Effector-mediated relocalization of a maize lipoxygenase protein triggers susceptibility to Ustilago maydis.

As the gall-inducing smut fungus Ustilago maydis colonizes maize (Zea mays) plants, it secretes a complex effector blend that suppresses host defense responses, including production of reactive oxygen species (ROS) and redirects host metabolism to facilitate colonization. We show that the U. maydis effector ROS burst interfering protein 1 (Rip1), which is involved in pathogen-associated molecular pattern (PAMP)-triggered suppression of host immunity, is functionally conserved in several other monocot-infecting smut fungi. We also have identified a conserved C-terminal motif essential for Rip1-mediated PAMP-triggered suppression of the ROS burst. The maize susceptibility factor lipoxygenase 3 (Zmlox3) bound by Rip1 was relocalized to the nucleus, leading to partial suppression of the ROS burst. Relocalization was independent of its enzymatic activity, revealing a distinct function for ZmLox3. Most importantly, whereas Zmlox3 maize mutant plants showed increased resistance to U. maydis wild-type strains, rip1 deletion strains infecting the Zmlox3 mutant overcame this effect. This could indicate that Rip1-triggered host resistance depends on ZmLox3 to be suppressed and that lox3 mutation-based resistance of maize to U. maydis requires functional Rip1. Together, our results reveal that Rip1 acts in several cellular compartments to suppress immunity and that targeting of ZmLox3 by Rip1 is responsible for the suppression of Rip1-dependent reduced susceptibility of maize to U. maydis.

Postmodification with Polycations Enhances Key Properties of Alginate-Based Multicomponent Microcapsules.

Postmodification of alginate-based microspheres with polyelectrolytes (PEs) is commonly used in the cell encapsulation field to control microsphere stability and permeability. However, little is known about how different applied PEs shape the microsphere morphology and properties, particularly in vivo. Here, we addressed this question using model multicomponent alginate-based microcapsules postmodified with PEs of different charge and structure. We found that the postmodification can enhance or impair the mechanical resistance and biocompatibility of microcapsules implanted into a mouse model, with polycations surprisingly providing the best results. Confocal Raman microscopy and confocal laser scanning microscopy (CLSM) analyses revealed stable interpolyelectrolyte complex layers within the parent microcapsule, hindering the access of higher molar weight PEs into the microcapsule core. All microcapsules showed negative surface zeta potential, indicating that the postmodification PEs get hidden within the microcapsule membrane, which agrees with CLSM data. Human whole blood assay revealed complex behavior of microcapsules regarding their inflammatory and coagulation potential. Importantly, most of the postmodification PEs, including polycations, were found to be benign toward the encapsulated model cells.

The Pleiades are a cluster of fungal effectors that inhibit host defenses.

Biotrophic plant pathogens secrete effector proteins to manipulate the host physiology. Effectors suppress defenses and induce an environment favorable to disease development. Sequence-based prediction of effector function is impeded by their rapid evolution rate. In the maize pathogen Ustilago maydis, effector-coding genes frequently organize in clusters. Here we describe the functional characterization of the pleiades, a cluster of ten effector genes, by analyzing the micro- and macroscopic phenotype of the cluster deletion and expressing these proteins in planta. Deletion of the pleiades leads to strongly impaired virulence and accumulation of reactive oxygen species (ROS) in infected tissue. Eight of the Pleiades suppress the production of ROS upon perception of pathogen associated molecular patterns (PAMPs). Although functionally redundant, the Pleiades target different host components. The paralogs Taygeta1 and Merope1 suppress ROS production in either the cytoplasm or nucleus, respectively. Merope1 targets and promotes the auto-ubiquitination activity of RFI2, a conserved family of E3 ligases that regulates the production of PAMP-triggered ROS burst in plants.

Many ways to TOPLESS - manipulation of plant auxin signalling by a cluster of fungal effectors.

Plant biotrophic pathogens employ secreted molecules, called effectors, to suppress the host immune system and redirect the host's metabolism and development in their favour. Putative effectors of the gall-inducing maize pathogenic fungus Ustilago maydis were analysed for their ability to induce auxin signalling in plants. Using genetic, biochemical, cell-biological, and bioinformatic approaches we functionally elucidate a set of five, genetically linked effectors, called Topless (TPL) interacting protein (Tips) effectors that induce auxin signalling. We show that Tips induce auxin signalling by interfering with central corepressors of the TPL family. CRISPR-Cas9 mutants and deletion strain analysis indicate that the auxin signalling inducing subcluster effectors plays a redundant role in virulence. Although none of the Tips seem to have a conserved interaction motif, four of them bind solely to the N-terminal TPL domain and, for Tip1 and Tip4, we demonstrate direct competition with auxin/indole-3-acetic acid transcriptional repressors for their binding to TPL class of corepressors. Our findings reveal that TPL proteins, key regulators of growth-defence antagonism, are a major target of the U. maydis effectome.

Characterization of insecticidal Cry1Cb2 protein from Bacillus thuringiensis toxic to Myzus persicae (Sulzer).

The toxins produced by Bacillus thuringiensis (Bt) are well known for their insecticidal activity against Lepidoptera, Diptera and Coleoptera; however, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. We describe the aphicidal effect of Cry toxin from Bt strain GP919 against one of the most pernicious hemipterans in the agricultural environment, Myzus persicae. The mortality bioassay shows that the strain cause mortality rates above 80% at concentration of 10 ng/µl with a LC50 of 9.01 ng/µl; whereas it showed no lethal toxicity against the lepidopteran Spodoptera frugiperda. The mayor protein (∼130 kDa) expressed by this strain was subjected to purification, solubilization and trypsin digestion, the band of âˆ¼ 65 kDa which was obtained from trypsin digestion was purified by ion-exchange chromatography and was used to feed the aphid. The bioassay shows mortality rates above 85% at concentration of 10 ng/µl and the LC50 was 6.58 ng/µl. The resulting fragment from the digestion was identified by mass spectrometry and the candidate protein showed an overall 100% amino acid sequence identity to the reported Cry1Cb2 (WP 033698561.1) protein from Bt. Koch's postulated also was carried out with the GP919 strain and also, we document the signs of infection caused by this strain. This is the first report of a Cry1Cb2 protein that is toxic to a sucking insect and this protein may become a promising environmentally friendly tool for the control of M. persicae and possible also for other sap sucking insect pests.

Proteomic analysis of Rickettsia akari proposes a 44 kDa-OMP as a potential biomarker for Rickettsialpox diagnosis.

BACKGROUND: Rickettsialpox is a febrile illness caused by the mite-borne pathogen Rickettsia akari. Several cases of this disease are reported worldwide annually. Nevertheless, the relationship between the immunogenicity of R. akari and disease development is still poorly understood. Thus, misdiagnosis is frequent. Our study is aiming to identify immunogenic proteins that may improve disease recognition and enhance subsequent treatment. To achieve this goal, two proteomics methodologies were applied, followed by immunoblot confirmation. RESULTS: Three hundred and sixteen unique proteins were identified in the whole-cell extract of R. akari. The most represented protein groups were found to be those involved in translation, post-translational modifications, energy production, and cell wall development. A significant number of proteins belonged to amino acid transport and intracellular trafficking. Also, some proteins affecting the virulence were detected. In silico analysis of membrane enriched proteins revealed 25 putative outer membrane proteins containing beta-barrel structure and 11 proteins having a secretion signal peptide sequence. Using rabbit and human sera, various immunoreactive proteins were identified from which the 44 kDa uncharacterized protein (A8GP63) has demonstrated a unique detection capability. It positively distinguished the sera of patients with Rickettsialpox from other rickettsiae positive human sera. CONCLUSION: Our proteomic analysis certainly contributed to the lack of knowledge of R. akari pathogenesis. The result obtained may also serve as a guideline for a more accurate diagnosis of rickettsial diseases. The identified 44 kDa uncharacterized protein can be certainly used as a unique marker of rickettsialpox or as a target molecule for the development of more effective treatment.

A framework for model analysis across multiple experiment regimes: Investigating effects of zinc on Xylella fastidiosa as a case study.

Mathematical models are ubiquitous in analyzing dynamical biological systems. However, it might not be possible to explicitly account for the various sources of uncertainties in the model and the data if there is limited experimental data and information about the biological processes. The presence of uncertainty introduces problems with identifiability of the parameters of the model and determining appropriate regions to explore with respect to sensitivity and estimates of parameter values. Since the model analysis is likely dependent on the numerical estimates of the parameters, parameter identifiability should be addressed beforehand to capture biologically relevant parameter space. Here, we propose a framework which uses data from different experiment regimes to identify a region in the parameter space over which subsequent mathematical analysis can be conducted. Along with building confidence in the parameter estimates, it provides us with variations in the parameters due to changes in the experimental conditions. To determine significance of these variations, we conduct global sensitivity analysis, allowing us to make testable hypothesis for effects of changes in the experimental conditions on the biological system. As a case study, we develop a model for growth dynamics and biofilm formation of a bacterial plant pathogen, and use our framework to identify possible effects of zinc on the bacterial populations in different metabolic states. The framework reveals underlying issues with parameter identifiability and identifies a suitable region in the parameter space, sensitivity analysis over which informs us about the parameters that might be affected by addition of zinc. Moreover, these parameters prove to be identifiable in this region.

Phytohormone sensing in the biotrophic fungus Ustilago maydis - the dual role of the transcription factor Rss1.

The phenolic compound salicylic acid (SA) is a key signalling molecule regulating local and systemic plant defense responses, mainly against biotrophs. Many microbial organisms, including pathogens, share the ability to degrade SA. However, the mechanism by which they perceive SA is unknown. Here we show that Ustilago maydis, the causal agent of corn smut disease, employs a so far uncharacterized SA sensing mechanism. We identified and characterized the novel SA sensing regulator, Rss1, a binuclear zinc cluster protein with dual functions as putative SA receptor and transcriptional activator regulating genes important for SA and tryptophan degradation. Rss1 represents a major component in the identified SA sensing pathway during the fungus' saprophytic stage. However, Rss1 does not have a detectable impact on virulence. The data presented in this work indicate that alternative or redundant sensing cascades exist that regulate the expression of SA-responsive genes in U. maydis during its pathogenic development.

Zinc Detoxification Is Required for Full Virulence and Modification of the Host Leaf Ionome by Xylella fastidiosa.

Zinc (Zn) is an essential element for all forms of life because it is a structural or catalytic cofactor of many proteins, but it can have toxic effects at high concentrations; thus, microorganisms must tightly regulate its levels. Here, we evaluated the role of Zn homeostasis proteins in the virulence of the xylem-limited bacterium Xylella fastidiosa, causal agent of Pierce's disease of grapevine, among other diseases. Two mutants of X. fastidiosa 'Temecula' affected in genes which regulate Zn homeostasis (zur) and Zn detoxification (czcD) were constructed. Both knockouts showed increased sensitivity to Zn at physiologically relevant concentrations and increased intracellular accumulation of this metal compared with the wild type. Increased Zn sensitivity was correlated with decreased growth in grapevine xylem sap, reduced twitching motility, and downregulation of exopolysaccharide biosynthetic genes. Tobacco plants inoculated with either knockout mutant showed reduced foliar symptoms and a much reduced (czcD) or absent (zur) modification of the leaf ionome (i.e., the mineral nutrient and trace element composition), as well as reduced bacterial populations. The results show that detoxification of Zn is crucial for the virulence of X. fastidiosa and verifies our previous findings that modification of the host leaf ionome correlates with bacterial virulence.

A Two-Dimensional Multiphase Model of Biofilm Formation in Microfluidic Chambers.

The bacterial pathogen Xylella fastidiosa is the causal agent of many pathological conditions of economically important agricultural crops. There is no known cure for X. fastidiosa diseases, and management of the problem is based solely in controlling the population of insect vectors, which is somewhat effective. The bacterium causes disease by forming biofilms inside the vascular system of the plant, a process that is poorly understood. In microfluidic chambers, used as artificial xylem vessels, this bacterium has been observed to reproducibly cluster into a distinct, regular pattern of aggregates, spatially separated by channels of non-biofilm components. We develop a multiphase model in two dimensions, which recapitulates this spatial patterning, suggesting that bacterial growth and attachment/detachment processes are strongly influential modulators of these patterns. This indicates plausible strategies, such as the addition of metals and chelators, for mitigating the severity of diseases induced by this bacterial pathogen.

Response of Xylella fastidiosa to zinc: decreased culturability, increased exopolysaccharide production, and formation of resilient biofilms under flow conditions.

The bacterial plant pathogen Xylella fastidiosa produces biofilm that accumulates in the host xylem vessels, affecting disease development in various crops and bacterial acquisition by insect vectors. Biofilms are sensitive to the chemical composition of the environment, and mineral elements being transported in the xylem are of special interest for this pathosystem. Here, X. fastidiosa liquid cultures were supplemented with zinc and compared with nonamended cultures to determine the effects of Zn on growth, biofilm, and exopolysaccharide (EPS) production under batch and flow culture conditions. The results show that Zn reduces growth and biofilm production under both conditions. However, in microfluidic chambers under liquid flow and with constant bacterial supplementation (closer to conditions inside the host), a dramatic increase in biofilm aggregates was seen in the Zn-amended medium. Biofilms formed under these conditions were strongly attached to surfaces and were not removed by medium flow. This phenomenon was correlated with increased EPS production in stationary-phase cells grown under high Zn concentrations. Zn did not cause greater adhesion to surfaces by individual cells. Additionally, viability analyses suggest that X. fastidiosa may be able to enter the viable but nonculturable state in vitro, and Zn can hasten the onset of this state. Together, these findings suggest that Zn can act as a stress factor with pleiotropic effects on X. fastidiosa and indicate that, although Zn could be used as a bactericide treatment, it could trigger the undesired effect of stronger biofilm formation upon reinoculation events.

Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae.

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7-11 (cadherin repeats 7-11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7-11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7-11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7-11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.

Sustainable Filtering Systems to Reduce Microfiber Emissions from Textiles during Household Laundering.

During laundering, synthetic textiles (polyester, polyamide, etc.) can release small fiber debris with a length of <5 mm. These are a type of microplastics (MPs), usually referred to as microfibers (MFs), which are considered high-concern pollutants due to their continuous and cumulative entrance into the environment. Currently, as far as we know, there are no feasible alternatives to remove them. In this work, four new and sustainable filtering systems are proposed to retain the MFs emitted from domestic washing machines. The filters contain a replaceable cartridge partially filled with recycled low-density polyethylene pellets. The four designed filtering systems of different sizes were tested in a household washing machine determining the retention efficiency of the MFs after several washing cycles. It was found that all four assessed filter arrangements have a good performance for retaining MFs from the washers' effluents. Filter F1 (diameter of 4 cm and a height of 30 cm) started retaining more than 50% of the MFs, at the 10th washing cycle, the retention climbed to 66%, while in the 20th washing cycle, its retention was greater than 80%. MFs retention was higher for filter F2 (diameter of 6.3 cm and a height of 41 cm), achieving a performance greater than 90% in the 20th washing cycle. Filter F3 was arranged by turning the F1 model flow upside down and the retention efficiency is higher compared with filter F1 values, reaching a retention efficiency of almost 100% in the 15th washing cycle. Finally, filter F4 arrangement was developed using the existing washing machine filter, obtaining better performance than the F1 and F2 filters, reaching efficiencies higher than 90% at the 20th washing cycle. In summary, depending on the arrangement, the microfiber retention efficiency was estimated between 52% and 86% in the 1st washing cycle and up to 83% to 99% in the 20th. Additionally, all arrangements demonstrated that the cartridges may last for more than 30 washing cycles before needing to be replaced.

[Primary sarcoma of the mammary gland]. / Sarcoma primario de la glándula mamaria.

BACKGROUND: Breast cancer occupies the chief place in incidence after the cervix. This gland sarcomas are rare with less than 1% at this location. Its diagnosis is difficult and its evolution is aggressive. Primary breast osteogenic sarcomas are a subset of lower frequency so that their behavior, evolution, prognosis ytratamiento no much experience and often are diagnosed as benign tumors. CASE REPORT: We report the case of a woman of 59 years which initially arose from a tumor in the right breast was performed with tru-cut biopsy with inconclusive results, using the tumor 7 months after the presence of a multilobulated solid tumor 20 cm, tumor compatible with mammography Phylodes (BIRADS 2). It was decided to perform right total mastectomy and pectoral muscle resection reconstruction with latissimus dorsi more and histochemical review pathology diagnosed with malignant mesenchymal neoplasm, osteosarcoma osteoblast osteoclast type and complemented the treatment with radiotherapy and chemotherapy. The prognosis is poor because the five-year survival is less than 40%. CONCLUSION: In the fast-growing breast tumors and large mammographic calcifications with signs and antecedentesde irradiation, trauma and Phyllodes tumor must be aware of this disease and that early diagnosis improves survival. In the surgical treatment of axillary dissection is not indicated and adjuvant treatment with radiotherapy and chemotherapy.

Novel Treatment to Immobilize and Use Textiles Microfibers Retained in Polymeric Filters through Their Incorporation in Composite Materials.

Microplastics (MPs, size < 5 mm) are among the most environmentally challenging pollutants. Their continuous and cumulative inflow or generation in the environment is what makes them drastically problematic. These pollutants can come from a wide variety of sources; hence, they are potential vectors that pose extensive risks to environmental and human health. Microfibers (MFs) are one type of MPs. Among the most well-known types of MFs are those detached from textile articles from household laundering or industrial processes. Currently, there are many ways to retain the MFs detached from textile articles. However, as far we know, there are no methods of valorizing the retained MFs. As such, we propose a novel and sustainable treatment method to immobilize MFs in a polymeric matrix, turning them into a composite. To determine the mechanical properties of the expected composites, different proportions of polyester MFs were mixed with low-density polyethylene, which is the material proposed for the immobilization of MFs. The results show that the optimum manufacturing composition was 10% (v/v) polyester MFs in the polymeric matrix. This composition improved some of the tensile mechanical properties of the polymeric matrix. Once the composites are obtained, these can be used for different purposes.

Towards Circular Economy by the Valorization of Different Waste Subproducts through Their Incorporation in Composite Materials: Ground Tire Rubber and Chicken Feathers.

Incorporation of residua into polymeric composites can be a successful approach to creating materials suitable for specific applications promoting a circular economy approach. Elastomeric (Ground Tire Rubber or GTR) and biogenic (chicken feathers or CFs) wastes were used to prepare polymeric composites in order to evaluate the tensile, acoustic and structural differences between both reinforcements. High-density polyethylene (HDPE), polypropylene (PP) and ethylene vinyl acetate (EVA) polymeric matrices were used. EVA matrix defines better compatibility with both reinforcement materials (GTR and CFs) than polyolefin matrices (HDPE and PP) as it has been corroborated by Fourier transform infrared spectroscopy (FTIR), termogravimetric analysis (TGA) and scanning electron microscopy (SEM). In addition, composites reinforced with GTR showed better acoustic properties than composites reinforced with CFs, due to the morphology of the reinforcing particles.

TOPLESS promotes plant immunity by repressing auxin signaling and is targeted by the fungal effector Naked1.

In plants, the antagonism between growth and defense is hardwired by hormonal signaling. The perception of pathogen-associated molecular patterns (PAMPs) from invading microorganisms inhibits auxin signaling and plant growth. Conversely, pathogens manipulate auxin signaling to promote disease, but how this hormone inhibits immunity is not fully understood. Ustilago maydis is a maize pathogen that induces auxin signaling in its host. We characterized a U. maydis effector protein, Naked1 (Nkd1), that is translocated into the host nucleus. Through its native ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif, Nkd1 binds to the transcriptional co-repressors TOPLESS/TOPLESS-related (TPL/TPRs) and prevents the recruitment of a transcriptional repressor involved in hormonal signaling, leading to the de-repression of auxin and jasmonate signaling and thereby promoting susceptibility to (hemi)biotrophic pathogens. A moderate upregulation of auxin signaling inhibits the PAMP-triggered reactive oxygen species (ROS) burst, an early defense response. Thus, our findings establish a clear mechanism for auxin-induced pathogen susceptibility. Engineered Nkd1 variants with increased expression or increased EAR-mediated TPL/TPR binding trigger typical salicylic-acid-mediated defense reactions, leading to pathogen resistance. This implies that moderate binding of Nkd1 to TPL is a result of a balancing evolutionary selection process to enable TPL manipulation while avoiding host recognition.

Circular Economy Assessment in Recycling of LLDPE Bags According to European Resolution, Thermal and Structural Characterization.

According to the Circular Economy Package promoted by the European directive, plastic bags companies must use in their formulations a percentage of polyethylene waste (industrial and/or domestic) greater than 70%. Following that regulation requires an understanding of its consequences in the final product from an industrial point of view. This manuscript analyzes the thermal and morphological changes related to the tear resistance of linear-low density polyethylene (LLDPE) samples from industrial waste generated by the company Sphere Spain subjected to the degradation produced by the recycling cycles. The process is analogue to the industrial, starts from samples in pellets then a film by blow extrusion is obtained (odd steps) and posteriorly this film is recycled to pellets again (even steps). The results obtained show that the LLDPE samples develop two crystalline structures (CS1 and CS2) which evolve differently through the recycling cycles with a tendency to decrease in crystallinity due to degradation that is not the same for the process of obtaining film or recycling to pellet. The molecules with a more linear structure and a longer chain break and branch. The more branched structure increases and tends to crosslinking. This leads to a decrease in tear strength in the longitudinal direction, which is not so evident in the transversal direction. The samples could admit four recycling cycles with and acceptable tear resistance. The longitudinal tear strength value decreases by 40% for each film and 20% in the case of tearing in the transverse direction. The results obtained in this research work show that the regulations included in the cited circular economy package can be applied in the manufacture of consumer bags, helping also to reduce the dependence of manufacturers on fluctuations in delivery by collapses in shipping.

A high-throughput screening method to identify proteins involved in unfolded protein response of the endoplasmic reticulum in plants.

BACKGROUND: The unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their host's UPR to facilitate infection. RESULTS: We developed a high-throughput screening assay to identify proteins that interfere with UPR signaling in planta. A set of 35 genes from a library of secreted proteins from the maize pathogen Ustilago maydis were transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress in Nicotiana benthamiana plants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method. CONCLUSIONS: We have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.