Internalization of Flax Rust Avirulence Proteins into Flax and Tobacco Cells Can Occur in the Absence of the Pathogen

Translocation of pathogen effector proteins into the host cell cytoplasm is a key determinant for the pathogenicity of many bacterial and oomycete plant pathogens. A number of secreted fungal avirulence (Avr) proteins are also inferred to be delivered into host cells, based on their intracellular recognition by host resistance proteins, including those of flax rust (Melampsora lini). Here, we show by immunolocalization that the flax rust AvrM protein is secreted from haustoria during infection and accumulates in the haustorial wall. Five days after inoculation, the AvrM protein was also detected within the cytoplasm of a proportion of plant cells containing haustoria, confirming its delivery into host cells during infection. Transient expression of secreted AvrL567 and AvrM proteins fused to cerulean fluorescent protein in tobacco (Nicotiana tabacum) and flax cells resulted in intracellular accumulation of the fusion proteins. The rust Avr protein signal peptides were functional in plants and efficiently directed fused cerulean into the secretory pathway. Thus, these secreted effectors are internalized into the plant cell cytosol in the absence of the pathogen, suggesting that they do not require a pathogen-encoded transport mechanism. Uptake of these proteins is dependent on signals in their N-terminal regions, but the primary sequence features of these uptake regions are not conserved between different rust effectors.     

Improved strategies for electrochemical 1,4-NAD(P)H2 regeneration: A new era of bioreactors for industrial biocatalysis

Industrial enzymatic reactions requiring 1,4-NAD(P)H₂ to perform redox transformations often require convoluted coupled enzyme regeneration systems to regenerate 1,4-NAD(P)H₂ from NAD(P) and recycle the cofactor for as many turnovers as possible. Renewed interest in recycling the cofactor via electrochemical means is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). We review various literature strategies for mitigating adventitious product formation by electrochemical cofactor regeneration systems, and offer insight as to how a successful electrochemical bioreactor system could be constructed to engineer efficient 1,4-NAD(P)H₂-dependent enzyme reactions of interest to the industrial biocatalysis community.     

The low C5 convertase activity of the C4A6 allotype of human complement component C4.

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.     

An autoactive mutant of the M flax rust resistance protein has a preference for binding ATP, whereas wild-type M protein binds ADP

Resistance (R) proteins are key regulators of the plant innate immune system and are capable of pathogen detection and activation of the hypersensitive cell death immune response. To understand the molecular mechanism of R protein activation, we undertook a phenotypic and biochemical study of the flax nucleotide binding (NB)-ARC leucine-rich repeat protein, M. Using Agrobacterium-mediated transient expression in flax cotyledons, site-directed mutations of key residues within the P-loop, kinase 2, and MHD motifs within the NB-ARC domain of M were shown to affect R protein function. When purified using a yeast expression system and assayed for ATP and ADP, these mutated proteins exhibited marked differences in the quantity and identity of the bound nucleotide. ADP was bound to recombinant wild-type M protein, while the nonfunctional P-loop mutant did not have any nucleotides bound. In contrast, ATP was bound to an autoactive M protein mutated in the highly conserved MHD motif. These data provide direct evidence supporting a model of R protein function in which the "off" R protein binds ADP and activation of R protein defense signaling involves the exchange of ADP for ATP.     

A generalized model of social and biological contagion

We present a model of contagion that unifies and generalizes existing models of the spread of social influences and microorganismal infections. Our model incorporates individual memory of exposure to a contagious entity (e.g. a rumor or disease), variable magnitudes of exposure (dose sizes), and heterogeneity in the susceptibility of individuals. Through analysis and simulation, we examine in detail the case where individuals may recover from an infection and then immediately become susceptible again (analogous to the so-called SIS model). We identify three basic classes of contagion models which we call epidemic threshold, vanishing critical mass, and critical mass classes, where each class of models corresponds to different strategies for prevention or facilitation. We find that the conditions for a particular contagion model to belong to one of the these three classes depend only on memory length and the probabilities of being infected by one and two exposures, respectively. These parameters are in principle measurable for real contagious influences or entities, thus yielding empirical implications for our model. We also study the case where individuals attain permanent immunity once recovered, finding that epidemics inevitably die out but may be surprisingly persistent when individuals possess memory.     

Enhancing beneficial antioxidants in fruits: a plant physiological perspective

The aim of this review is to present an outline of the physiological perspectives of beneficial antioxidant production in fruit. The drive to enhance the consumption of fruit and vegetables in the human diet is linked with positive effects of beneficial antioxidants impacting on health promotion. We briefly outline our physiological understanding of environmental processes which induce the production of reactive oxygen species and how antioxidants prevent plant cellular damage. More specifically, we describe the impact that environmental stresses, such as drought and radiation, have on the production of endogenous antioxidants and how these stresses can be incorporated into novel experimental crop growing systems to achieve high antioxidant concentrations in fruits. This includes in particular the use of irrigation application techniques and enhanced light reflectance to increase the concentrations of bioactive compounds such as ellagic acid and ascorbic acid.