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Inhibiting Virus Infection by RNA Interference of the Eight Functional Genes of the Potato Virus Y Genome
Author(s) -
Chen Xuemei,
Liu Jing,
Xu Li,
Jiang Fang,
Xie Xueying,
Zhu Changxiang,
Wen Fujiang
Publication year - 2010
Publication title -
journal of phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.53
H-Index - 60
eISSN - 1439-0434
pISSN - 0931-1785
DOI - 10.1111/j.1439-0434.2010.01701.x
Subject(s) - biology , complementary dna , tobacco etch virus , virology , gene , genetics , potato virus y , virus , genome , expression vector , microbiology and biotechnology , recombinant dna , plant virus , potyvirus
The genome of Potato virus Y is a single‐stranded, positive‐sense RNA molecule that encodes a single large polypeptide that is cleaved by self‐encoded proteases into 10 functional proteins. Using specific primers designed based on the cloned genome sequence of the necrotic strain of Potato virus Y (PVY N ), we amplified 400 bp and 500 bp cDNA fragments from the 3′ ends of P1, HC‐Pro, P3, CI, Vpg, NIa, NIb and CP genes. These cDNA fragments were then inserted into the binary vector pROKII to construct the vectors pROK‐P1, pROK‐HC‐Pro, pROK‐P3, pROK‐CI, pROK‐NIa‐Vpg, pROK‐NIa‐Pro, pROK‐NIb and pROK‐CP, all of which contained hairpin RNAs (hpRNAs). These recombinant binary vectors were introduced into Agrobacterium tumefaciens strain LBA4404 and then into Nicotiana tabacum L. var. NC89 plants, respectively. Virus challenge inoculation indicated that the plants transformed with each construct were resistant to PVY infection, and the percentage of resistant plants obtained ranged from 33–64%. Northern blot showed an inverse correlation between transgenic transcript accumulation and virus resistance. siRNAs could be detected in all the resistant plants. We also studied the relationship between the secondary structure and the gene‐silencing efficiency and found a positive correlation between local free energies (ΔG loc ) and the virus‐resistance ratio. For each construct, one line of progeny T 1 was randomly selected to analyse the inheritance of the transgene and the resistance. All transgenic single locus lines were completely resistant, and this resistance could be stably inherited.