William R. Belknap

Isolation of a polyubiquitin promoter and its expression in transgenic potato plants

A polyubiquitin clone (ubi7) was isolated from a potato (Solanum tuberosum) genomic library using a copy-specific probe from a stress-induced ubiquitin cDNA. The genomic clone contained a 569-bp intron immediately 5[prime] to the initiation codon for the first ubiquitin-coding unit. Two chimeric [beta]-glucuronidase (GUS) fusion transgenes were introduced into potato. The first contained GUS fused to a 1156-bp promoter fragment containing only 5[prime] flanking and 5[prime] untranslated sequences from ubi7. The second transgene contained GUS translationally fused to the carboxy terminus of the first ubiquitin-coding unit and thus included the intron present in the 5[prime] untranslated region of the polyubiquitin gene. Both ubi7-GUS transgenes were activated by wounding in tuber tissue and in leaves by application of exogenous methyl jasmonate. They were also expressed constitutively in the potato tuber peel (outer 1–2 mm). Both transgenes were actively expressed in mature leaves. Exceptionally high levels of expression were observed in senescent leaves. Transgenic clones containing the ubi7 intron and the first ubiquitin-coding unit showed GUS expression levels at least 10 times higher than clones containing GUS fused to the intronless promoter.

Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants.

A genomic clone encoding the potato homolog of the yeast ubiquitin-ribosomal protein fusion gene ubi3 was isolated and characterized. Chimeric genes containing the ubi3 promoter (920 bp of 5′ to the ubiquitin start codon) were constructed in which the reporter gene β-glucuronidase (GUS) was either fused directly to the promoter, or introduced as a translational fusion to the ubiquitin-coding region. After introduction into the potato by Agrobacterium-mediated transformation, GUS activities were measured in leaves and in tubers of transgenic clones. GUS activity was 5- to 10-fold higher in clones expressing the ubiquitin-GUS translational fusion than in clones containing GUS fused directly to the ubi3 promoter. For both types of constructs, GUS activity was highest in meristematic leaves and declined during leaf expansion, then rose again to near the meristematic levels during senescence. GUS activity in tubers was similar to that in young leaves. In contrast to the native ubi3 genes, the chimeric ubi3-GUS transgenes were not activated in the tuber by wounding.

The role of ubiquitin in plant senescence and stress responses

Ubiquitin is a small, highly conserved protein found in all eukaryotes. Within the cell, ubiquitin is covalently linked to substrate proteins, often targeting them for degradation via the ubiquitin pathway. This pathway has been demonstrated to be required for both the bulk degradation of cellular proteins and the targeted proteolysis of specific regulatory proteins. There is increasing biochemical and molecular evidence for involvement of the ubiquitin pathway in plant senescence and in response to stress. It appears that the role of ubiquitin during senescence could be to facilitate bulk protein degradation for nitrogen recycling. In the wound response, the pathway could have a role in altering the overall protein profile.

Expression of stress-responsive ubiquitin genes in potato tubers

The stress-induced expression of four different ubiquitin-encoding cDNAs was characterized in potato tuber tissue. The four clones exhibited differences in both structure and expression. The first cDNA encoded a single ubiquitin unit fused to an 80 amino acid ribosomal extension protein identical to the extension protein from tomato. Accumulation of the fusion transcript was induced by injury or ethylene, but not by heat shock. The three remaining ubiquitin cDNAs encoded polyubiquitins with 6 to 7 ubiquitin repeats. The first polyubiquitin gene was induced by injury, heat, or ethylene treatments. The second was induced also by injury or heat, with limited ethylene-dependent accumulation of transcript. Transcript levels of the third polyubiquitin gene were highest in control tubers and decreased markedly with injury, heat shock, or ethylene treatment. The data demonstrate the independent regulation of the different members of the ubiquitin gene family in response to stress and exogenous ethylene.

Characterization of Repetitive DNA Elements in Arabidopsis

Abstract. We have applied computational methods to the available database and identified several families of repetitive DNA elements in the Arabidopsis thaliana genome. While some of the elements have features expected of either miniature inverted-repeat transposable elements (MITEs) or retrotransposons, the most abundant class of repetitive elements, the AthE1 family, is structurally related to neither. The AthE1 family members are defined by conserved 5′ and 3′ sequences, but these terminal sequences do not represent either inverted or direct repeats. AthE1 family members with greater than 98% identity are easily identified on different Arabidopsis chromosomes. Similar to nonautonomous DNA-based transposon families, the AthE1 family contains members in which the conserved terminal domains flank unrelated sequences. The primary utility of characterizing repetitive sequences is in defining, at least in part, the evolutionary architecture of specific Arabidopsis loci. The repetitive elements described here make up approximately 1% of the available Arabidopsis thaliana genomic sequence.

Identification of putative nonautonomous transposable elements associated with several transposon families inCaenorhabditis elegans

Putative nonautonomous transposable elements related to the autonomous transposons Tc1, Tc2, Tc5, andmariner were identified in theC. elegans database by computational analysis. These elements are found throughout theC. elegans genome and are defined by terminal inverted repeats with regions of sequence similarity, or identity, to the autonomous transposons. Similarity between loci containing related nonautonomous elements ends at, or near, the boundaries of the terminal inverted repeats. In most cases the terminal inverted repeats of the putative nonautonomous transposable elements are flanked by potential target-site duplications consistent with the associated autonomous elements. The nonautonomous elements identified vary considerably in size (from 100 by to 1.5 kb in length) and copy number in the available database and are localized to introns and flanking regions of a wide variety ofC. elegans genes.

The use of in vitro-grown microtuber discs inAgrobacterium-mediated transformation of Russet Burbank and Lemhi Russet potatoes

We have usedin vitro-grown microtuber discs in the transformation of Russet Burbank and Lemhi Russet potato (Solanum tuberosum L.) cultivars byAgrobacterium-mediated gene transfer. Transformed plants were selected by their resistance to kanamycin and identified by β-glucuronidase activity. Northern blot analysis confirmed the presence of the corresponding messenger RNA. The ability to transform these two cultivars promises significant improvements to agronomically important varieties.

Comparison of the expression of several stress-responsive genes in potato tubers

Changes in gene expression induced by mechanical injury and heat shock were studied by comparing the expression of several stress-responsive gene families in potato tubers. The steady-state levels of mRNA-encoding ubiquitin, HSP70, and phenylalanine ammonia-lyase (PAL) increased and patatin transcript levels decreased within 45 minutes of impact injury. Nuclear runoff assays were used to demonstrate that the changes in steady-state transcript levels were due, at least in part, to changes in the rate of transcription for these genes. The observed changes in transcript levels were confined to the injured portion of the tuber. Treatment of tubers with exogenous ethylene elicited the same changes in the steady-state transcript levels as impact injury, indicating a potential role for this hormone in the injury-induced regulation of these genes. Two other forms of physical stress, heat shock and cutting injury, resulted in patterns of gene expression that are different from those induced by impact injury. The stress-induced regulation of these four gene families is complex, even though several characteristics of their expression are similar.

A modified method for routine Agrobacterium-mediated transformation of in vitro grown potato microtubers

SummaryIn vitro-grown potato (Solanum tuberosum L.) microtubers were used as an explant source in the production of transgenic plants by Agrobacterium-mediated gene transfer. In this study we tested four diverse potato cultivars, Lemhi Russet, Russet Burbank, Wauseon, and Yankee Chipper on various levels of zeatin riboside and 3-indoleacetyl-DL-aspartic acid for their ability to regenerate transgenic plants after infection with Agrobacterium tumefaciens. Culturing microtuber blocks from the medullary area separately from cortex and epidermal tissue containing the eyes resulted in fewer transgenic plants, with transgenic shoots arising only from the tissue with the eyes. Lemhi and Russet Burbank microtuber discs were also transformed with a chimeric gene, CLaSP, designed to increase resistance to blackspot bruise in the tuber. This method resulted in transformed plants in every experiment, with an efficiency that appeared to be genotype dependent.

Gene Rpi-bt1 from Solanum bulbocastanum Confers Resistance to Late Blight in Transgenic Potatoes

Phytophthora infestans, the causal agent of late blight is the most devastating pathogen of cultivated potato worldwide. Utilizing map based cloning; a genomic region containing a cluster of six nucleotide binding site-leucine-rich repeat resistance gene analogs was isolated from the wild potato species Solanum bulbocastanum. Four genes were pseudogenes, with coding sequences interrupted by either frame shift mutations or premature stop codons. However, neither of the two uninterrupted genes conferred resistance to P. infestans when introduced into susceptible potatoes. Specific primers for one of the pseudogenes were used to amplify an uninterrupted cDNA from P. infestans-infected S. bulbocastanum leaves. A corresponding gDNA was amplified from a late blight-resistant bulbocastanum–tuberosum introgression line (Rpi-bt1). The Rpi-bt1 gene under transcriptional control of the constitutive potato Ubi3 promoter was found to confer resistance to P. infestans in several transgenic potato lines in a whole plant greenhouse assay.ResumenPhytophthora infestans, el agente causal del tizón tardío, es el patógeno mas devastador de la papa cultivada en el mundo. Mediante la clonación basada en mapas, se aisló de la especie silvestre de papa Solanum bulbocastanum una región genómica que incluía un grupo de repetición con seis análogos de genes de resistencia de sitios de unión de nucleótidos ricos en leucina. Cuatro genes fueron pseudogenes, con secuencias de codificación interrumpidas ya fuera por mutaciones por cambio de marcos o por codones de terminación prematura. No obstante, ninguno de los dos genes sin interrupción confirió resistencia a P. infestans cuando se introdujeron a papas susceptibles. Se utilizaron iniciadores específicos para uno de los pseudogenes para amplificar un ADNc ininterrumpido de hojas de S. bulbocastanum infectadas con P. infestans. También se amplificó un ADNg correspondiente de una línea de introgresión (Rpi-bt1) de bulbocastanum-tuberosum resistente al tizón tardío. Se encontró que el gen Rpi-bt1 bajo control transcripcional del promotor de papa Ubi3 confiere resistencia a P. infestans en varias líneas de papa transgénica en un ensayo con planta completa bajo invernadero.