James E. Lincoln

Expression of the antiapoptotic baculovirus p35 gene in tomato blocks programmed cell death and provides broad-spectrum resistance to disease

The sphinganine analog mycotoxin, AAL-toxin, induces a death process in plant and animal cells that shows apoptotic morphology. In nature, the AAL-toxin is the primary determinant of the Alternaria stem canker disease of tomato, thus linking apoptosis to this disease caused by Alternaria alternata f. sp. lycopersici. The product of the baculovirus p35 gene is a specific inhibitor of a class of cysteine proteases termed caspases, and naturally functions in infected insects. Transgenic tomato plants bearing the p35 gene were protected against AAL-toxin-induced death and pathogen infection. Resistance to the toxin and pathogen co-segregated with the expression of the p35 gene through the T3 generation, as did resistance to A. alternata, Colletotrichum coccodes, and Pseudomonas syringae pv. tomato. The p35 gene, stably transformed into tomato roots by Agrobacterium rhizogenes, protected roots against a 30-fold greater concentration of AAL-toxin than control roots tolerated. Transgenic expression of a p35 binding site mutant (DQMD to DRIL), inactive against animal caspases-3, did not protect against AAL-toxin. These results indicate that plants possess a protease with substrate-site specificity that is functionally equivalent to certain animal caspases. A biological conclusion is that diverse plant pathogens co-opt apoptosis during infection, and that transgenic modification of pathways regulating programmed cell death in plants is a potential strategy for engineering broad-spectrum disease resistance in plants.

Diverse mechanisms for the regulation of ethylene-inducible gene expression

SummaryWe have investigated the mechanism of action of the plant hormone ethylene by analyzing the expression of ethylene-inducible genes isolated from tomato (Lycopersicon esculentum). We have found that the expression of each cloned gene is regulated by ethylene in a unique manner. That is, for certain genes ethylene affects transcriptional processes, while for another gene it affects both transcriptional and post-transcriptional processes. Furthermore, induction of gene transcription by ethylene is organ specific for one gene, while for others it is not. In addition, we have measured gene expression as a function of ethylene concentration and have found that each gene displays a unique ethylene dose-response curve. Our results suggest that ethylene modulates gene expression by a variety of mechanisms.

Regulation of Gene Expression by Ethylene

In plants, many processes are regulated by a small number of hormones: auxin, abscisic acid, cytokinin, gibberellin, and ethylene (for review, see Wareing and Phillips, 1981). Plant hormones, like animal hormones, are active in extremely small quantities and influence the growth and differentiation of tissues and organs. However, certain important aspects about plant hormone action are unique. That is, each plant hormone is synthesized in many parts of the plant and each regulates a wide variety of different processes depending on the organ or tissue. Furthermore, plants regulate many processes by modulating both hormone concentration and sensitivity to the hormone (Trewavas, 1982; Trewavas et al., 1983). Thus, understanding the mechanism of plant hormone action involves knowing how both hormone concentration and tissue sensitivity regulate cellular differentiation.

Plant and animal PR1 family members inhibit programmed cell death and suppress bacterial pathogens in plant tissues: PR1 inhibits programmed cell death

A role for programmed cell death (PCD) has been established as the basis for plant-microbe interactions. A functional plant-based cDNA library screen identified possible anti-PCD genes, including one member of the PR1 family, designated P14a, from tomato. Members of the PR1 family have been subject to extensive research in view of their possible role in resistance against pathogens. The PR1 family is represented in every plant species studied to date and homologues have been found in animals, fungi and insects. However, the biological function of the PR1 protein from plants has remained elusive in spite of extensive research regarding a role in the response of plants to disease. Constitutive expression of P14a in transgenic tomato roots protected the roots against PCD triggered by Fumonisin B1, as did the human orthologue GLIPR1, indicating a kingdom crossing function for PR1. Tobacco plants transformed with a P14a-GFP fusion construct and inoculated with Pseudomonas syringae pv. tabaci revealed that the mRNA was abundant throughout the leaves, but the fusion protein was restricted to the lesion margins, where cell death and bacterial spread were arrested. Vitus vinifera grapes expressing the PR1 homologue P14a as a transgene were protected against the cell death symptoms of Pierces disease. A pull-down assay identified putative PR1-interacting proteins, including members of the Rac1 immune complex, known to function in innate immunity in rice and animal systems. The findings herein are consistent with a role of PR1 in the suppression of cell death-dependent disease symptoms and a possible mode of action.

Construction of a Plant Transient Expression Vector which Coexpresses the Marker β-glucuronidase

We have developed a plant transient expression vector that simultaneously expresses β-glucuronidase from a Cauliflower mosaic virus promoter, and a test gene from a Figwort mosaic virus promoter. This vector, which is manipulated in E. coli, allows the testing of cell death inducing genes in plant cells. We have demonstrated the capability of this vector by expressing diphtheria toxin.