Inflammatory caspases: key regulators of inflammation and cell death.

The innate immune system represents the first line of defence against infectious agents, and co-ordinates cellular and molecular mechanisms that result in effective inflammatory and anti-microbial responses against pathogens. Infection and cellular stress trigger assembly of canonical and noncanonical inflammasome complexes that activate the inflammatory caspases-1 and -11, respectively. These inflammatory caspases play key roles in innate immune responses by inducing pyroptosis to halt intracellular replication of pathogens, and by engaging the extracellular release of pro-inflammatory cytokines and danger signals. In addition, the inflammatory caspases-4, -5 and -11 were recently shown to directly bind microbial components. Although the immune roles of caspase-12 are debated, it was proposed to dampen inflammatory responses by interfering with caspase-1 activation and other innate immune pathways. Here, we recapitulate the reported roles of inflammatory caspases with an emphasis on recent insights into their biological functions.

Symptomless Host and Nonhost Responses of Paulownia (Paulownia spp.) to Olive-Defoliating Verticillium dahliae.

Symptomless host and nonhost responses of Paulownia spp. to olive-defoliating (D) Verticillium dahliae is reported for the first time. Two paulownia clones, Paulownia elongata 'PC-2' and P. elongata × P. fortunei 'PC-3', were inoculated with a V. dahliae isolate representative of the D pathotype by either root dip or stem injection with a conidial suspension, repeated transplanting to a V. dahliae-infested soil mixture, or root dip in the conidial suspension followed by transplanting to the infested soil mixture. 'Picual' olive and 'Sugar Baby' watermelon were included in all experiments as susceptible standards to show that the inoculation procedures and incubation conditions were successful. Plants were incubated under conditions optimal for Verticillium wilt that caused severe disease in 'Picual' olive and 'Sugar Baby' watermelon in the growth chamber, shade house, and field microplots for 30 to 57 weeks in three independent experiments. No foliar symptoms developed on paulownia, whose stems were found free of V. dahliae both by isolation on semiselective NP-10 medium as well as by a nested-polymerase chain reaction assay using total genomic DNA from inoculated plants that effectively detected D V. dahliae in olive stems. V. dahliae was isolated to a limited extent from roots of PC-3 paulownia plants after 30 weeks of growth in the infested soil mixture but not from those that were root-dip inoculated or from PC-2 plants regardless the method of inoculation. The symptomless host and nonhost responses of Paulownia spp. to D V. dahliae may have practical applications in the use of fertile soils in southern Spain, particularly in those that are highly infested with the highly virulent D pathotype, as well as a replacement crop for Verticillium wilt-affected olive orchards in that region.

In planta and soil quantification of Fusarium oxysporum f. sp. ciceris and evaluation of Fusarium wilt resistance in chickpea with a newly developed quantitative polymerase chain reaction assay.

Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris can be managed by risk assessment and use of resistant cultivars. A reliable method for the detection and quantification of F. oxysporum f. sp. ciceris in soil and chickpea tissues would contribute much to implementation of those disease management strategies. In this study, we developed a real-time quantitative polymerase chain reaction (q-PCR) protocol that allows quantifying F. oxysporum f. sp. ciceris DNA down to 1 pg in soil, as well as in the plant root and stem. Use of the q-PCR protocol allowed quantifying as low as 45 colony forming units of F. oxysporum f. sp. ciceris per gram of dry soil from a field plot infested with several races of the pathogen. Moreover, the q-PCR protocol clearly differentiated susceptible from resistant chickpea reactions to the pathogen at 15 days after sowing in artificially infested soil, as well as the degree of virulence between two F. oxysporum f. sp. ciceris races. Also, the protocol detected early asymptomatic root infections and distinguished significant differences in the level of resistance of 12 chickpea cultivars that grew in that same field plot infested with several races of the pathogen. Use of this protocol for fast, reliable, and cost-effective quantification of F. oxysporum f. sp. ciceris in asymptomatic chickpea tissues at early stages of the infection process can be of great value for chickpea breeders and for epidemiological studies in growth chambers, greenhouses and field-scale plots.

Molecular and Pathogenic Characterization of Fusarium redolens, a New Causal Agent of Fusarium Yellows in Chickpea.

The association of Fusarium redolens with wilting-like symptoms in chickpea in Lebanon, Morocco, Pakistan, and Spain is reported for the first time, together with the molecular and pathogenic characterization of isolates of the pathogen from chickpea of diverse geographic origin. Maximum parsimony analysis of sequences of the translation elongation factor 1α (TEF-1α) gene grouped all F. redolens isolates from chickpea in the same main clade. Pathogenicity assays using three chickpea cultivars and isolates from different geographic origins indicated that F. redolens is mildly virulent on chickpea. Moreover, infection of chickpea by F. redolens induces a disease syndrome similar to that caused by the yellowing pathotype of F. oxysporum f. sp. ciceris, including leaf yellowing and necrosis that develop upward from the stem base, and premature senescence of the plant. In contrast, F. redolens does not cause discoloration of the vascular tissues in chickpea but does cause brown necrotic lesions in the tap root and necrosis of lateral roots. F. redolens is not easily differentiated from F. oxysporum f. sp. ciceris using morphology-based diagnosis, and the two species cause similar symptoms on chickpea; therefore, the use of molecular protocols should help to avoid misdiagnoses of Fusarium yellows in chickpea.

Strategies to Target ISG15 and USP18 Toward Therapeutic Applications.

The interferon (IFN)-stimulated gene product 15 (ISG15) represents an ubiquitin-like protein (Ubl), which in a process termed ISGylation can be covalently linked to target substrates via a cascade of E1, E2, and E3 enzymes. Furthermore, ISG15 exerts functions in its free form both, as an intracellular and as a secreted protein. In agreement with its role as a type I IFN effector, most functions of ISG15 and ISGylation are linked to the anti-pathogenic response. However, also key roles in other cellular processes such as protein translation, cytoskeleton dynamics, exosome secretion, autophagy or genome stability and cancer were described. Ubiquitin-specific protease 18 (USP18) constitutes the major ISG15 specific protease which counteracts ISG15 conjugation. Remarkably, USP18 also functions as a critical negative regulator of the IFN response irrespective of its enzymatic activity. Concordantly, lack of USP18 function causes fatal interferonopathies in humans and mice. The negative regulatory function of USP18 in IFN signaling is regulated by various protein-protein interactions and its stability is controlled via proteasomal degradation. The broad repertoire of physiological functions and regulation of ISG15 and USP18 offers a variety of potential intervention strategies which might be of therapeutic use. Due to the high mutation rates of pathogens which are often species specific and constantly give rise to a variety of immune evasion mechanisms, immune effector systems are under constant evolutionarily pressure. Therefore, it is not surprising that considerable differences in ISG15 with respect to function and sequence exist even among closely related species. Hence, it is essential to thoroughly evaluate the translational potential of results obtained in model organisms especially for therapeutic strategies. This review covers existing and conceptual assay systems to target and identify modulators of ISG15, ISGylation, USP18 function, and protein-protein interactions within this context. Strategies comprise mouse models for translational perspectives, cell-based and biochemical assays as well as chemical probes.

The in vivo ISGylome links ISG15 to metabolic pathways and autophagy upon Listeria monocytogenes infection.

ISG15 is an interferon-stimulated, ubiquitin-like protein, with anti-viral and anti-bacterial activity. Here, we map the endogenous in vivo ISGylome in the liver following Listeria monocytogenes infection by combining murine models of reduced or enhanced ISGylation with quantitative proteomics. Our method identifies 930 ISG15 sites in 434 proteins and also detects changes in the host ubiquitylome. The ISGylated targets are enriched in proteins which alter cellular metabolic processes, including upstream modulators of the catabolic and antibacterial pathway of autophagy. Computational analysis of substrate structures reveals that a number of ISG15 modifications occur at catalytic sites or dimerization interfaces of enzymes. Finally, we demonstrate that animals and cells with enhanced ISGylation have increased basal and infection-induced autophagy through the modification of mTOR, WIPI2, AMBRA1, and RAB7. Taken together, these findings ascribe a role of ISGylation to temporally reprogram organismal metabolism following infection through direct modification of a subset of enzymes in the liver.

Quantitative and microscopic assessment of compatible and incompatible interactions between chickpea cultivars and Fusarium oxysporum f. sp. ciceris races.

BACKGROUND: Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris, a main threat to global chickpea production, is managed mainly by resistant cultivars whose efficiency is curtailed by Fusarium oxysporum f. sp. ciceris races. METHODOLOGY: We characterized compatible and incompatible interactions by assessing the spatial-temporal pattern of infection and colonization of chickpea cvs. P-2245, JG-62 and WR-315 by Fusarium oxysporum f. sp. ciceris races 0 and 5 labeled with ZsGreen fluorescent protein using confocal laser scanning microscopy. FINDINGS: The two races colonized the host root surface in both interactions with preferential colonization of the root apex and subapical root zone. In compatible interactions, the pathogen grew intercellularly in the root cortex, reached the xylem, and progressed upwards in the stem xylem, being the rate and intensity of stem colonization directly related with the degree of compatibility among Fusarium oxysporum f. sp. ciceris races and chickpea cultivars. In incompatible interactions, race 0 invaded and colonized 'JG-62' xylem vessels of root and stem but in 'WR-315', it remained in the intercellular spaces of the root cortex failing to reach the xylem, whereas race 5 progressed up to the hypocotyl. However, all incompatible interactions were asymptomatic. CONCLUSIONS: The differential patterns of colonization of chickpea cultivars by Fusarium oxysporum f. sp. ciceris races may be related to the operation of multiple resistance mechanisms.