scholarly journals Stability of Transgenic Resistance Against Plant Viruses

10.5772/33133 ◽  
2012 ◽  
Author(s):  
Nikon Vassilakos
Keyword(s):  
Plant Viruses ◽  
Transgenic Resistance

scholarly journals A critical appraisal of non conventional resistance to plant viruses

2002 ◽  
Vol 38 (SI 1 - 6th Conf EFPP 2002) ◽  
pp. S15-S20
Author(s):  
G.P. Martelli
Keyword(s):  
Plant Pathogens ◽  
Resistance Mechanisms ◽  
Plant Viruses ◽  
Natural Resistance ◽  
Transcriptional Gene Silencing ◽  
Infectious Agents ◽  
Transgenic Resistance ◽  
Genetic Incompatibility ◽  
Post Transcriptional Gene Silencing ◽  
Pathogen Derived Resistance

Among natural resistance mechanisms to plant pathogens, cultivar resistance has been extensively used in plant breeding to introduce what can be defined as “conventional” resistance to a number of them, including viruses. The necessity of overcoming the constraints of genetic incompatibility, so as to widen the range of possibile use of genetic control of infectious agents, has propitiated the utilization of biotechnological procedures, whereby “non conventional” or transgenic resistance was developed. Transgenic resistance to plant viruses encompasses the identification, cloning and tranferring into the recipient host of single viral genes, which gives rise to what is known as “pathogen-derived resistance” (PDR). Of the hypothesized mechanisms underlying expression of PDR, post-transcriptional gene silencing has been most extensively investigated in recent years. Despite of the success that virus-resistant cropping of transgenic plants begins to enjoy, in Europe there is still a widespread sentiment against agricultural biotechnologies and the use of genetically modified plants in particular. Yet, experimental evidence is accumulating that, in the case of PDR, the feared risks associated with genetic trasformation are minimal, if not negligible

scholarly journals Suppression of Potyvirus Infection by a Closterovirus Protein

2002 ◽  
Author(s):  
Valerian V. Dolja ◽  
Amit Gal-On ◽  
Victor Gaba
Keyword(s):  
Transgenic Plants ◽  
Molecular Mechanisms ◽  
Plant Viruses ◽  
Transgenic Resistance ◽  
Long Distance ◽  
Virus Family ◽  
Suggested Approach ◽  
Systemic Spread ◽  
Wide Range ◽  
Trans Gene

The plant virus family Polyviridae is the largest and most destructive of all plant viruses. Despite the continuous effort to develop resistant plant varieties, there is a desperate need for novel approaches conferring wide-range potyvirus resistance. Based on experiments with the tobacco etch potyvirus (TEV)-derived gene expression vector, we suggested approach for screening of the candidate resistance genes. This approach relies on insertion of the genes into a virus vector and evaluation of the phenotypes of the resulting recombinant viruses. The genes which suppress infection by the recombinant virus are selected as candidates for engineering transgenic resistance. Our analysis of the TEV variants expressing proteins of the beet yellows closterovirus (BYV) revealed that one of those, the leader proteinase (L-Pro), strongly and specifically interfered with the hybrid TEV infection. Since closterovirus L-Pro is evolutionary related to potyviral helper component-proteinase (HC-Pro), we suggested that the L-Pro interfered with HC-Pro function via a trans-dominant inhibitory effect. Based on these findings, we proposed to test two major hypotheses. First, we suggested that L-Pro-mediated suppression of potyvirus infection is a general phenomenon effective against a range of potyviruses. The second hypothesis stated that the suppression effect can be reproduced in transgenic plants expressing L-Pro, and can be utilized for generation of resistance to potyviruses. In accord with these hypotheses, we developed two original objectives of our proposal: A) to determine the range of the closterovirus-derived suppression of potyviral infection, and B) to try and utilize the L-Pro-mediated suppression for the development of transgenic resistance to potyviruses. In the first phase of the project, we have developed all major tools and technologies required for successful completion of the proposed research. These included TEV and ZYMV vectors engineered to express several closteroviral L-Pro variants, and generation of the large collection of transgenic plants. To our satisfaction, characterization of the infection phenotypes exhibited by chimeric TEV and ZYMV variants confirmed our first hypothesis. For instance, similar to TEV-L- Pro(BYV) chimera, ZYMV-L-Pro(LIYV) chimera was debilitated in its systemic spread. In contrast, ZYMV-GUS chimera (positive control) was competent in establishing vigorous systemic infection. These and other results with chimeric viruses indicated that several closteroviral proteinases inhibit long-distance movement of the potyviruses upon co-expression in infected plants. In order to complete the second objective, we have generated ~90 tobacco lines transformed with closteroviral L-Pro variants, as well as ~100 lines transformed with BYV Hsp70-homolog (Hsp70h; a negative control). The presence and expression of the trans gene in each line was initially confirmed using RT-PCR and RNA preparations isolated from plants. However, since detection of the trans gene-specific RNA can not guarantee production of the corresponding protein, we have also generated L-Pro- and Hsp70h-specific antisera using corresponding synthetic peptides. These antisera allowed us to confirm that the transgenic plant lines produced detectable, although highly variable levels of the closterovirus antigens. In a final phase of the project, we tested susceptibility of the transgenic lines to TEV infection. To this end, we determined that the minimal dilution of the TEV inoculum that is still capable of infecting 100% of nontransgenic plants was 1:20, and used 10 plants per line (in total, ~2,000 plants). Unfortunately, none of the lines exhibited statistically significant reduction in susceptibility. Although discouraging, this outcome prompted us to expand our experimental plan and conduct additional experiments. Our aim was to test if closteroviral proteinases are capable of functioning in trans. We have developed agroinfection protocol for BYV, and tested if co- expression of the L-Pro is capable of rescuing corresponding null-mutant. The clear-cut, negative results of these experiments demonstrated that L-Pro acts only in cis, thus explaining the lack of resistance in our transgenic plants. We have also characterized a collection of the L-Pro alanine- scanning mutants and found direct genetic evidence of the requirement for L-Pro in virus systemic spread. To conclude, our research supported by BARD confirmed one but not another of our original hypotheses. Moreover, it provided an important insight into functional specialization of the viral proteinases and generated set of tools and data with which we will be able to address the molecular mechanisms by which these proteins provide a variety of critical functions during virus life cycle.

Ectodesmata as Revealed By Freeze-Etch Preparations of Plant Epidermal Cell Walls

1973 ◽  
Vol 31 ◽  
pp. 468-469
Author(s):  
N.C. Lyon ◽  
W. C. Mueller
Keyword(s):  
Electron Microscopy ◽  
Acetic Acid ◽  
Nitric Acid ◽  
Oxalic Acid ◽  
Light Microscopy ◽  
Epidermal Cell ◽  
Cell Walls ◽  
Plant Viruses ◽  
Plant Leaves ◽  
Thin Sections

Schumacher and Halbsguth first demonstrated ectodesmata as pores or channels in the epidermal cell walls in haustoria of Cuscuta odorata L. by light microscopy in tissues fixed in a sublimate fixative (30% ethyl alcohol, 30 ml:glacial acetic acid, 10 ml: 65% nitric acid, 1 ml: 40% formaldehyde, 5 ml: oxalic acid, 2 g: mecuric chloride to saturation 2-3 g). Other workers have published electron micrographs of structures transversing the outer epidermal cell in thin sections of plant leaves that have been interpreted as ectodesmata. Such structures are evident following treatment with Hg++ or Ag+ salts and are only rarely observed by electron microscopy. If ectodesmata exist without such treatment, and are not artefacts, they would afford natural pathways of entry for applied foliar solutions and plant viruses.

Solid-phase immunoelectron microscopy for rapid diagnosis of enteroviruses

1984 ◽  
Vol 42 ◽  
pp. 226-227 ◽  
Author(s):  
K. Pegg-Feige ◽  
F. W. Doane
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Immunoelectron Microscopy ◽  
Detection Method ◽  
Solid Phase ◽  
Protein A ◽  
Plant Viruses ◽  
Rapid Diagnosis ◽  
Virus Diagnosis ◽  
Antiviral Antibody

Immunoelectron microscopy (IEM) applied to rapid virus diagnosis offers a more sensitive detection method than direct electron microscopy (DEM), and can also be used to serotype viruses. One of several IEM techniques is that introduced by Derrick in 1972, in which antiviral antibody is attached to the support film of an EM specimen grid. Originally developed for plant viruses, it has recently been applied to several animal viruses, especially rotaviruses. We have investigated the use of this solid phase IEM technique (SPIEM) in detecting and identifying enteroviruses (in the form of crude cell culture isolates), and have compared it with a modified “SPIEM-SPA” method in which grids are coated with protein A from Staphylococcus aureus prior to exposure to antiserum.

A Sensitive On-Grid Immunoelectron Microscopic Procedure for Diagnosis of Rotavirus Infection

1980 ◽  
Vol 38 ◽  
pp. 506-507
Author(s):  
M. F. Miller ◽  
A. R. Rubenstein
Keyword(s):  
Electron Microscopy ◽  
Specific Antibody ◽  
Immune Complexes ◽  
Acute Gastroenteritis ◽  
Plant Viruses ◽  
Aggregation Process ◽  
Microscopic Technique ◽  
Fecal Material ◽  
Present Communication ◽  
Number Of Particles

Studies of rotavirus particles in humans, monkeys and various non-primates with acute gastroenteritis have involved detection of virus in fecal material by electron microscopy. The EM techniques most commonly employed have been the conventional negative staining (Fig. 1) and immune aggregation (Fig. 2) procedures. Both methods are somewhat insensitive and can most reliably be applied to samples containing large quantities of virus either naturaLly or as a result of concentration by ultracentrifugation. The formation of immune complexes by specific antibody in the immune aggregation procedures confirms the rotavirus diagnosis, but the number of particles per given microscope field is effectively reduced by the aggregation process. In the present communication, we describe use of an on-grid immunoelectron microscopic technique in which rotavirus particles are mounted onto microscope grids that were pre-coated with specific antibody. The technique is a modification of a method originalLy introduced by Derrick (1) for studies of plant viruses.

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