Thermomorphogenesis of the Arabidopsis thaliana root: Flexible cell division, constrained elongation, and the role of cryptochrome.

Understanding how plants respond to temperature is relevant for agriculture in a warming world. Responses to temperature of the shoot have been characterized more fully than those of the root. Previous work on thermomorphogenesis in roots established that for Arabidopsis thaliana (Columbia) seedlings grown continuously at a given temperature, the root meristem produces cells at the same rate at 15 as at 25ºC and the root's growth zone is the same length. To uncover the pathway(s) underlying this constancy, we screened 34 A. thaliana genotypes for parameters related to growth and division. No line failed to respond to temperature. Behavior was little affected by mutations in phytochrome or other genes that underly thermomorphogenesis in shoots. However, a mutant in cryptochrome2 was disrupted substantially in both cell division and elongation, specifically at 15ºC. Among the 34 lines, cell production rate varied extensively and was associated only weakly with root growth rate; in contrast, parameters relating to elongation were stable. Our data are consistent with models of root growth that invoke cell non-autonomous regulation for establishing boundaries between meristem, elongation zone, and mature zone.

Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM).

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.

Mother trees, altruistic fungi, and the perils of plant personification.

There are growing doubts about the true role of the common mycorrhizal networks (CMN or wood wide web) connecting the roots of trees in forests. We question the claims of a substantial carbon transfer from 'mother trees' to their offspring and nearby seedlings through the CMN. Recent reviews show that evidence for the 'mother tree concept' is inconclusive or absent. The origin of this concept seems to stem from a desire to humanize plant life but can lead to misunderstandings and false interpretations and may eventually harm rather than help the commendable cause of preserving forests. Two recent books serve as examples: The Hidden Life of Trees and Finding the Mother Tree.

Mycoviruses in the Rust Fungus Uromyces fabae.

Uromyces fabae, the causal agent of broad bean rust, is a major cause of yield losses in North and East Africa, China, and Australia. It has also served as an important model species for research on rust fungi. Early EST sequencing in U. fabae showed that viruses might be present in this species; however, no follow-up investigations were conducted. In order to identify these viruses, we performed purification of dsRNA followed by Illumina sequencing. We also used ultracentrifugation followed by negative staining electron microscopy to visualize virus particles. We identified 20 viral sequences, which we termed Ufvss. A phylogenetic analysis was performed that grouped Ufvss into totiviruses, polymycoviruses, and virgaviruse; three sequences could not be included in the phylogeny. We also found isometric particles. Our findings contribute to the knowledge of mycoviral diversity in rust fungi and point to the importance of further investigation of these viruses.

Cellulose assembles into helical bundles of uniform handedness in cell walls with abnormal pectin composition.

Plant cells and organs grow into a remarkable diversity of shapes, as directed by cell walls composed primarily of polysaccharides such as cellulose and multiple structurally distinct pectins. The properties of the cell wall that allow for precise control of morphogenesis are distinct from those of the individual polysaccharide components. For example, cellulose, the primary determinant of cell morphology, is a chiral macromolecule that can self-assemble in vitro into larger-scale structures of consistent chirality, and yet most plant cells do not display consistent chirality in their growth. One interesting exception is the Arabidopsis thaliana rhm1 mutant, which has decreased levels of the pectin rhamnogalacturonan-I and causes conical petal epidermal cells to grow with a left-handed helical twist. Here, we show that in rhm1 the cellulose is bundled into large macrofibrils, unlike the evenly distributed microfibrils of the wild type. This cellulose bundling becomes increasingly severe over time, consistent with cellulose being synthesized normally and then self-associating into macrofibrils. We also show that in the wild type, cellulose is oriented transversely, whereas in rhm1 mutants, the cellulose forms right-handed helices that can account for the helical morphology of the petal cells. Our results indicate that when the composition of pectin is altered, cellulose can form cellular-scale chiral structures in vivo, analogous to the helicoids formed in vitro by cellulose nano-crystals. We propose that an important emergent property of the interplay between rhamnogalacturonan-I and cellulose is to permit the assembly of nonbundled cellulose structures, providing plants flexibility to orient cellulose and direct morphogenesis.

BUZZ: an essential gene for postinitiation root hair growth and a mediator of root architecture in Brachypodium distachyon.

Here, we discover a player in root development. Recovered from a forward-genetic screen in Brachypodium distachyon, the buzz mutant initiates root hairs but they fail to elongate. In addition, buzz roots grow twice as fast as wild-type roots. Also, lateral roots show increased sensitivity to nitrate, whereas primary roots are less sensitive to nitrate. Using whole-genome resequencing, we identified the causal single nucleotide polymorphism as occurring in a conserved but previously uncharacterized cyclin-dependent kinase (CDK)-like gene. The buzz mutant phenotypes are rescued by the wild-type B. distachyon BUZZ coding sequence and by an apparent homolog in Arabidopsis thaliana. Moreover, T-DNA mutants in A. thaliana BUZZ have shorter root hairs. BUZZ mRNA localizes to epidermal cells and develops root hairs and, in the latter, partially colocalizes with the NRT1.1A nitrate transporter. Based on qPCR and RNA-Seq, buzz overexpresses ROOT HAIRLESS LIKE SIX-1 and -2 and misregulates genes related to hormone signaling, RNA processing, cytoskeletal, and cell wall organization, and to the assimilation of nitrate. Overall, these data demonstrate that BUZZ is required for tip growth after root hair initiation and root architectural responses to nitrate.

Major proliferation of transposable elements shaped the genome of the soybean rust pathogen Phakopsora pachyrhizi.

With >7000 species the order of rust fungi has a disproportionately large impact on agriculture, horticulture, forestry and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. A key example is Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease, one of the world's most economically damaging agricultural diseases. Despite P. pachyrhizi's impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. Here, we sequence three independent P. pachyrhizi genomes and uncover a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. We study the incursion and dominant impact of these TEs on the genome and show how they have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity.

Ethylene represses the promoting influence of cytokinin on cell division and expansion of cotyledons in etiolated Arabidopsis thaliana seedlings.

The plant hormones ethylene and cytokinin influence many processes; sometimes they act cooperatively, other times antagonistically. To study their antagonistic interaction, we used the cotyledons of etiolated, intact seedlings of Arabidopsis thaliana. We focused on cell division and expansion, because both processes are quantified readily in paradermal sections. Here, we show that exogenous cytokinins modestly stimulate cell division and expansion in the cotyledon, with a phenyl-urea class compound exerting a larger effect than benzyl-adenine. Similarly, both processes were stimulated modestly when ethylene response was inhibited, either chemically with silver nitrate or genetically with the eti5 ethylene-insensitive mutant. However, combining cytokinin treatment with ethylene insensitivity was synergistic, strongly stimulating both cell division and expansion. Evidently, ethylene represses the growth promoting influence of cytokinin, whether endogenous or applied. We suggest that the intact etiolated cotyledon offers a useful system to characterize how ethylene antagonizes cytokinin responsiveness.

The miR156 juvenility factor and PLETHORA 2 form a regulatory network and influence timing of meristem growth and lateral root emergence.

Plants develop throughout their lives: seeds become seedlings that mature and form fruits and seeds. Although the underlying mechanisms that drive these developmental phase transitions have been well elucidated for shoots, the extent to which they affect the root is less clear. However, root anatomy does change as some plants mature; meristems enlarge and radial thickening occurs. Here, in Arabidopsis thaliana, we show that overexpressing miR156A, a gene that promotes the juvenile phase, increased the density of the root system, even in grafted plants in which only the rootstock had the overexpression genotype. In the root, overexpression of miR156A resulted in lower levels of PLETHORA 2, a protein that affects formation of the meristem and elongation zone. Crossing in an extra copy of PLETHORA 2 partially rescued the effects of miR156A overexpression on traits affecting root architecture, including meristem length and the rate of lateral root emergence. Consistent with this, PLETHORA 2 also inhibited the root-tip expression of another miR156 gene, miR156C. We conclude that the system driving phase change in the shoot affects developmental progression in the root, and that PLETHORA 2 participates in this network.

Host-Induced Gene Silencing Using BPMV on Soybean to Study Genes in the Soybean Rust Fungus Phakopsora pachyrhizi.

To obtain direct evidence for the influence of an effector on the virulence or pathogenicity of a pathogen, it is necessary to knock out, knock down, or silence the respective gene. Since genetic transformation is not yet possible for rust fungi, silencing the gene is the only option. Posttranscriptional gene silencing uses RNAi. RNAi in plant pathogens can be accomplished by introducing dsRNA either by direct application of in vitro synthesized dsRNA or through positive-strand or double-strand RNA plant viruses. For studying effectors in Phakopsora pachyrhizi, we have implemented a host-induced silencing procedure based on virus-induced gene silencing using the bean pod mottle virus system. Here, procedures and interpretations of results are described and limitations of the system are discussed.

Isolation, Identification, and Biocontrol Potential of Root Fungal Endophytes Associated with Solanaceous Plants against Potato Late Blight (Phytophthora infestans).

Late blight of potato caused by Phytophthora infestans is one of the most damaging diseases affecting potato production worldwide. We screened 357 root fungal endophytes isolated from four solanaceous plant species obtained from Kenya regarding their in vitro antagonistic activity against the potato late blight pathogen and evaluated their performance in planta. Preliminary in vitro tests revealed that 46 of these isolates showed potential activity against the pathogen. Based on their ITS-sequences, 37 out of 46 endophytes were identified to species level, three isolates were connected to higher taxa (phylum or genus), while two remained unidentified. Confrontation assays, as well as assays for volatile or diffusible organic compounds, resulted in the selection of three endophytes (KB1S1-4, KA2S1-42, and KB2S2-15) with a pronounced inhibitory activity against P. infestans. All three isolates produce volatile organic compounds that inhibit mycelial growth of P. infestans by up to 48.9%. The addition of 5% extracts obtained from KB2S2-15 or KA2S1-42 to P. infestans sporangia entirely suppressed their germination. A slightly lower inhibition (69%) was achieved using extract from KB1S1-4. Moreover, late blight symptoms and the mycelial growth of P. infestans were completely suppressed when leaflets were pre-treated with a 5% extract from these endophytes. This might suggest the implementation of such biocontrol candidates or their fungicidal compounds in late blight control strategies.

Oxygen uptake rates have contrasting responses to temperature in the root meristem and elongation zone.

Growing at either 15 or 25°C, roots of Arabidopsis thaliana, Columbia accession, produce cells at the same rate and have growth zones of the same length. To determine whether this constancy is related to energetics, we measured oxygen uptake by means of a vibrating oxygen-selective electrode. Concomitantly, the spatial distribution of elongation was measured kinematically, delineating meristem and elongation zone. All seedlings were germinated, grown, and measured at a given temperature (15 or 25°C). Columbia was compared to lines where cell production rate roughly doubles between 15 and 25°C: Landsberg and two Columbia mutants, er-105 and ahk3-3. For all genotypes and temperatures, oxygen uptake rate at any position was highest at the root cap, where mitochondrial density was maximal, based on the fluorescence of a reporter. Uptake rate declined through the meristem to plateau within the elongation zone. For oxygen uptake rate integrated over a zone, the meristem had steady-state Q10 values ranging from 0.7 to 2.1; by contrast, the elongation zone had values ranging from 2.6 to 3.3, implying that this zone exerts a greater respiratory demand. These results highlight a substantial energy consumption by the root cap, perhaps helpful for maintaining hypoxia in stem cells, and suggest that rapid elongation is metabolically more costly than is cell division.

Establishment of a quadruplex real-time PCR assay to distinguish the fungal pathogens Diaporthe longicolla, D. caulivora, D. eres, and D. novem on soybean.

Diaporthe species are fungal plant pathogens of many important crops. Seed decay is one of the most important diseases on soybean. It is caused by various species of the genus Diaporthe and responsible for significant economic damage. In central Europe the four species D. longicolla, D. caulivora, D. eres, and D. novem are considered the principal species of Diaporthe on soybean. Fast and accurate detection of these pathogens is of utmost importance. In this study four species-specific TaqMan primer-probe sets that can be combined into a quadruplex assay were designed based on TEF sequences. The specificity and efficiency of the primer-probe sets were tested using PCR products and genomic DNA from pure cultures of the four Diaporthe species and other soybean fungal pathogens. Our results indicate that the primer-probe sets DPCL, DPCC, DPCE, and DPCN allow discrimination of D. longicolla, D. caulivora, D. eres, and D. novem, respectively, and can be used to detect and quantify these four Diaporthe species in parallel using quadruplex real-time PCR. In addition, the quadruplex real-time PCR assay was evaluated on different plant materials including healthy and infected soybean seeds or seed lots, soybean stems, and soybean leaves. This assay is a rapid and effective method to detect and quantify Diaporthe species from samples relevant for disease control.

Testing reference genes for transcript profiling in Uromyces appendiculatus during urediospore infection of common bean.

Uromyces appendiculatus is a major pathogen on common bean. Like other rust fungi, it uses effectors to influence its host plant. Effectors are assumed to possess characteristic expression profiles, reflecting their activity during the infection process. In order to determine expression profiles using RT-qPCR, stably expressed reference genes are necessary for normalization. These reference genes need to be tested. Using samples representing seven different developmental stages of the urediospore-based infection process we employed RT-qPCR to measure the expression of 14 candidate reference genes and determined the most suitable ones based on the range of Cq values and comparative calculations using the geNorm and NormFinder algorithms. Among the tested genes RPS14 had the smallest Cq range, followed by Elf1a and Elf3; geNorm rated Tub and UbcE2 best with CytB as a third and NormFinder found UbcE2, Tub and Elf3 as best reference genes. Combining these findings using equal weight for the rankings UbcE2, Elf3 and Tub can be considered the best reference genes. A combination of either two reference genes, UbcE2 and Tub or three reference genes, UbcE2, Tub, and Elf3 is recommended for normalization. However, differences between most genes were relatively small, so all tested genes can be considered suitable for normalization with the exception of RPS9, SDH, Ubc and PDK.

Positioning the Root Elongation Zone Is Saltatory and Receives Input from the Shoot.

In the root, meristem and elongation zone lengths remain stable, despite growth and division of cells. To gain insight into zone stability, we imaged individual Arabidopsis thaliana roots through a horizontal microscope and used image analysis to obtain velocity profiles. For a root, velocity profiles obtained every 5 min over 3 h coincided closely, implying that zonation is regulated tightly. However, the position of the elongation zone saltated, by on average 17 µm every 5 min. Saltation was apparently driven by material elements growing faster and then slower, while moving through the growth zone. When the shoot was excised, after about 90 min, growth zone dynamics resembled those of intact roots, except that the position of the elongation zone moved, on average, rootward, by several hundred microns in 24 h. We hypothesize that mechanisms determining elongation zone position receive input from the shoot.

RTIP: A FULLY AUTOMATED ROOT TIP TRACKER FOR MEASURING PLANT GROWTH WITH INTERMITTENT PERTURBATIONS.

RTip is a tool to quantify plant root growth velocity using high resolution microscopy image sequences at sub-pixel accuracy. The fully automated RTip tracker is designed for high-throughput analysis of plant phenotyping experiments with episodic perturbations. RTip is able to auto-skip past these manual intervention perturbation activity, i.e. when the root tip is not under the microscope, image is distorted or blurred. RTip provides the most accurate root growth velocity results with the lowest variance (i.e. localization jitter) compared to six tracking algorithms including the top performing unsupervised Discriminative Correlation Filter Tracker and the Deeper and Wider Siamese Network. RTip is the only tracker that is able to automatically detect and recover from (occlusion-like) varying duration perturbation events.

Candidate Effectors From Uromyces appendiculatus, the Causal Agent of Rust on Common Bean, Can Be Discriminated Based on Suppression of Immune Responses.

Rust fungi are devastating pathogens for several important crop plants. The biotrophic lifestyle of rust fungi requires that they influence their host plants to create a favorable environment for growth and reproduction. Rust fungi secrete a variety of effector proteins that manipulate host target proteins to alter plant metabolism and suppress defense responses. Because of the obligate biotrophic lifestyle of rust fungi, direct evidence for effector function is difficult to obtain, and so suites of experiments utilizing expression in heterologous systems are necessary. Here, we present results from a yeast cell death suppression assay and assays for suppression of PAMP-triggered immunity (PTI) and effector triggered immunity (ETI) based on delivery of effectors through the bacterial type III secretion system. In addition, subcellular localization was tested using transient expression of GFP fusion proteins in Nicotiana benthamiana through Agrobacterium infiltration. We tested 31 representative effector candidates from the devastating common bean rust pathogen Uromyces appendiculatus. These effector candidates were selected based on features of their gene families, most important lineage specificity. We show that several of our effector candidates suppress plant defense. Some of them also belong to families of effector candidates that are present in multiple rust species where their homologs probably also have effector functions. In our analysis of candidate effector mRNA expression, some of those effector candidates that gave positive results in the other assays were not up-regulated during plant infection, indicating that either these proteins have functions at multiple life stages or that strong up-regulation of RNA level in planta may not be as important a criterion for identifying effectors as previously thought. Overall, our pipeline for selecting effector candidates based on sequence features followed by screening assays using heterologous expression systems was successful in discriminating effector candidates. This work lays the foundation for functional characterization of U. appendiculatus effectors, the identification of effector targets, and identification of novel sources for resistance in common bean.

Kinematic Characterization of Root Growth by Means of Stripflow.

Kinematic methods for studying root growth are powerful but underutilized. To carry out kinematic analysis, the Baskin laboratory, in collaboration with computer scientists, developed software called Stripflow that quantifies the velocity of motion of points in the root, a quantification that is required for subsequent kinematic analysis. The first half of this chapter overviews concepts that underlie kinematic analysis of root growth; the second half provides a step-by-step guide for using Stripflow.

Analysis of Mitochondrial Dimensions and Cristae Structure in Pluripotent Stem Cells Using Transmission Electron Microscopy.

Dynamic alterations to mitochondrial structure and function regulate cell fate decisions and underlie multiple age-related and genetic diseases that are modeled using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Transmission electron microscopy (TEM) can be used to obtain high-resolution micrographs of mitochondria, but mitochondrial ultrastructure is easily distorted during specimen processing. This unit describes a method that preserves mitochondrial membrane structure from adherent ESC cultures for TEM sample preparation. This procedure is useful for assessing ultrastructural changes to mitochondria during differentiation, reprogramming, or experimental manipulation of ESC metabolism. We provide comprehensive protocols for: (1) preparation of ESC cultures for TEM; (2) retrieval of thin sections from individual ESCs; and (3) contrast staining and morphometric analysis of mitochondria and cristae. This unit also describes an alternative procedure for samples with low cell numbers and a supporting protocol for morphometric image analysis. Collectively, these protocols allow for the observation and quantitative analysis of mitochondria in ESCs. © 2018 by John Wiley & Sons, Inc.

The subspecies concept in Geocorinae: an integrated taxonomic case study on Geocoris (Piocoris) erythrocephalus (Lepeletier Serville, 1825) (Hemiptera: Heteroptera: Geocoridae).

The validity and necessity of subspecies as a taxonomic category and the implications of the subspecies concept in various taxa of animals is subject of debates since its very existence. In case of the species of the lygaeoid subfamily Geocorinae there are multiple examples of species consisting of up to nine subspecies which were mostly described as varieties or forms before the middle of 20th century, but upgraded to valid subspecies subsequently, usually without providing any arguments. As part of an integrated taxonomic study on the Palaearctic representatives of Geocorinae, the status of the subspecies of Geocoris (Piocoris) erythrocephalus (Lepeletier Serville, 1825) was revisited. A critical review of the literature available and our studies, involving analysis of COI sequences, morphological examination and distribution data processing with the use of Geographic Information System, concluded that two of the three subspecies, G. e. erythrocephalus and G. e. marginellus Horváth, 1907, can be considered valid, while G. e. litoreus Horváth, 1895, is merely a phenotypically manifested infrasubspecific genetic variety of G. e. erythrocephalus, with no taxonomic value as it was suggested by earlier study of another author. Besides the interpretation of evidence, the applicability of the subspecies concept in Geocorinae is discussed as well.