Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes

A hammerhead ribozyme [R(-)] targeting the minus strand RNA of potato spindle tuber viroid (PSTVd) and a mutated nonfunctional ribozyme [mR(-)] were designed, cloned, and transcribed. As predicted, both monomer and dimer transcripts of the active R(-) ribozyme gene could cleave the PSTVd minus strand dimer RNA into three fragments of 77, 338, and 359 bases in vitro at 25 and 50°C. The tandem dimer genes of R(-) and mR(-) were subcloned separately into the plant expression vector pROK2. Transgenic potato plants (cultivar Desirée) were generated by Agrobacterium tumefaciens-mediated transformation. Twenty-three of 34 independent transgenic plant lines expressing the active ribozyme R(-) resulted in having high levels of resistance to PSTVd, being free of PSTVd accumulation after challenge inoculation with PSTVd, but the remaining lines showed weaker levels of resistance to PSTVd with low levels of PSTVd accumulation. In contrast, 59 of 60 independent transgenic lines expressing the mutated ribozyme mR(-) were susceptible to PSTVd inoculation and had levels of PSTVd accumulation similar to that of the control plants transformed with the empty vector. The resistance against PSTVd replication was stably inherited to the vegetative progenies.

Precisely full length, circularizable, complementary RNA: An infectious form of potato spindle tuber viroid

The replication of many viral and subviral pathogens as well as the amplification of certain cellular genes proceeds via a rolling circle mechanism. For potato spindle tuber (PSTVd) and related viroids, the possible role of a circular (−)strand RNA as a template for synthesis of (+)strand progeny is unclear. Infected plants appear to contain only multimeric linear (−)strand RNAs, and attempts to initiate infection with multimeric (−)PSTVd RNAs generally have failed. To examine critically the infectivity of monomeric (−)strand viroid RNAs, we have developed a ribozyme-based expression system for the production of precisely full length (−)strand RNAs whose termini are capable of undergoing facile circularization in vitro. Mechanical inoculation of tomato seedlings with electrophoretically purified (−)PSTVd RNA led to a small fraction of plants becoming infected whereas parallel assays with an analogous tomato planta macho viroid (−)RNA resulted in a much larger fraction of infected plants. Ribozyme-mediated production of (−)PSTVd RNA in transgenic plants led to the appearance of monomeric circular (−)PSTVd RNA and large amounts of (+)PSTVd progeny. No monomeric circular (−)PSTVd RNA could be detected in naturally infected plants by using either ribonuclease protection or electrophoresis under partially denaturing conditions. Although not a component of the normal replicative pathway, precisely full length (−)PSTVd RNA appears to contain all of the structural and regulatory elements necessary for initiation of viroid replication.

Mexican papita viroid: putative ancestor of crop viroids.

The potato spindle tuber disease was first observed early in the 20th century in the northeastern United States and shown, in 1971, to be incited by a viroid, potato spindle tuber viroid (PSTVd). No wild-plant PSTVd reservoirs have been identified; thus, the initial source of PSTVd infecting potatoes has remained a mystery. Several variants of a novel viroid, designated Mexican papita viroid (MPVd), have now been isolated from Solanum cardiophyllum Lindl. (papita güera, cimantli) plants growing wild in the Mexican state of Aguascalientes. MPVd's nucleotide sequence is most closely related to those of the tomato planta macho viroid (TPMVd) and PSTVd. From TPMVd, MPVd may be distinguished on the basis of biological properties, such as replication and symptom formation in certain differential hosts. Phylogenetic and ecological data indicate that MPVd and certain viroids now affecting crop plants, such as TPMVd, PSTVd, and possibly others, have a common ancestor. We hypothesize that commercial potatoes grown in the United States have become viroid-infected by chance transfer of MPVd or a similar viroid from endemically infected wild solanaceous plants imported from Mexico as germplasm, conceivably from plants known to have been introduced from Mexico to the United States late in the 19th century in efforts to identify genetic resistance to the potato late blight fungus, Phytophthora infestans.

Mutagenesis of the Glucose-1-Phosphate-Binding Site of Potato Tuber ADP-Glucose Pyrophosphorylase1

Lysine (Lys)-195 in the homotetrameric ADP-glucose pyrophosphorylase (ADPGlc PPase) from Escherichia coli was shown previously to be involved in the binding of the substrate glucose-1-phosphate (Glc-1-P). This residue is highly conserved in the ADPGlc PPase family. Site-directed mutagenesis was used to investigate the function of this conserved Lys residue in the large and small subunits of the heterotetrameric potato (Solanum tuberosum) tuber enzyme. The apparent affinity for Glc-1-P of the wild-type enzyme decreased 135- to 550-fold by changing Lys-198 of the small subunit to arginine, alanine, or glutamic acid, suggesting that both the charge and the size of this residue influence Glc-1-P binding. These mutations had little effect on the kinetic constants for the other substrates (ATP and Mg2+ or ADP-Glc and inorganic phosphate), activator (3-phosphoglycerate), inhibitor (inorganic phosphate), or on the thermal stability. Mutagenesis of the corresponding Lys (Lys-213) in the large subunit had no effect on the apparent affinity for Glc-1-P by substitution with arginine, alanine, or glutamic acid. A double mutant, SK198RLK213R, was also obtained that had a 100-fold reduction of the apparent affinity for Glc-1-P. The data indicate that Lys-198 in the small subunit is directly involved in the binding of Glc-1-P, whereas they appear to exclude a direct role of Lys-213 in the large subunit in the interaction with this substrate.

The Role of Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato Tuber Formation in Vitro1

The effects of plant hormones and sucrose (Suc) on potato (Solanum tuberosum L.) tuberization were studied using in vitro cultured single-node cuttings. Tuber-inducing (high Suc) and -noninducing (low Suc or high Suc plus gibberellin [GA]) media were tested. Tuberization frequencies, tuber widths, and stolon lengths were measured during successive stages of development. Endogenous GAs and abscisic acid (ABA) were identified and quantified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Exogenous GA4/7 promoted stolon elongation and inhibited tuber formation, whereas exogenous ABA stimulated tuberization and reduced stolon length. Indoleacetic acid-containing media severely inhibited elongation of stolons and smaller sessile tubers were formed. Exogenous cytokinins did not affect stolon elongation and tuber formation. Endogenous GA1 level was high during stolon elongation and decreased when stolon tips started to swell under inducing conditions, whereas it remained high under noninducing conditions. GA1 levels were negatively correlated with Suc concentration in the medium. We conclude that GA1 is likely to be the active GA during tuber formation. Endogenous ABA levels decreased during stolon and tuber development, and ABA levels were similar under inducing and noninducing conditions. Our results indicate that GA is a dominant regulator in tuber formation: ABA stimulates tuberization by counteracting GA, and Suc regulates tuber formation by influencing GA levels.

Actin Depolymerization Affects Stress-Induced Translational Activity of Potato Tuber Tissue1

Changes in polymerized actin during stress conditions were correlated with potato (Solanum tuberosum L.) tuber protein synthesis. Fluorescence microscopy and immunoblot analyses indicated that filamentous actin was nearly undetectable in mature, quiescent aerobic tubers. Mechanical wounding of postharvest tubers resulted in a localized increase of polymerized actin, and microfilament bundles were visible in cells of the wounded periderm within 12 h after wounding. During this same period translational activity increased 8-fold. By contrast, low-oxygen stress caused rapid reduction of polymerized actin coincident with acute inhibition of protein synthesis. Treatment of aerobic tubers with cytochalasin D, an agent that disrupts actin filaments, reduced wound-induced protein synthesis in vivo. This effect was not observed when colchicine, an agent that depolymerizes microtubules, was used. Neither of these drugs had a significant effect in vitro on run-off translation of isolated polysomes. However, cytochalasin D did reduce translational competence in vitro of a crude cellular fraction containing both polysomes and cytoskeletal elements. These results demonstrate the dependence of wound-induced protein synthesis on the integrity of microfilaments and suggest that the dynamics of the actin cytoskeleton may affect translational activity during stress conditions.

Age-Induced Protein Modifications and Increased Proteolysis in Potato Seed-Tubers1

Long-term aging of potato (Solanum tuberosum) seed-tubers resulted in a loss of patatin (40 kD) and a cysteine-proteinase inhibitor, potato multicystatin (PMC), as well as an increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases (75, 90, and 100 kD) appeared in the oldest tubers. Over 90% of the total proteolytic activity in aged tubers was sensitive to trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged tubers were also inhibited by crude extracts or purified PMC from young tubers, suggesting that the loss of PMC was responsible for the age-induced increase in proteinase activity. Nonenzymatic oxidation, glycation, and deamidation of proteins were enhanced by aging. Aged tubers developed “daughter” tubers that contained 3-fold more protein than “mother” tubers, with a polypeptide profile consistent with that of young tubers. Although PMC and patatin were absent from the older mother tubers, both proteins were expressed in the daughter tubers, indicating that aging did not compromise the efficacy of genes encoding PMC and patatin. Unlike the mother tubers, proteinase activity in daughter tubers was undetectable. Our results indicate that tuber aging nonenzymatically modifies proteins, which enhances their susceptibility to breakdown; we also identify a role for PMC in regulating protein turnover in potato tubers.

Involvement of Ethylene in Potato Microtuber Dormancy

Potato (Solanum tuberosum L.) single-node explants undergoing in vitro tuberization produced detectable amounts of ethylene throughout tuber development, and the resulting microtubers were completely dormant (endodormant) for at least 12 to 15 weeks. The rate of ethylene production by tuberizing explants was highest during the initial 2 weeks of in vitro culture and declined thereafter. Continuous exposure of developing microtubers to the noncompetitive ethylene antagonist AgNO3 via the culture medium resulted in a dose-dependent increase in precocious sprouting. The effect of AgNO3 on the premature loss of microtuber endodormancy was observed after 3 weeks of culture. Similarly, continuous exposure of developing microtubers to the competitive ethylene antagonist 2,5-norbornadiene (NBD) at concentrations of 2 mL/L (gas phase) or greater also resulted in a dose-dependent increase in premature sprouting. Exogenous ethylene reversed this response and inhibited the precocious sprouting of NBD-treated microtubers. NBD treatment was effective only when it was begun within 7 d of the start of in vitro explant culture. These results indicate that endogenous ethylene is essential for the full expression of potato microtuber endodormancy, and that its involvement may be restricted to the initial period of endodormancy development.

Stress Induction of Mitochondrial Formate Dehydrogenase in Potato Leaves1

In higher plants formate dehydrogenase (FDH, EC 1.2.1.2.) is a mitochondrial, NAD-dependent enzyme. We previously reported that in potato (Solanum tuberosum L.) FDH expression is high in tubers but low in green leaves. Here we show that in isolated tuber mitochondria FDH is involved in formate-dependent O2 uptake coupled to ATP synthesis. The effects of various environmental and chemical factors on FDH expression in leaves were tested using the mitochondrial serine hydroxymethyltransferase as a control. The abundance of FDH transcripts is strongly increased under various stresses, whereas serine hydroxymethyltransferase transcripts decline. The application of formate to leaves strongly enhances FDH expression, suggesting that it might be the signal for FDH induction. Our experiments using glycolytic products suggest that glycolysis may play an important role in formate synthesis in leaves in the dark and during hypoxia, and in tubers. Of particular interest is the dramatic accumulation of FDH transcripts after spraying methanol on leaves, as this compound is known to increase the yields of C3 plants. In addition, although the steady-state levels of FDH transcript increase very quickly in response to stress, protein accumulation is much slower, but can eventually reach the same levels in leaves as in tubers.

Mutagenesis of the potato ADPglucose pyrophosphorylase and characterization of an allosteric mutant defective in 3-phosphoglycerate activation.

ADPglucose pyrophosphorylase (glucose-1-phosphate adenylyltransferase; ADP:alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) catalyzes a key regulatory step in alpha-glucan synthesis in bacteria and higher plants. We have previously shown that the expression of the cDNA sequences of the potato tuber large (LS) and small (SS) subunits yielded a functional heterotetrameric enzyme capable of complementing a mutation in the single AGP (glgC) structural gene of Escherichia coli. This heterologous complementation provides a powerful genetic approach to obtain biochemical information on the specific roles of LS and SS in enzyme function. By mutagenizing the LS cDNA with hydroxylamine and then coexpressing with wild-type SS in an E. coli glgC- strain, >350 mutant colonies were identified that were impaired in glycogen production. One mutant exhibited enzymatic and antigen levels comparable to the wild-type recombinant enzyme but required 45-fold greater levels of the activator 3-phosphoglycerate for maximum activity. Sequence analysis identified a single nucleotide change that resulted in the change of Pro-52 to Leu. This heterologous genetic system provides an efficient means to identify residues important for catalysis and allosteric functioning and should lead to novel approaches to increase plant productivity.

Chrysanthemum chlorotic mottle viroid: Unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes

The causal agent of chrysanthemum chlorotic mottle (CChM) disease has been identified, cloned, and sequenced. It is a viroid RNA (CChMVd) of 398–399 nucleotides. In vitro transcripts with the complete CChMVd sequence were infectious and induced the typical symptoms of the CChM disease. CChMVd can form hammerhead structures in both polarity strands. Plus and minus monomeric CChMVd RNAs self-cleaved during in vitro transcription and after purification as predicted by these structures, which are stable and most probably act as single hammerhead structures as in peach latent mosaic viroid (PLMVd), but not in avocado sunblotch viroid (ASBVd). Moreover, the plus CChMVd hammerhead structure also appears to be active in vivo, because the 5′ terminus of the linear plus CChMVd RNA isolated from infected tissue is that predicted by the corresponding hammerhead ribozyme. Both hammerhead structures of CChMVd display some peculiarities: the plus self-cleaving domain has an unpaired A after the conserved A9 residue, and the minus one has an unusually long helix II. The most stable secondary structure predicted for CChMVd is a branched conformation that does not fulfill the rod-like or quasi-rod-like model proposed for the in vitro structure of most viroids with the exception of PLMVd, whose proposed secondary structure of lowest free energy also is branched. The unusual conformation of CChMVd and PLMVd is supported by their insolubility in 2 M LiCl, in contrast to ASBVd and a series of representative non-self-cleaving viroids that are soluble under the same high salt conditions. These results support the classification of self-cleaving viroids into two subgroups, one formed by ASBVd and the other one by PLMVd and CChMVd.

Endosperm balance number manipulation for direct in vivo germplasm introgression to potato from a sexually isolated relative (Solanum commersonii Dun.)

Diploid (2n = 2x = 24) Solanum species with endosperm balance number (EBN) = 1 are sexually isolated from diploid 2EBN species and both tetraploid (2n = 4x = 48, 4EBN) and haploid (2n = 2x = 24, 2EBN) S. tuberosum Group Tuberosum. To sexually overcome these crossing barriers in the diploid species S. commersonii (1EBN), the manipulation of the EBN was accomplished by scaling up and down ploidy levels. Triploid F1 hybrids between an in vitro-doubled clone of S. commersonii (2n = 4x = 48, 2EBN) and diploid 2EBN clones were successfully used in 3x × 4x crosses with S. tuberosum Group Tuberosum, resulting in pentaploid/near pentaploid BC1 progenies. This provided evidence of 2n (3x) egg formation in the triploid female parents. Two selected BC1 pentaploid hybrids were successfully backcrossed both as male and as female parents with S. tuberosum Group Tuberosum. The somatic chromosome number varied greatly among the resulting BC2 progenies, which included hyperaneuploids, but also a number (4.8%) of 48-chromosome plants. The introgression of S. commersonii genomes was confirmed by the presence of S. commersonii-specific randomly amplified polymorphic DNA markers in the BC2 population analyzed. The results clearly demonstrate the feasibility of germplasm introgression from sexually isolated diploid 1EBN species into the 4x (4EBN) gene pool of the cultivated potato using sexual hybridization. Based on the amount and type of genetic variation generated, cumbersomeness, general applicability, costs, and other factors, it would be interesting to compare the approach reported here with other in vitro or in vivo, direct or indirect, approaches previously reported.

Cleavage Furrows Formed between Centrosomes Lacking an Intervening Spindle and Chromosomes Contain Microtubule Bundles, INCENP, and CHO1 but Not CENP-E

PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-cell video, immunofluorescence, and electron microscopy to compare the structure and composition of normal equatorial furrows with that of ectopic furrows formed between spindles. As in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also contained the microtubule-bundling protein CHO1 and the chromosomal passenger protein INCENP. However, CENP-E, another passenger protein, was not found in ectopic furrows but was always present in controls. We also examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundles as well as INCENP and CHO1. Together these data suggest that the mechanism defining the site of furrow formation during mitosis in vertebrates does not depend on the presence of underlying microtubule bundles and chromosomes or on the stable association of INCENP or CHO1. The data also suggest that the completion of cytokinesis requires the presence of microtubule bundles and specific proteins (e.g., INCENP, CHO1, etc.) that do not include CENP-E.

New Alleles of the Yeast MPS1 Gene Reveal Multiple Requirements in Spindle Pole Body Duplication

In Saccharomyces cerevisiae, the Mps1p protein kinase is critical for both spindle pole body (SPB) duplication and the mitotic spindle assembly checkpoint. The mps1–1 mutation causes failure early in SPB duplication, and because the spindle assembly checkpoint is also compromised, mps1–1 cells proceed with a monopolar mitosis and rapidly lose viability. Here we report the genetic and molecular characterization of mps1–1 and five new temperature-sensitive alleles of MPS1. Each of the six alleles contains a single point mutation in the region of the gene encoding the protein kinase domain. The mutations affect several residues conserved among protein kinases, most notably the invariant glutamate in subdomain III. In vivo and in vitro kinase activity of the six epitope-tagged mutant proteins varies widely. Only two display appreciable in vitro activity, and interestingly, this activity is not thermolabile under the assay conditions used. While five of the six alleles cause SPB duplication to fail early, yielding cells with a single SPB, mps1–737 cells proceed into SPB duplication and assemble a second SPB that is structurally defective. This phenotype, together with the observation of intragenic complementation between this unique allele and two others, suggests that Mps1p is required for multiple events in SPB duplication.

Spc98p Directs the Yeast γ-Tubulin Complex into the Nucleus and Is Subject to Cell Cycle-dependent Phosphorylation on the Nuclear Side of the Spindle Pole Body

In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the γ-tubulin complex containing the γ-tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.