Systems and methods for gene targeting in plants, including systems and methods that include the use of geminiviruses and customizable endonucleases.

The invention provides a plus-sense single-stranded RNA viral RNA molecule, comprising a segment encoding a movement protein, a segment encoding a coat protein and a segment that comprises a guide RNA (gRNA), wherein said RNA molecule can be translated, in infected cells, to a polyprotein comprising the movement protein and the coat protein.

Methods of preparing plant-derived proteins or suprastructure proteins, are provided. The method may comprise obtaining a plant, or plant matter comprising apoplast-localized proteins, or suprastructure proteins, producing a protoplast/spheroplast fraction and apoplast fraction from the plant or plant matter, and recovering the apoplast fraction. The apoplast fraction comprises plant-derived proteins or suprastructure proteins. Alternatively, the proteins, or suprastructure proteins, may be obtained from plant or plant matter comprising plant-derived proteins or suprastructure proteins, by digesting the plant matter using a cell wall degrading enzyme composition to produced a digested fraction. The digested fraction is filtered to produced a filtered fraction, and the plant-derived proteins or suprastructure proteins, are recovered from the filtered fraction.

Provided are more efficient conditions for allowing a characteristic regulator to function, by using an inter-family-graftable plant of a specific family, such as Solanaceae, as a medium, in a plant belonging to a different family from that of the medium. A plant virus for producing a characteristic regulator is selected as the delivery medium, and is delivered to a different-family plant through an inter-family-graftable plant of a specific family, such as Solanaceae.

An isolated expression enhancer active in a plant, portion of a plant or plant cell, the expression enhancer is provided. The isolated expression enhancer may be selected from the group consisting of nbMT78 (SEQ ID NO:1); nbATL75 (SEQ ID NO:2); nbDJ46 (SEQ ID NO:3); nbCHP79 (SEQ ID NO:4); nbEN42 (SEQ ID NO:5); atHSP69 (SEQ ID NO:6); atGRP62 (SEQ ID NO:7); atPK65 (SEQ ID NO:8); atRP46 (SEQ ID NO:9); nb30S72 (SEQ ID NO:10); nbGT61 (SEQ ID NO:11); nbPV55 (SEQ ID NO:12); nbPPI43 (SEQ ID NO:13); nbPM64 (SEQ ID NO:14); and nbH2A86 (SEQ ID NO:15). Methods for using the isolated expression enhancer are also provided.

The present application discloses an application of OsAO gene for improving rice resistance to rice stripe disease, rice black-streaked dwarf disease or other rice or corn virus diseases caused by homologous virus of rice black-streaked dwarf virus. The present application also provides an application of OsAO gene, the protein encoded by the gene or a recombinant vector containing the gene in regulating plant resistance to rice stripe disease, rice black-streaked dwarf disease or other rice and corn virus diseases caused by homologous virus of rice black-streaked dwarf virus, said protein has the amino acid sequence as shown in Seq 4. The experiment proved that the plant disease resistance is increased in rice overexpressing OsAO gene, indicating that the OsAO protein encoded by this gene plays an important role in rice resistance to rice stripe disease and rice black-streaked dwarf disease.

Improved plant transient expression systems using optimized geminiviral vectors that efficiently produce heteromultimeric proteins are described herein. Examples of high yields are shown herein, including two, three, or four fluorescent proteins coexpressed simultaneously. Various antibodies were produced using the optimized vectors with special focus given to the creation and production of a chimeric broadly neutralizing anti-flavivirus antibody. The variable regions of this murine antibody, 2A10G6, were codon optimized and fused to a human IgG1. Analysis of the chimeric antibody showed that it was efficiently expressed in plants, can be purified to near homogeneity by a simple one-step purification process, retains its ability to recognize the Zika virus envelope protein, and induce an immune response against Zika virus. Two other monoclonal antibodies were produced at similar levels. This technology is versatile tool for the production of a wide spectrum of pharmaceutical multi-protein complexes in a fast, powerful, and cost-effective way.

The invention is based on an expression enhancer sequence derived from the RNA-2 genome segment of a bipartite RNA virus, in which a target initiation site in the RNA-2 genome segment has been mutated. Deletion of appropriate start codons upstream of the main RNA2 translation initiation can greatly increase in foreign protein accumulation without the need for viral replication. Also provided are methods, vectors and systems, including the ‘hyper-translatable’ Cowpea Mosaic Virus (“CPMV-HT”) based protein expression system.

The present invention provides plant virus vectors developed from the Sugarcane mosaic virus (SCMV). The vectors include a nucleic acid sequence encoding an infectious Sugarcane mosaic virus (SCMV) operably linked to one or more regulatory elements functional in a plant. The plant virus vectors may be used to infect monocot plants, such as maize, for gene expression applications.

An expression system is provided, including a first nucleic acid fragment containing a long intergenic region (LIR) derived from geminivirus, a small intergenic region (SIR) derived from geminivirus, and an expression cassette of a target protein linked between the LIR and the SIR and a second nucleic acid fragment containing an expression cassette of a Rep/RepA protein derived from geminivirus, in which the expression cassette of the target protein includes a promoter, a nucleic acid fragment encoding the target protein, and two or more linked terminators in this order.