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Use of RNAi technology to develop a PRSV-resistant transgenic papaya.

ABSTRACT: Papaya ringspot virus (PRSV) seriously limits papaya (Carica papaya L.) production in tropical and subtropical areas throughout the world. Coat protein (CP)- transgenic papaya lines resistant to PRSV isolates in the sequence-homology-dependent manner have been developed in the U.S.A. and Taiwan. A previous investigation revealed that genetic divergence among Hainan isolates of PRSV has allowed the virus to overcome the CP-mediated transgenic resistance. In this study, we designed a comprehensive RNAi strategy targeting the conserved domain of the PRSV CP gene to develop a broader-spectrum transgenic resistance to the Hainan PRSV isolates. We used an optimized particle-bombardment transformation system to produce RNAi-CP-transgenic papaya lines. Southern blot analysis and Droplet Digital PCR revealed that line 474 contained a single transgene insert. Challenging this line with different viruses (PRSV I, II and III subgroup) under greenhouse conditions validated the transgenic resistance of line 474 to the Hainan isolates. Northern blot analysis detected the siRNAs products in virus-free transgenic papaya tissue culture seedlings. The siRNAs also accumulated in PRSV infected transgenic papaya lines. Our results indicated that this transgenic papaya line has a useful application against PRSV in the major growing area of Hainan, China.

SUBMITTER: Jia R

PROVIDER: S-EPMC5626737 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Use of RNAi technology to develop a PRSV-resistant transgenic papaya.

Jia Ruizong R Zhao Hui H Huang Jing J Kong Hua H Zhang Yuliang Y Guo Jingyuan J Huang Qixing Q Guo Yunling Y Wei Qing Q Zuo Jiao J Zhu Yun J YJ Peng Ming M Guo Anping A

Scientific reports 20171003 1

Papaya ringspot virus (PRSV) seriously limits papaya (Carica papaya L.) production in tropical and subtropical areas throughout the world. Coat protein (CP)- transgenic papaya lines resistant to PRSV isolates in the sequence-homology-dependent manner have been developed in the U.S.A. and Taiwan. A previous investigation revealed that genetic divergence among Hainan isolates of PRSV has allowed the virus to overcome the CP-mediated transgenic resistance. In this study, we designed a comprehensive ...[more]

PMID: 28974762

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Project description:BackgroundThe safety of genetically transformed plants remains a subject of scrutiny. Genomic variants in PRSV resistant transgenic papaya will provide evidence to rationally address such concerns.ResultsIn this study, a total of more than 74 million Illumina reads for progenitor 'Sunset' were mapped onto transgenic papaya 'SunUp' reference genome. 310,364 single nucleotide polymorphisms (SNPs) and 34,071 small Inserts/deletions (InDels) were detected between 'Sunset' and 'SunUp'. Those variations have an uneven distribution across nine chromosomes in papaya. Only 0.27% of mutations were predicted to be high-impact mutations. ATP-related categories were highly enriched among these high-impact genes. The SNP mutation rate was about 8.4 × 10- 4 per site, comparable with the rate induced by spontaneous mutation over numerous generations. The transition-to-transversion ratio was 1.439 and the predominant mutations were C/G to T/A transitions. A total of 3430 nuclear plastid DNA (NUPT) and 2764 nuclear mitochondrial DNA (NUMT) junction sites have been found in 'SunUp', which is proportionally higher than the predicted total NUPT and NUMT junction sites in 'Sunset' (3346 and 2745, respectively). Among all nuclear organelle DNA (norgDNA) junction sites, 96% of junction sites were shared by 'SunUp' and 'Sunset'. The average identity between 'SunUp' specific norgDNA and corresponding organelle genomes was higher than that of norgDNA shared by 'SunUp' and 'Sunset'. Six 'SunUp' organelle-like borders of transgenic insertions were nearly identical to corresponding sequences in organelle genomes (98.18 ~ 100%). None of the paired-end spans of mapped 'Sunset' reads were elongated by any 'SunUp' transformation plasmid derived inserts. Significant amounts of DNA were transferred from organelles to the nuclear genome during bombardment, including the six flanking sequences of the three transgenic insertions.ConclusionsComparative whole-genome analyses between 'SunUp' and 'Sunset' provide a reliable estimate of genome-wide variations and evidence of organelle-to-nucleus transfer of DNA associated with biolistic transformation.

Dataset Information

Use of RNAi technology to develop a PRSV-resistant transgenic papaya.

Publications

Use of RNAi technology to develop a PRSV-resistant transgenic papaya.

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