Virus-induced silencing of sterol biosynthetic genes: identification of a Nicotiana tabacum L. obtusifoliol-14alpha-demethylase (CYP51) by genetic manipulation of the sterol biosynthetic pathway in Nicotiana benthamiana L.
Obtusifoliol-14alpha-demethylase (CYP51) is implicated in plant sterol biosynthesis. An Arabidopsis expressed sequence tag encoding a CYP51 was used as a probe to isolate Nicotiana tabacum L. cDNAs. Two types of cDNA clones were identified. Nt CYP51-1 and Nt CYP51-2 shared 97% identity together and around 75% with other plant CYP51s. The function of the encoded enzyme has been demonstrated in planta by manipulating the sterol biosynthetic pathway at the gene level. The endogenous CYP51 of Nicotiana benthamiana was silenced upon inoculation of the plantlets with POTATO VIRUS X::Nt CYP51-1 transcripts. This resulted in the accumulation of obtusifoliol, the substrate of CYP51, and other 14alpha-methyl sterols, with a concomitant growth reduction phenotype. Virus-induced gene silencing was also applied to another steroidogenic enzyme, the Delta7-sterol-C5(6)-desaturase, and this resulted in the accumulation of Delta7-sterols in infected plants instead of the pathway end-products Delta5-sterols.
Burger
Céline
C
Institut de Biologie Moléculaire des Plantes du CNRS, Département Isoprénoïdes, Institut de Botanique, 28 rue Goethe, F-67083 Strasbourg, France.
Rondet
Sabine
S
Benveniste
Pierre
P
Schaller
Hubert
H
D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010840:Phytosterols / Q000096:biosynthesis* / Q000737:chemistry; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D017863:Potexvirus / Q000254:growth & development; D017422:Sequence Analysis, DNA; D058886:Sterol 14-Demethylase; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000821:virology
D018076:DNA, Complementary; D010840:Phytosterols; D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase