A pipeline for rapidly evaluating activity and inferring mechanisms of action of prospective antifungal compounds.

BACKGROUND: Fungal phytopathogens are a significant threat to crops and food security, and there is a constant need to develop safe and effective compounds that antagonize them. In-planta assays are complex and tedious and are thus not suitable for initial high-throughput screening of new candidate antifungal compounds. We propose an in vitro screening pipeline that integrates five rapid quantitative and qualitative methods to estimate the efficacy and mode of action of prospective antifungal compounds. RESULTS: The pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4-diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)-labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds' inhibition of both conidial germination and hyphal elongation. We then employed FUN-1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)-detection assay, we were able to quantify ROS production in response to compound application. CONCLUSIONS: Collectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Short- and long-term effects of continuous compost amendment on soil microbiome community.

Organic amendment, and especially the use of composts, is a well-accepted sustainable agricultural practice. Compost increases soil carbon and microbial biomass, changes enzymatic activity, and enriches soil carbon and nitrogen stocks. However, relatively little is known about the immediate and long-term temporal dynamics of agricultural soil microbial communities following repeated compost applications. Our study was conducted at two field sites: Newe Ya'ar (NY, Mediterranean climate) and Gilat (G, semi-arid climate), both managed organically over 4 years under either conventional fertilization (0, zero compost) or three levels of compost amendment (20, 40 and 60 m3/ha or 2, 4, 6 L/m2). Microbial community dynamics in the soils was examined by high- and low-time-resolution analyses. Annual community composition in compost-amended soils was significantly affected by compost amendment levels in G (first, second and third years) and in NY (third year). Repeated sampling at high resolution (9-10 times over 1 year) showed that at both sites, compost application initially induced a strong shift in microbial communities, lasting for up to 1 month, followed by a milder response. Compost application significantly elevated alpha diversity at both sites, but differed in the compost-dose correlation effect. We demonstrate higher abundance of taxa putatively involved in organic decomposition and characterized compost-related indicator taxa and a compost-derived core microbiome at both sites. Overall, this study describes temporal changes in the ecology of soil microbiomes in response to compost vs. conventional fertilization.

Pectin Induced Colony Expansion of Soil-Derived Flavobacterium Strains.

The genus Flavobacterium is characterized by the capacity to metabolize complex organic compounds and a unique gliding motility mechanism. Flavobacteria are often abundant in root microbiomes of various plants, but the factors contributing to this high abundance are currently unknown. In this study, we evaluated the effect of various plant-associated poly- and mono-saccharides on colony expansion of two Flavobacterium strains. Both strains were able to spread on pectin and other polysaccharides such as microcrystalline cellulose. However, only pectin (but not pectin monomers), a component of plant cell walls, enhanced colony expansion on solid surfaces in a dose- and substrate-dependent manner. On pectin, flavobacteria exhibited bi-phasic motility, with an initial phase of rapid expansion, followed by growth within the colonized area. Proteomic and gene expression analyses revealed significant induction of carbohydrate metabolism related proteins when flavobacteria were grown on pectin, including selected SusC/D, TonB-dependent glycan transport operons. Our results show a positive correlation between colony expansion and the upregulation of proteins involved in sugar uptake, suggesting an unknown linkage between specific operons encoding for glycan uptake and metabolism and flavobacterial expansion. Furthermore, within the context of flavobacterial-plant interactions, they suggest that pectin may facilitate flavobacterial expansion on plant surfaces in addition to serving as an essential carbon source.

Effects of tillage practices on soil microbiome and agricultural parameters.

No-tillage (NT) is a common soil-conservation management practice with known agricultural advantages and drawbacks. However, its short- and long-term effects on the soil microbiome have not been well established. Here, we compared conventional (CT), minimal (MT) and NT practices in two agricultural fields in the north of Israel over a period of 3 years. Edaphic properties, plant-associated pests, weed species abundance and soil microbial community structure were assessed to examine the effects of tillage. Tillage significantly altered physical and chemical soil properties, and a significant increase in hydrolytic and redox microbial activities was observed in NT soils from both sites. Consistent with this, the microbial community structure of NT samples diverged significantly over time from those of CT samples. Repetitive tillage and even a single tillage event caused significant changes in the relative abundance of microorganisms at taxonomic levels ranging from phylum to OTU. However, no significant difference between treatments was found in microbial community alpha-diversity or crop yield. Conversely, higher levels of weed diversity and some pests number were found in NT samples. Overall, we demonstrate that tillage plays a major role in shaping microbial community structure, and in influencing additional environmental, ecological and agricultural soil parameters.

Circadian Clock Control by Polyamine Levels through a Mechanism that Declines with Age.

Polyamines are essential polycations present in all living cells. Polyamine levels are maintained from the diet and de novo synthesis, and their decline with age is associated with various pathologies. Here we show that polyamine levels oscillate in a daily manner. Both clock- and feeding-dependent mechanisms regulate the daily accumulation of key enzymes in polyamine biosynthesis through rhythmic binding of BMAL1:CLOCK to conserved DNA elements. In turn, polyamines control the circadian period in cultured cells and animals by regulating the interaction between the core clock repressors PER2 and CRY1. Importantly, we found that the decline in polyamine levels with age in mice is associated with a longer circadian period that can be reversed upon polyamine supplementation in the diet. Our findings suggest a crosstalk between circadian clocks and polyamine biosynthesis and open new possibilities for nutritional interventions against the decay in clock's function with age.

The PXDLS linear motif regulates circadian rhythmicity through protein-protein interactions.

The circadian core clock circuitry relies on interlocked transcription-translation feedback loops that largely count on multiple protein interactions. The molecular mechanisms implicated in the assembly of these protein complexes are relatively unknown. Our bioinformatics analysis of short linear motifs, implicated in protein interactions, reveals an enrichment of the Pro-X-Asp-Leu-Ser (PXDLS) motif within circadian transcripts. We show that the PXDLS motif can bind to BMAL1/CLOCK and disrupt circadian oscillations in a cell-autonomous manner. Remarkably, the motif is evolutionary conserved in the core clock protein REV-ERBα, and additional proteins implicated in the clock's function (NRIP1, CBP). In this conjuncture, we uncover a novel cross talk between the two principal core clock feedback loops and show that BMAL/CLOCK and REV-ERBα interact and that the PXDLS motif of REV-ERBα participates in their binding. Furthermore, we demonstrate that the PXDLS motifs of NRIP1 and CBP are involved in circadian rhythmicity. Our findings suggest that the PXDLS motif plays an important role in circadian rhythmicity through regulation of protein interactions within the clock circuitry and that short linear motifs can be employed to modulate circadian oscillations.

Circadian clocks and feeding time regulate the oscillations and levels of hepatic triglycerides.

Circadian clocks play a major role in orchestrating daily physiology, and their disruption can evoke metabolic diseases such as fatty liver and obesity. To study the role of circadian clocks in lipid homeostasis, we performed an extensive lipidomic analysis of liver tissues from wild-type and clock-disrupted mice either fed ad libitum or night fed. To our surprise, a similar fraction of lipids (∼17%) oscillated in both mouse strains, most notably triglycerides, but with completely different phases. Moreover, several master lipid regulators (e.g., PPARα) and enzymes involved in triglyceride metabolism retained their circadian expression in clock-disrupted mice. Nighttime restricted feeding shifted the phase of triglyceride accumulation and resulted in ∼50% decrease in hepatic triglyceride levels in wild-type mice. Our findings suggest that circadian clocks and feeding time dictate the phase and levels of hepatic triglyceride accumulation; however, oscillations in triglycerides can persist in the absence of a functional clock.

Translation- and SRP-independent mRNA targeting to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae.

mRNAs encoding secreted/membrane proteins (mSMPs) are believed to reach the endoplasmic reticulum (ER) in a translation-dependent manner to confer protein translocation. Evidence exists, however, for translation- and signal recognition particle (SRP)-independent mRNA localization to the ER, suggesting that there are alternate paths for RNA delivery. We localized endogenously expressed mSMPs in yeast using an aptamer-based RNA-tagging procedure and fluorescence microscopy. Unlike mRNAs encoding polarity and secretion factors that colocalize with cortical ER at the bud tip, mSMPs and mRNAs encoding soluble, nonsecreted, nonpolarized proteins localized mainly to ER peripheral to the nucleus (nER). Synthetic nontranslatable uracil-rich mRNAs were also demonstrated to colocalize with nER in yeast. This mRNA-ER association was verified by subcellular fractionation and reverse transcription-PCR, single-molecule fluorescence in situ hybridization, and was not inhibited upon SRP inactivation. To better understand mSMP targeting, we examined aptamer-tagged USE1, which encodes a tail-anchored membrane protein, and SUC2, which encodes a soluble secreted enzyme. USE1 and SUC2 mRNA targeting was not abolished by the inhibition of translation or removal of elements involved in translational control. Overall we show that mSMP targeting to the ER is both translation- and SRP-independent, and regulated by cis elements contained within the message and trans-acting RNA-binding proteins (e.g., She2, Puf2).

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae.

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with mitochondria in vivo. This supports earlier cell fractionation and microarray-based studies that proposed mMP association with the mitochondrial fraction. Interestingly, a number of mMPs showed a dependency on the mitochondrial Puf3 RNA-binding protein, as well as nonessential proteins of the translocase of the outer membrane (TOM) complex import machinery, for normal colocalization with mitochondria. We examined the specific determinants of ATP2 and OXA1 mRNA localization and found a mutual dependency on the 3' UTR, Puf3, Tom7, and Tom70, but not Tom20, for localization. Tom6 may facilitate the localization of specific mRNAs as OXA1, but not ATP2, mRNA was mislocalized in tom6Δ cells. Interestingly, a substantial fraction of OXA1 and ATP2 RNA granules colocalized with the endoplasmic reticulum (ER) and a deletion in MDM10, which mediates mitochondria-ER tethering, resulted in a significant loss of OXA1 mRNA localization with ER. Finally, neither ATP2 nor OXA1 mRNA targeting was affected by a block in translation initiation, indicating that translation may not be essential for mRNA anchoring. Thus, endogenously expressed mRNAs are targeted to the mitochondria in vivo, and multiple factors contribute to mMP localization.

Addressing mRNAs to the ER: cis sequences act up!

Translation-coupled protein translocation requires that mRNAs encoding secreted and membrane proteins (mSMPs) reach the ER membrane. The classical view is that the signal recognition particle (SRP) pathway delivers translating signal sequence-containing proteins to the SRP receptor present on the ER surface and engages the translocation machinery. However, recent studies demonstrate both SRP- and translation-independent mRNA recruitment to the ER, and that mRNAs encoding non-signal sequence-containing cytosolic proteins (mCPs) might be full-time residents of ER membranes. Furthermore, translation-independent cis-acting sequence elements present in both mCPs and mSMPs appear to govern the ability of mRNAs to associate with ER. Thus, a more complex picture of how and why mRNAs target the ER is emerging.

Protein-tyrosine phosphatase epsilon regulates Shc signaling in a kinase-specific manner: increasing coherence in tyrosine phosphatase signaling.

Individual protein tyrosine kinases and phosphatases target multiple substrates; this may generate conflicting signals, possibly within a single pathway. Protein-tyrosine phosphatase epsilon (PTPepsilon) performs two potentially opposing roles: in Neu-induced mammary tumors, PTPepsilon activates Src downstream of Neu, whereas in other systems PTPepsilon can indirectly down-regulate MAP kinase signaling. We now show that the latter effect is mediated at least in part via the adaptor protein Shc. PTPepsilon binds and dephosphorylates Shc in vivo, reducing the association of Shc with Grb2 and inhibiting downstream ERK activation. PTPepsilon binds Shc in a phosphotyrosine-independent manner mediated by the Shc PTB domain and aided by a sequence of 10 N-terminal residues in PTPepsilon. Surprisingly, PTPepsilon dephosphorylates Shc in a kinase-dependent manner; PTPepsilon targets Shc in the presence of Src but not in the presence of Neu. Using a series of point mutants of Shc and Neu, we show that Neu protects Shc from dephosphorylation by binding the PTB domain of Shc, most likely competing against PTPepsilon for binding the same domain. In agreement, PTPepsilon dephosphorylates Shc in mouse embryo fibroblasts but not in Neu-induced mammary tumor cells. We conclude that in the context of Neu-induced mammary tumor cells, Neu prevents PTPepsilon from targeting Shc and from reducing its promitogenic signal while phosphorylating PTPepsilon and directing it to activate Src in support of mitogenesis. In so doing, Neu contributes to the coherence of the promitogenic role of PTPepsilon in this system.