Thomas E. Clemente

Abrogation of disease development in plants expressing animal antiapoptotic genes

An emerging topic in plant biology is whether plants display analogous elements of mammalian programmed cell death during development and defense against pathogen attack. In many plant–pathogen interactions, plant cell death occurs in both susceptible and resistant host responses. For example, specific recognition responses in plants trigger formation of the hypersensitive response and activation of host defense mechanisms, resulting in restriction of pathogen growth and disease development. Several studies indicate that cell death during hypersensitive response involves activation of a plant-encoded pathway for cell death. Many susceptible interactions also result in host cell death, although it is not clear how or if the host participates in this response. We have generated transgenic tobacco plants to express animal genes that negatively regulate apoptosis. Plants expressing human Bcl-2 and Bcl-xl, nematode CED-9, or baculovirus Op-IAP transgenes conferred heritable resistance to several necrotrophic fungal pathogens, suggesting that disease development required host–cell death pathways. In addition, the transgenic tobacco plants displayed resistance to a necrogenic virus. Transgenic tobacco harboring Bcl-xl with a loss-of-function mutation did not protect against pathogen challenge. We also show that discrete DNA fragmentation (laddering) occurred in susceptible tobacco during fungal infection, but does not occur in transgenic-resistant plants. Our data indicate that in compatible plant–pathogen interactions apoptosis-like programmed cell death occurs. Further, these animal antiapoptotic genes function in plants and should be useful to delineate resistance pathways. These genes also have the potential to generate effective disease resistance in economically important crops.

Copy Number Variation of Multiple Genes at Rhg1 Mediates Nematode Resistance in Soybean

Resistance to a damaging disease of soybean is conferred by a cluster of linked genes present in multiple copies. The rhg1-b allele of soybean is widely used for resistance against soybean cyst nematode (SCN), the most economically damaging pathogen of soybeans in the United States. Gene silencing showed that genes in a 31-kilobase segment at rhg1-b, encoding an amino acid transporter, an α-SNAP protein, and a WI12 (wound-inducible domain) protein, each contribute to resistance. There is one copy of the 31-kilobase segment per haploid genome in susceptible varieties, but 10 tandem copies are present in an rhg1-b haplotype. Overexpression of the individual genes in roots was ineffective, but overexpression of the genes together conferred enhanced SCN resistance. Hence, SCN resistance mediated by the soybean quantitative trait locus Rhg1 is conferred by copy number variation that increases the expression of a set of dissimilar genes in a repeated multigene segment. Soybean crops, which supply valuable protein, oil, and renewable fuel, are under attack by a nematode for which there is no effective pesticide. Instead, agriculture relies on resistance derived from a genetic locus, which is now represented in most of the soybean crops cultivated in the United States. Cook et al. (p. 1206, published online 11 October) elucidated the mechanisms by which this rhg1-b allele protects against disease. The region carries several genes, none of which resemble other known immune receptor genes. Experiments silencing one or another of the genes showed that the genes work as a cluster. However, one set of the genes is not enough: Plants need multiple repeats of the locus to acquire resistance.

Soybean Oil: Genetic Approaches for Modification of Functionality and Total Content

World consumption of soybean ( Glycine max ) in 2008 was over 221 million metric tons, with approximately 50% of this supply coming from U.S. production, where soybean plantings on an annual basis are over 77 million ha. Soybeans are desired on the marketplace as a valuable source of protein and oil

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.

Dicamba resistance: enlarging and preserving biotechnology-based weed management strategies.

The advent of biotechnology-derived, herbicide-resistant crops has revolutionized farming practices in many countries. Facile, highly effective, environmentally sound, and profitable weed control methods have been rapidly adopted by crop producers who value the benefits associated with biotechnology-derived weed management traits. But a rapid rise in the populations of several troublesome weeds that are tolerant or resistant to herbicides currently used in conjunction with herbicide-resistant crops may signify that the useful lifetime of these economically important weed management traits will be cut short. We describe the development of soybean and other broadleaf plant species resistant to dicamba, a widely used, inexpensive, and environmentally safe herbicide. The dicamba resistance technology will augment current herbicide resistance technologies and extend their effective lifetime. Attributes of both nuclear- and chloroplast-encoded dicamba resistance genes that affect the potency and expected durability of the herbicide resistance trait are examined.

The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean

The soybean transformation procedure using the Agrobacterium-cotyledonary node transformation system and the bar gene as the selectable marker coupled with glufosinate as a selective agent is described. Soybean cotyledonary explants were derived from 5 day old seedlings and co-cultivated with Agrobacterium tumefaciens for 3 days. Explants were cultured on Gamborgs B5 medium supplemented with 1.67 mg l-1 BAP and glufosinate at levels of 3.3 mg l-1 or 5.0 mg l-1 for 4 weeks. After 4 weeks explants were subcultured to medium containing MS major and minor salts and B5 vitamins (MS/B5) supplemented with 1.0 mg l-1 zeatin-riboside, 0.5 mg l-1 GA3 and 0.1 mg l-1 IAA amended with 1.7 mg l-1 or 2.0 mg l-1 glufosinate. Elongated shoots were rooted on a MS/B5 rooting medium supplemented with 0.5 mg l-1 NAA without further glufosinate selection. Plantlets were transplanted to soil and grown to maturity and set seed in the greenhouse. Primary transformants and their progeny were characterized by Southern blot analysis and a leaf paint assay.

Parallel domestication of the Shattering1 genes in cereals

A key step during crop domestication is the loss of seed shattering. Here, we show that seed shattering in sorghum is controlled by a single gene, Shattering1 (Sh1), which encodes a YABBY transcription factor. Domesticated sorghums harbor three different mutations at the Sh1 locus. Variants at regulatory sites in the promoter and intronic regions lead to a low level of expression, a 2.2-kb deletion causes a truncated transcript that lacks exons 2 and 3, and a GT-to-GG splice-site variant in the intron 4 results in removal of the exon 4. The distributions of these non-shattering haplotypes among sorghum landraces suggest three independent origins. The function of the rice ortholog (OsSh1) was subsequently validated with a shattering-resistant mutant, and two maize orthologs (ZmSh1-1 and ZmSh1-5.1+ZmSh1-5.2) were verified with a large mapping population. Our results indicate that Sh1 genes for seed shattering were under parallel selection during sorghum, rice and maize domestication.

New frontiers in oilseed biotechnology: meeting the global demand for vegetable oils for food, feed, biofuel, and industrial applications.

Vegetable oils have historically been a valued commodity for food use and to a lesser extent for non-edible applications such as detergents and lubricants. The increasing reliance on biodiesel as a transportation fuel has contributed to rising demand and higher prices for vegetable oils. Biotechnology offers a number of solutions to meet the growing need for affordable vegetable oils and vegetable oils with improved fatty acid compositions for food and industrial uses. New insights into oilseed metabolism and its transcriptional control are enabling biotechnological enhancement of oil content and quality. Alternative crop platforms and emerging technologies for metabolic engineering also hold promise for meeting global demand for vegetable oils and for enhancing nutritional, industrial, and biofuel properties of vegetable oils.

Construction of a Derivative of Agrobacterium tumefaciens C58 That Does Not Mutate to Tetracycline Resistance

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain. By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.

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.