Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-10-18
2004-11-30
Nolan, Patrick J. (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S388300, C530S388400, C530S388500, C530S388600, C530S387300
Reexamination Certificate
active
06825325
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to gene constructs suitable for expressing agents to protect a plant against pathogens and the suitable proteins for such plant protection. These agents are named “molecular pathogenicides”. This invention is related to the genetic engineering of plants and to means and methods for conferring pathogen resistance on a plant using a gene or genes encoding: a pathogen specific antibody and a pathogen specific antibody including a toxic activity which blocks stages of the pathogen life cycle, pathogen replication or pathogen movement within a plant or pathogen transmission from plant to plant. The means and methods are given for soluble expression of recombinant antibodies, antibody fusion proteins and antibody protein complexes in different plant cell compartments or the immobilisation of recombinant antibodies, antibody fusion proteins and antibody protein complexes in cellular membranes in different orientations and the display of recombinant proteins on the plant cell membrane. This invention also describes novel methods and protein binding partners for assembling protein complexes from individual polypeptide chains during expression of these proteins in vivo. Also given are the methods for activation of the molecular pathogenicides by in vivo proteolytic cleavage.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturers specifications, instructions, etc.) are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention.
BACKGROUND OF THE INVENTION
Plant disease constitutes a major and ongoing threat to human food stocks and animal feed. Most crop plants are regularly exposed to one or more pathogen(s) that can cause incredible damage resulting in substantial economical losses every year. Attack by pathogens, such as viruses, bacteria, fungi, nematodes and insects and is a severe economic problem, which impacts all economically important crops, for example rice, soybean, sweet potato, wheat, potato, grape, maize and ornamental plants. Current protective measures rely heavily on chemical control measures for pathogen vectors, which have undesirable environmental consequences.
A more effective approach to protecting plants from pathogen attack is to create plants that are endogenously resistant to pathogens. However, plant breeders have limited sources of resistance genes against plant diseases. This can now be achieved using genetic engineering techniques, by providing the plant with genetic information required for affecting the pathogens and for being resistant to the disease caused by the pathogen. For example, in the case of a viral pathogen, the host plant is resistant if it has the ability to inhibit or retard the replication of a virus, the symptoms of viral infection or the life cycle of the virus, including its transmission. “Resistant” is the opposite of “susceptible” and may be divided into three levels:
1) Full,
2) Medium,
3) Partial resistance,
A plant may be considered fully resistant when it shows no symptoms on infection and there is no evidence of pathogen replication or reproduction. The host plant may be resistant to the establishment of infection, pathogen reproduction and/or pathogen movement and transmission.
In recent years, the advances in plant molecular virology have enhanced the understanding of pathogen genome organisation and gene function. Moreover, genetic engineering of plants for virus resistance has recently provided new strategies for control of viral disease (Baulcombe, 1994), (Gadani et al., 1990), (Wilson, 1993). The following genes were expressed in transgenic plants in order to confer resistance: viral coat proteins, non-structural proteins of viral genomes, viral anti-sense transcripts, viral satellite RNAs, ribozymes and interferon genes (Baulcombe, 1994), (Gadani et al., 1990), (Wilson, 1993), (Harrison et al., 1987), (Namba et al., 1991), (Anderson et al., 1992). Although most of these approaches have been effective for attenuating infections, resistance was not complete and confined to a small spectrum of viral pathogens (Falk and Bruening, 1994), (Wilson, 1993) and bears significant risks (Palukaitis and Roossinck, 1996).
The major disadvantages of these methods are:
1) Host range is limited.
2) Pathogen range is limited.
3) Resistance is partial and though symptoms are delayed infection still results in the disease.
4) Resistance could be broken in case of coat protein mediated resistance and ribozyme mediated resistance.
5) Expression of viral proteins can lead to enhanced pathogen activity. For example, in the case of viral coat protein mediated resistance, cross encapsidation of mild non-pathogenic strains of virus by the expressed coat protein can occur which then leads to development of a more severe disease.
An alternative way to protect plants against pathogen infection is the generation and expression of recombinant antibodies (rAbs), which are often referred to as “Plantibodies”. Pathogen-specific recombinant antibodies targeted to different compartments of plant cells or different plant organs overcome many of the problems mentioned before and confer a broader spectrum of resistance to disease (Baulcombe, 1994). To achieve this, recombinant antibodies (Plückthun, 1991), (Winter and Milstein, 1991) against the target proteins have to be generated by cloning the corresponding antibody heavy and light chain genes from hybridoma cells, synthetic, semi-synthetic and immunocompetent phage display or ribosome display libraries; or by the generation of fully synthetic designer antibodies. This is followed by subsequent modification and rAb expression in different compartments of heterologous hosts such as bacteria, yeast, algae, baculovirus infected insect cells, mammalian cells and plants. For example, antibodies and antibody-fusion proteins binding to conserved functional domains of viral coat proteins, movement proteins, replicases or transmission factors can be used to inactivate such targets inside or outside the plant cell through immunomodulation. The feasibility of expressing recombinant antibodies (Plückthun, 1991), (Winter and Milstein, 1991) for the generation of resistance has been shown recently for both animal (Chen et al., 1994), (Duan et al., 1994), (Marasco et al., 1993) and plant viruses (Tavladoraki et al., 1993), (Voss et al., 1995), (Zimmermann et al., 1998). Single chain antibody fragments derived from monoclonal antibodies (scFvs) (Bird et al., 1988) directed against Rev (Duan et al., 1994) and gp120 (Chen et al., 1994) (Marasco et al., 1993) of HIV, inhibited HIV-replication, virion assembly and syncytia formation when expressed intracellularly, or within the ER of human cells.
Interestingly, intracellular expression of an scFv specific for the artichoke mottled crinkle virus coat protein in transgenic Tobacco caused a reduction of infection and a delay in symptom development (Tavladoraki et al., 1993). Targeting of TMV-specific full-size antibodies to the intercellular space of Tobacco plants inhibited viral infections up to 70% (Voss et al., 1995). In the latter case, plant produced antibodies showed the same specificity and affinity for TMV (Fischer et al., 1998) as the parental murine antibody. Cytosolic expression of an engineered scFv derived from this anti-TMV antibody yielded fully resistant Tobacco plants, even under systemic infection conditions (Zimmermann et al., 1998). These studies demonstrate the potential of heterologously expressed recombinant antibodies to combat pathogens via intra- or extra-cellular modulation of pathogen proteins.
Plant cells can synthesise large amounts of antibodies that are functionally indistinguishable from the source monoclonal. For example, full-size antibodies (During et al., 1990), (Hiatt et al., 1989), (Voss et al., 1995), Fab-fragments (De Neve et al., 1993), scFvs (Owen et al., 1992; Zimmermann et al., 1998), (Tavladoraki et al., 1993), scFv fusion protein
Emans Neil
Fischer Rainer
Liao Yu-Cai
Monecke Michael
Nähring Jörg
Birch & Stewart Kolasch & Birch, LLP
Fraunhofer Gesellschaft zur Forderung der angewandten Forschung
Nolan Patrick J.
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