Articles
THE DEVELOPMENT OF GENETIC RESISTANCE TO PLUM POX VIRUS IN TRANSGENIC NICOTIANA BENTHAMIANA AND PRUNUS DOMESTICA
Article number
839_91
Pages
665 – 672
Language
English
Abstract
Plum pox virus (PPV) is a member of the genus Potyvirus in the family Potyviridae. It is the causal agent of the most devastating viral disease on many stone-fruit spp.
The viral disease, known as Sharka in Europe, was recently found in the United States and Canada.
Genetic resistance is considered the most effective means to control PPV, but despite over 60 years of extensive screening for germplasm resistant to PPV, few resistant resources have been identified.
Here we report engineering genetic resistance against PPV through the hairpin-mediated RNA silencing (RNAi) approach.
Two highly conserved regions of the PPV genome corresponding to portions of viral RNA coding for P1 and CP, respectively, were cloned into a plant transformation vector under the control of the double Cauliflower mosaic virus 35S promoter as inverted repeats spanned by an intron from the peach endo-polygalacturonase genomic DNA. These two constructs (pAWp1 and pAWcp) were initially tested in Nicotiana benthamiana. Over 50 transgenic lines transformed with pAWp1, pAWcp and an empty plasmid pAWck (as a control) were generated.
The transgenic N. benthamiana plants were mechanically inoculated with PPV. ELISA and RT-PCR analysis revealed that 49.1% of pAWp1-derived lines and 41.2% of pAWcp-derived lines were PPV-negative and 100% of control lines (transformed with pAWck) showed PPV-positive.
The pAWp1 plasmid was used to transform plum (Prunus domestica L.). Transgenic plum plants were challenged with PPV by chip-bud inoculation.
PPV was detected in all the pAWck-derived plum lines but was not detectable in 50% of pAWp1-derived transgenic lines.
Two species of PPV-specific small interfering RNAs, a hallmark of posttranscriptional gene silencing (PTGS), were present in the resistant plum lines, suggesting PTGS is responsible for resistance.
The PPV-resistant transgenic plum generated in this study may provide an alternative resistant source for the control of PPV.
The viral disease, known as Sharka in Europe, was recently found in the United States and Canada.
Genetic resistance is considered the most effective means to control PPV, but despite over 60 years of extensive screening for germplasm resistant to PPV, few resistant resources have been identified.
Here we report engineering genetic resistance against PPV through the hairpin-mediated RNA silencing (RNAi) approach.
Two highly conserved regions of the PPV genome corresponding to portions of viral RNA coding for P1 and CP, respectively, were cloned into a plant transformation vector under the control of the double Cauliflower mosaic virus 35S promoter as inverted repeats spanned by an intron from the peach endo-polygalacturonase genomic DNA. These two constructs (pAWp1 and pAWcp) were initially tested in Nicotiana benthamiana. Over 50 transgenic lines transformed with pAWp1, pAWcp and an empty plasmid pAWck (as a control) were generated.
The transgenic N. benthamiana plants were mechanically inoculated with PPV. ELISA and RT-PCR analysis revealed that 49.1% of pAWp1-derived lines and 41.2% of pAWcp-derived lines were PPV-negative and 100% of control lines (transformed with pAWck) showed PPV-positive.
The pAWp1 plasmid was used to transform plum (Prunus domestica L.). Transgenic plum plants were challenged with PPV by chip-bud inoculation.
PPV was detected in all the pAWck-derived plum lines but was not detectable in 50% of pAWp1-derived transgenic lines.
Two species of PPV-specific small interfering RNAs, a hallmark of posttranscriptional gene silencing (PTGS), were present in the resistant plum lines, suggesting PTGS is responsible for resistance.
The PPV-resistant transgenic plum generated in this study may provide an alternative resistant source for the control of PPV.
Authors
A. Wang, L. Tian, T.-S. Huang, D.C.W. Brown, A.M. Svircev, L.W. Stobbs, B. Miki, H. Sanfaçon
Keywords
PPV, Sharka, gene silencing, transgenic plum, small interfering RNA, hairpin RNA, PTGS
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