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Virus resistant crops - new viral pathogens from transgenic
Table of Contents:
Author: Pia Malnoë
Tree diffierent events, RNA recombination, heterologous
encapsidation and sequence variability have been
identified as potential risks when transgenic virus resistant plants are
used in an agrosystem. Recombination is considered as the
most serious problem because a permanent change is introduced into the viral
genome. However, it is likely that recombination between a viral transgenic
mRNA and a viral genomic RNA is less frequent than recombination between
two different viral genomic RNA molecules co?infecting an untransformed
plant. In this case no increased risk factor is introduced into the fields.
However, to diminish the risk of recombination, only minimal sequence
necessary to induce resistance should be used and one should avoid introducing
into the plant the 3' untranslated region of the viral genome. Usually,
heterologous encapsidation is considered as a less sever problem because it
is limited to a single transfer. In order to avoid heterologous encapsidation
a biological containment system is recommended where the amino acids
responsible for aphid transmission and virion assembly are either mutated
In 1986 it was shown for the first time that, by expressing a viral coat
protein (CP) gene in a plant cell, it was possible to increase the resistance
of the plant against the infection of the homologous virus . Since then it
has been possible to obtain a good coat protein mediated protection (CPMT)
to at least 50 diffierent viruses belonging to 12 virus groups in 12 different
plant species . Commonly, the level of protection is proportional to the
degree of relatedness between the infecting virus and the source of the
transgene. CPMP is usually effective against different isolates or closely
related strains. Lately it has been shown that non structural viral genes,
like the viral replicase genes, also induce resistance.
The level of resistance obtained using a transgenic approach is comparable
to that conferred by host resistance genes. The possibility to induce virus
resistance into a susceptible variety without afflecting the intrinsic properties
of the cultivar is of high agronomic value. This year, a transgenic squash
resistant to zuchini yellow mosaic virus (ZYMV) has been commercialized
in the USA. Several other CPMP resistant plants are expected on the
market in the near future. Hence, it is now more and more urgent to
consider the eventual risks the use of transgenic plants expressing a viral
sequence may cause to our eco? and agrosystem. The principal question
one need to ask is "may the presence of such plants create new
viruses with altered host andlor vector ranges or may the transmission
rate and symptom induction be altered?" These effects could be
the results of recombination, heterologous encapsidation or sequence
variations. AD these events occur naturally in untransformed plants but the
frequency of the events may be different in a transgenic plant.
The genome of more than 70% of the plant viruses consists of one or
several RNA molecules which in most cases can serve as a niRNA (+sens
RNA). Transmission of the virus from one plantto another requires mechanical
inoculation or vectors such as insects, nematodes or fungi. Once the virus has
entered the host cell it is decapsidated. The replication and movement
of the virus within the plant is depending on the presence of viral and host
RNA viruses have highly variable genomes since the replication of the
genome is carried out by a RNA dependent RNA polymerase lacking error
reading functions. The error rate ranges within the limits of 10-3 to 10-5
which correspond to one mutation per 10 kilobases . Hence a virus strain
consists of a population of closely related RNA genomes. There is a strong
selection on these populations depending on the environment they have to
adapt to. Most mutations will be deleterious and only the ones given the
highest fitness in a specific host and environment will be conserved .
Sequence variability and recombination between the viral genomes are
considered as the main tools for the evolution of a viral population.
Recombination in plant viruses is less well understood than in animal
viruses . It seems that recombination occurs in both systems by template
switching between either homologous or non-homologous regions .
Sequence and structural similarities as well as subcellular concentrations
and locations are important factors for the recombination events .
Natural recombination between viral genomes has been described very
rarely [8, 9]. Lately it has been possible to study recombination in planta
during an infection. The data from these experiments indicates that RNA
recombination takes place and functional chimeric genomes can be generated
through this process. The example of abutflon mosaic virus is interesting.
In this case the viral genome contained dispersed point mutations in
different essential genes. Mutated genomes were able to complement
each other so that the virus population could be propagated at a low
frequency. At a later stage a single functional genome was obtained through
recombination and the mutated forms were lost . There is several
others example where selection has been used to generate new viral
particles through both homologous and nonhomologous recombination
[11, 12, 13, 14]. Without selection it is almost impossible to detect a
Potato with Y virus
We have compared the nucleotide sequences of several different strains
of potato virus Y (PVY) . This study shows that the evolution of the
strains is relying not only on sequence variability but also on recombination.
Recently a new highly virulent strain called PVY?NTN has emmerged in different
parts of Europe. This strain causes necroses on tubers resulting in important
economical losses for the farmers. The complet sequence of a Hungarian
isolate of the PVY?NTN strain has been puplished  and reveals a
combination of PVY?N and O sequences.
Recombination in CPMP
Recombination between a transgenic viral mRNA and an infecting viral
genomic RNA is considered as being the most prominent problem because
the changes resulting from a recombination event are permanent. It is
important to establish whether the frequency of recombination is different
during the infection of a transgenic plant compared to the double infection
of an untransformed plant. The following aspects may be considered:
This far we do not have enough knowledge to be able to give a definate
answer. However, research concerning these questions is taking place in
many laboratories. In order to attempt to elucidate this problem, several
factors are required:
- Is the overall rate of recombination changed because of an
- Is the rate proportional to the concentration of the two parental
- Is the site of synthesis important?
- Can the recombinant compete with the parental virus?
- a better knowledge of resistance mechanisms in
the transgenic plants,
- a well defined system to follow specific recombination events and
- a biological assay to be able to determine not only the recombination
frequency but also the fitness of a recombined virus, with and without
The mechanisms involved in coat protein mediated virus resistance can be
grouped into two main classes. In one class, the resistance depends on the
synthesis of viral coat protein in the plant. The efficiency of protection is
generally correlated with the abundance of the said protein. An example of
this type of resistance is illustrated by TMV . The authors have shown
that an efficient resistance to TMV is obtained if the CP accumulates in the
tissue which was initially infected. The virion disassembly seems to be affected
by the presence of the transgenic CP and the decapsidation of the infecting
virion might be inhibited . The transgenic CP may also interfere with
the long distance transport of the virus particles.
In the second class, described as RNA-mediated resistance, the situation is
reversed. The degree of resistance is negatively correlated with the amount
of transgenic mRNA and coat protein present in the cell. This is the case for
the viruses belonging to the potyvirus group. RNA-mediated virus resistance
cannot be explained easily using our present knowledge, but it shares common
features with a phenomenon observed in transgenic plants referred to as
co-suppression or silencing of gene expression.
The concentration of the transgenic viral mRNA in the cell is different
depending on the type of resistance. In the case of TMV resistance, the
concentration of the CP mRNA is relatively high but still at least ten times
inferior to the concentration of viral genomic RNA during an infection. In a
transgenic plant resistant to PVY, there is very little transgenic mRNA present
in the cytoplasm. However, if for any reason the resistance is lost, the
amount of CP mRNA may increase in the cell.
The replication of most plant RNA viruses takes place in the cytoplasm and
often in specialized compartments within the cell. These compartments are
formed by viral proteins as illustrated by the formation of the cytoplasmic
inclusion bodies during a PVY infection. Such kind of compartmentalization
could prevent the cytoplasmic mRNA from entering into direct contact with
the genomic viral RNA and at the same time prevent recombination.
RNA recombination is supposed to take place by template switching during
RNA replication [6, 22]. There is a possibility that the replicase complex of
an infecting virus is able to recognize the 3' untranslated region in a
CP RNA molecule and use it as a template for RNA synthesis. If the
template switching occurs from the mRNA to the viral genomic RNA strand,
a full length hybrid RNA could be created. Such a situation can be avoided
by deleting the Yuntranslated region from the CP construct introduced into
the plant genorne. If template switching occurs from the genon-fic RNA to
the viral mRNA a non infectious molecule will be obtained. To obtained a
full length infectious recombined RNA molecule a second recombination
event is required.
This data indicates that it is likely that recombination between a viral
transgenic mRNA and a viral genomic RNA is less frequent than recombination
between two different viral genomic RNA molecules infecting an untransformed
plant. However, proving it experimentally will require a very well defined
system with an infectious cDNA copy of the viral genome.
When two viruses infect the same plant, different kinds of interactions can
take place . For example,
This is a phenomenon called heterologous encapsidation [24, 25p 261 which
covers two different situations: (i) phenotypic mixing, where the genome
of virus A is encapsidated by coat proteins of type A and B, or
(ii) transcapsidation, where the genome of virus A is encapsidated only by
the coat protein from virus B.
- virus A may suppress the expression of virus B. this is referred to
as cross protection.
- Virus A may enhance the expression of virus B or
- virus A and B may act synergetically.
- In some cases the genome of virus A may be encapsidated by the
coat protein of virus B.
Heterologous encapsidation has been studied under field conditions for
PLRV  and two potyviruses, ZYMV and PRSV, . The efficiency of
heterologous encapsidation is variable, depending on the viruses involved.
Phenotypic mixing occurs mainly between closely related strains.
Transcapsidation occurs between less related strain and even between
different viruses although not necessarily in both directions. This has been
well documented for barley yellow darf virus (BYDV) .
The risks associated with heterologous encapsidation in CP resistant plants
have been discussed in detail by Tepfer  and Palukaitis . In infected
transgenic plants the situation is not quite the same as in plants with double
infection. In the latter case the CP of virus A and B as well as their genornic
RNA will be present in the cell and there will be a preference for a
homologous encapsidation. In the CPW plant the transgenic coat protein
will be present but not the corresponding genomic RNA, which means that
there will be an excess of free transgenic CP. Hence, it is possible that the
frequency of transcapsidation is higher in an infected transgenic plant than
in a plant infected by two viruses.
During our study using a transgenic potato line resistant to PVY-N, we were
able to show that a phenotypic mixing took place when the plants were
infected with a related PVY-O strain . Lecoq and coworkers  have
also been able to show that a non aphid transmissible strain of the
potyvirus ZYMV could be transmitted by aphids after having infected a
transgenic plant expressing the coat protein of PLRV. A third example is
the transencapsidation of CMV by the coat protein of AIMV . The two
last examples demonstrate a significant rate of heterologous encapsidation
between two unrelated viruses in transgenic plants.
Consequently, there is no doubts that heterologous encapsidation occurs in
untransformed and transformed plants but the main question is "what are
the potential risks of heterologous encapsidation in CP resistant plants".
The general opinion does not consider heterologous encapsidation as a
problem because the phenomenon is limited to a single transfer . The
transcapsidated virus becomes defective with regards to the new host and
should not be able to propagate without a helper virus. However, the fact
that transcapsidation in transgenic plants may contribute to the introduction
of a new virus into a new ecological niche can not be excluded .
For safety reasons it is recommend
- not to express a coat protein in a plant that is not its natural host.
- to create a biological containment system. For example, in the
potyvirus group it has been shown that an amino acid triplet (DAG)
in the N-terminal part of the CP determines aphid transnmission.
Deletion of the sequence encoding these amino acids in the CP
gene will create a transgenic coat protein that cannot be transmitted
to another plant. Jagadish and collaborators  have been able to
determine the amino acids in the core region of the coat protein of
a potyvirus that are essential for assembly of the viral particle.
Deletions or mutations of these amino acids in the transgenic CP
would be another way of hindering transcapsidation.