A novel method for efficient and abundant production of Phytophthora brassicae zoospores on Brussels sprout leaf discs

<p>Abstract</p> <p>Background</p> <p><it>Phytophthora </it>species are notorious oomycete pathogens that cause diseases on a wide range of plants. Our understanding how these pathogens are able to infect their host plants will benefit greatly from information obtained from model systems representative for plant-<it>Phytophthora </it>interactions. One attractive model system is the interaction between Arabidopsis and <it>Phytophthora brassicae</it>. Under laboratory conditions, Arabidopsis can be easily infected with mycelial plugs as inoculum. In the disease cycle, however, sporangia or zoospores are the infectious propagules. Since the current <it>P. brassicae </it>zoospore isolation methods are generally regarded as inefficient, we aimed at developing an alternative method for obtaining high concentrations of <it>P. brassicae </it>zoospores.</p> <p>Results</p> <p><it>P. brassicae </it>isolates were tested for pathogenicity on Brussels sprout plants (<it>Brassica oleracea </it>var.<it> gemmifera</it>). Microscopic examination of leaves, stems and roots infected with a GFP-tagged transformant of <it>P. brassicae </it>clearly demonstrated the susceptibility of the various tissues. Leaf discs were cut from infected Brussels sprout leaves, transferred to microwell plates and submerged in small amounts of water. In the leaf discs the hyphae proliferated and abundant formation of zoosporangia was observed. Upon maturation the zoosporangia released zoospores in high amounts and zoospore production continued during a period of at least four weeks. The zoospores were shown to be infectious on Brussels sprouts and Arabidopsis.</p> <p>Conclusion</p> <p>The <it>in vitro </it>leaf disc method established from <it>P. brassicae </it>infected Brussels sprout leaves facilitates convenient and high-throughput production of infectious zoospores and is thus suitable to drive small and large scale inoculation experiments. The system has the advantage that zoospores are produced continuously over a period of at least one month.</p

Development and individual differences in transitive reasoning: A fuzzy trace theory approach

Fuzzy trace theory explains why children do not have to use rules of logic or premise information to infer transitive relationships. Instead, memory of the premises and performance on transitivity tasks is explained by a verbatim ability and a gist ability. Until recently, the processes involved in transitive reasoning and memory of the premises were studied by comparing mean performance in fixed-age groups. In this study, an individual-difference model of fuzzy trace theory for transitive reasoning was formulated and tested on a sample (N = 409) of 4- to 13-year-old children. Tasks were used which differed with respect to presentation ordering and position ordering. From this individual- difference model expectations could be derived about the individual performance on memory and transitivity test-pairs. The multilevel latent class model was used to fit the formalized individual-difference fuzzy trace theory to the sample data. The model was shown to fit the data to a large extent. The results showed that verbatim ability and gist ability drove the activation of verbatim and gist traces, respectively, and that children used combinations of these traces to solve memory tasks (testing memory of the premises) and transitivity tasks. Task format had a stronger effect on transitivity task performance than on memory of the premises. Development of gist ability was found to be faster than development of verbatim ability. Another important finding was that some children remembered the premise information correctly but were not able to infer the transitive relationship, even though the premises provided all the necessary information. This contradicts Trabasso’s linear ordering theory which posits that memory of the premises is sufficient to infer transitive relationships

Entanglement generation by adiabatic navigation in the space of symmetric multi-particle states

We propose a technique for robust and efficient navigation in the Hilbert space of entangled symmetric states of a multiparticle system with externally controllable linear and nonlinear collective interactions. A linearly changing external field applied along the quantization axis creates a network of well separated level crossings in the energy diagram of the collective states. One or more transverse pulsed fields applied at the times of specific level crossings induce adiabatic passage between these states. By choosing the timing of the pulsed field appropriately, one can transfer an initial product state of all N spins into (i) any symmetric state with n spin excitations and (ii) the N-particle analog of the Greenberger-Horne-Zeilinger state. This technique, unlike techniques using pulses of specific area, does not require precise knowledge of the number of particles and is robust against variations in the interaction parameters. We discuss potential applications in two-component Bose condensates and ion-trap systems.Comment: 7 pages, 6 figure

The Lectin Receptor Kinase LecRK-I.9 Is a Novel Phytophthora Resistance Component and a Potential Host Target for a RXLR Effector

In plants, an active defense against biotrophic pathogens is dependent on a functional continuum between the cell wall (CW) and the plasma membrane (PM). It is thus anticipated that proteins maintaining this continuum also function in defense. The legume-like lectin receptor kinase LecRK-I.9 is a putative mediator of CW-PM adhesions in Arabidopsis and is known to bind in vitro to the Phytophthora infestans RXLR-dEER effector IPI-O via a RGD cell attachment motif present in IPI-O. Here we show that LecRK-I.9 is associated with the plasma membrane, and that two T-DNA insertions lines deficient in LecRK-I.9 (lecrk-I.9) have a ‘gain-of-susceptibility’ phenotype specifically towards the oomycete Phytophthora brassicae. Accordingly, overexpression of LecRK-I.9 leads to enhanced resistance to P. brassicae. A similar ‘gain-of-susceptibility’ phenotype was observed in transgenic Arabidopsis lines expressing ipiO (35S-ipiO1). This phenocopy behavior was also observed with respect to other defense-related functions; lecrk-I.9 and 35S-ipiO1 were both disturbed in pathogen- and MAMP-triggered callose deposition. By site-directed mutagenesis, we demonstrated that the RGD cell attachment motif in IPI-O is not only essential for disrupting the CW-PM adhesions, but also for disease suppression. These results suggest that destabilizing the CW-PM continuum is one of the tactics used by Phytophthora to promote infection. As countermeasure the host may want to strengthen CW-PM adhesions and the novel Phytophthora resistance component LecRK-I.9 seems to function in this process

Effector Genomics Accelerates Discovery and Functional Profiling of Potato Disease Resistance and Phytophthora Infestans Avirulence Genes

Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Effector Trafficking: RXLR-dEER as Extra Gear for Delivery into Plant Cells

When driving a car with automatic transmission, one hardly notices that extra gears give more power to the car. But in a car with manual transmission, one is constantly aware that even one gear shift helps to reach your goal much more efficiently. For Phytophthora pathogens, a domain characterized by the amino acid motifs RXLR and dEER seems to function as a special gear. In this issue of The Plant Cell, Dou et al. (2008b; pages 1930¿1947) report that an RXLR-dEER domain embodies the complete machinery that the pathogen needs to deliver effectors into host cells. This is in contrast with the bacterial type III secretion system, which requires a multitude of proteins to accomplish this task (see figure panels A and B)