ISSN 1022-7954, Russian Journal of Genetics, 2007, Vol. 43, No. 7, pp. 831–833. © Pleiades Publishing, Inc., 2007.
Original Russian Text © S.S. Sangaev, E.A. Trifonova, S.E. Titov, A.V. Romanova, Ya.S. Kolodyazhnaya, M.L. Komarova, M.V. Sapotsky, V.I. Malinovsky, A.V. Kochetov,
V.K. Shumny, 2007, published in Genetika, 2007, Vol. 43, No. 7, pp. 1002–1005.
Analysis of the pathogen-resistance mechanisms
lead to the understanding that extracellular space com-
ponents played the key role in the interaction between
the plants and pathogens . It is suggested that extra-
cellular ribonucleases, recently described in plants, and
induced in response to wounding, can be the compo-
nents of this system [2, 3].
In the extracellular space, extracellular RNases
demonstrate complex proﬁles of specialized activities.
For instance, S-RNases of gametophytic self-incompat-
ible plants from the families Solanaceae, Rosaceae, and
Scrophulariaceae, have the recognition function, and at
the same time, are cytotoxic agents. These properties
provide allele-speciﬁc inhibition of the pollen tube
growth, thereby, preventing inbreeding . Extracellu-
lar S-like RNases are involved in the defense activity
against fungal pathogens [5, 6].
We have suggested that at the early stages of infec-
tion, wounding-induced extracellular plant RNases
served as active components of virus-defense mecha-
nism . At some stages of infection, extracellular
RNases can directly hydrolyze viral genomic RNA. On
the other hand, it is possible that extracellular RNases
penetrate into infection- or wounding-damaged cells,
causing death of the virus and host cell.
It was reported that adding of active RNase A to the
inoculum substantially decreases the plant damage
upon infection of the tobacco leaves by tobacco mosaic
virus by means of either mechanical injury . Rapid
induction of extracellular S-like RNases in response to
wounding was demonstrated in
Nicotiana glutinosa, Zinnia elegans,
[2, 8–10]. In
ing induced the activity of extracellular RNase RNS1,
as well as at least of three more nucleases. Furthermore,
RNS1 was detected not only at the site of wounding
(local), but also in distant uninjured tissues (systemic).
Systemic induction of RNS1 in response to wounding
suggests that this RNase can participate in the phyto-
pathogen defense activity . In
NGR3 induced by TMV inoculation was described .
Unlike wounding-induced RNases, this RNase was
induced after 48 h upon wounding.
Construction of transgenic plants with altered activ-
ities of certain RNases is one of the promising
approaches towards the analysis of the physiological
role of these enzymes .
In this study, the heterologous gene, encoding extra-
cellular RNase of
, ZRNase II, was used.
It was demonstrated that activity of this enzyme was
induced by wounding .
Total RNA was extracted from mechanically injured
after 2.5 h upon wounding. Total
cDNA was synthesized by reverse transcription with
oligo(dT) primers (Sileks M, Russia) and ampliﬁed.
Ampliﬁcation primers were chosen based on the known
cDNA sequence of ZRNase II . (P1: 5'-ACACTC-
GAGCACACAAACATGAAGA and P2: 5'-
procedures were performed using the synthesized prim-
ers with inserted recognition sites for the
restriction endonucleases. The ampliﬁed cDNA frag-
ment was cloned into plasmid pRT104  under con-
trol of the cauliﬂower mosaic virus 35S RNA promoter
at the recognition sites of the
cleases. After that, cDNA containing promoter, protein-
encoding part of the RNase gene with leader peptide
sequence, and polyadenilation signal was ampliﬁed in
the polymerase chain reaction with the primers (pRTf:
5'-CAGGTCGACATGGTGGAGCA and pRTr: 5'-
Effective Expression of the Gene Encoding
an Extracellular Ribonuclease of
in the SR1
S. S. Sangaev
, E. A. Trifonova
, S. E. Titov
, A. V. Romanova
, Ya. S. Kolodyazhnaya
M. L. Komarova
, 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
Received July 4, 2006; in ﬁnal form, October 4, 2006
—Complementary DNA for the extracellular RNase of
was cloned under control of
the cauliﬂower mosaic virus 35S RNA constitutive promoter and transferred into the
plants. Primary tobacco transformants were characterized by a high level of RNase activity.