Grapevine virome and production of healthy plants by somatic embryogenesis

Grapevine (Vitis spp.) is a widespread fruit tree hosting many viral entities that interact with the plant modifying its responses to the environment. The production of virus-free plants is becoming increasingly crucial for the use of grapevine as a model species in different studies. Using high-throughput RNA sequencing, the viromes of seven mother plants grown in a germplasm collection vineyard were sequenced. In addition to the viruses and viroids already detected in grapevine, we identified 13 putative new mycoviruses. The different spread among grapevine tissues collected in vineyard, greenhouse and in vitro conditions suggested a clear distinction between viruses/viroids and mycoviruses that can successfully be exploited for their identification. Mycoviruses were absent in in vitro cultures, while plant viruses and viroids were particularly accumulated in these plantlets. Somatic embryogenesis applied to the seven mother plants was effective in the elimination of the complete virome, including mycoviruses. However, different sanitization efficiencies for viroids and grapevine pinot gris virus were observed among genotypes. The absence of mycoviruses in in vitro plantlets, associated with the absence of all viral entities in somaclones, suggested that this regeneration technique is also effective to eradicate endophytic/epiphytic fungi, resulting in gnotobiotic or pseudo-gnotobiotic plants.     

Wild type and mutants of the HET‐s(218–289) prion show different flexibility at fibrillar ends: A simulation study

The C‐terminal segment (residues 218–289) of the HET‐s protein of the filamentous fungus Podosporina anserina is a prion‐forming domain. The structural model of the HET‐s(218–289) amyloid fibril based on solid‐state nuclear magnetic resonance (NMR) restraints shows a β solenoid topology which is comprised of a β‐sheet core and interconnecting loops. For the single‐point mutants Phe286Ala and Trp287Ala, slower aggregation rates in vitro and loss of prionic infectivity have been reported recently. Here we have used molecular dynamics to compare the flexibility of the mutants and wild type. The simulations, initiated from a trimeric aggregate extracted from the NMR structural model, show structural stability on a 100‐ns time scale for wild type and mutants. Analysis of the fluctuations along the simulations reveals that the mutants are less flexible than the wild type in the C‐terminal segment at only one of the two external monomers. Analysis of interaction energy and buried accessible surface indicates that residue Phe286 in particular is stabilized in the Trp287Ala mutant. The simulation results provide an atomistic explanation of the suggestion (based on indirect experimental evidence) that flexibility at the protofibril end(s) is required for fibril elongation. Moreover, they provide further evidence that the growth of the HET‐s amyloid fibril is directional. Proteins 2014; 82:399–404. © 2013 Wiley Periodicals, Inc.     

Phyllosphere grapevine yeast Aureobasidium pullulans reduces Aspergillus carbonarius (sour rot) incidence in wine-producing vineyards in Greece

Phyllosphere yeasts were isolated from leaves of vine (Vitis vinifera L.) canes and evaluated in a detached berry assay for their ability to suppress Aspergillus carbonarius (Order: Eurotiales, Family: Trichocomaceae) growth. Seventeen of the 21 yeast isolates significantly reduced A. carbonarius growth, i.e. sour rot infection compared to untreated controls in laboratory tests. The most effective yeast isolate Aureobasidium pullulans (Order: Dothideales, Family: Dothioraceae), isolate Y-1, was field tested on two varieties of red grape, Grenache Rouge and Agiorgitiko located on the Island of Rhodes and in Corinthos County, Greece. It was demonstrated that A. pullulans Y-1 was as effective as the commercial fungicide fludioxonil + cyprodinil, in reducing sour rot infection, A. carbonarius presence on berries at harvest and ochratoxin A contamination in must.     

A rapid microbore HPLC method for determination of primary structure of β-lactoglobulin genetic variants

A rapid method for determination of the primary structures for β-lactoglobulin (β-LG) genetic variants is described. This included rapid microbore HPLC, amino acid analyses, and wherever necessary, direct peptide sequencing. Two novel variants of β-LG have been identified, bovine β-LG W and ovine β-LG C. The proteins were oxidized, digested with trypsin and separated using RP-HPLC. All peptides were recovered in a single run. Peptides with amino acid exchanges were identified by retention time and subjected to amino acid and sequence analyses. Ovine β-LG C differs from the ovine β-LG A variant by a single amino acid exchange at position 148 where Arg is replaced by Gln. Bovine β-LG W differs from bovine β-LG B by having Leu at position 56 instead of Ile. The method described here is reliable and can be used for mapping of 20–1000 pmol of material.     

Spring Usurpation Restlessness: A Wasp Social Parasite Adapts its Seasonal Activity to the Host Cycle

In animals, restlessness is a strategy to fulfil a goal within a narrow seasonal window when ecological and physiological constraints require the performance of certain fundamental events. Although restlessness can be expected in parasitic species that have their biological cycle similar to that of the host, no 'urge to parasitize' has thus far been demonstrated in any host-parasite pair. The paper wasp, Polistes sulcifer , is a social parasite that has lost the nest-building ability and marked by the absence of the worker caste; it depends on a congeneric host species to reproduce. Host colony usurpation is successful when it occurs in a well-defined seasonal window. The short time available for host-nest usurpation might lead to parasite restlessness that overlaps this period. Under controlled laboratory conditions, we demonstrated that the parasite females exhibit 'usurpation restlessness' during the period in which field usurpations occur. Additional experiments showed that both this period and laboratory temperature influence the parasite's 'urge to usurp'.     

PARP1 ADP-ribosylates lysine residues of the core histone tails

The chromatin-associated enzyme PARP1 has previously been suggested to ADP-ribosylate histones, but the specific ADP-ribose acceptor sites have remained enigmatic. Here, we show that PARP1 covalently ADP-ribosylates the amino-terminal histone tails of all core histones. Using biochemical tools and novel electron transfer dissociation mass spectrometric protocols, we identify for the first time K13 of H2A, K30 of H2B, K27 and K37 of H3, as well as K16 of H4 as ADP-ribose acceptor sites. Multiple explicit water molecular dynamics simulations of the H4 tail peptide into the catalytic cleft of PARP1 indicate that two stable intermolecular salt bridges hold the peptide in an orientation that allows K16 ADP-ribosylation. Consistent with a functional cross-talk between ADP-ribosylation and other histone tail modifications, acetylation of H4K16 inhibits ADP-ribosylation by PARP1. Taken together, our computational and experimental results provide strong evidence that PARP1 modifies important regulatory lysines of the core histone tails.