Department of Genetics
Biochemical Genetics and Biotechnology Research Unit
Areas of research
last update 2010-10-6
The group of Biochemical Genetics and Biotechnology is involved in the study of
the microbial control of insect pests using Bacillus
thuringiensis and baculovirus. In the following sections, there are some general ideas on our field
of work, as well as the specific lines of research on which we are working.
1) Bacillus
thuringiensis AND BACULOVIRUS FOR THE MICROBIAL CONTROL OF INSECT PESTS
One of the agents most used in the microbial control of pests is the
bacterium Bacillus thuringiensis. During sporulation,
this bacterium synthesises especial proteins that aggregate to form a parasporal crystal which can reach, in some strains, the
same size as the spore. When insects ingest the sporulated
bacterium, in addition to the spore they also ingest the crystal. This is
dissolved in the intestine where it releases its protein components (protoxins) which, normally, have to be activated by
intestine proteases to their toxic final form. The toxin then binds to
receptors in the epithelial membrane of the intestine forming pores in the
cells which eventually kills the insect. This poisoning facilitates germination
of the spore and invasion of its host.
The other agents, baculovirus, are viruses that infect invertebrates, mostly
insects from the order Lepidoptera. Also known as nucleopolyhedroviruses, they are obligate pathogens that
are characterized by their rod-shaped nucleocapsids,
each of which contains a single circular molecule of double-stranded DNA. The nucleocapsid is wrapped in a lipoprotein membrane to form virions, that
are occluded by a protein matrix to form the occlusion body (
Some
advantages of using these pathogens in the fight against insects are, on the
one hand, their specificity, since one can target the pest insect without
affecting the beneficial insect populations and, on the other hand, they are
totally innocuous to organisms other than insects, including humans. These
characteristics make the insecticides based on B.
thuringiensis and baculovirus a resource very appreciated from the standpoint
of the organic farming. As a matter of fact, they are of the very few
insecticides allowed in this type of agriculture. Another advantage which B. thuringiensis has is that, because the toxin is a protein, genes can be manipulated
in the laboratory in such a way that the mutant protein becomes more efficient
or even acquires toxicity to control new pests. However, the reason why this
bacterium is so popular is because of its use in genetic engineering, since its
genes can be integrated in plant genomes and then these plants produce the
insecticidal protein and become resistant to the insects that normally feed on
them.
The search
for new B. thuringiensis genes that codify for new insecticidal proteins
will allow to broaden the spectrum of action of B.
thuringiensis-based
insecticides. This is of great interest not just to fight pests that are not
controlled at present by this type of insecticides, but to obtain toxins that
will maintain their effectiveness to control insect populations when these
start to develop resistance to toxins currently in use from this bacterium.
The problem
of insect resistance is a very important one as it can render an insecticide
obsolete in few years. This problem does not only affect chemical insecticides,
but it also affects microbial insecticides. An understanding of the mechanisms
by which B. thuringiensis toxins or baculovirus
act, and by which an insect can become resistant to them, can help to design strategies
to prevent resistance from appearing. The results of this type of research are
very useful to design strategies for the utilisation of B. thuringiensis-based
insecticides to minimise the possibility of resistance appearing and, in this
way, to ensure that this insecticide, which has such useful characteristics,
continues to be useable. But this understanding is especially relevant in the
context of the design of insect resistant transgenic plants, since the correct
selection of genes (which codify toxins) to be introduced in a plant will
depend on their mode of action. A plant expressing two toxins that use the same
target site for their action will provoke the appearance of resistance in the
pest insects much faster than one expressing two toxins that use different
target sites.
2) RESEARCH
LINES AND GOALS
The main goal of this research group consists on the control of insect
pests by the use of microbial insecticides based on the bacterium Bacillus thuringiensis and baculovirus. The group works on three research
lines and their objectives are summarised below.
1. Resistance to bioinsecticides
General
objective: To study the potential of insects to develop resistance to toxins
from B. thuringiensis and the genetic and biochemical basis of this resistance.
This is a basic line of research, though with an applied projection. It was
with this line that the most senior members of the group began to work with B.
thuringiensis, at the end of 1989.
One of the
objectives pursued within this line of research is the study of the mode of
action of the toxins from B. thuringiensis to, in this way, be able to
understand the mechanisms of resistance that the insects can generate against
these toxins. The understanding of how these toxins act in a particular pest also
allows us to make predictions on the possible appearance of cross-resistance to
other B. thuringiensis toxins. Another objective is the detailed
understanding of the resistance mechanism occurring in insect strains resistant
to B. thuringiensis formulations.
The species
which we have most studied, and the one that we are still most actively
studying, is the diamondback moth, Plutella
xylostella, which is the only species to date to evolve resistance to B.
thuringiensis in the field (though not to transgenic crops). However, our
interest covers any species that has evolved resistance to B. thuringiensis
toxins. So far we have worked with laboratory resistant strains of the
lepidopterans Plodia interpunctella
(Indianmeal moth), Trichoplusia
ni (cabbage looper), Ostrinia nubilalis
(corn borer) and Heliothis virescens (tobacco
budworm), and we have performed studies on the mode of action in other
lepidopterans, such as Helicoverpa armigera, Phthorimaea
operculella (potato tuber moth), Mamestra brassicae, Spodoptera exigua and Cacyreus marshalli (geranium
bronze). We have also worked with the dipteran Bactrocera oleae
(olive fly).
2. Development of novel bioinsecticides
General
objective: To find B. thuringiensis strains and genes with novel
insecticidal properties. These are activities concerned with practical
applications, since the final goal is to obtain industry-transferable results.
This research line was started in 1994.
One of the
objectives is the development of bioinsecticides
based on new strains of B. thuringiensis with high activity against pest
insects. We plan to use natural strains with high activity or with a broader
insecticidal spectrum, as well as hybrid strains obtained by means of plasmid
transfer. We have currently a collection of around 650 isolates of B. thuringiensis. Another objective is the isolation and characterisation of novel genes
in this bacterium codifying proteins with high insecticidal properties.
In
collaboration with the Departamento de Producción Agraria of the
Universidad Pública de Navarra,
we have found and characterised five new B.
thuringiensis serovars, whose respective type strains have been deposited
in the
With this
line of research we collaborate with a number of companies, within and outside
3. Insect-pathogen interaction
Insects
represent one of the most successful examples of adaptive evolution, being able
to colonize almost all ecological niches. Consequently, they had to cope with
an extremely large variety of pathogens, including baculovirus
and B. thuringiensis. The evolution of insects as well as the evolution in
their pathogens has followed parallel pathways. Changes in the insect that
contributed to reduce the effect of the pathogen have been responded by
adaptations of the pathogen trying to escape to the insect defence. In order to
overcome, or at least, to minimize the pathological effect, insects have
developed different mechanism. Among the known mechanisms, some of them promote
the activation of detoxification and damage repair mechanisms. Other mechanisms
include the synthesis and secretion of antimicrobial peptides and degradation
enzymes, phagocytosis, melanization,
cell apoptosis, or cell sloughing, among others (some of them probably still
unknown). The activation of these mechanisms reduces the chances of pathogens
to extend their infection and can be responsible of the development of insect
resistance.
By
combination of SSH (Suppressive Subtractive Hybridization) with DNA-microarray we are trying to identify the insect’s elements
involved in the response to these pathogen and as consequence, determining the
mechanism of insect response to pathogens. Information about the mechanism of
insect defence can be very useful for the identification of the possible
mechanisms of insect resistance to baculovirus or B.
thuringiensis. Moreover, the obtained information can be used for optimizing
the use of these pathogens as a pest control agent. In collaborations with the
group of Dr. Ruud de Maagd
(Plant Research International, The Netherlands) we are currently analyzing the
interaction between S. exigua and B. thuringiensis
(or its toxins). Similarly, in collaboration with Dr. Karl Gordon (CSIRO
Entomology,
3) FUNDING
SOURCES AND RESEARCH PROJECTS:
The projects
on which we work have been funded by the European Union within all Framework
Programmes since the third. The group has also received non-interrupted funding
from the Ministry of Science and Education (or from that of Science and
Technology) and from the Generalitat Valenciana (Autonomic Government) competing with
competitive projects. Other sources of funding have been contracts and
agreements with private companies such as Bayer BioScience,
Industrias AFRASA and Biológicas
Canarias, among others. The most relevant projects in
the last 10 years are shown below:
Title of the project: Introduction of resistance to Cacyreus marshalli in Pelargonium
zonale by genetic transformation with Bacillus
thuringiensis
Funding agency: Ministerio de Educación y Ciencia (Proyectos de I+D
del programa FEDER) (FD1997-0917-C02-01).
Participant Centres: Universitat
de València, Universidad Politécnica de Valencia and
Fundación PROMIVA (Manises, Valencia)
Title of the project: Bacillus
thuringiensis resistance management in
cotton pests.
Funding agency: Ministerio de Educación y Cultura (Plan Nacional de
I+D, Programa Nacional de Recursos y Tecnología Agroalimentarias)
(AGL2000-0840-C03-01).
Participant Centres: Universitat
de València, Universidad de Córdoba and Universidad
Pública de Navarra.
Title of the
project: Engineering transgenic pelargoniums for resistance to fungi and
insects
Funding agency: Ministerio de
Ciencia y Tecnología (Plan Nacional I+D+T, Programa de Fomento de la
Investigación Técnica, PROFIT) (FIT-010000-2001-87).
Participant Centres: Fundación PROMIVA (Majadahonda,
Madrid), Instituto de Biología Molecular (CSIC, Barcelona) and
Universitat de València.
Title of the project: Engineering
transgenic ornamental Pelargonium resistant to the infection by pathogenic
fungi or to insect attack
Funding agency: Ministerio de
Ciencia y Tecnología (Plan Nacional I+D+T, Programa de Fomento de la
Investigación Técnica, PROFIT) (FIT-010000-2002-13).
Participant Centres: Fundación PROMIVA (Majadahonda,
Madrid), Instituto de Biología Molecular (CSIC, Barcelona) and
Universitat de València.
Title of the project: Protecting the
benefits of Bt-toxins from insect resistance development by monitoring and
management.
Funding agency: Comisión
Europea (Programa Quality
of life and management of living resources). (QLTR-2001-01969)
Participant Centres: Aachen University of Technology (Aachen,
Germany), CIB-CSIC (Madrid), INRA La Minière (Guillantcourt, France), Università
degli Studi (Milán, Italy), Slovak Agricultural Unviersity
(Nitra, Slovakia), Aristotle University of Thessaloniki (Salónica, Greece), Fraunhofer Institute for Molecular BIology
and Applied Ecology (Schmallenberg, Germany),
University of Melbourne (Parkville, Australia) and Universitat
de València.
Title of the project: Microbial alternatives to agrochemicals in plant
protection.
Funding source: Oficina de
Ciencia y Tecnología de la Generalitat Valenciana (OCYTGV). (CTGA/2002/03/01)
Participant Centres: Universitat de València.
Title of the project: Biochemical and
genetic analysis of resistance to Bacillus thuringiensis toxins in Plutella
xylostella.
Funding agency: Consejo Nacional
de Ciencia y Tecnología de Méjico (CONACYT) (Ref. 38040-N).
Participant Centres: Universidad Autónoma de Nuevo León
(Méjico) and Universitat de
València.
Title of the project: Development of
a new bioinsecticidal product from a novel authoctonous strain of Bacillus
thuringiensis.
Funding agency: Ministerio de
Ciencia y Tecnología (Proyecto de Estímulo a la Transferencia de Resultados de
Investigación, PETRI) (PTR1995-0633-OP).
Participant Centres: Universitat de València and
Title of the project: Bases of
resistance to Bt toxins in lepidopteran pests.
Funding agency: Ministerio de Ciencia y Tecnología (Plan Nacional de
I+D+I, Programa Nacional de Recursos y Tecnología Agroalimentarias)
(AGL2003-09282-C03-01).
Participant Centres: Universitat
de València, Universidad de Córdoba and Universidad
Pública de Navarra.
Title of the
project: Grant for research groups.
Funding agency: Conselleria de Cultura, Educació
i Esport de la Generalitat Valenciana
(GRUPOS2004-21).
Participant Centres: Universitat
de València.
Title of the project: Resistance
bases to the insecticidal crystal proteins from Bacillus thuringiensis in lepidopteran populations.
Funding agency: Conselleria de Cultura, Educació
i Esport de la Generalitat Valenciana (GV04B-165).
Participant Centres: Universitat
de València.
Title of the project: Prevention of resistance to baculovirus in Spodoptera
exigua.
Funding agency: Ministerio de Educación y Ciencia (Plan Nacional de
I+D+I, Programa Nacional de Recursos y Tecnología Agroalimentarias)
(AGL2005-0709-C03-03/AGR).
Participant Centres: Universitat de València and
Universidad Pública de Navarra
(
Title of the project: Bases of
resistance to Bacillus thuringiensis
toxins in lepidopteran pests.
Funding agency: Conselleria de Empresa, Universidad y Ciencia de la
Generalitat Valenciana, programa de Ayudas
complementarias para proyectos de I+D+I (ACOMP06/133).
Participant Centres: Universitat de
València, Universidad de Córdoba and Universidad
Pública de Navarra (Pamplona).
Title of the
project: Resistance management to Bacillus thuringiensis insecticidal proteins.
Funding agency: Ministerio de Educación y Ciencia (Plan Nacional de
I+D+I, Programa Nacional de Recursos y Tecnología Agroalimentarias)
(AGL2006-11914).
Entitats participants: Universitat
de València.
Title of the project: Building a net
of research groups and centres to assist the scientific-technical needs of
companies in the Valencian Comunity (Red Valenciana de Investigación
Vinculada).
Funding agency: Conselleria de Empresa, Universidad y Ciencia de la
Generalitat Valenciana (ref. ARVIV/2007/090).
Participant Centres: Universitat de València and other
universities and research centres from the Comunidad Valenciana.
Title of the project: Host response to baculovirus infection in Helicoverpa
armigera.
Funding agency: Unión Europea (programa Structuring the ERA).
Participant Centres: Universitat de València
Title of the project: Stablishing collaboration links in the research on Bacillus
thuringiensis based bioinsecticides.
Funding agency: Programa de Cooperación Interuniversitaria de la
Agencia Española de Cooperación Internacional (AECI) el marco del PCI-Mediterráneo
2007.
Participant Centres: Universitat
de València and Centre de
Biotechnologie de Sfax (Tunisia)
Title of the project:. Specific binding of Bt toxins to
midgut membranes from sweetpotato
weevil, Cylas spp.
Funding agency:
The Rockefeller Foundation (07-ENT-367314-VAL).
Participant Centres: Universitat de València, Auburn University (USA) and Centro Internacional de la Papa (
Title of the project: Mode of action
of Bacillus thuringiensis VIP
proteins in Helicoverpa armigera and
Spodoptera exigua.
Funding agency: Unión
Europea (programa
Structuring the ERA).
Participant Centres: Universitat de València
Title of the project: Genes of
response to the baculovirus of Spodoptera
exigua, applications for the improvement of its
insecticidal properties.
Funding agency: Ministerio de
Ciencia e Innovación (Plan Nacional de Investigación Científica y Desarrollo e
Innovación Tecnológica) (AGL2008-05456-C03-03).
Participant Centres: Universitat de València and Universidad Pública de Navarra.
Title of the
project: Development
of a novel bioinsecticide based on Pseudomonas fluorescens.
Funding agency: Conselleria de Empresa, Universidad y Ciencia de la
Generalitat Valenciana (ref. GVPRE/2008).
Participant Centres: Universitat de València.
Title of the project: Secretable toxins from Bacillus
thuringiensis for the control of lepidopterans.
Funding agency: Programa de Cooperación Interuniversitaria de la Agencia Española de Cooperación Internacional para el Desarrollo (AECID) en el marco del PCI-Mediterráneo 2008 (A/017413/08).
Participant Centres: Universitat
de València and Centre de
Biotechnologie de Sfax (Tunisia).
Title of the project: Second
generation insecticidal proteins from Bacillus
thuringiensis.
Funding agency: Ministerio de Ciencia e Innovación, Programa Hispano-Brasileño de Cooperación Interuniversitaria (PHB2008-0113-PC).
Participant Centres: Universitat de València and Universidade Estadual Paulista (Brasil).
Title of the project: Resistance
to African sweetpotato weevils using biotechnology.
Funding agency:
The Rockefeller Foundation (08-EPP-368722-UV).
Participant Centres: Universitat de València, Auburn
University (USA) and Centro Internacional de la Papa
(
Title of the project: Resistance management of Bacillus thuringiensis insecticidal
proteins.
Funding
agency: Conselleria d’Educació de la Generalitat Valenciana, programa de Ayudas
complementarias de I+D+i a grupos de calidad contrastada para completar
el desarrollo de proyectos de I+D vigentes (ref.
ACOMP/2009/313).
Participant Centres: Universitat
de València.
Title of the project: Mode of action of secretable insecticidal proteins from Bacillus
thuringiensis.
Funding agency:
Ministerio de Ciencia e Innovación (Plan Nacional de I+D+I,
Programa Nacional de Investigación Fundamental) (AGL2009-13340-C02-01).
Participant
Centres: Universitat de València y
Universidad Pública de Navarra.
Title of the project: Resistance management
of Bacillus thuringiensis
insecticidal proteins.
Funding agency:
Conselleria d’Educació de la Generalitat
Valenciana, programa de Ayudas para grupos de calidad contrastada, del programa Gerónimo
Forteza.
(ref. FPA/2010/011).
Participant Centres: Universitat de València
Title of the project: Sweet Potato
Action for Security and Health in Africa (SASHA)
Funding agency: Bill and Melinda
Gates Foundation (BMGF)
Participant Centres: Universitat de València, Centro Internacional de la Papa (CIP), consorcio de países africanos