Publisher Summary Cucumber mosaic virus (CMV), the type member of the cucumovirus group, was first reported in 1916 as the causal agent of a disease of cucumber and muskmelon in Michigan and cucumber in New York. Since then, CMV has been found in most countries of the world, predominantly in the temperate zones, but increasingly more often in the tropical countries. CMV has the largest host range of any virus. The number of plant species identified as hosts for CMV has increased steadily over the past 60 years. The highlights of the more recent research include the following: (1) the complete nucleotide sequence of the genome of three strains of CMV has been determined, as well as nucleotide sequences of individual RNAs of eight other CMV strains, (2) the CMV replicase has been purified to homogeneity, and it functions in vitro to synthesize CMV RNA de novo , (3) infectious transcripts have been synthesized from full-length cDNA clones of the three strains of CMV, (4) these biologically active cDNAs are being used to map sequences involved in replication, movement, pathogenesis, resistance, and vector transmission. Biologically active cDNA clones of the satellite RNAs of CMV have been produced in seven laboratories and sequences involved in replication and pathogenicity have/are being identified, (5) finally, transgenic plants have been produced expressing either the CMV coat protein gene or satellite RNA sequences that show to protect such plants from infection by CMV. This chapter, while focusing on the more recent developments in CMV biology and biochemistry, also covers some of the same ground albeit in brief. The chapter presents a comprehensive review that can be used as a reference work for general virologists and plant pathologists, as well as those specializing in the molecular biology of CMV and/or other multicomponent plant viruses.

Cucumber Mosaic Virus

The invention provides for identification and use of certain chloroplast transit peptides for efficient processing and localization of dicamba monooxygenase (DMO) enzyme in transgenic plants. Methods for producing dicamba tolerant plants, methods for controlling weed growth, and methods for producing food, feed, and other products are also provided, as well as seed that confers tolerance to dicamba when it is applied pre- or post-emergence.

Chloroplast transit peptides for efficient targeting of DMO and uses thereof

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

Progeny of transgenic tobacco plants expressing the cucumber mosaic virus (CMV) coat protein (CP +) or its antisense transcript (antisense–CP) were protected from CMV infection. Symptom development and virus accumulation were reduced or absent in CP+ plants, independent of the strength of the inoculum. Antisense–CP plants were protected only at low inoculum concentrations. These results demonstrate that although it is possible to obtain protection in transgenic plants using antisense–CP message, this protection is not as effective as the CP–mediated protection.

Viral Protection in Transgenic Tobacco Plants Expressing the Cucumber Mosaic Virus Coat Protein or its Antisense RNA

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.

Exogenous application of pokeweed antiviral protein (PAP), a ribosome-inhibiting protein found in the cell walls of Phytolacca americana (pokeweed), protects heterologous plants from viral infection. A cDNA clone for PAP was isolated and introduced into tobacco and potato plants by transformation with Agrobacterium tumefaciens. Transgenic plants that expressed either PAP or a double mutant derivative of PAP showed resistance to infection by different viruses. Resistance was effective against both mechanical and aphid transmission. Analysis of the vacuum infiltrate of leaves expressing PAP showed that it is enriched in the intercellular fluid. Analysis of resistance in transgenic plants suggests that PAP confers viral resistance by inhibiting an early event in infection. Previous methods for creating virus-resistant plants have been specific for a particular virus or closely related viruses. To protect plants against more than one virus, multiple genes must be introduced and expressed in a single transgenic line. Expression of PAP in transgenic plants offers the possibility of developing resistance to a broad spectrum of plant viruses by expression of a single gene.

https://www.pnas.org/content/pnas/90/15/7089.full.pdf

Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein

Transgenic tobacco plants engineered to express either the potato virus X (PVX) coat protein (CP+) or the antisense coat protein transcript (CP-antisense) were protected from infection by PVX, as indicated by reduced lesion numbers on inoculated leaves, delay or absence of systemic symptom development and reduction in virus accumulation in both inoculated and systemic leaves. The extent of protection observed in CP+ plants primarily depended upon the level of expression of the coat protein. Plants expressing antisense RNA were protected only at low inoculum concentrations. The extent of this protection was even lower than that observed in plants expressing low levels of CP. In contrast to previous reports for plants expressing tobacco mosaic virus or alfalfa mosaic virus CP, inoculation of plants expressing high levels of PVX CP with PVX RNA did not overcome the protection. Specifically, lesion numbers on inoculated leaves and PVX levels on inoculated and systemtic leaves of the CP+ plants were reduced to a similar extent in both virus and RNA inoculated plants. Although these results do not rule out that CP-mediated protection involves inhibition of uncoating of the challenge virus, they suggest that PVX CP (or its RNA) can moderate early events in RNA infection by a different mechanism.

Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA.

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.

Wojciech K. Kaniewski

Papers

Viral Protection in Transgenic Tobacco Plants Expressing the Cucumber Mosaic Virus Coat Protein or its Antisense RNA

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

Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein

Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA.

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