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Abstract Although genome-wide A-to-I editing mediated by adenosine-deaminase-acting-on-tRNA (ADAT) occurs during sexual reproduction in the presence of stage-specific cofactors, RNA editing is not known to occur during vegetative growth in filamentous fungi. Here we identified 33 A-to-I RNA editing events in vegetative hyphae of Fusarium graminearum and functionally characterized one conserved hyphal-editing site. Similar to ADAT-mediated editing during sexual reproduction, majority of hyphal-editing sites are in coding sequences and nonsynonymous, and have strong preference for U at -1 position and hairpin loops. Editing at TA437G, one of the hyphal-specific editing sites, is a premature stop codon correction (PSC) event that enables CHE1 gene to encode a full-length zinc fingertranscription factor. Manual annotations showed that this PSC site is conserved in CHE1 orthologs from closely-related Fusarium species. Whereas the che1 deletion and CHE1 TAA (G438 to A) mutants had no detectable phenotype, the CHE1 TGG (A437 to G) mutant was defective in hyphal growth, conidiation, sexual reproduction, and plant infection. However, the CHE1 TGG mutant was increased in tolerance against oxidative stress and editing of TA437G in CHE1 was stimulated by H2O2 treatment in F. graminearum. These results indicate that fixation of the premature stop codon in CHE1 has a fitness cost on normal hyphal growth and reproduction but provides a benefit to tolerance against oxidative stress. Taken together, A-to-I editing events, although rare (not genome-wide), occur during vegetative growth and editing in CHE1 plays a role in response to oxidative stress in F. graminearum and likely in other fungal pathogens.

Abstract Polyamines, such as putrescine, spermidine, and spermine, are crucial for plant defense against both abiotic and biotic stresses. Putrescine is also known as a significant inducer of deoxynivalenol (DON) production in Fusarium graminearum, the primary causal agent of Fusarium head blight (FHB). However, the impact of other polyamines on DON production and whether modifying polyamine biosynthesis could improve wheat resistance to FHB are currently unknown. In this study, we demonstrate that key precursor components of putrescine synthesis, including arginine, ornithine, and agmatine, can induce DON production, albeit to a lesser extent than putrescine in trichothecene biosynthesis-inducing (TBI) culture under the same total nitrogen conditions. Intriguingly, spermidine and spermine, downstream products of putrescine in the polyamine biosynthesis pathway, do not induce DON production under the same conditions. Additionally, externally applying either spermidine or spermine to wheat heads significantly reduces the diseased spikelet number caused by F. graminearum. Furthermore, our results show that overexpression of the wheat spermidine synthase (SPDS) gene TaSPDS-7D1 significantly enhances the spermidine content and wheat resistance to FHB. In addition, the TaSPDS-7D1-overexpressing line OE3 exhibited a 1000-grain weight and plant height increase compared to the wild type. Our findings reveal that overexpression of the spermidine synthase gene can enhance wheat resistance to FHB without compromising wheat yield.

Abstract A-to-I mRNA editing in animals is mediated by ADARs, but the mechanism underlying sexual stage-specific A-to-I mRNA editing in fungi remains unknown. Here, we show that the eukaryotic tRNA-specific heterodimeric deaminase FgTad2-FgTad3 is responsible for A-to-I mRNA editing in Fusarium graminearum. This editing capacity relies on the interaction between FgTad3 and a sexual stage-specific protein called Ame1. Although Ame1 orthologs are widely distributed in fungi, the interaction originates in Sordariomycetes. We have identified key residues responsible for the FgTad3-Ame1 interaction. The expression and activity of FgTad2-FgTad3 are regulated through alternative promoters, alternative translation initiation, and post-translational modifications. Our study demonstrates that the FgTad2-FgTad3-Ame1 complex can efficiently edit mRNA in yeasts, bacteria, and human cells, with important implications for the development of base editors in therapy and agriculture. Overall, this study uncovers mechanisms, regulation, and evolution of RNA editing in fungi, highlighting the role of protein-protein interactions in modulating deaminase function.

Fusarium head blight (FHB), mainly caused by the fungal pathogen Fusarium graminearum, is one of the most destructive wheat diseases. Besides directly affecting the yield, the mycotoxin residing in the kernel greatly threatens the health of humans and livestock. Xinong 979 (XN979) is a widely cultivated wheat elite with high yield and FHB resistance. However, its resistance mechanism remains unclear. In this study, we studied the expression of genes involved in plant defense in XN979 by comparative transcriptomics. We found that the FHB resistance in XN979 consists of two lines of defense. The first line of defense, which is constitutive, is knitted via the enhanced basal expression of lignin and jasmonic acid (JA) biosynthesis genes. The second line of defense, which is induced upon F. graminearum infection, is contributed by the limited suppression of photosynthesis and the struggle of biotic stress-responding genes. Meanwhile, the effective defense in XN979 leads to an inhibition of fungal gene expression, especially in the early infection stage. The formation of the FHB resistance in XN979 may coincide with the breeding strategies, such as selecting high grain yield and lodging resistance traits. This study will facilitate our understanding of wheat–F. graminearum interaction and is insightful for breeding FHB-resistant wheat.

Abstract Deoxynivalenol (DON) is the most frequently detected mycotoxin in cereal grains and processed food or feed. Two transcription factors, Tri6 and Tri10, are essential for DON biosynthesis in Fusarium graminearum. In this study we conduct stranded RNA-seq analysis with tri6 and tri10 mutants and show that Tri10 acts as a master regulator controlling the expression of sense and antisense transcripts of TRI6 and over 450 genes with diverse functions. TRI6 is more specific for regulating TRI genes although it negatively regulates TRI10. Two other TRI genes, including TRI5 that encodes a key enzyme for DON biosynthesis, also have antisense transcripts. Both Tri6 and Tri10 are essential for TRI5 expression and for suppression of antisense-TRI5. Furthermore, we identify a long non-coding RNA (named RNA5P) that is transcribed from the TRI5 promoter region and is also regulated by Tri6 and Tri10. Deletion of RNA5P by replacing the promoter region of TRI5 with that of TRI12 increases TRI5 expression and DON biosynthesis, indicating that RNA5P suppresses TRI5 expression. However, ectopic constitutive overexpression of RNA5P has no effect on DON biosynthesis and TRI5 expression. Nevertheless, elevated expression of RNA5P in situ reduces TRI5 expression and DON production. Our results indicate that TRI10 and TRI6 regulate each other’s expression, and both are important for suppressing the expression of RNA5P, a long non-coding RNA with cis-acting inhibitory effects on TRI5 expression and DON biosynthesis in F. graminearum.

ABSTRACT Anthracnose diseases caused by Colletotrichum species are among the most common fungal diseases. These symptoms typically manifest as dark, sunken lesions on leaves, stems, and fruit. In China, mango anthracnose seriously affects fruit yield and quality. Genome sequencing of several species shows the presence of mini-chromosomes. These are thought to contribute to virulence, but their formation and activity remain to be fully elucidated. Here, we assembled 17 Colletotrichum genomes (16 isolated from mango plus one from persimmon) through PacBio long-read sequencing. Half of the assembled scaffolds had telomeric repeats at both ends indicating full-length chromosomes. Based on comparative genomics analysis at interspecies and intraspecies levels, we identified extensive chromosomal rearrangements events. We analyzed mini-chromosomes of Colletotrichum spp. and found large variation among close relatives. In C. fructicola, homology between core chromosomes and mini-chromosomes suggested that some mini-chromosomes were generated by recombination of core chromosomes. In C. musae GZ23-3, we found 26 horizontally transferred genes arranged in clusters on mini-chromosomes. In C. asianum FJ11-1, several potential pathogenesis-related genes on mini-chromosomes were upregulated, especially in strains with highly pathogenic phenotypes. Mutants of these upregulated genes showed obvious defects in virulence. Our findings provide insights into the evolution and potential relationships to virulence associated with mini-chromosomes. IMPORTANCE Colletotrichum is a cosmopolitan fungal genus that seriously affects fruit yield and quality of many plant species. Mini-chromosomes have been found to be related to virulence in Colletotrichum. Further examination of mini-chromosomes can help us elucidate some pathogenic mechanisms of Colletotrichum. In this study, we generated novel assemblies of several Colletotrichum strains. Comparative genomic analyses within and between Colletotrichum species were conducted. We then identified mini-chromosomes in our sequenced strains systematically. The characteristics and generation of mini-chromosomes were investigated. Transcriptome analysis and gene knockout revealed pathogenesis-related genes located on mini-chromosomes of C. asianum FJ11-1. This study represents the most comprehensive investigation of chromosome evolution and potential pathogenicity of mini-chromosomes in the Colletotrichum genus.

Abstract Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is one of the most devastating diseases in wheat and barley worldwide. In addition to causing severe yield losses, F. graminearum produces deoxynivalenol (DON), a trichothecene mycotoxin which is harmful to human health and serves as an important virulence factor. Currently, changes in global climate and tillage systems have made FHB epidemics more frequent and severe. During the past decade, considerable efforts have been deployed to reveal the pathogenic mechanisms of F. graminearum, identify resistance genes in wheat, and breed FHB-resistant varieties. In this review, we highlight recent advances in FHB pathogenesis, F. graminearum-wheat interaction, and wheat defense mechanisms. This review contains four main sections: (1) signal sensing and transduction associated with the pathogenesis of F. graminearum; (2) regulation and functions of DON during wheat infection; (3) roles of F. graminearum-secreted enzymes and effectors in facilitating pathogen infection of wheat; (4) wheat components involved in interactions with F. graminearum. A comprehensive understanding of the molecular interactions between F. graminearum and wheat will contribute to the development of novel and efficient strategies for controlling FHB.

Abstract Application‐oriented assembly of two‐dimensional nanosheets with uniform nanochannels is critical for fabricating sophisticated, high‐performance membranes for water treatment. However, fabricating the desired membranes by a simple, fast, and effective method is a challenge as most of the previous methods are based on batch processes rather than a continuous roll‐to‐roll process. Here, a simple Meyer rod‐coating approach to continuously fabricate large‐size and flat MXene membranes at a scale up to 5 m is introduced. This study demonstrated that a high MXene concentration, above 10 mg mL−1, is critical in processability due to the desired viscosity, surface tension, and viscoelastic properties. The as‐made MXene membranes show that shearing and solutal‐Marangoni flow can considerably improve the ordering of the stacked MXene nanoflakes. Thus, the rod‐coated MXene membranes demonstrate a smaller surface roughness and interlayer distance compared to the MXene membranes fabricated by the most commonly vacuum‐assisted filtration. The rod‐costed MXene membranes show superior performance in dye and mono/divalent cation separation. The proposed roll‐to‐roll Meyer rod‐coating method can also be used to fabricate MXene‐based composites, such as MXene/carbon nanotubes and MXene/polymer, using the inks containing high concentration MXene and other desired compositions. This roll‐to‐roll method will promote an industry‐level fabrication and application of MXene‐based membranes.

Abstract Mitogen-activated protein kinase (MAPK) cascades are activated by external stimuli and convert signals to cellular changes. Individual MAPKs have been characterized in a number of plant pathogenic fungi for their roles in pathogenesis and responses to biotic or abiotic stresses. However, mutants deleted of all the MAPK genes have not been reported in filamentous fungi. To determine the MAPK-less effects in a fungal pathogen, in this study we generated and characterized mutants deleted of all three MAPK genes in the wheat scab fungus Fusarium graminearum. The Gpmk1 mgv1 Fghog1 triple mutants had severe growth defects and was non-pathogenic. It was defective in infection cushion formation and DON production. Conidiation was reduced in the triple mutant, which often produced elongated conidia with more septa than the wild-type conidia. The triple mutant was blocked in sexual reproduction due to the loss of female fertility. Lack of any MAPKs resulted in an increased sensitivity to various abiotic stress including cell wall, osmotic, oxidative stresses, and phytoalexins, which are likely related to the defects of the triple mutant in environmental adaptation and plant infection. The triple mutant also had increased sensitivity to the biocontrol bacterium Bacillus velezensis and fungus Clonostachys rosea. In co-incubation assays with B. velezensis, the Gpmk1 mgv1 Fghog1 mutant had more severe growth limitation than the wild type and was defective in conidium germination and germ tube growth. In confrontation assays, the triple mutant was defective in defending against mycoparasitic activities of C. rosea and the latter could grow over the mutant but not wild-type F. graminearum. RNA-seq and metabolomics analyses showed that the MAPK triple mutant was altered in the expression of many ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes and the accumulation of metabolites related to arachidonic acid, linoleic acid, and alpha-linolenic acid metabolisms. Overall, as the first study on mutants deleted of all three MAPKs in fungal pathogens, our results showed that although MAPKs are not essential for growth and asexual reproduction, the Gpmk1 mgv1 Fghog1 triple mutant was blocked in plant infection and sexual reproductions. It also had severe defects in responses to various abiotic stresses and bacterial- or fungal-fungal interactions.

ABSTRACT Adenosine-to-inosine (A-to-I) RNA editing independent of adenosine deaminase acting on RNA (ADAR) enzymes was discovered in fungi recently, and shown to be crucial for sexual reproduction. However, the underlying mechanism for editing is unknown. Here, we combine genome-wide comparisons, proof-of-concept experiments, and machine learning to decipher cis-regulatory elements of A-to-I editing in Fusarium graminearum. We identified plenty of RNA primary sequences and secondary structural features that affect editing specificity and efficiency. Although hairpin loop structures contribute importantly to editing, unlike in animals, the primary sequences have more profound influences on editing than secondary structures. Nucleotide preferences at adjacent positions of editing sites are the most important features, especially preferences at the −1 position. Unexpectedly, besides the number of positions with preferred nucleotides, the combination of preferred nucleotides with depleted ones at different positions are also important for editing. Some cis-sequence features have distinct importance for editing specificity and efficiency. Machine learning models built from diverse sequence and secondary structural features can accurately predict genome-wide editing sites but not editing levels, indicating that the cis-regulatory principle of editing efficiency is more complex than that of editing specificity. Nevertheless, our model interpretation provides insights into the quantitative contribution of each feature to the prediction of both editing sites and levels. We found that efficient editing of FG3G34330 transcripts depended on the full-length RNA molecule, suggesting that additional RNA structural elements may also contribute to editing efficiency. Our work uncovers multidimensional cis-regulatory elements important for A-to-I RNA editing in F. graminearum, helping to elucidate the fungal editing mechanism. IMPORTANCE A-to-I RNA editing is a new epigenetic phenomenon that is crucial for sexual reproduction in fungi. Deciphering cis-regulatory elements of A-to-I RNA editing can help us elucidate the editing mechanism and develop a model that accurately predicts RNA editing. In this study, we discovered multiple RNA sequence and secondary structure features important for A-to-I editing in Fusarium graminearum. We also identified the cis-sequence features with distinct importance for editing specificity and efficiency. The potential importance of full-length RNA molecules for editing efficiency is also revealed. This study represents the first comprehensive investigation of the cis-regulatory principles of A-to-I RNA editing in fungi.

ObjectiveThis study aimed to analyze the cerebrospinal fluid (CSF) parameters affecting the outcomes of patients with tuberculous meningitis (TBM).MethodsThis is a multi-center, retrospective, cohort study involving 81 patients who were diagnosed with TBM and treated in Haihe Clinical College of Tianjin Medical University, Tianjin Medical University General Hospital, and General Hospital of Air Force PLA from January 2016 to December 2019. Baseline data, Glasgow Coma Scale (GCS) score, and clinical presentations of all patients were collected at admission. CSF samples were collected at 48 h, 1, 2, and 3 weeks after admission. CSF lactate, adenosine deaminase, chloride, protein, glucose levels and intracranial pressure were measured. After a follow-up of 16.14 ± 3.03 months, all patients were assessed using the modified Rankin Scale (mRS) and divided into good (mRS scores of 0–2 points) and poor outcome groups (mRS scores of 3–6 points). The differences in patients' baseline data, GCS score, clinical presentations, and levels of CSF parameters detected at 48 h, 1, 2, and 3 weeks after admission between two groups were compared. Statistically significant variables were added to the binary logistic regression model to identify the factors impacting the outcomes of patients with TBM. Receiver operating characteristic (ROC) curve was used to assess the predictive ability of the model.ResultsThe CSF lactate level exhibited a decreasing trend within 3 weeks of admission in the two groups. For the within-group comparison, statistically significant differences in the lactate level was found in both groups between four different time points. A binary logistic regression model revealed that CSF lactate level at 48 h after admission, age, and GSC score on admission were independently associated with the outcomes of patients with TBM. ROC curve analysis showed that the area under the ROC curve (AUC) was 0.786 for the CSF lactate level (48 h), 0.814 for GCS score, and 0.764 for age.ConclusionHigh CSF lactate level at 48 h after admission is one of the important factors for poor outcomes in patients with TBM.

Abstract Background The production of cereal crops is frequently affected by diseases caused by Fusarium graminearum and Magnaporthe oryzae, two devastating fungal pathogens. To improve crop resistance, many studies have focused on understanding the mechanisms of host defense against these two fungi individually. However, our knowledge of the common and different host defenses against these pathogens is very limited. Results In this study, we employed Brachypodium distachyon as a model for cereal crops and performed comparative transcriptomics to study the dynamics of host gene expression at different infection stages. We found that infection with either F. graminearum or M. oryzae triggered massive transcriptomic reprogramming in the diseased tissues. Numerous defense-related genes were induced with dynamic changes during the time course of infection, including genes that function in pattern detection, MAPK cascade, phytohormone signaling, transcription, protein degradation, and secondary metabolism. In particular, the expression of jasmonic acid signaling genes and proteasome component genes were likely specifically inhibited or manipulated upon infection by F. graminearum. Conclusions Our analysis showed that, although the affected host pathways are similar, their expression programs and regulations are distinct during infection by F. graminearum and M. oryzae. The results provide valuable insight into the interactions between B. distachyon and two important cereal pathogens.

Fusarium graminearum is a major fungal pathogen of cereals. Here the authors show that F. graminearum secretes an effector, Osp24, that induces degradation of the wheat TaSnRK1α kinase to promote disease while an orphan wheat protein, TaFROG1, can compete with Osp24 for binding to TaSnRK1α and protect it from degradation

BackgroundTo construct and validate a nomogram for predicting the risk of esophageal fistula in esophageal cancer patients receiving radiotherapy.MethodsA retrospective nested case–control study was performed, in which a total of 81 esophageal fistula patients and 243 controls from 2014 to 2020 in the First Affiliated Hospital of Anhui Medical University were enrolled. Factors included in the nomogram were determined by univariate and multiple logistic regression analysis. The following methods including ROC curve, C-index, calibration curves, Brier score, and decision curve analysis (DCA) were adopted to evaluate this nomogram.ResultsMultivariate logistic regression analysis showed that T4 stage, level 4 stenosis, ulcerative esophageal cancer, prealbumin, and maximum diameters of GTV and NLR were the independent risk factors of esophageal fistula. Accordingly, a nomogram incorporating the aforementioned six parameters was constructed. The AUC was 0.848 (95% CI 0.901–0.895), indicating a high prediction accuracy of this nomogram. Further evaluation of this model showed that the C-index was 0.847, while the bias-corrected C-index after internal validation was 0.833. The Brier score was 0.127. The calibration curves presented good concordance, and the DCA revealed promising clinical application.ConclusionsThe nomogram presents accurate and applicable prediction for the esophageal fistula risk in esophageal cancer patients receiving radiotherapy.

Background: Cachexia is a multifactorial disorder characterized by weight loss and muscle wasting, making up for about 20% of cancer-related death. However, there are no effective drugs to combat cachexia at present.Methods: In this study, the effect of CT26 exosomes on C2C12 myotubes was observed. We compared serum HMGB1 level in cachexia and non-cachexia colon cancer patients. We further explored HMGB1 expression level in CT26 exosome. We added recombinant HMGB1 to C2C12 myotubes to observe the effects of HMGB1 on C2C12 myotubes and detected the expression level of the muscle atrophy-related proteins. Then, we used the HMGB1 inhibitor glycyrrhizin to reverse the effects of HMGB1 on C2C12 myotubes. Finally, HMGB1 inhibitor glycyrrhizin was utilized to relieve cachexia in CT26 cachexia mouse model.Results: Exosomes containing HMGB1 led to muscle atrophy with significantly decreased myotube diameter and increased expression of muscle atrophy-related proteins Atrogin1 and MuRF1. Further, we detected that HMGB1 induced the muscle atrophy mainly via TLR4/NF-κB pathway. Administration of the HMGB1 inhibitor glycyrrhizin could relieve muscle wasting in vitro and attenuate the progression of cachexia in vivo.Conclusion: These findings demonstrate the cachectic role of HMGB1, whether it is soluble form of HMGB1 or secreted from tumor cells as part of exosomes. HMGB1 inhibitor glycyrrhizin might be a promising drug in colon cancer cachexia.

Fusarium head blight (FHB), caused by the fungal pathogen Fusarium graminearum, is a destructive disease worldwide. Ascospores are the primary inoculum of F. graminearum, and sexual reproduction is a critical step in its infection cycle. In this study, we characterized the functions of FgCsn12. Although the ortholog of FgCsn12 in budding yeast was reported to have a direct interaction with Csn5, which served as the core subunit of the COP9 signalosome, the interaction between FgCsn12 and FgCsn5 was not detected through the yeast two-hybrid assay. The deletion of FgCSN12 resulted in slight defects in the growth rate, conidial morphology, and pathogenicity. Instead of forming four-celled, uninucleate ascospores, the Fgcsn12 deletion mutant produced oval ascospores with only one or two cells and was significantly defective in ascospore discharge. The 3′UTR of FgCsn12 was dispensable for vegetative growth but essential for sexual reproductive functions. Compared with those of the wild type, 1204 genes and 2240 genes were up- and downregulated over twofold, respectively, in the Fgcsn12 mutant. Taken together, FgCsn12 demonstrated an important function in the regulation of ascosporogenesis in F. graminearum.

Abstract Background Cancer cachexia is a cancer-induced multifactorial debilitating syndrome directly accounting for 20% of cancer deaths without effective therapeutic approaches. It is extremely urgent to explore effective anti-cachexia drugs to ameliorate muscle and fat loss in cachexia patients. Methods Lewis lung carcinoma bearing C57BL/6 mice were applied as the animal model to examine the therapeutic effect of Coix seed oil (CSO) on cancer cachexia. The food intake and body weight change were monitored every 3 days throughout the experiment. The IL-6 and TNF-α levels in serum were detected by ELISA assay. Several key proteins involved in muscle wasting and fat lipolysis were tested by Western blot to identify the potential mechanism of CSO. Results Administration of CSO through gavage significantly prevented body weight loss and ameliorated systemic inflammation without affecting food intake and tumor size. The weight and histological morphology of gastrocnemius muscle and epididymal adipose tissue in CSO-treated mice were also improved. In mechanism, we found that CSO decreased the expression of MuRF1 and the ratio of phospho-p65 (Ser536) to p65 in muscle tissue. Meanwhile, cancer-induced activation of HSL and AMPK was also inhibited by CSO administration. Conclusion Coix seed oil exerts an anti-cachexia pharmaceutical effect by counteracting muscle and adipose tissue loss most likely through regulating NF-κB-MuRF1 and AMPK-HSL pathway.

Sodium/glucose cotransporter 1 (SGLT1), an essential active glucose transport protein that helps maintain high intracellular glucose levels, was previously shown to interact with epidermal growth factor receptor (EGFR); the SGLT1–EGFR interaction maintains intracellular glucose levels to promote survival of cancer cells. Here, we explore the role of SGLT1 in triple‐negative breast cancer (TNBC), which is the most aggressive type of breast cancer. We performed TCGA analysis coupled to in vitro experiments in TNBC cell lines as well as in vivo xenografts established in the mammary fat pad of female nude mice. Tissue microarrays of TNBC patients with information of clinical–pathological parameters were also used to investigate the expression and function of SGLT1 in TNBC. We show that high levels of SGLT1 are associated with greater tumour size in TNBC. Knockdown of SGLT1 compromises cell growth in vitro and in vivo. We further demonstrate that SGLT1 depletion results in decreased levels of phospho‐EGFR, and as a result, the activity of downstream signalling pathways (such as AKT and ERK) is inhibited. Hence, targeting SGLT1 itself or the EGFR–SGLT1 interaction may provide novel therapeutics against TNBC.

Abstract Plasmodiophora brassicae is an important biotrophic eukaryotic plant pathogen and a member of the rhizarian protists. This biotrophic pathogen causes clubroot in cruciferous plants via novel intracellular mechanisms that are markedly different from those of other biotrophic organisms. To date, genomes from six single spore isolates of P. brassicae have been sequenced. An accurate description of the evolutionary status of this biotrophic protist, however, remains lacking. Here, we determined the draft genome of the P. brassicae ZJ-1 strain. A total of 10,951 protein-coding genes were identified from a 24.1 Mb genome sequence. We applied a comparative genomics approach to prove the Rhizaria supergroup is an independent branch in the eukaryotic evolutionary tree. We also found that the GPCR signaling pathway, the versatile signal transduction to multiple intracellular signaling cascades in response to extracellular signals in eukaryotes, is significantly enriched in P. brassicae-expanded and P. brassicae-specific gene sets. Additionally, treatment with a GPCR inhibitor relieved the symptoms of clubroot and significantly suppressed the development of plasmodia. Our findings suggest that GPCR signal transduction pathways play important roles in the growth, development, and pathogenicity of P. brassicae.

Histone acetylation, balanced by histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes, affects dynamic transitions of chromatin structure to regulate transcriptional accessibility. However, little is known about the interplay between HAT and HDAC complexes in Fusarium graminearum, a causal agent of Fusarium Head Blight (FHB) that uniquely contains chromosomal regions enriched for house-keeping or infection-related genes. In this study, we identified the ortholog of the human inhibitor of growth (ING1) gene in F. graminearum (FNG1) and found that it specifically interacts with the FgEsa1 HAT of the NuA4 complex. Deletion of FNG1 led to severe growth defects and blocked conidiation, sexual reproduction, DON production, and plant infection. The fng1 mutant was normal in H3 acetylation but significantly reduced in H4 acetylation. A total of 34 spontaneous suppressors of fng1 with faster growth rate were isolated. Most of them were still defective in sexual reproduction and plant infection. Thirty two of them had mutations in orthologs of yeast RPD3, SIN3, and SDS3, three key components of the yeast Rpd3L HDAC complex. Four mutations in these three genes were verified to suppress the defects of fng1 mutant in growth and H4 acetylation. The rest two suppressor strains had a frameshift or nonsense mutation in a glutamine-rich hypothetical protein that may be a novel component of the FgRpd3 HDAC complex in filamentous fungi. FgRpd3, like Fng1, localized in euchromatin. Deletion of FgRPD3 resulted in severe growth defects and elevated H4 acetylation. In contract, the Fgsds3 deletion mutant had only a minor reduction in growth rate but FgSIN3 appeared to be an essential gene. RNA-seq analysis revealed that 48.1% and 54.2% of the genes with altered expression levels in the fng1 mutant were recovered to normal expression levels in two suppressor strains with mutations in FgRPD3 and FgSDS3, respectively. Taken together, our data showed that Fng1 is important for H4 acetylation as a component of the NuA4 complex and functionally related to the FgRpd3 HDAC complex for transcriptional regulation of genes important for growth, conidiation, sexual reproduction, and plant infection in F. graminearum.

Abstract Background Cachexia is a multifactorial syndrome that is highly prevalent in advanced cancer patients and leads to progressive functional impairments. The classification of cachexia stages is essential for diagnosing and treating cachexia. However, there is a lack of simple tools with good discrimination for classifying cachexia stages. Therefore, our study aimed to develop a clinically applicable cachexia staging score (CSS) and validate its discrimination of clinical outcomes for different cachexia stages. Methods Advanced cancer patients were enrolled in our study. A CSS comprising the following five components was developed: weight loss, a simple questionnaire of sarcopenia (SARC‐F), Eastern Cooperative Oncology Group, appetite loss, and abnormal biochemistry. According to the CSS, patients were classified into non‐cachexia, pre‐cachexia, cachexia, and refractory cachexia stages, and clinical outcomes were compared among the four groups. Results Of the 297 participating patients, data from 259 patients were ultimately included. Based on the CSS, patients were classified into non‐cachexia (n = 69), pre‐cachexia (n = 68), cachexia (n = 103), and refractory cachexia (n = 19) stages. Patients with more severe cachexia stages had lower skeletal muscle indexes (P = 0.002 and P = 0.004 in male and female patients, respectively), higher prevalence of sarcopenia (P = 0.017 and P = 0.027 in male and female patients, respectively), more severe symptom burden (P

Abstract Ascospores are the primary inoculum in the wheat scab fungus Fusarium graminearum that was recently shown to have sexual stage-specific A-to-I RNA editing. One of the genes with premature-stop-codons requiring A-to-I editing to encode full-length functional proteins is AMD1 that encodes a protein with a major facilitator superfamily (MFS) domain. Here, we characterized the functions of AMD1 and its UAG to UGG editing event. The amd1 deletion mutant was normal in growth and conidiation but defective in ascospore discharge due to the premature breakdown of its ascus wall in older perithecia, which is consistent with the specific expression of AMD1 at later stages of sexual development. Expression of the wild-type or edited allele of AMD1 but not un-editable allele rescued the defects of amd1 in ascospore discharge. Furthermore, Amd1-GFP localized to the ascus membrane and Amd1 orthologs are only present in ascocarp-forming fungi that physically discharge ascospores. Interestingly, deletion of AMD1 results in the up-regulation of a number of genes related to transporter activity and membrane functions. Overall, these results indicated that Amd1 may play a critical role in maintaining ascus wall integrity during ascus maturation, and A-to-I editing of its transcripts is important for ascospore discharge in F. graminearum.

A Correction to this paper has been published: https://doi.org/10.1038/s41419-021-03530-0

Fusarium graminearum, the major causal agent of Fusarium head blight (FHB), causes serious wheat yield losses and a threat to human and animal health. The main efforts to combat the disease are the research of pathogenesis mechanisms and breeding for disease resistance plants. The efficiency of these actions could be evaluated by reliable inoculation assay, which is performed by accurate and repeatable inoculation methods. Hence, a standard procedure of effective wheat inoculation should improve the accuracy of pathogenicity evaluation. Here, we present a protocol for wheat spike inoculation with fungal conidial suspensions or fungus agar discs. These methods show highly reproducibility and accuracy on wheat infection experiment in laboratory conditions.

Trichothecene mycotoxins, such as deoxynivalenol (DON) produced by the fungal pathogen, Fusarium graminearum, are not only important for plant infection but are also harmful to human and animal health. Trichothecene targets the ribosomal protein Rpl3 that is conserved in eukaryotes. Hence, a self-defense mechanism must exist in DON-producing fungi. It is reported that TRI (trichothecene biosynthesis) 101 and TRI12 are two genes responsible for self-defense against trichothecene toxins in Fusarium. In this study, however, we found that simultaneous disruption of TRI101 and TRI12 has no obvious influence on DON resistance upon exogenous DON treatment in F. graminearum, suggesting that other mechanisms may be involved in self-defense. By using RNA-seq, we identified 253 genes specifically induced in DON-treated cultures compared with samples from cultures treated or untreated with cycloheximide, a commonly used inhibitor of eukaryotic protein synthesis. We found that transporter genes are significantly enriched in this group of DON-induced genes. Of those genes, 15 encode major facilitator superfamily transporters likely involved in mycotoxin efflux. Significantly, we found that genes involved in the metabolism of gamma-aminobutyric acid (GABA), a known inducer of DON production in F. graminearum, are significantly enriched among the DON-induced genes. The GABA biosynthesis gene PROLINE UTILIZATION 2-2 (PUT2-2) is downregulated, while GABA degradation genes are upregulated at least twofold upon treatment with DON, resulting in decreased levels of GABA. Taken together, our results suggest that transporters influencing DON efflux are important for self-defense and that GABA mediates the balance of DON production and self-defense in F. graminearum.

Ascospores act as the primary inoculum of Fusarium graminearum, which causes the destructive disease Fusarium head blight (FHB), or scab. MicroRNAs (miRNAs) have been reported in the F. graminearum vegetative stage, and Fgdcl2 is involved in microRNA-like RNA (milRNA) biogenesis but has no major impact on vegetative growth, abiotic stress or pathogenesis. In the present study, we found that ascospore discharge was decreased in the Fgdcl1 deletion mutant, and completely blocked in the double-deletion mutant of Fgdcl1 and Fgdcl2. Besides, more immature asci were observed in the double-deletion mutant. Interestingly, the up-regulated differentially expressed genes (DEGs) common to ΔFgdcl1 and ΔFgdcl1/2 were related to ion transmembrane transporter and membrane components. The combination of small RNA and transcriptome sequencing with bioinformatics analysis predicted 143 novel milRNAs in wild-type perithecia, and 138 of these milRNAs partly or absolutely depended on Fgdcl1, while only 5 novel milRNAs were still obtained in the Fgdcl1 and Fgdcl2 double-deletion mutant. Furthermore, 117 potential target genes were predicted. Overall, Fgdcl1 and Fgdcl2 genes were partly functionally redundant in ascospore discharge and perithecium-specific milRNA generation in F. graminearum, and these perithecium-specific milRNAs play potential roles in sexual development.

Coniothyrium minitans is an important mycoparasite of Sclerotinia sclerotiorum. In addition, it also produces small amounts of antifungal substances. ZS-1TN1812, an abnormal mutant, was originally screened from a T-DNA insertional library. This mutant showed abnormal growth phenotype and could significantly inhibit the growth of S. sclerotiorum when dual-cultured on a PDA plate. When spraying the filtrate of ZS-1TN1812 on the leaves of rapeseed, S. sclerotiorum infection was significantly inhibited, suggesting that the antifungal substances produced by this mutant were effective on rapeseed leaves. The thermo-tolerant antifungal substances could specifically suppress the growth of S. sclerotiorum, but could not significantly suppress the growth of another fungus, Colletotrichum higginsianum. However, C. higginsianum was more sensitive to proteinous antibiotics than S. sclerotiorum. The T-DNA insertion in ZS-1TN1812 activated the expression of CmSIT1, a gene involved in siderophore-mediated iron transport. It was also determined that mutant ZS-1TN1812 produced hypha with high iron levels. In the wild-type strain ZS-1, CmSIT1 was expressed only when in contact with S. sclerotiorum, and consistent overexpression of CmSIT1 showed similar phenotypes as ZS-1TN1812. Therefore, activated expression of CmSIT1 leads to the enhanced antifungal ability, and CmSIT1 is a potential gene for improving the control ability of C. minitans.

In the rice blast fungus Magnaporthe oryzae, the cAMP-PKA pathway regulates surface recognition, appressorium turgor generation, and invasive growth. However, deletion of CPKA failed to block appressorium formation and responses to exogenous cAMP. In this study, we generated and characterized the cpk2 and cpkA cpk2 mutants and spontaneous suppressors of cpkA cpk2 in M. oryzae. Our results demonstrate that CPKA and CPK2 have specific and overlapping functions, and PKA activity is essential for appressorium formation and plant infection. Unlike the single mutants, the cpkA cpk2 mutant was significantly reduced in growth and rarely produced conidia. It failed to form appressoria although the intracellular cAMP level and phosphorylation of Pmk1 MAP kinase were increased. The double mutant also was defective in plant penetration and Mps1 activation. Interestingly, it often produced fast-growing spontaneous suppressors that formed appressoria but were still non-pathogenic. Two suppressor strains of cpkA cpk2 had deletion and insertion mutations in the MoSFL1 transcription factor gene. Deletion of MoSFL1 or its C-terminal 93-aa (MoSFL1ΔCT) was confirmed to suppress the defects of cpkA cpk2 in hyphal growth but not appressorium formation or pathogenesis. We also isolated 30 spontaneous suppressors of the cpkA cpk2 mutant in Fusarium graminearum and identified mutations in 29 of them in FgSFL1. Affinity purification and co-IP assays showed that this C-terminal region of MoSfl1 was essential for its interaction with the conserved Cyc8-Tup1 transcriptional co-repressor, which was reduced by cAMP treatment. Furthermore, the S211D mutation at the conserved PKA-phosphorylation site in MoSFL1 partially suppressed the defects of cpkA cpk2. Overall, our results indicate that PKA activity is essential for appressorium formation and proper activation of Pmk1 or Mps1 in M. oryzae, and phosphorylation of MoSfl1 by PKA relieves its interaction with the Cyc8-Tup1 co-repressor and suppression of genes important for hyphal growth.

Clubroot disease is a serious threat to cruciferous plants worldwide, especially to oilseed rape. However, knowledge on pathogenic molecular mechanisms and host interaction is limited. We presume that the recognition between Arabidopsis thaliana and Plasmodiophora brassicae occurs at the early stage of infection and within a relatively short period. In this study, we demonstrated changes on gene expression and pathways in A. thaliana during early infection with P. brassicae using transcriptome analysis. We identified 1,903 and 1,359 DEGs at 24 and 48 h post-inoculation (hpi), respectively. Flavonoids and the lignin synthesis pathways were enhanced, glucosinolates, terpenoids, and proanthocyanidins accumulated and many hormonal- and receptor-kinase related genes were expressed, caused by P. brassicae infection during its early phase. Therefore, the early interaction between A. thaliana and P. brassicae plays an important role in the entire infection process. The results provide a new contribution to a better understanding of the interaction between host plants and P. brassicae, as well as the development of future measures for the prevention of clubroot.

Clubroot caused by Plasmodiophora brassicae, is a severe disease of cruciferous crops that causes large hypertrophic galls in the roots. The plant microbiome is important for growth promotion and disease suppression. In this study, using 16S rRNA and internal transcribed spacer (ITS) sequencing techniques, we compared the endosphere microbiome of symptomatic and asymptomatic B. napus roots infected with P. brassicae collected from the same natural clubroot field. The results showed that the microbial population and its relative abundance in the asymptomatic roots was far higher than that in the symptomatic roots, and that many microorganisms in asymptomatic roots have biological control and plant growth promotion functions that may be related to clubroot symptoms. These results suggest the importance of the endosphere microbiome in clubroot disease and provide potential bio-control resources for its prevention.

PRP4 encodes the only kinase among the spliceosome components. Although it is an essential gene in the fission yeast and other eukaryotic organisms, the Fgprp4 mutant was viable in the wheat scab fungus Fusarium graminearum. Deletion of FgPRP4 did not block intron splicing but affected intron splicing efficiency in over 60% of the F. graminearum genes. The Fgprp4 mutant had severe growth defects and produced spontaneous suppressors that were recovered in growth rate. Suppressor mutations were identified in the PRP6, PRP31, BRR2, and PRP8 orthologs in nine suppressor strains by sequencing analysis with candidate tri-snRNP component genes. The Q86K mutation in FgMSL1 was identified by whole genome sequencing in suppressor mutant S3. Whereas two of the suppressor mutations in FgBrr2 and FgPrp8 were similar to those characterized in their orthologs in yeasts, suppressor mutations in Prp6 and Prp31 orthologs or FgMSL1 have not been reported. Interestingly, four and two suppressor mutations identified in FgPrp6 and FgPrp31, respectively, all are near the conserved Prp4-phosphorylation sites, suggesting that these mutations may have similar effects with phosphorylation by Prp4 kinase. In FgPrp31, the non-sense mutation at R464 resulted in the truncation of the C-terminal 130 aa region that contains all the conserved Prp4-phosphorylation sites. Deletion analysis showed that the N-terminal 310-aa rich in SR residues plays a critical role in the localization and functions of FgPrp4. We also conducted phosphoproteomics analysis with FgPrp4 and identified S289 as the phosphorylation site that is essential for its functions. These results indicated that FgPrp4 is critical for splicing efficiency but not essential for intron splicing, and FgPrp4 may regulate pre-mRNA splicing by phosphorylation of other components of the tri-snRNP although itself may be activated by phosphorylation at S289.

Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.

Tyrosine kinases (TKs) specifically catalyze the phosphorylation of tyrosine residues in proteins and play essential roles in many cellular processes. Although TKs mainly exist in animals, recent studies revealed that some organisms outside the Opisthokont clade also contain TKs. The fungi, as the sister group to animals, are thought to lack TKs. To better understand the origin and evolution of TKs, it is important to investigate if fungi have TK or TK-related genes. We therefore systematically identified possible TKs across the fungal kingdom by using the profile hidden Markov Models searches and phylogenetic analyses. Our results confirmed that fungi lack the orthologs of animal TKs. We identified a fungi-specific lineage of protein kinases (FslK) that appears to be a sister group closely related to TKs. Sequence analysis revealed that members of the FslK clade contain all the conserved protein kinase sub-domains and thus are likely enzymatically active. However, they lack key amino acid residues that determine TK-specific activities, indicating that they are not true TKs. Phylogenetic analysis indicated that the last common ancestor of fungi may have possessed numerous members of FslK. The ancestral FslK genes were lost in Ascomycota and Ustilaginomycotina and Pucciniomycotina of Basidiomycota during evolution. Most of these ancestral genes, however, were retained and expanded in Agaricomycetes. The discovery of the fungi-specific lineage of protein kinases closely related to TKs helps shed light on the origin and evolution of TKs and also has potential implications for the importance of these kinases in mushroom fungi.

Genome sequence of viruses can contribute greatly to the study of viral evolution, diversity and the interaction between viruses and hosts. Traditional molecular cloning methods for obtaining RNA viral genomes are time-consuming and often difficult because many viruses occur in extremely low titers. DsRNA viruses in the families, Partitiviridae, Totiviridae, Endornaviridae, Chrysoviridae, and other related unclassified dsRNA viruses are generally associated with symptomless or persistent infections of their hosts. These characteristics indicate that samples or materials derived from eukaryotic organisms used to construct cDNA libraries and EST sequencing might carry these viruses, which were not easily detected by the researchers. Therefore, the EST databases may include numerous unknown viral sequences. In this study, we performed in silico cloning, a procedure for obtaining full or partial cDNA sequence of a gene by bioinformatics analysis, using known dsRNA viral sequences as queries to search against NCBI Expressed Sequence Tag (EST) database. From this analysis, we obtained 119 novel virus-like sequences related to members of the families, Endornaviridae, Chrysoviridae, Partitiviridae, and Totiviridae. Many of them were identified in cDNA libraries of eukaryotic lineages, which were not known to be hosts for these viruses. Furthermore, comprehensive phylogenetic analysis of these newly discovered virus-like sequences with known dsRNA viruses revealed that these dsRNA viruses may have co-evolved with respective host supergroups over a long evolutionary time while potential horizontal transmissions of viruses between different host supergroups also is possible. We also found that some of the plant partitiviruses may have originated from fungal viruses by horizontal transmissions. These findings extend our knowledge of the diversity and possible host range of dsRNA viruses and offer insight into the origin and evolution of relevant viruses with their hosts.

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study.

Meiosis is essential for generating genetic diversity and sexual spores, but the regulation of meiosis and ascosporogenesis is not clear in filamentous fungi, in which dikaryotic and diploid cells formed inside fruiting bodies are not free living and independent of pheromones or pheromone receptors. In this study, Gia1, a non-pheromone GPCR (G protein-coupled receptor) with sexual-specific expression in Fusarium graminearum, is found to be essential for ascosporogenesis. The gia1 mutant was normal in perithecium development, crozier formation, and karyogamy but failed to undergo meiosis, which could be partially rescued by a dominant active mutation in GPA1 and activation of the Gpmk1 pathway. GIA1 orthologs have conserved functions in regulating meiosis and ascosporogenesis in Sordariomycetes. GIA1 has a paralog, GIP1, in F. graminearum and other Hypocreales species which is essential for perithecium formation. GIP1 differed from GIA1 in expression profiles and downstream signaling during sexual reproduction. Whereas the C-terminal tail and IR3 were important for intracellular signaling, the N-terminal region and EL3 of Gia1 were responsible for recognizing its ligand, which is likely a protein enriched in developing perithecia, particularly in the gia1 mutant. Taken together, these results showed that GIA1 encodes a non-pheromone GPCR that regulates the entry into meiosis and ascosporogenesis via the downstream Gpmk1 MAP kinase pathway in F. graminearum and other filamentous ascomycetes. [ABSTRACT FROM AUTHOR]