Comparative transcriptome analysis provides insights into the resistance regulation mechanism and inhibitory effect of fungicide phenamacril in Fusarium asiaticum.

Fusarium asiaticum is a destructive phytopathogenic fungus that causes Fusarium head blight of wheat (FHB), leading to serious yield and economic losses to cereal crops worldwide. Our previous studies indicated that target-site mutations (K216R/E, S217P/L, or E420K/G/D) of Type I myosin FaMyo5 conferred high resistance to phenamacril. Here, we first constructed one sensitive strain H1S and three point mutation resistant strains HA, HC and H1R. Then we conducted comparative transcriptome analysis of these F. asiaticum strains after 1 and 10 µg·mL-1 phenamacril treatment. Results indicated that 2135 genes were differentially expressed (DEGs) among the sensitive and resistant strains. The DEGs encoding ammonium transporter MEP1/MEP2, nitrate reductase, copper amine oxidase 1, 4-aminobutyrate aminotransferase, amino-acid permease inda1, succinate-semialdehyde dehydrogenase, 2, 3-dihydroxybenzoic acid decarboxylase, etc., were significantly up-regulated in all the phenamacril-resistant strains. Compared to the control group, a total of 1778 and 2097 DEGs were identified in these strains after 1 and 10 µg·mL-1 phenamacril treatment, respectively. These DEGs involved in 4-aminobutyrate aminotransferase, chitin synthase 1, multiprotein-bridging factor 1, transcriptional regulatory protein pro-1, amino-acid permease inda1, ATP-dependent RNA helicase DED1, acetyl-coenzyme A synthetase, sarcoplasmic/endoplasmic reticulum calcium ATPase 2, etc., showed significantly down-regulated expression in phenamacril-sensitive strain but not in resistant strains after phenamacril treatment. In addition, cyanide hydratase, mating-type protein MAT-1, putative purine nucleoside permease, plasma membrane protein yro2, etc., showed significantly co-down-regulated expression in all the strains after phenamacril treatment. Taken together, This study provides deep insights into the resistance regulation mechanism and the inhibitory effect of fungicide phenamacril and these new annotated proteins or enzymes are worth for the discovery of new fungicide targets.

Parabacteroides distasonis ameliorates insulin resistance via activation of intestinal GPR109a.

Gut microbiota plays a key role in insulin resistance (IR). Here we perform a case-control study of Chinese adults (ChiCTR2200065715) and identify that Parabacteroides distasonis is inversely correlated with IR. Treatment with P. distasonis improves IR, strengthens intestinal integrity, and reduces systemic inflammation in mice. We further demonstrate that P. distasonis-derived nicotinic acid (NA) is a vital bioactive molecule that fortifies intestinal barrier function via activating intestinal G-protein-coupled receptor 109a (GPR109a), leading to ameliorating IR. We also conduct a bioactive dietary fiber screening to induce P. distasonis growth. Dendrobium officinale polysaccharide (DOP) shows favorable growth-promoting effects on P. distasonis and protects against IR in mice simultaneously. Finally, the reduced P. distasonis and NA levels were also validated in another human type 2 diabetes mellitus cohort. These findings reveal the unique mechanisms of P. distasonis on IR and provide viable strategies for the treatment and prevention of IR by bioactive dietary fiber.

Comprehensive evaluation of the prebiotic properties of Dendrobium officinale polysaccharides, ß-glucan, and inulin during in vitro fermentation via multi-omics analysis.

Dietary fiber is crucial for human health mainly due to its impact on gut microbiota structure and metabolites. This study aimed to investigate the impact of Dendrobium officinale polysaccharides (DOP) and two common fibers (ß-glucan and inulin) on the gut microbiome structure and metabolic profile in vitro. Fecal samples were obtained from 30 healthy volunteers, which were then individually subjected to fermentation with each type of fiber. The results revealed that all fibers were efficiently degraded by gut microbiota, with DOP exhibiting a slower fermentation rate compared to ß-glucan and inulin. The fermentation of all fibers led to a significant increase in the production of short-chain fatty acids (SCFAs) and a reduction in branched-chain fatty acids (BCFAs), sulfides, phenols, and indole. Moreover, the abundance of unclassified Enterobacteriaceae, which was positively correlated with sulfide, phenols, and indole levels, was significantly reduced by all fibers. Additionally, DOP specifically promoted the growth of Parabacteroides, while ß-glucan and inulin promoted the growth of Bifidobacterium and Faecalibacterium. Taken together, these findings enhance our understanding of the role of DOP, ß-glucan, and inulin in modulating gut microbiota and metabolites, where the fermentation with fecal bacteria from different volunteers could provide valuable insights for personalized therapeutic approaches.

Comparative transcriptome analysis reveals the resistance regulation mechanism and fungicidal activity of the fungicide phenamacril in Fusarium oxysporum.

Fusarium oxysporum (Fo) is an important species complex of soil-borne pathogenic fungi that cause vascular wilt diseases of agricultural crops and some opportunistic diseases of humans. The fungicide phenamacril has been extensively reported to have antifungal activity against Fusarium graminearum and Fusarium fujikuroi. In this study, we found that the amino acid substitutions (V151A and S418T) in Type I myosin FoMyo5 cause natural low resistance to phenamacril in the plant pathogenic Fo isolates. Therefore, we compared the transcriptomes of two phenamacril-resistant Fo isolates FoII5, Fo1st and one phenamacril-sensitive isolate Fo3_a after 1 µg/mL phenamacril treatment. Among the 2728 differentially expressed genes (DEGs), 14 DEGs involved in oxidation-reduction processes and MFS transporters, were significantly up-regulated in phenamacril-resistant isolates. On the other hand, 14 DEGs involved in ATP-dependent RNA helicase and ribosomal biogenesis related proteins, showed significantly down-regulated expression in both phenamacril-resistant and -sensitive isolates. These results indicated that phenamacril not only seriously affected the cytoskeletal protein binding and ATPase activity of sensitive isolate, but also suppressed ribosome biogenesis in all the isolates. Hence, this study helps us better understand resistance regulation mechanism and fungicidal activity of phenamacril and provide reference for the development of new fungicides to control Fo.

Gut microbial utilization of xylan and its implication in gut homeostasis and metabolic response.

Xylan as the second most abundant indigestible carbohydrate found in nature attracts great interests of researchers, nutritionist and consumers due to its various health benefits. However, accumulated studies indicate the interactions with gut microbiota greatly affect these benefits, and significant progress has been made over the past few years to understand how microbes utilize xylan at gene level. In this review, we focused on gut xylanolytic microbes and xylan's physico-chemical features, summarized the xylanases needed for complete xylan decomposition, their substrate specificity and the presence in gut microbes, as well as microbial degradation of xylan in single strain mode and cooperation mode. Xylan utilization system were discussed with different phyla. Furthermore, the implications on intestinal homeostasis and metabolic response were reviewed with clinical effects emphasized, and highlight is placed on specific gut microbes and the complexity of xylan structure to provide a clue for the inconsistent results in human studies. CHEMICAL COMPOUNDS: xylan; arabinoxylan, glucuronoxylans; glucuronoarabinoxylans; xylo-oligosaccharides; arabinoxylo-oligosaccharides.

Genome­wide characterization of the Gα subunit gene family in Rosaceae and expression analysis of PbrGPAs under heat stress.

The Gα subunit is an important component of the heterotrimeric G-protein complex and an integral component of several signal transduction pathways. It plays crucial roles in the diverse processes of plant growth and development, including the response to abiotic stress, regulation of root development, involvement in stomatal movement, and participation in hormone responses, which have been well investigated in many species. However, no comprehensive analysis has identified and explored the evolution, expression pattern characteristics and heat stress response of the Gα subunit genes in Rosaceae. In this study, 52 Gα subunit genes were identified in eight Rosaceae species; these genes were divided into three subfamilies (I, II, and III) based on their phylogenetic, conserved motif, and structural characteristics. Whole genome and dispersed duplication events were found to have contributed significantly to the expansion of the Gα subunit gene family, and purifying selection to have played a key role in the evolution of Gα subunit genes. An expression analysis identified some PbrGPA genes that were highly expressed in leaf, root, and fruit, and exhibited diverse spatiotemporal expression models in pear. Under abiotic stress conditions, the mRNA transcript levels of PbrGPA genes were up-regulated in response to high temperature treatment in leaves. Furthermore, three Gα subunit genes were shown to be located in the plasma membrane and nucleus in pear. In conclusion, the study of the Gα subunit gene family will help us to better understand its evolutionary history and expression patterns, while facilitating further investigations into the function of the Gα subunit gene in response to heat stress.

FoMyo5 motor domain substitutions (Val151 to Ala and Ser418 to Thr) cause natural resistance to fungicide phenamacril in Fusarium oxysporum.

Fusarium oxysporum (Fo) is an important genus of filamentous fungi that causes many devastating diseases of agronomical plants and some opportunistic diseases of humans. Previous studies have indicated that mutations in myosin5 acquired resistance to phenamacril in Fusarium graminearum (Fg). Here, we need to determine the residues of FoMyo5 involved in the natural resistance of plant pathogenic Fo strains. Six kinds of Fo reference strains from different hosts were studied. Fungicide susceptibility testing showed that these Fo strains demonstrated different resistance or susceptibility to phenamacril, which is Fusarium-specific antifungal compound, compared with Fg species. When aligned these homologous myosin5 motor domains of these strains, we found that the substitutions (Val151 to Ala and Ser418 to Thr) in FoMyo5 cause natural resistance to phenamacril in the plant pathogenic Fo strains. And we confirmed this result by gene replacement strategy. Such a phenomenon impeded the practical development of this fungicide for controlling vascular wilt diseases.

Ankyrin-Like Protein AnkB Interacts with CatB, Affects Catalase Activity, and Enhances Resistance of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola to Phenazine-1-Carboxylic Acid.

Xanthomonas oryzae pv. oryzae, which causes rice bacterial leaf blight, and Xanthomonas oryzae pv. oryzicola, which causes rice bacterial leaf streak, are important plant-pathogenic bacteria. A member of the adaptor protein family, ankyrin protein, has been investigated largely in humans but rarely in plant-pathogenic bacteria. In this study, a novel ankyrin-like protein, AnkB, was identified in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. The expression of ankB was significantly upregulated when these bacteria were treated with phenazine-1-carboxylic acid (PCA). ankB is located 58 bp downstream of the gene catB (which encodes a catalase) in both bacteria, and the gene expression of catB and catalase activity were reduced following ankB deletion in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Furthermore, we demonstrated that AnkB directly interacts with CatB by glutathione S-transferase (GST) pulldown assays. Deletion of ankB increased the sensitivity of X. oryzae pv. oryzae and X. oryzae pv. oryzicola to H2O2 and PCA, decreased bacterial biofilm formation, swimming ability, and exopolysaccharide (EPS) production, and also reduced virulence on rice. Together our results indicate that the ankyrin-like protein AnkB has important and conserved roles in antioxidant systems and pathogenicity in X. oryzae pv. oryzae and X. oryzae pv. oryzicola.IMPORTANCE This study demonstrates that the ankyrin protein AnkB directly interacts with catalase CatB in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola. Ankyrin protein AnkB can affect the gene expression of catB, catalase activity, and sensitivity to H2O2 In Xanthomonas spp., the locations of genes ankB and catB and the amino acid sequence of AnkB are highly conserved. It is suggested that in prokaryotes, AnkB plays a conserved role in the defense against oxidative stress.

F240 of ß2-Tubulin Explains why Fusarium graminearum is Less Sensitive to Carbendazim than Botrytis cinerea.

ß-Tubulin is the target of benzimidazole fungicides, the most widely used of which is carbendazim (methyl benzimidazol-2-ylcarbamate [MBC]). MBC sensitivity is determined by the differential affinity of MBC for ß-tubulins. However, the mechanism of less sensitivity of Fusarium graminearum to MBC compared with other fungi, including Botrytis cinerea, Colletotrichum gloeosporioides, and Sclerotinia sclerotiorum, remains exclusive. Alignment of ß-tubulin amino acid sequences showed that position 240 of ß-tubulins is leucine (L) in most pathogenic fungi but is phenylalanine (F) in the Fgß2-tubulin of the F. graminearum wild type. The effective concentration resulting in 50% inhibition (EC50) value of MBC against the Fgß2F240L mutant of F. graminearum is 0.047 µg/ml, which was 10-fold lower than that of wild-type strain 2021. Moreover, The EC50 value of MBC against the BcßL"240"F (actually position 232) mutant of Botrytis cinerea was 0.44 µg/ml, which was ninefold higher than that of B. cinerea wild-type strain Bt4-1. In response to MBC treatment (0.15 µg/ml), microtubules were clearly visible in Fgß2-enhanced green fluorescent protein (EGFP) but not in Fgß2F240L-EGFP. Moreover, a molecular docking assay indicated that F240L mutation created a pi-pi interaction between Fgß2-tubulin and MBC and increased the binding affinity of Fgß2-tubulin to MBC. Our results suggest that F240 is responsible for the naturally less MBC sensitivity in F. graminearum compared with B. cinerea, C. gloeosporioides, and S. sclerotiorum by decreasing the binding affinity between Fgß2-tubulin and MBC.

The septins FaCdc3 and FaCdc12 are required for cytokinesis and affect asexual and sexual development, lipid metabolism and virulence in Fusarium asiaticum.

Septins are a highly conserved family of GTP-binding proteins that contribute to many cellular and metabolic functions, including cell polarity, cytokinesis, cell morphogenesis and pathogenesis. In this study, we characterized the septins FaCdc3 and FaCdc12 in the filamentous fungus Fusarium asiaticum. The functions of FaCdc3 and FaCdc12 were evaluated by constructing deletion mutants of FaCdc3 and FaCdc12, designated ΔFaCdc3-5 and ΔFaCdc12-71, respectively. The deletion mutants exhibited a reduced rate of mycelial growth, increased aerial hyphae formation, irregularly shaped hyphae, reduced conidiation and a lack of sexual reproduction in wheat kernels. Histochemical analysis revealed that the conidia and hyphae of ΔFaCdc3-5 and ΔFaCdc12-71 formed large lipid droplets (LDs). ΔFaCdc3-5 and ΔFaCdc12-71 also exhibited increased resistance to agents that induce osmotic stress and damage the cell membrane and cell wall. In addition, the hyphae and conidia of the two mutants formed fewer septa than those of the wild-type and exhibited aberrant nuclear distribution. Pathogenicity assays showed that ΔFaCdc3-5 and ΔFaCdc12-71 exhibited reduced virulence on wheat spikelets, which was indirectly correlated with a reduced level of deoxynivalenol accumulation. All of these defects were restored by genetic complementation of the two mutants with the parental FaCdc3 and FaCdc12. These results indicate that FaCdc3 and FaCdc12 play a critical role in various cellular processes in F. asiaticum.

Myosins FaMyo2B and Famyo2 Affect Asexual and Sexual Development, Reduces Pathogenicity, and FaMyo2B Acts Jointly with the Myosin Passenger Protein FaSmy1 to Affect Resistance to Phenamacril in Fusarium asiaticum.

We previously reported that mutations occurred in the gene myosin5 were responsible for resistance to the fungicide phenamacril in Fusarium graminearum. Here, we determined whether there is a functional link between phenamacril resistance and the myosin proteins FaMyo2B and Famyo2 in Fusarium asiaticum, which is the major causal agent of Fusarium head blight in China. We found that FaMyo2B acts jointly with FaSmy1 to affect resistance to phenamacril in F. asiaticum. We also found that FaMyo2B disruption mutant and Famyo2 deletion mutant were defective in hyphal branching, conidiation, and sexual reproduction. ΔFamyo2 also had an enhanced sensitivity to cell wall damaging agents and an abnormal distribution of septa and nuclei. In addition, the FaMyo2B and Famyo2 mutants had reduced pathogenicity on wheat coleoptiles and flowering wheat heads. Taken together, these results reveal that FaMyo2B and Famyo2 are required for several F. asiaticum developmental processes and activities, which help us better understand the resistance mechanism and find the most effective approach to control FHB.

Genotypes and Characteristics of Phenamacril-Resistant Mutants in Fusarium asiaticum.

Fusarium asiaticum is a critical pathogen of Fusarium head blight (FHB) in the southern part of China. The fungicide phenamacril has been extensively used for controlling FHB in recent years, which reduced both FHB severity and mycotoxin production. Our previous report indicated that resistance of F. asiaticum to phenamacril was related to mutations in myosin5. A recent article revealed that the resistance level of phenamacril-resistant mutants was associated with the genotypes of myosin5 in these mutants. In total, we obtained 239 resistant isolates by fungicide domestication, and 82 resistant mutants were randomly selected for further study. Of these mutants, 25.6, 7.3, and 67.1% showed low resistance (LR), moderate resistance (MR), and high resistance (HR), respectively, to phenamacril determined by 50% effective concentration values. Point mutations A135T, V151M, P204S, I434M, A577T, R580G/H, or I581F led to LR. Point mutations S418R, I424R, and A577G were responsible for MR and point mutations K216R/E, S217P/L, or E420K/G/D conferred HR. Interestingly, all of the mutations concentrated in the myosin5 motor domain and mutations conferring HR occurred at codon 217 and 420, which we called the core region. Homology modeling revealed that mutations far from the core region led to a lower resistance degree. Phenotype assays revealed that the most highly resistant mutants did not significantly change pathogenicity but decreased conidia production compared with the wild type, which may slow down the formation of the resistant pathogen population in the fields.

Whole-genome sequencing reveals that mutations in myosin-5 confer resistance to the fungicide phenamacril in Fusarium graminearum.

To determine the mechanism of resistance to the fungicide phenamacril (JS399-19) in Fusarium graminearum, the causal agent of Fusarium head blight, we sequenced and annotated the genome of the resistant strain YP-1 (generated by treating the F. graminearum reference strain PH-1 with phenamacril). Of 1.4 million total reads from an Illumina-based paired-end sequencing assay, 92.80% were aligned to the F. graminearum reference genome. Compared with strain PH-1, strain YP-1 contained 1,989 single-nucleotide polymorphisms that led to amino acid mutations in 132 genes. We sequenced 22 functional annotated genes of another F. graminearum sensitive strain (strain 2021) and corresponding resistant strains. The only mutation common to all of the resistant mutants occurred in the gene encoding myosin-5 (point mutations at codon 216, 217, 418, 420, or 786). To confirm whether the mutations in myosin-5 confer resistance to phenamacril, we exchanged the myosin-5 locus between the sensitive strain 2021 and the resistant strain Y2021A by homologous double exchange. The transformed mutants with a copy of the resistant fragment exhibited resistance to phenamacril, and the transformed mutant with a copy of the sensitive fragment exhibited sensitivity to phenamacril. These results indicate that mutations in myosin-5 confers resistance to phenamacril in F. graminearum.

Transcription factors spt3 and spt8 are associated with conidiation, mycelium growth, and pathogenicity in Fusarium graminearum.

Fusarium graminearum (teleomorph: Gibberella zeae), the dominant pathogen of Fusarium head blight (FHB) on wheat, can cause substantial economic losses. The Spt-Ada-Gcn5-acetyltransferase (SAGA) transcription coactivator plays multiple roles in regulating transcription because of the presence of functionally independent modules of subunits within the complex. The transcription factors spt3 and spt8 are components of the SAGA complex and they are important in yeasts and filamentous fungi including F. graminearum. In this study, we identified Fgspt3 and Fgspt8, homologs of Saccharomyces cerevisiae spt3 and spt8 from F. graminearum using the blastp program. The aim of the present study was to investigate the functions of Fgspt3 and Fgspt8 in F. graminearum. The deletion mutants grew significantly more slowly than the wild-type parent and did not produce conidia. Expression of the sporulation-related genes FgFlbC and FgRen1 were significantly down-regulated in the mutants. The mutants exhibited no sexual reproduction on infected wheat kernels and a 90% decrease in virulence on wheat. Pigment formation was also greatly altered in the mutants. All of the defects were restored by genetic complementation of the mutant with wild-type Fgspt3 and Fgspt8 genes. Overall, Fgspt3 and Fgspt8 are essential genes in F. graminearum.

FgFim, a key protein regulating resistance to the fungicide JS399-19, asexual and sexual development, stress responses and virulence in Fusarium graminearum.

Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia and fibroblast filopodia. Its homologue Sac6p has been shown to play a critical role in endocytosis and diverse cellular processes in Saccharomyces cerevisiae. FgFim from the wheat scab pathogenic fungus Fusarium graminearum strain Y2021A, which is highly resistant to the fungicide JS399-19, was identified by screening a mutant library generated by HPH-HSV-tk cassette-mediated integration. The functions of FgFim were evaluated by constructing a deletion mutant of FgFim, designated ΔFgFim-15. The deletion mutant exhibited a reduced rate of mycelial growth, reduced conidiation, delayed conidium germination, irregularly shaped hyphae, a lack of sexual reproduction on autoclaved wheat kernels and a dramatic decrease in resistance to JS399-19. ΔFgFim-15 also exhibited increased sensitivity to diverse metal cations, to agents that induce osmotic stress and oxidative stress, and to agents that damage the cell membrane and cell wall. Pathogenicity assays showed that the virulence of the FgFim deletion mutant on flowering wheat heads was impaired, which was consistent with its reduced production of the toxin deoxynivalenol in host tissue. All of these defects were restored by genetic complementation of the mutant with the parental FgFim gene. Quantitative real-time polymerase chain reaction (PCR) assays showed that the basal expression of three Cyp51 genes, which encode sterol 14α-demethylase, was significantly lower in the mutant than in the parental strain. The results of this study indicate that FgFim plays a critical role in the regulation of resistance to JS399-19 and in various cellular processes in F. graminearum.

Involvement of the anucleate primary sterigmata protein FgApsB in vegetative differentiation, asexual development, nuclear migration, and virulence in Fusarium graminearum.

The protein ApsB has been shown to play critical roles in the migration and positioning of nuclei and in the development of conidiophores in Aspergillus nidulans. The functions of ApsB in Fusarium graminearum, a causal agent of Fusarium head blight in China, are largely unknown. In this study, we used the blastp program at the Broad Institute to identify FgApsB, an F. graminearum homolog of A. nidulansApsB. The functions of FgApsB were evaluated by constructing a deletion mutant of FgApsB, designated ΔFgApsB-28. Conidiation and mycelial growth rate are reduced in ΔFgApsB-28. The hyphae of ΔFgApsB-28 are thinner than those of the wild type and have a different branching angle. ΔFgApsB-28 exhibited reduced aerial hyphae formation, but increased production of rubrofusarin. Whereas nuclei are evenly distributed in germ tubes and hyphae of the wild type, they are clustered and irregularly distributed in ΔFgApsB-28. The mutant exhibited increased resistance to cell wall-damaging agents, but reduced virulence on flowering wheat heads, which is consistent with its reduced production of the toxin deoxynivalenol. All of the defects in ΔFgApsB-28 were restored by genetic complementation with the parental FgApsB gene. Taken together, the results indicate that FgApsB is important for vegetative differentiation, asexual development, nuclear migration, and virulence in F. graminearum.

Proteomic analysis of Fusarium graminearum treated by the fungicide JS399-19.

JS399-19 (2-cyano-3-amino-3-phenylancryic acetate), a novel cyanoacrylate fungicide, has powerful inhibition against Fusarium species, especially to Fusarium graminearum. Treated with JS399-19, mycelium of F. graminearum was distorted and swelled. The embranchment increased. In order to investigate the effect of JS399-19 on protein expression of F. graminearum, total protein of F. graminearum cultured in normal condition and that treated with 0.5 µg/mL (EC90 value) JS399-19 were extracted respectively and proteomic analysis was performed using two-dimensional gel electrophoresis. The expression levels of 38 proteins varied quantitatively at least twofold. 33 proteins out of the 38 were successfully identified by MALDI-TOF-MS/MS and MASCOT. According to the classification of physiological functions from Conserved Domain Database analysis, 19, 5, 2, 3, 2 and 2 proteins were respectively associated with metabolism, regulation, motility, defense, signal transduction, and unknown function, which indicated that energy metabolism, the synthesis and transport of proteins and DNA of F. graminearum were inhibited by JS399-19 in different degrees. The expression levels of the genes were further confirmed by quantitative real-time PCR analyses. This study represents the first proteomic analysis of F. graminearum treated by JS399-19 and will provide some useful information to find the mode of action of the fungicide against F. graminearum.