Histone Deacetylase Complex 1 and histone 1 epigenetically moderate stress responsiveness of Arabidopsis thaliana seedlings.

Early responses of plants to environmental stress factors prevent damage but can delay growth and development in fluctuating conditions. Optimising these trade-offs requires tunability of plant responsiveness to environmental signals. We have previously reported that Histone Deacetylase Complex 1 (HDC1), which interacts with multiple proteins in histone deacetylation complexes, regulates the stress responsiveness of Arabidopsis seedlings, but the underlying mechanism remained elusive. Here, we show that HDC1 attenuates transcriptome re-programming in salt-treated seedlings, and we identify two genes (LEA and MAF5) that inhibit seedling establishment under salt stress downstream of HDC1. HDC1 attenuates their transcriptional induction by salt via a dual mechanism involving H3K9/14 deacetylation and H3K27 trimethylation. The latter, but not the former, was also abolished in a triple knockout mutant of the linker histone H1, which partially mimics the hypersensitivity of the hdc1-1 mutant to salt stress. Although stress-induced H3K27me3 accumulation required both H1 and HDC1, it was not fully recovered by complementing hdc1-1 with a truncated, H1-binding competent HDC1 suggesting other players or independent inputs. The combined findings reveal a dual brake function of HDC1 via regulating both active and repressive epigenetic marks on stress-inducible genes. This natural 'anti-panic' device offers a molecular leaver to tune stress responsiveness in plants.

The Histone Deacetylase Complex 1 Protein of Arabidopsis Has the Capacity to Interact with Multiple Proteins Including Histone 3-Binding Proteins and Histone 1 Variants.

Intrinsically disordered proteins can adopt multiple conformations, thereby enabling interaction with a wide variety of partners. They often serve as hubs in protein interaction networks. We have previously shown that the Histone Deacetylase Complex 1 (HDC1) protein from Arabidopsis (Arabidopsis thaliana) interacts with histone deacetylases and quantitatively determines histone acetylation levels, transcriptional activity, and several phenotypes, including abscisic acid sensitivity during germination, vegetative growth rate, and flowering time. HDC1-type proteins are ubiquitous in plants, but they contain no known structural or functional domains. Here, we explored the protein interaction spectrum of HDC1 using a quantitative bimolecular fluorescence complementation assay in tobacco (Nicotiana benthamiana) epidermal cells. In addition to binding histone deacetylases, HDC1 directly interacted with histone H3-binding proteins and corepressor-associated proteins but not with H3 or the corepressors themselves. Surprisingly, HDC1 also was able to interact with variants of the linker histone H1. Truncation of HDC1 to the ancestral core sequence narrowed the spectrum of interactions and of phenotypic outputs but maintained binding to a H3-binding protein and to H1. Thus, HDC1 provides a potential link between H1 and histone-modifying complexes.

Histone deacetylase complex1 expression level titrates plant growth and abscisic acid sensitivity in Arabidopsis.

Histone deacetylation regulates gene expression during plant stress responses and is therefore an interesting target for epigenetic manipulation of stress sensitivity in plants. Unfortunately, overexpression of the core enzymes (histone deacetylases [HDACs]) has either been ineffective or has caused pleiotropic morphological abnormalities. In yeast and mammals, HDACs operate within multiprotein complexes. Searching for putative components of plant HDAC complexes, we identified a gene with partial homology to a functionally uncharacterized member of the yeast complex, which we called Histone Deacetylation Complex1 (HDC1). HDC1 is encoded by a single-copy gene in the genomes of model plants and crops and therefore presents an attractive target for biotechnology. Here, we present a functional characterization of HDC1 in Arabidopsis thaliana. We show that HDC1 is a ubiquitously expressed nuclear protein that interacts with at least two deacetylases (HDA6 and HDA19), promotes histone deacetylation, and attenuates derepression of genes under water stress. The fast-growing HDC1-overexpressing plants outperformed wild-type plants not only on well-watered soil but also when water supply was reduced. Our findings identify HDC1 as a rate-limiting component of the histone deacetylation machinery and as an attractive tool for increasing germination rate and biomass production of plants.

Identification of the domains of cauliflower mosaic virus protein P6 responsible for suppression of RNA silencing and salicylic acid signalling.

Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded.

Deubiquitinating enzymes AtUBP12 and AtUBP13 and their tobacco homologue NtUBP12 are negative regulators of plant immunity.

• Signalling by ubiquitination is implicated in diverse aspects of the plant lifecycle, and enzymes of ubiquitin metabolism are overrepresented in the Arabidopsis genome compared with other model eukaryotes. Despite the importance of ubiquitination in the regulation of signalling, little is known about deubiquitinating enzymes, which reverse the process of ubiquitination. • Transgenic RNA interference-based cosuppression and the isolation of Atubp12/13 double mutants collectively provides the first report that AtUBP12 and AtUBP13 are functionally redundant and are required for immunity against virulent Pseudomonas syringae pv tomato in Arabidopsis. The Solanaceous AtUBP12 orthologue NtUBP12 was identified. Viral-induced gene silencing and transient gain-of-function assays were employed to establish that the NtUBP12 protein functions as a negative regulator of the Cf-9-triggered hypersensitive response. • Here, we demonstrate that NtUBP12 and AtUBP12 are bona fide deubiquitinating enzymes capable of cleaving lysine-48-linked ubiquitin chains. AtUBP12 and NtUBP12 are functionally interchangeable and their deubiquitinating activity is required to suppress plant cell death. • Overall, our data implicate AtUBP12- and NtUBP12-dependent deubiquitination in the stabilization of common substrates across Solanaceae and Brassicaceae which regulate disease resistance.

Rapid Development of a Tool for Prioritizing Patients with Coronavirus Disease 2019 for Intensive Care.

OBJECTIVES: To explain and demonstrate a new approach for rapidly developing a decision-support tool for prioritizing patients with coronovirus 2019 disease for admission to ICUs. DESIGN: An expert group used multi-criteria decision analysis methods to specify criteria and weights, representing their relative importance, for prioritizing patients with coronovirus 2019 disease with respect to likely clinical benefit. Specialized multi-criteria decision analysis software, implementing the "Potentially All Pairwise RanKings of all possible Alternatives" method to determine the weights, was used. Social equity considerations for prioritizing patients were also identified as important. SETTING: The prioritization tool was developed in New Zealand. SUBJECTS: An expert group comprising specialists from intensive care medicine and nursing, Maori (New Zealand's indigenous population) health, infectious diseases, and neonatology was formed. The group's work was supported by health economists and decision analysts and overseen by an ethicist and a senior representative from the New Zealand Ministry of Health. INTERVENTIONS: Multi-criteria decision analysis to create a prioritization tool. MEASUREMENTS AND MAIN RESULTS: The prioritization tool comprised eight criteria with respect to likely clinical benefit. In decreasing order of importance (weights in parentheses): Sequential Organ Failure Assessment score (15.7%), preexisting cardiovascular conditions (15.7%), functional capacity (15.7%), age (12.4%), preexisting respiratory conditions (11.1%), immunocompromised (11.1%), body mass index (9.2%), and other relevant medical conditions (9.2%). Two social equity considerations were also included in the overarching decision framework to be used alongside the clinical criteria: prioritizing Maori and Pacific people (and, potentially, other at-risk groups), and healthcare and other frontline workers. CONCLUSIONS: The criteria and weights in the prioritization tool can be easily revised as new evidence emerges. The approach for developing the tool could be used in other countries whose ICUs are at risk of being overwhelmed by the coronavirus disease 2019 pandemic to rapidly develop their own prioritization tools. In the event that future crises threaten to overload ICUs, other prioritization tools could also be rapidly developed.

Cryptic variation in RNA-directed DNA-methylation controls lateral root development when auxin signalling is perturbed.

Maintaining the right balance between plasticity and robustness in biological systems is important to allow adaptation while maintaining essential functions. Developmental plasticity of plant root systems has been the subject of intensive research, but the mechanisms underpinning robustness remain unclear. Here, we show that potassium deficiency inhibits lateral root organogenesis by delaying early stages in the formation of lateral root primordia. However, the severity of the symptoms arising from this perturbation varies within a natural population of Arabidopsis and is associated with the genetic variation in CLSY1, a key component of the RNA-directed DNA-methylation machinery. Mechanistically, CLSY1 mediates the transcriptional repression of a negative regulator of root branching, IAA27, and promotes lateral root development when the auxin-dependent proteolysis pathway fails. Our study identifies DNA-methylation-mediated transcriptional repression as a backup system for post-translational protein degradation which ensures robust development and performance of plants in a challenging environment.

Testing VHF/GPS collar design and safety in the study of free-roaming horses.

Effective and safe monitoring techniques are needed by U.S. land managers to understand free-roaming horse behavior and habitat use and to aid in making informed management decisions. Global positioning system (GPS) and very high frequency (VHF) radio collars can be used to provide high spatial and temporal resolution information for detecting free-roaming horse movement. GPS and VHF collars are a common tool used in wildlife management, but have rarely been used for free-roaming horse research and monitoring in the United States. The purpose of this study was to evaluate the design, safety, and detachment device on GPS/VHF collars used to collect free-roaming horse location and movement data. Between 2009 and 2010, 28 domestic and feral horses were marked with commercial and custom designed VHF/GPS collars. Individual horses were evaluated for damage caused by the collar placement, and following initial observations, collar design was modified to reduce the potential for injury. After collar modifications, which included the addition of collar length adjustments to both sides of the collar allowing for better alignment of collar and neck shapes, adding foam padding to the custom collars to replicate the commercial collar foam padding, and repositioning the detachment device to reduce wear along the jowl, we observed little to no evidence of collar wear on horses. Neither custom-built nor commercial collars caused injury to study horses, however, most of the custom-built collars failed to collect data. During the evaluation of collar detachment devices, we had an 89% success rate of collar devices detaching correctly. This study showed that free-roaming horses can be safely marked with GPS and/or VHF collars with minimal risk of injury, and that these collars can be a useful tool for monitoring horses without creating a risk to horse health and wellness.

Cauliflower mosaic virus protein P6 inhibits signaling responses to salicylic acid and regulates innate immunity.

Cauliflower mosaic virus (CaMV) encodes a multifunctional protein P6 that is required for translation of the 35S RNA and also acts as a suppressor of RNA silencing. Here we demonstrate that P6 additionally acts as a pathogenicity effector of an unique and novel type, modifying NPR1 (a key regulator of salicylic acid (SA)- and jasmonic acid (JA)-dependent signaling) and inhibiting SA-dependent defence responses We find that that transgene-mediated expression of P6 in Arabidopsis and transient expression in Nicotiana benthamiana has profound effects on defence signaling, suppressing expression of representative SA-responsive genes and increasing expression of representative JA-responsive genes. Relative to wild-type Arabidopsis P6-expressing transgenics had greatly reduced expression of PR-1 following SA-treatment, infection by CaMV or inoculation with an avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst). Similarly transient expression in Nicotiana benthamiana of P6 (including a mutant form defective in translational transactivation activity) suppressed PR-1a transcript accumulation in response to Agrobacterium infiltration and following SA-treatment. As well as suppressing the expression of representative SA-regulated genes, P6-transgenic Arabidopsis showed greatly enhanced susceptibility to both virulent and avirulent Pst (titres elevated 10 to 30-fold compared to non-transgenic controls) but reduced susceptibility to the necrotrophic fungus Botrytis cinerea. Necrosis following SA-treatment or inoculation with avirulent Pst was reduced and delayed in P6-transgenics. NPR1 an important regulator of SA/JA crosstalk, was more highly expressed in the presence of P6 and introduction of the P6 transgene into a transgenic line expressing an NPR1:GFP fusion resulted in greatly increased fluorescence in nuclei even in the absence of SA. Thus in the presence of P6 an inactive form of NPR1 is mislocalized in the nucleus even in uninduced plants. These results demonstrate that P6 is a new type of pathogenicity effector protein that enhances susceptibility to biotrophic pathogens by suppressing SA- but enhancing JA-signaling responses.