A functional RNase P protein subunit of bacterial origin in some eukaryotes

RNase P catalyzes 5′-maturation of tRNAs. While bacterial RNase P comprises an RNA catalyst and a protein cofactor, the eukaryotic (nuclear) variant contains an RNA and up to ten proteins, all unrelated to the bacterial protein. Unexpectedly, a nuclear-encoded bacterial RNase P protein (RPP) homolog is found in several prasinophyte algae including Ostreococcus tauri. We demonstrate that recombinant O. tauri RPP can functionally reconstitute with bacterial RNase P RNAs (RPRs) but not with O. tauri organellar RPRs, despite the latter’s presumed bacterial origins. We also show that O. tauri PRORP, a homolog of Arabidopsis PRORP-1, displays tRNA 5′-processing activity in vitro. We discuss the implications of the striking diversity of RNase P in O. tauri, the smallest known free-living eukaryote.Ministerio de Ciencia e Innovación European Regional Fund BFU2007-60651Junta de Andalucía P06-CVI-01692National Science Foundation MCB-0238233 MCB-0843543European Union ASSEMBLE 22779

Selective nucleolus organizer inactivation in Arabidopsis is a chromosome position-effect phenomenon

International audienc

Geminivirus AL2 and L2 Proteins Suppress Transcriptional Gene Silencing and Cause Genome-Wide Reductions in Cytosine Methylation▿ †

Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S-adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibition. However, at one exceptional locus, ADK inhibition was insufficient and TGS reversal required the transcriptional activation domain of AL2. Using restriction-sensitive PCR and bisulfite sequencing, we showed that AL2-mediated TGS suppression is accompanied by reduced cytosine methylation. Finally, using a methylation-sensitive single-nucleotide extension assay, we showed that transgenic expression of AL2 or L2 causes global reduction in cytosine methylation. Our results provide further evidence that viral chromatin methylation is an important host defense and allow us to propose that as a countermeasure, geminivirus proteins reverse TGS by nonspecifically inhibiting cellular transmethylation reactions. To our knowledge, this is the first report that viral proteins can inhibit TGS

A Complex Containing SNF1-Related Kinase (SnRK1) and Adenosine Kinase in Arabidopsis

<div><p>SNF1-related kinase (SnRK1) in plants belongs to a conserved family that includes sucrose non-fermenting 1 kinase (SNF1) in yeast and AMP-activated protein kinase (AMPK) in animals. These kinases play important roles in the regulation of cellular energy homeostasis and in response to stresses that deplete ATP, they inhibit energy consuming anabolic pathways and promote catabolism. Energy stress is sensed by increased AMP:ATP ratios and in plants, 5′-AMP inhibits inactivation of phosphorylated SnRK1 by phosphatase. In previous studies, we showed that geminivirus pathogenicity proteins interact with both SnRK1 and adenosine kinase (ADK), which phosphorylates adenosine to generate 5′-AMP. This suggested a relationship between SnRK1 and ADK, which we investigate in the studies described here. We demonstrate that SnRK1 and ADK physically associate in the cytoplasm, and that SnRK1 stimulates ADK <i>in vitro</i> by an unknown, non-enzymatic mechanism. Further, altering SnRK1 or ADK activity in transgenic plants altered the activity of the other kinase, providing evidence for <i>in vivo</i> linkage but also revealing that <i>in vivo</i> regulation of these activities is complex. This study establishes the existence of SnRK1-ADK complexes that may play important roles in energy homeostasis and cellular responses to biotic and abiotic stress.</p></div

Endogenous <i>N. benthamiana</i> SnRK1 activity co-purifies with expressed ADK.

<p>(A) HA₂His₆-tagged Arabidopsis ADK and HA₂His₆-GFP (control) were expressed in <i>N. benthamiana</i> and enriched using nickel-NTA resin. Proteins were fractionated by SDS-PAGE, and Coomassie stained samples are shown. (B) GST-SAMS and γ³²P-ATP were added to ADK or GFP preparations (3 µg protein). Samples were separated by SDS-PAGE and exposed to a phosphor-imager for 2 days. The Coomassie stained panel is a loading control for GST-SAMS and GST-SAMA, while the immunoblot (bottom panel, probed with anti-HA) is a loading control for ADK and GFP.</p

ADK is phosphorylated by SnRK1 <i>in vitro</i>.

<p>Kinase assays were conducted using γ³²P-ATP with SnRK1-KD or SnRK1-KD-K49R either alone or with ADK. With the exception of autophosphorylation assays, SnRK1-KD and SnRK1-KD-K49R were pre-incubated with unlabeled ATP to obscure autophosphorylation. Samples were fractioned by SDS-PAGE and signals detected using a phosphor-imager. Immunoblots were probed with anti-ADK to detect ADK, or with anti-HA to detect SnRK1-KD or SnRK1-KD-K49R. (A) Kinase assays with SnRK1-KD (10 ng) and SnRK1-KD-K49R (30 ng) were performed without pre-incubation with unlabeled ATP. (B) ADK protein (3 µg) was incubated with SnRK1-KD (10 ng) or SnRK1-KD-K49R (30 ng). Note that SnRK1-KD autophosphorylation (in the lane lacking ADK) was nearly undetectable due to pre-incubation of SnRK1 with unlabeled ATP. The same pre-incubated SnRK1-KD and SnRK1-KD-K49R preparations were used to perform the GST-SAMS phosphorylation experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087592#pone-0087592-g001" target="_blank">Figure 1C</a>. Activity data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087592#pone.0087592.s006" target="_blank">Table S3</a>.</p

The <i>in vitro</i> SnRK1 assay.

<p>(A) Diagrams of GST-SAMS and GST-SAMA substrates. The target serine in GST-SAMS, containing a SNF1/AMPK/SnRK1 consensus site, is replaced by alanine in GST-SAMA (residues underlined). The line connecting the SAMS/SAMA sequence to GST indicates a flexible polyglycine linker. These substrates were expressed and purified from <i>E. coli</i> cells. (B) Autophosphorylation assay confirmed that SnRK1-KD, but not SnRK1-KD-K49R, expressed in <i>N. benthamiana</i> is active. HA₂His₆-tagged SnRK1-KD and SnRK1-KD-K49R (inactive mutant, negative control) expressed and purified from <i>N. benthamiana</i> were incubated with γ³²P-ATP alone or with GST-SAMS or GST-SAMA. Aliquots were fractionated on polyacrylamide gels containing SDS (SDS-PAGE) and exposed to a phosphor-imager to detect labeled proteins. For loading controls, GST-SAMS and GST-SAMA were monitored by Coomassie staining, while immunoblots were probed with anti-HA (α-HA) to detect the kinases. (C) Kinase assays in which GST-SAMS or GST-SAMA (3 µg) were incubated with varying amounts of SnRK1-KD or SnRK1-KD-K49R, as indicated. (D) A linear correlation (R² = 0.9664) was observed between the intensity of labeled GST-SAMS signal and the amount of SnRK1-KD added in the range tested. The data shown are representative of three experiments, and were normalized to activity observed with 5 ng SnRK1-KD. (E) Extracts from <i>N. benthamiana</i> cells expressing full-length SnRK1, SnRK1-KD, or GUS (control for endogenous activity) from a plasmid vector were assayed following the addition of γ²P-ATP and GST-SAMS. SnRK1-KD activity data (autophosphorylation and phosphorylation of GST-SAMS) is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087592#pone.0087592.s004" target="_blank">Table S1</a>.</p

Not Available

Not AvailableGlobally, soil salinity has been on the rise owing to various factors that are both human and environmental. The abiotic stress caused by soil salinity has become one of the most damaging abiotic stresses faced by crop plants, resulting in significant yield losses. Salt stress induces physiological and morphological modifications in plants as a result of significant changes in gene expression patterns and signal transduction cascades. In this comprehensive review, with a major focus on recent advances in the field of plant molecular biology, we discuss several approaches to enhance salinity tolerance in plants comprising various classical and advanced genetic and genetic engineering approaches, genomics and genome editing technologies, and plant growth-promoting rhizobacteria (PGPR)-based approaches. Furthermore, based on recent advances in the field of epigenetics, we propose novel approaches to create and exploit heritable genome-wide epigenetic variation in crop plants to enhance salinity tolerance. Specifically, we describe the concepts and the underlying principles of epigenetic recombinant inbred lines (epiRILs) and other epigenetic variants and methods to generate them. The proposed epigenetic approaches also have the potential to create additional genetic variation by modulating meiotic crossover frequency.NASF/CRISPR-Cas-7003/2018-19/GATES/Bill & Melinda Gates Foundation/United State