AEM Accepted Manuscript Posted Online 16 January 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.03992-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Title: Molecular and Biological Characterization of a Novel Hypovirulence-Associated
2
RNA Mycovirus in the Plant Pathogenic Fungus Botrytis cinerea
3
Lin Yu,a Wen Sang,b Ming-De Wu,a Jing Zhang,a Long Yang,a Ying-Jun Zhou,a Wei-Dong
4
Chen,c Guo-Qing Lia*
5
The State Key Laboratory of Agricultural Microbiology and The Key Laboratory of Plant
6
Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, Chinaa;
7
Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, Institute
8
of Insect Resources, Huazhong Agricultural University, Wuhan 430070, Chinab; and United
9
States Department of Agriculture, Agricultural Research Service, Washington State University,
10
Pullman, WA, USAc
11
*Corresponding author: Dr. Guo-Qing Li, E-mail address: [email protected]
12
Running title: A novel RNA mycovirus in B. cinerea
13 14
MS information:
15
Abstract: 218 words; Main text (excluding abstracts, key words, acknowledgements, references,
16
tables and figure legends): 7,188 words; Figures, 9; Tables, 2; Supplemental material includes
17
one table and seven figures.
18 19 20 21 1
22
ABSTRACT Botrytis cinerea is a pathogenic fungus causing gray mold on numerous
23
economically important crops and ornamental plants. This study was conducted to characterize
24
the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1
25
(BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp
26
long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a
27
hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A
28
-1 frameshifting region (designated as the KNOT element) containing a shifty heptamer, a
29
heptanucleotide spacer and an H-type pseudoknot, was predicted in the junction region of
30
ORF1 and ORF2. The -1 frameshifting role of the KNOT element was experimentally
31
confirmed through determining production of the fusion protein RFP-GFP by the plasmid
32
containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a
33
taxonomically unassigned dsRNA mycovirus group. It is closely related to
34
Grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in
35
strain BerBc-1 was found capable of being transmitted vertically through macroconidia and
36
horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was
37
found to be positively correlated with hypovirulence in B. cinerea with the attenuation effects
38
of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation
39
level of BcRV1.
40
Keywords: Botrytis cinerea; hypovirulence; BcRV1/BerBc-1; dsRNA; mycovirus; -1
41
frameshifting.
42 2
43 44
INTRODUCTION Mycoviruses or fungal viruses are viruses infecting filamentous fungi and yeasts (1, 2).
45
Most mycoviruses reported so far are either positive single-stranded RNA (+ssRNA) viruses,
46
including mycoviruses in the families Hypoviridae and Narnaviridae, or double-stranded RNA
47
(dsRNA) viruses, including mycoviruses in the families Chrysoviridae, Megabirnaviridae,
48
Partitiviridae, Reoviridae and Totiviridae (1, 3). Recently, mycoviruses with negative
49
single-stranded RNA genomes (-ssRNA) were reported (4, 5). Moreover, Yu et al. (6) reported
50
a single-stranded DNA (ssDNA) mycovirus, namely Sclerotinia sclerotiorum
51
hypovirulence-associated DNA virus 1 (SsHADV-1), in Sclerotinia sclerotiorum, the causal
52
agent of Sclerotinia stem rot of oilseed rape (Brassica napus).
53
Mycoviruses are widespread in all major taxonomic groups of fungi including many plant
54
pathogens (2). In most cases, mycovirus infection appears symptomless on the host fungi,
55
usually called latent or cryptic infection (1). However, infection by some mycoviruses in the
56
families Hypoviridae, Megabirnaviridae, Narnaviridae, Partitiviridae and Reoviridae or by the
57
unassigned -ssRNA and ssDNA mycoviruses can cause visible abnormal symptoms on the host
58
fungi, including reduced mycelial growth, reduced production of spores and/or sclerotia,
59
suppressed biosynthesis of secondary metabolites, and attenuated aggressiveness or virulence
60
(2, 3, 5–7). Some hypovirulence-causing mycoviruses have been reported to be promising
61
agents for control of plant pathogenic fungi such as the +ssRNA mycovirus Cryphonectria
62
hypovirus 1 (CHV1) against Cryphonectria parasitica (8, 9), the causal agent of chestnut
63
blight, and the ssDNA mycovirus SsHADV-1 against S. sclerotiorum (6, 10). Moreover, 3
64
detailed studies on interactions between mycoviruses and fungal hosts can provide novel
65
insights into molecular mechanisms involved in pathogenesis of plant pathogenic fungi (7).
66
Botrytis cinerea Pers.: Fr. [teleomorph: Botryotinia fuckeliana (de Bary) Whetzel] is a
67
ubiquitous phytopathogenic fungus causing gray mold disease. It infects leaves, stems,
68
blossoms, and/or fruits of more than 200 plant species, including ornamentals (e.g. carnation,
69
rose), vegetables (e.g. tomato, cucumber), fruits (e.g. grapes, strawberry) and some field crops
70
(e.g. oilseed rape), resulting in substantial economic losses (11). Given the importance of B.
71
cinerea and the problems of fungicide resistance and residues, the possibility of using
72
mycoviruses as biological control agents has attracted the interests of many researchers (12,
73
13).
74
Mycoviruses are common in B. cinerea (14–24). A few RNA mycoviruses in B. cinerea
75
have been sequenced (17, 18, 20, 23, 24). They include +ssRNA mycoviruses, such as Botrytis
76
cinerea mitovirus 1 (BcMV1, GenBank Acc. No. EF580100), Botrytis virus F (BVF,
77
AF238884) and Botrytis virus X (BVX, AY055762), and dsRNA mycoviruses, such as Botrytis
78
cinerea CCg378 virus 1 (Bc378V1, KF201714), Botryotinia fuckeliana totivirus 1 (BfTV1,
79
AM491608) and Botryotinia fuckeliana partitivirus 1 (BfPV1, AM491609 and AM 491610).
80
Among these mycoviruses, BcMV1 is closely associated with hypovirulence of B. cinerea (23,
81
24). Moreover, Xiao et al. (25) reported that the dsRNA mycovirus Sclerotinia sclerotiorum
82
partitivirus 1 (SsPV1) in strain WF-1 of S. sclerotiorum can infect B. cinerea, resulting in
83
reduced virulence, suppressed mycelial growth and inhibited elongation of conidial germ tubes.
84
Previous studies showed that vegetative incompatibility is an obstacle to the application of 4
85
mycoviruses as biological control agents in controlling plant fungal diseases, as it restricts
86
transmission of mycoviruses from mycovirus-infected strains to mycovirus-free strains through
87
hyphal contact or anastomosis (8, 9). Vegetative incompatibility has been detected in
88
populations of B. cinerea (26, 27). Wu et al. (23, 24) reported that through hyphal contact,
89
BcMV1 could be transmitted to single-conidium virulent isolates of B. cinerea strain CanBc-1,
90
from which BcMV1 was originally isolated, but could not be transmitted to a different virulent
91
strain CanBc-2 of B. cinerea. Therefore, screening of mycoviruses capable of overcoming
92
hyphal incompatibility in transmission is a prerequisite for applications of mycoviruses to
93
control B. cinerea. Previous studies also showed that hyphal incompatibility can not restrict
94
horizontal transmission of the mycoviruses in C. parasitica and the basidiomycetous fungus
95
Heterobasidion annosums (28–30).
96
A hypovirulent strain of B. cinerea designated as strain BerBc-1 was isolated from
97
Berberis sp. in Wuhan of China. A dsRNA element of approximately 10 kb in size was detected
98
in strain BerBc-1, hereby designated as Botrytis cinerea RNA virus 1 (BcRV1). According to
99
the recent review by Pearson and Bailey (13), dsRNA elements or mycoviruses in B. cinerea
100
with such large size have not been previously characterized either at the biological level or at
101
the molecular level (14, 15, 17, 18, 20, 23, 24). It might represent the genome of a novel
102
mycovirus in B. cinerea. Therefore, we conducted this study to fulfill the following three
103
objectives: (i) to characterize the molecular properties of BcRV1; (ii) to determine the
104
transmissibility of BcRV1; and (iii) to investigate the effects of BcRV1 on pathogenicity of B.
105
cinerea. 5
106
MATERIALS AND METHODS
107
Fungal strains.
108
were isolated in Wuhan of China from Berberis sp. in 2008, Lagenaria siceraria in 2009 and
109
Rosa chinensis in 2004, respectively. They were stored in 20% (v/v) glycerol solution at
110
-80 °C.
111
Strains BerBc-1, LagBc-1 and RoseBc-3 of B. cinerea used in this study
Extraction and identification of dsRNA.
Mycelia of each strain of B. cinerea were
112
collected from 3-day-old cultures (20 °C) on autoclaved cellophane films placed on PDA in
113
Petri dishes, and stored at -80 °C until use. DsRNA was extracted and purified from the
114
mycelia using the procedures described by Wu et al. (23), and detected by agarose gel (1%, w/v)
115
electrophoresis (23). The nature of the dsRNA was confirmed by digestion of the extracts with
116
RNase A (TaKaRa Biotechnology Co., Ltd., Dalian, China), RQ1 RNase-free DNase (Promega,
117
Madison, USA) and S1 nuclease (TaKaRa) (23, 24, 31). The molecules that can be digested by
118
RNase A, but not by DNase and S1 nuclease were considered to be dsRNAs (23, 24, 31).
119
Extraction of total RNA and genomic DNA.
Total RNA was extracted from 3-day-old
120
mycelia (20 °C) of each strain of B. cinerea using the RNAiso Plus kit (TaKaRa) following the
121
procedures recommended by the manufacturer. It was purified by removing the contaminating
122
DNA with RQ1 RNase-free DNase (Promega). Concentration of the purified RNA (1.8
6595
HP
RdRp
>765
ND
ND
GAAAAAC
7
+
SsNsV-L (JQ513382)
9124
HP
RdRp
1088
54
+
GAAAAAC
14
+
FgV3 (GQ140626)
9098
HP
RdRp
865
44
+
GAAAAAC
2
+
FvV1 (JN671444)
9402
SP
RdRp
1267
45
+
AAAAAAC
43
+
FvV2 (JN671443)
9327
SP
RdRp
1043
131
+
AAAAAAC
24
+
PgV2 (AM111097)
>8727
HP
RdRp
ND
47
+
GGAAAAC
6
+
PiRV3 (JN603241)
8112
HP
RdRp
848
65
–
GUUAAAC
12
+
DsRV1 (EU547739)
5018
HP
RdRp
29
186
+
–
–
–
Abbreviations: ORF, open reading frame; ND, not detected due to the incomplete genome sequence; HP, hypothetical protein; SP, structural protein; See Figure 5 for abbreviations of the virus names.
824 825
Virus (GenBank Acc. No.) a
b
The symbols “+” and “–” indicate the presence and absence of certain structure, respectively.
41
1
TABLE 1 Summary of the BLASTP search results of the polypeptide P1 encoded by ORF1 and
2
RNA-dependent RNA polymerase (RdRp) encoded by ORF2 of Botrytis cinerea RNA virus 1
3
(BcRV1) Virus (GenBank Acc. No.) a
Size (aa)
% Identity
Overlap
Bit score
E-value
GaTV2 (ADO60932)
1313
93
1227/1314
2439
0.0
SsNsV-L (YP_006331064)
1305
77
1018/1317
2085
0.0
FgV3 (YP_003288788)
1369
45
587/1304
1040
0.0
FvV1 (AEZ54147)
1311
31
327/1054
378
7e-107
FvV2 (AEZ54145)
1347
32
253/794
358
1e-99
PgV2 (CAJ34334)
1696
26
112/427
109
5e-21
PiRV3 (AEX87901)
1296
22
124/556
71.2
2e-09
SsNsV-L (YP_006331065)
1338
71
956/1338
1961
0.0
GaTV2 (ADO60933) b
> 613
93
571/613
1162
0.0
FgV3 (ACY56323)
1311
45
592/1327
1067
0.0
FvV1 (AEZ54148)
1289
33
412/1266
568
8e-176
FvV2 (AEZ54146)
1310
33
426/1274
565
3e-174
DsRV1 (ACD91658)
1110
31
329/1064
410
4e-119
PgV2 (CAJ34335)
1153
37
230/614
341
5e-95
PiRV3 (AEX87902)
1011
32
193/605
249
2e-65
P1
RdRp
4
a
See Figure 5 for abbreviations of the virus names.
5
b
The RdRp sequence of GaTV2 is incomplete in the NCBI database.
1
2
TABLE 2 Comparison of Botrytis cinerea RNA virus 1 (BcRV1) with related viruses in genome size and other putative structures
a
Genome (bp)
ORF1
ORF2
5’ UTR (bp)
3’ UTR (bp)
S7
Shifty heptamer
Spacer (nt)
Pseudoknot
BcRV1 (KJ549662)
8952
HP a
RdRp
878
66
+b
GAAAAAC
7
+
GaTV2 (GU108594)
>6595
HP
RdRp
>765
ND
ND
GAAAAAC
7
+
SsNsV-L (JQ513382)
9124
HP
RdRp
1088
54
+
GAAAAAC
14
+
FgV3 (GQ140626)
9098
HP
RdRp
865
44
+
GAAAAAC
2
+
FvV1 (JN671444)
9402
SP
RdRp
1267
45
+
AAAAAAC
43
+
FvV2 (JN671443)
9327
SP
RdRp
1043
131
+
AAAAAAC
24
+
PgV2 (AM111097)
>8727
HP
RdRp
ND
47
+
GGAAAAC
6
+
PiRV3 (JN603241)
8112
HP
RdRp
848
65
–
GUUAAAC
12
+
DsRV1 (EU547739)
5018
HP
RdRp
29
186
+
–
–
–
Abbreviations: ORF, open reading frame; ND, not detected due to the incomplete genome sequence; HP, hypothetical protein; SP, structural protein; See Figure 5 for abbreviations of the virus names.
3 4
Virus (GenBank Acc. No.) a
b
The symbols “+” and “–” indicate the presence and absence of certain structure, respectively.
1
Title: Molecular and Biological Characterization of a Novel Hypovirulence-Associated
2
RNA Mycovirus in the Plant Pathogenic Fungus Botrytis cinerea
3
Lin Yu,a Wen Sang,b Ming-De Wu,a Jing Zhang,a Long Yang,a Ying-Jun Zhou,a Wei-Dong
4
Chen,c Guo-Qing Lia*
5
The State Key Laboratory of Agricultural Microbiology and The Key Laboratory of Plant
6
Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, Chinaa;
7
Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, Institute
8
of Insect Resources, Huazhong Agricultural University, Wuhan 430070, Chinab; and United
9
States Department of Agriculture, Agricultural Research Service, Washington State University,
10
Pullman, WA, USAc
11
*Corresponding author: Dr. Guo-Qing Li, E-mail address: [email protected]
12
Running title: A novel RNA mycovirus in B. cinerea
13 14
MS information:
15
Abstract: 218 words; Main text (excluding abstracts, key words, acknowledgements, references,
16
tables and figure legends): 7,188 words; Figures, 9; Tables, 2; Supplemental material includes
17
one table and seven figures.
18 19 20 21 1
22
ABSTRACT Botrytis cinerea is a pathogenic fungus causing gray mold on numerous
23
economically important crops and ornamental plants. This study was conducted to characterize
24
the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1
25
(BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp
26
long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a
27
hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A
28
-1 frameshifting region (designated as the KNOT element) containing a shifty heptamer, a
29
heptanucleotide spacer and an H-type pseudoknot, was predicted in the junction region of
30
ORF1 and ORF2. The -1 frameshifting role of the KNOT element was experimentally
31
confirmed through determining production of the fusion protein RFP-GFP by the plasmid
32
containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a
33
taxonomically unassigned dsRNA mycovirus group. It is closely related to
34
Grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in
35
strain BerBc-1 was found capable of being transmitted vertically through macroconidia and
36
horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was
37
found to be positively correlated with hypovirulence in B. cinerea with the attenuation effects
38
of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation
39
level of BcRV1.
40
Keywords: Botrytis cinerea; hypovirulence; BcRV1/BerBc-1; dsRNA; mycovirus; -1
41
frameshifting.
42 2
43 44
INTRODUCTION Mycoviruses or fungal viruses are viruses infecting filamentous fungi and yeasts (1, 2).
45
Most mycoviruses reported so far are either positive single-stranded RNA (+ssRNA) viruses,
46
including mycoviruses in the families Hypoviridae and Narnaviridae, or double-stranded RNA
47
(dsRNA) viruses, including mycoviruses in the families Chrysoviridae, Megabirnaviridae,
48
Partitiviridae, Reoviridae and Totiviridae (1, 3). Recently, mycoviruses with negative
49
single-stranded RNA genomes (-ssRNA) were reported (4, 5). Moreover, Yu et al. (6) reported
50
a single-stranded DNA (ssDNA) mycovirus, namely Sclerotinia sclerotiorum
51
hypovirulence-associated DNA virus 1 (SsHADV-1), in Sclerotinia sclerotiorum, the causal
52
agent of Sclerotinia stem rot of oilseed rape (Brassica napus).
53
Mycoviruses are widespread in all major taxonomic groups of fungi including many plant
54
pathogens (2). In most cases, mycovirus infection appears symptomless on the host fungi,
55
usually called latent or cryptic infection (1). However, infection by some mycoviruses in the
56
families Hypoviridae, Megabirnaviridae, Narnaviridae, Partitiviridae and Reoviridae or by the
57
unassigned -ssRNA and ssDNA mycoviruses can cause visible abnormal symptoms on the host
58
fungi, including reduced mycelial growth, reduced production of spores and/or sclerotia,
59
suppressed biosynthesis of secondary metabolites, and attenuated aggressiveness or virulence
60
(2, 3, 5–7). Some hypovirulence-causing mycoviruses have been reported to be promising
61
agents for control of plant pathogenic fungi such as the +ssRNA mycovirus Cryphonectria
62
hypovirus 1 (CHV1) against Cryphonectria parasitica (8, 9), the causal agent of chestnut
63
blight, and the ssDNA mycovirus SsHADV-1 against S. sclerotiorum (6, 10). Moreover, 3
64
detailed studies on interactions between mycoviruses and fungal hosts can provide novel
65
insights into molecular mechanisms involved in pathogenesis of plant pathogenic fungi (7).
66
Botrytis cinerea Pers.: Fr. [teleomorph: Botryotinia fuckeliana (de Bary) Whetzel] is a
67
ubiquitous phytopathogenic fungus causing gray mold disease. It infects leaves, stems,
68
blossoms, and/or fruits of more than 200 plant species, including ornamentals (e.g. carnation,
69
rose), vegetables (e.g. tomato, cucumber), fruits (e.g. grapes, strawberry) and some field crops
70
(e.g. oilseed rape), resulting in substantial economic losses (11). Given the importance of B.
71
cinerea and the problems of fungicide resistance and residues, the possibility of using
72
mycoviruses as biological control agents has attracted the interests of many researchers (12,
73
13).
74
Mycoviruses are common in B. cinerea (14–24). A few RNA mycoviruses in B. cinerea
75
have been sequenced (17, 18, 20, 23, 24). They include +ssRNA mycoviruses, such as Botrytis
76
cinerea mitovirus 1 (BcMV1, GenBank Acc. No. EF580100), Botrytis virus F (BVF,
77
AF238884) and Botrytis virus X (BVX, AY055762), and dsRNA mycoviruses, such as Botrytis
78
cinerea CCg378 virus 1 (Bc378V1, KF201714), Botryotinia fuckeliana totivirus 1 (BfTV1,
79
AM491608) and Botryotinia fuckeliana partitivirus 1 (BfPV1, AM491609 and AM 491610).
80
Among these mycoviruses, BcMV1 is closely associated with hypovirulence of B. cinerea (23,
81
24). Moreover, Xiao et al. (25) reported that the dsRNA mycovirus Sclerotinia sclerotiorum
82
partitivirus 1 (SsPV1) in strain WF-1 of S. sclerotiorum can infect B. cinerea, resulting in
83
reduced virulence, suppressed mycelial growth and inhibited elongation of conidial germ tubes.
84
Previous studies showed that vegetative incompatibility is an obstacle to the application of 4
85
mycoviruses as biological control agents in controlling plant fungal diseases, as it restricts
86
transmission of mycoviruses from mycovirus-infected strains to mycovirus-free strains through
87
hyphal contact or anastomosis (8, 9). Vegetative incompatibility has been detected in
88
populations of B. cinerea (26, 27). Wu et al. (23, 24) reported that through hyphal contact,
89
BcMV1 could be transmitted to single-conidium virulent isolates of B. cinerea strain CanBc-1,
90
from which BcMV1 was originally isolated, but could not be transmitted to a different virulent
91
strain CanBc-2 of B. cinerea. Therefore, screening of mycoviruses capable of overcoming
92
hyphal incompatibility in transmission is a prerequisite for applications of mycoviruses to
93
control B. cinerea. Previous studies also showed that hyphal incompatibility can not restrict
94
horizontal transmission of the mycoviruses in C. parasitica and the basidiomycetous fungus
95
Heterobasidion annosums (28–30).
96
A hypovirulent strain of B. cinerea designated as strain BerBc-1 was isolated from
97
Berberis sp. in Wuhan of China. A dsRNA element of approximately 10 kb in size was detected
98
in strain BerBc-1, hereby designated as Botrytis cinerea RNA virus 1 (BcRV1). According to
99
the recent review by Pearson and Bailey (13), dsRNA elements or mycoviruses in B. cinerea
100
with such large size have not been previously characterized either at the biological level or at
101
the molecular level (14, 15, 17, 18, 20, 23, 24). It might represent the genome of a novel
102
mycovirus in B. cinerea. Therefore, we conducted this study to fulfill the following three
103
objectives: (i) to characterize the molecular properties of BcRV1; (ii) to determine the
104
transmissibility of BcRV1; and (iii) to investigate the effects of BcRV1 on pathogenicity of B.
105
cinerea. 5
106
MATERIALS AND METHODS
107
Fungal strains.
108
were isolated in Wuhan of China from Berberis sp. in 2008, Lagenaria siceraria in 2009 and
109
Rosa chinensis in 2004, respectively. They were stored in 20% (v/v) glycerol solution at
110
-80 °C.
111
Strains BerBc-1, LagBc-1 and RoseBc-3 of B. cinerea used in this study
Extraction and identification of dsRNA.
Mycelia of each strain of B. cinerea were
112
collected from 3-day-old cultures (20 °C) on autoclaved cellophane films placed on PDA in
113
Petri dishes, and stored at -80 °C until use. DsRNA was extracted and purified from the
114
mycelia using the procedures described by Wu et al. (23), and detected by agarose gel (1%, w/v)
115
electrophoresis (23). The nature of the dsRNA was confirmed by digestion of the extracts with
116
RNase A (TaKaRa Biotechnology Co., Ltd., Dalian, China), RQ1 RNase-free DNase (Promega,
117
Madison, USA) and S1 nuclease (TaKaRa) (23, 24, 31). The molecules that can be digested by
118
RNase A, but not by DNase and S1 nuclease were considered to be dsRNAs (23, 24, 31).
119
Extraction of total RNA and genomic DNA.
Total RNA was extracted from 3-day-old
120
mycelia (20 °C) of each strain of B. cinerea using the RNAiso Plus kit (TaKaRa) following the
121
procedures recommended by the manufacturer. It was purified by removing the contaminating
122
DNA with RQ1 RNase-free DNase (Promega). Concentration of the purified RNA (1.8
6595
HP
RdRp
>765
ND
ND
GAAAAAC
7
+
SsNsV-L (JQ513382)
9124
HP
RdRp
1088
54
+
GAAAAAC
14
+
FgV3 (GQ140626)
9098
HP
RdRp
865
44
+
GAAAAAC
2
+
FvV1 (JN671444)
9402
SP
RdRp
1267
45
+
AAAAAAC
43
+
FvV2 (JN671443)
9327
SP
RdRp
1043
131
+
AAAAAAC
24
+
PgV2 (AM111097)
>8727
HP
RdRp
ND
47
+
GGAAAAC
6
+
PiRV3 (JN603241)
8112
HP
RdRp
848
65
–
GUUAAAC
12
+
DsRV1 (EU547739)
5018
HP
RdRp
29
186
+
–
–
–
Abbreviations: ORF, open reading frame; ND, not detected due to the incomplete genome sequence; HP, hypothetical protein; SP, structural protein; See Figure 5 for abbreviations of the virus names.
824 825
Virus (GenBank Acc. No.) a
b
The symbols “+” and “–” indicate the presence and absence of certain structure, respectively.
41
1
TABLE 1 Summary of the BLASTP search results of the polypeptide P1 encoded by ORF1 and
2
RNA-dependent RNA polymerase (RdRp) encoded by ORF2 of Botrytis cinerea RNA virus 1
3
(BcRV1) Virus (GenBank Acc. No.) a
Size (aa)
% Identity
Overlap
Bit score
E-value
GaTV2 (ADO60932)
1313
93
1227/1314
2439
0.0
SsNsV-L (YP_006331064)
1305
77
1018/1317
2085
0.0
FgV3 (YP_003288788)
1369
45
587/1304
1040
0.0
FvV1 (AEZ54147)
1311
31
327/1054
378
7e-107
FvV2 (AEZ54145)
1347
32
253/794
358
1e-99
PgV2 (CAJ34334)
1696
26
112/427
109
5e-21
PiRV3 (AEX87901)
1296
22
124/556
71.2
2e-09
SsNsV-L (YP_006331065)
1338
71
956/1338
1961
0.0
GaTV2 (ADO60933) b
> 613
93
571/613
1162
0.0
FgV3 (ACY56323)
1311
45
592/1327
1067
0.0
FvV1 (AEZ54148)
1289
33
412/1266
568
8e-176
FvV2 (AEZ54146)
1310
33
426/1274
565
3e-174
DsRV1 (ACD91658)
1110
31
329/1064
410
4e-119
PgV2 (CAJ34335)
1153
37
230/614
341
5e-95
PiRV3 (AEX87902)
1011
32
193/605
249
2e-65
P1
RdRp
4
a
See Figure 5 for abbreviations of the virus names.
5
b
The RdRp sequence of GaTV2 is incomplete in the NCBI database.
1
2
TABLE 2 Comparison of Botrytis cinerea RNA virus 1 (BcRV1) with related viruses in genome size and other putative structures
a
Genome (bp)
ORF1
ORF2
5’ UTR (bp)
3’ UTR (bp)
S7
Shifty heptamer
Spacer (nt)
Pseudoknot
BcRV1 (KJ549662)
8952
HP a
RdRp
878
66
+b
GAAAAAC
7
+
GaTV2 (GU108594)
>6595
HP
RdRp
>765
ND
ND
GAAAAAC
7
+
SsNsV-L (JQ513382)
9124
HP
RdRp
1088
54
+
GAAAAAC
14
+
FgV3 (GQ140626)
9098
HP
RdRp
865
44
+
GAAAAAC
2
+
FvV1 (JN671444)
9402
SP
RdRp
1267
45
+
AAAAAAC
43
+
FvV2 (JN671443)
9327
SP
RdRp
1043
131
+
AAAAAAC
24
+
PgV2 (AM111097)
>8727
HP
RdRp
ND
47
+
GGAAAAC
6
+
PiRV3 (JN603241)
8112
HP
RdRp
848
65
–
GUUAAAC
12
+
DsRV1 (EU547739)
5018
HP
RdRp
29
186
+
–
–
–
Abbreviations: ORF, open reading frame; ND, not detected due to the incomplete genome sequence; HP, hypothetical protein; SP, structural protein; See Figure 5 for abbreviations of the virus names.
3 4
Virus (GenBank Acc. No.) a
b
The symbols “+” and “–” indicate the presence and absence of certain structure, respectively.
