Starch, a major storage metabolite in plants, positively affects the agricultural yield of a number of crops. Its biosynthetic reactions use adenosine diphosphate glucose (ADPGlc) as a substrate; ADPGlc pyrophosphorylase, the enzyme involved in ADPGlc formation, is regulated by allosteric effectors. Evidence that this plastidial enzyme catalyzes a rate-limiting reaction in starch biosynthesis was derived by expression in plants of a gene that encodes a regulatory variant of this enzyme. Allosteric regulation was demonstrated to be the major physiological mechanism that controls starch biosynthesis. Thus, plant and bacterial systems for starch and glycogen biosynthesis are similar and distinct from yeast and mammalian systems, wherein glycogen synthase has been demonstrated to be the rate-limiting regulatory step.

https://science.sciencemag.org/content/sci/258/5080/287.full.pdf

Regulation of the Amount of Starch in Plant Tissues by ADP Glucose Pyrophosphorylase

lhe Sainsbury Laboratory, John lnnes Centre, Colney, Norwich NR4 7UH, United Kingdom INTRODUCTION In 1985, Sanford and Johnston developed the simple and ele- gant concept of parasite- or pathogen-derived resistance (Sanford and Johnston, 1985). Subsequently, there have been numerous attempts to generate virus resistance in transgenic plants based on this concept through the expression of virus- derived genes or genome fragments (Beachy, 1993; Wilson, 1993; Baulcombe, 1994b; Lomonossoff, 1995). Many of these attempts have been successful, and some have led to the de- velopment of virus-resistant potato and squash cultivars for commercial application. In this article, the major emphasis is on the mechanisms of pathogen-derived resistance rather than on the practicali- ties of using this technology for crop improvement. These mechanisms have proven difficult to unravel largely because a resistance mechanism related to transgene silencing can override the direct phenotype of virus-derived transgenes. lhe examples discussed first are those in which it is clear whether the mechanism involves gene silencing. In later sections, some less well understood examples are reviewed. The final sec- tion anticipates likely future developments in pathogen-derived resistance.

Mechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants.

The common phenotype of different resistant plants are described and individual examples are reviewed in order to compare and contrast the characteristics of resistance to different viruses. The limited number of experiments that explore the fundamental basis of resistance are described. Ideas are presented for the types of experiments that are needed to more completely understand the mechanisms of resistance and to extend the resistance to other important plant viruses. Topics covered include: definition; strategy to create coat protein-mediated resistance; phenotype of coat protein-mediated resistance; examples of coat protein-mediated resistance; understanding the mechanisms of CP-mediated resistance; field tests of coat protein-mediated resistance; and conclusions and future directions

Coat Protein-Mediated Resistance Against Virus Infection

Plant defense responses to pathogen infection involve the production of active oxygen species, including hydrogen peroxide (H2O2). We obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H2O2 when glucose is oxidized. H2O2 levels were elevated in both leaf and tuber tissues of these plants. Transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora subsp carotovora, and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H2O2, because the resistance could be counteracted by exogenously added H2O2-degrading catalase. The transgenic plants with increased levels of H2O2 also exhibited enhanced resistance to potato late blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of an active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum disease resistance in plants.

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Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants.

We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/Trps16:PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regenera- tion system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastid-expressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1999.00508.x

Stable chloroplast transformation in potato : use of green fluorescent protein as a plastid marker

Potato virus X (PVX) and potato virus Y (PVY) infection in potato may result in the loss of certification of seed potatoes and affect quality and yield of potatoes in commercial production. We transformed a major commercial cultivar of potato, Russet Burbank, with the coat protein genes of PVX and PVY. Transgenic plants that expressed both CP genes were resistant to infection by PVX and PVY by mechanical inoculation. One line was also resistant when PVY was inoculated with viruliferous green peach aphids. These experiments demonstrate that CP protection is effective against mixed infection by two different viruses and against mechanical and aphid transmission of PVY.

Engineering resistance to mixed virus infection in a commercial potato cultivar: resistance to potato virus X and potato virus Y in transgenic Russet Burbank.

Transgenic Russet Burbank potato plants expressing the coat protein genes of potato virus X (PVX) and potato virus Y (PVY) were transplanted into the field after propagation in tissue culture. Virus resistance, plant growth and tuber yield were determined. Our results show that expression of PVX and PVY CP genes confer a very high level of resistance to PVX and PVY infection in clone 303, one of the four different transgenic potato clones tested. After inoculation with PVX and PVY, tuber yield of control Russet Burbank plants decreased, while the yield of clone 303 was unaffected. Four different uninoculated transgenic clones had tuber yields as high as control Russet Burbank. These results confirm that resistance to PVX and PVY is effective in the field and can prevent yield losses due to dual infection by these viruses.

Field resistance of transgenic russet burbank potato to effects of infection by potato virus X and potato virus Y

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. 'Russet Burbank'. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100 mg/l kanamycin monosulfate. Regenerated shoots were assayed for kanamycin resistance by placing stem segments on callus induction medium containing an increased level of kanamycin. Of a total 255 regenerated shoots, 47 (18%) were kanamycin resistant. Of the kanamycin resistant shoots, 25 (53%) expressed the PVX or PVY coat protein genes as assayed by enzyme-linked immunosorbent assay or Western immunoblot analysis.

Agrobacterium-mediated transformation of Solanum tuberosum L. cv. 'Russet Burbank'.

Transgenic plants expressing the coat protein (CP) of alfalfa mosaic virus (AIMV) are resistant to infection by AIMV. A mutation was introduced into the second amino acid of the cDNA for the CP of AIMV. Three different transgenic tobacco lines expressing the mutant CP and two different transgenic tobacco lines expressing the wild-type CP at similar levels were challenged with AIMV virions and viral RNA. Whereas the lines expressing the wild-type CP were highly resistant to infection by AIMV virions and viral RNA, the lines expressing the mutant CP were susceptible to infection by both. The binding affinity of the mutant and the wild-type CPs for the 3' terminal protein binding site on AIMV RNAs was similar, as determined by electrophoretic mobility shift assay. A mixture of AIMV genomic RNAs 1-3 was infectious on the plants expressing the mutant CP but not on vector control plants or plants expressing the wild-type CP, indicating that the mutant CP can activate the AIMV genomic RNAs for infection. These results demonstrate that the second amino acid of the AIMV CP is critical for protection from AIMV but not for the initial interaction between the AIMV RNA and CP, suggesting that this initial interaction does not play a major role in CP-mediated protection.

Lisa Haley

Papers

Engineering resistance to mixed virus infection in a commercial potato cultivar: resistance to potato virus X and potato virus Y in transgenic Russet Burbank.

Field resistance of transgenic russet burbank potato to effects of infection by potato virus X and potato virus Y

Agrobacterium-mediated transformation of Solanum tuberosum L. cv. 'Russet Burbank'.

The second amino acid of alfalfa mosaic virus coat protein is critical for coat protein-mediated protection.

Genetically engineered resistance: transgenic plants.