Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported, but little attention has been paid so far to their sensitivity and reliability for diagnostic purposes. We therefore compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large scale performance test ten datasets of 21-24 nt small (s)RNA sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty to detect viral agents when they are novel and/or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases and (iv) the significant level of scientific expertise needed when interpreting pipelines results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.

In the fission yeast Schizosaccharomyces pombe, several genes including cdc15+, spo12+, fin1+, slp1+, ace2+ and plo1+ are periodically expressed during M phase. The products of these genes control various aspects of cell cycle progression including sister chromatid separation, septation and cytokinesis. We demonstrate that periodic expression of these genes is regulated by a common promoter sequence element, named a PCB. In a genetic screen for cell cycle regulators we have identified a novel forkhead transcription factor, Fkh2p, which is periodically phosphorylated in M phase. We show that Fhk2p and another forkhead transcription factor, Sep1p, are necessary for PCB-driven M-phase-specific transcription. In a previous report we identified a complex by electrophoretic mobility shift assay, which we termed PBF, that binds to a 150 bp region of the cdc15+ promoter that contains the PCB element. We have identified Mbx1p, a novel MADS box protein, as a component of PBF. However, although Mbx1p is periodically phosphorylated in M phase, Mbx1p is not required for periodic gene transcription in M phase. Moreover, although PBF is absent in strains bearing a C-terminal epitope tag on Fkh2p, simultaneous deletion of fkh2+ and sep1+ does not abolish PBF binding activity. This suggests that Mbx1p binds to gene promoters, but is not required for transcriptional activation. Together these results suggest that the activation of the Fkh2p and Sep1p forkhead transcription factors triggers mitotic gene transcription in fission yeast.

We have analyzed the histone acetyltransferase enzymes obtained from a series of yeast hat1, hat2, and gcn5 single mutants and hat1,hat2 and hat1,gcn5 double mutants. Extracts prepared from both hat1 and hat2 mutant strains specifically lack the following two histone acetyltransferase activities: the well known cytoplasmic type B enzyme and a free histone H4-specific histone acetyltransferase located in the nucleus. The catalytic subunits of both cytoplasmic and nuclear enzymes have identical molecular masses (42 kDa), the same as that of HAT1. However, the cytoplasmic complex has a molecular mass (150 kDa) greater than that of the nuclear complex (110 kDa). The possible functions of HAT1 and HAT2 in the yeast nucleus are discussed. In addition, we have detected a yeast histone acetyltransferase not previously described, designated HAT-A4. This enzyme is located in the nucleus and is able to acetylate free and nucleosome-bound histones H3 and H4. Finally, we show that the hat1,gcn5 double mutant is viable and does not exhibit a new phenotype, thus suggesting the existence of several histone acetyltransferases with overlapping functions.

Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.