Endoplasmic reticulum-quality control chaperones facilitate the biogenesis of Cf receptor-like proteins involved in pathogen resistance of tomato

Cf proteins are receptor-like proteins (RLPs) that mediate resistance of tomato (Solanum lycopersicum) to the foliar pathogen Cladosporium fulvum. These transmembrane immune receptors, which carry extracellular leucine-rich repeats that are subjected to posttranslational glycosylation, perceive effectors of the pathogen and trigger a defense response that results in plant resistance. To identify proteins required for the functionality of these RLPs, we performed immunopurification of a functional Cf-4-enhanced green fluorescent protein fusion protein transiently expressed in Nicotiana benthamiana, followed by mass spectrometry. The endoplasmic reticulum (ER) heat shock protein70 binding proteins (BiPs) and lectin-type calreticulins (CRTs), which are chaperones involved in ER-quality control, were copurifying with Cf-4-enhanced green fluorescent protein. The tomato and N. benthamiana genomes encode four BiP homologs and silencing experiments revealed that these BiPs are important for overall plant viability. For the three tomato CRTs, virus-induced gene silencing targeting the plant-specific CRT3a gene resulted in a significantly compromised Cf-4-mediated defense response and loss of full resistance to C. fulvum. We show that upon knockdown of CRT3a the Cf-4 protein accumulated, but the pool of Cf-4 protein carrying complex-type N-linked glycans was largely reduced. Together, our study on proteins required for Cf function reveals an important role for the CRT ER chaperone CRT3a in the biogenesis and functionality of this type of RLP involved in plant defense.     

No evidence for binding between resistance gene product Cf-9 of tomato and avirulence gene product AVR9 of Cladosporium fulvum.

The gene-for-gene model postulates that for every gene determining resistance in the host plant, there is a corresponding gene conditioning avirulence in the pathogen. On the basis of this relationship, products of resistance (R) genes and matching avirulence (Avr) genes are predicted to interact. Here, we report on binding studies between the R gene product Cf-9 of tomato and the Avr gene product AVR9 of the pathogenic fungus Cladosporium fulvum. Because a high-affinity binding site (HABS) for AVR9 is present in tomato lines, with or without the Cf-9 resistance gene, as well as in other solanaceous plants, the Cf-9 protein was produced in COS and insect cells in order to perform binding studies in the absence of the HABS. Binding studies with radio-labeled AVR9 were performed with Cf-9-producing COS and insect cells and with membrane preparations of such cells. Furthermore, the Cf-9 gene was introduced in tobacco, which is known to be able to produce a functional Cf-9 protein. Binding of AVR9 to Cf-9 protein produced in tobacco was studied employing surface plasmon resonance and surface-enhanced laser desorption and ionization. Specific binding between Cf-9 and AVR9 was not detected with any of the procedures. The implications of this observation are discussed.     

Red light imaging for programmed cell death visualization and quantification in plant–pathogen interactions

Studies on plant–pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant–pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.     

Western Diet Triggers NLRP3-Dependent Innate Immune Reprogramming

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Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction.

Rheumatoid arthritis is a chronic inflammatory joint disease, leading to cartilage and bone destruction. In this study, we investigated the effects of local IL-4 application, introduced by a recombinant human type 5 adenovirus vector, in the knee joint of mice with collagen-induced arthritis. One intraarticular injection with an IL-4-expressing virus caused overexpression of IL-4 in the mouse knee joint. Enhanced onset and aggravation of the synovial inflammation were found in the IL-4 group. However, despite ongoing inflammation, histologic analysis showed impressive prevention of chondrocyte death and cartilage erosion. In line with this, chondrocyte proteoglycan synthesis was enhanced in the articular cartilage. This was quantified with ex vivo 35S-sulfate incorporation in patellar cartilage and confirmed by autoradiography on whole knee joint sections. Reduction of cartilage erosion was further substantiated by lack of expression of the stromelysin-dependent cartilage proteoglycan breakdown neoepitope VDIPEN in the Ad5E1 mIL-4-treated knee joint. Reduced metalloproteinase activity was also supported by markedly diminished mRNA expression of stromelysin-3 in the synovial tissue. Histologic analysis revealed marked reduction of polymorphonuclear cells in the synovial joint space in the IL-4-treated joints. This was confirmed by immunolocalization studies on knee joint sections using NIMP-R14 staining and diminished mRNA expression of macrophage-inflammatory protein-2 in the synovium tissue. mRNA levels of TNF-alpha and IL-1beta were suppressed as well, and IL-1beta and nitric oxide production by arthritic synovial tissue were strongly reduced. Our data show an impressive cartilage-protective effect of local IL-4 and underline the feasibility of local gene therapy with this cytokine in arthritis.     

Locomotor Dysfunction and Pain: The Scylla and Charybdis of Fiber Sprouting After Spinal Cord Injury

Injury to the spinal cord (SCI) can produce a constellation of problems including chronic pain, autonomic dysreflexia, and motor dysfunction. Neuroplasticity in the form of fiber sprouting or the lack thereof is an important phenomenon that can contribute to the deleterious effects of SCI. Aberrant sprouting of primary afferent fibers and synaptogenesis within incorrect dorsal horn laminae leads to the development and maintenance of chronic pain as well as autonomic dysreflexia. At the same time, interruption of connections between supraspinal motor control centers and spinal cord output cells, due to lack of successful regenerative sprouting of injured descending fiber tracts, contributes to motor deficits. Similarities in the molecular control of axonal growth of motor and sensory fibers have made the development of cogent therapies difficult. In this study, we discuss recent findings related to the degradation of inhibitory barriers and promotion of sprouting of motor fibers as a strategy for the restoration of motor function and note that this may induce primary afferent fiber sprouting that can contribute to chronic pain. We highlight the importance of careful attentiveness to off-target molecular- and circuit-level modulation of nociceptive processing while moving forward with the development of therapies that will restore motor function after SCI.