David R. Rockhold

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.

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.

pBINPLUS/ARS: an improved plant transformation vector based on pBINPLUS

Binary plant transformation vectors are widely used for introduction of transgenes into plants via Agrobacterium tumefaciens-mediated transformation. We report the construction of a binary plant vector pBINPLUS/ARS based on the pBINPLUS vector. Improvements introduced into pBINPLUS/ARS include the use of nonproprietary (ubiquitin-3 gene of Solanum tuberosum) promoter and terminator sequences for transcription of the NptII selectable marker and introduction of rare 8-bp restriction enzyme sites flanking both the NptII coding sequence (PmeI) and the entire selectable marker gene (FseI). This vector offers all of the advantages of its predecessor pBINPLUS and its helper plasmid pUCAP, which use the proprietary nopaline synthase promoter and terminator, while allowing for facile modification of selectable marker sequences in complex binary vector constructs. pBINPLUS/ARS has been used to introduce transgenes into potato and other crop species and is available to all researchers in academic, government, and industrial laboratories for proof-of-principle and commercial applications.

Field performance of transgenic Russet Burbank and Lemhi Russet potatoes

Transformed Russet Burbank and Lemhi Russet clones which contained three different transgene constructs were evaluated for performance under field conditions in Idaho. The transgenic lines were characterized over two growing seasons, using plants grown from both greenhouse produced minitubers (first year) and field grown seed (second year). Individual clones were evaluated for a variety of agronomic and quality properties. Many of the transformed clones showed reduced yield and increases in percent malformed and undersized tubers. Other characteristics, such as specific gravity and fry color, showed less variability. The variation observed in non-transgenic clones regenerated from tissue culture was less than that of the transformed lines. Out of an original population of 57 transgenic lines in tissue culture, maintenance of key agronomic and quality properties of the parental material was observed in only four clones. These results suggest that experiments designed to generate transgenic lines for the marketplace should be initiated with a large number of transgenic clones.CompendioClones transformados de Russet Burbank y Lemhi Russet, que contenían tres diferentes prototipos de transgenes fueron evaluados para comportamiento bajo condiciones de campo en Idaho. Las líneas transgénicas se caracterizaron en dos temporadas de cultivo, utilizando plantas obtenidas tanto de minituberculos producidos en invernadero (primer año) como semilla producida en el campo (segundo año). Se evaluaron clones individuales para diversas propiedades agronomicas y de calidad. Muchos de los clones transformados mostraron rendimientos reducidos y un incremento en el porcentaje de tubérculos deformesy de pequeño tamaño. Otras características, tales como la gravedad específica y el color a la fritura, mostraron una menor variabilidad. La variacíon observada en los clones no transgénicos regenerados de cultivo de tejidos fue menor que aquella de las líneas transformadas. De una población original de 57 líneas transgénicas en cultivo de tejidos, se observó que sólo cuatro de ellas mantenían las principales propiedades agronómicas y de calidad del material progenitor. Estos resultados sugieren que los experimentos diseñados para generar líneas transgenicas para el mercado deberán iniciarse con un gran número de clones transgenicos.

Structure of Two Solanum bulbocastanum Polyubiquitin Genes and Expression of Their Promoters in Transgenic Potatoes

Two polyubiquitin genes, bul409 and bul427, were isolated from a Solanum bulbocastanum BAC library. The bul409 and bul427 genes encode hexameric and heptameric polyproteins, respectively. bul427 exhibits a number of features suggestive of a pseudogene: (1) The last ubiquitin monomer of bul427 is interrupted by a frame shift mutation. (2) The coding sequence is flanked 3′ by mitochondrial and chloroplast sequences and 5′ by a protein kinase pseudogene. However, characterization of cDNAs amplified using bul427-based primers demonstrated that bul427 is transcriptionally active. Chimeric transgenes encoding β-glucuronidase (GUS) translationally fused to the first ubiquitin-coding units of bul409, a truncated version 409s, and bul427 were constructed and introduced into potato. In transgenic potato lines both S. bulbocastanum promoters were weakly transcribed in tubers and efficiently transcribed in leaves. In leaves, bul409s was wound-induced, while bul427 was not. In tubers both promoters were wound-induced. In unwounded leaves and tubers, the steady state mRNA levels from both bul promoters were lower than the steady state mRNA levels from the Cauliflower Mosaic Virus 35S promoter. However, in the leaves and tubers of many of the transgenic lines the GUS activity was significantly higher in the bul lines compared to the 35S lines. The apparent inconsistency of higher enzymatic activity correlated with lower steady state levels of mRNA demonstrates the enhanced protein expression observed with ubiquitin fusion proteins.ResumenDos genes de poliubiquitina, bul409 y bul427, fueron aislados de una librería BAC de Solanum bulbocastanum. Los genes bul409 y bul427 codifican poliproteínas hexámera y heptámera, respectivamente. El bul427 exhibe varios rasgos que sugieren un seudo gen: (1) El último monómero de bul427 es interrumpido por una mutación con desplazamiento de la pauta de lectura. (2) La secuencia codificante es flanqueada en 3′ por secuencias mitocondriales y de cloroplastos y en 5′ por un seudogen de proteína quinasa. Sin embargo, la caracterización de cDNAs amplificadas usando iniciadores basados en bul427 demostró que bul427 es transcripcionalmente activo. Se construyeron e introdujeron en papa transgenes quiméricos que codifican β-glucuronidasa (GUS) translacionalmente fusionados a las primeras unidades que codifican ubiquitina de bul409, una versión truncada de 409s y bul427. En las líneas de papa transgénica de S. bulbocastanum, los promotores fueron débilmente transcritos en los tubérculos y eficientemente transcritos en hojas. En hojas el bul409s fue inducido por heridas, mientras que bul427 no fue. En tubérculos, ambos promotores fueron inducidos por heridas. En hojas y tubérculos sin herir, los niveles mRNA en estado de equilibrio para ambos promotores bul fueron más bajos que los niveles de mRNA en estado de equilibrio del promotor 35S del virus del Mosaico de la Coliflor. Sin embargo, en hojas y tubérculos de muchas líneas transgénicas, la actividad de GUS fue significativamente más alta en las líneas bul en comparación con las 35S. La aparente inconsistencia de una actividad enzimática mayor, correlacionada con bajos niveles de mRNA en estado de equilibrio demuestra el incremento de la expresión de proteína observada con proteínas fusión ubiquitina.

Blackspot bruise dependent changes in enzyme activity and gene expression in Lemhi Russet potato.

Blackspot bruise-induced changes in enzyme activity and gene expression were examined in the bruise susceptible cultivar Lemhi. The activities of several enzymes involved with the synthesis and metabolism of phenolic intermediates were examined over the time course of blackspot bruise development in tubers. A large, transient increase (200-fold) in phenylalanine ammonia lyase (PAL) activity was observed, while other enzymes examined (chorismate mutase, tyrosine ammonia-lyase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, and polyphenol oxidase) showed either modest or no activation. Maximal PAL activity was observed 48 hours after bruise induction, well after the discoloration reactions were complete. The change in PAL activity was associated with a transient increase in messenger RNA coding for PAL. In addition, messenger RNAs encoding two stress-induced genes, ubiquitin and the 70 kD heat shock protein, were transiendy induced by bruising. Physical impact also induced a marked decrease, followed by recovery, in the messenger RNA level for patatin, a primary storage protein in the tuber. These results suggest that the stress resulting in blackspot bruising elicits a wound response similar to those observed in other injuries to plant tissues.

Characterizing the citrus cultivar Carrizo genome through 454 shotgun sequencing.

The citrus cultivar Carrizo is the single most important rootstock to the US citrus industry and has resistance or tolerance to a number of major citrus diseases, including citrus tristeza virus, foot rot, and Huanglongbing (HLB, citrus greening). A Carrizo genomic sequence database providing approximately 3.5×genome coverage (haploid genome size approximately 367 Mb) was populated through 454 GS FLX shotgun sequencing. Analysis of the repetitive DNA fraction indicated a total interspersed repeat fraction of 36.5%. Assembly and characterization of abundant citrus Ty3/gypsy elements revealed a novel type of element containing open reading frames encoding a viral RNA-silencing suppressor protein (RNA binding protein, rbp) and a plant cytokinin riboside 5′-monophosphate phosphoribohydrolase-related protein (LONELY GUY, log). Similar gypsy elements were identified in the Populus trichocarpa genome. Gene-coding region analysis indicated that 24.4% of the nonrepetitive reads contained genic regions. The depth of genome coverage was sufficient to allow accurate assembly of constituent genes, including a putative phloem-expressed gene. The development of the Carrizo database (http://citrus.pw.usda.gov/) will contribute to characterization of agronomically significant loci and provide a publicly available genomic resource to the citrus research community.

Generation of PVY Coat Protein siRNAs in Transgenic Potatoes Resistant to PVY

Global climate change has the potential to quickly alter the distribution and profile of crop pests and pathogens. Ready tools for mobilization of resistance into elite crops will be part of an integrated strategy to maintain agricultural productivity. Engineered virus resistance through expression of coat protein is well established. The mechanisms of virus suppression by small RNAs (sRNAs) have only recently been elucidated. Understanding the role of sRNAs in gene regulation and development is rapidly evolving. High throughput sequencing (HTS) can generate large data sets of sRNA sequences which can be analyzed to recognize the abundant sRNAs that result from transcript degradation and to identify the regulatory micro RNAs (miRNA) involved in gene regulation and small inhibitory RNAs (siRNA) that confer virus resistance. Transgenic Solanum tuberosum that are glass house or field grown and that express the potato virus Y coat protein (PVY-CP) inverted hairpin RNA (ihRNA) from the 409s promoter with GBSS6 intron as a spacer exhibited resistance to PVY. Southern blots of genomic DNA indicated one or two copies of the transgene and analysis of HTS data of the small RNA population indicated high levels of siRNA production from the transgenic hairpin construct. Up to 47 of the top 200 most frequent sRNA reads were attributable to the PVY-CP transgenic construct in Ranger Russet line #5. This combination of transgene and transcription strength is functional for constructing efficient resistance cassettes for crop protection. Up to 95 of the top 200 sRNA sequences were 21 nucleotides in length with as many as 19 sequences corresponding to known miRNA transcripts. Additional endogenous sRNAs with homology to resistance gene analogs may be indicative of additional or complimentary resistance mechanisms produced in concert with the PVY siRNAs arising from PVY-CP RNA transcription.ResumenEl cambio climático global tiene el potencial de alterar rápidamente la distribución y el perfil de plagas y patógenos de los cultivos. Las herramientas listas para movilización de la resistencia a cultivos elite será parte de una estrategia integrada para mantener la productividad agrícola. Esta bien establecida la ingeniería de la resistencia a virus a través de la expresión de la cubierta proteica. Recientemente se han elucidado los mecanismos de la supresión del virus mediante pequeños ARNs (sRNAs). Esta evolucionando rápidamente el entendimiento del papel de los sRNAs en la regulación y desarrollo de genes. La obtención de alta secuenciación (HTS) puede generar grandes grupos de datos de secuencias de sRNA que se pueden analizar para reconocer la abundancia de sRNAs que resultan de la degradación de la transcripción e identificar los micro RNAs regulatorios (miRNA) involucrados en la regulación de genes y pequeños ARN inhibitorios (siRNA) que confieren resistencia a virus. Solanum tuberosum transgenicas que se cultivan en invernadero o campo y que expresan la horquilla invertida de RNA (ihRNA) de la cubierta proteica del virus Y de la papa (PVY-CP) del promotor 409s con el intron GBSS6 como un espaciador, exhibieron resistencia al PVY. Las técnicas de manchas sureñas (southern blots) de ADN genómico indicaron una o dos copias del transgene y el análisis de datos de HTS de una población de pequeño ARN indicó altos niveles de producción de siRNA de la construcción transgénica de la horquilla. Hasta 47 de las 200 mas frecuentes lecturas de sRNA fueron atribuibles a la construcción transgénica de PVY-CP en Ranger Russet línea #5. Esta combinación de transgene y fuerza de transcripción es funcional en la construcción de cartuchos de resistencia eficientes para la protección de los cultivos. Hasta 95 de las mejores 200 secuencias de sRNA fueron de una longitud de 21 nucleótidos con tantas como 19 secuencias correspondientes a transcripciones miRNA conocidas. sRNAs endógenos adicionales con homología a genes de resistencia análogos, pudieran ser indicativos de mecanismos de resistencia adicionales o complementarios producidos en concordancia con los PVY siRNAs surgidos de la transcripción de ARN PVY-CP.

Structure of Two Solanum tuberosum Steroidal Glycoalkaloid Glycosyltransferase Genes and Expression of their Promoters in Transgenic Potatoes

The Sgt2 gene in potato encodes a solanidine glucosyltransferase and is present as two distinct alleles expressed in cultivated potatoes. Promoter regions of both steroidal glycoalkaloid biosynthetic gene alleles, Sgt2.1 and Sgt2.2, were isolated from Solanum tuberosum cv. Russet Burbank genomic DNA. The genomic sequences of Sgt2.1 and Sgt2.2 were isolated by PCR amplification using a conserved region of Sgt2 and artificial upstream primers. The longest sequences for each allele were used to create β-glucuronidase (GUS) reporter gene fusions. Fusion constructs were mobilized into stable transgenic lines for analysis of promoter expression in leaves and tubers under control and wounded conditions. S. tuberosum promoters from Sgt2.1 and from Sgt2.1 produced GUS activity in transgenic potato leaves and tubers comparable to GUS activity produced by the CaMV35S promoter. The CaMV35S promoter is a strong promoter frequently used in plant biotechnology. Both Sgt2 promoters exhibited activities similar to the CaMV35S promoter in tubers and lower relative activities in leaves. On average the Sgt2.2 promoter exhibited higher activity in both leaves and tubers relative to the Sgt2.1. There was no consistent effect of wounding on GUS activity from the Sgt2.2 promoter in leaves or tubers. The Sgt2.1 promoter supported higher transgene activity in tubers versus leaves and exhibited small but consistent increases in response to wounding in tubers only. This may be due to the presence of a MITE sequence in the Sgt2.1 promoter.ResumenEl gen Sgt2 codifica para una solanidina-glucosiltransferasa y está presente como dos alelos distintos expresados en papas cultivadas. Las regiones promotoras de estos dos genes alelos biosinteticos de glicoalcaloides steroidales, Sgt2.1 y Sgt2.2, se aislaron de ADN genómico de Solanum tuberosum cv. Russet Burbank. Se aislaron las secuencias genómicas de Sgt2.1 y Sgt2.2 mediante amplificación por PCR usando una región conservada de Sgt2 y con iniciadores artificiales de partes superiores. Se usaron las secuencias más largas para cada alelo para crear fusiones de genes reporteros de β-glucuronidasa (GUS). Las fusiones generadas se movilizaron hacia líneas transgénicas estables para el análisis de la expresión del promotor en hojas y tubérculos bajo condiciones de control y de heridas. Los promotores de S. tuberosum de Sgt2.1 y de Sgt2.2 produjeron actividad GUS en hojas de papa transgénica y en tubérculos comparable con la actividad GUS producida por el promotor CaMV35S. Éste es un fuerte promotor que se usa frecuentemente en biotecnología de plantas. Los dos promotores de Sgt2 exhibieron actividades similares a las del promotor CaMV35S en tubérculos y actividades relativas más bajas en hojas. En promedio, el promotor Sgt2.2 exhibió mayor actividad tanto en hojas como en tubérculos en relación con el Sgt2.1. No hubo efecto consistente de heridas en la actividad de GUS del promotor Sgt2.2 en hojas o tubérculos. El promotor Sgt2.1 resistió más alta actividad transgénica en tubérculos contra hojas, y mostró aumentos pequeños pero consistentes en respuesta a heridas solamente en tubérculos. Esto pudo deberse a la presencia de una secuencia MITE en el promotor Sgt2.1.