Innovative sustainable bioreactor-in-a-granule formulation of Trichoderma asperelloides.

The advancement of fungal biocontrol agents depends on replacing cereal grains with low-cost agro-industrial byproducts for their economical mass production and development of stable formulations. We propose an innovative approach to develop a rice flour-based formulation of the beneficial biocontrol agent Trichoderma asperelloides CMAA1584 designed to simulate a micro-bioreactor within the concept of full biorefinery process, affording in situ conidiation, extended shelf-life, and effective control of Sclerotinia sclerotiorum, a devastating pathogen of several dicot agricultural crops worldwide. Rice flour is an inexpensive and underexplored byproduct derived from broken rice after milling, capable of sustaining high yields of conidial production through our optimized fermentation-formulation route. Conidial yield was mainly influenced by nitrogen content (0.1% w/w) added to the rice meal coupled with the fermentor type. Hydrolyzed yeast was the best nitrogen source yielding 2.6 × 109 colony-forming units (CFU)/g within 14 days. Subsequently, GControl, GLecithin, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru formulations were obtained by extrusion followed by air-drying and further assessed for their potential to induce secondary sporulation in situ, storage stability, and efficacy against Sclerotinia. GControl, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru stood out with the highest number of CFU after sporulation upon re-hydration on water-agar medium. Shelf-life of formulations GControl and GBentonite remained consistent for > 3 months at ambient temperature, while in GBentonite and GOrganic compost+Break-Thru formulations remained viable for 24 months during refrigerated storage. Formulations exhibited similar efficacy in suppressing the myceliogenic germination of Sclerotinia irrespective of their concentration tested (5 × 104 to 5 × 106 CFU/g of soil), resulting in 79.2 to 93.7% relative inhibition. Noteworthily, all 24-month-old formulations kept under cold storage successfully suppressed sclerotia. This work provides an environmentally friendly bioprocess method using rice flour as the main feedstock to develop waste-free granular formulations of Trichoderma conidia that are effective in suppressing Sclerotinia while also improving biopesticide shelf-life. KEY POINTS: • Innovative "bioreactor-in-a-granule" system for T. asperelloides is devised. • Dry granules of aerial conidia remain highly viable for 24 months at 4 °C. • Effective control of white-mold sclerotia via soil application of Trichoderma-based granules.

Volatilome changes during black truffle (Tuber melanosporum) ontogeny.

The aroma is critical in the reproductive biology of truffles and in their commercial quality. However, previous research has almost exclusively focused on characterizing ripe ascocarps. We characterized the volatilome of the highly-prized black truffle (Tuber melanosporum) ascocarps from July, in an early development stage, to March, in the late harvesting season, and investigated the relationships among aroma, ascocarp growth and morphogenetic development. The aroma profile was analyzed using a head space gas chromatography technique coupled with mass spectrometer. Seventy-one volatile compounds were identified and three development stages were clearly distinguished according to the volatile profile. In unripe ascocarps of July-September, the profile was dominated by methanethiol (19 %), 4-penten-2-ol (11 %) and acetone (11 %), the monthly mean weight of ascocarps ranged 2-20 g, and morphogenetic stages 4-6a were prevalent. In unripe ascocarps of October-December, the most abundant volatiles were 4-penten-2-ol (21 %), methanethiol (20 %) and ethanol (13 %), the monthly mean ascocarp weight ranged 28-43 g, and morphogenetic stages 6a, 6b-c were prevalent. In ripe ascocarps (December-March), the most abundant volatiles were 4-penten-2-ol (17 %), dimethyl sulfide (16 %) and ethanol (10 %), ascocarp weight did not increase significantly, and 6b-c was practically the sole morphogenetic stage. Thirty volatiles were associated to one of these three development stages. Amongst those with higher occurrence, 4-penten-2-ol, dimethyl sulfide, ethyl acetate, 2-pentanol and 2-butanone were associated to ripe truffles, whereas methanethiol, isobutyl isobutyrate, butanedione and 3-methylanisole were associated to unripe truffles.

PARylation of 14-3-3 proteins controls the virulence of Magnaporthe oryzae.

Magnaporthe oryzae is a devastating fungal pathogen that causes the rice blast disease worldwide. The post-translational modification of ADP-ribosylation holds significant importance in various fundamental biological processes. However, the specific function of this modification in M. oryzae remains unknown. This study revealed that Poly(ADP-ribosyl)ation (PARylation) executes a critical function in M. oryzae. M. oryzae Poly(ADP-ribose) polymerase 1 (PARP1) exhibits robust PARylation activity. Disruption of PARylation by PARP1 knock-out or chemical inhibition reveals its involvement in M. oryzae virulence, particularly in appressorium formation. Furthermore, we identified two M. oryzae 14-3-3 proteins, GRF1 and GRF2, as substrates of PARP1. Deletion of GRF1 or GRF2 results in delayed and dysfunctional appressorium, diminished plant penetration, and reduced virulence of the fungus. Biochemical and genetic evidence suggest that PARylation of 14-3-3s is essential for its function in M. oryzae virulence. Moreover, PARylation regulates 14-3-3 dimerization and is required for the activation of the mitogen-activated protein kinases (MAPKs), Pmk1 and Mps1. GRF1 interacts with both Mst7 and Pmk1, and bridges their interaction in a PARylation-dependent manner. This study unveils a distinctive mechanism that PARylation of 14-3-3 proteins controls appressorium formation through MAPK activation, and could facilitate the development of new strategies of rice blast disease control.

Microbial Biotransformation of 1-Methyl-L-tryptophan into Herbicidal Indole Alkaloids by Endophytic Fungus Nigrospora chinensis GGY-3.

Indole alkaloids are privileged secondary metabolites, and their production may be achieved by the microbial biotransformation of tryptophan analogues. By feeding 1-methyl-L-tryptophan (1-MT) into the culture of endophytic Nigrospora chinensis GGY-3, six novel (1-6) and seven known indole alkaloids (7-13) were generated. Their structures were elucidated by means of NMR spectroscopy, experimental electronic circular dichroism (ECD) spectra, and X-ray crystallography analysis. A Friedel-Crafts reaction was proposed as the key reaction responsible for the formation of the new compounds. Racemates 4 and 6 were separated into isomers by chiral HPLC, with their absolute configurations determined by X-ray and ECD calculation. Compounds 3, 4, and 8 display good herbicidal activity against dicotyledon weed Eclipta prostrata, of which 4 and 8 exhibited 88.50% and 100% inhibition rates on the radicle at 200 µg/mL, respectively, a similar effect compared to the positive control penoxsulam.

Antagonistic Mechanism Analysis of Bacillus velezensis JLU-1, a Biocontrol Agent of Rice Pathogen Magnaporthe oryzae.

Magnaporthe oryzae, the causal agent of rice blast, is a fungal disease pathogen. Bacillus spp. have emerged as the most promising biological control agent alternative to chemical fungicides. In this study, the bacterial strain JLU-1 with significant antagonistic activity isolated from the rhizosphere soil of rice was identified as Bacillus velezensis through whole-genome sequencing, average nucleotide identity analysis, and 16S rRNA gene sequencing. Twelve gene clusters for secondary metabolite synthesis were identified in JLU-1. Furthermore, 3 secondary metabolites were identified in JLU-1, and the antagonistic effect of secondary metabolites against fungal pathogens was confirmed. Exposure to JLU-1 reduced the virulence of M. oryzae, and JLU-1 has the ability to induce the reactive oxygen species production of rice and improve the salt tolerance of rice. All of these results indicated that JLU-1 and its secondary metabolites have the promising potential to be developed into a biocontrol agent to control fungal diseases.

Discovery of a Hybrid Molecule with Phytotoxic Activity by Genome Mining, Heterologous Expression, and OSMAC Strategy.

Genome mining in association with the OSMAC (one strain, many compounds) approach provides a feasible strategy to extend the chemical diversity and novelty of natural products. In this study, we identified the biosynthetic gene cluster (BGC) of restricticin, a promising antifungal agent featuring a reactive primary amine, from the fungus Aspergillus sclerotiorum LZDX-33-4 by genome mining. Combining heterologous expression and the OSMAC strategy resulted in the production of a new hybrid product (1), along with N-acetyl-restricticin (2) and restricticinol (3). The structure of 1 was determined by spectroscopic data, including optical rotation and electronic circular dichroism (ECD) calculations, for configurational assignment. Compound 1 represents a fusion of restricticin and phytotoxic cichorin. The biosynthetic pathway of 1 was proposed, in which the condensation of a primary amine of restricticin with a precursor of cichorine was postulated. Compound 1 at 5 mM concentration inhibited the growth of the shoots and roots of Lolium perenne, Festuca arundinacea, and Lactuca sativa with inhibitory rates of 71.3 and 88.7% for L. perenne, 79.4 and 73.0% for F. arundinacea, and 58.2 and 52.9% for L. sativa. In addition, compound 1 at 25 µg/mL showed moderate antifungal activity against Fusarium fujikuroi and Trichoderma harzianum with inhibition rates of 22.6 and 31.6%, respectively. These results suggest that heterologous expression in conjunction with the OSMAC approach provides a promising strategy to extend the metabolite novelty due to the incorporation of endogenous metabolites from the host strain with exogenous compounds, leading to the production of more complex compounds and the acquisition of new physiological functions.

Unravelling the secondary metabolome and biocontrol potential of the recently described species Bacillus nakamurai.

In the prospect of novel potential biocontrol agents, a new strain BDI-IS1 belonging to the recently described Bacillus nakamurai was selected for its strong in vitro antimicrobial activities against a range of bacterial and fungal phytopathogens. Genome mining coupled with metabolomics revealed that BDI-IS1 produces multiple non-ribosomal secondary metabolites including surfactin, iturin A, bacillaene, bacillibactin and bacilysin, together with some some ribosomally-synthesized and post-translationally modified peptides (RiPPs) such as plantazolicin, and potentially amylocyclicin, bacinapeptin and LCI. Reverse genetics further showed the specific involvement of some of these compounds in the antagonistic activity of the strain. Comparative genomics between the five already sequenced B. nakamurai strains showed that non-ribosomal products constitute the core metabolome of the species while RiPPs are more strain-specific. Although the secondary metabolome lacks some key bioactive metabolites found in B. velezensis, greenhouse experiments show that B. nakamurai BDI-IS1 is able to protect tomato and maize plants against early blight and northern leaf blight caused by Alternaria solani and Exserohilum turcicum, respectively, at levels similar to or better than B. velezensis QST713. The reduction of these foliar diseases, following root or leaf application of the bacterial suspension demonstrates that BDI-IS1 can act by direct antibiosis and by inducing plant defence mechanisms. These findings indicate that B. nakamurai BDI-IS1 can be considered as a good candidate for biocontrol of plant diseases prevailing in tropical regions, and encourage further research into its spectrum of activity, its requirements and the conditions needed to ensure its efficacy.

Integrative workflows for the characterization of hydrophobin and cerato-platanin in the marine fungus Paradendryphiella salina.

Hydrophobins (HFBs) and cerato-platanins (CPs) are surface-active extracellular proteins produced by filamentous fungi. This study identified two HFB genes (pshyd1 and pshyd2) and one CP gene (pscp) in the marine fungus Paradendryphiella salina. The proteins PsCP, PsHYD2, and PsHYD1 had molecular weights of 12.70, 6.62, and 5.98 kDa, respectively, with isoelectric points below 7. PsHYD1 and PsHYD2 showed hydrophobicity (GRAVY score 0.462), while PsCP was hydrophilic (GRAVY score - 0.202). Stability indices indicated in-solution stability. Mass spectrometry identified 2,922 proteins, including CP but not HFB proteins. qPCR revealed differential gene expression influenced by developmental stage and substrate, with pshyd1 consistently expressed. These findings suggest P. salina's adaptation to marine ecosystems with fewer hydrophobin genes than other fungi but capable of producing surface-active proteins from seaweed carbohydrates. These proteins have potential applications in medical biocoatings, food industry foam stabilizers, and environmental bioremediation.

De Novo Genome Assembly of Toniniopsis dissimilis (Ramalinaceae, Lecanoromycetes) from Long Reads Shows a Comparatively High Composition of Biosynthetic Genes Putatively Involved in Melanin Synthesis.

Lichens have developed numerous adaptations to optimize their survival in various environmental conditions, largely by producing secondary compounds by the fungal partner. They often have antibiotic properties and are involved in protection against intensive UV radiation, pathogens, and herbivores. To contribute to the knowledge of the arsenal of secondary compounds in a crustose lichen species, we sequenced and assembled the genome of Toniniopsis dissimilis, an indicator of old-growth forests, using Oxford Nanopore Technologies (ONT, Oxford, UK) long reads. Our analyses focused on biosynthetic gene clusters (BGCs) and specifically on Type I Polyketide (T1PKS) genes involved in the biosynthesis of polyketides. We used the comparative genomic approach to compare the genome of T. dissimilis with six other members of the family Ramalinaceae and twenty additional lichen genomes from the database. With only six T1PKS genes, a comparatively low number of biosynthetic genes are present in the T. dissimilis genome; from those, two-thirds are putatively involved in melanin biosynthesis. The comparative analyses showed at least three potential pathways of melanin biosynthesis in T. dissimilis, namely via the formation of 1,3,6,8-tetrahydroxynaphthalene, naphthopyrone, or YWA1 putative precursors, which highlights its importance in T. dissimilis. In addition, we report the occurrence of genes encoding ribosomally synthesized and posttranslationally modified peptides (RiPPs) in lichens, with their highest number in T. dissimilis compared to other Ramalinaceae genomes. So far, no function has been assigned to RiPP-like proteins in lichens, which leaves potential for future research on this topic.

Discovery, Expression, and In Silico Safety Evaluation of Honey Truffle Sweetener, a Sweet Protein Derived from Mattirolomyces terfezioides and Produced by Heterologous Expression in Komagataella phaffii.

Honey truffle sweetener (HTS), a 121 amino acid protein is identified as a high-intensity sweetener found naturally occurring in the Hungarian Sweet Truffle Mattirolomyces terfezioides, an edible mushroom used in regional diets. The protein is intensely sweet, but the truffle is difficult to cultivate; therefore, the protein was systematically characterized, and the gene coding for the protein was expressed in a commonly used host yeast Komagataella phaffii. The heterologously expressed protein maintained the structural characteristics and sweet taste of the truffle. Preliminary safety evaluations for use as a food ingredient were performed on the protein including digestibility and in silico approaches for predicting the allergenicity and toxicity of the protein. HTS is predicted to be nonallergenic, nontoxic, and readily digestible. This protein is readily produced by precision fermentation of the host yeast, making it a potential replacement for both added sugars and small molecule high-intensity sweeteners in food.

N6-methyladenosine RNA methyltransferase CpMTA1 mediates CpAphA mRNA stability through a YTHDF1-dependent m6A modification in the chestnut blight fungus.

In eukaryotic cells, N6-methyladenosine (m6A) is the most prevalent RNA epigenetic modification that plays crucial roles in multiple biological processes. Nevertheless, the functions and regulatory mechanisms of m6A in phytopathogenic fungi are poorly understood. Here, we showed that CpMTA1, an m6A methyltransferase in Cryphonectria parasitica, plays a crucial role in fungal phenotypic traits, virulence, and stress tolerance. Furthermore, the acid phosphatase gene CpAphA was implicated to be a target of CpMTA1 by integrated analysis of m6A-seq and RNA-seq, as in vivo RIP assay data confirmed that CpMTA1 directly interacts with CpAphA mRNA. Deletion of CpMTA1 drastically lowered the m6A level of CpAphA and reduced its mRNA expression. Moreover, we found that an m6A reader protein CpYTHDF1 recognizes CpAphA mRNA and increases its stability. Typically, the levels of CpAphA mRNA and protein exhibited a positive correlation with CpMTA1 and CpYTHDF1. Importantly, site-specific mutagenesis demonstrated that the m6A sites, A1306 and A1341, of CpAphA mRNA are important for fungal phenotypic traits and virulence in C. parasitica. Together, our findings demonstrate the essential role of the m6A methyltransferase CpMTA1 in C. parasitica, thereby advancing our understanding of fungal gene regulation through m6A modification.

Discovery of Two Novel Phthalide Phytotoxins from the Wheat Tan Spot Fungal Pathogen Pyrenophora tritici-repentis.

The Dothideomycete fungal pathogen Pyrenophora tritici-repentis (Ptr) is the causal agent of the tan spot disease of wheat. The proteinaceous necrotrophic effectors ToxA and ToxB are well characterized. A nonproteinaceous effector called ToxC has also been partially characterized. Ptr produces a number of other small molecular weight compounds, but these remain poorly characterized. In this study, two novel compounds, designated ToxE1 and ToxE2, capable of inducing chlorotic symptoms on wheat leaves in a cultivar-specific manner, were purified from Ptr liquid cultures. There is no evidence that these compounds correspond to ToxC. Most isolates produced ToxE1, ToxE2, or both, and both compounds were detected in infected wheat leaves. The structures of both analogues were elucidated by NMR spectroscopy and comprise a phthalide core structure with an amide moiety. We postulate that these compounds have a general phytotoxic effect and may have an ancillary role in disease development.

Rhamnolipids and fengycins interact differently with biomimetic lipid membrane models of Botrytis cinerea and Sclerotinia sclerotiorum: Lipidomics profiles and biophysical studies.

Rhamnolipids (RLs) and Fengycins (FGs) are biosurfactants with very promising antifungal properties proposed to reduce the use of synthetic pesticides in crops. They are amphiphilic molecules, both known to target the plasma membrane. They act differently on Botrytis cinerea and Sclerotinia sclerotiorum, two close Sclerotiniaceae phytopathogenic fungi. RLs are more efficient at permeabilizing S. sclerotiorum, and FGs are more efficient at permeabilizing B. cinerea mycelial cells. To study the link between the lipid membrane composition and the activity of RLs and FGs, we analyzed the lipid profiles of B. cinerea and S. sclerotiorum. We determined that unsaturated or saturated C18 and saturated C16 fatty acids are predominant in both fungi. We also showed that phosphatidylethanolamine (PE), phosphatidic acid (PA), and phosphatidylcholine (PC) are the main phospholipids (in this order) in both fungi, with more PA and less PC in S. sclerotiorum. The results were used to build biomimetic lipid membrane models of B. cinerea and S. sclerotiorum for all-atom molecular dynamic simulations and solid-state NMR experiments to more deeply study the interactions between RLs or FGs with different compositions of lipid bilayers. Distinctive effects are exerted by both compounds. RLs completely insert in all the studied model membranes with a fluidification effect. FGs tend to form aggregates out of the bilayer and insert individually more easily into the models representative of B. cinerea than those of S. sclerotiorum, with a higher fluidification effect. These results provide new insights into the lipid composition of closely related fungi and its impact on the mode of action of very promising membranotropic antifungal molecules for agricultural applications.

Hybrid de novo whole genome assembly of lipopeptide producing novel Bacillus thuringiensis strain NBAIR BtAr exhibiting antagonistic activity against Sclerotium rolfsii.

Bacillus thuringiensis Berliner is recognized as a predominant bioinsecticide but its antifungal potential has been relatively underexplored. A novel B. thuringiensis strain NBAIR BtAr was isolated and morphologically characterized using light and scanning electron microscopy, revealing presence of bipyramidal, cuboidal, and spherical parasporal crystals. The crude form of lipopeptides was extracted from NBAIR BtAr and assessed for its antagonistic activity in vitro, and demonstrated 100 % inhibition of Sclerotium rolfsii Sacc. at a minimum inhibitory concentration of 50 µL of the crude lipopeptide extract per mL of potato dextrose agar. To identify the antagonistic genes responsible, we performed whole genome sequencing of NBAIR BtAr, revealing the presence of circular chromosome of 5,379,913 bp and 175,362 bp plasmid with 36.06 % guanine-cytosine content and 5814 protein-coding sequences. Average nucleotide identity and whole genome phylogenetic analysis delineated the NBAIR BtAr strain as konkukian serovar. Gene ontology analysis revealed associations of 1474, 1323, and 1833 genes with biological processes, molecular function, and cellular components, respectively. Antibiotics & secondary metabolite analysis shell analysis of the whole genome yielded secondary metabolites biosynthetic gene clusters with 100 %, 85 %, 40 %, and 35 % similarity for petrobactin, bacillibactin, fengycin, and paenilamicin, respectively. Also, novel biosynthetic gene clusters, along with antimicrobial genes, including zwittermicin A, chitinase, and phenazines, were identified. Moreover, the presence of eight bacteriophage sequences, 18 genomic islands, insertion sequences, and one CRISPR region indicated prior occurrences of genetic exchange and thus improved competitive fitness of the strain. Overall, the whole genome sequence of NBAIR BtAr is presented, with its taxonomic classification and critical genetic attributes that contribute to its strong antagonistic activity against S. rolfsii.

VmSom1 is essential for growth, development, maintenance of cell wall integrity and virulence in Valsa mali.

Apple Valsa canker disease, caused by Valsa mali Miyabe et Yamada, severely endangers the healthy growth of apple trees. The Som1, located downstream of the cyclic AMP-dependent protein kinase A (cAMP-PKA) pathway, plays crucial roles in the growth, development, morphological differentiation, and virulence of filamentous fungi. In this study, we identify and functionally characterize VmSom1, a homolog of Som1, in Valsa mali. The VmSom1 gene is located on chromosome 12, encoding an 824 amino acid protein. Phylogenetic analysis reveals VmSom1 as a fungal Som1 homolog. The VmSom1 deletion mutants exhibit slower growth rates and fail to produce pycnidia. Additionally, their hyphal growth is significantly inhibited on media containing Calcofluor White, Congo Red, NaCl, and sorbitol. The growth rate of VmSom1 deletion mutants is reduced on maltose, lactose, sucrose and fructose media but increases on glucose medium. Moreover, the mycelial growth rate of the VmSom1 deletion mutant is significantly lower than that of the wild-type strain in peptone, NH4SO4, NaNO3, and no nitrogen. Notably, the distances between the septa increase, and chitin concentration shifts to the hyphal tip in the VmSom1 deletion mutant. Furthermore, compared with the wild-type strain, the VmSom1 deletion mutant exhibits fewer diseased spots on apple fruit and branches. Overall, our findings demonstrate that VmSom1 is involved in regulating the growth and development, colony surface hydrophobicity, osmotic stress, cell wall integrity maintenance, carbon and nitrogen source utilization, septa formation, and virulence of V. mali.

A single septin from a polyextremotolerant yeast recapitulates many canonical functions of septin hetero-oligomers.

Morphological complexity and plasticity are hallmarks of polyextremotolerant fungi. Septins are conserved cytoskeletal proteins and key contributors to cell polarity and morphogenesis. They sense membrane curvature, coordinate cell division, and influence diffusion at the plasma membrane. Four septin homologues are conserved from yeasts to humans, the systems in which septins have been most studied. But there is also a fifth family of opisthokont septins that remain biochemically mysterious. Members of this family, Group 5 septins, appear in the genomes of filamentous fungi, but are understudied due to their absence from ascomycete yeasts. Knufia petricola is an emerging model polyextremotolerant black fungus that can also serve as a model system for Group 5 septins. We have recombinantly expressed and biochemically characterized KpAspE, a Group 5 septin from K. petricola. This septin--by itself in vitro--recapitulates many functions of canonical septin hetero-octamers. KpAspE is an active GTPase that forms diverse homo-oligomers, binds shallow membrane curvatures, and interacts with the terminal subunit of canonical septin hetero-octamers. These findings raise the possibility that Group 5 septins govern the higher-order structures formed by canonical septins, which in K. petricola cells form extended filaments, and provide insight into how septin hetero-oligomers evolved from ancient homomers.

The Tet-on system for controllable gene expression in the rock-inhabiting black fungus Knufia petricola.

Knufia petricola is a black fungus that colonizes sun-exposed surfaces as extreme and oligotrophic environments. As ecologically important heterotrophs and biofilm-formers on human-made surfaces, black fungi form one of the most resistant groups of biodeteriorating organisms. Due to its moderate growth rate in axenic culture and available protocols for its transformation and CRISPR/Cas9-mediated genome editing, K. petricola is used for studying the morpho-physiological adaptations shared by extremophilic and extremotolerant black fungi. In this study, the bacteria-derived tetracycline (TET)-dependent promoter (Tet-on) system was implemented to enable controllable gene expression in K. petricola. The functionality i.e., the dose-dependent inducibility of TET-regulated constructs was investigated by using GFP fluorescence, pigment synthesis (melanin and carotenoids) and restored uracil prototrophy as reporters. The newly generated cloning vectors containing the Tet-on construct, and the validated sites in the K. petricola genome for color-selectable or neutral insertion of expression constructs complete the reverse genetics toolbox. One or multiple genes can be expressed on demand from different genomic loci or from a single construct by using 2A self-cleaving peptides, e.g., for localizing proteins and protein complexes in the K. petricola cell or for using K. petricola as host for the expression of heterologous genes.

The Velvet transcription factor PnVeA regulates necrotrophic effectors and secondary metabolism in the wheat pathogen Parastagonospora nodorum.

The fungus Parastagonospora nodorum causes septoria nodorum blotch on wheat. The role of the fungal Velvet-family transcription factor VeA in P. nodorum development and virulence was investigated here. Deletion of the P. nodorum VeA ortholog, PnVeA, resulted in growth abnormalities including pigmentation, abolished asexual sporulation and highly reduced virulence on wheat. Comparative RNA-Seq and RT-PCR analyses revealed that the deletion of PnVeA also decoupled the expression of major necrotrophic effector genes. In addition, the deletion of PnVeA resulted in an up-regulation of four predicted secondary metabolite (SM) gene clusters. Using liquid-chromatography mass-spectrometry, it was observed that one of the SM gene clusters led to an accumulation of the mycotoxin alternariol. PnVeA is essential for asexual sporulation, full virulence, secondary metabolism and necrotrophic effector regulation.

Biodegradation and adsorption of benzo[a]pyrene by fungi-bacterial coculture.

In this work, the relationship and kinetics of biodegradation and bio-adsorption of benzo[a]pyrene (BaP) by Bacillus and Ascomycota were explored, and the metabolites of BaP under mixed microbial coculture were analyzed and characterized. The results show that BaP was removed through both biosorption and biodegradation. Under mixed microbial coculture, biosorption played a significant role in the early stage and biodegradation was predominant in the later stage. During the removal of BaP, the fungi exhibited remarkable adsorption capabilities for BaP with an adsorption efficiency (AE) of 38.14 %, while bacteria had a best degradation for BaP with a degradation efficiency (DE) of 56.13 %. Under the mixed microbial culture, the removal efficiency (RE) of BaP by the synergistic action of fungi and bacteria reached up to 76.12 % within 15 days. Kinetics analysis illustrated that the degradation and adsorption process of BaP were well fit to the first-order and the pseudo-second-order kinetic models, respectively. The research on the relationship between degradation and adsorption during microbial removal of BaP, as well as the synergistic effects of fungi and bacteria, will provide a theoretical guidance for two or even synthetic microbial communities.

MoRgs3 functions in intracellular reactive oxygen species perception-integrated cAMP signaling to promote appressorium formation in Magnaporthe oryzae.

Regulator of G-protein signaling (RGS) proteins exhibit GTPase-accelerating protein activities to govern G-protein function. In the rice blast fungus Magnaporthe oryzae, there is a family of at least eight RGS and RGS-like proteins (MoRgs1 to MoRgs8), each exhibiting distinct or shared functions in the growth, appressorium formation, and pathogenicity. MoRgs3 recently emerged as one of the crucial regulators that senses intracellular oxidation during appressorium formation. To explore this unique regulatory mechanism of MoRgs3, we identified the nucleoside diphosphate kinase MoNdk1 that interacts with MoRgs3. MoNdk1 phosphorylates MoRgs3 under induced intracellular reactive oxygen species levels, and MoRgs3 phosphorylation is required for appressorium formation and pathogenicity. In addition, we showed that MoRgs3 phosphorylation determines its interaction with MoCrn1, a coronin-like actin-binding protein homolog, which regulates MoRgs3 internalization. Finally, we provided evidence demonstrating that MoRgs3 functions in MoMagA-mediated cAMP signaling to regulate normal appressorium induction. By revealing a novel signal perception mechanism, our studies highlighted the complexity of regulation during the appressorium function and pathogenicity of the blast fungus. IMPORTANCE: We report that MoRgs3 becomes phosphorylated in an oxidative intracellular environment during the appressorium formation stage. We found that this phosphorylation is carried out by MoNdk1, a nucleoside diphosphate kinase. In addition, this phosphorylation leads to a higher binding affinity between MoRgs3 and MoCrn1, a coronin-like actin-binding protein that was implicated in the endocytic transport of several other RGS proteins of Magnaporthe oryzae. We further found that the internalization of MoRgs3 is indispensable for its GTPase-activating protein function toward the Gα subunit MoMagA. Importantly, we characterized how such cellular regulatory events coincide with cAMP signaling-regulated appressorium formation and pathogenicity in the blast fungus. Our studies uncovered a novel intracellular reactive oxygen species signal-transducing mechanism in a model pathogenic fungus with important basic and applied implications.