PLANT VIRUS BIOTECHNOLOGY

Personnel:

 

Viruses offer a myriad of opportunities to be targets or tools of plant biotechnology. On one hand viruses are intracellular pathogens impossible to combat chemically, hence fighting against them requires different approaches frequently involving biotechnological components. A clear example is the exploitation of molecular-based technologies for virus detection, diagnosis, and/or typing. Deployment of natural or transgenic resistances to viruses also demands a wide range of molecular technologies, useful for structural genomic characterizations of plant varieties in this regard, too. On the other hand plant viruses are organisms developed to interact with plant components. These interactions often lead to host physiological or developmental alterations, yet they have the built-in advantage of being a useful lead to understanding and potentially modifying both the interaction itself (infection symptoms), and plant physiology or development with different purposes. Finally viruses can be efficient vehicles for heterologous gene expression in plants exploited as biofactories, and its encapsidated forms (virions) are but highly complex and stable nanostructures, the basis for development of multiple nanobiotechnological applications.

Our group tries to exploit this formidable multilevel virus potential for its biotechnological development.

 

Relevant results (2010-2016)

Viral diagnosis and typing. Structural genomics of viruses, pathogens, and plant varieties

New molecular markers of tomato linked to pathogen resistance genes (including viral pathogens) were obtained and validated with an aim to be applied in distinctness, uniformity and stability (DUS) testing for Plant Breeders Rights (PBR) applications.

Plant-virus interactions

For our studies of the interactions with higher plants, underlying or induced by the infection of viruses, we have mostly exploited over the last few years the pathosystem formed by the model plant Arabidopsis thaliana and Turnip mosaic virus (TuMV), a potyvirus. TuMV makes most of its proteins through the processing of a viral encoded polyprotein. During the past five years our results in this area have focused on the viral determinants of pathogenicity, the disease side of the viral infection, and the response of the infected plant to the infection by the virus.

Viruses are intracellular pathogens frequently inducing disease in the infected plant. Different viruses induce different diseases, this holding true even for different strains of the same virus. The combined use of viral infectious clones (cloned copies of viral genomes), mutagenesis, and genomic interchanges between different viruses or viral strains (chimeric viruses) allows the precise determination of viral pathogenicity determinants for specific disease symptoms. For TuMV the selection of virulent variants overcoming resistances that involve plant genes of the family of translation initiation factors, showed the involvement of the region coding for the viral genome-linked protein (VPg) in determining the specificity of the resistances. The results also suggest that the virulent TuMV variants could use an eIF4F-independent pathway, which would represent a novel pathway for potyviruses.

Two different TuMV strains were used to characterize the different symptomatology and strong differences in oxidative status induced by them. Early differences in several senescence-associated genes linked to specific regulatory networks were found, as well as persistent divergence in the production of reactive oxygen species (ROS) and scavenging systems. However, at a later stage, both strains induced nutrient competition, indicating that senescence rates are influenced by different mechanisms upon viral infections. It was also found that differential senescence progression and ROS accumulation between strains rely on an intact salicylic acid pathway.

Effects of different Turnip mosaic virus strains on Arabidopsis Col-0 development. Comparison of A buffer-inoculated, B JPN 1-infected and C UK1-infected plants. For better size and global architecture comparisons, pictures were taken with pots leaning to one side and plants were placed not to show creeping effects. D A JPN 1-infected plant showing the induced creeping effect. E A close-up of a UK 1-infected plant showing an extremely short flower stalkand flowers forming a bunch. Bar = 5 cm.

 

TuMV infections also affect many plant developmental traits, an aspect which has not received much attention so far in plant-virus interactions studies. We found that the same two TuMV strains previously cited, differentially affect plant development including many developmental traits. One of these traits is flower stalk elongation. We have mapped the main component of this differential strain effect to the P3 cistron of the viral polyprotein, one of the most variable genomic regions in the virus genome. Transcriptomic and interactomic analyses at different stages of plant development revealed large differences in the number of genes affected by the different infections at medium infection times but no significant differences at very early times.

Viral vectors and bionanotechnologies

Elongated and flexuous recombinant nanoparticles were derived from TuMV to be used as bioscaffolds for increased peptide immunogenicity and peptide-specific antibody sensing.

For this purpose, a 20-amino acid peptide derived from human vascular endothelial growth factor receptor 3 (VEGFR-3) was fused to the N-terminal region of Turnip mosaic virus coat protein (CP) by genetic insertion. Systemic infections of the recombinant constructs were achieved in plants which generated identifiable viral nanoparticles (VNPs).

When these VNPs were used to immunize mice, a very high boost of immune response towards the VEFGR-3 peptide was found. The same VNPs also showed log increases in their ability to detect VEGFR-3antibodies in sera, an application developed for the first time.

Viral TuMV particles can also act as bioscaffolds for nanoimmnobilization of enzymes in such a way that the enzymatic activity is preserved, and even increased. This has been proven by the group through the immobilization of lipase B from Candida Antarctica on nanonets derived from the virus, onto which enzyme aggregates were covalently attached.

Electron micrographs of non-recombinant and VEGFR3-recombinant TuMV VNP preparations. A low magnification (×8000, panel a) shows that VEGFR3-recombinant has the typical morphology of potyvirus particles. Higher magnifications show that non-recombinant TuMV virions are not decorated with a polyclonal antibody against theVEGFR-3 peptide (b, ×120,000), whereas VEGFR3-TuMV are heavily decorated (c, ×200,000), revealing the presence of the peptide on the VNP surface.

 

R+D Funding

The group was funded during the last ten years by grants from the following sources:

Spanish National Plan of R+D

Program for Agricultural Resources and Technologies (RTA program)
Program of Integrated Actions (bilateral cooperation with other countries)

Spanish Ministry for Environment and Rural and Maritime Affairs (MARM)

Agreement INIA-MARM for technical support in issues related to plant varieties

European Union funding

Program for Cooperation in Science and Technology (COST program)
Program of the Community Plant Variety Office (CPVO)

Recent Publications (2007-2016)

Viral diagnosis and typing. Structural genomics of viruses, pathogens, and plant varieties

Plant-virus interactions

Viral vectors and nanobiotechnologies

Divulgation and opinion papers


 

Centro de Biotecnología y Genómica de Plantas UPM – INIA Parque Científico y Tecnológico de la U.P.M. Campus de Montegancedo
Autopista M-40, Km 38 - 28223 Pozuelo de Alarcón (Madrid) Tel.: +34 91 4524900 ext. 1806 / +34 91 3364539 Fax: +34 91 7157721. Contacto

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