Shirley Sato

RDR6 Has a Broad-Spectrum but Temperature-Dependent Antiviral Defense Role in Nicotiana benthamiana

ABSTRACT SDE1/SGS2/RDR6, a putative RNA-dependent RNA polymerase (RdRP) from Arabidopsis thaliana, has previously been found to be indispensable for maintaining the posttranscriptional silencing of transgenes, but it is seemingly redundant for antiviral defense. To elucidate the antiviral role of this RdRP in a different host plant and to evaluate whether plant growth conditions affect its role, we down-regulated expression of the Nicotiana benthamiana homolog, NbRDR6, and examined the plants for altered susceptibility to various viruses at different growth temperatures. The results we describe here clearly show that plants with reduced expression of NbRDR6 were more susceptible to all viruses tested and that this effect was more pronounced at higher growth temperatures. Diminished expression of NbRDR6 also permitted efficient multiplication of tobacco mosaic virus in the shoot apices, leading to serious disruption with microRNA-mediated developmental regulation. Based on these results, we propose that NbRDR6 participates in the antiviral RNA silencing pathway that is stimulated by rising temperatures but suppressed by virus-encoded silencing suppressors. The relative strengths of these two factors, along with other plant defense components, critically influence the outcome of virus infections.

Rapid and reproducible Agrobacterium-mediated transformation of sorghum

A rapid and reproducible Agrobacterium-mediated transformation protocol for sorghum has been developed. The protocol uses the nptII selectable marker gene with either of the aminoglycosides geneticin or paromomycin. A screen of various A. tumefaciens strains revealed that a novel C58 nopaline chromosomal background carrying the chrysanthopine disarmed Ti plasmid pTiKPSF2, designated NTL4/Chry5, was most efficient for gene transfer to sorghum immature embryos. A NTL4/Chry5 transconjugant harboring the pPTN290 binary plasmid, which carries nptII and GUSPlusTM expression cassettes, was used in a series of stable transformation experiments with Tx430 and C2-97 sorghum genotypes and approximately 80% of these transformation experiments resulted in the recovery of at least one transgenic event. The transformation frequencies among the successful experiments ranged from 0.3 to 4.5%, with the average transformation frequency being approximately 1% for both genotypes. Over 97% of the transgenic events were successfully established in the greenhouse and were fully fertile. Co-expression of GUSPlusTM occurred in 89% of the transgenic T0 events. Seed set for the primary transgenic plants ranged from 145 to 1400 seed/plant. Analysis of T1 progeny demonstrated transmission of the transgenes in a simple Mendelian fashion in the majority of events.

THE USE OF THE TWO T-DNA BINARY SYSTEM TO DERIVE MARKER-FREE TRANSGENIC SOYBEANS

SummaryA binary vector, pPTN133, was assembled that harbored two separate T-DNAs. T-DNA one contained a bar cassette, while T-DNA two carried a GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strain EHA101. Mature soybean cotyledonary node explants were inoculated and regenerated on medium amended with glufosinate. Transgenic soybeans were grown to maturity in the greenhouse. Fifteen primary transformants (T0) representing 10 independent events were characterized. Seven of the 10 independent T0 events co-expressed GUS. Progeny analysis was conducted by sowing the T1 seeds and monitoring the expression of the GUS gene after 21 d. Individual T1 plants were subsequently scored for herbicide tolerance by leaf painting a unifoliate leaf with a 100 mgl−1 solution of glufosinate and scoring the leaf 5 d post application. Herbicide-sensitive and GUS-positive individuals were observed in four of the 10 independent events. Southern blot analysis confirmed the absence of the bar gene in the GUS positive/herbicide-sensitive individuals. These results demonstrate that simultaneous integration of two T-DNAs followed by their independent segregation in progeny is a viable means to obtain soybeans that lack a selectable marker.

MutS HOMOLOG1 Is a Nucleoid Protein That Alters Mitochondrial and Plastid Properties and Plant Response to High Light

This work provides evidence, using genetic perturbation of the MSH1 nuclear gene in five plant species, that MSH1 functions within the mitochondrion and plastid to influence organellar genome behavior and plant growth patterns. Mitochondrial-plastid interdependence within the plant cell is presumed to be essential, but measurable demonstration of this intimate interaction is difficult. At the level of cellular metabolism, several biosynthetic pathways involve both mitochondrial- and plastid-localized steps. However, at an environmental response level, it is not clear how the two organelles intersect in programmed cellular responses. Here, we provide evidence, using genetic perturbation of the MutS Homolog1 (MSH1) nuclear gene in five plant species, that MSH1 functions within the mitochondrion and plastid to influence organellar genome behavior and plant growth patterns. The mitochondrial form of the protein participates in DNA recombination surveillance, with disruption of the gene resulting in enhanced mitochondrial genome recombination at numerous repeated sequences. The plastid-localized form of the protein interacts with the plastid genome and influences genome stability and plastid development, with its disruption leading to variegation of the plant. These developmental changes include altered patterns of nuclear gene expression. Consistency of plastid and mitochondrial response across both monocot and dicot species indicate that the dual-functioning nature of MSH1 is well conserved. Variegated tissues show changes in redox status together with enhanced plant survival and reproduction under photooxidative light conditions, evidence that the plastid changes triggered in this study comprise an adaptive response to naturally occurring light stress.

Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean

Enterotoxigenic Escherichia coli (ETEC) cause acute diarrhea in humans and farm animals, and can be fatal if the host is left untreated. As a potential alternative to traditional needle vaccination of cattle, we investigated the feasibility of expressing the major K99 fimbrial subunit, FanC, in soybean (Glycine max) for use as an edible subunit vaccine. As a first step in this developmental process, a synthetic version of fanC was optimized for expression in the cytosol and transferred to soybean via Agrobacterium-mediated transformation. Western analysis of T0 events revealed the presence of a peptide with the expected mobility for FanC in transgenic protein extracts, and immunofluorescense confirmed localization to the cytosol. Two T0 lines, which accumulated FanC to levels near 0.5% of total soluble protein, were chosen for further molecular characterization in the T1 and T2 generations. Mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing synthetic FanC developed significant antibody titers against bacterially derived FanC and produced antigen-specific CD4+ T lymphocytes, demonstrating the ability of transgenic FanC to function as an immunogen. These experiments are the first to demonstrate the expression and immunogenicity of a model subunit antigen in the soybean system, and mark the first steps toward the development of a K99 edible vaccine to protect against ETEC.

Robust RNAi-Based Resistance to Mixed Infection of Three Viruses in Soybean Plants Expressing Separate Short Hairpins from a Single Transgene

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.

Modulation of kernel storage proteins in grain sorghum( Sorghum bicolor (L.) Moench)

Sorghum prolamins, termed kafirins, are categorized into subgroups α, β, and γ. The kafirins are co-translationally translocated to the endoplasmic reticulum (ER) where they are assembled into discrete protein bodies that tend to be poorly digestible with low functionality in food and feed applications. As a means to address the issues surrounding functionality and digestibility in sorghum, we employed a biotechnology approach that is designed to alter protein body structure, with the concomitant synthesis of a co-protein in the endosperm fraction of the grain. Wherein perturbation of protein body architecture may provide a route to impact digestibility by reducing disulphide bonds about the periphery of the body, while synthesis of a co-protein, with known functionality attributes, theoretically could impact structure of the protein body through direct association and/or augment end-use applications of sorghum flour by stabilizing ß-sheet formation of the kafirins in sorghum dough preparations. This in turn may improve viscoelasticity of sorghum dough. To this end, we report here on the molecular and phenotypic characterizations of transgenic sorghum events that are down-regulated in γ- and the 29-kDa α-kafirins and the expression of a wheat Dy10/Dx 5 hybrid high-molecular weight glutenin protein. The results demonstrate that down-regulation of γ-kafirin alone does not alter protein body formation or impacts protein digestibility of cooked flour samples. However, reduction in accumulation of a predicted 29-kDa α-kafirin alters the morphology of protein body and enhances protein digestibility in both raw and cooked samples.

Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastids

Plastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll-specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath-specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon-optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid-related studies of wild-type and mutant maize plants and provide material from which different plastid types may be isolated.

IDENTIFICATION OF AN ELITE SORGHUM GENOTYPE WITH HIGH IN VITRO PERFORMANCE CAPACITY

SummaryThe use of plant genetic engineering to augment plant breeding programs is significantly strengthened if novel trait(s) can be introduced directly into elite germplasm. Implementing this technology to sorghum breeding programs has been hampered by the lack of an efficient and transferable protocol that is suitable with elite genotypes. This study was conducted to identify parameters that maximize in vitro culture performance in sorghum targeting a specific elite genotype, C2-97, which possesses enhanced agronomic characteristics. Three different tissue culture media formulations, MS, N6, and M11 were evaluated. M11 medium contains approximately 16% and 85% more total nitrogen and sevenfold and threefold higher levels of potassium phosphate than MS and N6 formulations, respectively. Culture performance of C2-97 across the three media formulations was compared to sorghum genotypes that were previously reported to be amenable to genetic engineering, namely Tx430, P898012. Bwheatland, and C401. Maximum embryogenesis induction was observed on M11 medium for all genotypes tested, with greater than 70% embryogenic calluses occurring on immature embryos derived from the C2-97 genotype cultured on M11 medium.

Expression of the rice CDPK-7 in sorghum: molecular and phenotypic analyses

Sorghum (Sorghum bicolor (L.) Moench) is an important source for food, feed, and possesses many agronomic attributes attractive for a biofuels feedstock. A warm season crop originating from the semi-arid tropics, sorghum is relatively susceptible to both cold and freezing stress. Enhancing the ability of sorghum to tolerate cold and freezing offers a route to expand the acreage for production, and provides a potential drought avoidance strategy during flowering, an important parameter for protection of yield. Targeted perturbation of the signal transduction pathway, that is triggered by exposure to abiotic stress in plants, has been demonstrated in model systems as an avenue to augment tolerance. Calcium-dependent protein kinases (CDPKs) are key players in a plant’s response to environmental assaults. To test the impact of modulating CDPK activity in sorghum as a means to enhanced abiotic stress tolerance, we introduced a constitutively expressed rice CDPK-7 (OsCDPK-7) gene construct. Sorghum transformants carrying this cassette, were not improved in cold or salt stress under the conditions tested. However, a lesion mimic phenotype and up-regulation of a number of pathogen related proteins, along with transcripts linked to photosynthesis were observed. These results demonstrate that modulating the Ca signaling cascade in planta via unregulated enhanced CDPK activity can lead to off-type effects likely due to the broadly integrated nature of these enzymes in signaling.