What is cost-efficient phenotyping? Optimizing costs for different scenarios

Progress in remote sensing and robotic technologies decreases the hardware costs of phenotyping. Here, we first review cost-effective imaging devices and environmental sensors, and present a trade-off between investment and manpower costs. We then discuss the structure of costs in various real-world scenarios. Hand-held low-cost sensors are suitable for quick and infrequent plant diagnostic measurements. In experiments for genetic or agronomic analyses, (i) major costs arise from plant handling and manpower; (ii) the total costs per plant/microplot are similar in robotized platform or field experiments with drones, hand-held or robotized ground vehicles; (iii) the cost of vehicles carrying sensors represents only 5–26% of the total costs. These conclusions depend on the context, in particular for labor cost, the quantitative demand of phenotyping and the number of days available for phenotypic measurements due to climatic constraints. Data analysis represents 10–20% of total cost if pipelines have already been developed. A trade-off exists between the initial high cost of pipeline development and labor cost of manual operations. Overall, depending on the context and objsectives, “cost-effective” phenotyping may involve either low investment (“affordable phenotyping”), or initial high investments in sensors, vehicles and pipelines that result in higher quality and lower operational costs

An essential role for the VASt domain of the Arabidopsis VAD1 protein in the regulation of defense and cell death in response to pathogens

Several regulators of programmed cell death (PCD) have been identified in plants which encode proteins with putative lipid -binding domains. Among them, VAD1 (Vascular Associated Death) contains a novel protein domain called VASt (VAD1 analog StAR-related lipid transfer) still uncharacterized. The Arabidopsis mutant vadl-1 has been shown to exhibit a lesion mimic phenotype with light-conditional appearance of propagative hypersensitive response -like lesions along the vascular system, associated with defense gene expression and increased resistance to Pseudomonas strains. To test the potential of ectopic expression of VAD1 to influence HR cell death and to elucidate the role of the VASt domain in this function, we performed a structure -function analysis of VAD1 by transient over -expression in Nicotiana benthamiana and by complementation of the mutant vadl-1. We found that (i) overexpression of VAD1 controls negatively the HR cell death and defense expression either transiently in Nicotiana benthamania or in Arabidopsis plants in response to avirulent strains of Pseudomonas syringae, (ii) VAD1 is expressed in multiple subcellular compartments, including the nucleus, and (iii) while the GRAM domain does not modify neither the subcellular localization of VAD1 nor its immunorepressor activity, the domain VASt plays an essential role in both processes. In conclusion, VAD1 acts as a negative regulator of cell death associated with the plant immune response and the VASt domain of this unknown protein plays an essential role in this function, opening the way for the functional analysis of VASt-containing proteins and the characterization of novel mechanisms regulating PCD

Patterns of Sequence and Expression Diversification Associate Members of the PADRE Gene Family With Response to Fungal Pathogens

Pathogen infection triggers extensive reprogramming of the plant transcriptome, including numerous genes the function of which is unknown. Due to their wide taxonomic distribution, genes encoding proteins with Domains of Unknown Function (DUFs) activated upon pathogen challenge likely play important roles in disease. InArabidopsis thaliana, we identified thirteen genes harboring a DUF4228 domain in the top 10% most induced genes after infection by the fungal pathogenSclerotinia sclerotiorum.Based on functional information collected through homology and contextual searches, we propose to refer to this domain as the pathogen and abiotic stress response, cadmium tolerance, disordered region-containing (PADRE) domain. Genome-wide and phylogenetic analyses indicated that PADRE is specific to plants and diversified into 10 subfamilies early in the evolution of Angiosperms. PADRE typically occurs in small single-domain proteins with a bipartite architecture. PADRE N-terminus harbors conserved sequence motifs, while its C-terminus includes an intrinsically disordered region with multiple phosphorylation sites. A pangenomic survey ofPADREgenes expression uponS. sclerotioruminoculation inArabidopsis, castor bean, and tomato indicated consistent expression across species within phylogenetic groups. Multi-stress expression profiling and co-expression network analyses associated AtPADRE genes with the induction of anthocyanin biosynthesis and responses to chitin and to hypoxia. Our analyses reveal patterns of sequence and expression diversification consistent with the evolution of a role in disease resistance for an uncharacterized family of plant genes. These findings highlightPADREgenes as prime candidates for the functional dissection of mechanisms underlying plant disease resistance to fungi

An essential role for the VASt domain of the Arabidopsis VAD1 protein in the regulation of defense and cell death in response to pathogens

<div><p>Several regulators of programmed cell death (PCD) have been identified in plants which encode proteins with putative lipid-binding domains. Among them, VAD1 (Vascular Associated Death) contains a novel protein domain called VASt (VAD1 analog StAR-related lipid transfer) still uncharacterized. The Arabidopsis mutant <i>vad1-1</i> has been shown to exhibit a lesion mimic phenotype with light-conditional appearance of propagative hypersensitive response-like lesions along the vascular system, associated with defense gene expression and increased resistance to <i>Pseudomonas</i> strains. To test the potential of ectopic expression of <i>VAD1</i> to influence HR cell death and to elucidate the role of the VASt domain in this function, we performed a structure-function analysis of VAD1 by transient over-expression in <i>Nicotiana benthamiana</i> and by complementation of the mutant <i>vad1-1</i>. We found that (i) overexpression of <i>VAD1</i> controls negatively the HR cell death and defense expression either transiently in <i>Nicotiana benthamania</i> or in Arabidopsis plants in response to avirulent strains of <i>Pseudomonas syringae</i>, (ii) VAD1 is expressed in multiple subcellular compartments, including the nucleus, and (iii) while the GRAM domain does not modify neither the subcellular localization of VAD1 nor its immunorepressor activity, the domain VASt plays an essential role in both processes. In conclusion, VAD1 acts as a negative regulator of cell death associated with the plant immune response and the VASt domain of this unknown protein plays an essential role in this function, opening the way for the functional analysis of VASt-containing proteins and the characterization of novel mechanisms regulating PCD.</p></div

Overexpression of <i>VAD1</i>, but not <i>VAD1</i> lacking the VASt domain, leads to suppression of cell death and defense phenotypes in <i>vad1-1</i> during plant development and in response to bacterial avirulent pathogens.

<p>(A) Four-week old <i>vad1-1</i> plants and transgenic <i>vad1-1</i> plants containing the <i>35S</i>::<i>RFP</i>::<i>ΔVASt</i> construct show typical cell death lesions (marked by white arrows), while transgenic <i>vad1-1</i> plants containing the <i>35S</i>::<i>RFP</i>::<i>VAD1</i> construct or the <i>35S</i>::<i>RFP</i>::<i>ΔGRAM</i> construct show a wild type phenotype. (B-C) Five-week old plants have been inoculated with suspensions (2.10⁶ colony-forming units (CFU/mL) of <i>Pst</i> strain DC3000 expressing AvrRpm1. Wild type and transgenic <i>35S</i>::<i>RFP</i>::<i>VAD1</i> and <i>35S</i>::<i>RFP</i>::<i>ΔGRAM</i> plants showed typical HR lesions, while <i>vad1-1</i> and transgenic plants containing the construct <i>35S</i>::<i>RFP</i>::<i>ΔVASt</i> construct showed run away cell death. These phenotypes were observed 3 days post-inoculation (B) or 6 days post-inoculation (C). (D) Quantification of cell death by measuring electrolyte leakage 24 (grey bars) and 48h (black bars) after infiltration of leaves with <i>Pst</i> strain DC3000 AvrRpm1 (2x10⁷ colony-forming units (CFU/mL). Data are expressed relative to WS-4 1h after sampling. One transgenic line is shown per construct, out of 2–3 analyzed with similar results. Statistically significant differences were determined using Kruskal and Wallis one-way analysis of variance followed by nonparametric multiple comparison (* indicates P < 0.05).</p

Structure-function analysis of <i>VAD1</i> effects on HR cell death and defense in response to bacterial avirulence effectors.

<p>(A) Schematic representation of VAD1 and constructs. GRAM (Glucosyltransferases, Rab-like GTPase Activators, Myotubularins) domain; VASt (VAD1 analog of START) domain; TM, transmembrane helix; CC: Coiled-coil. Residue number corresponds to amino acid position of VAD1 domains. (B) Observation of HR induced by AvrRpt2 after agroinfiltration of <i>N</i>. <i>benthamiana</i> leaves alone or co-expressed with the constructs <i>35S</i>::<i>VAD1</i>, <i>35S</i>::<i>ΔGRAM</i>::<i>VAD1</i>, <i>35S</i>::<i>ΔVASt</i>::<i>VAD1</i> or <i>35S</i>::<i>SYMREM1</i>. Observations were made 24h post-infiltration. (C) Quantification of cell death by measuring electrolyte leakage 24h after agroinfiltration of <i>N</i>. <i>benthamiana</i> leaves with the indicated strains (OD 0.3). Data are expressed relative to AvrRpt2 data at 1 h after sampling. Statistically significant differences were determined using Kruskal and Wallis one-way analysis of variance (letters indicate P < 0.05). (D and E) Expression analysis of the <i>PR1a</i> defense gene and <i>HSR203J</i> gene in <i>N</i>. <i>benthamiana</i> leaves after agroinfiltration with the indicated strains (OD 0.3) Data are presented as means ± SE (n ≥ 12), and error bars represent standard error. Letters indicate a significant difference between tested construct and control at P<0.05 by Kruskal and Wallis one-way analysis of variance test.</p

Overexpression of <i>VAD1</i>, but not <i>VAD1</i> lacking the VASt domain, leads to enhanced susceptibility in <i>vad1-1</i> in response to the virulent strain <i>Pst</i> DC3000.

<p>(A) Four-week old wild type and transgenic <i>vad1-1</i> plants containing the <i>35S</i>::<i>RFP</i>::<i>VAD1</i> construct or the <i>35S</i>::<i>RFP</i>::<i>ΔGRAM</i> construct show a typical chlorosis 72h after inoculation with suspensions (5x10⁶ colony-forming units (CFU/mL) of <i>Pst</i> strain DC3000. <i>vad1-1</i> plants and transgenic <i>vad1-1</i> plants containing the <i>35S</i>::<i>RFP</i>::<i>ΔVASt</i> construct do not show (or very faint) symptoms. (B) Growth of <i>Pst</i> DC3000 in the different lines indicated. Leaves of four-week old plants were inoculated with a bacterial suspension (2x10⁵ CFU/mL) of <i>Pst</i> strain DC3000 and bacterial growth was measured 0 (grey bars) and 3 (black bars) days after inoculation. Data were collected from four independent experiments with five individual plants (four leaves/plant) per point. Statistical differences using Kruskal and Wallis one-way analysis of variance followed by the Conover's-test for multiple comparisons analysis of variance (P value < 0.05) are indicated by letters.</p

Inactivation of Thioredoxin Reductases Reveals a Complex Interplay between Thioredoxin and Glutathione Pathways in Arabidopsis Development[W]

NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem

VAD1 localizes in multiple subcellular compartments and a VASt domain deletion excludes VAD1 from the nucleus.

<p>(A) Transient co-expression by agroinfiltration of <i>N</i>. <i>benthamiana</i> leaves of <i>35S</i>::<i>RFP</i>::<i>VAD1</i> construct (left panels), with subcellular markers (central panels, ERD2–GFP for the Golgi, γ-TIP–GFP for the tonoplast, GFP for the cytoplasm, MYB30-GFP for the nucleus, PMA4–GFP for the plasma membrane). Co-localization of VAD1 with the different subcellular markers is shown in the merge panel (right). Confocal images were observed two days after agroinfiltration. Scale bars = 20μM. (B) Transient co-expression by agroinfiltration of <i>N</i>. <i>benthamiana</i> leaves of <i>35S</i>::<i>RFP</i>::<i>ΔVASt</i> construct (left panels), with subcellular markers (central panels, ERD2–GFP labels the Golgi, γ-TIP–GFP labels the tonoplast, GFP labels the cytoplasm, MYB30-GFP labels the nucleus, PMA4–GFP labels the plasma membrane (Plasma m.). Co-localization of VAD1 with the different subcellular markers is shown in the merge panel (right). Localization of <i>35S</i>::<i>RFP</i>::<i>ΔVASt</i> is not observed in the nucleus. Confocal images were observed two days after agroinfiltration. Bars = 20 μM.</p

Percentage of leaves with lesions in the different plant lines, at several times (17, 21, 24 and 28 days post-transplanting) of plant development, under normal growth conditions.

<p>Percentage of leaves with lesions in the different plant lines, at several times (17, 21, 24 and 28 days post-transplanting) of plant development, under normal growth conditions.</p