Cynipid gall formation is achieved by an insect-plant interaction whereby cynipid gallwasps redirect host-plant development to form novel structures to protect and nourish the developing larvae. Work was carried out to investigate the molecular mechanisms involved in this interaction, and extend the understanding of plant tissue development. Cytological changes of the inner-gall tissue throughout the development of several gall species was investigated and the developmental stages of gall formation defined, to reveal two different patterns of development followed by the galls tested. Fluorescent in situ hybridization demonstrated many of the inner-gall cells to be polytenized. Comparisons between inner-gall and non-gall tissue protein signatures by Schonrogge et al. (Plant, Cell and Environment 23, 215-222, 2000) have demonstrated the variation between gall and non-gall protein signatures, and identified a number of inner-gall proteins. Further analysis of one of these inner-gall proteins involved in lipid synthesis, putative biotin carboxyl carrier protein (BCCP), revealed differential expression throughout development, and showed this expression to be concentrated in the inner-gall tissue in all the gall species tested.

A horizontal transmission of a geminiviral DNA sequence, into the germ line of an ancestral Nicotiana, gave rise to multiple repeats of geminivirus-related DNA, GRD, in the genome. We follow GRD evolution in Nicotiana tabacum (tobacco), an allotetraploid, and its diploid relatives, and show GRDs are derived from begomoviruses. GRDs occur in two families: the GRD5 family's ancestor integrated into the common ancestor of three diploid species, Nicotiana kawakamii, Nicotiana tomentosa and Nicotiana tomentosiformis, on homeologous group 4 chromosomes. The GRD3 family was acquired more recently on chromosome 2 in a lineage of N. tomentosiformis, the paternal ancestor of tobacco. Both GRD families include individual members that are methylated and diverged. Using relative rates of synonymous and nonsynonymous nucleotide substitutions, we tested for evidence of selection on GRD units and found none within the GRD3 and GRD5 families. However, the substitutions between GRD3 and GRD5 do show a significant excess of synonymous changes, suggesting purifying selection and hence a period of autonomous evolution between GRD3 and GRD5 integration. We observe in the GRD3 family, features of Helitrons, a major new class of putative rolling-circle replicating eukaryotic transposon, not found in the GRD5 family or geminiviruses. We speculate that the second integration event, resulting in the GRD3 family, involved a free-living geminivirus, a Helitron and perhaps also GRD5. Thus our data point towards recurrent dynamic interplay between geminivirus and plant DNA in evolution.

Cotton, the major cash crop in Pakistan, suffers 30% losses to cotton leaf curl disease, caused by the geminivirus, cotton leaf curl virus DNA A, plus a satellite component, DNA beta responsible for symptom development with plants failing to produce cotton bolls. We constructed transgenic tobacco expressing sense and antisense RNAs representing: [i] the 5' half of the viral DNA replication gene, AC1, [ii] the 3' half of AC1, [iii] two overlapping genes, AC2, a transcription activator, and AC3, a replication enhancer. In contrast to controls, 25% of 72 transgenic tobacco lines tested showed heritable resistance [T(1) - T(3) generations]: symptom-free and no replication of DNA A or DNA beta even after 120 days of continuous exposure to viruliferous whiteflies. As geminiviral and transgene RNAs are not detected in resistant lines following infection, and selected uninfected resistant tobacco sense lines reveal double-stranded and small interfering RNAs, the most likely mechanism is via post-transcriptional gene silencing.

Nicotiana tabacum (tobacco) is a natural allotetraploid. The maternal genome donor is not controversial and is probably derived from an ancestor of N. sylvestris. The paternal, T-genome donor has been less clear, with N. tomentosiformis, N. otophora, or an introgression hybrid proposed. Here we provide evidence that the T genome of N. tabacum is derived from a particular lineage of N. tomentosiformis. We show that the repetitive sequences of geminiviral origin, GRD53 and GRD3, are present in the genomes of N. tabacum cultivars, a tobacco cell suspension culture TBY-2, and N. tomentosiformis ac. NIC 479/84. Surprisingly, they are not present in another three varieties of N. tomentosiformis. A detailed cytogenetic analysis also revealed that N. tomentosiformis ac. NIC 479/84 most closely resembles the N. tabacum T genome in the location of other tandem repetitive sequences. Thus, tobacco formed after divergence within N. tomentosiformis, and the spectrum of potential donors of the paternal genome can be narrowed to a genotype of N. tomentosiformis characterized by the presence of GRD53 and GRD3 repeats. It is clear that future paternity studies in tobacco should use N. tomentosiformis ac. NIC 479/84 rather than any other accession.

Bruchins, which are lipids extracted from the pea weevil (Bruchus pisorum), can stimulate cell division and callus development on pods of Pisum sativum plants carrying the dominant gene Np (Refs 1,2). This neoplastic-like response can be accompanied by tissue browning around the application site. Such changes resemble classic defense responses 2, and have in this case been shown to protect the plant from the insect at least partially by making it more difficult for larvae to invade the pod 1.

Cytosine methylation levels and susceptibility to drug-induced hypomethylation have been studied in several Nicotiana tabacum (tobacco) DNA repetitive sequences. It has been shown using HapII, MspI, BamHI and Sau3AI methylation-sensitive restriction enzymes that the degree of 5′-mCmCG-3′ methylation varied significantly between different repeats. There were almost saturation levels of 5-methylcytosine at the inner (3′) cytosine position and variable degrees of methylation at the outer (5′) cytosine at the enzyme recognition sites. The non-transcribed high copy satellite sequences (HRS60, GRS) displayed significant heterogeneity in methylation of their basic units while middle repetitive sequences (R8.1, GRD5, 5S rDNA) were more uniformly modified at both cytosine residues. Dihydroxypropyladenine (DHPA) treatment, which is thought to reduce DNA methyltransferase activity by increasing S-adenosylhomocysteine levels, resulted in extensive demethylation of the outer cytosine in all repeats, and the partial hypomethylation of cytosines at the inner positions in less densely methylated repeats such as HRS60 and GRS. The results suggest that hypomethylation of 5′-mCmCG-3′ sites with DHPA is a gradual non-random process proceeding in the direction mCmCG→CmCG→CCG. The 18S-5.8S-25S rDNA was remarkably hypomethylated relative to the 5S rDNA at all restriction sites studied. Fluorescence in-situ hybridization showed that DNA decondensation within and between the 18S-5.8S-25S and 5S rDNA loci was variable in different nuclei. All nuclei had condensed and decondensed sequence. The chromatin of 18S-5.8S-25S rDNA was more readily digested with micrococcal nuclease than the 5S rDNA suggesting that the overall levels of decondensation were higher for 18S-5.8S-25S rDNA. Variable decondensation patterns within and between loci were also observed for GRS and HRS60. Cytosine methylation of the tobacco repeats is discussed with respect to transcription, overall levels of condensation and overall structure.

ABSTRACTCynipid galls are examples of induced plant development, where the gall inducer is in control of cell differentiation and morphogenesis of a new plant organ. This study concentrates on the tissues of the larval chamber common to all cynipid galls. The protein content of the inner gall tissue was compared to that of non‐gall plant tissues. We investigated three oak and two rose galls and their respective host plants. Total protein signatures of inner gall tissues were different from those of non‐gall plant tissues, and among the five galls. N‐terminal sequences were obtained for two abundant proteins from the inner gall tissues of D. spinosa and A. quercuscalicis, which were common to all galls, at 62 and 43 kDa. Database queries suggest the 62 kDa protein to be homologous to a protein disulphide isomerase (PDI), and the 43 kDa protein to be homologous to NAD‐dependent formate dehydrogenase (FDH). A naturally biotinylated protein was detected at 33 kDa during Western analyses with streptavidin. Western analyses revealed the presence of the biotinylated protein and PDI in the inner gall tissues of all five galls, while FDH was only detected in A. quercuscalicis and A. fecundator. PDI was also common to all non‐gall tissues, while FDH was not detected in non‐gall tissues, and the biotinylated protein was only detected in seeds. The proteins identified in the inner gall tissue suggest that (a) inner gall tissues in some galls are under respiratory stress, and (b) cynipid gall formation might involve the ectopic expression of seed‐specific proteins.

. We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nico-tiana sylvestris (2 n =2 x =24) and N. tomentosiformis (2 n =2 x =24) and compared these with patterns in N. tabacum (tobacco, 2 n =4 x =48). We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris . The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N. sylvestris type (from ca. 75% based on the sum of the rDNA copy numbers in the parents). Since the active genes are likely to be of an N. tomentosiformis type, the N. sylvestris type units are presumably contained within inactive loci (i.e. on chromosome S12). Nicotiana sylvestris has approximately three times as much rDNA as the other two species, resulting in much condensed rDNA at interphase. This species also has three classes of IGS, indicating gene conversion has not homogenised repeat length in this species. The results suggest that methylation and/or DNA condensation has reduced or prevented gene conversion from occurring at inactive genes at rDNA loci. Alternatively, active undermethylated units may be vulnerable to gene conversion, perhaps because they are decondensed and located in close proximity within the nucleolus at interphase. In TBY-2, restriction enzymes showed hybridisation patterns that were similar to, but different from, those of N. tabacum. In addition, TBY-2 has elevated rDNA copy number and variable numbers of rDNA loci, all indicating rDNA evolution in culture.

Each species of cynipid gall wasps induce the growth of unique galls which are both structurally distinct and anatomically novel structures to their host plant. Although much has been written on the anatomy of cynipid galls, little is known about the molecular mechanisms responsible for gall initiation and growth. Presumably the gall wasps send signals to the host plant to bring about gall formation. Here we present the first results of two approaches to identify these signals.First, we are trying to identify molecular markers to distinguish between gall and non-gall plant tissue. If such markers can be established, it should also be possible to use marker expression in a bioassay to find and characterise the compounds which serve as signal molecules. We have identified two proteins, expressed in galls of Diplolepis spinosa on Rosa rugosa, which could prove useful as molecular markers. One protein (90kDa) was found to be exceptionally more abundant in the inner gall tissue, compared to non-gall tissues, such as leaf and stem tissues. The second protein (60kDa) was not detected in the non galled tissues, and appears to be differentially produced in the inner gall tissue, making it an excellent molecular marker.Nod-factors are signal molecules involved in the interaction/communication between nitrogen fixing bacteria, Rhizobium spp., and their legume hosts. To address whether gall wasps use similar signals we are also searching for homologues to nodC, a gene known to be involved in the synthesis of all Nod-factors, in gall wasp genomic DNA. A homologue to nodC in gall wasp DNA suggests that the signal molecules used by cynipids to induce galls might be of a Nod-factor nature. In a polymerase-chain-reaction we used oligonucleotide primers designed from NodC and DG42 (a nodC homologue from Xenopus laevis) amino acid sequences on gall wasp template DNA. Both sets of primers amplified a fragment of 400bp length from the gall wasp DNA. However, the primers designed from DG42, also amplified two more fragments of 440bp and 1kb length.

Mutations in the terminal 8-bp (5'-T(1)G(2)T(3)G(4)G(5)G(6)C(7)G(8)-3') of the inverted repeats of the bacterial transposon, Tn7, were analysed by measuring Tn7 transposition to the attachment site, attTn7. The mutation, C-2, present at either end of Tn7 reduces transposition only threefold, but in the double mutant, with C-2 at both ends of Tn7, no transposition is detected. C-6 mutations have no effect on transposition frequency. Replacement with 5'-A(3)C(4)G(5)C(6)G(7)C(8)-3' at the right end of Tn7 apparently abolishes transposition; yet in the double mutant, where the inverted repeats are restored by substituting this sequence at both ends of Tn7, transposition is partially rescued. This suggests that the mechanism of Tn7 transposition requires communication between the two ends. Tn7 transposition has always been seen to generate a 5-bp target duplication. This is presumed to result from a staggered cut, plus repair synthesis during transposition. We found that two of our right-end mutants, C-2 and C-6, sometimes yielded a 6-bp target duplication. This observation implies that cleavage of the target site might also involve interaction with the donor ends which, when mutant, relax the specificity for target-site cleavage.

The bacterial transposon Tn7 encodes five trans-acting transposition genes, tnsA, B, C, D, and E. Tn7 requires four of these genes, tnsA, B, C, and D, for a novel transposition pathway: high-efficiency site-specific transposition to a chromosomal attachment site, attTn7. Plasmids that individually allow inducible overexpression of proteins from the first initiation codon of four of these genes were constructed. Escherichia coli strains carrying these plasmids were used to overexpress the TnsA, B, C, and D proteins. The abundance and the apparent relative molecular mass of these proteins were examined and the latter was compared to those predicted from wild-type Tn7. The functionality of these proteins, encoded by an overexpression construct, was demonstrated by the fact that they could efficiently trans-complement a defective mini-Tn7 carrying only the cis-essential Tn7 termini in an in vivo assay for transposition to attTn7.

Conference Article| November 01 1992 Antisense genes as tools to engineer virus resistance in plants E. R. Bejarano; E. R. Bejarano † *Department of Biochemistry, Imperial College of Science, Technology & Medicine, Exhibition Road, London SW7 2AZ, U.K. Search for other works by this author on: This Site PubMed Google Scholar A. G. Day; A. G. Day ‡ Search for other works by this author on: This Site PubMed Google Scholar V. Paranjape; V. Paranjape *Department of Biochemistry, Imperial College of Science, Technology & Medicine, Exhibition Road, London SW7 2AZ, U.K. Search for other works by this author on: This Site PubMed Google Scholar C. P. Lichtenstein C. P. Lichtenstein § *Department of Biochemistry, Imperial College of Science, Technology & Medicine, Exhibition Road, London SW7 2AZ, U.K. §To whom correspondence should be addressed. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1992) 20 (4): 757–761. https://doi.org/10.1042/bst0200757 Article history Received: July 22 1992 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation E. R. Bejarano, A. G. Day, V. Paranjape, C. P. Lichtenstein; Antisense genes as tools to engineer virus resistance in plants. Biochem Soc Trans 1 November 1992; 20 (4): 757–761. doi: https://doi.org/10.1042/bst0200757 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: TGMV, Tomato Golden Mosaic Virus, ORF, open reading frame, Ti, tumour inducing, T-DNA, transfer DNA, ssDNA, single strand DNA, dsDNA, double strand DNA, CLV, Cassava latent virus, BCTV, beet curly top virus This content is only available as a PDF. © 1992 Biochemical Society1992 Article PDF first page preview Close Modal You do not currently have access to this content.

Transgenic tobacco plants carrying a genetic cassette including an antisense DNA sequence of the virally encoded AL1 gene of the geminivirus tomato golden mosaic virus (TGMV) were constructed; AL1 encodes a protein absolutely required for TGMV DNA replication. These genetic cassettes also contained, on the same transcription unit, a gene encoding hygromycin resistance, which allowed selection for concomitant expression of the antisense gene. In transgenic lines, RNA transcripts of the predicted size and strand specificity were detected in antisense plants and sense controls. After infection of plants with TGMV, by agroinoculation, the frequency of symptom development was very significantly reduced in a number of antisense lines and correlated, broadly, with the abundance of antisense RNA transcript and with a reduction in viral DNA harvested from infected leaf tissue. We used an in vitro assay to study viral DNA replication in the absence of cell-to-cell spread; no replication was seen in five of the six antisense lines studied, in contrast to controls.

A new assay for the detection of the enzyme neomycin phosphotransferase II (NPTII) in crude cell extracts is described. The method is based on the chromatographic separation of the compounds resulting from the reaction of NPTII with kanamycin (Km) and [gamma-32P]ATP; the labelled Km.phosphate is subsequently detected by autoradiography. Chromatography is carried out on polyethyleneimine cellulose plates. This assay has been tested with bacterial, plant and animal crude extracts and comparisons with the assays in current use have been made. Our assay has several advantages: it is simple, rapid, sensitive and safe.