Molecular and ecological analysis of LOX3- and NPR1-dependent defense responses in plant-herbivore interactions

JA-dependent LOX3 and SA-dependent NPR1 perform vital defense functions in Nicotiana attenuata. LOX3 impose stringent growth responses in insects and NPR1 act as a signalling modulator of plant defenses. We found that JA-dependent LOX3-dependent defense responses/metabolites decrease food digestibility in early-instar larvae which prevent them from consuming more food in the later stages. On the other hand, NPR1 serves as a signal modulator and is vital in determining the outcome of JA-dependent LOX3-dependent defense responses. SA was found to have an inhibitory effect on JA- dependent LOX3-dependent defense responses. Therefore, NPR1 serves as a negative regulator of SA biosynthesis in response to herbivore damage in order to activate the JA pathway, and helps the plant finetune an appropriate defense response to herbivores

Large-scale phenomics identifies primary and fine-tuning roles for CRKs in responses related to oxidative stress

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.Peer reviewe

Plant development is affected in several <i>crk</i> mutants.

<p><b>(A)</b> Representative pictures of 17-day old seedlings of Col-0 wild type and <i>crk2</i>. Complementation of <i>crk2</i> with 35S::<i>CRK2-CDS</i>:YFP rescued the growth defect of the mutant. Plants were grown under the following conditions: 250 μmol m^-2 s^-1 light intensity under 12 h-day length (day: 23°C, 70% relative humidity; night: 18°C, 90% relative humidity). Bar = 1 cm. Pictures are representative of three independent experiments. <b>(B)</b> A selection of <i>crk</i> mutant lines showing earlier senescence compared to Col-0 wild type. Results are means ± SE (<i>n</i> = 8). <b>(C)</b> Several <i>crk</i> mutants flowered earlier compared to wild type while <i>crk2</i> flowered later. Results are means ± SE (<i>n</i> = 8). <b>(D)</b> Time course analysis of endosperm rupture showed delayed germination in several <i>crk</i> mutants compared to wild type. Results represent means from three independent biological experiments (<i>n</i> = 30). Testa and endosperm rupture were assessed every 5 hours up to 51 hours of imbibition. A seed was considered as germinated when the radicle protruded through both envelopes. <b>(E)</b> Several <i>crk</i> mutants exhibit a lower pavement cell density (number of pavement cells / mm²) in cotyledons. Results are means ± SE (<i>n</i> = 15). <b>(F)</b> Three <i>crks</i> showed slightly longer roots compared to wild type (measured eight days after stratification). Results are means ± SE (<i>n</i> = 16). (<b>B-F)</b> Differences between mutants and Col-0 wild type were compared and analysed using one-way-ANOVA (<i>post hoc</i> Dunnett, asterisks indicate statistical significance at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001) for <b>(B, C, E</b>) and linear model with single step p-value adjustment (<b>F</b>). All experiments were repeated three times with similar results.</p

Phenotypic analysis of the <i>Arabidopsis thaliana</i> CRK protein family.

<p>A T-DNA insertion collection for the CRK family was compiled and subjected to phenotyping addressing aspects of plant development, biotic and abiotic stress responses, photosynthesis as well as stomatal regulation. Length of red and blue bars in the five phenotyping sections is representative of the number of <i>crk</i> lines found to have phenotypes in the thematic area. Information about the sections in the pie chart is displayed in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.g001" target="_blank">1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s003" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s004" target="_blank">S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s026" target="_blank">S1 Table</a>. The red outline in the pie chart highlights the lines included in the analyses and figures throughout the manuscript. The gray scale bar serves as a reference for comparison. The length of the scale bar corresponds to ten lines.</p

Integrated cluster analysis of <i>crk</i> mutant phenotypes.

<p>An age-matched collection of T-DNA insertion lines in <i>CRK</i> genes was analyzed for developmental and stress-related phenotypes. <b>(A)</b> Analysis of developmental phenotypes of <i>crk</i> mutant lines: senescence, germination (endosperm rupture), epidermal cell segmentation, bolting, flowering, and root length. <b>(B)</b> Analysis of abiotic stresses phenotypes of <i>crk</i> lines: germination of <i>crk</i> lines on medium containing NaCl, cell death (measured by electrolyte leakage) in response to Xanthine-Xanthine Oxidase (X+XO), ultraviolet light (UV-AB), ozone (O₃), or light stress. <b>(C)</b> Analysis of photosynthesis responses upon treatment with DCMU or methyl viologen (MV). <b>(D)</b> Pathogen phenotypes. ROS production in response to treatment with the bacterial elicitor flagellin (flg22). Stomatal aperture ratio in response to flg22 and chitin treatments and <i>crk</i> susceptibility to the hemibiotrophic bacterial pathogen <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 (<i>Pto</i> infection) or the biotrophic fungal pathogens <i>Golovinomyces orontii</i> (<i>Go</i>) (virulent on <i>Arabidopsis</i>) or <i>Blumeria graminis</i> f.sp. <i>hordei</i> (<i>Bgh</i>, a barley pathogen, non-pathogenic on <i>Arabidopsis</i>). <b>(E)</b> Analysis of stomatal parameters: fresh weight (for determination of water loss), density, length, aperture, stomatal aperture in response to ABA treatment, steady state stomatal conductance, stomatal closure in response to elevated CO₂, O₃, and darkness. Experiments were made comparable by bootstrap sampling to <i>n</i> = 15 followed by averaging over bootstrap estimates. Red and blue indicate statistically significant increase or decrease in response compared to Col-0 wild type, respectively, while white indicates a response that is similar to wild type Col-0. The intensity of color is proportional to the Benjamini-Hochberg false discovery rate (FDR) adjusted Z statistic which takes the estimated means and their variation into account. As a rough guideline, |Z|>1.67 corresponds to a FDR<10% (shown with light hue), and |Z|<2.6 to a strong FDR<1% (intense color). White: non-significant response; grey: not measured. A corresponding plot displaying the adjusted Z statistics without thresholding is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s025" target="_blank">S25 Fig</a>.</p

Models of CRK function and how they could provide specificity of stomatal aperture regulation.

<p><b>(A)</b> CRKs might act as pathway-specific or multi-pathway regulators of stomatal aperture in response to the PAMPs flg22 and chitin but also the stress hormone ABA and the abiotic stimuli O₃, darkness and CO₂. The figure has been created from data presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.g010" target="_blank">Fig 10</a>. <b>(B)</b> CRKs are involved in the response to pathogens downstream of extracellular ROS production. PAMPs are recognized by pattern recognition receptor complexes. Subsequently, intracellular signalling leads to activation of extracellular superoxide production by NADPH oxidases. ROS perception subsequently leads to intracellular signalling and ultimately stomatal closure. CRKs are implicated in linking extracellular ROS production to intracellular signalling and might regulate and/or interact directly with the recognition receptor complexes.</p