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Abstract Non-self recognition is a fundamental aspect of life, serving as a crucial mechanism for mitigating proliferation of molecular parasites within fungal populations. However, studies investigating the potential interference of plants with fungal non-self recognition mechanisms are limited. Here, we demonstrate a pronounced increase in the efficiency of horizontal mycovirus transmission between vegetatively incompatible Sclerotinia sclerotiorum strains in planta as compared to in vitro. This increased efficiency is associated with elevated proline concentration in plants following S. sclerotiorum infection. This surge in proline levels attenuates the non-self recognition reaction among fungi by inhibition of cell death, thereby facilitating mycovirus transmission. Furthermore, our field experiments reveal that the combined deployment of hypovirulent S. sclerotiorum strains harboring hypovirulence-associated mycoviruses (HAVs) together with exogenous proline confers substantial protection to oilseed rape plants against virulent S. sclerotiorum. This unprecedented discovery illuminates a novel pathway by which plants can counteract S. sclerotiorum infection, leveraging the weakening of fungal non-self recognition and promotion of HAVs spread. These promising insights provide an avenue to explore for developing innovative biological control strategies aimed at mitigating fungal diseases in plants by enhancing the efficacy of horizontal HAV transmission.

Mycoviral infection can either be asymptomatic or have marked effects on fungal hosts, influencing them either positively or negatively. To fully understand the effects of mycovirus infection on the fungal host, transcriptomic profiling of four Beauveria bassiana isolates, including EABb 92/11-Dm that harbors mycoviruses, was performed 48 h following infection of Tenebrio molitor via topical application or injection. Genes that participate in carbohydrate assimilation and transportation, and those essential for fungal survival and oxidative stress tolerance, calcium uptake, and iron uptake, were found to be overexpressed in the virus-infected isolate during the mid-infection stage. Mycotoxin genes encoding bassianolide and oosporein were switched off in all isolates. However, beauvericin, a mycotoxin capable of inducing oxidative stress at the molecular level, was expressed in all four isolates, indicating an important contribution to virulence against T. molitor. These observations suggest that detoxification of immune-related (oxidative) defenses and nutrient scouting, as mediated by these genes, occurs in mid-infection during the internal growth phase. Consequently, we observe a symbiotic relationship between mycovirus and fungus that does not afflict the host; on the contrary, it enhances the expression of key genes leading to a mycovirus-mediated hypervirulence effect.

Mycoviruses are viruses that infect fungi and are widespread across all major fungal taxa, exhibiting great biological diversity. Since their discovery in the 1960s, researchers have observed a myriad of fungal phenotypes altered due to mycoviral infection. In this review, we examine the nuanced world of mycoviruses in the context of the medically and agriculturally important fungal genus, Aspergillus. The advent of RNA sequencing has revealed a previous underestimate of viral prevalence in fungi, in particular linear single-stranded RNA viruses, and here we outline the diverse viral families known to date that contain mycoviruses infecting Aspergillus. Furthermore, we describe these novel mycoviruses, highlighting those with peculiar genome structures, such as a split RNA dependent RNA polymerase gene. Next, we delineate notable mycovirus-mediated phenotypes in Aspergillus, in particular reporting on observations of mycoviruses that affect their fungal host’s virulence and explore how this may relate to virus-mediated decreased stress tolerance. Furthermore, mycovirus effects on microbial competition and antifungal resistance are discussed. The factors that influence the manifestation of these phenotypes, such as temperature, fungal life stage, and infection with multiple viruses, among others, are also evaluated. In addition, we attempt to elucidate the molecular mechanisms that underpin these phenotypes, examining how mycoviruses can be targets, triggers, and even suppressors of RNA silencing and how this can affect fungal gene expression and phenotypes. Finally, we highlight the potential therapeutic applications of mycoviruses and how, in an approach analogous to bacteriophage therapy, their ability to produce hypovirulence in Aspergillus might be used to attenuate invasive aspergillosis infections in humans.

Background: The placenta exerts a crucial role in fetus growth and development during gestation, protecting the fetus from maternal drugs and chemical exposure. However, diverse drugs and chemicals (xenobiotics) can penetrate the maternal placental barrier, leading to deleterious, adverse effects concerning fetus health. Moreover, placental enzymes can metabolize drugs and chemicals into more toxic compounds for the fetus. Thus, evaluating the molecular mechanisms through which drugs and chemicals transfer and undergo metabolism across the placental barrier is of vital importance. In this aspect, this comprehensive literature review aims to provide a holistic approach by critically summarizing and scrutinizing the potential molecular processes and mechanisms governing drugs and chemical transfer and metabolism across the placental barrier, which may lead to fetotoxicity effects, as well as analyzing the currently available experimental methodologies used to assess xenobiotics placental transfer and metabolism. Methods: A comprehensive and in-depth literature review was conducted in the most accurate scientific databases such as PubMed, Scopus, and Web of Science by using relevant and effective keywords related to xenobiotic placental transfer and metabolism, retrieving 8830 published articles until 5 February 2024. After applying several strict exclusion and inclusion criteria, a final number of 148 relevant published articles were included. Results: During pregnancy, several drugs and chemicals can be transferred from the mother to the fetus across the placental barrier by either passive diffusion or through placental transporters, resulting in fetus exposure and potential fetotoxicity effects. Some drugs and chemicals also appear to be metabolized across the placental barrier, leading to more toxic products for both the mother and the fetus. At present, there is increasing research development of diverse experimental methodologies to determine the potential molecular processes and mechanisms of drug and chemical placental transfer and metabolism. All the currently available methodologies have specific strengths and limitations, highlighting the strong demand to utilize an efficient combination of them to obtain reliable evidence concerning drug and chemical transfer and metabolism across the placental barrier. To derive the most consistent and safe evidence, in vitro studies, ex vivo perfusion methods, and in vivo animal and human studies can be applied together with the final aim to minimize potential fetotoxicity effects. Conclusions: Research is being increasingly carried out to obtain an accurate and safe evaluation of drug and chemical transport and metabolism across the placental barrier, applying a combination of advanced techniques to avoid potential fetotoxic effects. The improvement of the currently available techniques and the development of novel experimental protocols and methodologies are of major importance to protect both the mother and the fetus from xenobiotic exposure, as well as to minimize potential fetotoxicity effects.

ABSTRACT The unique, recently discovered fungus Didymella theifolia specifically infects local varieties of tea plant Camellia sinensis in China, and therefore, the characterization of its mycoviruses is important. Three double-stranded (ds) RNAs (1, 2, and 3, with 6,338, 5,910, and 727 bp in size, respectively) were identified in the avirulent D. theifolia strain CJP4-1, which exhibits normal growth and morphology. Characterization of these double-stranded RNAs (dsRNAs) revealed that the two largest elements are the genomic components of a novel botybirnavirus, tentatively named Didymella theifolia botybirnavirus 1 (DtBRV1). Conversely, dsRNA3 shares no detectable similarity with sequences deposited in public databases but has high similarity with the 5′-terminal regions of dsRNAs 1 and 2 and contains a duplicated region encoding a putative small peptide. All three dsRNAs are encapsidated in isometric virions ca. 40 nm in diameter, supporting the notion that dsRNA3 is a DtBRV1 satellite. SDS-polyacrylamide gel electrophoresis in combination with peptide mass fingerprint analysis revealed that the DtBRV1 capsid protein consists of polypeptides encoded by the 5′-terminal regions of both genomic components dsRNA1 and dsRNA2. Vertical transmission of DtBRV1 through conidia is efficient, while its horizontal transmission from CJP4-1 to other strains was not detected. DtBRV1, with or without dsRNA3, has no obvious effects on fungal growth and virulence, as illustrated following transfection of the virulent D. theifolia strain JYC1-6. In summary, DtBRV1 exhibits unique molecular traits and contributes to our understanding of mycovirus diversity. IMPORTANCE A novel botybirnavirus, infecting the tea plant pathogen Didymella theifolia and tentatively named Didymella theifolia botybirnavirus 1 (DtBRV1), together with an additional double-stranded RNA (dsRNA), was characterized. DtBRV1 comprises two dsRNAs (1 and 2) encapsidated in isometric virions, while dsRNA3 is a satellite. The satellite represents a unique specimen since it contains a duplicated region and has high similarity to the two botybirnavirus dsRNAs, supporting the notion that it most likely originated from a deficient genomic component. The biological characteristics of DtBRV1 were further determined. With their unique molecular traits, DtBRV1 and its related dsRNA expand our understanding of virus diversity, taxonomy, and evolution.

Mycoviruses are widely present in all major groups of fungi but those in entomopathogenic Metarhizium spp. remain understudied. In this investigation, a novel double-stranded (ds) RNA virus is isolated from Metarhizium majus and named Metarhizium majus partitivirus 1 (MmPV1). The complete genome sequence of MmPV1 comprises two monocistronic dsRNA segments (dsRNA 1 and dsRNA 2), which encode an RNA-dependent RNA polymerase (RdRp) and a capsid protein (CP), respectively. MmPV1 is classified as a new member of the genus Gammapartitivirus in the family Partitiviridae based on phylogenetic analysis. As compared to an MmPV1-free strain, two isogenic MmPV1-infected single-spore isolates were compromised in terms of conidiation, and tolerance to heat shock and UV-B irradiation, while these phenotypes were accompanied by transcriptional suppression of multiple genes involved in conidiation, heat shock response and DNA damage repair. MmPV1 attenuated fungal virulence since infection resulted in reduced conidiation, hydrophobicity, adhesion, and cuticular penetration. Additionally, secondary metabolites were significantly altered by MmPV1 infection, including reduced production of triterpenoids, and metarhizins A and B, and increased production of nitrogen and phosphorus compounds. However, expression of individual MmPV1 proteins in M. majus had no impact on the host phenotype, suggesting insubstantive links between defective phenotypes and a single viral protein. These findings indicate that MmPV1 infection decreases M. majus fitness to its environment and its insect-pathogenic lifestyle and environment through the orchestration of the host conidiation, stress tolerance, pathogenicity, and secondary metabolism.

Abstract To date, viroids have been found to naturally infect only plants, resulting in substantial losses for some crops. Whether viroids or viroid‐like RNAs naturally infect non‐plant hosts remains unknown. Here the existence of a set of exogenous, single‐stranded circular RNAs, ranging in size from 157 to 450 nucleotides, isolated from the fungus Botryosphaeria dothidea and nominated B. dothidea RNAs (BdcRNAs) is reported. BdcRNAs replicate autonomously in the nucleus via a rolling‐circle mechanism following a symmetric pathway. BdcRNA infection induces symptoms, because BdcRNAs can apparently modulate, to different degrees, specific biological traits (e.g., alter morphology, decrease growth rate, attenuate virulence, and increase or decrease tolerance to osmotic and oxidative stress) of the host fungus. Overall, BdcRNAs have genome characteristics similar to those of viroids and exhibit pathogenic effects on fungal hosts. It is proposed that these novel fungus infecting RNAs should be termed mycoviroids. BdcRNA(s) may be considered additional inhabitants at the frontier of life in terms of genomic complexity, and represent a new class of acellular entities endowed with regulatory functions, and novel epigenomic carriers of biological information.

Background: The Mediterranean diet (MD) exerts a protective effect against cancer development and progression; however, the evaluation of its impact on gastric cancer still remains quite scarce. The present study aims to evaluate the association of MD adherence during the lifespan with disease progression characteristics, lifestyle factors and overall survival in gastric carcinoma patients. Methods: This is an observational, cross-sectional study conducted on 186 gastric cancer patients followed up for a median time interval of 57 months or until death due to cancer disease. Tumor histopathological characteristics were retrieved from patients’ medical records, while validated questionnaires assessing, immediately after the time of diagnosis, health-related quality of life, physical activity levels, sleep quality, depression, anxiety and MD adherence during the lifespan were used. Results: Higher MD adherence during the lifespan was significantly associated with younger patients (p = 0.0106), regular smoking (p < 0.0001), abnormal BMI status (p < 0.0001), intestinal-type gastric carcinoma (p = 0.0111), high tumor histopathological grade (p < 0.0001) and earlier disease stage (p < 0.0001). Moreover, patients with elevated MD adherence during their lifespan showed significantly better health-related quality of life (p < 0.0001), higher physical activity levels (p < 0.0001), more adequate sleep quality (p < 0.0001) and lower prevalence of depression (p = 0.0003) and anxiety (p = 0.0006) compared to those with reduced MD adherence. In multiple regression analysis, elevated MD compliance during the lifespan was independently correlated with longer overall patient survival after adjustment for several confounders (Cox regression analysis, p = 0.0001). Conclusions: Higher MD adherence during the lifespan was associated with less advanced tumor histopathology characteristics and favorable mental and physical lifestyle factors. Moreover, higher MD adherence during the lifespan was also independently correlated with longer overall survival in gastric carcinoma patients. Thus, adopting a healthy dietary pattern like the MD during the lifespan may act as a preventive agent in combination with a healthy lifestyle against gastric cancer development and progression.

Summary: CRISPR-associated Rossmann fold (CARF) domain signaling underpins modulation of CRISPR-Cas nucleases; however, the RtcR CARF domain controls expression of two conserved RNA repair enzymes, cyclase RtcA and ligase RtcB. Here, we demonstrate that RtcAB are required for RtcR-dependent transcription activation and directly bind to RtcR CARF. RtcAB catalytic activity is not required for complex formation with CARF, but is essential yet not sufficient for RtcRAB-dependent transcription activation, implying the need for an additional RNA repair-dependent activating signal. This signal differs from oligoadenylates, a known ligand of CARF domains, and instead appears to originate from the translation apparatus: RtcB repairs a tmRNA that rescues stalled ribosomes and increases translation elongation speed. Taken together, our data provide evidence for an expanded range for CARF domain signaling, including the first evidence of its control via in trans protein-protein interactions, and a feed-forward mechanism to regulate RNA repair required for a functioning translation apparatus.

ABSTRACT Phenotypic heterogeneity in clonal bacterial batch cultures has been shown for a range of bacterial systems; however, the molecular origins of such heterogeneity and its magnitude are not well understood. Under conditions of extreme low-nitrogen stress in the model diazotroph Klebsiella oxytoca, we found remarkably high heterogeneity of nifHDK gene expression, which codes for the structural genes of nitrogenase, one key enzyme of the global nitrogen cycle. This heterogeneity limited the bulk observed nitrogen-fixing capacity of the population. Using dual-probe, single-cell RNA fluorescent in situ hybridization, we correlated nifHDK expression with that of nifLA and glnK-amtB, which code for the main upstream regulatory components. Through stochastic transcription models and mutual information analysis, we revealed likely molecular origins for heterogeneity in nitrogenase expression. In the wild type and regulatory variants, we found that nifHDK transcription was inherently bursty, but we established that noise propagation through signaling was also significant. The regulatory gene glnK had the highest discernible effect on nifHDK variance, while noise from factors outside the regulatory pathway were negligible. Understanding the basis of inherent heterogeneity of nitrogenase expression and its origins can inform biotechnology strategies seeking to enhance biological nitrogen fixation. Finally, we speculate on potential benefits of diazotrophic heterogeneity in natural soil environments. IMPORTANCE Nitrogen is an essential micronutrient for both plant and animal life and naturally exists in both reactive and inert chemical forms. Modern agriculture is heavily reliant on nitrogen that has been “fixed” into a reactive form via the energetically expensive Haber-Bosch process, with significant environmental consequences. Nitrogen-fixing bacteria provide an alternative source of fixed nitrogen for use in both biotechnological and agricultural settings, but this relies on a firm understanding of how the fixation process is regulated within individual bacterial cells. We examined the cell-to-cell variability in the nitrogen-fixing behavior of Klebsiella oxytoca, a free-living bacterium. The significance of our research is in identifying not only the presence of marked variability but also the specific mechanisms that give rise to it. This understanding gives insight into both the evolutionary advantages of variable behavior as well as strategies for biotechnological applications.

Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) affects Aspergillus fumigatus Af293’s growth in vitro, iron metabolism, resistance in intermicrobial competition with Pseudomonas aeruginosa, resistance to osmotic stress, and resistance to the chitin synthase inhibitor nikkomycin Z. Here, we show that response to high temperature, Congo Red-induced stress, and hydrogen peroxide are also dependent on the viral infection status of A. fumigatus. AfuPmV-1- infected Af293 was more susceptible than virus-free Af293 to growth inhibition by high temperature, hydrogen peroxide, Congo Red exposure, and nutrient restriction. Increased resistance of virus-free fungus was observed when cultures were started from conidia but, in the case of high temperature and hydrogen peroxide, not when cultures were started from hyphae. This indicates that the virus impairs the stress response during the growth phase of germination of conidia and development into hyphae. In conclusion, our work indicates that AfuPmV-1 infection in A. fumigatus impairs host responses to stress, as shown by exposure to high temperature, oxidative stress such as hydrogen peroxide, and some cell wall stresses, as shown by exposure to Congo Red (in agreement with our previous observations using nikkomycin Z) and nutrient restriction.

Transcription noise in bacteria is often attributed to burstiness, but the mechanisms are unclear. Here, the authors show that the transition from low to high expression can be regulated via burst size or burst frequency, depending on the mode of transcription initiation determined by different sigma factors.

Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) weakens the resistance of biofilms of common A. fumigatus reference strain Af293 in intermicrobial competition with Pseudomonas aeruginosa, and sensitizes A. fumigatus for antifungal effects of nikkomycin Z. We compared the sensitivity of two virus-infected (VI) and one virus-free (VF) Af293 strains to hypertonic salt. Salt stress impairs the growth of VI and VF at all times; VF control growth always exceeds VI, and VF growth in salt always exceeds VI. Since VF growth exceeds VI in the presence and absence of salt, we also examined growth in salt as a percentage of control growth. Initially, as a percentage of control, VI exceeded VF, but at 120 h VF began to exceed VI consistently even by this measure; thus, at that time the growth of VF in salt surges in relation to control growth, or, alternatively, its growth in salt persists compared to the relative inhibition of VI. In summary, virus infection impairs the response of A. fumigatus to several different stresses, including hypertonic salt.

Abstract Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin‐regulating antisense RNA (micF). In bacteria, σ70, together with activators, controls the majority of genes by recruiting RNA polymerase (RNAP) to the promoter regions. RNAP and σ70 form a holoenzyme that recognizes ‐35 and ‐10 promoter DNA consensus sites. Many activators bind upstream from the holoenzyme and can be broadly divided into two classes. RamA acts as a class I activator on acrA and class II activator on micF, respectively. The authors present biochemical and structural data on RamA in complex with RNAP‐σ70 at the two promoters and the data reveal the molecular basis for how RamA assembles and interacts with core RNAP and activates transcription that contributes to antibiotic resistance. Further, comparing with CAP/TAP complexes reveals common and activator‐specific features in activator binding and uncovers distinct roles of the two C‐terminal domains of RNAP α subunit.

Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) weakens resistance of Aspergillus fumigatus common reference strain Af293 biofilms in intermicrobial competition with Pseudomonas aeruginosa. We compared the sensitivity of two infected and one virus-free Af293 strains to antifungal drugs. All three were comparably sensitive to drugs affecting fungal membranes (voriconazole, amphotericin) or cell wall glucan synthesis (micafungin, caspofungin). In contrast, forming biofilms of virus-free Af293 were much more resistant than AfuPmV-1-infected Af293 to nikkomycin Z (NikZ), a drug inhibiting chitin synthase. The IC50 for NikZ on biofilms was between 3.8 and 7.5 µg/mL for virus-free Af293 and 0.94–1.88 µg/mL for infected strains. The IC50 for the virus-free A. fumigatus strain 10AF was ~2 µg/mL in most experiments. NikZ also modestly affected the planktonic growth of infected Af293 more than the virus-free strain (MIC 50%, 2 and 4 µg/mL, respectively). Virus-free Af293 biofilm showed increased metabolism, and fungus growing as biofilm or planktonically showed increased growth compared to infected; these differences do not explain the resistance of the virus-free fungus to NikZ. In summary, AfuPmV-1 infection sensitized A. fumigatus to NikZ, but did not affect response to drugs commonly used against A. fumigatus infection. Virus infection had a greater effect on NikZ inhibition of biofilm than planktonic growth.

Alternaria alternata virus 1 (AaV1) has been identified in the saprophytic fungus Alternaria alternata strain EGS 35–193. AaV1 has four genomic double-stranded (ds)RNA segments (dsRNA1–4) packaged in isometric particles. The 3' end of each coding strand is polyadenylated (36–50nt), but the presence of a cap structure at each 5' end has not previously been investigated. Here, we have characterized the AaV1 genome and found that it has unique features among the mycoviruses. We confirmed the existence of cap structures on the 5' ends of the AaV1 genomic dsRNAs using RNA dot blots with anti-cap antibodies and the oligo-capping method. Polyclonal antibodies against purified AaV1 particles specifically bound to an 82kDa protein, suggesting that this protein is the major capsid component. Subsequent Edman degradation indicated that the AaV1 dsRNA3 segment encodes the major coat protein. Two kinds of defective AaV1 dsRNA2, which is 2,794bp (844 aa) in length when intact, appeared in EGS 35–193 during subculturing, as confirmed by RT-PCR and northern hybridization. Sequence analysis revealed that one of the two defective dsRNA2s contained a 231bp deletion, while the other carried both the 231bp deletion and an additional 465bp deletion in the open reading frame. Both deletions occurred in-frame, resulting in predicted proteins of 767 aa and 612 aa. The fungal isolates carrying virions with the defective dsRNA2s showed impaired growth and abnormal pigmentation. To our best knowledge, AaV1 is the first dsRNA virus to be identified with both 5' cap and 3'poly(A) structures on its genomic segments, as well as the specific deletions of dsRNA2.

Phoma stem canker (blackleg) is one of the most important diseases of winter oilseed rape (Brassica napus) worldwide and is caused by a complex that comprises at least two species: Leptosphaeria maculans and L. biglobosa. Screening a panel of field Leptosphaeria isolates from B. napus for the presence of mycoviruses revealed the presence of a novel double-stranded RNA quadrivirus in L. biglobosa and no viruses in L. maculans. Following elimination of the mycovirus, virus-infected and virus-free isogenic lines of L. biglobosa were created. A direct comparison of the growth and virulence of these isogenic lines illustrated that virus infection caused hypervirulence and resulted in induced systemic resistance toward L. maculans in B. napus following lower leaf preinoculation with the virus-infected isolate. Analysis of the plant transcriptome suggests that the presence of the virus leads to subtle alterations in metabolism and plant defenses. For instance, transcripts involved in carbohydrate and amino acid metabolism are enriched in plants treated with the virus-infected isolate, while pathogenesis-related proteins, chitinases and WRKY transcription factors are differentially expressed. These results illustrate the potential for deliberate inoculation of plants with hypervirulent L. biglobosa to decrease the severity of Phoma stem canker later in the growing season.[Graphic: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Free-living nitrogen-fixing bacteria can improve growth yields of some non-leguminous plants and, if enhanced through bioengineering approaches, have the potential to address major nutrient imbalances in global crop production by supplementing inorganic nitrogen fertilisers. However, nitrogen fixation is a highly resource-costly adaptation and is de-repressed only in environments in which sources of reduced nitrogen are scarce. Here we investigate nitrogen fixation (nif) gene expression and nitrogen starvation response signaling in the model diazotroph Klebsiella oxytoca (Ko) M5a1 during ammonium depletion and the transition to growth on atmospheric N2. Exploratory RNA-sequencing revealed that over 50% of genes were differentially expressed under diazotrophic conditions, among which the nif genes are among the most highly expressed and highly upregulated. Isotopically labelled QconCAT standards were designed for multiplexed, absolute quantification of Nif and nitrogen-stress proteins via multiple reaction monitoring mass spectrometry (MRM-MS). Time-resolved Nif protein concentrations were indicative of bifurcation in the accumulation rates of nitrogenase subunits (NifHDK) and accessory proteins. We estimate that the nitrogenase may account for more than 40% of cell protein during diazotrophic growth and occupy approximately half the active ribosome complement. The concentrations of free amino acids in nitrogen-starved cells were insufficient to support the observed rates of Nif protein expression. Total Nif protein accumulation was reduced 10-fold when the NifK protein was truncated and nitrogenase catalysis lost (nifK1–1203), implying that reinvestment of de novo fixed nitrogen is essential for further nif expression and a complete diazotrophy transition. Several amino acids accumulated in non-fixing ΔnifLA and nifK1–1203 mutants, while the rest remained highly stable despite prolonged N starvation. Monitoring post-translational uridylylation of the PII-type signaling proteins GlnB and GlnK revealed distinct nitrogen regulatory roles in Ko M5a1. GlnK uridylylation was persistent throughout the diazotrophy transition while a ΔglnK mutant exhibited significantly reduced Nif expression and nitrogen fixation activity. Altogether, these findings highlight quantitatively the scale of resource allocation required to enable the nitrogen fixation adaptation to take place once underlying signaling processes are fulfilled. Our work also provides an omics-level framework with which to model nitrogen fixation in free-living diazotrophs and inform rational engineering strategies.

In the original publication [...]

Polymycoviridae is a growing family of mycoviruses whose members typically have non-conventional capsids and multi-segmented, double-stranded (ds) RNA genomes. Beauveria bassiana polymycovirus (BbPmV) 1 is known to enhance the growth and virulence of its fungal host, the entomopathogenic ascomycete and popular biological control agent B. bassiana. Here we report the complete sequence of BbPmV-3, which has six genomic dsRNA segments. Phylogenetic analysis of RNA-dependent RNA polymerase (RdRp) protein sequences revealed that BbPmV-3 is closely related to the partially sequenced BbPmV-2 but not BbPmV-1. Nevertheless, both BbPmV-3 and BbPmV-1 have similar effects on their respective host isolates ATHUM 4946 and EABb 92/11-Dm, affecting pigmentation, sporulation, and radial growth. Production of conidia and radial growth are significantly enhanced in virus-infected isolates as compared to virus-free isogenic lines on Czapek-Dox complete and minimal media that contain sucrose and sodium nitrate. However, this polymycovirus-mediated effect on growth is dependent on the carbon and nitrogen sources available to the host fungus. Both BbPmV-3 and BbPmV-1 increase growth of ATHUM 4946 and EABb 92/11-Dm when sucrose is replaced by lactose, trehalose, glucose, or glycerol, while the effect is reversed on maltose and fructose. Similarly, both BbPmV-3 and BbPmV-1 decrease growth of ATHUM 4946 and EABb 92/11-Dm when sodium nitrate is replaced by sodium nitrite, potassium nitrate, or ammonium nitrate. In conclusion, the effects of polymycoviruses on B. bassiana are at least partially mediated via its metabolic pathways.

The plant pathogen Pseudomonas syringae pv. tomato DC3000 uses a type III secretion system (T3SS) to transfer effector proteins into the host. The expression of T3SS proteins is controlled by the HrpL σ factor. Transcription of hrpL is σ54-dependent and bacterial enhancer-binding proteins HrpR and HrpS coactivate the hrpL promoter. The HrpV protein imposes negative control upon HrpR and HrpS through direct interaction with HrpS. HrpG interacts with HrpV and relieves such negative control. The sequence alignments across Hrp group I-type plant pathogens revealed conserved HrpV and HrpG amino acids. To establish structure–function relationships in HrpV and HrpG, either truncated or alanine substitution mutants were constructed. Key functional residues in HrpV and HrpG are found within their C-terminal regions. In HrpG, L101 and L105 are indispensable for the ability of HrpG to directly interact with HrpV and suppress HrpV-dependent negative regulation of HrpR and HrpS. In HrpV, L108 and G110 are major determinants for interactions with HrpS and HrpG. We propose that mutually exclusive binding of HrpS and HrpG to the same binding site of HrpV governs a transition from negative control to activation of the HrpRS complex leading to HrpL expression and pathogenicity of P. syringae.

Bacteriophage T7 infects Escherichia coli and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics.

A virus-free (VF) A. fumigatus isolate has been shown to be resistant in competition with Pseudomonas as compared to the isogenic line infected with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1), and this phenotype was apparently related to alterations in iron metabolism. Here we investigated further the mechanisms underpinning this phenotype. The extracellular siderophore profiles of five isogenic VF and virus-infected (VI) strains were sampled at 24, 31, 48, 54, and 72 h in submerged cultures and quantitatively examined by liquid chromatography and mass spectrometry. Intracellular profiles of conidia and cultures at the stationary growth phase were defined. VF A. fumigatus demonstrated the best fitness represented by the fastest onset of its exponential growth when grown on an iron-limited mineral medium. The exponential phase and transitional production phase of the extracellular triacetylfusarinine C (TafC) were achieved at 24 and 31 h, respectively, contrary to VI strains, which acted more slowly. As a result, the TafC reservoir was consumed sooner in the VF strain. Additionally, the VF strain had lower ferricrocin and higher hydroxyferricrocin content in the pellet during the stationary phase. All of these differences were significant (Kruskal–Wallis, p < 0.01). In our study, the siderophore reservoir of a VF strain was consumed sooner, improving the fitness of the VF strain in competition with P. aeruginosa.

Aspergillus and Pseudomonas compete in nature, and are the commonest bacterial and fungal pathogens in some clinical settings, such as the cystic fibrosis lung. Virus infections of fungi occur naturally. Effects on fungal physiology need delineation. A common reference Aspergillus fumigatus strain, long studied in two (of many) laboratories, was found infected with the AfuPmV-1 virus. One isolate was cured of virus, producing a virus-free strain. Virus from the infected strain was purified and used to re-infect three subcultures of the virus-free fungus, producing six fungal strains, otherwise isogenic. They were studied in intermicrobial competition with Pseudomonasaeruginosa. Pseudomonas culture filtrates inhibited forming or preformed Aspergillus biofilm from infected strains to a greater extent, also seen when Pseudomonas volatiles were assayed on Aspergillus. Purified iron-chelating Pseudomonas molecules, known inhibitors of Aspergillus biofilm, reproduced these differences. Iron, a stimulus of Aspergillus, enhanced the virus-free fungus, compared to infected. All infected fungal strains behaved similarly in assays. We show an important consequence of virus infection, a weakening in intermicrobial competition. Viral infection may affect the outcome of bacterial–fungal competition in nature and patients. We suggest that this occurs via alteration in fungal stress responses, the mechanism best delineated here is a result of virus-induced altered Aspergillus iron metabolism.

Hu-antigen R (HuR) is an RNA-binding posttranscriptional regulator that belongs to the Hu/ELAV family. HuR expression levels are modulated by a variety of proteins, microRNAs, chemical compounds, or the microenvironment, and in turn, HuR affects mRNA stability and translation of various genes implicated in breast cancer formation, progression, metastasis, and treatment. The aim of the present review is to critically summarize the role of HuR in breast cancer development and its potential as a prognosticator and a therapeutic target. In this aspect, all the existing English literature concerning HuR expression and function in breast cancer cell lines, in vivo animal models, and clinical studies is critically presented and summarized. HuR modulates many genes implicated in biological processes crucial for breast cancer formation, growth, and metastasis, whereas the link between HuR and these processes has been demonstrated directly in vitro and in vivo. Additionally, clinical studies reveal that HuR is associated with more aggressive forms of breast cancer and is a putative prognosticator for patients' survival. All the above indicate HuR as a promising drug target for cancer therapy; nevertheless, additional studies are required to fully understand its potential and determine against which types of breast cancer and at which stage of the disease a therapeutic agent targeting HuR would be more effective.

The cosmopolitan insect-pathogenic fungus and popular biocontrol agent Beauveria bassiana can be used to control Anopheles mosquito populations and restrict the spread of malaria, the deadliest vector-borne infectious disease in the world caused by the protozoan parasite Plasmodium. Here, we establish that infection with a double-stranded (ds)RNA mycovirus, Beauveria bassiana polymycovirus (BbPmV)-1, significantly reduces B. bassiana virulence against A. coluzzii, the main vector of malaria. The BbPmV-1-mediated hypovirulence can be at least partially attributed to slow fungal growth on the mosquitos. Analysis of the dual next-generation sequencing of the B. bassiana and A. coluzzii transcriptomes provided insight into the molecular mechanisms of the BbPmV-1-mediated effects. BbPmV-1-free B. bassiana has a wide impact on the A. coluzzii transcriptome, affecting immunity and metabolism, and led to the identification of novel immune response proteins. BbPmV-1 regulates the gene expression profile of its fungal host, directing the use of available resources towards sporulation and suppressing the mosquito immune system. Additionally, BbPmV-1-infected and -free B. bassiana strains differentially modulate mosquito gut microbiota; the former reduces the bacterial genus Elizabethkingia and the latter Serratia. Co-transfection of mosquitos with B. bassiana and P. berghei revealed a reduction of ookinetes in the presence of BbPmV-1, potentially due to the upregulation of a mycotoxin. Finally, BbPmV-1-mediated hypovirulence is at least partially dependent on the A. coluzzii RNAi pathway, and silencing of the dicer-2 gene restores virulence. Taken together, our data clearly demonstrate the crucial role of mycovirus infection in mediating B. bassiana virulence against A. coluzzii and suggest that BbPmV-1 protects A. coluzzii from B. bassiana, the mosquito’s own immune system, potentially harmful gut microbiota, and Plasmodium parasites.

This article describes reproducibility of a single-step automated β-galactosidase, and the equivalence of its data to the traditional assay (“Experiments in Molecular Genetics” [1]). This was done via a pairwise comparison of both methods using strains with Miller Unit [MU] values ranging from 0 to over 2000. The data presented in this article is associated with the research article entitled “A single-step method for mid to high throughput β-galactosidase assays in Escherichia coli using a microplate reader” [2]. Keywords: LacZ, B-galactosidase, β-Galactosidase, B-Gal, β-Gal, Microplate reader

Fungi, similar to all species, are susceptible to viral infection. Aspergillus is arguably the most well studied fungal genus because of its medical, ecological and economical significance. Mycoviruses were initially detected in Aspergillus species almost 50 years ago and the field continues to be active today with ground-breaking discoveries. The aim of the present review is to cover the scientific progress in all aspects of mycovirology as exemplified by Aspergillus-focused research. Initially an overview of the population studies illustrating the presence of mycoviruses in numerous important Aspergillus species, such as A. niger, A. flavus, and A. fumigatus with be presented. Moreover the intricacies of mycovirus transmission, both inter- and intra-species, will be discussed together with the methodologies used to investigate viral dispersion in a laboratory setting. Subsequently, the genomic features of all molecularly characterized mycoviruses to date will be analyzed in depth. These include members of established viral families, such as Partitiviridae, Chrysoviridae and Totiviridae, but also more recent, novel discoveries that led to the proposal of new viral families, such as Polymycoviridae, Alternaviridae and, in the context of the present review, Exartaviridae. Finally, the major issue of phenotypic effects of mycoviral infection on the host is addressed, including aflatoxin production in A. flavus, together with growth and virulence in A. fumigatus. Although the molecular mechanisms behind these phenomena are yet to be elucidated, recent studies suggest that by implication, RNA silencing may be involved.

Banana trees, citrus fruit trees, pome fruit trees, grapevines, mango trees, and stone fruit trees are major fruit trees cultured worldwide and correspond to nearly 90% of the global production of woody fruit trees. In light of the above, the present manuscript summarizes the viruses that infect the major fruit trees, including their taxonomy and morphology, and highlights selected viruses that significantly affect fruit production, including their genomic and biological features. The results showed that a total of 163 viruses, belonging to 45 genera classified into 23 families have been reported to infect the major woody fruit trees. It is clear that there is higher accumulation of viruses in grapevine (80/163) compared to the other fruit trees (each corresponding to less than 35/163), while only one virus species has been reported infecting mango. Most of the viruses (over 70%) infecting woody fruit trees are positive-sense single-stranded RNA (+ssRNA), and the remainder belong to the -ssRNA, ssRNA-RT, dsRNA, ssDNA and dsDNA-RT groups (each corresponding to less than 8%). Most of the viruses are icosahedral or isometric (79/163), and their diameter ranges from 16 to 80 nm with the majority being 25−30 nm. Cross-infection has occurred in a high frequency among pome and stone fruit trees, whereas no or little cross-infection has occurred among banana, citrus and grapevine. The viruses infecting woody fruit trees are mostly transmitted by vegetative propagation, grafting, and root grafting in orchards and are usually vectored by mealybug, soft scale, aphids, mites or thrips. These viruses cause adverse effects in their fruit tree hosts, inducing a wide range of symptoms and significant damage, such as reduced yield, quality, vigor and longevity.

Approximately a year ago, when I accepted the offer to act as a Guest Editor for the Special Issue ‘Mycoviruses’ organised by the MDPI journal Viruses, I dared not expect that ‘Mycoviruses’ would include such a large number of manuscripts [...]

The entomopathogenic fungus Beauveria bassiana has a wide host range and is used as a biocontrol agent against arthropod pests. Mycoviruses have been described in phytopathogenic fungi while in entomopathogenic fungi their presence has been reported only rarely. Here we show that 21.3% of a collection of B. bassiana isolates sourced from worldwide locations, harbor dsRNA elements. Molecular characterization of these elements revealed the prevalence of mycoviruses belonging to the Partitiviridae and Totiviridae families, the smallest reported virus to date, belonging to the family Narnaviridae, and viruses unassigned to a family or genus. Of particular importance is the discovery of members of a newly proposed family Polymycoviridae in B. bassiana. Polymycoviruses, previously designated as tetramycoviruses, consist of four non-conventionally encapsidated capped dsRNAs. The presence of additional non-homologous genomic segments in B. bassiana polymycoviruses and other fungi illustrates the unprecedented dynamic nature of the viral genome. Finally, a comparison of virus-free and virus-infected isogenic lines derived from an exemplar B. bassiana isolate revealed a mild hypervirulent effect of mycoviruses on the growth of their host isolate and on its pathogenicity against the greater wax moth Galleria mellonella, highlighting for the first time the potential of mycoviruses as enhancers of biocontrol agents.

Here we report the molecular characterisation of a novel dsRNA virus isolated from the filamentous, plant pathogenic fungus Leptosphaeria biglobosa and known to cause significant alterations to fungal pigmentation and growth and to result in hypervirulence, as illustrated by comparisons between virus-infected and -cured isogenic fungal strains. The virus forms isometric particles approximately 40–45 nm in diameter and has a quadripartite dsRNA genome structure with size ranges of 4.9 to 4 kbp, each possessing a single ORF. Sequence analysis of the putative proteins encoded by dsRNAs 1–4, termed P1–P4, respectively, revealed modest similarities to the amino acid sequences of equivalent proteins predicted from the nucleotide sequences of known and suspected members of the family Quadriviridae and for that reason the virus was nominated Leptosphaeria biglobosa quadrivirus-1 (LbQV-1). Sequence and phylogenetic analysis using the P3 sequence, which encodes an RdRP, revealed that LbQV-1 was most closely related to known and suspected quadriviruses and monopartite totiviruses rather than other quadripartite mycoviruses including chrysoviruses and alternaviruses. Of the remaining encoded proteins, LbQV-1 P2 and P4 are structural proteins but the function of P1 is unknown. We propose that LbQV-1 is a novel member of the family Quadriviridae.

The use of mycoviruses to manipulate the virulence of entomopathogenic fungi employed as biocontrol agents may lead to the development of novel methods to control attacks by insect pests. Such approaches are urgently required, as existing agrochemicals are being withdrawn from the market due to environmental and health concerns. The aim of this work is to investigate the presence and diversity of mycoviruses in large panels of entomopathogenic fungi, mostly from Spain and Denmark. In total, 151 isolates belonging to the genera Beauveria, Metarhizium, Lecanicillium, Purpureocillium, Isaria, and Paecilomyces were screened for the presence of dsRNA elements and 12 Spanish B. bassiana isolates were found to harbor mycoviruses. All identified mycoviruses belong to three previously characterised species, the officially recognised Beauveria bassiana victorivirus 1 (BbVV-1) and the proposed Beauveria bassiana partitivirus 2 (BbPV-2) and Beauveria bassiana polymycovirus 1 (BbPmV-1); individual B. bassiana isolates may harbor up to three of these mycoviruses. Notably, these mycovirus species are under distinct selection pressures, while recombination of viral genomes increases population diversity. Phylogenetic analysis of the RNA-dependent RNA polymerase gene sequences revealed that the current population structure in Spain is potentially a result of both vertical and horizontal mycovirus transmission. Finally, pathogenicity experiments using the Mediterranean fruit fly Ceratitis capitata showed no direct correlation between the presence of any particular mycovirus and the virulence of the B. bassiana isolates, but illustrated potentially interesting isolates that exhibit relatively high virulence, which will be used in more detailed virulence experimentation in the future.