Conserved secreted effectors contribute to endophytic growth and multihost plant compatibility in a vascular wilt fungus.

Fungal interactions with plant roots, either beneficial or detrimental, have a crucial impact on agriculture and ecosystems. The cosmopolitan plant pathogen Fusarium oxysporum (Fo) provokes vascular wilts in more than a hundred different crops. Isolates of this fungus exhibit host-specific pathogenicity, which is conferred by lineage-specific Secreted In Xylem (SIX) effectors encoded on accessory genomic regions. However, such isolates also can colonize the roots of other plants asymptomatically as endophytes or even protect them against pathogenic strains. The molecular determinants of endophytic multihost compatibility are largely unknown. Here, we characterized a set of Fo candidate effectors from tomato (Solanum lycopersicum) root apoplastic fluid; these early root colonization (ERC) effectors are secreted during early biotrophic growth on main and alternative plant hosts. In contrast to SIX effectors, ERCs have homologs across the entire Fo species complex as well as in other plant-interacting fungi, suggesting a conserved role in fungus-plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence and rapid activation of plant immune responses, while ERC deletion in a nonpathogenic isolate led to impaired root colonization and biocontrol ability. Strikingly, some ERCs contribute to Fo infection on the nonvascular land plant Marchantia polymorpha, revealing an evolutionarily conserved mechanism for multihost colonization by root infecting fungi.

Enhanced Resistance to Viruses in Nicotiana edwardsonii 'Columbia' Is Dependent on Salicylic Acid, Correlates with High Glutathione Levels, and Extends to Plant-Pathogenic Bacteria and Abiotic Stress.

Our earlier research showed that an interspecific tobacco hybrid (Nicotiana edwardsonii 'Columbia' [NEC]) displays elevated levels of salicylic acid (SA) and enhanced resistance to localized necrotic symptoms (hypersensitive response [HR]) caused by tobacco mosaic virus (TMV) and tobacco necrosis virus (TNV), as compared with another interspecific hybrid (Nicotiana edwardsonii [NE]) derived from the same parents. In the present study, we investigated whether symptomatic resistance in NEC is indeed associated with the inhibition of TMV and TNV and whether SA plays a role in this process. We demonstrated that enhanced viral resistance in NEC is manifested as both milder local necrotic (HR) symptoms and reduced levels of TMV and TNV. The presence of an adequate amount of SA contributes to the enhanced defense response of NEC to TMV and TNV, as the absence of SA resulted in seriously impaired viral resistance. Elevated levels of subcellular tripeptide glutathione (GSH) in NEC plants in response to viral infection suggest that in addition to SA, GSH may also contribute to the elevated viral resistance of NEC. Furthermore, we found that NEC displays an enhanced resistance not only to viral pathogens but also to bacterial infections and abiotic oxidative stress induced by paraquat treatments. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A three-step process of manganese acquisition and storage in the microalga Chlorella sorokiniana.

Metabolism of metals in microalgae and adaptation to metal excess are of significant environmental importance. We report a three-step mechanism that the green microalga Chlorella sorokiniana activates during the acquisition of and adaptation to manganese (Mn), which is both an essential trace metal and a pollutant of waters. In the early stage, Mn2+ was mainly bound to membrane phospholipids and phosphates in released mucilage. The outer cell wall was reorganized and lipids were accumulated, with a relative increase in lipid saturation. Intracellular redox settings were rapidly altered in the presence of Mn excess, with increased production of reactive oxygen species that resulted in lipid peroxidation and a decrease in the concentration of thiols. In the later stage, Mn2+ was chelated by polyphosphates and accumulated in the cells. The structure of the inner cell wall was modified and the redox milieu established a new balance. Polyphosphates serve as a transient Mn2+ storage ligand, as proposed previously. In the final stage, Mn was stored in multivalent Mn clusters that resemble the structure of the tetramanganese-calcium core of the oxygen-evolving complex. The present findings elucidate the bioinorganic chemistry and metabolism of Mn in microalgae, and may shed new light on water-splitting Mn clusters.

Volumetric 3D reconstruction of plant leaf cells using SEM, ion milling, TEM, and serial sectioning.

MAIN CONCLUSION: Focused ion beam scanning electron microscopy is well suited for volumetric extractions and 3D reconstructions of plant cells and its organelles. The three-dimensional (3D) reconstruction of individual plant cells is an important tool to extract volumetric data of organelles and is necessary to fully understand ultrastructural changes and adaptations of plants to their environment. Methods such as the 3D reconstruction of cells based on light microscopical images often lack the resolution necessary to clearly reconstruct all cell compartments within a cell. The 3D reconstruction of cells through serial sectioning transmission electron microscopy (ssTEM) and focused ion beam scanning electron microscopy (FIB-SEM) are powerful alternatives but not widely used in plant sciences. Here, we present a method for the 3D reconstruction and volumetric extraction of plant cells based on FIB milling and compare the results with 3D reconstructions obtained with ssTEM. When compared to 3D reconstruction based on ssTEM, FIB-SEM delivered similar results. The data extracted in this study demonstrated that tobacco cells were larger (31410 µm3) than pumpkin cells (20697 µm3) and contained more chloroplasts (175 vs. 124), mitochondria (1317 vs. 291) and peroxisomes (745 vs. 79). While individual chloroplasts, mitochondria, peroxisomes were larger in pumpkin plants (25, 53, and 50%, respectively) they covered more total volume in tobacco plants (5390, 395, 374 µm3, respectively) due to their higher number per cell when compared to pumpkin plants (4762, 134, 59 µm3, respectively). While image acquisition with FIB-SEM was automated, software controlled, and less difficult than ssTEM, FIB milling was slower and sections could not be revised or re-imaged as they were destroyed by the ion beam. Nevertheless, the results in this study demonstrated that both, FIB-SEM and ssTEM, are powerful tools for the 3D reconstruction of and volumetric extraction from plant cells and that there were large differences in size, number, and organelle composition between pumpkin and tobacco cells.

Marchantia polymorpha model reveals conserved infection mechanisms in the vascular wilt fungal pathogen Fusarium oxysporum.

Root-infecting vascular fungi cause wilt diseases and provoke devastating losses in hundreds of crops. It is currently unknown how these pathogens evolved and whether they can also infect nonvascular plants, which diverged from vascular plants over 450 million years ago. We established a pathosystem between the nonvascular plant Marchantia polymorpha (Mp) and the root-infecting vascular wilt fungus Fusarium oxysporum (Fo). On angiosperms, Fo exhibits exquisite adaptation to the plant xylem niche as well as host-specific pathogenicity, both of which are conferred by effectors encoded on lineage-specific chromosomes. Fo isolates displaying contrasting lifestyles on angiosperms - pathogenic vs endophytic - are able to infect Mp and cause tissue maceration and host cell killing. Using isogenic fungal mutants we define a set of conserved fungal pathogenicity factors, including mitogen activated protein kinases, transcriptional regulators and cell wall remodelling enzymes, that are required for infection of both vascular and nonvascular plants. Markedly, two host-specific effectors and a morphogenetic regulator, which contribute to vascular colonisation and virulence on tomato plants are dispensable on Mp. Collectively, these findings suggest that vascular wilt fungi employ conserved infection strategies on nonvascular and vascular plant lineages but also have specific mechanisms to access the vascular niche of angiosperms.

Comparison of skeletal muscle ultrastructural changes between normal and blood flow-restricted resistance exercise: A case report.

NEW FINDINGS: What is the main observation in this case? The main observation of this case report is that blood flow-restricted exercise can cause myofibrils to have an aberrant wave-like appearance that is accompanied by irregular pockets of sarcoplasm in the intermyofibrillar space, while traditional forms of damage to the Z-discs and contractile elements are not as apparent. What insights does it reveal? Our findings indicate that blood flow restriction-mediated fluid pooling might cause alterations in skeletal muscle ultrastructure after exercise that might be directly related to myofibre swelling. ABSTRACT: The acute effects of blood flow-restricted (BFR) exercise training on skeletal muscle ultrastructure are poorly understood owing to inconsistent findings and the use of largely imprecise systemic markers for indications of muscle damage. The purpose of this study was to compare myofibrillar ultrastructure before and 30 min after normal and BFR resistance exercise using transmission electron microscopy in a single individual to evaluate the feasibility of this more nuanced approach. One apparently healthy male with 13 years of resistance exercise completed six sets of both BFR [30% of one-repetition maximum (1-RM)] and normal non-occluded (70% of 1-RM) unilateral angled leg press on the contralateral leg, as a control, after assessment of 1-RM 72 h before. Vastus lateralis muscle biopsies were collected before and 30 min after each exercise session. The lengths and widths of 250 sarcomeres and the sarcoplasmic area were assessed via 20 individual transmission electron photomicrographs. Analysis revealed that BFR training (1.769 ± 0.12 µm) increased sarcomere length when compared with normal exercise (1.64 ± 0.17 µm; P < 0.001), without differences in sarcomere width between conditions (BFR, 0.90 ± 0.26 µm; normal, 0.93 ± 0.27 µm; P = 0.172). Furthermore, there were no significant interaction (P = 0.168) or condition effects between BFR (25.98 ± 4.17%) and normal (27.3 ± 6.49%) resistance exercise for sarcoplasmic area (P = 0.229). Exercise also increased sarcoplasmic area within the myofibril (pre-exercise, 24.42 ± 5.13%; postexercise, 28.95 ± 5.92%) for both conditions (P = 0.001). This case study demonstrates a unique BFR training-induced alteration in myofibril ultrastructure that appeared wave like and was accompanied by intracellular abnormalities that appeared to be fluid pockets of sarcoplasm disrupting the surrounding myofibrils.

Quantifying the effect of shade on cuticle morphology and carbon isotopes of sycamores: present and past.

PREMISE: Reconstructing the light environment and architecture of the plant canopy from the fossil record requires the use of proxies, such as those derived from cell wall undulation, cell size, and carbon isotopes. All approaches assume that plant taxa will respond predictably to changes in light environments. However, most species-level studies looking at cell wall undulation only consider "sun" or "shade" leaves; therefore, we need a fully quantitative taxon-specific method. METHODS: We quantified the response of cell wall undulation, cell size, and carbon isotopes of Platanus occidentalis using two experimental setups: (1) two growth chambers at low and high light and (2) a series of outdoor growth experiments using green and black shade cloth at different densities. We then developed and applied a proxy for daily light integral (DLI) to fossil Platanites leaves from two early Paleocene floras from the San Juan Basin in New Mexico. RESULTS: All traits responded to light environment. Cell wall undulation was the most useful trait for reconstructing DLI in the geological record. Median reconstructed DLI from early Paleocene leaves was ~44 mol m-2 d-1 , with values from 28 to 54 mol m-2 d-1 . CONCLUSIONS: Cell wall undulation of P. occidentalis is a robust, quantifiable measurement of light environment that can be used to reconstruct the paleo-light environment from fossil leaves. The distribution of high DLI values from fossil leaves may provide information on canopy architecture; indicating that either (1) most of the canopy mass is within the upper portion of the crown or (2) leaves exposed to more sunlight are preferentially preserved.

Mechanisms of detoxification of high copper concentrations by the microalga Chlorella sorokiniana.

Microalgae have evolved mechanisms to respond to changes in copper ion availability, which are very important for normal cellular function, to tolerate metal pollution of aquatic ecosystems, and for modulation of copper bioavailability and toxicity to other organisms. Knowledge and application of these mechanisms will benefit the use of microalgae in wastewater processing and biomass production, and the use of copper compounds in the suppression of harmful algal blooms. Here, using electron microscopy, synchrotron radiation-based Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, and X-ray absorption fine structure spectroscopy, we show that the microalga Chlorella sorokiniana responds promptly to Cu2+ at high non-toxic concentration, by mucilage release, alterations in the architecture of the outer cell wall layer and lipid structures, and polyphosphate accumulation within mucilage matrix. The main route of copper detoxification is by Cu2+ coordination to polyphosphates in penta-coordinated geometry. The sequestrated Cu2+ was accessible and could be released by extracellular chelating agents. Finally, the reduction in Cu2+ to Cu1+ appears also to take place. These findings reveal the biochemical basis of the capacity of microalgae to adapt to high external copper concentrations and to serve as both, sinks and pools of environmental copper.

Arabidopsis glutathione reductase 2 is indispensable in plastids, while mitochondrial glutathione is safeguarded by additional reduction and transport systems.

A highly negative glutathione redox potential (EGSH ) is maintained in the cytosol, plastids and mitochondria of plant cells to support fundamental processes, including antioxidant defence, redox regulation and iron-sulfur cluster biogenesis. Out of two glutathione reductase (GR) proteins in Arabidopsis, GR2 is predicted to be dual-targeted to plastids and mitochondria, but its differential roles in these organelles remain unclear. We dissected the role of GR2 in organelle glutathione redox homeostasis and plant development using a combination of genetic complementation and stacked mutants, biochemical activity studies, immunogold labelling and in vivo biosensing. Our data demonstrate that GR2 is dual-targeted to plastids and mitochondria, but embryo lethality of gr2 null mutants is caused specifically in plastids. Whereas lack of mitochondrial GR2 leads to a partially oxidised glutathione pool in the matrix, the ATP-binding cassette (ABC) transporter ATM3 and the mitochondrial thioredoxin system provide functional backup and maintain plant viability. We identify GR2 as essential in the plastid stroma, where it counters GSSG accumulation and developmental arrest. By contrast a functional triad of GR2, ATM3 and the thioredoxin system in the mitochondria provides resilience to excessive glutathione oxidation.

A barley powdery mildew fungus non-autonomous retrotransposon encodes a peptide that supports penetration success on barley.

Pathogens overcome plant immunity by means of secreted effectors. Host effector targets often act in pathogen defense, but might also support fungal accommodation or nutrition. The barley ROP GTPase HvRACB is involved in accommodation of fungal haustoria of the powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) in barley epidermal cells. We found that HvRACB interacts with the ROP-interactive peptide 1 (ROPIP1) that is encoded on the active non-long terminal repeat retroelement Eg-R1 of Bgh. Overexpression of ROPIP1 in barley epidermal cells and host-induced post-transcriptional gene silencing (HIGS) of ROPIP1 suggested that ROPIP1 is involved in virulence of Bgh. Bimolecular fluorescence complementation and co-localization supported that ROPIP1 can interact with activated HvRACB in planta. We show that ROPIP1 is expressed by Bgh on barley and translocated into the cytoplasm of infected barley cells. ROPIP1 is recruited to microtubules upon co-expression of MICROTUBULE ASSOCIATED ROP GTPase ACTIVATING PROTEIN (HvMAGAP1) and can destabilize cortical microtubules. The data suggest that Bgh ROPIP targets HvRACB and manipulates host cell microtubule organization for facilitated host cell entry. This points to a possible neo-functionalization of retroelement-derived transcripts for the evolution of a pathogen virulence effector.

A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors.

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize.

An assay for entry of secreted fungal effectors into plant cells.

Successful colonization of plants by prokaryotic and eukaryotic pathogens requires active effector-mediated suppression of defense responses and host tissue reprogramming. Secreted effector proteins can either display their activity in the apoplast or translocate into host cells and function therein. Although characterized in bacteria, the molecular mechanisms of effector delivery by fungal phytopathogens remain elusive. Here we report the establishment of an assay that is based on biotinylation of effectors in the host cytoplasm as hallmark of uptake. The assay exploits the ability of the bacterial biotin ligase BirA to biotinylate any protein that carries a short peptide (Avitag). It is based on the stable expression of BirA in the cytoplasm of maize plants and on engineering of Ustilago maydis strains to secrete Avitagged effectors. We demonstrate translocation of a number of effectors in the U. maydis-maize system and show data that suggest that the uptake mechanism could be rather nonspecific The assay promises to be a powerful tool for the classification of effectors as well as for the functional study of effector uptake mechanism not only in the chosen system but more generally for systems where biotrophic interactions are established.

Compartment-specific investigations of antioxidants and hydrogen peroxide in leaves of Arabidopsis thaliana during dark-induced senescence.

The aim of this study was to gain insight into the compartment-specific roles of ascorbate and glutathione in leaf senescence in Arabidopsis thaliana. The subcellular distribution of ascorbate, glutathione, and hydrogen peroxide (H2O2) was analyzed by transmission electron microscopy and correlated with the activity of antioxidative enzymes in wildtype plants and the ascorbate- and glutathione-deficient mutants vtc2-1 and pad2-1, respectively. Both mutants showed earlier and stronger senescence than the wildtype indicating the importance of a functioning ascorbate and glutathione cycle in the induction and regulation of senescence. Glutathione levels dropped drastically and up to 93 % in all cell compartments of wildtype plants and the vtc2-1 mutant within the first day of dark-induced senescence while ascorbate contents remained unchanged until the very end. Glutathione contents in mitochondria of pad2-1 mutants decreased more slowly over the first 7 days than compared to the other plants indicating an important role of glutathione in mitochondria in this mutant during senescence. The strongest decrease (84 %) of glutathione contents in wildtype plants at this time point was found in mitochondria indicating an important role of mitochondria for the induction of senescence and cell death events. Due to the general decrease of the antioxidative capacity, a strong accumulation of H2O2 was observed in cell walls, plastids, and the cytosol in all plants. Activities of glutathione reductase, dehydroascorbate reductase and catalase were strongly reduced while ascorbate peroxidase and monodehydroascorbate reductase were increased. The initial rapid drop of glutathione levels seemed to be the trigger for senescence, while ascorbate appeared to be the key factor in regulating senescence through controlling H2O2 levels by the oxidation of reduced ascorbate to monodehydroascorbate and the subsequent reduction to ascorbate by monodehydroascorbate reductase.

Ultraviolet-B component of sunlight stimulates photosynthesis and flavonoid accumulation in variegated Plectranthus coleoides leaves depending on background light.

We used variegated Plectranthus coleoides as a model plant with the aim of clarifying whether the effects of realistic ultraviolet-B (UV-B) doses on phenolic metabolism in leaves are mediated by photosynthesis. Plants were exposed to UV-B radiation (0.90 W m(-2) ) combined with two photosynthetically active radiation (PAR) intensities [395 and 1350 µmol m(-2) s(-1) , low light (LL) and high light (HL)] for 9 d in sun simulators. Our study indicates that UV-B component of sunlight stimulates CO2 assimilation and stomatal conductance, depending on background light. UV-B-specific induction of apigenin and cyanidin glycosides was observed in both green and white tissues. However, all the other phenolic subclasses were up to four times more abundant in green leaf tissue. Caffeic and rosmarinic acids, catechin and epicatechin, which are endogenous peroxidase substrates, were depleted at HL in green tissue. This was correlated with increased peroxidase and ascorbate peroxidase activities and increased ascorbate content. The UV-B supplement to HL attenuated antioxidative metabolism and partly recovered the phenolic pool indicating stimulation of the phenylpropanoid pathway. In summary, we propose that ortho-dihydroxy phenolics are involved in antioxidative defence in chlorophyllous tissue upon light excess, while apigenin and cyanidin in white tissue have preferentially UV-screening function.

The mutualistic fungus Piriformospora indica colonizes Arabidopsis roots by inducing an endoplasmic reticulum stress-triggered caspase-dependent cell death.

In Arabidopsis thaliana roots, the mutualistic fungus Piriformospora indica initially colonizes living cells, which die as the colonization proceeds. We aimed to clarify the molecular basis of this colonization-associated cell death. Our cytological analyses revealed endoplasmic reticulum (ER) swelling and vacuolar collapse in invaded cells, indicative of ER stress and cell death during root colonization. Consistent with this, P. indica-colonized plants were hypersensitive to the ER stress inducer tunicamycin. By clear contrast, ER stress sensors bZIP60 and bZIP28 as well as canonical markers for the ER stress response pathway, termed the unfolded protein response (UPR), were suppressed at the same time. Arabidopsis mutants compromised in caspase 1-like activity, mediated by cell death-regulating vacuolar processing enzymes (VPEs), showed reduced colonization and decreased cell death incidence. We propose a previously unreported microbial invasion strategy during which P. indica induces ER stress but inhibits the adaptive UPR. This disturbance results in a VPE/caspase 1-like-mediated cell death, which is required for the establishment of the symbiosis. Our results suggest the presence of an at least partially conserved ER stress-induced caspase-dependent cell death pathway in plants as has been reported for metazoans.

Two and three dimensional characterization of Zucchini Yellow Mosaic Virus induced structural alterations in Cucurbita pepo L. plants.

Infection of plants by Zucchini Yellow Mosaic Virus (ZYMV) induces severe ultrastructural changes. The aim of this study was to investigate ultrastructural changes during ZYMV-infection in Cucurbita pepo L. plants on the two and three dimensional (2D and 3D) level and to correlate these changes with the spread of ZYMV throughout the plant by transmission electron microscopy (TEM) and image analysis. This study revealed that after inoculation of the cotyledons ZYMV moved into roots [3 days post inoculation (dpi)], then moved upwards into the stem and apical meristem (5 dpi), then into the first true leaf (7 dpi) and could finally be found in all plant parts (9 dpi). ZYMV-infected cells contained viral inclusion bodies in the form of cylindrical inclusions (CIs). These CIs occurred in four different forms throughout the cytosol of roots and leaves: scrolls and pinwheels when cut transversely and long tubular structures and bundles of filaments when cut longitudinally. 3D reconstruction of ZYMV-infected cells containing scrolls revealed that they form long tubes throughout the cytosol. The majority has a preferred orientation and an average length and width of 3 µm and 120 nm, respectively. Image analysis revealed an increased size of cells and vacuoles (107% and 447%, respectively) in younger ZYMV-infected leaves leading to a similar ratio of cytoplasm to vacuole (about 1:1) in older and younger ZYMV-infected leaves which indicates advanced cell growth in younger tissues. The collected data advances the current knowledge about ZYMV-induced ultrastructural changes in Cucurbita pepo.

Alternative cell death mechanisms determine epidermal resistance in incompatible barley-Ustilago interactions.

Programmed cell death is a key feature of epidermal plant immunity, which is particularly effective against biotrophic microbes that depend on living host tissue. The covered smut fungus Ustilago hordei establishes a compatible biotrophic interaction with its host plant barley. The maize smut U. maydis triggers a nonhost response in barley, which results in epidermal cell death. Similarly, Ustilago mutants being deleted for pep1, a gene encoding a secreted effector, are blocked upon host penetration. We studied the epidermal responses of barley to incompatible Ustilago strains. Molecular and cellular analyses were used to test the impact of Bax inhibitor-1 (BI-1), a suppressor of programmed cell death, on the barley nonhost resistance to U. maydis as well as Ustilago Δpep1 mutants. Overexpression of BI-1 resulted in partial break of barley nonhost resistance to U. maydis. By contrast, the epidermal cell death response triggered by pep1 deletion mutants was not impaired by BI-1. Hypersensitive-response-like cell death caused by U. maydis wild-type infection showed features of necrotic cell death, while Δpep1 mutant-induced host responses involved hallmarks of autophagy. Therefore, we propose that the mechanisms of epidermal cell death in response to different types of incompatible pathogens depend on spatial and temporal appearance of cell-death-triggering stimuli.

The Ustilago maydis effector Pep1 suppresses plant immunity by inhibition of host peroxidase activity.

The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1) as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H2O2 strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction.

Scanning Electron Microscopy and Energy-Dispersive X-ray Spectroscopy of Staphylococcus aureus Biofilms.

Understanding the dynamics of biofilm formation and its elemental composition is crucial for developing effective strategies against biofilm-associated infections. In this study, we employed scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) to investigate the morphological changes and elemental compositions of Staphylococcus aureus biofilms. SEM images revealed distinct stages of biofilm development, from initial aggregation to the formation of mature and aged biofilms. EDS analysis consistently showed elevated levels of sodium (Na), oxygen (O), and phosphorus (P) in the biofilm matrix, indicating its high negative charge and the presence of anionic biopolymers. The incorporation of extracellular DNA (eDNA) into the biofilm matrix, leading to significant retention of sodium ions, underscored the importance of electrostatic interactions in biofilm formation and stability. Our findings highlight the potential of EDS analysis in quantifying elemental compositions and elucidating the role of anionic biopolymers in biofilm development.