Roger N. Beachy

Particle bombardment and the genetic enhancement of crops: myths and realities

DNA transfer by particle bombardment makes use of physical processes to achieve the transformation of crop plants. There is no dependence on bacteria, so the limitations inherent in organisms such as Agrobacterium tumefaciens do not apply. The absence of biological constraints, at least until DNA has entered the plant cell, means that particle bombardment is a versatile and effective transformation method, not limited by cell type, species or genotype. There are no intrinsic vector requirements so transgenes of any size and arrangement can be introduced, and multiple gene cotransformation is straightforward. The perceived disadvantages of particle bombardment compared to Agrobacterium-mediated transformation, i.e. the tendency to generate large transgene arrays containing rearranged and broken transgene copies, are not borne out by the recent detailed structural analysis of transgene loci produced by each of the methods. There is also little evidence for major differences in the levels of transgene instability and silencing when these transformation methods are compared in agriculturally important cereals and legumes, and other non-model systems. Indeed, a major advantage of particle bombardment is that the delivered DNA can be manipulated to influence the quality and structure of the resultant transgene loci. This has been demonstrated in recently reported strategies that favor the recovery of transgenic plants containing intact, single-copy integration events, and demonstrating high-level transgene expression. At the current time, particle bombardment is the most efficient way to achieve plastid transformation in plants and is the only method so far used to achieve mitochondrial transformation. In this review, we discuss recent data highlighting the positive impact of particle bombardment on the genetic transformation of plants, focusing on the fate of exogenous DNA, its organization and its expression in the plant cell. We also discuss some of the most important applications of this technology including the deployment of transgenic plants under field conditions.

Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants.

A chimeric gene encoding the alfalfa mosaic virus (AlMV) coat protein was constructed and introduced into tobacco and tomato plants using Ti plasmid‐derived plant transformation vectors. The progeny of the self‐fertilized transgenic plants were significantly delayed in symptom development and in some cases completely escaped infection after inoculated with AlMV. The inoculated leaves of the transgenic plants had significantly reduced numbers of lesions and accumulated substantially lower amounts of coat protein due to virus replication than the control plants. These results show that high level expression of the chimeric viral coat protein gene confers protection against AlMV, which differs from other plant viruses in morphology, genome structure, gene expression strategy and early steps in viral replication. Based on our results with AlMV and those reported earlier for tobacco mosaic virus, it appears that genetically engineered cross‐protection may be a general method for preventing viral disease in plants.

Degradation of Tobacco Mosaic Virus Movement Protein by the 26S Proteasome

ABSTRACT Cell-to-cell spread of tobacco mosaic virus is facilitated by the virus-encoded 30-kDa movement protein (MP). This process involves interaction of viral proteins with host components, including the cytoskeleton and the endoplasmic reticulum (ER). During virus infection, high-molecular-weight forms of MP were detected in tobacco BY-2 protoplasts. Inhibition of the 26S proteasome by MG115 andclasto-lactacystin-β-lactone enhanced the accumulation of high-molecular-weight forms of MP and led to increased stability of the MP. Such treatment also increased the apparent accumulation of polyubiquitinated host proteins. By fusion of MP with the jellyfish green fluorescent protein (GFP), we demonstrated that inhibition of the 26S proteasome led to accumulation of the MP-GFP fusion preferentially on the ER, particularly the perinuclear ER. We suggest that polyubiquitination of MP and subsequent degradation by the 26S proteasome may play a substantial role in regulation of virus spread by reducing the damage caused by the MP on the structure of cortical ER.

Reduced Photosystem II Activity and Accumulation of Viral Coat Protein in Chloroplasts of Leaves Infected with Tobacco Mosaic Virus.

We previously reported (A Reinero, RN Beachy 1986 Plant Mol Biol 6:291-301) that coat protein (CP) of tobacco mosaic virus (TMV) accumulates in chloroplasts of systemically infected leaves. To determine the significance of such interaction we examined electron transport rates in chloroplasts containing different levels of TMV-CP. Tobacco (Nicotiana tabacum L.) plants were infected with either a TMV strain inducing chlorosis or with a strain inducing mild symptoms, and both the accumulation pattern of TMV-CP inside chloroplasts as well as the rates of photosynthetic electron transport were followed. The CP of the TMV strain inducing chlorosis was detected inside chloroplasts 3 days after infection, and thereafter accumulated at a rapid rate, first in the stroma and then in the thylakoid membranes. On the other hand, the CP of the TMV strain that caused only mild symptoms accumulated in chloroplasts to lower levels and little CP was associated with the thylakoids. In vivo and in vitro measurements of electron transport revealed that photosystem II activity was inhibited in plants infected with the aggressive TMV strain while no reduction was observed in plants infected with the mild strain. The capacity of chloroplasts to synthesize proteins was equivalent in organelles isolated from healthy and virus-infected leaves. The possibility that a large accumulation of TMV-CP inside chloroplasts may affect photosynthesis in virus-infected plants by inhibiting photosystem II activity is discussed.

Functional organization of the cassava vein mosaic virus (CsVMV) promoter

Cassava vein mosaic virus (CsVMV) is a pararetrovirus that infects cassava plants in Brazil. A promoter fragment isolated from CsVMV, comprising nucleotides -443 to +72, was previously shown to direct strong constitutive gene expression in transgenic plants. Here we report the functional architecture of the CsVMV promoter fragment. A series of promoter deletion mutants were fused to the coding sequence of uidA reporter gene and the chimeric genes were introduced into transgenic tobacco plants. Promoter activity was monitored by histochemical and quantitative assays of β-glucuronidase activity (GUS). We found that the promoter fragment is made up of different regions that confer distinct tissue-specific expression of the gene. The region encompassing nucleotides -222 to -173 contains cis elements that control promoter expression in green tissues and root tips. Our results indicate that a consensus as1 element and a GATA motif located within this region are essential for promoter expression in those tissues. Expression from the CsVMV promoter in vascular elements is directed by the region encompassing nucleotides -178 to -63. Elements located between nucleotides -149 and -63 are also required to activate promoter expression in green tissues suggesting a combinatorial mode of regulation. Within the latter region, a 43 bp fragment extending from nucleotide -141 to -99 was shown to interact with a protein factor extracted from nuclei of tobacco seedlings. This fragment showed no sequence homology with other pararetrovirus promoters and hence may contain CsVMV-specific regulatory cis elements.

Complete nucleotide sequence of the geminivirus tomato yellow leaf curl virus, Thailand isolate.

The complete genome of a Thailand isolate of the geminivirus tomato yellow leaf curl virus (TYLCV-Th) has been cloned and the nucleotide sequence determined. The genome consists of two DNAs each slightly greater than 2700 nucleotides in length and designated A-DNA and B-DNA. The A-DNA contains six open reading frames (ORFs) capable of encoding proteins with M(r)s greater than 10K; two ORFs were located on the B-DNA. The predicted ORFs were found at positions on the genome similar to those identified in other geminiviruses. Based upon computer-assisted sequence comparisons with other geminiviruses, TYLCV-Th maintained the greatest degree of similarity with a tomato pathogen isolated in Australia, tomato leaf curl virus. Furthermore, sequence comparisons with a large number of geminiviruses confirm further divisions of the geminiviruses based upon geographical proximity of the viruses. Agroinoculation of Nicotiana benthamiana with a TYLCV-Th A-DNA clone containing a mutation in the capsid protein ORF demonstrated that the coat protein is not essential for TYLCV-Th infection but enhances the progression of the disease.

Rice tungro disease is caused by an RNA and a DNA virus.

We present evidence that rice tungro spherical virus (RTSV) has a genome of polyadenylated single-stranded RNA of about 10 kb whereas rice tungro bacilliform virus (RTBV) contains double-stranded circular DNA. RTBV DNA has been mapped and shown to have two discontinuities, one in each strand, at specific sites; it thus resembles that of the caulimoviruses. Gel electrophoresis of RTSV preparations revealed two protein bands (Mr 35K and 26K). RTBV yielded two major protein bands of 37K and 33K together with several minor species of higher and lower Mr which react with antiviral serum.

The TMV movement protein: Role of the C-terminal 73 amino acids in subcellular localization and function

The role of the C-terminal one-third of the tobacco mosaic virus (TMV) 30-kDa movement protein (MP) on its subcellular localization and on virus spread was investigated. We have constructed eight cDNAs encoding MPs with variable size deletions from the C-terminal end. Expression of the truncated proteins was verified in recombinant yeast using an antiserum directed to a synthetic peptide corresponding to 21 amino acids near the N-terminal end of the MP. In transgenic tobacco plants, MP from which more than 55 amino acids were deleted no longer accumulated in the cell wall fraction of a cellular extract, where the complete MP accumulates. Dye diffusion studies showed that both unmodified and modified MPs that accumulate in the cell wall fraction are able to alter plasmodesmatal size exclusion limits. Biological function of the modified MPs was tested in the transgenic plants with the TMV thermosensitive mutant Ls1 and a TMV genomic RNA transcript lacking a functional MP. There was a correlation between the cell wall localization of the modified MPs and its ability to potentiate virus spread. The results presented here demonstrate the dispensability of the C-terminal 55 amino acids of the MP in its subcellular localization in tobacco plants and its role in virus movement. Moreover, our results show that a stretch of 19 amino acids (195 to 213) is essential for localization of the MP to the cell wall fraction of plant cells.

Development of Tobacco Mosaic Virus Infection Sites in Nicotiana benthamiana

To monitor infection of Nicotiana benthamiana by tobacco mosaic virus (TMV), leaves were inoculated with viral constructs expressing the green fluorescent protein (GFP) from jellyfish (Aequorea victoria) fused to the movement protein (MP) of TMV (MP:GFP) or as a free GFP in place of the coat protein (CP). Infection sites produced by TMV expressing the MP:GFP appeared as fluorescent rings larger in diameter and less fluorescent than fluorescent disks induced by constructs encoding free GFP. These results suggest that protein expression driven by the MP subgenomic promoter (sgp) initiates and ends earlier and is at lower level than that observed for proteins driven by the CP sgp. Similarly, analyses of cross sections through the infection sites revealed that in different cell types the accumulation of MP:GFP was regulated differently than the accumulation of free GFP. Immunocytochemistry and electron microscopy showed that near the leading edge of the fluorescent ring the MP:GFP and the viral 126 kDa and 18...

Partial desiccation of mature embryo-derived calli, a simple treatment that dramatically enhances the regeneration ability of indica rice.

Regeneration of indica rice varieties remains a limiting factor for researchers undertaking rice Iransformation experiments. As reported for japonica rice and other crops, partial desiccation of indica rice calli dramatically promotes organogenesis and leads to high regeneration ability. We are now able to obtain 66.5%, 61.1% and 73.7% of calli that regenerate plants for the indica varieties TN1, IR72 and IR64 whereas in non desiccated controls only 30.0%, 15.5% and 18.7% of calli regenerated, respectively. Plants obtained were phenotypically normal and 50% were highly fertile. Partial desiccation is a reliable and simple method for improving indica rice regeneration. It also shortens the time of in vitro culture.