terminal sequence of dsRNA-2, respectively 167
The PCR primers and their sequences mentioned above are listed in Table S1 (in Supplemental 168
material). The full-length cDNA sequence for dsRNA-1 or dsRNA-2 was obtained by assembling 169
of the partial sequences in the different cDNA clones (Fig. S1 in Supplemental material). Open 170
reading frames (ORFs) in each full-length cDNA sequence were deduced using the ORF Finder 171
program in the website of the National Center for Biotechnology Information (NCBI) 172
(http://www.ncbi.nlm. nih.gov/gorf/ gorf.html) with the standard codon usages. Database 173
searches of the full-length cDNA sequences of dsRNA-1 and dsRNA-2 and deduced polypeptides 174
from the two cDNA sequences were performed in the public database at NCBI using the 175
programs of BLASTn and BLASTp, respectively. Motifs present in the deduced polypeptide 176
sequences were searched in three databases, including the CDD database 177
(http://www.ncbi.nlm.nih.gov/Structure /cdd/wrpsb.cgi), the Pfam database 178
(http://pfam.sanger.ac.uk/) (15) and the PROSITE database (http://www.expasy.ch/). The 179
deduced polypeptide sequences encoded by the two dsRNAs were also BLAST-searched in the 180
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MEROPS protease database (http://merops.sanger.ac.uk/) (40). Multiple alignments of the 181
sequences of RNA-dependent RNA polymerase (RdRp) was accomplished using the 182
CLUSTAL_X program (48). A phylogenetic tree was constructed using the neighbor joining (NJ) 183
method and tested with a bootstrap of 1,000 replicates to ascertain the reliability of a given 184
branch pattern in the NJ tree. Potential secondary structures at the 3’- and 5’-terminal sequences 185
of the positive strand of dsRNA-1 and dsRNA-2 were predicted using the RNAstructure software 186
(version 4.6) (32). The proline frequency on the two polypeptides deduced from dsRNA-ORF I 187
on dsRNA-1 and ORF II on dsRNA-2 was calculated using Microsoft Office Excel 2007 as 188
described by Spear et al. (46). The threshold value of 0.33 was used to indicate the minimum 189
proline frequency required for generation a PPII-like helix (PxxPxxPxx) over a nine-amino acids 190
span. 191
Northern hybridization. Northern hybridization was performed to verify the authenticity of 192
the cDNA sequences generated from dsRNA-1 (6.5 kb) and dsRNA-2 (6.2 kb) in strain Bc-72 of 193
B. porri. Meanwhile, it was also used to confirm the high nucleotide sequence similarity between 194
the two dsRNAs at the 5’-terminus (Fig. 1A). The dsRNAs from strain Bc-72 were separated by 195
agarose gel electrophoresis. They were then transferred from the agarose gel to an 196
Immobilon™-Ny+ membrane (Millipore, Bedford, MA, USA) using the procedures described by 197
Jiang and Ghabrial (25). Three DNA probes (423 bp for probes 1, 506 bp for probe 2 and 451 bp 198
for probe 3) were designed on the basis of the full-length cDNA sequences of dsRNA-1 and 199
dsRNA-2. They were generated by reverse-transcription PCR (RT-PCR) using dsRNA-1 or 200
dsRNA-2 as template with the specific primer pairs listed in Table S1 (in Supplemental material). 201
The probes were individually labelled with digoxigenin (DIG)-11-dUTP in the DIG High Prime 202
DNA Labeling and Detection Starter Kit I (Roche Diagnostics GmbH, Roche Applied Science, 203
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Mannheim, Germany) and used to hybridize with the dsRNAs blotted on the nylon membrane. 204
The hybridization signals were detected by enzymatic immunoassay using the reagents and the 205
procedures recommended by the manufacturer (Roche Applied Science). 206
Purification of viral particles. Strain Bc-72 of B. porri was grown at 20 ºC for 6 days on 207
sterilized cellophane films placed on PDA. Mycelia were harvested and a sample of 10 g fresh 208
mycelia was ground to fine powder in liquid nitrogen using sterilized mortar and pestle. The 209
powder was transferred to a 1,000 mL-blender containing 150 mL extraction solution [0.1 M 210
sodium phosphate and 3% (w/v) Triton X100, pH 7.0]. The suspension was blended for 30 s and 211
then transferred to a plastic tube (200 mL), followed by centrifuging at 10,000 × g for 20 min to 212
remove the hyphal cell debris. The supernatant was then carefully pipetted out to another plastic 213
tube, which was then subject to a 2-h-long ultra-centrifuge at 119,000 × g under 4 ºC to 214
precipitate all the particles including the virus particles in the supernatant. The pellet was 215
re-suspended in 4 mL sodium phosphate buffer (0.1 M) and the resulting suspension was 216
centrifuged at 16,000 × g for 20 min to precipitate large particles. Then, the supernatant 217
containing the virus particles was overlayed on a centrifuge tube containing sucrose solutions 218
with a concentration gradient ranging from 10 to 40% (w/v) and centrifuged at 70,000 × g under 219
4 ºC for 2 h. Fractions in the middle portion of the tube were individually measured for the 220
presence of the virus particles by detection of the dsRNA segments using the agarose gel 221
electrophoresis. The fraction containing virus particles was carefully collected and suspended in 222
100 µL sodium phosphate buffer (0.05 M, pH 7.0). The virus particles were stained with 223
phosphotungstic acid solution [20 g/L (w/v), pH 7.4] and observed under TEM. dsRNA from 224
viral particles was sequentially extracted with phenol, phenol-chloroform, and 225
chloroform-isoamyl alcohol and fractionated by electrophoresis on an agarose gel (1%, w/v). The 226
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SPs from viral particles were detected by electrophoresis on a polyacrylamide gel (12%, w/v) 227
amended with sodium dodecyl sulfate (SDS) (1%, w/v). 228
Peptide mass fingerprinting (PMF). The virus particle suspension, derived from B. porri 229
strain Bc-72 was loaded on a 12% polyacrylamide gel amended with 1% SDS. After 230
electrophoresis, the gel was stained with Coomassie brilliant blue G250. The protein bands on the 231
gel were individually excised to make different gel samples. Each gel sample was sequentially 232
destained with NH4HCO3 (100 mM) and acetonitrile (ACN) (30%, w/v). The gel samples were 233
lyophilized, followed by rehydration in 5 µL NH4HCO3 solution (50 mM) amended with 50 ng 234
trypsin (Sequencing Grade, Promega, Madison, WI, USA). The mixtures in small tubes were 235
bath-incubated at 37 ºC overnight to digest the proteins. The resulting peptides from each gel 236
sample were extracted three times with 1 mL of extraction buffer containing 0.1% trifluoroacetic 237
acid (TFA, w/v) and 60% ACN. The aqueous phase of the extracts for each sample was pooled 238
and lyophilized. The lyophilized peptides mixture for each protein was dissolved in 0.1% TFA. 239
The resulting solution was desalted and concentrated using ZipTip® pipette tips (Millipore 240
Bedford, MA, USA). Then, the peptides were dissolved in R-cyano-4-hydroxycinnamic acid (5 241
mg/mL) amended with 0.1% TFA and 50% ACN and analyzed by matrix-assisted laser 242
desorption/ionization–time of flight mass spectrometry (MALDI-TOF-MS) and MALDI-TOF 243
tandem MS on a 4800 Plus MALDI TOF/TOFTM Analyzer (Applied Biosystems, Foster City, 244
USA). Molecular weight of each peptide was compared to that deduced from the nucleotide 245
sequences of ORF I on dsRNA-1 or ORF II on dsRNA-2 using the MASCOT program (Matrix 246
Science Ltd., London, UK). 247
Protoplast transfection. The virus-free strain Bc-38 of B. porri was used as the recipient in 248
this experiment. Hypovirulence-associated dsRNAs in strain Bc-72 could not be transmitted to 249
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from strain Bc-72 to strain Bc-38 in the horizontal transmission experiment of this study. 250
Protoplasts were prepared from actively-growing mycelia of strain Bc-38 using the procedures 251
described by Yelton et al. (54). The method described by Kanematsu et al. (27) was used to 252
transfect protoplasts of strain Bc-38 with the purified virus particles from strain Bc-72 in the 253
presence of polyethylene glycol (PEG) 4000. The transfected protoplast suspension was 254
incubated without shaking at 20 °C in the dark for 2 days and plated on YCGA media (0.1% 255
yeast extracts, 0.1% casein hydrolysate, 0.5 M glucose and 1.5% agar in Petri dishes (9 cm 256
diameter), this was followed by incubation at 20 °C for 3 days. Agar plugs containing mycelia 257
generated from the protoplasts were individually transferred to PDA plates, one plug per plate. 258
The dishes were incubated at 20 °C in the dark for 12 days, and the resulting cultures were 259
identified and screened for abnormal colonies (slow growth and formation of mycelial sectors). A 260
representative virus-transfected isolate 38T was tested for mycelial growth rate (20 °C) on PDA, 261
pathogenicity on detached garlic leaves (20 °C, 72 h) and the presence of the dsRNAs in mycelia. 262
The parental strain Bc-38 was used as a control strain in these tests. 263
Statistical analysis. Data on mycelial growth rate and leaf lesion length caused by strains of 264
B. porri were analyzed using analysis of variance (ANOVA) in SAS V8.0 (SAS Institute, Cary, 265
NC, USA, 1999). Treatment means on each of these two parameters for the tested strains/isolates 266
were compared using the Least Significant Difference Test at α = 0.05. Significant differences 267
between strains Bc-38 and 38T of B. porri in each of the two parameters were detected using 268
Student’s t-test at α = 0.01. 269
RESULTS 270
Virus particles. The virus particles purified from strain Bc-72 of Botrytis porri were 271
isometric and ~35 nm in diameter (Figs. 2A, B). The virus particles accommodate two dsRNA 272
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segments with similar sizes to dsRNA-1 and dsRNA-2 extracted from mycelia of strain Bc-72 273
(Fig. 2C). SDS/PAGE of the purified particles revealed the presence of three major proteins with 274
estimated molecular mass of 70, 80 and 85 kDa designated as p70, p80 and p85, respectively 275
(Fig. 2D). It is worth noting that the virion dsRNA segments showed similar brightness to the two 276
dsRNAs extracted from the mycelia, suggesting that the two dsRNAs occur at the similar molar 277
ratios in both cases. 278
Nucleotide sequences of dsRNA-1 and dsRNA-2. A total of 21 cDNA clones were 279
obtained using random hexameric primers. Assembly sequences of these cDNA clones generated 280
two contigs. Contig 1 comprised 13 clones belonging to dsRNA-1 and contig-2 comprised 8 281
clones belonging to dsRNA-2 (Fig. S1 in Supplemental material). No other cDNA clones were 282
found to make additional contigs, suggesting that no other dsRNAs in strain Bc-72 have the 283
similar size to dsRNA-1 or dsRNA-2. The full-length cDNA sequences for dsRNA-1 (GenBank 284
Acc. No. JF716350) and dsRNA-2 (GenBank Acc. No. JF716351) were 6,215 and 5,879 bp in 285
length, respectively (Fig. 3). Interestingly, we observed that the nucleotide sequences of 286
dsRNA-1 and dsRNA-2 at the 3’-termin (80-bp long) and the 5’-termini (500-bp long) shared 287
sequence identities of 62 and 95%, respectively (Fig. 1A, B). Moreover, the extreme terminal 288
sequences of dsRNAs are strictly conserved. Results of the Northern hybridization analysis 289
confirmed the high nucleotide sequence identity at the 5’-termini of dsRNA-1 and dsRNA-2 (Fig. 290
3A). Probe 1 (423 bp) corresponding to sequences at the 5’-terminus of dsRNA-1 simultaneously 291
hybridized with both dsRNA-1 and dsRNA-2. However, probe 2 (506 bp) corresponding to 292
sequences at the middle region of dsRNA-1 hybridized only with dsRNA-1, but did not hybridize 293
with dsRNA-2. Similarly, probe 3 (451 bp) corresponding to sequences at the middle region of 294
dsRNA-2 hybridized only with dsRNA-2. 295
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The 5’- and 3’-untranslated regions (UTR) of dsRNA-1 and dsRNA-2 were examined for 296
potential secondary structures using the RNA structure software (version 4.6) (32). Results 297
showed that formation of a stem-loop structure was detected at the 3’- and 5’-terminal regions of 298
dsRNA-1, and at the 5’-terminal region of dsRNA-2, but was not detected at the 3’-terminial 299
region of dsRNA-2 (Fig. 1C). Results also showed that both the 3’- and 5’-terminal regions of 300
dsRNA-1 and dsRNA-2 lack inverted complementarity required for formation of any panhandle 301
structures. 302
Analysis of the putative polypeptides. Both strands of the full-length cDNAs of dsRNA-1 303
and dsRNA-2 were examined for open reading frames (ORFs). Two ORFs, designated as ORF I 304
and ORF II, were found on the positive strand of dsRNA-1 (404–6112) and dsRNA-2 305
(405–5771), respectively (Fig. 3B). ORF I was deduced to encode a polypeptide with 1902 amino 306
acids. It has a proline-rich region at positions 221–264 (Fig. S2 in Supplemental material), 307
corresponding to the nucleotide (nt) position at 1067–1196 (Fig. 3B). It also contains 308
RNA-dependent RNA polymerase (RdRp)_4 surperfamily domain at nt positions 4625–5455 309
(Fig. 3). The predicted RdRp sequence includes the eight conserved motifs (I to VIII) similar to 310
the RdRp sequences of viruses in the families Totiviridae, Chrysoviridae, Megabirnaviridae and 311
a few unassigned virus taxa (Fig. 4). Therefore, dsRNA-1 and dsRNA-2 are proposed as the 312
genome segments of a mycovirus. We designated this mycovirus as Botrytis porri RNA virus 1 313
(BpRV1). Results from BLASTp analysis showed that RdRp sequence has low levels of 314
sequence similarity (< 25%) to the RdRp sequences of viruses in the families Totiviridae, 315
Chrysoviridae, Megabirnaviridae and as well as to those of a few unassigned virus taxa (Table 316
2). With the exception of the RdRp sequence, no proteins homologous to the remaining part of 317
ORF I-encoded polypeptide were identified in the database of NCBI. Moreover, the polypeptide 318
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of ORF I was searched for homologous proteases in the MEROPS protease database. However, 319
no hits with E values less than 0.01 were detected. 320
ORF II on dsRNA-2 was deduced to encode a polypeptide with 1788 amino acids (Fig. 3B). 321
The polypeptide sequence was detected to contain numerous phosphorylation/glycosylation sites. 322
No conserved proteins homologous to this polypeptide were found in the database of NCBI. 323
Moreover, no hits indicating the presence of any known homologous proteases on this 324
polypeptide were detected in the MEROPS protease database. 325
The three proteins (p70, p80 and p85) detected in the purified virus particles of BpRV1 were 326
individually cleaved with trypsin and the resulting peptide fragments for each protein were 327
subject to PMF analysis. Results showed that p70, p80 and p85 generated a total of 13, 23 and 22 328
peptide fragments, respectively (Tables S2, S3, S4 in Supplemental material). Of these peptide 329
fragments, 11 from p70 matched the partial peptide sequence encoded by ORF II at amino acid 330
positions 200–850, accounting for 30.7% of the entire coverage (651 amino acids). Four peptide 331
fragments matched the deduced sequences HSVNLWSYRPTSAQSR (319–334), 332
AVNTGIGYTSPR (337–348), TDLSIVPTYYTYQTDIR (505–521) and YLEETFTTGR 333
(714–723), with the ion scores higher than 46 (> 95% confidence) (Table S4 in Supplemental 334
material). Twenty one peptide fragments from p80 and 18 peptide fragments from p85 matched 335
the partial peptide sequence encoded by ORF I at amino acid positions 200–1050 (amino acid 336
position) accounting for 41.9 and 30.7% of the entire coverage (851 amino acid residues), 337
respectively. Seven peptide fragments from these two proteins matched the deduced sequences, 338
including DTIAYNVPFGMSVYNR (407–422), FLLPVEDGDDAPIRK (501–515), 339
ITTYVTAGPGAPDLAVHSTIDLYVPEQR (552–579), FTLCPLNGSELIEER (580–594), 340
YLTAHNLWQQFPVMR (616–630), QILQQEYCTER (807–817), and 341
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VAQENVGAVTYYQHEILPSESVSDTPVVR (829–857), with the ion scores higher than 46. 342
Therefore, p80 might be derived from p85 via post-translational modification in a comparable 343
manner to the related capsid proteins of the victorivirus Helminthosporium victoriae virus 190S 344
(HvV190S) (24). 345
Phylogenetic analysis. A phylogenetic tree based on RdRp sequences of BpRV1 and 38 346
selected RNA viruses was generated using the neighbor joining method (Fig. 5). As no members 347
of the reported bipartite dsRNA viral families Birnaviridae, Partitiviridae and Picobirnaviridae 348
were indentified through BLAST search (Table 2), these viruses were not selected to establish the 349
phylogenetic tree. Phylogenetic analysis revealed that BpRV1 appears to be more closely related 350
to members of the genera Totivirius and Victorivirus (both in the family Totiviridae) than to 351
members of the families Chrysoviridae and Megabirnaviridae. BpRV1 clustered with two 352
unassigned insect viruses, Spissistilus festinus virus 1 (SFV1) and Cirulifer tenellus virus 1 353
(CiTV1), forming the BpRV1-SFV1-CiTV1 clade, which was more closely related to Ustilago 354
maydis virus H1 (UmVH1) than to ScV-L-A and ScV-LBC, forming the 355
BpRV1-SFV1-CiTV1-UmVH1 clade. However, the bootstrap support for generation of the two 356
clades was weak, 408 and 343 (1,000 replicates) for support of the clades of 357
BpRV1-SFV1-CiTV1 and BpRV1-SFV1-CiTV1-UMVH1, respectively. 358
Hypovirulence, abnormal colony morphology and dsRNAs. Strains Bc-16, Bc-38 and 359
Bc-95 of B. porri, which lack detectable dsRNAs, caused large necrotic lesions on garlic leaves 360
(20 ºC, 72 h) with the average lesion length of 12 to 17 mm (Fig. 6A). On PDA, they grew at 361
radial growth rates ranging from 11.4 to 13.2 mm/d under 20 ºC (Fig. 6B), and formed 362
grey-whitish colonies and black cerebriform sclerotia after incubation for 17 days (Fig. 6C). In 363
contrast, strain Bc-72 of B. porri harboring two dsRNA segments caused small lesions on garlic 364
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leaves with the average lesion length of 2.3 mm (Fig. 6A). On PDA, it grew at a radial growth 365
rate of 2.3 mm/d under 20 ºC (Fig. 6B) and produced grey-brownish colonies with the formation 366
of numerous mycelial sectors at the colony margin (Fig. 6C). No sclerotia were formed on the 367
colonies of strain Bc-72 after incubation for 17 days (Fig. 6C). 368
Hyphae of strains Bc-16 and Bc-72 were sampled both from the colony margin area and 369
from the colony centre area. Ultra-thin hyphal sections for each strain were observed under TEM. 370
The cytoplasm of hyphal compartments in strain Bc-72 showed a degeneration syndrome 371
manifested by formation of abundant vacuole-like membranous structures and small membranous 372
vesicles (Fig. 7A, B, C). In most cases, the membranous vacuoles/vesicles aggregated and 373
merged. In contrast, the cytoplasm of hyphal compartments of comparable age from strain Bc-16 374
was densely stained and evenly-distributed without formation of abundant and aggregated 375
membranous vacuoles/vesicles (Fig. 7D). Moreover, virus-like particles with the similar size to 376
the virus particles of BpRV1 were observed in the hyphal cells of Bc-72 (Fig. 8). 377
Vertical transmission of hypovirulence-associated dsRNAs. Thirty-five single-conidial 378
(SC) isolates derived from strain Bc-72 were obtained. Thirty-four SC isolates (GP72SC01 to 379
GP72SC34) were detected to be BpRV1-positive and only one SC isolate named GP72SC35 380
(shortened as SC35) was detected to be BpRV1-negative (Fig. 9). On PDA, all the 381
BpRV1-postive SC isolates grew at the average mycelial growth rates ranging from 1.0 to 6.4 382
mm/d, significantly lower (P < 0.05) than the BpRV1-free strain Bc-16 and isolate SC35 (9.9 383
mm/d). On garlic leaves, they produced no lesions or small lesions with the average length 384
ranging from 0 to 3.6 mm, significantly shorter (P < 0.05) than the average lesion length for 385
strain Bc-16 (15.1 mm) and isolate SC35 (11.7 mm). We also found that the average lesion 386
length caused by the BpRV1-positve SC isolates were not significantly correlated with the 387
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average mycelial growth rates of these isolates (r 2 = 1 × 10-7, P > 0.01). Therefore, BpRV1 in 388
strain Bc72 can be vertically transmitted from mycelia to conidia and the hypovirulence 389
phenotype appears just qualitatively correlated with the transmission of BpRV1. 390
Horizontal transmission of hypovirulence-associated dsRNAs. Results of the horizontal 391
transmission experiment showed that in the single cultures, strains Bc-38, Bc-95 and SC35 grew 392
rapidly on PDA and colonized the entire dishes after incubation for 8 days (20 ºC), whereas strain 393
Bc-72 grew slowly and formed small abnormal colonies (Fig. 10). In the contact cultures of 394
Bc-72/Bc-38 and Bc-72/Bc-95, strains Bc-38 and Bc-95 still grew rapidly and colonized the 395
entire dishes after 8 days. In the contact cultures of Bc-72/SC35, strain SC35 grew rapidly in the 396
first three days, followed by reduced growth and formation of mycelial sectors at the colony 397
margin (Fig. 10). However, strain SC35 still failed to colonize the entire dishes in the contact 398
cultures of Bc-72/SC-35 after incubation for 8 days. 399
Three mycelial derivative isolates of strain SC35 (72-35-1, 72-35-2 and 72-35-3) were 400
obtained from three recipient colonies of SC35 in the three contact cultures of Bc-72/SC35 (Fig. 401
10). They were similar to strain Bc-72 in mycelial growth on PDA with the average growth rates 402
of 2.2–3.4 mm/d and in pathogenicity on garlic leaves with the average leaf lesion length of 403
1.1–2.4 mm (Fig. S3 in Supplemental material). However, these derivatives differed greatly from 404
their parental strain SC35, which grew at 10.2 mm/d on PDA and caused lesions of 12.3 mm in 405
length on garlic leaves. Both dsRNA-1 and dsRNA-2 were detected in 72-35-1, 72-35-2 and 406
72-35-3, as well as in strain Bc-72, but were not detected in strain SC35. Therefore, BpRV1 in 407
strain Bc-72 can be horizontally transmitted to strain SC35 through hyphal contact and the 408
hypovirulence phenotype appears qualitatively correlated with virus transmission. 409
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Three mycelial derivative isolates of strain Bc-38 (72-38-1, 72-38-2 and 72-38-3) were 410
obtained from the three recipient colonies of strain Bc-38 in the contact cultures of Bc-72/Bc-38 411
(data not shown). Three mycelial derivatives of strain Bc-95 (72-95-1, 72-95-2 and 72-95-3) were 412
obtained from the three recipient colonies of strain Bc-95 in the contact cultures of Bc-72/Bc-95 413
(Fig. 10). All these mycelial derivative isolates were similar to the corresponding parental strains 414
Bc-38 and Bc-95 both in mycelial growth rate on PDA (20 ºC) and in leaf lesion length on garlic 415
leaves (Fig. S3 in Supplemental material). No dsRNA was detected in these derivative isolates, as 416
well as in their parental strains Bc-38 and Bc-95. Therefore, BpRV1 in strain Bc-72 can not be 417
horizontally transmitted to strains Bc-38 or Bc-95 through hyphal contact. 418
Virulence attenuation as a consequence of transfection with purified virions. The 419
contact culture experiments aiming at co-transmission of the hypovirulence trait and the 420
hypovirulence-associated dsRNAs from strain Bc-72 to strain Bc-38 (BpRV1 free) was 421
unsuccessful (Fig. S3 in Supplemental material). As an alternative, we tried to transfect 422
protoplasts of strain Bc-38 with the purified BpRV1 particles in the presence of PEG 4000. A 423
derivative isolate 38T was obtained from the generated protoplasts of strain Bc-38. It grew at the 424
average radial rate of 4.3 mm/d on PDA at 20 °C with formation of abnormal whitish compacted 425
colonies (Fig. 11A) and was hypovirulent on garlic leaves with the average lesion length of 3.3 426
mm (20 °C, 72 h) (Fig. 11). Two dsRNA segments present in strain Bc-72 were consistently 427
detected in the mycelia of 38T (data not shown). The identity of the dsRNAs in 38T as the 428
genome of BpRV1 was confirmed using RT-PCR with the BpRV1-specific primers 429
BVP1F/BVP1R (Table S1 in Supplemental material). 430
DISCUSSION 431
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The present study reveals that hypovirulence of strain Bc-72 of Botrytis porri is caused by 432
infection with a novel bipartite dsRNA mycovirus (BpRV1). To our knowledge, this is the first 433
report about the co-occurrence of a dsRNA mycovirus and hypovirulence in B. porri. This study 434
provided three lines of experimental evidence supporting the conclusion that BpRV1 is the causal 435
agent of hypovirulence in B. porri. The first evidence is based on the vertical transmission 436
experiment. Thirty-four BpRV1-infected single-conidium (SC) isolates of strain Bc-72 were 437
hypovirulent on garlic leaves, whereas the BpRV1-free SC isolate SC35 became virulent on 438
garlic leaves (Fig. 9). The second evidence is derived from the horizontal transmission 439
experiment, in which BpRV1 was transmitted to the virus-cured strain SC35 through hyphal 440
contact (Fig. 10). The three derivative isolates of strain SC35 showed reduced mycelial growth 441
rates on PDA and debilitated virulence on garlic leaves (Fig. S3 in Supplemental material). The 442
third evidence is from the transfection experiment, in which the purified BpRV1 particles were 443
successfully introduced into the protoplasts of the BpRV1-free virulent strain Bc-38 of B. porri. 444
The transfected derivative isolate 38T showed debilitation symptoms, including reduced mycelial 445
growth on PDA and hypovirulence on garlic leaves (Fig. 11A). 446
It is of interest that the 34 BpRV1-infected hypovirulent SC isolates of strain Bc-72 varied 447
greatly both in mycelial growth rates (1.0 to 6.4 mm/d) on PDA and in leaf lesion length (0 to 3.6 448
mm) on garlic leaves. We interpret this kind of variation as inter-isolate variation, which might be 449
caused by different levels of virus accumulation in different SC isolates. We also observed that 450
there was no significant correlation (P > 0.01) between the two parameters. This result might be 451
due to the unstable cultural characteristics for some SC isolates. We call this kind of variation as 452
intra-isolate variation, which might be caused by the uneven distribution of BpRV1 in hyphal 453
cytoplasm. We observed that the subcultures of even the same SC isolate differed greatly in 454
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colony morphology on PDA, colonies of some subcultures showing normal appearance (slightly 455
attenuated) and colonies of some subcultures showing very abnormal appearance (severely 456
attenuated) (M.D. Wu and G.Q. Li, unpublished data). The intra-isolate variability may mask or 457
obscure the correlative relationship between the mycelial growth rates and the leaf lesion length 458
values. We also observed that the hypovirulent isolate 38T infected by BpRV1 (protoplast 459
infection) showed dramatic differences from the hypovirulent strain GarlicBc-72 and its 460
hypovirulent derivatives, such as 72-35-1 (Fig. 11) in colony morphology on PDA, although 461
mycelial growth of all these hypovirulent isolates was impaired. The difference between the 462
hypovirulent strains 38T and 72-35-1 in colony morphology might be caused by different genetic 463
backgrounds. 464
Results of the horizontal transmission experiment showed that co-transmission of the 465
hypovirulence trait and BpRV1 was successful from strain Bc-72 to virulent strain SC35 of B. 466
porri, but was unsuccessful from Bc-72 to two other virulent strains (Bc-38 and Bc-95) of B. 467
porri. Therefore, the strain-specific transmission manner appears similar to the horizontal 468
transmission of Botrytis cinerea mitovirus 1 (BcMV1) (51, 52). The unsuccessful horizontal 469
transmission of BpRV1 might be due to the vegetative incompatibility between the hypovirulent 470
strain Bc-72 and virulent strains Bc-38 and Bc-95. Strain SC35, a single-conidium isolate of 471
strain Bc-72, is isogenic to strain Bc-72 and they may have the same or similar alleles at the 472
vegetative incompatibility (vic) loci, conferring a compatible interaction between hyphal cells of 473
strains Bc-72 and SC35. In contrast, strains Bc-38, Bc-72 and Bc-95 were collected from 474
different geographic locations in China (Table 1). They might have different alleles at the vic 475
loci, thus maybe conferring an incompatible interaction between hyphal cells and failure of 476
BpRV1 transmission. 477
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Previous studies showed that it is often difficult to establish the etiology of a mycovirus as 478
an agent for hypovirulence in phytopathogenic fungi due to the lack of a proper infection method 479
to complete the Koch’s postulates (9). Two approaches have been successfully used to solve this 480
problem. The first approach is to prepare virus-derived cDNA transcripts (7) or to construct 481
infectious viral cDNA clones (10, 28). Both the synthetic cDNA transcripts and the infectious 482
viral cDNA clones can initiate mycovirus infection in host fungi. This approach has been 483
successfully applied in the hypovirus-C. parasitica system to ascertain the determinant role of 484
hypovirus in hypovirulence of C. parasitica (7, 10, 28). Another approach is to transfect cells or 485
protoplasts of host fungi with purified virus particles. A PEG-mediated transfection method with 486
virus particles has been developed for a mycovirus in B. cinerea (6), mycoreviruses in 487
Cryphonectria parasitica (20) and in Rosellinia necatrix (43), a megabirnavirus (9) and a 488
partitivirus (42) in R. necatrix, and an ssDNA mycovirus in Sclerotinia sclerotiorum (55). In this 489
study, we successfully introduced the purified BpRV1 particles into virus-free strain Bc-38 (Fig. 490
11). This provides an additional example showing the potential of using the PEG-mediated 491
mycovirus transfection technique to study the etiology of mycoviruses for fungal hypovirulence. 492
This technique might even be useful for determining the experimental host range of BpRV1 in 493
the future. 494
Sequence analysis indicates that dsRNA-1 and dsRNA-2 in strain Bc-72 comprise the 495
genome of a bipartite virus. Only dsRNA-1 encodes an RdRp, implying that the two dsRNAs are 496
not derived from two independent mycoviruses. Previous studies showed that many multipartite 497
RNA viruses have conserved terminal regions (9, 25, 49). This phenomenon was observed in the 498
two dsRNAs of BpRV1, which has conserved terminal regions (Fig. 1). This result suggests that 499
dsRNA-2 of BpRV1 may depend on the RdRp encoded by ORF I on dsRNA-1 of BpRV1 for 500
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replication. We observed that the remaining regions on the two dsRNAs of BpRV1 have low 501
sequence similarity (< 40%), implying that dsRNA-2 is not a defective RNA of dsRNA-1. In 502
addition, the genome size of BpRV1 appears different from that of other reported bipartite 503
dsRNA viruses, including those in the Birnaviridae (hosts: vertebrates and invertebrates), 504
Partitiviridae (hosts: fungi and plants), Picobirnaviridae (hosts: vertebrates) and 505
Megabirnaviridae (hosts: fungi). The genome size of BpRV1 is ~12 kb, smaller than that of 506
Megabirnaviridae (~17 kb) (9), but larger than those of Partitiviridae (2.8–4.6 kb) (16), 507
Picobirnaviridae (~4 kb) (9) and Birnaviridae (~6 kb) (9). 508
The RdRp of BpRV1 contains eight conserved motifs, which appear similar to the conserved 509
motifs of the viral RdRps in Totiviridae, Chrysoviridae, Megabirnaviridae and a few 510
taxonomically-unassigned viruses, including SFV1 and CiTV1 (Fig. 4), but are different from the 511
RdRp motifs of other bipartite viruses (Birnaviridae, Partitiviridae and Picobirnaviridae). 512
However, the RdRp sequence of BpRV1 shows low sequence similarity levels (< 25%) to the 513
RdRp sequences of the mycoviruses in Totiviridae, Chrysoviridae and Megabirnaviridae (Table 514
2). In the phylogenetic tree inferred from the viral RdRp sequences (Fig. 5), BpRV1 formed a 515
separate clade distinct from those for Totiviridae, Chrysoviridae, Megabirnaviridae and a few 516
taxonomically-unassigned virus taxa. It has a closer phylogenetic relationship to SFV1, CiTV1 517
and UmVH1 than with RnMBV1. However, BpRV1 with two dsRNA segments differs from 518
these three viruses in the number of dsRNA segments since genomes of SFV1, CiTV1 and 519
UmVH1 are monosegmented (26, 46). Although BpRV1 and RnMBV1 have bipartite dsRNA 520
genomes and their RdRps share sequence similarities, BpRV1 differs from RnMBV1 in the 521
expression strategy for generation of RdRp and the structural proteins. For BpRV1, the RdRp of 522
BpRV1 is more likely expressed as a fusion protein containing a part of the structural proteins 523
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(p80/p85) (Fig. 3). The other structural protein component (p70) is more likely encoded by ORF 524
II. However, RnMBV1 has two ORFs on dsRNA-1, ORF 1 for capsid protein and ORF 2 for 525
RdRp (9). 526
Taking together the information about the number of dsRNA segments, the RNA genome 527
size, the genetic organization and the RdRp sequences, we believe that BpRV1 is a novel 528
bipartite dsRNA mycovirus, which can not be assigned to any known mycovirus families. 529
Furthermore, BpRV1 has been detected in a strain of Botrytis squamosa Walker, the causal agent 530
of onion leaf blight (M. D. Wu, unpublished data). The BpRV1-infected strain of B. squamosa 531
was collected from Tongshan County of Hubei Province, China, ~500 kilometers away from 532
Zhushan County, where strain Bc-72 of B. porri was originally collected. Moreover, a bipartite 533
dsRNA mycovirus infecting Sclerotinia sclerotiorum, the causal agent of sclerotinia stem rot of 534
oilseed rape, was recently identified in China. The sequences of the two dsRNAs were identical 535
to dsRNA-1 and dsRNA-2 of BpRV1 by 91 and 97%, respectively (L. J. Liu and D. H. Jiang, 536
unpublished data). These results suggest that besides B. porri, BpRV1 can infect B. squamosa 537
and S. sclerotiorum. It might be widely distributed in B. porri-, B. squamosa- and S. 538
sclerotiorum-occurring regions. 539
BpRV1 particles are approximately 35 nm in diameter. The size is similar to that of the 540
families Chrysoviridae (30 to 40 nm), Partitiviridae (30 to 40 nm) and Totiviridae (30 to 40 nm), 541
but is smaller than that of RnMBV1 in Megabirnaviridae (~50 nm) and Mycoreovirus (~80 nm). 542
Whether or not the two dsRNAs of BpRV1 are encapsidated in separate virus particles or within 543
the same virus particles remains unknown. The dsRNA segments for the genomes of the viruses 544
in Chrysoviridae and Partitiviridae are separately encapsidated (17, 18, 41). In addition, the two 545
dsRNA segments for RnMBV1 were thought to be separately encapsidated based on the finding 546
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that the unequal molar ratios between dsRNA-1 and dsRNA-2 extracted directly from host 547
mycelia and the purified virus particles (9). However, no significant molar ratio variation was 548
observed for BpRV1 dsRNA-1 and dsRNA-2 (Fig. 2C). To address the question about the 549
particle assembly in BpRV1, we calculated the gross volumes of virus particles of dsRNA viruses 550
in all recognized genera based on diameter of virus particles (Table S5 in Supplemental material). 551
Then, the values of the dsRNA length encapsidated within one volume unit of a virus particle 552
were inferred to indicate the dsRNA assembly density. For almost all dsRNA viruses, the density 553
of dsRNA in one particle is within the range of 0.03–0.28 bp/nm3. For BpRV1, the values for the 554
dsRNA density are 0.28 bp/nm3 under separate encapsidation and 0.53 bp/nm3 under combined 555
encapsidation. From this point of view, the two dsRNAs of BpRV1 are likely to be separately 556
encapsidated, as the virus particles might not be spacious enough for simultaneous packaging of 557
the two dsRNAs. 558
Results of the PMF analysis of the three proteins (p70, p80 and p85) in BpRV1 indicate that 559
there may be two structural proteins (SPs) for BpRV1 (Fig. S4 and Tables S2, S3, S4 in 560
Supplemental material). The protein component p70 is encoded by ORF II on dsRNA-2, whereas 561
p80 and p85 are encoded by ORF I at a comparable region on dsRNA-1. There are two 562
possibilities for generation of p80 and p85. The first possibility is that p80 might be derived from 563
p85 via post-translational modification in a comparable manner to the related capsid proteins of 564
HvV190S (24). Another possibility is that p80 and p85 are generated via different events in 565
processing the ORF I-encoded polyprotein. In the PMF analysis, we also found several peptide 566
sequences matched the C-terminal regions, rather than the N-terminal regions, on the ORF I- and 567
ORF II-encoded polypepetides (Fig. S4 in Supplemental material). They are probably random 568
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matches, as the ion scores for these peptide sequences are lower than 46 (Table S2, S3, S4 in 569
Supplemental materials). 570
The genomes of RNA viruses are susceptible to chemical or physical damages from 571
environmental factors. They also encounter immune response or antiviral defense of their hosts. 572
To overcome these challenges, many RNA viruses have developed series of self-defense 573
mechanisms (4). In the interaction between hypoviruses and Cryphonectria parasitica, the 574
hypovirus-encoded papain-like protease p29 acts as an inhibitor of the RNA silencing-induced 575
antiviral defense by C. parasitica, thus enhancing viral RNA accumulation and vertical 576
transmission (44, 45, 47). Moreover, hypovirus infection of C. parasitica causes proliferation of 577
vesicle formation within hyphal cells (11, 35, 36) and these vesicles were determined to be sites 578
for viral replication (13, 14). The replication of RNA viruses in these membranous lipid vesicles 579
might also offer protection from nucleases (31). 580
In addition, Spear et al. (46) proposed that the proline-alanine rich protein (PArp) encoded 581
by SFV1 and CiTV1 may act like a scaffold protein to interact with some membrane components 582
in insect cells, as many animal viruses can utilize a protein scaffold containing proline-rich “late” 583
domain motifs to hijack host proteins involved in the vacuolar protein sorting pathway, resulting 584
in formation of multivesicular bodies (8). In the present study, formation of abundant 585
vacuole-like membranous structures and small membranous vesicles in hyphal cells infected with 586
BpRV1 were observed (Fig. 7) and a proline-rich region (PrR) was detected on putative 587
polypeptide encoded by ORF I on dsRNA-1. Therefore, further studies for elucidation of the 588
function of PrR-containing polypeptide in virus accumulation of BpRV1 and for understanding of 589
the expression strategy of dsRNA-1 and dsRNA-2 are warranted. 590
ACKNOWLEDGMENTS 591
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This research was funded by the Natural Science Foundation of China (Grant No. 592
31070122). The authors greatly appreciate the kind help of Dr. S. A. Ghabrial of the University 593
of Kentucky (USA) for critical reading of the manuscript and providing useful literature on 594
mycovirus taxonomy. We also thank Dr. N. Suzuki at Okayama University (Japan) in providing a 595
protocol for the transfection experiment with the purified virus particles of BpRV1, and Professor 596
N. Hong of Huazhong Agricultural University (China) for her advice to purify virus particles of 597
BpRV1. 598
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764
765
766
767
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Figure legends 769
FIG. 1. Alignment of the 5’-terminal (A) and 3’- terminal (B) sequences of the coding strand of 770
dsRNA-1 and dsRNA-2 of Botrytis porri RNA virus 1 (BpRV1). Black shades are used to 771
highlight the identical nucleotides. The “•” symbol represents the missing nucleotide. The start 772
codon (AUG) at the 5’-terminus for both dsRNA-1 and dsRNA-2 is indicated with a black bar 773
above the two nucleotide sequences; (C) The predicted secondary structures for the terminal 774
regions of dsRNA-1 and dsRNA-2 of BpRV1. 775
FIG. 2. BpRV1 particles from strain GarlicBc-72 of Botrytis porri. (A) and (B) Transmission 776
electron microscopic graphs (negative straining) of the virus particles of BpRV1; (C) Agarose gel 777
electrophoresis of the dsRNAs extracted from the purified virus particles of BpRV1, and from 778
mycelia of strains GarlicBc-72 and GarlicBc-38. Marker = 1 kb DNA ladder. Note the similar 779
size for the dsRNAs from the purified virus particles and from the mycelia; (D) SDS-PAGE 780
analysis of the purified virus particles of BpRV1 showing three protein components (p85, p80 781
and p70). 782
FIG. 3. Molecular characteristics of dsRNA-1 and dsRNA-2 of Botrytis porri RNA virus 1 783
(BpRV1). (A) Northern blot detection of dsRNA-1 and dsRNA-2 using digoxigenin-labeled 784
probes 1, 2 and 3. The locations of these probes are indicated in graph B; (B) Schematic diagrams 785
of the genetic organization of BpRV1 dsRNA-1 and dsRNA-2. The coding strand of dsRNA-1 is 786
6,215 bp long and comprises one large open reading frame (ORF), designated as ORF I, which 787
encodes a polyprotein of 1902 amino acid residues. The coding strand of dsRNA-2 is 5,788 bp 788
long and also comprises one large ORF, designated as ORF II, which encodes a polypeptide of 789
1788 amino acid residues. p70, p80 and p85 are the structural proteins of BpRV1 inferred from 790
peptide fingerprinting analyses. 791
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FIG. 4. Multiple alignment of the RdRp sequences of BpRV1 and other RNA viruses showing 792
the structure of the eight-motifs (I to VIII). The alignment was made using CLUSTAL_ X. “*” 793
indicates identical amino acid residues; “:” and “.” indicate high and low chemically-similar 794
amino acid residues, respectively. UmVH1 = Ustilago maydis virus H1 (GenBank Acc. No. 795
NC_003823), ScV-L-A = Saccharomyces cerevisiae virus L-A (J04692), ScV-L-BC = 796
Saccharomyces cerevisiae virus L-BC (La) (U01060), HvV145S = Helminthosporium victoriae 797
virus 145S (AF297176), PcV = Penicillium chrysogenum virus (AF296339), FoCV1 = Fusarium 798
oxysporum chrysovirus 1 (EF152346), AfV = Aspergillus fumigatus chrysovirus (FN178512), 799
CnV1 = Cryphonectria nitschkei chrysovirus 1 (GQ290650), ACDACV = Amasya cherry 800
disease-associated chrysovirus (NC_009947), VcV = Verticillium chrysogenum virus 801
(HM004067), AbV1 = Agaricus bisporus virus 1 (X94361), CCRSCV = cherry chlorotic rusty 802
spot-associated chrysovirus (AJ781397), Hm17V = Helicobasidium mompa no. 17 dsRNA virus 803
(AB085814), SsRV1 = Sphaeropsis sapinea RNA virus 1 (NC_001963), MoV1 = Magnaporthe 804
oryzae virus 1 (AB176964), GaVL1 = Gremmeniella abetina RNA virus L1 (AF337175), BfTV1 805
= Botryotinia fuckeliana totivirus 1 (AM491608), HvV190S = Helminthosporium victoriae virus 806
190S (U41345), RnBMV1 = Rosellinia necatrix megabirnavirus 1 (AB512282), SFV1 = 807
Spissistilus festinus virus 1 (GU979419), CiTV1 = Cirulifer tenellus virus 1 (GU979420), PGV1 808
= Phlebiopsis gigantea mycovirus dsRNA-1 (AM111096), FgV3 = Fusarium graminearum 809
dsRNA mycovirus-3 (NC_013469), DsRV1 = Diplodia scrobiculata RNA virus 1 (NC_013699), 810
PGV2 = Phlebiopsis gigantea mycovirus dsRNA 2 (AM111097), LEV = Lentinula edodes 811
mycovirus HKB (AB429554), PRV = Porcine rotavirus (Q85036), HTV = Human rotavirus 812
(Q91E95), ARV = Avian rotavirus PO-13 (O55590), CMV = Cocksfoot mottle virus (Q66150), 813
RYMV = Rice yellow mottle virus (Q86519), SCMV = Southern cowpea mosaic virus (P21405), 814
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SBMV = Southern bean mosaic virus (O72157), LTSV = Lucerne transient streak virus 815
(Q83093), BWYV = Beet western yellows virus (P09507), CAYV = Cucurbit aphid-borne 816
yellows virus (Q65969), PLV = Potato leafroll virus (P11623), PEMV-1 = Pea enation mosaic 817
virus-1 (P29154). 818
FIG. 5. Phylogenetic analysis of BpRV1 and 38 selected RNA viruses presented in a 819
neighbor-joining (NJ) tree inferred from the RdRp sequences. Animal viruses (rotaviruses) and 820
plant viruses (poleroviruses, enamoviruses and sobemoviruses) with their RdRps belonging to 821
RdRp _4 superfamily were included as outgroups. Numbers at the nodes are bootstrap values out 822
of 1,000 replicates. Bootstrap values lower than 700 are not shown in the figure. The taxonomic 823
status of the viruses labeled with the question mark (?) has not been determined. See the legend 824
for definitions of the virus name abbreviations in FIGURE 4. 825
FIG. 6. Leaf lesion length on garlic, mycelial growth rate on potato dextrose agar and colony 826
morphology of different strains of Botrytis porri. (A) Average lesion length caused by strains 827
GarlicBc-16, GarlicBc-38, GarlicBc-72 and OnionBc-95 on detached garlic leaves (20 °C, 72 h); 828
(B) Average radial growth rates of these strains of B. porri on potato dextrose agar (PDA) at 20 829
°C; and (C) Cultures of strains GarlicBc-16 and GarlicBc-72 on PDA (20 °C, 17 days). In the 830
histograms A and B, results are expressed as arithmetic means ± standard errors of means. Bars 831
in each histogram labeled with the same letters are not significantly different (P > 0.05) 832
according to Least Significant Difference Test. dsRNA– = dsRNA negative, dsRNA+ = dsRNA 833
positive; V = Virulent, HV = Hypovirulent 834
FIG. 7. Transmission electron microscopic graphs showing the cytoplasmic characteristics of the 835
hypovirulent strain GarlicBc-72 and the virulent strain of Botrytis porri. The mycelial samples 836
examined were collected from the colony margin area of each strain. (A, B, C) Three hyphal cells 837
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of strain GarlicBc-72 showing a degeneration sign characterized by formation of abundant 838
vacuole-like membranous structures and small membranous vesicles; (D) A hyphal cell of strain 839
GarlicBc-16 with dense and evenly-distributed cytoplasm; V = vacuoles or vesicles, M = 840
mitochondrion, scale bars = 1 µm. 841
FIG. 8. Transmission electron microscopic graphs of the hyphal cells of the hypovirulent strain 842
GarlicBc-72 containing virus-like particles inside the vesicles (arrowheads). 843
FIG. 9. Lesion length on detached garlic leaves (20 ºC, 72 h) (A) and mycelial growth rates on 844
potato dextrose agar at 20 ºC (B) for strains GarlicBc-16 and GarlicBc-72 of Botrytis porri and 845
35 single-conidium isolates (GP72SC01–GP72SC35) of strain GarlicBc-72. The results were 846
expressed as the arithmetic means ± standard errors of means. In each histogram, bars labeled 847
with the same letters are not significantly different (P > 0.05) according to Least Significant 848
Difference Test. The symbols “+” and “–” indicate the presence and the absence of 849
dsRNA-1/dsRNA-2, respectively. The dsRNAs were extracted from the mycelia of each 850
strain/isolate and detected by agarose gel electrophoresis. 851
FIG. 10. Transmission of the hypovirulence trait and BpRV1 from the hypovirulent strain 852
GarlicBc-72 to the virulent strains GP72SC35 of Botrytis porri on potato dextrose agar. All the 853
dishes were incubated at 20 °C for 8 days. In the single cultures (the top row), strains GP72SC35 854
and OnionBc-95 colonized the entire dishes, whereas strain GarlicBc-72 formed a small colony. 855
In the contact culture of OnionBc-95/GarlicBc-72 (bottom row), OnionBc-95 colonized the entire 856
dish, indicating unsuccessful transmission, as the contact of the hyphae of GarlicBc-72 with the 857
hyphae of GarlicBc-95 had no effect on mycelial growth of GarlicBc-95. However, in the contact 858
culture of GP72SC35/GarlicBc-72, GP72SC35 colonized only a part of the dish with formation 859
of mycelial sector along the colony margin, indicating successful transmission, as the contact of 860
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the hyphae of GarlicBc-72 with the hyphae of GP72SC35 produced a dramatic effect on mycelial 861
growth of GP72SC35. The symbol “*” indicates the place to remove a mycelial agar plug for 862
generation of a derivative isolate for GP72SC35 or OnionBc-95 transfected by BpRV1. 863
FIG. 11. Phenotypic effects of BpRV1 on fungal colony morphology, mycelial growth rate and 864
virulence in strains GarlicBc-38 and GP72SC35 of Botrytis porri. (A) Comparison of the colony 865
morphology between the BpRV1-free strain GarlicBc-38 (left) and the BpRV1-transfected isolate 866
38T (right) (20 °C, 17 days); (B) Comparison of the colony morphology between BpRV1-free 867
isolate GP72SC35 (left) and the BpRV1-infected isolate 72-35-1 (right) (20 °C, 17 days); (C) 868
The mycelial growth rates of strains GP72SC35, 72-35-1, GarlicBc-38 and 38T on PDA (20 °C); 869
(D) Lesion length caused by strains GP72SC35, 72-35-1, GarlicBc-38 and 38Ton garlic leaves 870
(20 °C, 72 h). Results in each histogram are expressed as arithmetic means ± standard errors of 871
means. “**” indicates significant difference (P < 0.01) between strains GarlicBc-38 and 38T, and 872
between strains GP72SC35 and 72-35-1, in mycelial growth rate or lesion length according to 873
Student’s t-test. 874
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GCAAAUAAAAGAGCUGCC .U UUUGAAGCCUUUUAUUUGUAAGGAAAAUUU
AUAUUGAUCGUCAUUCAACGAUCCCCGCUC
GCAAAUAAAAGAGCUGCCUAUUUGAAGCUUUUUAUUUGCAAAGAAAAUGU
AUUUUGAUCGUCAUUAAACGAUCCCCGCUAGGGUUAGAAAGCACCACAAG
CGCAGUGACGATCGCUUGUGAGUAGUAGUCACACUAGAUGGUAAAUUCUA
UUUGGAACCU
GGGUUAGAAAGCGCUACGAA
CGAAGUGACGAACGUACGUAGAUAGUAGUCACACUAAAUGGUAAAUUCUA
UUUGGAACCUCGGACACUCUCAAACCGCCUUGUCAGGAUAUCAGCGGAUG
UGAAUAGACGUGUGGAACACAACGUGCCCACCACAUCUUC
CGGACACUCUCAAACCGCCUUGUCAGGAUAUCAGCGGAUG
UGAAUAGACGUGUGGAACACAACGUGCCCACCACAUCUUCAACUGCUCCU
GUUAGUGUAUAAAUCAGAAGGAUAGGUAAACCGAGAUCUCUAGAGAAG
GCUACCUGGCACUGCUGACG
AACUGCUCCU
GUUAGUGUAUAAAUCAGAAGGAUAGGUAAACCGAGAUCUCUAGAGAAG
GCUACCUGGCACUGCUGACGGGCCGUAGUAUUCUACGAUAUCGGGAGGAA
UUGAAGCCUUCUGUAAUCCCCCAGUACCCAGGCUCUCUAGGGUAAAGUGA
GGCCGUAGUAUUCUACGAUAUCGGGAGGAA
UUGAAGCCUUCUGUAAUCCCCCAGUACCCAGGCUCUCUAGGGUAAAGUGA
GAGAAUGAAAUUCAAAAUGCCUUACAACACACAAACCGCCUCCUCG
AAGAGGACAACAGAGUGGAGAAUGUUGCUA
GAGAAUGAAAUUCAAAAUGCCUUACAACACACAAACCGCCUCCUCG
AAGAGGACAACAGAGUGGAGAAUGUUGCUAAAUCAGCUCCACAAAACAU
AACUUGCCUGCAGAAUUGUG
AAUCAUCUCCACGCAAAA
GCUCAACCUAAGAGCAAGAA
UUACAACAUGCAGUUGGUA..AGUAUAUAGGGUGAUUAGGCUAUCACAUGCAGUGAGGAGUAAAGGAUGAGUUAACCAGG
G.UUAA
CCCCAUAAUUUACAGGGAGAAGUGAAUAGAUAGC
GGCUAA
UCCCAUAUUAUAACGGAAGGAGUAAUUACAGAGC58796215
A
B
dsRNA-1dsRNA-2
AGAG
GCGCGCGC
CCA
The 5’ terminus sequence alignment of the two dsRNA segments
The 3’ terminus sequence alignment of the two dsRNA segments
dsRNA-1dsRNA-2
dsRNA-1dsRNA-2
dsRNA-1dsRNA-2
dsRNA-1dsRNA-2
dsRNA-1dsRNA-2
dsRNA-1dsRNA-2
dsRNA-1dsRNA-2
dsRNA-1dsRNA-2
dsRNA-1dsRNA-2
dsRNA-1dsRNA-2
dsRNA-1dsRNA-2
dsRNA-1dsRNA-2
4950
99100
149150
199200
249250
299300
349350
399400
449450
499500
519520
58456181
A UAU
A
GGGU
GAUUA G G
GUU
AACCC
CAUA A
UUUAC
AGGG A
GAA
GUGAAUG
CUUAC
AACAU G
CA
GUUG
GUAAGU AGATAGC
dsRNA-1
10
20
30
GCAAAUAAA
A
GAGCU
GCCUA U
UUG
AAGCUU
UUUAUUUGC
dsRNA-2
5’ terminus 5’ terminus3’ terminusΔG= -13.2 (kcal/mol) ΔG= -16.4 (kcal/mol) ΔG= -14.7 (kcal/mol)
6137
6147
6157
6167
6177
6187 6207
1
10
20
30
GCAAAUAAAAG
AGCU
GCCU U U
UG
AAGC
CUUUUAUUUGU1
C
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.org/D
ownloaded from
200 nm 100 nm
50 nm
GarlicB
c-38
Virus p
articl
es
GarlicB
c-72
Marker
(kb) Virus p
articl
es
Marker
(kDa)
8654
1501201008570
60
p85
A B C D
p80
p70
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ownloaded from
404 6112
5’ 3’
62151
405 5771
3’5’1 5879
RdRp_4
4625 5455
dsRNA-1
dsRNA-2
ORF I (1902 aa)
ORF II (1788 aa)
B
A
dsRNA-1dsRNA-2
Probe 1 Probe 2
Probe 3
Probe 2 Probe 1 Probe 3
1 kb
1067 1196
Proline-rich region
Estimated coding regionfor p85/80
Estimated coding regionfor p70
on June 24, 2018 by guesthttp://jvi.asm
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LAKLVSS[82]LAKIVSS[82]LAKLVSS[82]***:***
SFHVGF[66]SFHVGF[66]SFHVGY[66]*****:
SN-GESRQLKFGRKTIFSTKKNSN-GESRQLKFGKKTIFSTKKNSN-GILREINFGGQKVRSTKKN** * *:::** :.: *****
[33]RTRIIFILPYE[119][33]RTRIIFILPYE[119][32]RTRAIFILPYQ[115]
*** ******:
AVASGEKQTKAANSIANLALI[18]AVASGEKQTKAANSIANLALI[18]ATASGEKQTKIMNSIANKALI[18]*.******** ***** ***
RVDGDDNYAVL[18]RYIYSRMNAK-----VKALVSTVG[56]RVDGDDNYAVL[18]RDTYSRMNAK-----VKALVSTVG[56]RVDGDDNYAIV[18]RSYYSEMNVK-----VKALASLTG[56]*********:: * **.**.* ****.* .*
FILTKIIFILTKIMFILTKIC******
QMTSVAITQMTSVAITQMTSIKIT****: **
SCMVSBMVLTSVCMVRYMV
BWYVCAYVPLVPEMV-1
MVEMGLC[36]LVRRGLC[36]LVRLGLC[36]LVKRGLC[36]LVEKGLC[36]
LVRNGLC[36]LVRRGLC[36]LVQEGLC[36]AVKDGLV[36] *: **
RMLFGA[14]KP-GMGLSVI-HQADAIFRDLRRMLFGP[14]KP-GMGLSLR-QQAKSLFDDLRRLLFGP[14]KP-GMGLSQK-WQFEALWKDLQRMLFGP[14]KP-GMGLSKA-SQVALLWEDLARMLFGP[14]KP-GMGLLTP-EQIRLVWDDVF
RVLFQN[14]KP-GFGLSTD-EQVLDFVESLARVLFQN[14]KP-GFGLSTD-EQVAEFMQILSRVLFQN[14]KP-GFGLSTD-TQTAEFLECLQRMLFRD[14]KP-GLGFSQD-HQVLAFTESVA*:** :: ** *:*:* * *. * : :
[15]FDWSVQDWELW[046][15]FDWSVQDWELW[046][15]FDWSVQKWELE[046][15]FDWSVQDWELW[046][15]FDWSVQDWELW[046]
[25]FDWSVADWMLH[047][25]FDWSVSDWLLE[046][26]FDWSVAYWMLE[047][25]FDWSVPMWLLE[047]
*****. *:*.
IMKSGSYCTSSTNSRIRCLMA[09]IAMGDDSVEGFIMKSGSYCTSSTNSRIRCLMA[09]IAMGDDSVEGWLMKSGSYCTSSTNSRIRCLMA[09]IAMGDDSVEGYLMKSGSYCTSSSNSRIRCLMA[09]IAMGDDSVEGWLMKSGSYCTSSTNSRIRCLMG[09]IAMGDDSVEGW
VQKSGSYNTSSSNSRIRVMAA[09]MAMGDDALESNVQKSGSYNTSSSNSRIRVMAA[09]MAMGDDALESAVQKSGSYNTSSSNSRIRVMAA[09]MAMGDDALEAPIQKSGSFNTSSTNSRMRYMLA[09]VTMGDDALESV:*****:****:***:* * * ::*******:
[19]PCATTPTGQL-----YAVEFCSHV[05]AFLTSWPKTLYRFLS[19]PCATTISGRL-----YEVEFCSHV[05]CWLASWPKTLFKYLS[19]PCAAEG-EIL-----KEVNFCSHS[05]CYLQSWAKTLFRYLE[19]ACPVLPNGDL-----KEVSFCSHL[05]AELETWPKCLFRYLS[19]PCKTR-GREL-----LEFNFCSHL[05]AELTSWPKALFRFLS
[07]KKLGFKVEVS-----GQLEFCSHI[06]ALPVNENKMIYKLIY[07]KSLGFKVEES-----SKLEFCSHI[06]AIPVNKAKMLYKLIH[07]KTLGFKVEVG-----RELEFCSHI[06]AVPVNTNKMLYKLIH[07]ARLGIKCERA-----EEFDFCSHL[06]VIPKNLEKMVYGLLS
:::****: .:* * **:* *:
ScV-L-A L--MNRG[60]ScV-L-BC L--ENGV[58]BfTV1 L--QGRF[60]HvV190S L--QGRY[61]MoV1 L--QGRG[60]Hm17V L--QGRM[62]GaVL1 L--LGRA[65]
UmVH1 L--YGRG[66]
WVPGGSIMPGGSWCVNGSWCVNGSWCVNGAWCVNGSWAVNGA
WLVSGS
[49]KY-EWG--KQ-RAIYGTDLRST[44][50]KY-EWG--KV-RALYGCDFSSH[43][45]KL-EAG--KD-RAIFACDTRSY[47][45]KL-ENG--KD-RAIFACDTRSY[47][50]KL-EHG--KT-RAIFACDTRSY[47][50]KL-EHG--KT-RAIFACDTQNY[47][48]KL-EAG--KT-RAIFACDTVNY[47]
[58]KLNETGG-KA-RAIYGVTLWHY[47]
FDYDDFNSQHS[052]YDFDDFNSQHS[052]LDYDNFNSHHS[045]LDYDNFNSQHS[045]LDYDDFNSQHS[045]LDYDDFNSHHA[046]LDYDDFNSHHS[046]
YDYPDFNSMHT[064]
TLLSGWRLTTFMNTVLTLFSGWRLTTFFNTALTLASGHRGTSFINSLLTLMSGHRATTFTNSVLTLMSGHRGTTFFNSILTMMSGHRGTMFINSVLTLMSGHRGTTFINSVL
GLYSGDRDTTLINTLL
NWAYM[14]VHNGDDVMISL[18]RAQPAKCNYCYL[12]LHNGDDVFAGI[18]RANTTKMNAAYI[14]LHAGDDVYIRA[18]RMNPTKQNAAYI[14]LHAGDDVYLRL[18]RMNPTKQNGVYI[14]MHTGDDVYMRL[18]RLNPTKQNAVYI[14]LHAGDDVYAVL[18]RMNSTKQNKAYL[14]LHVGDDVYFGV[18]RMNRMKQ
NIAYA[19]LCHGDDIITVH[18]KGQESKL
NL------FSISEFLRV[12]QYLSRSCATLVHSRINI------GTIAEFLRV[10]QYLTRGIATFTHSRVSIG-----FRNAEFLRL[08]GYVARTISTLVAGNWSIG-----YTGAEFLRL[08]GYLCRAIASLVSGSWSIG-----YKHAEFLRV[08]GYAARSIAALASGNWSVG-----VVCGEFLRM[08]GYVCRSIASFISGNWSVG-----HVSTEFLRN[08]GYFARAVASTVSGNW
MID-----HKHHEYLRI[09]GCLARCVATYVNGNW
ACDACV1 L--VGRR[73]CCRSCV L--VGRR[73]HvV145S L--LGRR[73]FoCV1 L--LGKE[73]PcV1 L--VGRG[73]
AbV1 L--TGRP[93] * *:
WLTKGSWLTKGSWMTKGSWLTKGSWLTKGG
SQVPGN*
[61]KLNECGY-[61]KLNECGY-[61]KLNENGH-[61]KLDENGH-[62]KY-EVGK-
[69]KH-EVGKN* * *
KD-RTLLPGSLFHY[44] FDKD-RTLLPGSLFHY[44] FDKD-RVLLPGGLLHY[44] YDKR-RALLPGSLMHY[43] FDKD-RTLLPGTLVHF[44] YD
AS-RSLWPAHLVHY[45] MD * * *. *.*: *
WANFNAFHSWANFNAFHSWANFNVQHSWANFNLYHSWADFNEQHS
YANFNEQHS:*:** **
[049]GLYSGWRGTSFLNSVLNSCYT[19]DHGGDDIDGGI[049]GLYSGWRGTSFLNSVLNSCYT[19]DHGGDDIDGGI[049]GLYSGWRGTTWDNTVLNGCYM[19]DQGGDDVDQEF[049]GLYSGWRGTTWINTVLNCVYT[19]DGGGDDHDGGL[049]GLYSGWRGTTWINTVLNFCYV[19]DHGGDDIDLGL
[053]GLLSGWRCTAYINNLINIAQY[19]DTGGDDGCADE ** **** *:: *.::* * ****
[18]EAQKIKQMIGI------DSEFFRI[08]GSATRALARFVSGNW[18]EAQKIKQMIGI------DSEFFRI[08]GSATRALARFVSGNW[18]EATKSKQMIGR------NSEFFRV[08]ASPVRGLATFVAGNW[18]KASAIKQMIAM------KSEFFRN[08]ASPVRTLTTFVNGKW[18]KANKWKQMFGT------RSEFFRN[08]ASPTRALASFVAGDW
[18]EFKDIKQLISS-----GTHEFFRL[08]GSVIRMLASAASGQW **::. **** .* * *: . *.*
LEV L--SGRA[73]PGV1 L--VGRA[79]RnBMV1 A--LGRF[72] **
LAPTGAVAPRGSFGSSGS... *:
[48]KV-ETG-LRS-RQIVPGPTKHW[51][48]KT-ESG-LRL-RQIIPGEIHQW[49][46]KR-EAG--KL-RQLLPGRIPHW[48]
* *:* : **::** :*
SDYADFNFLHTTDYADFNYLHTVDWADFNITHT *:**** **
[063]GLWSGWRTTSVI[064]SLWSGWRTTTMI[069]GLWSGWRTTSFI *****:.*
NNVFNEVYG[19]RRNGDDEDARA[18]DAQNNTMNLVYN[19]HVNGDDGDFEV[18]DVQNCSFNVAYC[19]VHAGDDFFGTY[18]EVN* :* .* *** :.:
PAKQKIGF-----TESEYTRV[08]NPIARGIASFASKQLLAA-----SHAEYLRI[08]GSIARSCASFAQKQLVGG-----EMGEFLRH[08]GSVMRSIGSF. ** :. .*: * ..: *. .**
TSSDLVGGDLVGSDL...**
Motif-I Motif-II Motif-III Motif-IV Motif-V Motif-VI Motif-VII Motif-VIII
Lute
o-v
irida
eS
obem
o-v
irus
Reo
-viri
dae
Totiv
irida
eC
hrys
oviri
dae
Una
ssig
ned
:*. *** *:** *. ** **** * :: *: ******.*** :**********:********. **********: ..**** . * :*.* *:::*.
SFV1 L--TGRS[91]CiTV1 S--FGRS[93]BpRV1 M--VGRP[67] **.
WIASGS[58]WMASGS[57]WMIKGS[59]*: .**
EKYENG--KA-RAIYGVEPMEKYENG--KA-RAIYGVAPMKGNEHG--KV-RAIYGSLYA: *:* *. *****
HY[49]LDYADFNRHHY[49]FDYADFNRHHY[47]LDYADFNAQ** :****** :
HT[053]GMFHT[053]GMFHS[055]GMF*: ***
SGTRSTDLINTLLNLAYF[20]VHQGDDVWVSN[18]SGTRSTDLLNTLLNLAYF[20]VHQGDDVWVSN[18]SGVRQTTLINTILNLTYH[20]YVLGDDGWVEF[18]**.*.* *:**:***:*. *** **.
IFQGSKQMFGP-----GRGEYLRV[08]GYFARSLANYLLRPIIFQKSKQMFGV-----GRGEYLRV[08]GYLGRAIANYILRPIEINAIKQLIGQ-----GKGEYLRL[09]GCPVRSLASFVHGNV :: **::* *:*****: * *::*.:: :
AfV L--VGRS[73]VcV L--IGRT[74]CnV1 L--VGRT[74]
WLTKGS[62]WLTKGS[63]WLTKGS[63]
KY-EVGN-KKY-ETGK-KKF-ETGR-K
D-RTLLPGTLVHF[44] YDD-RTLLPGSLIHF[44] YDD-RTLLPGTLIHF[44] YD
WANFNEQHS[049]WADFNEQHS[049]WADFNEQHT[049]
GLYSGWRGTTWVNTVLNFCYV[19]DHGGDDIVLGL[18]GLFSGWRGTTWINTVLNFCYS[19]DQGGDDIDSAL[18]GLYSGWRGTTWINSVLNFCYI[19]DHGGDDVDAAV[18]
KANKWKQMFGV------RSEFFRN[08]ASPTRALASFVAGDWNANAWKQMFST------RTEFFRN[08]ASPTRALASFCAGDWNANAWKQLFST------RSEFFRY[08]ASPTRALASFIAGDW
SaRV1 L--LGRA[61]WCVNGS[45]KL-EHG--KT-RAIFACDTRSY [47]LDFDDFNSHHS [045]TLPSGHRGTTIVNSVLNAAYI [14]LHTGDDVYIRA[18]RINPAKQSVG -----FGTGEFLRM[08]GYLARSVASFVSGNW
* . .*: * * * * **::. *: :*** *: : ** * * : *: ** * : ***: : : * : *:** * :: .
PGV2 L--PGPS[73]DSRV1 L--PGRN[73]FGV3 L--PGRS[72] * ** .
WAASGG[49]WAASGG[58]WAASGG[50]
******
SKFEKG--KR-RAIWNTAIEHY[54]PKFENG--KR-RIIWNTSLTHY[47]LKYEPG--KL-RSILNTSMEHY[47] *:* * * * * **:: **
WDFSDFNINHQ[064]WDYADFNINHS[062]WDFADFNINHT[063]**::******
CLASGERATSLASGERATSLQSGERAT.* ******
SFTNTILSRVYR[20]SQQGDDVFAAV[18]SFVNTVLSRAYR[20]DHQGDDVFATV[18]SWINTLRNHVDH[20]YKTGDDVFLTV[18]*: **: .:. : : ***** :*
AGQSYKVTLDY----APRGEFLRL[10]GYPIRAGLGLISGEFAGQLFKITCNH----TPSGEFLRQ[08]GYPIRSAMGLFSGEYAGQVSKIFVSYPELGGSRGEFVRY[10]GYPLRALTGLVHGEY*** *: .: . ***:* ***:*: **. **:
Meg
abirn
aviri
dae
**:* * .
.* . *
PRVARV HTV
on June 24, 2018 by guesthttp://jvi.asm
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ownloaded from
PcV
HvV145SFoCV1
CCRSCV ACDACV
AfV
VcV
CnV1
AbV1
GaVL1
HvV190SBfTV1 MoV1
Hm17V
SsRV1ScV-L-BC
ScV-L-A
UmVH1
RnBMV1
PGV1
LEV
BpRV1
SFV1
CiTV1
PGV2
DsRV1
FgV3
PRV
ARV
HTV
BWYV
CAYV
PLV
PEMV-1
SCMV
SBMV
LTSV
CMVRYMV
Chrysovirus
Rotavirus
Polerovirus
Sobem
oviru
s
Meg
abirn
aviru
s
Victorivirus
Totivirus
?
?
?
Enam
ovirus
1000
1000
1000
10001000
1000982
1000
1000
1000
1000
1000
1000
1000
1000 1000 1000
963
904
1000
0.1
704
706
on June 24, 2018 by guesthttp://jvi.asm
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ownloaded from
OnionBc-95 GarlicBc-38 GarlicBc-16 GarlicBc-72
Lesi
on L
engt
h (m
m)
Gro
wth
rate
(mm
/d)
V (dsRNA-) HV (dsRNA+)
Gar
licB
c-72
Gar
licB
c-16
A
B
C
0
6
12
18
24
0
4
8
12
16
n = 9
n = 5
aab
b
c
a abb
c
on June 24, 2018 by guesthttp://jvi.asm
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A B
C D
VV
M
M
V
V
M
M
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0
5
10
15
20
25
0
2
4
6
8
10
12
14
Gro
wth
rate
(mm
/d)
Lesi
on le
ngth
(mm
)
a
b
cccd
deeefe-gf-hg-ig-ih-jh-jh-jh-kh-k
i-li-lj-lj-lj-mj-mj-mk-nk-n
k-nl-nl-nl-nm-om-om-ok-n
noopp
a
b
ccdcd
c-ec-e dedee
e e e e e e e e e e e e e e e e
e
e e e e e
e
e e e e
GP
72S
C30
GP
72S
C28
GP
72S
C18
GP
72S
C12
GP
72S
C09
GP
72S
C14
GP
72S
C08
GP
72S
C03
GP
72S
C13
GP
72S
C16
GP
72S
C22
GP
72S
C02
GP
72S
C31
GP
72S
C11
GP
72S
C01
GP
72S
C33
GP
72S
C06
GP
72S
C24
GP
72S
C04
GP
72S
C34
GP
72S
C21
GP
72S
C32
GP
72S
C15
GP
72S
C20
GP
72S
C35
GP
72S
C07
GP
72S
C05
GP
72S
C17
GP
72S
C10
GP
72S
C29
GP
72S
C25
GP
72S
C23
GP
72S
C27
GP
72S
C26
Gar
licB
c-16
Gar
licB
c-72
GP
72S
C19
dsRNA-1dsRNA-2
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
--
--
n = 9
n = 5
A
B
on June 24, 2018 by guesthttp://jvi.asm
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ownloaded from
A
B
0
2
4
6
8
10
12
GarlicB
c-38
38T
GP72SC35
72-35
-1
Gro
wth
rate
(mm
/d)
0
3
6
9
12
15
18
GarlicB
c-38
38T
GP72SC35
72-35
-1
Lesi
on le
ngth
(mm
)
****
****
dsRNA-dsRNA+
C dsRNA-dsRNA+
D
on June 24, 2018 by guesthttp://jvi.asm
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ownloaded from
Table 1. The origin of strains/isolates of Botrytis porri used in this study
Strain/isolate Origin (host, place and isolation time ) Pathogenicity Reference
GarlicBc-72 Allium sativum, Zhushan, China, 2008 Hypovirulent This study
OnionBc-95 Allium fistulosum, Qianjiang, China, 2008 Virulent Zhang et al., 2010
GarlicBc-38 Allium sativum, Laifeng, China, 2007 Virulent Zhang et al., 2010
GarlicBc-16 Allium sativum, Zhushan, China, 2007 Virulent Zhang et al., 2009
GP72SC35 A single-conidium isolate of GarlicBc-72,
2009
Virulent This study
72-35-1 GP72SC35 in a pairing-culture of
GP72SC35 and GarlicBc-72, 2009
Hypovirulent This study
72-35-2 GP72SC35 in a pairing-culture of
GP72SC35 and GarlicBc-72, 2009
Hypovirulent This study
72-35-3 GP72SC35 in a pairing-culture of
GP72SC35 and GarlicBc-72, 2009
Hypovirulent This study
72-38-1 GarlicBc-38-1 in a pairing-culture of
GarlicBc-38-1 and GarlicBc-72, 2009
Virulent This study
72-38-2 GarlicBc-38-1 in a pairing-culture of
GarlicBc-38-1 and GarlicBc-72, 2009
Virulent This study
72-38-3 GarlicBc-38-1 in a pairing-culture of
GarlicBc-38-1 and GarlicBc-72, 2009
Virulent This study
72-95-1 OnionBc-95 in a pairing-culture of
OnionBc-95 and GarlicBc-72, 2009
Virulent This study
72-95-2 OnionBc-95 in a pairing-culture of
OnionBc-95 and GarlicBc-72, 2009
Virulent This study
72-95-3 OnionBc-95 in a pairing-culture of
OnionBc-95 and GarlicBc-72, 2009
Virulent This study
38T GarlicBc-38-1 infected by BpRV1, 2010 Hypovirulent This study
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TABLE 2. Summary of the results of a BLASTP search for RdRp encoded by ORF I on dsRNA-1 of Botrytis porri RNA virus 1 (BpRV 1).
a See the legend for definitions of the virus name abbreviations in FIGURE 4.
Virus a RdRp
size (aa)
RdRp_4 conserved region BLASTP GenBank
Acc. No. Motif (I-VIII) length (aa) % Identity Overlap Bit score E value
Totivirus (Totiviridae)
UmVH1 1820 1142–1530 389 23 132/506 154 1e-34 NC_003823
ScV-L-A 731 169–520 352 19 67/254 73.2 4e-10 J04692
ScV-L-BC 863 306–654 349 20 98/424 67.0 3e-08 U01060
Chrysovirus (Chrysoviridae)
HvV145S 1086 426–805 380 23 164/616 121 1e-24 AF297176
PcV 1117 453–832 380 23 145/563 115 1e-22 AF296339
FoCV1 858 360–738 379 21 133/527 99.8 4e-18 EF152346
AfV 1114 451–829 379 21 135/561 97.1 3e-17 FN178512
CnV1 962 442–822 381 20 99/412 89.4 5e-15 GQ290650
ACDACV 1087 427–806 380 21 121/474 91.7 1e-15 NC_009947
VcV 1108 443–823 381 20 122/513 87.8 1e-14 HM004067
AbV1 1078 372–785 414 20 62/238 67.8 2e-08 X94361
CCRSCV 1087 427–806 380 21 64/282 60.5 3e-06 AJ781397
Victorivirus (Totiviridae)
Hm17V 845 243–594 352 21 115/469 87.0 3e-14 AB085814
SsRV1 838 241–589 349 22 74/256 79.0 8e-12 NC_001963
MoV1 845 227–575 353 20 98/407 78.2 1e-11 AB176964
GaVL1 825 221–573 353 20 103/427 68.6 1e-08 AF337175
BfTV1 838 242–585 344 21 57/219 67.4 3e-08 AM491608
HvV190S 835 241–585 345 21 64/241 59.7 5e-06 U41345
Megabirnavirus (Megabirnaviridae)
RnBMV1 1111 378–765 388 21 95/389 95.9 6e-17 AB512282
Unassigned
SFV1 1326 537–941 405 25 125/468 139 7e-30 GU979419
CiTV1 1338 550–953 404 23 145/591 108 6e-21 GU979420
PGV1 1414 669–1061 393 20 79/287 96.3 5e-17 AM111096
FgV3 1311 383–774 392 19 97/367 93.6 3e-16 NC_013469
DsRV1 1110 227–620 394 20 89/367 67.4 2e-08 NC_013699
PGV2 1153 230–624 395 18 87/374 66.2 5e-08 AM111097
LEV 1245 491–878 388 18 64/287 52.8 6e-04 AB429554
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