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The translation initiation factor 4E (eIF4E) has been implicated in naturally-occurring resistance to the Potato Virus Y (PVY) determined by the pot-1 locus in tomato. The susceptible potato varieties Russet Norkotah, Silverton Russet, and Classic Russet, along with the Michigan State University breeding line MSE149-5Y, were transformed with the pot-1 gene from Solanum hirsutum to achieve PVY resistance. Transgenic potato events and their non-transgenic controls were screened for PVY resistance against three PVY strains (PVYO, PVYN:O, PVYNTN) by enzyme-linked immunosorbent assay (ELISA) in greenhouse and in field trials. The event A95.87.05 (Classic Russet) and MSE149.87.22 (MSE149-5Y) had resistance against all three PVY isolates tested in both foliage and tuber under either greenhouse or field conditions. In contrast, SR.87.62 (Silverton Russet) and RN1.87.09 (Russet Norkotah), displayed moderate susceptibility to PVY in foliage during the growing season and increased level of infection in tubers. Transformation of PVY-susceptible potato varieties with the tomato pot-1 gene can be a useful tool for improving PVY resistance.

Diploid (2n = 2x = 24) and tetraploid (2n = 4x = 48) germplasm are commonly used in potato breeding programs. Potato breeders need to efficiently and inexpensively differentiate between diploid and tetraploid progeny in dihaploid induction crosses as well as 2x-2x crosses where 2n gametes may occur in the parents. In this study, we compared the chloroplast count, genome-wide SNP genotyping and flow cytometry methods to determine ploidy. Twenty-eight clones were used as reference samples (14 diploid lines (2n = 2x = 24), 14 tetraploid (2n = 4x = 48) varieties and advanced breeding lines) to compare the three ploidy determination methods. The chloroplast count method was used to determine the ploidy level in the 28 reference samples, and 102 potato breeding lines derived from diploid (2x-2x) crosses. The Infinium 12 K V2 Potato Single Nucleotide Polymorphism (SNP) Array was used to examine the 28 reference samples and the 102 breeding lines. The results obtained from both chloroplasts counts and SNP genotyping techniques determined that there was a total of 84 diploid lines and 18 tetraploid lines. Flow cytometry was also used to determine ploidy level in a subset of 42 lines (28 reference lines set and 14 breeding lines). All three methods of ploidy determination (chloroplast counting, SNP genotyping and flow cytometry) agreed for all samples evaluated. These results demonstrate the usefulness of chloroplast counting as an efficient and inexpensive method for breeders to differentiate ploidy between diploid and tetraploid potato in applied breeding programs.

We describe the development of genetic tools (electroporation, conjugation, vector for targeted gene replacement) for use in the psychrophile Psychrobacter arcticus 273-4 to test hypotheses about cold adaptation. Successful electroporation only occurred with nonstandard parameters, such as: electrocompetent cells freshly prepared from stationary-phase cultures, high field strengths (25 kV cm(-1)), long recovery times (16-24 h), and selection with low concentrations of antibiotics. Transformation frequencies were greatly affected by a methylation-dependent restriction barrier homologous to DpnI. The vector pJK100 (which was self-transmissible and contained a Pir-dependent R6K origin of replication) proved effective as a suicide plasmid that could be used to recombine mutations into the P. arcticus 273-4 genome. We used this vector for targeted replacement of dctT, the substrate-binding periplasmic subunit of a TRAP (tripartite ATP-independent periplasmic) transporter (which we have named dctTUF), as it was more highly expressed at cold temperatures. The replacement of dctT (with kan) decreased the rate of growth at low temperatures in mineral medium with glutamate, acetate, butyrate, and fumarate, but not with pyruvate suggesting that DctTUF participates in the transport of glutamate, acetate, butyrate, and fumarate at cold temperatures. This is the first report to demonstrate the creation of site-specific mutants in the genus Psychrobacter, their affect on low-temperature growth, and a substrate range for TAXI proteins of TRAP transporters.

The objective of this study was to construct a single nucleotide polymorphism (SNP)-based genetic map at the cultivated tetraploid level to locate quantitative trait loci (QTL) contributing to economically important traits in potato (Solanum tuberosum L.). The 156 F1 progeny and parents of a cross (MSL603) between “Jacqueline Lee” and “MSG227-2” were genotyped using the Infinium 8303 Potato Array. Furthermore, the progeny and parents were evaluated for foliar late blight reaction to isolates of the US-8 genotype of Phytophthora infestans (Mont.) de Bary and vine maturity. Linkage analyses and QTL mapping were performed using a novel approach that incorporates allele dosage information. The resulting genetic maps contained 1972 SNP markers with an average density of 1.36 marker per cM. QTL mapping identified the major source of late blight resistance in “Jacqueline Lee.” The best SNP marker mapped ∼0.54 Mb from a resistance hotspot on the long arm of chromosome 9. For vine maturity, the major-effect QTL was located on chromosome 5 with allelic effects from both parents. A candidate SNP marker for this trait mapped ∼0.25 Mb from the StCDF1 gene, which is a candidate gene for the maturity trait. The identification of markers for P. infestans resistance will enable the introgression of multiple sources of resistance through marker-assisted selection. Moreover, the discovery of a QTL for late blight resistance not linked to the QTL for vine maturity provides the opportunity to use marker-assisted selection for resistance independent of the selection for vine maturity classifications.

Competition between spermatozoa of rival males to gain fertilizations has led to a wide array of modifications in sperm structure and function. Sperm cells of most muroid rodents have hook-shaped extensions in the apical–ventral tip of the head, but the function of this structure is largely unknown. These ‘hooks’ may facilitate aggregation of spermatozoa in so-called ‘trains’, as an adaptation to sperm competition, because sperm in trains may swim faster than free-swimming cells. However, there is controversy regarding the role of the hook in train formation, and in relation to whether it is selected by sperm competition. We examined spermatozoa from muroid rodents with varying levels of sperm competition to assess whether (i) sperm aggregates are common in these taxa, (ii) presence of a hook relates to the formation of sperm aggregations, and (iii) formation of sperm aggregations is explained by sperm competition. Our analyses in 25 muroid species revealed that > 92% of spermatozoa swim individually in all species, with the exception of the wood mouse, Apodemus sylvaticus, which has ~50% spermatozoa swimming freely. Species with hooked spermatozoa had higher sperm competition levels and longer sperm than species whose sperm lack a hook. Neither the presence of hook nor sperm competition levels were related to the percentage of sperm in aggregations. Thus, (i) sperm aggregates in muroid rodents are an exceptional trait found only in a few species, (ii) evolution of the sperm hook is associated to sperm competition levels, but (iii) the hook is unlikely to be related to the formation of sperm aggregates. The evolutionary significance of the sperm head hook thus remains elusive, and future studies should examine potential roles of this pervasive structure in sperm's hydrodynamic efficiency and sperm–female tract interactions., This study was funded by the Spanish Ministry of Economy and Competitiveness (grants CGL2011-26341 and CGL2012-37423). M.T. was supported by a ‘Juan de la Cierva’ postdoctoral fellowship (JCI-2011-10381), whereas D.Z.T. was supported by a graduate studentship (BES2012-2012-059401), both from the Spanish Ministry of Economy and Competitiveness.

Summary The CBF cold response pathway has a prominent role in cold acclimation. The pathway includes action of three transcription factors, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), that are rapidly induced in response to low temperature followed by expression of the CBF-targeted genes (the CBF regulon) that act in concert to increase plant-freezing tolerance. The results of transcriptome profiling and mutagenesis experiments, however, indicate that additional cold response pathways exist and may have important roles in life at low temperature. To further understand the roles that the CBF proteins play in configuring the low temperature transcriptome and to identify additional transcription factors with roles in cold acclimation, we used the Affymetrix GeneChip containing probe sets for approximately 24,000 Arabidopsis genes to define a core set of cold-responsive genes and to determine which genes were targets of CBF2 and 6 other transcription factors that appeared to be coordinately regulated with CBF2. A total of 514 genes were placed in the core set of cold-responsive genes, 302 of which were upregulated and 212 downregulated. Hierarchical clustering and bioinformatic analysis indicated that the 514 cold-responsive transcripts could be assigned to one of seven distinct expression classes and identified multiple potential novel cis-acting cold-regulatory elements. Eighty-five cold-induced genes and eight cold-repressed genes were assigned to the CBF2 regulon. An additional nine cold-induced genes and 15 cold-repressed genes were assigned to a regulon controlled by ZAT12. Of the 25 core cold-induced genes that were most highly upregulated (induced over 15-fold), 19 genes (84%) were induced by CBF2 and another two genes (8%) were regulated by both CBF2 and ZAT12. Thus, the large majority (92%) of the most highly induced genes belong to the CBF and ZAT12 regulons. Constitutive expression of ZAT12 in Arabidopsis caused a small, but reproducible, increase in freezing tolerance, indicating a role for the ZAT12 regulon in cold acclimation. In addition, ZAT12 downregulated the expression of the CBF genes indicating a role for ZAT12 in a negative regulatory circuit that dampens expression of the CBF cold response pathway.

Many cold-regulated genes of Arabidopsis are inducible by abscisic acid (ABA) as well as by cold. This has been thought to occur via two separate signaling pathways, with ABA acting via ABA-responsive promoter elements and low temperature activating the C-repeat element (CRT; dehydration-responsive) promoter element via CBF (DREB1) transcription factors. We show here that ABA is also capable of activating the CRT promoter element. Although the more recently discovered ABA-inducible CBF4 transcription factor might have accounted for this, we show here that CBF1-3 transcript levels also increase in response to elevated ABA levels. This increase in CBF1-3 transcript levels appears to be at least in part due to increased activity of the CBF promoters in response to ABA. A total of 125 bp of the CBF2 promoter, which has previously been shown to be sufficient for cold-, mechanical-, and cycloheximide-induced expression, was also sufficient for ABA-induced expression. However, the ABA-responsive promoter element-like motif within this region is not needed for ABA-induced expression. An observed increase in CBF protein levels after ABA treatment, together with previous data showing that increased CBF levels are sufficient for cold-regulated gene induction, suggests that ABA-induced increases in CBF1-3 transcript levels do have the potential to activate the CRT. Our data indicate therefore that activation of the CRT may also occur via a novel ABA-inducible signaling pathway using the normally cold-inducible CBFs.

The Arabidopsis CBF1, 2, and 3 genes (also known as DREB1b, c, and a, respectively) encode transcriptional activators that have a central role in cold tolerance. CBF1-3 are rapidly induced upon exposing plants to low temperature, followed by expression of CBF-targeted genes, the CBF regulon, resulting in an increase in plant freezing tolerance. At present, little is known about the cold-sensing mechanism that controls CBF expression. Results presented here indicate that this mechanism does not require a cold shock to bring about the accumulation of CBF transcripts, but instead, absolute temperature is monitored with a greater degree of input, i.e. lower temperature, resulting in a greater output, i.e. higher levels of CBF transcripts. Temperature-shift experiments also indicate that the cold-sensing mechanism becomes desensitized to a given low temperature, such as 4°C, and that resensitization to that temperature requires between 8 and 24 h at warm temperature. Gene fusion experiments identified a 125-bp section of the CBF2 promoter that is sufficient to impart cold-responsive gene expression. Mutational analysis of this cold-responsive region identified two promoter segments that work in concert to impart robust cold-regulated gene expression. These sequences, designated ICEr1 and ICEr2 (induction of CBF expression region 1 or 2), were also shown to stimulate transcription in response to mechanical agitation and the protein synthesis inhibitor, cycloheximide.

Transgenic Arabidopsis thaliana plants which express genes encoding insect, Dendroides canadensis, antifreeze proteins (AFP) were produced by Agrobacterium-mediated transformation. The antifreeze protein genes, both with and without the signal peptide sequence (for protein secretion), were expressed in transformed plants. Thermal hysteresis activity (indicating the presence of active AFPs) was present in protein extracts from plants expressing both proteins and was also detected in leaf apoplast fluid from plants expressing AFPs with the signal peptide. Transgenic lines did not demonstrate improved ability to survive freezing when compared to wild-type. However, when cooled under four different regimes, transgenic lines with AFPs in the apoplast fluid froze at significantly lower temperatures than did wild-type, especially in the absence of extrinsic nucleation events.

Many plants increase in freezing tolerance upon exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. A fundamental goal of cold acclimation research is to identify genes with key roles in this response. Here we review results from our laboratory regarding the discovery of a family of transcriptional activators in Arabidopsis (Arabidopsis thaliana) that regulates the expression of freezing tolerance genes. Specifically, we have identified 3 genes that encode nearly identical transcriptional activators that bind to the CRT (C-repeat)/DRE (dehydration responsive element) DNA regulatory element present in the promoters of many cold- and drought-inducible genes, including those designated COR (cold-regulated). These regulatory genes, CBF1, CBF2 and CBF3 (CRT/DRE binding factor), are located in tandem array on chromosome 4. Overexpression of the CBF genes in Arabidopsis induces expression of the entire battery of known COR genes and increases freezing tolerance without a low temperature stimulus. We have, therefore, proposed that the CBF genes are ‘master switches’ that activate a regulon of genes involved in cold acclimation. Significantly, the CBF genes themselves are responsive to low temperature; the levels of CBF transcripts begin increasing within 15 min of transferring plants to low temperature followed by accumulation of COR gene transcripts at 2–4 h. The CBF genes do not appear to be subject to autoregulation as the promoter regions have no evident CRT/DRE elements and overexpression of CBF1 does not induce expression of CBF3. Thus, we have suggested that COR gene induction involves a two-step cascade of transcriptional activators: the first step, CBF induction, involving an unknown activator present at normal growth temperature and the second step, COR gene induction, involving the action of the CBF activators.

Cold-induced expression of the Arabidopsis COR (cold-regulated) genes is mediated by a DNA regulatory element termed the CRT (C-repeat)/DRE (dehydration-responsive element). Recently, we identified a transcriptional activator, CBF1, that binds to the CRT/DRE and demonstrated that its overexpression in transgenic Arabidopsis plants at non-acclimating temperatures induces COR gene expression and increases plant freezing tolerance. Here we report that CBF1 belongs to a small family of closely related proteins which includes CBF2 and CBF3. DNA sequencing of an 8.7 kb region of the Arabidopsis genome along with genetic mapping experiments indicated that the three CBF genes are organized in direct repeat on chromosome 4 at 72.8 cM, closely linked to molecular markers PG11 and m600. Like CBF1, both CBF2 and CBF3 activated expression of reporter genes in yeast that contained the CRT/DRE as an upstream activator sequence. The transcript levels for all three CBF genes increased within 15 min of transferring plants to low temperature, followed by accumulation of COR gene transcripts at about 2 h. CBF transcripts also accumulated rapidly in response to mechanical agitation. The promoter regions of the CBF genes do not contain the CRT sequence, CCGAC, and overexpression of CBF1 did not have a detectable effect on CBF3 transcript levels, suggesting that the CBF gene family is not subject to autoregulation. We propose that cold-induced expression of CRT/DRE-containing COR genes involves a low temperature-stimulated signalling cascade in which CBF gene induction is an early event.

Like many plants, Arabidopsis thaliana increases in freezing tolerance in response to low non-freezing temperatures, a phenomenon known as cold acclimation. Associated with cold acclimation are a number of biochemical changes including the expression of COR (cold-regulated) genes. Here we summarize recent progress we have made in understanding the function and regulation of these genes. One significant finding regarding COR gene function is that constitutive expression of COR15a in transgenic Arabidopsis plants enhances the freezing tolerance of both chloroplasts and protoplasts. These results provide the first direct evidence for a COR gene having a role in freezing tolerance. The precise mechanism of COR15a action is not yet know, but current results indicate the gene has a role in stabilizing membranes against freeze-induced damage. In regards to COR gene regulation, we have isolated a cDNA for CBF1, the first identified transcriptional activator that binds to the CRT (C-repeat)/DRE (drought responsive element), a cold- and drought-responsive DNA regulatory element present in the promoters of COR genes. Our working hypothesis is that CBF1 binds to the CRT/DRE sequence and participates in the regulation of COR genes in response to low temperature and drought.

Oenothera plants homozygous for a recessive allele at the plastome mutator (pm) locus show non-Mendelian mutation frequencies that are 1000-fold higher than spontaneous levels. Characterization of RFLP sites in a collection of mutants indicates that insertion-deletion hot spots in the pm lines are defined by tandem direct repeats, implicating replication slippage or misalignment during recombination. Several sites known to contain very short direct repeats were examined, and all were found to have been targeted in one or more plants of the mutant collection. To determine if replication slippage was occurring, two oligo-A stretches in non-coding DNA were examined, and 3 of 12 plants were found to have an additional adenine in a 13-base track. To search for other mutations that would not be visible as restriction fragment length polymorphisms, PCR-amplification products of the psbB gene were digested with a restriction endonuclease, denatured, and examined for single-strand conformational polymorphisms. Among 21 mutants, one 4-bp insertion and one point mutation were identified in psbB. The discovery that the plastome mutator can cause base substitutions as well as repeat-mediated insertions and deletions points to a likely defect in a component of the cpDNA replication machinery.

The effects of selection on genome variation were investigated and visualized in tomato using a high-density single nucleotide polymorphism (SNP) array. 7,720 SNPs were genotyped on a collection of 426 tomato accessions (410 inbreds and 16 hybrids) and over 97% of the markers were polymorphic in the entire collection. Principal component analysis (PCA) and pairwise estimates of F(st) supported that the inbred accessions represented seven sub-populations including processing, large-fruited fresh market, large-fruited vintage, cultivated cherry, landrace, wild cherry, and S. pimpinellifolium. Further divisions were found within both the contemporary processing and fresh market sub-populations. These sub-populations showed higher levels of genetic diversity relative to the vintage sub-population. The array provided a large number of polymorphic SNP markers across each sub-population, ranging from 3,159 in the vintage accessions to 6,234 in the cultivated cherry accessions. Visualization of minor allele frequency revealed regions of the genome that distinguished three representative sub-populations of cultivated tomato (processing, fresh market, and vintage), particularly on chromosomes 2, 4, 5, 6, and 11. The PCA loadings and F(st) outlier analysis between these three sub-populations identified a large number of candidate loci under positive selection on chromosomes 4, 5, and 11. The extent of linkage disequilibrium (LD) was examined within each chromosome for these sub-populations. LD decay varied between chromosomes and sub-populations, with large differences reflective of breeding history. For example, on chromosome 11, decay occurred over 0.8 cM for processing accessions and over 19.7 cM for fresh market accessions. The observed SNP variation and LD decay suggest that different patterns of genetic variation in cultivated tomato are due to introgression from wild species and selection for market specialization.

Objective. Candida species represent the fourth cause of nosocomial bloodstream infections worldwide. Because Candida glabrata has become the second most frequently identified yeast and because the rate of fluconazole-resistant C. glabrata strains reaches 10% to 15%, initial antifungal therapy based on fluconazole in nonneutropenic hemodynamically stable patients, as recommended by current guidelines, may be an ineffective option. Our aim was to determine easy-to-identify risk factors for C. glabrata fungemia likely to guide and improve initial antifungal therapy. Design: Prospective multicenter cohort study. Setting: Five French intensive care units. Patients. Consecutive nonneutropenic patients without known Candida colonization who had blood culture-confirmed fungemia over a 4-yr period. Interventions: None. Measurements and Main Results: A total of 8206 patients were screened. One hundred fifty-four patients with blood culture-confirmed fungemia constituted the cohort, of whom 48 had C. glabrata fungemia and 106 had nonglabrata fungemia. Patients' baseline characteristics and in-intensive care unit events potentially related to C. glabrata fungemia were systematically recorded. Compared with patients with nonglabrata fungemia, patients with C. glabrata fungemia were older and more severely ill, had received more antibiotics, and were more likely to have undergone surgery. The stepwise logistic regression analysis identified six independent risk factors for C. glabrata fungemia: age >60 yrs, recent abdominal surgery, interval from intensive care unit admission to first positive blood culture ≤7 days, recent use of cephalosporins, solid tumor, and absence of diabetes mellitus. The model showed satisfying goodness of fit (Hosmer-Lemeshow statistic = .26) and discrimination (c statistic = .89). Conclusions. We found six early available and easy-to-identify risk factors for C. glabrata fungemia. When these factors are present, alternatives to fluconazole for initial antifungal therapy should be considered.

High voltage electroporation has been investigated as a method for rapid recovery of plasmid and chromosomal DNA from the cyanobacteria Nostoc PCC 7121, Synechococcus PCC 7002, and Anabaena PCC 7120. Pulses of 18 kV/cm and higher applied to concentrated Nostoc cells carrying a shuttle plasmid (pRL25) resulted in copious release of nucleic acids and phycobiliproteins into the suspending medium. Small portions of these supernatants, when electroporated with Escherichia coli, gave rise to hundreds of E. coli transformants which contained pRL25. Electroporation of Synechococcus carrying plasmid pAQE19 did not cause detectable release of macromolecules but did reveal a low-level, voltage independent ‘leakage’ of pAQE19 into the medium. Electroextraction of Nostoc or Anabaena followed by addition of E. coli and delivery of a second high-voltage pulse permitted direct, one-cuvette transfer of shuttle plasmids from these cyanobacteria into E. coli. Electroextraction of single cyanobacterial colonies, as shown for Nostoc, also released sufficient chromosomal DNA for amplification of specific sequences by the polymerase chain reaction.

We have investigated host restriction as a barrier to transformation and developed a method for gene transfer into the previously untransformable, heterotrophic cyanobacterium Nostoc PCC 7121. A restriction endonuclease, designated Nsp 7121I, has been partially purified by phosphocellulose chromatography of Nostoc cell extracts. Comparisons of Nsp 7121I digests of bacteriophage lambda and plasmid DNAs with computer-generated restriction fragment profiles showed that Nsp 7121I is an isoschizomer of restriction endonucleases, such as Asu I, Nsp 7524IV, Sau 96I, and Eco 47II, that recognize the sequence GGNCC. Cleavage by Nsp 7121I within this sequence was confirmed by sequence analysis of DNA fragments cleaved at a unique Nsp 7121I site. These data further suggested that cleavage occurs after the first G (5'-G/GNCC-3') in this site to generate a three base 5' overhang. Nsp 7121I degraded all plasmids used in previous transformation attempts but modification of these DNA molecules by Eco 47II methylase effectively prevented digestion by Nsp 7121I. Plasmids premethylated by passage through Escherichia coli carrying a plasmid encoded Eco 47II methylase have now been used in an electroporation procedure to transform Nostoc PCC 7121 to neomycin resistance at frequencies as high as one transformant per 10(3) viable cells. Transformation, and stable replication within Nostoc of one of the transforming plasmids (pRL25), was confirmed by recovery of pRL25, in its original form, from transformants. Conjugal transfer of pRL25 from E. coli into Nostoc was also possible but at much lower efficiency than by electroporation. These findings establish the basis for genetic analysis of Nostoc PCC 7121, from which genes for photosynthetic electron transport have been cloned.