J. Dunez

A highly sensitive immunocapture polymerase chain reaction method for plum pox potyvirus detection.

A highly sensitive assay, based on polymerase chain reaction amplification of cDNA synthesized from the viral RNA of antibody-captured viral particles, has been developed for plum pox potyvirus (PPV) detection. The reaction, called immunocapture/PCR (IC/PCR), yields a specific 243-bp product. The immunocapture step, by allowing the use of large sample volumes and by the viral particle prepurification it achieves, dramatically increases the sensitivity of the assay. As few as 8000 target viral particles per ml of plant extract could be detected by IC/PCR. When compared to direct PCR (Wetzel et al., 1991), molecular hybridization using 32P-labeled cRNA probes and ELISA, this result corresponds to a 250-fold, 625-fold and 5000-fold increased sensitivity, respectively. The high sensitivity of IC/PCR was confirmed during an indexing trial with field samples collected from naturally infected trees. This very powerful technique should have wide ranging applications for the detection of a number of other viruses and pathogens for which specific antisera and sequence data are available.

A polymerase chain reaction assay adapted to plum pox potyvirus detection.

A sensitive, polyvalent assay based on the polymerase chain reaction (PCR) was developed for plum pox potyvirus (PPV) detection. This technique was adapted for a single tube, the chemical denaturation and reverse transcription of the viral RNA followed by the PCR reaction yielding a 243-base-pair product. As few as 10 fg of purified viral RNA, corresponding to approximately 2000 viral particles, were detected in plant extracts. All PPV isolates tested were amplified, and the amplified fragments were analysed by restriction endonuclease digestion. An RsaI restriction site polymorphism in the amplified fragments allowed the discrimination of two groups of isolates. In a field indexing trial, the PCR assay proved to be more sensitive than molecular hybridization using 32P-labelled RNA probes for PPV detection.

Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene

SummaryPlum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and β-glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.

Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the D and M serotypes of plum pox potyvirus

ABSTRACT Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D- and PPV-M-specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M-specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.

Biological and molecular variability of lettuce mosaic virus isolates.

ABSTRACT Lettuce mosaic potyvirus (LMV) causes severe disease of commercial lettuce crops. LMV isolates show wide biological variability, particularly in their ability to overcome the resistance genes described in Lactuca sativa. For a better understanding of the molecular interaction between lettuce and LMV, biological and molecular characterization of a collection of 10 LMV isolates known to differ in virulence or aggressiveness was performed. The ability of these isolates to overcome the resistance genes was reevaluated under standardized conditions. To study the molecular variability of LMV, an immunocapture-reverse transcription-poly-merase chain reaction technique, coupled with direct sequencing, was used to obtain nucleotide sequence data from three short regions of the LMV genome. Clustering analysis was performed and compared to the biological properties of the 10 isolates. Three groups of LMV isolates were discriminated based on the molecular data. These groups appear to correlate with the geographic origin of the isolates rather than with their pathogenicity. Sequence comparison with California isolates clearly showed that the California isolates are related to the western European isolates, raising the possibility of past exchanges of LMV between western Europe and California.

Complete nucleotide sequence of the genome of a severe cherry isolate of apple chlorotic leaf spot trichovirus (ACLSV)

SummaryThe genome of the Balaton1 severe cherry isolate of apple chlorotic leaf spot trichovirus (ACLSV-Bal1) has been cloned and sequenced. The genomic RNA is 7 549 nucleotide long, excluding the poly A tail. The genomic organization, with three overlapping open reading frames (ORF), is similar to that of the other sequenced ACLSV isolates. Sequence comparisons indicate a high variability between ACLSV isolates, with overall nucleotide sequence homology levels between 76 and 82%. The coat protein, encoded internally inside a larger ORF, is the most conserved protein (identity levels between 87 and 93%) while the central ORF, encoding the putative movement protein, is the most divergent (77 to 85% identity).

Variable resistance to plum pox virus and potato virus Y infection in transgenic Nicotiana plants expressing plum pox virus coat protein

Abstract The plum pox virus (PPV) coat protein (CP) gene was transferred into three species of Nicotiana using an Agrobacterium tumefaciens based system. Transgenic Nicotiana benthamiana plants that express the PPV CP exhibited protection when inoculated with PPV plant sap. After a first stage where PPV was able to multiply in transgenic and control plants, the virus was progressively excluded from newly formed tissues and at 40 days after inoculation no PPV could be detected in the most resistant lines. This homologous resistance to PPV appears to be due both to the inhibition of viral long-distance transport and to the inhibition of viral replication. Transgenic Nicotiana clevalandii and Nicotiana tobacum cv. Xanthi expressing PPV CP exhibited partial levels of protection to infection by PPV or by potato virus Y (PVY) that were dependent on the inoculum concentration. Different interpretations of these resistance phenomena are discussed.

Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants.

Aphid transmission of a non-aphid-transmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) occurs in transgenic plants expressing the plum pox potyvirus (PPV) coat protein (CP) gene. Heteroencapsidation has been shown to be responsible for this modification in the epidemiological characteristics of the infecting virus. In order to prevent this biological risk, several modified PPV CP constructs were produced that were designed to interfere with heteroencapsidation itself or to block aphid transmission of heteroencapsidated virions. These constructs were first expressed in Escherichia coli in order to check for the accumulation of pseudo-particles by electron microscopy. Virus-like particles (VLPs) were found with the full-length CP and with a PPV CP lacking the DAG amino acid triplet involved in aphid transmission. However, no VLPs were observed with CP lacking R220, Q221 or D264, amino acids known to be essential for the assembly of other potyvirus CPs. Transgenic Nicotiana benthamiana lines expressing the different PPV CP constructs were infected with ZYMV-NAT. Aphid transmission assays performed with these plants demonstrated that the strategies developed here provide an effective means of minimizing the biological risks associated with heteroencapsidation.

Construction of full-length cDNA clones of lettuce mosaic virus (LMV) and the effects of intron-insertion on their viability in Escherichia coli and on their infectivity to plants

SummaryA full length cDNA copy of the genomic RNA of lettuce mosaic virus (LMV) was constructed under the control of an enhanced CaMV 35S promoter and of the NOS terminator. This construct was found infectious when inoculated to lettuce plants. The intron II of the bean nitrite reductase gene was engineered into the LMV FL cDNA in order to relieve possible deleterious effects of viral sequences to Escherichia coli cells and to evaluate the effects of the presence of the intron on the FL cDNA infectivity. The intron-less FL cDNA was found to be as stable as its intron-containing counterpart in E. coli. Sequence analysis of progeny RNA derived from plants inoculated with the intron-containing FL cDNA demonstrated that the inserted intron was perfectly spliced out. The symptoms induced in lettuce by either the intron-less or the intron-containing constructs were identical to those caused by the wild-type virus. However a slight delay in the establishment of infection in lettuce and a more obvious lag in Nicotiana benthamiana were observed with the intron-containing FL cDNA.

Genetically engineered resistance against grapevine chrome mosaic nepovirus.

Nepoviruses are a group of isometric plant viruses with a genome divided between two-single-stranded, positive-sense, RNA molecules. They are usually transmitted by nematodes and a number of them have significant economic impact, especially in perennial crops such as grapevine and fruit trees. Like all other picorna-like viruses, nepoviruses express their coat protein (CP) as part of a larger polyprotein which is further processed by a virus-encoded protease, a feature which poses specific problems when trying to express the viral coat protein in transgenic plants. A hybrid gene, driving the high-level expression of the CP of grapevine chrome mosaic nepovirus (GCMV) has been constructed and transferred to the genome of tobacco plants. Progeny of CP-expressing transformants show resistance against GCMV. When compared to control plants, fewer inoculated plants become infected and those that become infected accumulate reduced levels of viral RNAs. This protection was also shown to be efficient when plants are inoculated with purified viral RNA.