A low-cost, portable, dual-function readout device for amplification-based point-of-need diagnostics.

The first critical step in timely disease management is rapid disease identification, which is ideally on-site detection. Of all the technologies available for disease identification, nucleic acid amplification-based diagnostics are often used due to their specificity, sensitivity, adaptability, and speed. However, the modules to interpret amplification results rapidly, reliably, and easily in resource-limited settings at point-of-need (PON) are in high demand.

Changing the diagnostic paradigm for sugarcane: development of a mill-based diagnostic for ratoon stunting disease in crude cane juice.

The availability of efficient diagnostic methods is crucial to monitor the incidence of crop diseases and implement effective management strategies. One of the most important elements in diagnostics, especially in large acreage crops, is the sampling strategy as hundreds of thousands of individual plants can grow in a single farm, making it difficult to assess disease incidence in field surveys. This problem is compounded when there are no external disease symptoms, as in the case for the ratoon stunting disease (RSD) in sugarcane.

Rapid parasite detection utilizing a DNA dipstick.

Molecular diagnostics are powerful tools for disease detection but are typically confined to the laboratory environment due to the cumbersome methods required to extract nucleic acids from biological samples. Accurate diagnosis is essential for early detection of parasitic worm infections and for monitoring control programs, particularly during new transmission outbreaks to limit infection spread. We optimized the recently developed DNA dipstick technology to purify Schistosoma japonicum DNA from different life stages in <60 s.

Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks.

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids).

Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics.

A number of isothermal DNA amplification technologies claim to be ideal for point-of-need (PON) applications as they enable reactions to be performed using a single-temperature heat source (e.g. water bath).

A simple, robust and equipment-free DNA amplification readout in less than 30 seconds.

Molecular based diagnostic methods rely on the amplification of pathogen DNA but naked eye visualization of results is still challenging. We present here a simple and highly reliable DNA amplification readout system for naked eye detection of isothermally or PCR amplified DNA in less than 30 seconds. This system utilizes spermine to precipitate DNA amplicons and initiate bridging flocculation of a mix of charcoal and diatomaceous earth particles in suspension.

Correction: Nucleic acid purification from plants, animals and microbes in under 30 seconds.

[This corrects the article DOI: 10.1371/journal.pbio.

Nucleic acid purification from plants, animals and microbes in under 30 seconds.

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed.

LATERAL BRANCHING OXIDOREDUCTASE acts in the final stages of strigolactone biosynthesis in Arabidopsis.

Strigolactones are a group of plant compounds of diverse but related chemical structures. They have similar bioactivity across a broad range of plant species, act to optimize plant growth and development, and promote soil microbe interactions. Carlactone, a common precursor to strigolactones, is produced by conserved enzymes found in a number of diverse species.

Strigolactone Inhibition of Branching Independent of Polar Auxin Transport.

The outgrowth of axillary buds into branches is regulated systemically via plant hormones and the demand of growing shoot tips for sugars. The plant hormone auxin is thought to act via two mechanisms. One mechanism involves auxin regulation of systemic signals, cytokinins and strigolactones, which can move into axillary buds.

Sugar demand, not auxin, is the initial regulator of apical dominance.

For almost a century the plant hormone auxin has been central to theories on apical dominance, whereby the growing shoot tip suppresses the growth of the axillary buds below. According to the classic model, the auxin indole-3-acetic acid is produced in the shoot tip and transported down the stem, where it inhibits bud growth. We report here that the initiation of bud growth after shoot tip loss cannot be dependent on apical auxin supply because we observe bud release up to 24 h before changes in auxin content in the adjacent stem.

Functional characterization of type-B response regulators in the Arabidopsis cytokinin response.

Cytokinins play critical roles in plant growth and development, with the transcriptional response to cytokinin being mediated by the type-B response regulators. In Arabidopsis (Arabidopsis thaliana), type-B response regulators (ARABIDOPSIS RESPONSE REGULATORS [ARRs]) form three subfamilies based on phylogenic analysis, with subfamily 1 having seven members and subfamilies 2 and 3 each having two members. Cytokinin responses are predominantly mediated by subfamily 1 members, with cytokinin-mediated effects on root growth and root meristem size correlating with type-B ARR expression levels.

Strigolactones suppress adventitious rooting in Arabidopsis and pea.

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting.

Type-B response regulators ARR1 and ARR12 regulate expression of AtHKT1;1 and accumulation of sodium in Arabidopsis shoots.

Soil salinity affects a large proportion of the land worldwide, forcing plants to evolve a number of mechanisms to cope with salt stress. Cytokinin plays a role in the plant response to salt stress, but little is known about the mechanism by which cytokinin controls this process. We used a molecular genetics approach to examine the influence of cytokinin on sodium accumulation and salt sensitivity in Arabidopsis thaliana.

Type B response regulators of Arabidopsis play key roles in cytokinin signaling and plant development.

The type B Arabidopsis Response Regulators (ARRs) of Arabidopsis thaliana are transcription factors that act as positive regulators in the two-component cytokinin signaling pathway. We employed a mutant-based approach to perform a detailed characterization of the roles of ARR1, ARR10, and ARR12 in plant growth and development. The most pronounced phenotype was found in the arr1-3 arr10-5 arr12-1 triple loss-of-function mutant, which showed almost complete insensitivity to high levels of exogenously applied cytokinins.

Heterotrimeric G protein gamma subunits provide functional selectivity in Gbetagamma dimer signaling in Arabidopsis.

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Galpha and Gbeta subunit genes and two Ggamma subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Ggamma genes revealed specialized roles for each Ggamma subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.

A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway.

The plant hormone cytokinin regulates numerous growth and developmental processes. A signal transduction pathway for cytokinin has been elucidated that is similar to bacterial two-component phosphorelays. In Arabidopsis, this pathway is comprised of receptors that are similar to sensor histidine kinases, histidine-containing phosphotransfer proteins, and response regulators (ARRs).

Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis.

Type-B Arabidopsis thaliana response regulators (ARRs) are transcription factors that function in the final step of two-component signaling systems. To characterize their role in plant growth and development, we isolated T-DNA insertions within six of the genes (ARR1, ARR2, ARR10, ARR11, ARR12, and ARR18) from the largest subfamily of type-B ARRs and also constructed various double and triple combinations of these mutations. Higher order mutants revealed progressively decreased sensitivity to cytokinin, including effects on root elongation, lateral root formation, callus induction and greening, and induction of cytokinin primary response genes.

Type-B response regulators display overlapping expression patterns in Arabidopsis.

Two-component signaling systems, involving His kinases, His-containing phosphotransfer proteins, and response regulators, have been implicated in plant responses to hormones and environmental factors. Genomic analysis of Arabidopsis supports the existence of 22 response regulators (ARRs) that can be divided into at least two distinct groups designated type-A and type-B. Phylogenetic analysis indicates that the type-B family is composed of one major and two minor subfamilies.

Type-A Arabidopsis response regulators are partially redundant negative regulators of cytokinin signaling.

Type-A Arabidopsis (Arabidopsis thaliana) response regulators (ARRs) are a family of 10 genes that are rapidly induced by cytokinin and are highly similar to bacterial two-component response regulators. We have isolated T-DNA insertions in six of the type-A ARRs and constructed multiple insertional mutants, including the arr3,4,5,6,8,9 hextuple mutant. Single arr mutants were indistinguishable from the wild type in various cytokinin assays; double and higher order arr mutants showed progressively increasing sensitivity to cytokinin, indicating functional overlap among type-A ARRs and that these genes act as negative regulators of cytokinin responses.

Research note: Rapid isolation of total RNA and genomic DNA from Hakea actities.

Tissues of the Australian native plant species Hakea actities (Proteaceae) contain numerous metabolites and structural compounds that hinder the isolation of nucleic acids. Separate RNA and genomic DNA extraction procedures were developed to isolate high quality nucleic acids from H. actities.