Lothaire Pinck

High efficiency regeneration of grapevine plants transformed with the GFLV coat protein gene

Abstract Genetically transformed grapevines were obtained through co-cultivation of embryogenic cell suspensions with an engineered A. tumefaciens strain. Two economically important rootstocks, 41B and SO4, as well as a well-known grapevine vinifera variety, Chardonnay were regenerated. For the first time transformation of a scion variety is reported. A chimeric coat protein gene (CP) was integrated in order to protect grapevine against grapevine fanleaf virus (GFLV) infection. A neomycin phosphotransferase II (NPT II) gene allowed the selection of large number of transformed embryogenic calli and plants for the three varieties. Percentages of transformed material were first estimated with GUS activity. Presence of the CP gene was assessed by PCR and Southern analysis and gene expression by ELISA. Transformed calli have now been subcultured in vitro for 3 years without losing their embryogenic ability. GUS activity assays on leaves and roots of acclimatized plants showed transformation to be stable.

Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus RNA1

The nucleotide sequence of the genomic RNA1, 7342 nucleotides (nt) of grapevine fanleaf virus strain F13 (GFLV-F13) has been determined from cDNA clones. The complete sequence contained only one long open reading frame (ORF) of 6852 nucleotides extending from nucleotide 243 to 7101. The putative polyprotein encoded by this ORF is 2284 amino acids in length with an Mr of 253K. The location of genome-linked protein and comparison of the primary structure of the 253K polyprotein to that of other closely related viral proteins of the picronavirus-like family allows the proposal of a scheme for the genetic organization of GFLV-F13 RNA1. The primary structure of the polyprotein includes a putative RNA-dependent RNA polymerase of 92K and a cysteine protease of 25K. This protease shares not only major structural homologies, particularly in the substrate-binding pocket, with the trypsin-like serine proteases of other picorna-like viruses, but also their specificity in terms of cleavage. The large region of Mr 133K upstream of the VPg was found to contain at least two domains, one of which could be easily aligned with the NTP-binding sequence pattern and another which may have the characteristics of a protease cofactor. Thus, the 253K protein possesses the same general genetic organization as the corresponding protein of other picorna-like viruses.

Transformation of grapevine rootstocks with the coat protein gene of grapevine fanleaf nepovirus

SummaryControl of fanleaf disease induced by the Grapevine Fanleaf Nepovirus (GFLV) today is based on sanitary selection and soil disinfection with nematicides. This way of control is not always efficient and nematicides can be dangerous pollutants. Coat protein (CP) mediated protection could be an attractive alternative. We have transferred a chimeric CP gene of GFLV-F13 via Agrobacterium tumefaciens LBA4404 into two rootstock varieties: Vitis rupestris and 110 Richter (V. rupestris X V. Berlandieri). Transformation was performed on embryogenic callus obtained from anthers and on hypocotyl fragments from mature embryos. Success of the transformation was assessed by polymerase chain reaction and Southern analyses. Transformants with a number of copies of the CP gene varying from one to five were obtained. Enzyme-linked immunosorbent assay with virus-specific antibodies revealed various levels of expression of the coat protein in the different transformants.

Genome organization of grapevine fanleaf nepovirus RNA2 deduced from the 122K polyprotein P2 in vitro cleavage products

The full-length transcript of grapevine fanleaf virus (GFLV) RNA2 produces a primary product of 122K when translated in the rabbit reticulocyte system. This 122K polyprotein is completely processed in vitro by the RNA1-encoded 24K proteinase. The positions of the cleavage sites within the polyprotein have been mapped and the genome organization of GFLV-F13 RNA2 has been established. The order of mature proteins in the 122K polyprotein is the amino-terminal 28K protein, the 38K protein followed by the 56K coat protein at the carboxy terminus. These proteins represent the final cleavage products of the 122K polyprotein. A 66K protein which yields 28K and 38K proteins constitutes the major maturation intermediate. Microsequencing of the amino extremity of radioactively labelled 38K protein allowed identification of the Cys257/Ala258 site as the cleavage site recognized by the GFLV proteinase between the 28K and the 38K proteins in the 66K protein in addition to the Arg605/Gly606 site between the 38K protein and the coat protein.

The nine C-terminal residues of the grapevine fanleaf nepovirus movement protein are critical for systemic virus spread

The grapevine fanleaf virus (GFLV) RNA2-encoded polyprotein P2 is proteolytically cleaved by the RNA1-encoded proteinase to yield protein 2A, 2B(MP) movement protein and 2C(CP) coat protein. To further investigate the role of the 2B(MP) and 2C(CP) proteins in virus movement, RNA2 was engineered by alternatively replacing the GFLV 2B(MP) and 2C(CP) genes with their counterparts from the closely related Arabis mosaic virus (ArMV). Transcripts of all chimeric RNA2s were able to replicate in Chenopodium quinoa protoplasts and form tubules in tobacco BY-2 protoplasts in the presence of the infectious transcript of GFLV RNA1. Virus particles were produced when the GFLV 2C(CP) gene was replaced with its ArMV counterpart, but systemic virus spread did not occur in C. quinoa plants. In addition, chimeric RNA2 containing the complete ArMV 2B(MP) gene was neither encapsidated nor infectious on plants, probably because polyprotein P2 was incompletely processed. However, chimeric RNA2 encoding ArMV 2B(MP), in which the nine C-terminal residues were those of GFLV 2B(MP), formed virus particles and were infectious in the presence of GFLV but not ArMV 2C(CP). These results suggest that the nine C-terminal residues of 2B(MP) must be of the same virus origin as the proteinase for efficient proteolytic processing of polyprotein P2 and from the same virus origin as the 2C(CP) for systemic virus spread.

Cloning and in vitro characterization of the grapevine fanleaf virus proteinase cistron

The region of the genomic RNA-1 from grapevine fanleaf virus isolate F13 (GFLV-F13), containing the proteinase cistron and flanking sequences (nucleotides 3894 to 4789) of the GFLV polyprotein, was modified by PCR mutagenesis to create a start codon and cloned in a transcription vector. The transcripts from the resulting clone (pVP7) produced, upon translation in rabbit reticulocyte lysate, a 37.8-kDa protein which was subsequently cleaved to a stable 28-kDa product. Autocleavage was maximal at pH 7.0-8.5 and at 30 degrees. Inhibition of the activity was greater than 80% when translation was performed in the wheat germ system. In rabbit reticulocyte lysate, inhibition was also obtained with PMSF, EDTA, E-64, Ca+2, Zn+2, and Co+2. The pVP7 translation product acts in cis, in the case of its autocleavage, or in trans in the processing of the viral 122-kDa polyprotein from GFLV RNA-2 into a 66-kDa protein and the 56-kDa coat protein. The carboxy extremity of the complete pVP7 translation product, encoded by nucleotides 4633 to 4789 of RNA-1, was not required for the proteinase activity, at least in trans.

Effects of site-directed mutagenesis on the presumed catalytic triad and substrate-binding pocket of grapevine fanleaf nepovirus 24-kDa proteinase.

Grapevine fanleaf nepovirus (GFLV) has a bipartite plus-sense RNA genome. Its structural and functional proteins originate from polyprotein maturation by at least one virus-encoded proteinase. Here we describe the cloning of the 24-kDa proteinase cistron located between the virus-linked protein (VPg) and the RNA-dependent RNA polymerase cistron in GFLV RNA1 (nucleotides 3966 to 4622). Proteinase expressed from this clone is able to cleave GFLV polyprotein P2 in order to produce the coat protein and a 66-kDa protein which is further processed to the 38-kDa presumed movement protein. The GFLV 24-kDa proteinase sequence contains sequence similarities with other nepovirus and comovirus proteinases, particularly at the level of the conserved domains corresponding to the hypothetical catalytic triad and to the substrate-binding pocket (amino acids 192 to 200). Site-directed mutagenesis of residues His43, Glu87, and Leu197 abolished proteinase activity. Inactivation of the enzyme is also observed if the catalytic residue Cys179 was substituted by isoleucine, but replacement by a serine at the same position produced a mutant with an activity identical to that of native proteinase. All our data show that GFLV cysteine proteinase presents structure similarities to the proteinases of cowpea mosaic virus and potyviruses but is most closely related to trypsin.

Protection against virus infection in tobacco plants expressing the coat protein of grapevine fanleaf nepovirus

SummaryGrapevine fanleaf nepovirus (GFLV) is responsible for the economically significant “court-noué” disease in vineyards. Its genome is made up of two single-stranded RNA molecules (RNA1 and RNA2) which direct the synthesis of polyproteins P1 and P2 respectively. A chimeric coat protein gene derived from the C-terminal part of P2 was constructed and subsequently introduced into a binary transformation vector. Transgenic Nicotiana benthamiana plants expressing the coat protein under the control of the CaMV 35S promoter were engineered by Agrobacterium tumefaciens-mediated transformation. Protection against infection with virions or viral RNA was tested in coat protein-expressing plants. A significant delay of systemic invasion was observed in transgenic plants inoculated with virus compared to control plants. This effect was also observed when plants were inoculated with viral RNA. No coat protein-mediated cross-protection was observed when transgenic plants were infected with arabis mosaic virus (ArMV), a closely related nepovirus also responsible for a “court-noué” disease.

Replication of alfalfa mosaic virus RNAs

Abstract The RNAs constituting the genome of alfalfa mosaic virus (AMV) have been separated by electrophoresis on polyacrylamide gels and inoculated alone or in mixture upon susceptible plants. The isolation and analysis of replicative forms (RF) found in these plants revealed that the mixtures which induce the formation of RF are the same as those which give rise to local lesions on hypersensitive plants: that is, only the mixture of 4 species of AMV RNA, or the mixture of the 3 largest RNAs in addition to coat protein is able to induce the formation of RF, whereas no single RNA alone or with the addition of small amounts of coat protein gives rise to the corresponding replicative form. The problem of the existence of a replicative form of 12 S RNA was also examined. It was never possible to show any replicative form of 12 S RNA even when a strain producing much 12 S RNA (AMV425) was used. Some possibilities of 12 S RNA biosynthesis were examined.

Sequence homology at the 3′‐ends of alfalfa mosaic virus RNAs

We report that the four RNA molecules of alfalfa mosaic virus (AMV) have an extensive sequence homology at their 3’ends. Our sequence data were obtained by the new direct chemical sequencing method [l]_ AMV is a muItipa~ite~enome plant virus. The virus particles contain four RNA molecules, conventionally numbered l-4 in order of decreasing size. The first three constitute the true genome and contain copies of all the genes of the virus; for their replication either the coat protein or its messenger (RNA 4 in the v&ions) must be present [2]. aptly bromegrass mosaic virus contains four RNA molecules but here genome expression is not known to be dependent on coat protein or on its messenger RNA. These four RNAs were shown to contain a common tRNA&ke 3’end sequence that is 161 nueleotides long [3]. In AMV RNA 4, the sequence of 9 1 nucieotides [4], corresponding to a 3’-end fragment that interacts strongly with the viral coat protein, has recently been reported. This site may be required for proper recognition of the RNA 4 3’-terminus by the viral replicase [5]. Our observations extend this po~ibi~ity to the genomic AMV RNAs.