Fernando Ponz

A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens

A method for the detection of RNA viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (RT-PCR) and quantitation of the amplified products by fluorescence. The reverse transcription of the RNA is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. The whole procedure can be carried out in a microtiter plate. Its validity has been successfully confirmed for the detection of bean yellow mosaic virus, cherry leafroll virus, cucumber mosaic virus, citrus tristeza virus, grapevine fanleaf virus, potato leafroll virus, pepper mild mottle virus, and tomato spotted wilt virus, as well as the satellite RNA of cucumber mosaic virus and potato spindle tuber viroid. In this procedure virus-specific antibodies can be replaced by monoclonal antibodies against double-stranded RNA, thus offering the possibility of detection when no specific virus antibodies are available, or immunological methods are difficult to use (i.e., subviral pathogens like satellite-RNAs or viroids). The method described has the typical sensitivity of assays based on the polymerase chain reaction, it is not more laborious than ELISA, and an equivalent degree of automation is possible.

The Dual Role of the Potyvirus P3 Protein of Turnip mosaic virus as a Symptom and Avirulence Determinant in Brassicas

Two isolates of the potyvirus Turnip mosaic virus (TuMV), UK 1 and CDN 1, differ both in their general symptoms on the susceptible propagation host Brassica juncea and in their ability to infect B. napus lines possessing a variety of dominant resistance genes. The isolate CDN 1 produces a more extreme mosaic in infected brassica leaves than UK 1 and is able to overcome the resistance genes TuRB01, TuRB04, and TuRB05. The resistance gene TuRB03, in the B. napus line 22S, is effective against CDN 1 but not UK 1. The nucleic acid sequences of the UK 1 and CDN 1 isolates were 90% identical. The C-terminal half of the P3 protein was identified as being responsible for the differences in symptoms in B. juncea. A single amino acid in the P3 protein was found to be the avirulence determinant for TuRB03. Previous work already has identified the P3 as an avirulence determinant for TuRB04. Our results increase the understanding of the basis of plant-virus recognition, show the importance of the potyviral P3 gene as a symptom determinant, and provide a role in planta for the poorly understood P3 protein in a normal infection cycle.

The cylindrical inclusion gene of Turnip mosaic virus encodes a pathogenic determinant to the brassica resistance gene TuRB01.

The viral component of Turnip mosaic virus (TuMV) determining virulence to the Brassica napus TuRB01 dominant resistance allele has been identified. Sequence comparisons of an infectious cDNA clone of the UK 1 isolate of TuMV (avirulent on TuRB01) and a spontaneous mutant capable of infecting plants possessing TuRB01 suggested that a single nucleotide change in the cylindrical inclusion (CI) protein coding region (gene) of the virus was responsible for the altered phenotype. A second spontaneous mutation involved a different change in the CI gene. The construction of chimeric genomes and subsequent inoculations to plant lines segregating for TuRB01 confirmed the involvement of the CI gene in this interaction. Site-directed mutagenesis of the viral coat protein (CP) gene at the ninth nucleotide was carried out to investigate its interaction with TuRB01. The identity of this nucleotide in the CP gene did not affect the outcome of the viral infection. Both mutations identified in the CI gene caused amino acid changes in the C terminal third of the protein, outside any of the conserved sequences reported to be associated with helicase or cell-to-cell transport activities. This is the first example of a potyvirus CI gene acting as a determinant for a genotype-specific resistance interaction.

A fitness cost for Turnip mosaic virus to overcome host resistance.

The relative fitness of the Turnip mosaic virus (TuMV) isolate UK 1 was compared with that of two other wildtype isolates CZE 1 and CDN 1. The isolates CZE 1 and CDN 1 are able to overcome the effect of the resistance gene TuRB01 and at least three other resistance sources that are effective against UK 1. Comparisons were also made between the fitness of UK 1 and a recombinant virus with a single nucleotide change (v35Tunos +5570 A>G) conferring the ability to overcome TuRB01 resistance. Co-inoculation experiments were carried out where pairs of isolates were serially passaged over 5 months in a plant line possessing no known resistance genes in order to examine the relative fitness of the isolates. In each case, UK 1 dominated the mixture in the susceptible host background. It out-competed CZE 1 and v35Tunos +5570 A>G within four passages, and CDN 1 after one passage. The greater fitness of UK 1 suggests that there may be a fitness cost to TuMV overcoming resistance genes of brassica crops. This may shed some light on the frequency of naturally occurring isolates, in that pathotype 1 isolates are found much more frequently than isolates of other pathotypes. Implications for the deployment of TuRB01 are discussed.

Survey of Bovine Enterovirus in Biological and Environmental Samples by a Highly Sensitive Real-Time Reverse Transcription-PCR

ABSTRACT Animal enteroviruses shed in the feces of infected animals are likely environmental contaminants and thus can be used as indicators of animal fecal pollution. Previous work has demonstrated that bovine enterovirus (BEV) present in bovine feces contaminates waters adjacent to cattle herds and that BEV-like sequences are also present in shellfish and in deer feces from the same geographical area. However, little information is available about the prevalence, molecular epidemiology, and genomic sequence variation of BEV field isolates. Here we describe an optimized highly sensitive real-time reverse transcription-PCR method to detect BEV RNA in biological and environmental samples. A combination of the amplification procedure with a previously described filtration step with electropositive filters allowed us to detect up to 12 BEV RNA molecules per ml of water. The feasibility of using the method to detect BEV in surface waters at a high risk of fecal pollution was confirmed after analysis of water samples obtained from different sources. The method was also used to study the prevalence of BEV in different cattle herds around Spain, and the results revealed that 78% (78 of 100) of the fecal samples were BEV positive. BEV-like sequences were also detected in feces from sheep, goats, and horses. Nucleotide sequence analyses showed that BEV isolates are quite heterogeneous and suggested the presence of species-specific BEV-like variants. Detection of BEV-like sequences may help in the differentiation and characterization of animal sources of contamination.

Potato virus Y group C isolates are a homogeneous pathotype but two different genetic strains

Potato virus Y group C isolates (PVYC) have been characterized according to biological, molecular and genetic criteria. Two genetic strains, PVYC1 and PVYC2, were identified on the basis of genetic distances (among them and other PVY strains), host range (ability or inability to infect pepper), MAb response (ELISA recognition with MAb 10E3) and coat protein processing site. Some characteristics, such as aphid transmission and ELISA using other MAbs, did not correlate with classification into these two genetic strains. All isolates tested induced a hypersensitive response on potatoes bearing the Nc resistance gene, confirming the nature of PVYC isolates as a homogeneous pathotype.

Characterization of potato potyvirus Y (PVY) isolates from seed potato batches. Situation of the NTN, Wilga and Z isolates

AbstractA collection of 38 PVY isolates from seed potato batches, originating from several Western European countries, was characterized by using current biological, serological and molecular tools differentiating PVY strains and groups. The correlation between the three kinds of tests was good but not absolute. No single serological or PCR method was able to discriminate among the five isolate groups found. Twenty-nine isolates belonged to the PVYN strain and six to the PVYO strain. No PVYC was found. Two other isolates reacted serologically like PVYO, but were unable to elicit a hypersensitive response from the Nytbr gene and probably represent the PVYZ group. At the molecular level, these two isolates showed a combination of both PVYO and PVYN and could be recombinants of these strains. Another isolate reacted serologically like PVYO, but induced vein necrosis in tobacco, like PVYN-Wilga. Some PVYN isolates caused tuber ring necrosis in glasshouse conditions. These might belong to the PVYNTN group. The PVYNTN, PVYN-Wilga and PVYZ groups probably represent pathotypes within strains PVYN and PVYO, respectively. The present study also confirms previous reports showing a high genetic variation at the 5

INFECTIVITY OF TURNIP MOSAIC POTYVIRUS CDNA CLONES AND TRANSCRIPTS ON THE SYSTEMIC HOST ARABIDOPSIS THALIANA AND LOCAL LESION HOSTS

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