Transgene silencing in monocots.

Plant gene silencing was originally thought to be a quirk of transformation procedures, but is now recognized to be a facet of vitally important gene regulatory systems, present in all organisms. Monocot plants, especially the grasses, play a foremost role in the agricultural economy of all nations, and their biotechnological manipulation offers great potential for both developed and developing countries. Here, we review reported instances of transgene silencing in monocots and relate the processes of transcriptional and post-transcriptional gene silencing (TGS, PTGS) in perspective to the rapidly burgeoning knowledge of these phenomena in many organisms.

Virus recovery is induced in Brome mosaic virus p2 transgenic plants showing synchronous complementation and RNA-2-specific silencing.

Nicotiana benthamiana plants expressing Brome mosaic virus (BMV) p2 protein complemented replication of RNAs1 + 3 but, surprisingly, supported little or no replication of RNA-2. Despite this, the p2 transgenic plants were able to support systemic migration of RNAs-1 and -3. Kinetic analyses showed identical degradation rates for RNAs-2 and -3, greatly detracting from the concept of an induction of an RNA-2-specific degradation system.

Organizational complexity of a rice transgene locus susceptible to methylation-based silencing.

Molecular analyses of a rice (Oryza sativa L.) transgene locus introduced using biolistic techniques revealed the presence of multiple copies of rearranged fragments, as well as an intact copy of the supplied constructs. Both the gene of interest (35S-Btt cryIIIA) and the selectable marker used (Ubi1-bar) were methylated and silenced.

Longevity of 5-azacytidine-mediated gene expression and re-establishment of silencing in transgenic rice.

Epigenetic silencing of a bialaphos resistance (bar) gene in R1 progeny of a transgenic rice line was found to be meiotically stable since selfed (R2) progeny were also susceptible and the bar locus highly methylated. A high proportion of R2 seedlings germinated in the presence of 5-azacytidine (AzaC) were herbicide-resistant and also contained at least one unmethylated copy of the bar gene, further establishing the relationship between silencing and methylation. Restored bar gene expression was typically maintained for 20-50 days, but eventual methylation and silencing of the bar locus underscores the ability of the recipient genome to recognize and inactivate intrusive DNA.

Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice.

Despite a growing number of reports indicating non-Mendelian inheritance of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants generated by direct DNA-transfer techniques, making the cause for these observations difficult to determine. In this paper we describe the complex organization of Btt cryIIIA and bar transgenes in rice (Oryza sativa L.) that displayed aberrant segregation in R1 progeny.

The beta-phaseolin 5' matrix attachment region acts as an enhancer facilitator.

MARs found flanking the beta-phaseolin gene (phas) were tested for insulating activity in an enhancer blocking assay. True insulators should block enhancer dependent expression of a reporter gene when placed between the enhancer and a promoter. Insertion of phas 3' MAR or coding sequences lowered CaMV 35S enhancer driven GUS expression from the phas basal promoter, indicating a distance dependence of the 35S enhancer.

Analysis of kafirin promoter activity in transgenic tobacco seeds.

Sequences corresponding to 855 bp of 5' promoter region and the transit peptide from lambdaGK.1,a genomic clone encoding a 22 kDa alpha-kafirin seed protein from sorghum, were translationally fused to a cloned beta-glucuronidase (GUS) coding sequence from uidA and transferred to tobacco via Agrobacterium tumefaciens-mediated transformation. No GUS expression was detectable at any stage of growth in stems or leaves of these plants.

A 68 bp element of the beta-phaseolin promoter functions as a seed-specific enhancer.

In beans, expression of the beta-phaseolin gene (phas), encoding the major seed storage protein of bean (Phaseolus vulgaris) is confined to the cotyledons of developing embryos. Phaseolin has not been detected in the endosperm, which remains liquid and is lost early in development. However, fusion constructs between the phas promoter and the gus-coding region yield expression in both embryo and endosperm of developing seeds from transgenic tobacco (Nicotiana tabacum) plants.

Intron position affects expression from the tpi promoter in rice.

A series of promoter-GUS fusion constructs containing a portion of the rice triosephosphate isomerase (tpi) promoter, the first tpi intron, and the gene encoding bacterial beta-glucuronidase (GUS) were made. These constructs were electroporated into rice protoplasts and transient expression was monitored. Inclusion of the first intron from the rice tpi gene enhanced expression of the GUS gene from the tpi promoter when it was placed 5' of the GUS gene.

Interaction of host proteins with the plus-strand promoter of brome mosaic virus RNA-2.

The binding of five barley proteins (Mr: 37, 36, 35, 34, and 30 kDa) to the ICR2 motif present at the 5' end of brome mosaic virus (BMV) RNA-2 was identified using UV cross-linking. Evidence that the interaction is specific included the observation that these proteins did not recognize a similar-size RNA fragment transcribed from a nonviral (beta-glucuronidase) gene, nor did they bind to the 3' end of the plus strand of RNA-3. Replication-defective BMV RNA-2 mutants bearing substitution mutations at nucleotides 9 and 10 of the ICR2 motif were used to show that these positions were involved in the interaction of the five barley proteins with BMV RNA-2.

Complete sequence of the binary vector Bin 19.

Despite the widespread use of Bin 19 as a vector for plant transformation, detailed sequence information on its T-DNA region has only recently become available. We now show that the non-T-DNA region, like the T-DNA region, contains several superfluous insertions and find that some functional elements may not contain optimal sequences. Knowledge of the complete 11,777 bp sequence will aid in the construction of exceptionally efficient derivative vectors of approximately half this size.

Characterization of a rice gene family encoding root-specific proteins.

Two cDNA clones (RCc2 and RCc3) corresponding to mRNAs highly expressed only in root tissues of rice (Oryza sativa L.) seedlings were characterized. Respectively, they encode polypeptides of 146 (14.

Cyclophilins are encoded by a small gene family in rice.

cDNA clones were isolated and sequenced that encode two related but distinct rice cyclophilins, Cyp1 and Cyp2. The predicted amino acid sequences of each are 72% identical to human T-cell cyclophilin. Genomic DNA gel blot analysis suggests cyclophilins in rice are encoded by a small, 6-10-member gene family.

Identification of domains in brome mosaic virus RNA-1 and coat protein necessary for specific interaction and encapsidation.

Even though many single-stranded RNAs are present in the cytoplasm of infected cells, encapsidation by brome mosaic virus (BMV) coat protein is specific for BMV RNA. Although the highly conserved 3' region of each of the three BMV genomic RNAs is an attractive candidate for the site of recognition by the coat protein, band shift and UV cross-linking assays in the presence of specific and nonspecific competitors revealed only nonspecific interactions. However, BMV RNA-1 formed a retarded complex (complex I) with the coat protein in the absence of competitors, and two domains of RNA-1 that specifically bound coat protein in a small complex (complex II), presumably early in the encapsidation process, were identified.

Interference with brome mosaic virus replication by targeting the minus strand promoter.

Sense and antisense strategies for interfering with the replication of brome mosaic virus (BMV) were examined. The effects of 200 nucleotide-long sense and antisense transcripts, corresponding to the viral 3' end (-) strand promoter, on the accumulation of progeny viral RNAs were studied by co-inoculation with wildtype BMV RNAs. Progeny accumulation in barley protoplasts transfected with either sense or antisense transcripts of the (-) strand promoter and BMV RNAs-1 and -2 was decreased by more than 90%, and by 60 to 80% when RNA-3 was also present.

Contributions of the brome mosaic virus RNA-3 3'-nontranslated region to replication and translation.

Sequences upstream of the 3'-terminal tRNA-like structure of brome mosaic virus RNAs have been predicted to fold into several stem-loop and pseudoknot structures. To elucidate the functional role of this upstream region, a series of deletions was made in cDNA clones of RNA-3, a genomic component not required for replication. These deletion mutants were transcribed in vitro and cotransfected with RNA-1 and RNA-2 into barley protoplasts.

Cytosolic triosephosphate isomerase is a single gene in rice.

A cDNA clone encoding rice (Oryza sativa L.) cytosolic triosephosphate isomerase (TPI), an important glycolytic enzyme, was isolated and characterized. The clone (pRTPI-6) contains an open reading frame of 759 base pairs, encoding a polypeptide chain of 253 amino acid residues (M(r) 27,060).