Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format

Background

The Arabidopsis thaliana-Pseudomonas syringae model pathosystem is one of the most widely used systems to understand the mechanisms of microbial pathogenesis and plant innate immunity. Several inoculation methods have been used to study plant-pathogen interactions in this model system. However, none of the methods reported to date are similar to those occurring in nature and amicable to large-scale mutant screens.

Results

In this study, we developed a rapid and reliable seedling flood-inoculation method based on young Arabidopsis seedlings grown on MS medium. This method has several advantages over conventional soil-grown plant inoculation assays, including a shorter growth and incubation period, ease of inoculation and handling, uniform infection and disease development, requires less growth chamber space and is suitable for high-throughput screens. In this study we demonstrated the efficacy of the Arabidopsis seedling assay to study 1) the virulence factors of P. syringae pv. tomato DC3000, including type III protein secretion system (TTSS) and phytotoxin coronatine (COR); 2) the effector-triggered immunity; and 3) Arabidopsis mutants affected in salicylic acid (SA)- and pathogen-associated molecular pattern (PAMPs)-mediated pathways. Furthermore, we applied this technique to study nonhost resistance (NHR) responses in Arabidopsis using nonhost pathogens, such as P. syringae pv. tabaci, pv. glycinea and pv. tomato T1, and confirmed the functional role of FLAGELLIN-SENSING 2 (FLS2) in NHR.

Conclusions

The Arabidopsis seedling flood-inoculation assay provides a rapid, efficient and economical method for studying Arabidopsis-Pseudomonas interactions with minimal growth chamber space and time. This assay could also provide an excellent system for investigating the virulence mechanisms of P. syringae. Using this method, we demonstrated that FLS2 plays a critical role in conferring NHR against nonhost pathovars of P. syringae, but not to Xanthomonas campestris pv. vesicatoria. This method is potentially ideal for high-throughput screening of both Arabidopsis and pathogen mutants.     

EFFECTS OF CARBOHYDRATE SOURCE ON THE IN VITRO ASYMBIOTIC SEED GERMINATION OF THE TERRESTRIAL ORCHID HABENARIA MACROCERATITIS

The effects of the carbohydrates fructose, sucrose, and dextrose on the seed germination and protocorm development of the terrestrial orchid Habenaria macroceratitis were assessed in the presence or absence of banana powder. Malmgren Modified Terrestrial Orchid Medium was used as the basal medium. Each carbohydrate was screened at 50 mM with and without banana powder (15 g L^?1). No significant differences in germination or protocorm development among all treatments were observed after seven weeks. There were no significant differences in advanced development among carbohydrate treatments without banana powder after 21 weeks. However, the presence of banana powder in the medium suppressed advanced protocorm development in the fructose, dextrose, and control treatments. These data support the wide suitability of simple exogenous carbohydrates in supporting the germination and protocorm development of H. macroceratitis, and suggest that a wide range of carbohydrates are suitable for the culture of often difficult-to-germinate terrestrial orchids.     

The prevalence of salmonella infection in sheep at slaughter

The prevalence of subclinical Salmonella infection in sheep at slaughter was monitored at two slaughter-houses in the southern part of the North Island over a 17-month period during 1975 and 1976. The crude prevalence rate was 4.7% (96/2027). Prevalence rates were greatest in the 2 and 4-tooth age group, with maximum monthly rates of 30%and 32% during 1975 and 1976 respectively. The seasonal distribution of infections was similar in each age group, prevalence rates being greatest in March, April and May, and least in October, November and December.

Salmonella typhimurium was found in 78% of the infected sheep, and phage-type 1 caused 72% of these infections. Other serotypes isolated were S. bovis-morbificans, S. derby, S. newingion, S. saintpaul, S. anatutn and S. enieritidis. The organisms were all fully sensitive to nine antibiotics. On the grounds that 87% of the infected sheep had salmonellae in their intestinal lymph nodes, it was deemed likely that most of the infected sheep harboured their infections while still on the farms of origin.     

Extending ideas about animal welfare assessment to include ‘quality of life’ and related concepts

Abstract

Ideas within the animal welfare science arena have evolved continuously throughout the last 30 years, and will continue to do so. This paper outlines some of these developments. These included reformulation of the five freedoms concept into the five domains of potential welfare compromise. This accommodated weaknesses in the former by distinguishing between the physical/functional and the mental factors that contribute to an animal's welfare state. This development reflected a rising scientific acceptance that the mental experiences of animals were legitimate foci for study and highlighted that what the animal itself experiences represents its welfare status. Initially, most concepts of animal welfare emphasised predominantly negative subjective experiences, such as thirst, hunger and pain, and negative affective states or feelings including anxiety, fear and boredom, but today positive experiences or emotions such as satiety, vitality, reward, contentment, curiosity and playfulness are also considered to be important. During the same period, the focus shifted from evaluating the impacts of individual mental subjective experiences or emotions towards seeking a more comprehensive, multifactorial understanding. The five domains concept was specifically designed to achieve this. Subsequent notions about quality of life (QoL) had the same objective, and emphasised the importance of positive experiences. However, some approaches to QoL assessment relied heavily on empathetic speculation about what animals may experience subjectively and this raised concerns about inappropriate anthropomorphic projections. Such pitfalls may be minimised when informed personnel rigorously apply objectively based methodologies to QoL assessments limited to a short time frame. It is clear that both formal and somewhat less formal QoL assessments of this type are already used to guide decision-making about the ongoing care and therapeutic management of animals on a daily basis. However, application of the recently introduced concepts of ‘a life not worth living’, ‘a life worth avoiding’, ‘a life worth living’ and ‘a good life’ is problematical, because extending the assessment time scale to the whole of life is attended by a number of as yet unresolved difficulties. Accordingly, their value in the practical management of animals is limited so that, at present, reliance on the minimum standards and recommendations for best practice outlined in codes of practice or welfare will continue to be necessary and worthwhile. Nevertheless, these concepts have value in providing a contextual theme that strongly focuses attention on the promotion of a lifelong QoL with an overall balance that is positive.     

Detection of asinine gammaherpesviruses in association with pulmonary fibrosis in free-ranging donkeys

A mortality event among recently captured feral donkeys (Equus asinus) occurred in south-central Utah in 2016. The deaths were sporadic, and clinical signs were indicative of respiratory disease, likely associated with an infectious etiology. Ten of 13 donkeys autopsied had moderate-to-severe interstitial fibrosing pneumonia, and one had pyogranulomatous pneumonia. Consensus PCRs directed toward the DNA polymerase and DNA packaging terminase subunit 1 for herpesviruses were performed followed by sequencing of the PCR amplicons and phylogenetic analysis. Asinine herpesvirus 4 (AsHV4) and 5 (AsHV5) were consistently identified in lung tissues of affected donkeys. No other herpesviruses were identified, and herpesviral DNA was not detected in lung tissues of 2 donkeys without evidence of respiratory disease. The detection of asinine gammaherpesviruses may have been associated with the lesions described. AsHV4 and AsHV5 have been reported in previous studies as novel gammaherpesviruses based on sequences obtained from donkeys with interstitial pneumonia and marked syncytial cell formation. Our findings suggest that the association of asinine gammaherpesviruses with respiratory conditions in equids deserves further attention.     

Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase

The TFP1 gene of the yeast Saccharomyces cerevisiae encodes two proteins: the 69-kilodalton (kD) catalytic subunit of the vacuolar proton-translocating adenosine triphosphatase (H(+)-ATPase) and a 50-kD protein. The 69-kD subunit is encoded by the 5' and 3' thirds of the TFP1 coding region, whereas the 50-kD protein is encoded by the central third. Evidence is presented that both the 69-kD and 50-kD proteins are obtained from a single translation product that is cleaved to release the 50-kD protein and spliced to form the 69-kD subunit.