TRANSGENIC PLANTS OF TOBACCO WITH MODIFIED LEVELS OF RNASE

							TRANSGENIC PLANTS WITH A MODIFIED LEVEL OF
EXTRACELLULAR RIBONUCLEASE ACTIVITY AS A NEW
GENETIC MODEL
S.S. Sangaev1*, A.V. Romanova1, E.A. Trifonova1, M.V. Sapotsky2, V.I. Malinovsky2,
A.V. Kochetov1
1
  Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia
2
  Institute of Biology and Soil Science FEB RAS, Vladivostok, Russia
e-mail: sangaev@gorodok.net
*Corresponding author

Extracellular S-like RNases of plants are endoribonucleases with no absolute substrate
base specificity that are found in apoplastic compartment. Their expression is associated
with senescence, phosphate starvation and wounding, but the nature of their function is
largely unknown. It was previously shown that transgenic tobacco plants with high level
of bovine RNase activity in apoplast were characterized by lower accumulation of a
viral antigen and delayed symptoms when inoculated by tobacco mosaic virus (TMV).
Taking into account this fact and the fact that wounding is one of the major ways of
viral invasion, we have suggested that wound-inducible extracellular RNases could be
used to protect plants at the viral infection onset. However, the role of plant
ribonucleases in the mechanism of antiviral protection has not been studied yet.
The objective of this research concerns investigation of the functional role of plant
wound-inducible extracellular RNases in the mechanism of defense against viruses. For
this purpose, we planned to obtain model tobacco plants with modified activity of
certain wound-inducible extracellular RNases and to analyse their resistance against
TMV.
In order to obtain plants with increased activity of a wound-inducible RNase we
obtained tobacco plants expressing ZRNaseII gene of Zinnia elegans. In the host plant,
ZRNaseII is not detected in intact leaves and is induced in response to wounding. The
transgenic plants were characterized by a high level of RNase activity in apoplast.
Activity gel assay also revealed the presence of an additional RNase in the transgenic
plants.
To obtain plants with decreased level of wound-induced RNase we transformed tobacco
plants with genetic construction bearing a dsRNA suppressor of the tobacco RNase
gene Nk1. This RNase is particularly interesting as it is induced either by wounding or
by virus inoculation. Activity gel assay showed lack of one of proteins with RNase
activity compared to control plants.
Thus, we obtained tobacco plants with either increased or decreased levels of
extracellular RNase activity and these plants can be used as a model to study the
functional roles of apoplastic RNases.
Under normal conditions the expression of introduced genetic constructions had no
visible effect on the growth and development of the transgenic plants. We have obtained
some preliminary data concerning resistance of the plants bearing ZRNaseII gene
against TMV infection. These data agree with our hypothesis on the role of wound-
inducible ribonucleases in plant antiviral defense mechanisms.