Local expression of enzymatically active class I beta-1, 3-glucanase enhances symptoms of TMV infection in tobacco.

Mutant tobacco plants deficient for class I beta-1,3-glucanase (GLU I) are decreased in their susceptibility to virus infection. This is correlated with delayed virus spread, a reduction in the size exclusion limit of plasmodesmata and increased cell-wall deposition of the beta-1,3-glucan callose. To further investigate a role of GLU I during cell-to-cell movement of virus infection, we inserted the GLU I coding sequence into TMV for overexpression in infected cells. Compared with the size of local lesions produced on plants infected with virus expressing either an enzymatically inactive GLU I or a frameshift mutant of the gene, the size of local lesions caused by infection with virus expressing active GLU I was consistently increased. Viruses expressing antisense GLU I constructs led to lesions of decreased size. Similar effects were obtained for virus spread using plants grown at 32 degrees C to block the hypersensitive response. Together, these results indicate that enzymatically active GLU I expressed in cells containing replicating virus can increase cell-to-cell movement of virus. This supports the view that GLU I induced locally during infection helps to promote cell-to-cell movement of virus by hydrolyzing callose. Moreover, our results provide the first direct evidence that a biological function of a plant beta-1,3-glucanase depends on its catalytic activity.

Graft transmission of induced and spontaneous post-transcriptional silencing of chitinase genes.

Sense and antisense tobacco chitinase (CHN) transgenes, Luciferase-CHN transcriptional fusions, and promoterless CHN cDNAs were introduced biolistically into CHN transformants of tobacco that never exhibit spontaneous gene silencing. All of the constructs tested induced systemic silencing of the resident CHN transgene and endogenes. Nuclear run-on transcription assays showed that local introduction of additional gene copies triggers systemic post-transcriptional gene silencing (PTGS). Together, this provides evidence that additional transgene copies need not be either highly transcribed or produce sense transcripts to evoke production of systemic PTGS signals. CHN PTGS was transmitted by top grafting, but not by reciprocal grafting of mature stems or the exchange of tissue plugs. Thus, the commonly encountered difficulties in achieving graft-transmission could reflect the method used. Silencing in sense but not antisense transformants was transmitted by grafting to a high-expressing sense CHN scion suggesting that the elaboration of mobile signals may not be an essential feature of antisense-mediated gene silencing.

Movement of plant viruses is delayed in a beta-1,3-glucanase-deficient mutant showing a reduced plasmodesmatal size exclusion limit and enhanced callose deposition.

Susceptibility to virus infection is decreased in a class I beta-1,3-glucanase (GLU I)-deficient mutant (TAG4.4) of tobacco generated by antisense transformation. TAG4.4 exhibited delayed intercellular trafficking via plasmodesmata of a tobamovirus (tobacco mosaic virus), of a potexvirus (recombinant potato virus X expressing GFP), and of the movement protein (MP) 3a of a cucumovirus (cucumber mosaic virus). Monitoring the cell-to-cell movement of dextrans and peptides by a novel biolistic method revealed that the plasmodesmatal size exclusion limit (SEL) of TAG4.4 was also reduced from 1.0 to 0.85 nm. Therefore, GLU I-deficiency has a broad effect on plasmodesmatal movement, which is not limited to a particular virus type. Deposition of callose, a substrate for beta-1,3-glucanases, was increased in TAG4.4 in response to 32 degrees C treatment, treatment with the fungal elicitor xylanase, and wounding, suggesting that GLU I has an important function in regulating callose metabolism. Callose turnover is thought to regulate plasmodesmatal SEL. We propose that GLU I induction in response to infection may help promote MP-driven virus spread by degrading callose.

Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco.

To study the influence of genomic context on transgene expression, we have determined the T-DNA structure, flanking DNA sequences, and chromosomal location of four independent transgene loci in tobacco. Two of these loci were stably expressed in the homozygous condition over many generations, whereas the other two loci became unstable after several generations of homozygosity. The stably expressed loci comprised relatively simple T-DNA arrangements that were flanked on at least one side by plant DNA containing AT-rich regions that bind to nuclear matrices in vitro. Of the unstably expressed loci, one consisted of multiple incomplete T-DNA copies, and the second contained a single intact T-DNA; in both cases, however, binary vector sequences were directly contiguous to a right T-DNA border. Fluorescence in situ hybridization demonstrated that the two stably expressed inserts were present in the vicinity of telomeres. The two unstably expressed inserts occupied intercalary and paracentromeric locations, respectively. Results on the stability of transgene expression in F1 progeny obtained by intercrossing the four lines and the sensitivity of the four transgene loci to inactivation in the presence of an unlinked "trans-silencing" locus are also presented. The findings are discussed in the context of repetitive DNA sequences and the allotetraploid nature of the tobacco genome.

Structural instability of a transgene locus in tobacco is associated with aneuploidy.

This paper describes molecular and cytogenetic evidence for the stability of a transgene locus that is present on the triplicated chromosome in an aneuploid tobacco line. This instability was manifested in several ways in trisomics including a major chromosome rearrangement that was detectable cytogenetically, smaller scale DNA rearrangements that occurred both germinally and somatically, and methylation/epigenetic silencing. In a deletion derivative of the locus, DNA breakpoints were found in AT-rich regions. One of these regions binds to nuclear scaffolds in vitro, suggesting a possible role for aberrant topoisomerase II cleavage in destabilization of the locus. The implications of increased chromosome instability in aneuploids for plant karyotype evolution and human carcinogenesis are discussed.

In vivo observation of large foreign DNA molecules in host plant cells.

We have developed a system to monitor microscopically the fate of foreign DNA within plant cells in vivo. Fluorescein-11-dUTP was used to label DNA during target-specific amplification by polymerase chain reaction (PCR). Labeled DNA fragments of 1.5-3.5 kb were prepared and then transported into tobacco protoplasts by polyethylene glycol (PEG)-mediated direct gene transfer. We localized the foreign, labeled DNA within the cell by confocal laser scanning microscopy.

Transgenic Indica rice breeding line IR58 expressing a synthetic cryIA(b) gene from Bacillus thuringiensis provides effective insect pest control.

The Indica rice breeding line IR58 was transformed by particle bombardment with a truncated version of a synthetic cryIA(b) gene from Bacillus thuringiensis. This gene is expressed under control of the CaMV 35S promoter and allows efficient production of the lepidopteran specific delta-endotoxin. R0, R1 and R2 generation plants displayed a significant insecticidal effect on several lepidopterous insect pests. Feeding studies showed mortality rates of up to 100% for two of the most destructive insect pests of rice in Asia, the yellow stem borer (Scirpophaga incertulas) and the striped stem borer (Chilo suppressalis), and feeding inhibition of the two leaffolder species Cnaphalocrocis medinalis and Marasmia patnalis. Introduction of stem borer resistance into the germplasm of an Indica rice breeding line now makes this agronomically important trait available for conventional rice breeding programs.

Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity.

The promoter homology-dependent inactivation of a 35Spro-hygromycin phosphotransferase (hpt) gene, which is present at the H2 locus, by the multipurpose 271 silencing locus has been studied. The 271 locus can silence any gene under the control of the 35Spro as well as endogenous nitrite reductase (NiR) genes of tobacco because of the presence of a chimeric antisense gene (35Spro-RiN). All F1 progeny of a cross between homozygous H2 and 271 lines were sensitive to hygromycin and were chlorotic (a symptom of nitrogen deficiency). These phenotypes were accompanied by a reduction in the steady-state levels of Hyg and NiR transcripts. Transcriptional run-on experiments indicated, however, that while NiR silencing occurred post-transcriptionally, the hpt gene was inactivated at the transcriptional level; this was associated with increased methylation of the 35Spro of the hpt gene. NiR gene expression recovered uniformly to wild-type levels in first generation backcross (BC1) progeny that did not inherit the 271 locus. In contrast, hygromycin resistance was only partially and non-uniformly regained among adult BC1 plants. Moreover, substantial silencing of the hpt gene could persist into the BC2 generation. Genomic sequencing demonstrated that the meiotic heritability of hpt silencing in the absence of the 271 locus was correlated with cytosine methylation primarily at CpG and CpNpG residues. Despite this residual methylation, H2 loci weakened by an association with 271 did not acquire the ability to silence a 'naive' H2 locus. Fluorescence in situ hybridization revealed that the 271 locus was located at a telomere. The results strengthen the distinction between silencing effects involving homology restricted to coding or promoter regions, respectively. The former is a post-transcriptional process that is meiotically reversible; the latter is due to transcriptional inactivation and is associated with increased promoter methylation, which can lead to meiotically heritable reductions in target gene activity. The relevance of these data for the meiotic heritability of silencing, the non-transferability of silencing activity, and the basis of 271 silencing effects is discussed.

Transgenic Indica rice (Oryza sativa L.) plants obtained by direct gene transfer to protoplasts.

We have established a reproducible procedure for transformation of protoplasts and regeneration of transgenic plants for an improved Indica rice cultivar IR43. Mature embryo-derived calli were placed in liquid culture medium containing maltose to establish meristematically active, embryogenic cell suspension lines. In order to obtain transgenic plants, a chimeric hygromycin phosphotransferase hph gene under the control of the cauliflower mosaic virus CaMV 35S promoter was introduced into protoplasts from these cell suspension lines using polyethylene glycol. Protoplasts were cultured in maltose-containing medium. Hygromycin B selection was applied to 14-day-old dividing cell colonies. Resistant calli were readily obtained after 3 weeks of selection. Seventy-three plantlets were regenerated from resistant calli from several independent experiments, and a few of the 29 plants grown in the greenhouse reached maturity. Stable integration of the transgene in the genome of these plants was confirmed by Southern blot analysis and the expression of the transgene in plants by hygromycin phosphotransferase assay. The procedure described yielded 5 to 18 resistant colonies and approximately four transgenic plantlets per million treated protoplasts.

In vitro germination of wheat proembryos to fertile plants.

Wheat proembryos were excised 7 days after anthesis and germinated to normal fertile plants with an efficiency of 90%. These embryos had clearly differentiated shoot apical meristems and represent an interesting model system for studies of meristem biology. A combination of two media with different osmolarities allowed the continued development of the embryos before induction of germination. Normal germination occurred only after the first leaf primordium had already covered the shoot apical meristem.

Gene transfer by electroporation into intact scutellum cells of wheat embryos.

Gene transfer into intact cells was achieved by electroporating zygotic wheat embryos without any special pretreatment. Electroporation was tissue specific in so far as scutellum cells were found to be much more susceptible to gene transfer than other cell types of the embryo. The orientation of the embryos in the electroporation chamber also influenced the number of transformed scutellum cells; during electroporation, as in electrophoresis, the negatively charged plasmid DNA molecules seemed to move towards the positive electrode. Therefore, the embryos were arranged so that the scutella faced the negative electrode. The use of plasmids carrying either two chimeric anthocyanin regulatory genes or a chimeric gusA gene allowed clear identification of transformed cells in the scutellum. On some of the embryos, more than 100 transformed scutellum cells were found after electroporation with single electric pulses of 275 V/cm discharged from a 960-µF capacitor and with 100 µg DNA/ml electroporation buffer. Using the anthocyanin marker system, visibly transformed cells grew to produce red sectors.

Transgenic plants of tall fescue (Festuca arundinacea Schreb.) obtained by direct gene transfer to protoplasts.

Chimeric hygromycin phosphotransferase (hph) and phosphinothricin acetyltransferase (bar) genes were introduced, using polyethylene glycol treatment, into protoplasts isolated from embryogenic cell suspension cultures of tall fescue (Festuca arundinacea Schreb.), a graminaceous plant that is an important forage crop in temperate pastures. Colonies resistant to either 200 mg/l hygromycin or 100 mg/l phosphinothricin, respectively, were recovered upon selection using bead-type culture systems. Stable integration of the transgenes in the genomes of plants regenerated from resistant callus clones was shown by Southern hybridization analysis. In situ hybridization of a labeled transgene-probe to metaphase chromosomes is shown for one transgenic primary regenerant. Expression of the transgenes in mature plants was demonstrated by HPH enzyme assay or by phosphinothricin-herbicide spraying.