Molecular characterisation of integrated sequences of Banana streak virus in the banana plant genome.
HR 100%
The genome of banana(Musa sp.) harbours multiple integrationsof several species of Banana streak virus (BSV)known as endogenous pararetrovirus (EPRV) named eBSV in banana. Surprisingly, this pararetrovirus does not require integration for its replication. Some integrations, only existing in the Musa balbisiana genome (denoted B), are infectious by releasing virions in interspecific hybrids. Here we describe and analyze the organization of the integration for four BSV species(Goldfinger-BSGfV, Imové- BSImV, Mysore -BSMysVand Obinol’Ewai-BSOLV) present in the wild diploid M. balbisiana cv. Pisang Klutuk Wulung (PKW) where virus expression never occurred. This was undergone by studying both aMusa BAC libraryobtained from PKW and one interspecific genetic crossusing PKW as female parent.
Molecular characterisation of integrated sequences of Banana streak virus in the banana plant genome.
CLUSTER of viral integrants in PKW
eBSGfV(Gayral et al., July 2008 JVI vol 82 N°13 p6697-6710) : TWO types eBSImV: ONE type sofar
M. CHABANNES1, P. GAYRAL1, O. GUIDOLIN1, F-C. BAURENS2, S. SIDIBE BOCS2,
N. LABOUREAU1, M-L. ISKRA-CARUANA1.
1CIRAD, UMR BGPI, F-34398 Montpellier Cedex 5.
2CIRAD, UMR DAP, F-34398 Montpellier Cedex 5.
©Nathalie Le Gall
BSV
genomeMusa 15.8Kbp
•One and a half viral genome in continuity in the right orientation.
•Genotyping by PCR with 5 different markers of junctions plant-virus and virus-virus
ÎMonogenic Mendelian Segregation?
eBSImV BSImV+
(Diseased) BSImV- (Virus free) 74
68 N = 142
xOnly one eBSImV identified so far
xMono allelic?
eBSGfV-7 BAC 71C19
eBSGfV-9 BAC 94I16
Musa
genome 13.3Kbp 15.6Kbp
genomeMusa
Genotyping of both eBSVby 3 independent PCR methods : Subst., Indel, Structure
ÎMonogenic Mendelian Segregation
PKW (BB) x IDN110T (AAAA) 142 hybridsB(AA)
♀ X ♂ BB AAAA
BAA
+
eBSGfV-7 eBSGfV-9
53% 47%
eBSGfV-7 eBSGfV-9 BSGfV+
(Diseased) BSGfV- (Virus free)
17 0
58 67
N = 142
xWhich one is infectious?
•Virus detectionby IC-PCR
•Genotypingby nestedPCR-RFLP 97% Ntid
xDi allelic eBSV
eBSImV-1 BAC 68C24
PKW (BB) x IDN110T (AAAA) 142 hybridsB(AA)
♀ X ♂ BB AAAA
BAA
+
eBSImV eBSImV
eBSImV
48% 52%
23% 77%
7.5 Kbp
ORF1 ORF2
ORF3
IR (Intergenic region)
eBSGfV-7 eBSGfV-9
eBSMysV-1 BAC 29H14
eBSMysV-2 BAC 86I03
genomeMusa 11.3Kbp
genomeMusa
eBSMysV: TWO types
Sequencing in progress
eBSMysV-1seems not functional based on sequence analysis (a part of IntergenicRegion is never present in the integration)
PKW (BB) x IDN110T (AAAA) 142 hybridsB(AA)
♀ X ♂ BB AAAA
BAA
+
eBSMysV- 1 and/or 2 eBSMysV-1
eBSMysV-2 xPCR markers have to be
developed for the genotyping
eBSMysV- 1 and/or 2
xNo particles of BSMysV detected in the hybrid population
eBSOLV-1 BAC 31O07
eBSOLV-2 BAC 73B22
Musa genome
22.9 Kbp
23.2 Kbp
genomeMusa
eBSOLV: TWO types
•eBSOLV-1 could be functional based on sequence analysis (the whole viral genome is present)
•eBSOLV-2 seems not functional based on sequence analysis (Parts of IntergenicRegion and ORF3 are never present in the integration)
PKW (BB) x IDN110T (AAAA) 142 hybridsB(AA)
♀ X ♂ BB AAAA
BAA
+
eBSOLV-1 and/or 2 eBSOLV-1
ÎMonogenic Mendelian Segregation?
eBSOLV BSOLV+
(Diseased) BSOLV- (Virus free)
N = 142
xPCR markers have to be developped
for the genotyping xDi allelic?
52 % 48 %
eBSOLV-2
eBSOLV-1 and/or 2
xGenotyping : 5 PCR markers present in the whole hybrid population
xeBSGfV-7 is the only one to be infectious.
xOne heterozygous factor is required for eBSGfV-7 to become infectious.
ÎMonogenic Mendelian Segregation?
xDi allelic? ORF1ORF2
ORF3 eBSGfV-7
Empty site eBSGfV-7
eBSGfV-7
7.5 kpB HR
BSV Virion Musa balbisiana
eBSGfV-7
Musa balbisiana
eBSGfV-9
67 75
SILENT plant harbouring
eBSV
STRESS
(Geneticcross, Heatshock…)
INFECTED plant releasing BSV
The four eBSVdescribed in PKW genome suggest an allelic insertionresulting from a single integration event. Although we found only one eBSImVso far, the presence of the 5 PCR markers in the whole hybrid population suggest an allelic insertion too. Every eBSVis extensively rearrangedexcept for eBSImVwhere one and a half viral genome is present in continuity in the right orientation. In vivo validation of the infectious nature of each eBSVallele is under way. Finally, we are currently working on the mechanisms underlying EPRV activationby testing experimentally a model of activation based on homologous recombination(HR).