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Molecular characterisation of integrated sequences of Banana streak virus in the banana plant genome.

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Molecular characterisation of integrated sequences of Banana streak virus in the banana plant genome.

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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).

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