Single-molecule visualization of sequence-specific RNA binding by a designer PPR protein.

Pentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins, whereby each module can be modified to bind to a specific ssRNA nucleobase. As such, there is interest in developing 'designer' PPRs (dPPRs) for a range of biotechnology applications, including diagnostics or in vivo localization of ssRNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. To address this, we determined the structure of a dPPR bound to its target sequence and used two- and three-color single-molecule fluorescence resonance energy transfer to interrogate the mechanism of ssRNA binding to individual dPPRs in real time.

CRISPRi-based circuits to control gene expression in plants.

The construction of synthetic gene circuits in plants has been limited by a lack of orthogonal and modular parts. Here, we implement a CRISPR (clustered regularly interspaced short palindromic repeats) interference (CRISPRi)-based reversible gene circuit platform in plants. We create a toolkit of engineered repressible promoters of different strengths and construct NOT and NOR gates in Arabidopsis thaliana protoplasts.

Insights into U-to-C RNA editing from the lycophyte Phylloglossum drummondii.

The lycophyte Phylloglossum drummondii is the sole inhabitant of its genus in the Huperzioideae group and one of a small minority of plants which perform uridine to cytidine RNA editing. We assembled the P. drummondii chloroplast and mitochondrial genomes and used RNA sequence data to build a comprehensive profile of organellar RNA editing events.

Mitochondrial atp1 mRNA knockdown by a custom-designed pentatricopeptide repeat protein alters ATP synthase.

Spontaneous mutations are rare in mitochondria and the lack of mitochondrial transformation methods has hindered genetic analyses. We show that a custom-designed RNA-binding pentatricopeptide repeat (PPR) protein binds and specifically induces cleavage of ATP synthase subunit1 (atp1) mRNA in mitochondria, significantly decreasing the abundance of the Atp1 protein and the assembled F1Fo ATP synthase in Arabidopsis (Arabidopsis thaliana). The transformed plants are characterized by delayed vegetative growth and reduced fertility.

Applications of Synthetic Pentatricopeptide Repeat Proteins.

RNA-binding proteins play integral roles in the regulation of essential processes in cells and as such are attractive targets for engineering to manipulate gene expression at the RNA level. Expression of transcripts in chloroplasts and mitochondria is heavily regulated by pentatricopeptide repeat (PPR) proteins. The diverse roles of PPR proteins and their naturally modular architecture make them ideal candidates for engineering.

Alteration of Mitochondrial Transcript Expression in Using a Custom-Made Library of Pentatricopeptide Repeat Proteins.

Pentatricopeptide repeat (PPR) proteins are considered a potential tool for manipulating organelle gene expression in plants because they can recognise a wide range of different RNA sequences, and the molecular basis for this sequence recognition is partially known and understood. A library of redesigned PPR proteins related to restorer-of-fertility proteins was created and transformed into plants in order to target mitochondrial transcripts. Ninety different variants tested in vivo showed a wide range of phenotypes.

Environmental Factors Influencing Stem Rot Development in Peanut: Predictors and Action Thresholds for Disease Management.

Peanuts grown in tropical, subtropical, and temperate regions are susceptible to stem rot, which is a soilborne disease caused by . Due to the lack of reliable environmental-based scheduling recommendations, stem rot control relies heavily on fungicides that are applied at predetermined intervals. We conducted inoculated field experiments for six site-years in North Florida to examine the relationship between germination of sclerotia: the inoculum, stem rot symptom development in the peanut crop, and environmental factors such as soil temperature (ST), soil moisture, relative humidity (RH), precipitation, evapotranspiration, and solar radiation.

A unique C-terminal domain contributes to the molecular function of Restorer-of-fertility proteins in plant mitochondria.

Restorer-of-fertility (Rf) genes encode pentatricopeptide repeat (PPR) proteins that are targeted to mitochondria where they specifically bind to transcripts that induce cytoplasmic male sterility and repress their expression. In searching for a molecular signature unique to this class of proteins, we found that a majority of known Rf proteins have a distinct domain, which we called RfCTD (Restorer-of-fertility C-terminal domain), and its presence correlates with the ability to induce cleavage of the mitochondrial RNA target. A screen of 219 angiosperm genomes from 123 genera using a sequence profile that can quickly and accurately identify RfCTD sequences revealed considerable variation in RFL/RfCTD gene numbers across flowering plants.

Application of Hydrothermal Time Models to Predict Sclerotial Germination of .

, causal agent of "southern blight" disease, is a soilborne fungal pathogen with a wide host range of more than 500 species. This study's objectives were to (i) quantify the effects of two environmental factors, temperature and soil moisture, on germination of inoculum (sclerotia), which is a critical event for the onset of disease epidemics and (ii) predict the timing of sclerotial germination by applying population-based threshold-type hydrothermal time (HTT) models. We conducted in vitro germination experiments with three isolates of isolated from peanuts, which were tested at five temperatures (), ranging from 17 to 40°C, four matric potentials (Ψ) between -0.

Sensor-Based Quantification of Peanut Disease Defoliation Using an Unmanned Aircraft System and Multispectral Imagery.

Early leaf spot () and late leaf spot () are two of the most economically important foliar fungal diseases of peanut, often requiring seven to eight fungicide applications to protect against defoliation and yield loss. Rust () may also cause significant defoliation depending on season and location. Sensor technologies are increasingly being utilized to objectively monitor plant disease epidemics for research and supporting integrated management decisions.

Evolution of cox2 introns in angiosperm mitochondria and efficient splicing of an elongated cox2i691 intron.

In angiosperms, the mitochondrial cox2 gene harbors up to two introns, commonly referred to as cox2i373 and cox2i691. We studied the cox2 from 222 fully-sequenced mitogenomes from 30 angiosperm orders and analyzed the evolution of their introns. Unlike cox2i373, cox2i691 shows a distribution among plants that is shaped by frequent intron loss events driven by localized retroprocessing.

MSP1 encodes an essential RNA-binding pentatricopeptide repeat factor required for nad1 maturation and complex I biogenesis in Arabidopsis mitochondria.

Mitochondrial biogenesis relies on nuclearly encoded factors, which regulate the expression of the organellar-encoded genes. Pentatricopeptide repeat (PPR) proteins constitute a major gene family in angiosperms that are pivotal in many aspects of mitochondrial (mt)RNA metabolism (e.g.

Plant organellar RNA maturation.

Plant organellar RNA metabolism is run by a multitude of nucleus-encoded RNA-binding proteins (RBPs) that control RNA stability, processing, and degradation. In chloroplasts and mitochondria, these post-transcriptional processes are vital for the production of a small number of essential components of the photosynthetic and respiratory machinery-and consequently for organellar biogenesis and plant survival. Many organellar RBPs have been functionally assigned to individual steps in RNA maturation, often specific to selected transcripts.

Effects of Grass-Based Crop Rotation, Nematicide, and Irrigation on the Nematode Community in Cotton.

Plant-parasitic and free-living nematodes - bacterivores, fungivores, omnivores, predators - comprise the nematode community. Nematicide application and crop rotation are important tools to manage plant-parasitic nematodes, but effects on free-living nematodes and nematode ecological indices need further study. The nematicide fluopyram was recently introduced in cotton () production and its effects on the nematode community need assessment.

Synthetic PPR proteins as tools for sequence-specific targeting of RNA.

In native systems, gene expression is regulated by RNA binding proteins. Such proteins have been the target of a great deal of recent research interest, due to the potential for harnessing these regulatory effects for the construction of new biotechnological tools. In particular, focus has been targeted on building synthetic RNA binding proteins for sequence-specific targeting of new RNA transcripts.

The GENOMES UNCOUPLED1 protein has an ancient, highly conserved role but not in retrograde signalling.

The pentatricopeptide repeat protein GENOMES UNCOUPLED1 (GUN1) is required for chloroplast-to-nucleus signalling when plastid translation becomes inhibited during chloroplast development in Arabidopsis thaliana, but its exact molecular function remains unknown. We analysed GUN1 sequences in land plants and streptophyte algae. We tested functional conservation by complementation of the Arabidopsis gun1 mutant with GUN1 genes from the streptophyte alga Coleochate orbicularis or the liverwort Marchantia polymorpha.

Cofactor-independent RNA editing by a synthetic S-type PPR protein.

Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors and particularly prevalent in the lycophyte .

Triticeae genome sequences reveal huge expansions of gene families implicated in fertility restoration.

Breakthroughs in assembly of whole-genome sequencing and targeted sequence capture data have accelerated comparative genomics analyses in cereals with big and complex genomes such as wheat. This newly acquired information has revealed unexpected expansions in two large gene families linked to restoration of fertility in species that exhibit cytoplasmic male sterility. Extreme levels of copy-number and structural variation detected within and between species illustrate the genetic diversity among the family members and reveal the evolutionary mechanisms at work.

Deepred-Mt: Deep representation learning for predicting C-to-U RNA editing in plant mitochondria.

In land plant mitochondria, C-to-U RNA editing converts cytidines into uridines at highly specific RNA positions called editing sites. This editing step is essential for the correct functioning of mitochondrial proteins. When using sequence homology information, edited positions can be computationally predicted with high precision.

A synthetic RNA editing factor edits its target site in chloroplasts and bacteria.

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19).

Peanut disease epidemiology under dynamic microclimate conditions and management practices in North Florida.

Diverse field characteristics, weather patterns, and management practices can result in variable microclimates. The objective was to relate in-field microclimate conditions with peanut diseases and yield and determine the effect of irrigation and fungicides within these environments. Irrigation did not have a major impact on disease and yield.

Chromosome-scale genome assembly provides insights into rye biology, evolution and agronomic potential.

Rye (Secale cereale L.) is an exceptionally climate-resilient cereal crop, used extensively to produce improved wheat varieties via introgressive hybridization and possessing the entire repertoire of genes necessary to enable hybrid breeding. Rye is allogamous and only recently domesticated, thus giving cultivated ryes access to a diverse and exploitable wild gene pool.

The Pentatricopeptide Repeat Protein MEF100 Is Required for the Editing of Four Mitochondrial Editing Sites in .

In there are more than 600 C-to-U RNA editing events in the mitochondria and at least 44 in the chloroplasts. Pentatricopeptide repeat (PPR) proteins provide the specificity for these reactions. They recognize RNA sequences in a partially predictable fashion via key amino acids at the fifth and last position in each PPR motif that bind to individual ribonucleotides.

The genetic basis of cytoplasmic male sterility and fertility restoration in wheat.

Hybrid wheat varieties give higher yields than conventional lines but are difficult to produce due to a lack of effective control of male fertility in breeding lines. One promising system involves the Rf1 and Rf3 genes that restore fertility of wheat plants carrying Triticum timopheevii-type cytoplasmic male sterility (T-CMS). Here, by genetic mapping and comparative sequence analyses, we identify Rf1 and Rf3 candidates that can restore normal pollen production in transgenic wheat plants carrying T-CMS.

Predictive Modeling of Brown Rust of Sugarcane Based on Temperature and Relative Humidity in Florida.

Logistic regression models were developed from 5 years (2014 to 2018) of disease severity and weather data in an attempt to predict brown rust of sugarcane at the Everglades Research and Education Center in Belle Glade, Florida. Disease severity (percentage area of the top visible dewlap leaf covered by rust) was visually assessed in the field every 2 weeks for two varieties susceptible to brown rust. A total of 250 variables were derived from weather data for 10- to 40-day periods before each brown rust assessment day.