ISSN 10227954, Russian Journal of Genetics, 2015, Vol. 51, No. 7, pp. 715–719. © Pleiades Publishing, Inc., 2015.
Original Russian Text © E.A. Trifonova, A.V. Saveleva, A.V. Romanova, E.A. Filipenko, M.V. Sapotsky, V.I. Malinovsky, A.V. Kochetov, V.K. Shumny, 2015, published
in Genetika, 2015, Vol. 51, No. 7, pp. 835–840.
nuclease is a
nonspecific endonuclease, which cleaves DNA as
well as RNA, doublestranded or singlestranded, to
produce 5' phosphorylated (mono), di, tri and tet
ranucleotides. The protein is very stable towards ther
mal and chemical denaturation, active from 6 to 10 pH
with an optimum pH of 8–8.5, and requires Mg
cofactor . Its antiviral activity was shown in honey
bees and silkworms [2–4]; in plants the enzyme
increased the yield of virusfree potato .
Higher plants accumulate several kinds of patho
genesisrelated (PR) proteins in response to infection
by pathogens such as fungi or viruses . Currently,
there are distinguished 17 classes of the proteins,
mainly performing hydrolytic or inhibitory functions
[7, 8]. Recently it has been shown that the PR4 and
PR10 proteins are nucleases with DNase and RNase
activity located extracellularly [9, 10], and their anti
viral activity has been suggested in several conditions
[9, 11], so the
nuclease might be inter
preted as a functional analogue of the PR proteins. In
this paper we present data that transgenic plants pro
ducing chimeric, mutant, and intracellular mutant
variants of the
nuclease are more resis
tant to tobacco mosaic virus (TMV). The resistance
was assessed using the levels of expression of the dis
ease symptoms, and the levels of accumulation of the
virus antigen in leaves.
The article was translated by the authors.
extracellular nuclease gene  was
isolated from bacterial genomic DNA by polymerase
chain reaction (PCR). Primer sequences were chosen
from the known nucleotide sequence of the gene (acc.
no. GenBank M19495): SMNf (5'ATCGATGCGC
TTTAA CAACA AGAT3') and SMNr (5'TGGATC
CGTCAGTTTTTGCAGCCC3'). The amplified
DNA fragment was cloned in the pGEM TEasy
The chimeric nuclease gene included nucleotide
extracellular nuclease gene
without secretory peptide sequence and
secretory ribonuclease nucleotide sequence (acc.
no. GenBank U19924). The chimeric gene was con
structed by PCR splicing by overlap extension (SOE
ing) with primers: ZRNf (5'CACCATGAAGATG
CAATCCAACTATCC3'), SOE2 (5'AGCGT
GTCGGCCGCGGATAAA3'), and SOE3 (5'
TTTATCCGCGGCCGACACGCT3'). The resulting
chimeric nucleotide sequence was cloned in the
pENTR/DTOPO vector using pENTR/DTOPO
cloning kit (Invitrogen).
It was shown that one amino acid replacement
nuclease hydrolytically inactive . We have
changed the nucleotide sequence of
nuclease gene by twosided PCR splicing by overlap
extension and created mutant gene that cannot
hydrolyze DNA and RNA but still can bind them.
Transgenic Expression of
Native and Mutant
Nucleases Modulates Tobacco Mosaic Virus Resistance
E. A. Trifonova
, A. V. Saveleva
, A. V. Romanova
, E. A. Filipenko
, M. V. Sapotsky
V. I. Malinovsky
, A. V. Kochetov
, and V. K. Shumny
Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, 630090 Russia
Institute of Biology and Soil Science, Russian Academy of Sciences, Vladivostok, 690022 Russia
Department of Cytology and Genetics, Novosibirsk State University, Novosibirsk, 630090 Russia
Received August 19, 2014; in final form, October 10, 2014
nuclease is an extremely active enzyme which nonspecifically
degrades RNA and DNA. Its antiviral activity was previously shown both in animals and in plants when
applied exogenously. Transgenic tobacco plants (
L. cv. SR1) expressing
meric, mutant, and intracellular mutant nuclease gene variants were regenerated and challenged with
tobacco mosaic virus. The transgenic plants exhibited a higher level of resistance to the virus infection than
the control nontransgenic plants. The resistance was evidenced by the delay of the appearance of mosaic
symptoms and the retarded accumulation of viral antigen. Thus, these results reveal that modulations of both
extracellular nuclease activity and intracellular RNA/DNA binding can protect plants against viral diseases.