Translating ribosome affinity purification (trap) to investigate Arabidopsis thaliana root development at a cell type-specific scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation. As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained. However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq. TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation

Breakout — lateral root emergence in Arabidopsis thaliana

Lateral roots are determinants of plant root system architecture. Besides providing anchorage, they are a plant's means to explore the soil environment for water and nutrients. Lateral roots form post-embryonically and initiate deep within the root. On its way to the surface, the newly formed organ needs to grow through three overlying cell layers; the endodermis, cortex and epidermis. A picture is emerging that a tight integration of chemical and mechanical signalling between the lateral root and the surrounding tissue is essential for proper organogenesis. Here we review the latest progress made towards our understanding of the fascinating biology underlying lateral root emergence in Arabidopsis.</p

Breakout-lateral root emergence in Arabidopsis thaliana

Lateral roots are determinants of plant root system architecture. Besides providing anchorage, they are a plant's means to explore the soil environment for water and nutrients. Lateral roots form post-embryonically and initiate deep within the root. On its way to the surface, the newly formed organ needs to grow through three overlying cell layers; the endodermis, cortex and epidermis. A picture is emerging that a tight integration of chemical and mechanical signalling between the lateral root and the surrounding tissue is essential for proper organogenesis. Here we review the latest progress made towards our understanding of the fascinating biology underlying lateral root emergence in Arabidopsis

De-Novo Design of Antimicrobial Peptides for Plant Protection

This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 mg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 mg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of &quot;healthy&quot; food, these peptides might serve as templates for novel antibacterial and antifungal agents

Growth inhibition of phytopathogens on tomato leaves.

<p>Tomato leaves were inoculated with (A) virulent <i>P. syringae pv. tomato</i> DC3000 (10⁷ CFU/ml) or (B) spores of <i>A. alternata</i> or <i>C. herbarum</i> (10⁴ spores/ml). Afterwards different concentrations of antimicrobial peptides were sprayed onto the leaves. Bacterial growth was monitored 30 min after peptide treatment by determining colony-forming units per defined leaf area. Fungal growth was analysed 48 h after peptide treatment by quantification of fungal DNA content in the leave tissue. Fungal growth on leaves treated with peptide dilution buffer was set to 100%. Values represent the mean of at least three biological replicates ± standard error of the mean. *indicates significantly lower than the control treatment, <i>P</i><0.05.</p

Antibacterial activity of synthetic peptides in presence of apoplast fluid and in tomato fruits.

<p>(A) Approximately 10⁵ cfu/ml bacteria (<i>P. syringae</i> pv <i>tomato</i>) were incubated with 0 or 10 µg/ml peptide in the presence or absence of different concentrations (10 µg/ml or 30 µg/ml) of tomato apoplastic fluid. After 14–16 h the bacterial growth was determined by measuring OD_{600 nm}. APO, tomato apoplastic fluid. Values represent the mean of at least three biological replicates ± standard error of the mean. *indicates significantly different in comparison to the corresponding control treatment, <i>P</i><0.05. **indicates significantly different in comparison to the corresponding control treatment, <i>P</i><0.01. (B) <i>X. vesicatoria</i> (0.5×10⁵ cfu/ml) were treated with different concentrations of peptide SP10-5 and immediately injected into tomato fruits. After incubation for 5 d at room temperature infection symptoms were monitored. Above the values of incidence of infection symptoms is given in percentage. The total number of inoculation sides of three biological replicates were 22.</p

Bacterial membrane depolarization 60 minutes after AMP treatment.

<p>Depolarisation of bacterial membranes was determined by loading 5×10⁷ cfu/ml gram-positive <i>C. michiganensis</i> or gram-negative <i>P. syringae pv. syringae</i> with DiSC3(5) and measuring fluorescence intensity (FI) 60 min after addition of 0.5, 1, 5 or 10 µg/ml peptides (λ_ex: 622 nm λ_em: 670 nm). 1% Sodium dodecyl sulfate (SDS) and SP8 were used as positive and negative control, respectively. Shown are mean values ± standard error of the mean of three independent measurements normalized against FI after buffer treatment.</p

Antimicrobial activities (MIC) of designed first generation peptides against plant pathogens and hemolytic activities.

a<p>Shown are the peptide concentrations (µg/ml) leading to 25% hemoglobin release from human blood cells.</p><p>>200 describes a slight hemolytic activity at 200 µg/ml but still below the above mentioned threshold.</p

Effect of designed antimicrobial peptides on the viability of Arabidopsis mesophyll protoplasts <i>in-vitro</i>.

<p>Protoplasts were incubated for 1 h with different concentrations of SP1-1, SP10-2 and SP10-5 and analysed with a microscope (x 200). Cells with spherical shape without any sign of cytoplasmic degradation were defined as viable. A change of the cell shape, chloroplast release and/or agglomeration of protoplasts indicates a toxic effect of the peptides on plant cells (arrows).</p