Transient reprogramming of crop plants for agronomic performance

The development of a new crop variety is a time-consuming and costly process due to the reliance of plant breeding on gene shuffling to introduce desired genes into elite germplasm, followed by backcrossing. Here, we propose alternative technology that transiently targets various regulatory circuits within a plant, leading to operator-specified alterations of agronomic traits, such as time of flowering, vernalization requirement, plant height or drought tolerance. We redesigned techniques of gene delivery, amplification and expression around RNA viral transfection methods that can be implemented on an industrial scale and with many crop plants. The process does not involve genetic modification of the plant genome and is thus limited to a single plant generation, is broadly applicable, fast, tunable and versatile, and can be used throughout much of the crop cultivation cycle. The RNA-based reprogramming may be especially useful in plant pathogen pandemics but also for commercial seed production and for rapid adaptation of orphan crops

Clinical Study Clinical Safety and Immunogenicity of Tumor-Targeted, Plant-Made Id-KLH Conjugate Vaccines for Follicular Lymphoma

We report the first evaluation of plant-made conjugate vaccines for targeted treatment of B-cell follicular lymphoma (FL) in a Phase I safety and immunogenicity clinical study. Each recombinant personalized immunogen consisted of a tumor-derived, plantproduced idiotypic antibody (Ab) hybrid comprising the hypervariable regions of the tumor-associated light and heavy Ab chains, genetically grafted onto a common human IgG1 scaffold. Each immunogen was produced in Nicotiana benthamiana plants using twin magnICON vectors expressing the light and heavy chains of the idiotypic Ab. Each purified Ab was chemically linked to the carrier protein keyhole limpet hemocyanin (KLH) to form a conjugate vaccine. The vaccines were administered to FL patients over a series of ≥6 subcutaneous injections in conjunction with the adjuvant Leukine (GM-CSF). The 27 patients enrolled in the study had previously received non-anti-CD20 cytoreductive therapy followed by ≥4 months of immune recovery prior to first vaccination. Of 11 patients who became evaluable at study conclusion, 82% (9/11) displayed a vaccine-induced, idiotype-specific cellular and/or humoral immune response. No patients showed serious adverse events (SAE) related to vaccination. The fully scalable plant-based manufacturing process yields safe and immunogenic personalized FL vaccines that can be produced within weeks of obtaining patient biopsies

Airborne Signals from a Wounded Leaf Facilitate Viral Spreading and Induce Antibacterial Resistance in Neighboring Plants

Many plants release airborne volatile compounds in response to wounding due to pathogenic assault. These compounds serve as plant defenses and are involved in plant signaling. Here, we study the effects of pectin methylesterase (PME)-generated methanol release from wounded plants (“emitters”) on the defensive reactions of neighboring “receiver” plants. Plant leaf wounding resulted in the synthesis of PME and a spike in methanol released into the air. Gaseous methanol or vapors from wounded PME-transgenic plants induced resistance to the bacterial pathogen Ralstonia solanacearum in the leaves of non-wounded neighboring “receiver” plants. In experiments with different volatile organic compounds, gaseous methanol was the only airborne factor that could induce antibacterial resistance in neighboring plants. In an effort to understand the mechanisms by which methanol stimulates the antibacterial resistance of “receiver” plants, we constructed forward and reverse suppression subtractive hybridization cDNA libraries from Nicotiana benthamiana plants exposed to methanol. We identified multiple methanol-inducible genes (MIGs), most of which are involved in defense or cell-to-cell trafficking. We then isolated the most affected genes for further analysis: β-1,3-glucanase (BG), a previously unidentified gene (MIG-21), and non-cell-autonomous pathway protein (NCAPP). Experiments with Tobacco mosaic virus (TMV) and a vector encoding two tandem copies of green fluorescent protein as a tracer of cell-to-cell movement showed the increased gating capacity of plasmodesmata in the presence of BG, MIG-21, and NCAPP. The increased gating capacity is accompanied by enhanced TMV reproduction in the “receivers”. Overall, our data indicate that methanol emitted by a wounded plant acts as a signal that enhances antibacterial resistance and facilitates viral spread in neighboring plants

Plant Viral Vectors

X, 194 p. 33 illus., 19 illus. in color.online re

Self-fertile cybrids nicotiana tabacum (+hyoscyamus aureus) with a nucleo-plastome incompatibility

Cytoplasmic hybrids (cybrids) in a novel inter-generic combination, Nicotiana tabacum (+Hyoscyamus aureus), were generated by fusion of protoplasts from a plastome tobacco albino mutant (line R100a1) and %-irradiated green protoplasts of H. aureus. Cybrids possessed a plastome of H. aureus and a rearranged mitochondrial DNA. The cybrids displayed a syndrome of nucleo-plastome incompatibility expressed as a partial chlorophyll-deficiency of cotyledonary and true leaves at the early stage of vegetative development of plants grown from seeds in soil. During later development, the plants restored a normal green coloration. This character is phenotypically indistinguishable from the same syndrome in previously generated cybrids N. tabacum (+H. nigrum). In contrast to the cybrids N. tabacum (+H. nigrum), cybrids N. tabacum (+H. aureus) were self-fertile, and did not manifest other features that were interpreted as nucleo-mitochondrial incompatibilities in N. tabacum (+H. nigrum) plants. Therefore, the cybrids N. tabacum (+H. aureus) present a self-propagating system of Nicotiana (+Hyoscyamus) nucleo-plastome incompatibility in its pure form