The critical roles of the Zn2Cys6 transcription factor Fp487 in the development and virulence of Fusarium pseudograminearum: A potential target for Fusarium crown rot control.

Fusarium crown rot (FCR) caused by Fusarium pseudograminearum poses a significant threat to wheat production in the Huang-Huai-Hai region of China. However, the pathogenic mechanism of F. pseudograminearum is still poorly understood. Zn2Cys6 transcription factors, which are exclusive to fungi, play pivotal roles in regulating fungal development, drug resistance, pathogenicity, and secondary metabolism. In this study, we present the functional characterization of a Zn2Cys6 transcription factor F. pseudograminearum, designated Fp487. In F. pseudograminearum, Fp487 is shown to be required for mycelial growth through gene knockout and phenotypic analyses. Compared with wild-type CF14047, the ∆Fp487 mutant displayed a slight reduction in growth rate but a significant decrease in conidiogenesis, pathogenicity and 3-acetyl-deoxynivalenol (3AcDON) production. Moreover, the mutant exhibited heightened sensitivity to oxidative and cytomembrane stress. Furthermore, we synthesized dsRNA from the Fp487 gene in vitro, resulting in a reduction in the growth rate of F. pseudograminearum and its virulence on barley leaves through spray-induced gene silencing (SIGS). Notably, this study makes the first instance of inducing the expression of abundant dsRNA from F. pseudograminearum by engineering the Escherichia coli strain HT115 (DE3) and utilizing the SIGS technique to evaluate the virulence effect of dsRNA on F. pseudograminearum. In conclusion, our findings revealed the crucial role of Fp487 in regulating pathogenicity, stress responses, DON production, and conidiogenesis in F. pseudograminearum. Furthermore, Fp487 is a potential RNAi-based target for FCR control.

Genome Analyses Reveal the Secondary Metabolites that Potentially Influence the Geographical Distribution of Fusarium pseudograminearum Populations.

Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, significantly impacts wheat yield and quality in China's Huanghuai region. The rapid F. pseudograminearum epidemic and FCR outbreak within a decade remain unexplained. In this study, two high-quality, chromosome-level genomes of F. pseudograminearum strains producing 3-acetyl-deoxynivalenol (3AcDON) and 15-acetyl-deoxynivalenol (15AcDON) toxins were assembled. Additionally, 38 related strains were resequenced. Genomic differences such as single nucleotide polymorphisms (SNPs), insertions/deletions (indels), and structural variations (SVs) among F. pseudograminearum strains were analyzed. The whole-genome SNP locus-based population classification mirrored the toxin chemotype (3AcDON and 15AcDON)-based classification, indicating the presence of genes associated with the trichothecene toxin gene cluster. Further analysis of differential SNP, indel, and SV loci between the 3AcDON and 15AcDON populations revealed a predominant connection to secondary metabolite synthesis genes. Notably, the majority of the secondary metabolite biosynthesis gene cluster loci were located in SNP-dense genomic regions, suggesting high mutability and a possible contribution to F. pseudograminearum population structure and environmental adaptability. This study provides insightful perspectives on the distribution and evolution of F. pseudograminearum and for forecasting the spread of wheat FCR, thereby aiding in the development of preventive measures and control strategies.

Efficacy of cyclobutrifluram in controlling Fusarium crown rot of wheat and resistance risk of three Fusarium species to cyclobutrifluram.

Cyclobutrifluram (TYMIRIUM® technology), a new succinate dehydrogenase inhibitor (SDHI) fungicide, is currently being registered by SYNGENTA for controlling Fusarium crown rot (FCR) of wheat in China. The application of 15 or 30 g of active ingredient/100 kg seed of cyclobutrifluram significantly reduced pre-emergence damping-off, discoloration on the stem base and formation of whiteheads caused by FCR. The EC50 values of cyclobutrifluram for 60 isolates of F. pseudograminearum, 30 isolates of F. asiaticum and 30 isolates of F. graminearum ranged from 0.016 to 0.142 mg L-1, 0.010 to 0.041 mg L-1 and 0.012 to 0.059 mg L-1, respectively. One hundred and seven cyclobutrifluram-resistant (CR) mutants were obtained from three Fusarium species isolates, with ten types of mutations identified in Sdh genes. Three Fusarium species isolates exhibited similar resistance mechanisms, with the most prevalent mutations, SdhC1A83V and SdhC1R86K, accounting for 61.68% of mutants. The CR mutants possessed comparable or slightly impaired fitness compared to the corresponding parental isolates. The CR mutants carrying FpSdhBH248Y/Q/D exhibited increased sensitivity to fluopyram. An overall moderate risk of resistance development in three Fusarium species was recommended for cyclobutrifluram.

FgSdhC Paralog Confers Natural Resistance toward SDHI Fungicides in Fusarium graminearum.

Fusarium graminearum exhibited natural resistance to a majority of succinate dehydrogenase inhibitor fungicides (SDHIs) and the molecular mechanisms responsible for the natural resistance were still unknown. Succinate dehydrogenase subunit C (SdhC) is an essential gene for maintaining succinate-ubiquinone oxidoreductase (SQR) function in fungi. In F. graminearum, a paralog of FgSdhC named as FgSdhC1 was identified. Based on RNA-Seq and qRT-PCR assay, we found that the expression level of FgSdhC1 was very low but upregulated by SDHIs treatment. Based on reverse genetics, we demonstrated that FgSdhC1 was an inessential gene in normal growth but was sufficient for maintaining SQR function and conferred natural resistance or reduced sensitivity toward SDHIs. Additionally, we found that the standard F. graminearum isolate PH-1 had high sensitivity to a majority of SDHIs. A single nucleotide variation (C to T) in the FgSdhC1 of isolate PH-1, resulting in a premature termination codon (TAA) replacing the fourth amino acid glutamine (Q), led to the failure of FgSdhC1 to perform functions of conferring nature resistance. These results established that a dispensable paralogous gene determined SDHIs resistance in natural populations of F. graminearum.

Regulation of mRNA stability contributes to the function of innate lymphoid cells in various diseases.

Innate lymphoid cells (ILCs) are important subsets of innate immune cells that regulate mucosal immunity. ILCs include natural killer cells, innate lymphoid cells-1 (ILC1s), ILC2s, and ILC3s, which have extremely important roles in the immune system. In this review, we summarize the regulation of mRNA stability mediated through various factors in ILCs (e.g., cytokines, RNA-binding proteins, non-coding RNAs) and their roles in mediating functions in different ILC subsets. In addition, we discuss potential therapeutic targets for diseases such as chronic obstructive pulmonary disease, cancer, and pulmonary fibrosis by regulation of mRNA stability in ILCs, which may provide novel directions for future clinical research.

Hepatocyte growth factor-mediated apoptosis mechanisms of cytotoxic CD8+ T cells in normal and cirrhotic livers.

Intrahepatic stem/progenitor cells and cytotoxic CD8+ T cells (CD8+ T cells) in the cirrhotic liver undergo apoptosis, which potentially facilitates progression to cancer. Here, we report that hepatocyte growth factor (HGF) signaling plays an important role in promoting normal and damaged liver CD8+ T cell Fas-mediated apoptosis through its only receptor, c-Met. In addition to binding with HGF, c-Met also binds to Fas to form a complex. Using a diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis mouse model, immunostaining, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining, we found that HGF secretion was significantly higher at 10 weeks post-DEN, the liver cirrhotic phase (LCP), than at 3 weeks post-DEN, the liver fibrotic phase (LFP). Correspondingly, differences in CD8+ T cell proliferation and apoptosis were noted between the two phases. Interestingly, staining and TUNEL assays revealed lower smooth muscle actin (α-SMA)+ cell apoptosis, a marker for hepatic stellate cells (HSCs), in the LFP group than in the LCP group, which suggested a beneficial correlation among HGF, CD8+ T cells and HSCs in improving the fibrotic load during damaged liver repair. In cultures, when met different concentrations of recombinant HGF (rHGF), phytohemagglutinin (PHA)-stimulated naive mouse splenic CD8+ T cells (pn-msCD8+ T cells) responded differently; as increases in rHGF increased were associated with decreases in the clonal numbers of pn-msCD8+ T cells, and when the rHGF dose was greater than 200 ng/mL, the clonal numbers significantly decreased. In the presence of 400 ng/mL rHGF, the death-inducing signaling complex (DISC) can be directly activated in both nsCD8+ T cells and healthy human peripheral blood CD8+ T cells (hp-CD8+ T cells), as indicated by recruitment of FADD and caspase-8 because DISC forms via the recruitment of FADD and caspase-8, among others. These findings suggest that Fas-mediated apoptosis, may also indicate a regulatory role of HGF signaling in hepatic homeostasis.

TTP-mediated regulation of mRNA stability in immune cells contributes to adaptive immunity, immune tolerance and clinical applications.

Dendritic cells (DCs) form a sentinel network to induce protective immunity against pathogens or self-tolerance. mRNA stability is an important part of the post-transcriptional regulation (PTR) that controls the maturation and function of DCs. In this review, we summarize the effects of TTP-mediated regulation of mRNA stability in DCs, focusing on DC maturation and antigen presentation, T cell activation and differentiation, immune tolerance and inflammation. We also discuss the potential DC-based immune treatment for HIV+ patients through regulation of mRNA stability. This review proposes the regulation of mRNA stability as a novel immune therapy for various inflammatory diseases, such as arthritis and dermatitis.

The CHY-Type Zinc Finger Protein FgChy1 Regulates Polarized Growth, Pathogenicity, and Microtubule Assembly in Fusarium graminearum.

Microtubules (MTs), as transport tracks, play important roles in hyphal-tip growth in filamentous fungi, but MT-associated proteins involved in polarized growth remain unknown. Here, we found that one novel zinc finger protein, FgChy1, is required for MT morphology and polarized growth in Fusarium graminearum. The Fgchy1 mutant presented curved and directionless growth of hyphae. Importantly, the conidia and germ tubes of the Fgchy1 mutant exhibited badly damaged and less-organized beta-tubulin cytoskeletons. Compared with the wild type, the Fgchy1 mutant lost the ability to maintain polarity and was also more sensitive to the anti-MT drugs carbendazim and nocodazole, likely due to the impaired MT cytoskeleton. Indeed, the hyphae of the wild type treated with nocodazole exhibited a morphology consistent with that of the Fgchy1 mutant. Interestingly, the disruption of FgChy1 resulted in the off-center localization of actin patches and the polarity-related polarisome protein FgSpa2 from the hyphal-tip axis. A similar defect in FgSpa2 localization was also observed in the nocodazole-treated wild-type strain. In addition, FgChy1 is also required for conidiogenesis, septation, sexual reproduction, pathogenicity, and deoxynivalenol production. Overall, this study provides the first demonstrations of the functions of the novel zinc finger protein FgChy1 in polarized growth, development, and virulence in filamentous fungi.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Functional Characterization of Calcineurin-Responsive Transcription Factors Fg01341 and Fg01350 in Fusarium graminearum.

Ca2 +/calmodulin-dependent phosphatase calcineurin is one of the important regulators of intracellular calcium homeostasis and has been investigated extensively in Saccharomyces cerevisiae. However, only a few reports have explored the function of the Crz1 homolog in filamentous fungi, especially in Fusarium graminearum. In this study, we identified Fg01341 as a potential ortholog of yeast Crz1. Fg01341 could interact with calcineurin and initiate nuclear transport in a calcineurin-dependent manner. The ΔFg01341 mutant exhibited normal hyphal growth on basic medium and conidia formation, but sexual reproduction was partially blocked. Pathogenicity assays showed that the virulence of the ΔFg01341 mutant in flowering wheat heads and corn silks dramatically decreased and was thus consistent with the reduction in deoxynivalenol production. Unexpectedly, the sensitivity to osmotic stress of the deletion mutant and that of the wild-type strain did not present any differences. The deletion mutant showed higher sensitivity to tebuconazole than the wild-type strain. Results also showed that the transcription factor Fg01350 might be the calcineurin target and was independent of Crz1. Furthermore, ΔFg01350 showed defects in hyphal growth, sexual production, virulence, and deoxynivalenol production. Collectively, the results indicate that these two proteins functionally redundant and that the calcineurin-Crz1-independent pathway is particularly important in F. graminearum.

Mitochondrial phylogenomics of human-type Ascaris, pig-type Ascaris, and hybrid Ascaris populations.

Ascaris lumbricoides and Ascaris suum are parasitic nematodes in human and pig intestines. The two species can cross infect and produce hybrids, which contribute to the controversy concerning the taxonomy of A. lumbricoides and A. suum. The purpose of this study was to investigate the microevolutionary process and evolutionary history of human-type Ascaris, pig-type Ascaris, and hybrid Ascaris and provide a theoretical basis for the prevention and control of human and animal ascariasis. The mitochondrial phylogenomics of human-type Ascaris (n = 5), pig-type Ascaris (n = 6), and hybrid Ascaris (n = 6) populations were analyzed using high-throughput sequencing technology. The mitochondrial genomes of human-type Ascaris, pig-type Ascaris, and hybrid Ascaris contained 36 genes (atp8 was missing), including 12 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. All genes were located on the heavy chain. The initiation codons used for protein-coding genes were ATT and TTG and the termination codons were TAA and TAG. The base distribution showed obvious AT preference. The phylogenetic tree based on the Ascaris mitochondrial genomes showed three main clusters (A, B, and C). The Ascaris populations sequenced in this study were all gathered in cluster B. The human-type and hybrid Ascaris populations belonged to different sub-clusters, but the pig-type Ascaris population was more scattered. The mitochondrial genome sequences of the 17 Ascaris individuals in this study did not differ much. The results of this study indicate that Ascaris populations were geographically isolated before host shift. In addition, the data show that there are differences between hybrid Ascaris, human-type Ascaris, and pig-type Ascaris. The information has important theoretical significance and application value.

Fitness of three chemotypes of Fusarium graminearum species complex in major winter wheat-producing areas of China.

In China, Fusarium head blight is caused mainly by the Fusarium graminearum species complex (FGSC), which produces trichothecene toxins. The FGSC is divided into three chemotypes: 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and nivalenol (NIV). In order to predict the geographical changes in the distribution of these chemotype populations in major winter wheat-producing areas in China, the biological characteristics of twenty randomly selected isolates from each of the three chemotypes were studied. No significant difference was exhibited in the growth rate of 3-ADON, 15-ADON, and NIV isolates at 15°C. At 20°C and 25°C, the growth rate of 15-ADON isolates was the highest. At 30°C, the growth rate of NIV and 3-ADON isolates was significantly higher than that of 15-ADON isolates. The 15-ADON isolates produced the highest quantities of perithecia and two to three days earlier than the other two populations at each temperature, and released more ascospores at 18°C. The aggressiveness test on wheat seedlings and ears indicated there was no significant difference between the 3-ADON and 15-ADON isolates. However, the aggressiveness of NIV isolates was significantly lower than that of the 3-ADON and 15-ADON isolates. The DON content in grains from heads inoculated with the 3-ADON isolates was higher than the content of 15-ADON and NIV isolates. The results showed that 15-ADON population had the advantage in perithecia formation and ascospore release, and the 3-ADON population produced more DON in wheat grains. We suggested that distribution of these three chemotype populations may be related to these biological characteristics.

Homozygous and heterozygous point mutations in succinate dehydrogenase subunits b, c and d of Rhizoctonia cerealis conferring resistance to thifluzamide.

BACKGROUND: Thifluzamide, a succinate dehydrogenase inhibitor (SDHI) fungicide, is a promising fungicide for controlling wheat sharp eyespot (WSE). WSE is caused by Rhizoctonia cerealis. Information on the resistance mechanism of this pathogen to thifluzamide remains unavailable. RESULTS: We used selective reculturing and UV mutagenesis to generate thifluzamide-resistant mutants. Thifluzamide-resistant mutants were only generated through UV mutagenesis. Sequence analysis of succinate dehydrogenase (Sdh) genes revealed that two mutants had no mutation in RCSdhB, RCSdhC and RCSdhD, and the other 18 mutants all had at least one mutation in RCSdhB, RCSdhC or RCSdhD, either in a homozygous or heterozygous state. The majority of mutants included either RCSdhD-H116Y or RCSdhC-H139Y. They showed slight resistance to boscalid, bixafen and penflufen. Only one mutant possessed RCSdhB-H246Y, and it showed medium resistance to boscalid and penflufen and a slight resistance to bixafen. All the thifluzamide mutants were sensitive to flutolanil. Compared with their parental isolates, these mutants present no or minor fitness penalties. CONCLUSION: Homozygous and heterozygous point mutations in the succinate dehydrogenase subunits b, c and d of R. cerealis may be involved in thifluzamide resistance. © 2016 Society of Chemical Industry.

Analysis of simple sequence repeats in the Gaeumannomyces graminis var. tritici genome and the development of microsatellite markers.

Understanding the genetic structure of Gaeumannomyces graminis var. tritici is essential for the establishment of efficient disease control strategies. It is becoming clear that microsatellites, or simple sequence repeats (SSRs), play an important role in genome organization and phenotypic diversity, and are a large source of genetic markers for population genetics and meiotic maps. In this study, we examined the G. graminis var. tritici genome (1) to analyze its pattern of SSRs, (2) to compare it with other plant pathogenic filamentous fungi, such as Magnaporthe oryzae and M. poae, and (3) to identify new polymorphic SSR markers for genetic diversity. The G. graminis var. tritici genome was rich in SSRs; a total 13,650 SSRs have been identified with mononucleotides being the most common motifs. In coding regions, the densities of tri- and hexanucleotides were significantly higher than in noncoding regions. The di-, tri-, tetra, penta, and hexanucleotide repeats in the G. graminis var. tritici genome were more abundant than the same repeats in M. oryzae and M. poae. From 115 devised primers, 39 SSRs are polymorphic with G. graminis var. tritici isolates, and 8 primers were randomly selected to analyze 116 isolates from China. The number of alleles varied from 2 to 7 and the expected heterozygosity (He) from 0.499 to 0.837. In conclusion, SSRs developed in this study were highly polymorphic, and our analysis indicated that G. graminis var. tritici is a species with high genetic diversity. The results provide a pioneering report for several applications, such as the assessment of population structure and genetic diversity of G. graminis var. tritici.

Complete genome sequence of a novel endornavirus in the wheat sharp eyespot pathogen Rhizoctonia cerealis.

We report here the presence of a novel double-stranded RNA (dsRNA) virus in an isolate (R0959) of the fungus Rhizoctonia cerealis, the causal agent of sharp eyespot of wheat in China. Sequence analysis showed that the dsRNA segment is 17,486 bp long and contains a single open reading frame (ORF) with the potential to encode a protein of 5,747 amino acids. The predicted protein contains conserved motifs of putative viral methyltransferase, helicase 1, and RNA-dependent RNA polymerase. Sequence similarity and phylogenetic analysis clearly place it in a distinct species within the genus Endornavirus, family Endornaviridae, and therefore we propose its name to Rhizoctonia cerealis endornavirus 1 (RcEV1). This is the first report of the full-length genomic sequence of a dsRNA mycovirus in R. cerealis.

The heterogeneity of the rDNA-ITS sequence and its phylogeny in Rhizoctonia cerealis, the cause of sharp eyespot in wheat.

The sequence heterogeneity of the ribosomal internal transcribed spacer (ITS) region was investigated for Rhizoctonia cerealis isolates from the anastomosis group AG-DI. Although sequence variability of the ITS has been reported in a few multinucleate R. solani isolates, it has very rarely been reported in binucleate Rhizoctonia spp. isolates and has never been described in R. cerealis, the pathogen of wheat sharp eyespot. In this study, the ITS regions of 15 R. cerealis isolates were cloned and sequenced. The results revealed more than one different ITS sequence within each isolate. This is the first evidence of ITS sequence heterogeneity in R. cerealis. Based on these ITS sequences, different sequences of one isolate did not cluster in one clade, but all of the sequences of the 15 isolates were clustered in the anastomosis subgroup AG-DI, suggesting that the heterogeneity of the ITS did not affect the molecular identification of their anastomosis group. Haplotype analyses indicated that there might be three evolutionary origins of R. cerealis, or a recombination event could be the cause of different ITS sequences in one genome. This study demonstrates the variability and the evolution of Rhizoctonia, especially binucleate R. cerealis. These findings will help design disease control strategies.

[Population dynamics and antagonism toward Fusarium oxysporium f. sp. Vasinfectum. and Verticillium dahliae Kleb of endophytic bacteria from cotton].

OBJECTIVE: To explore population dynamics of endophytic bacteria and obtain antagonistic endophytic bacteria toward Verticillium dahliae Kleb (Vd), Fusarium oxysporium f. sp. Vasinfectum (Fov) from cotton. METHODS: Root, stem and leaf samples were surface-disinfested, and subsequently used to isolate endophytic bacteria by diluting plate counting method. We assayed antagonism of the isolated endophytic bacteria toward three pathogens: Vd (V107, which is a highly virulent defoliating isolate and V396, which is a mildly virulent non-defoliating isolate), Fov (F108) using a dual culture method, and analyzed the 16S rDNA sequence of doubly antagonistic endophytic bacteria (DAEB) isolates toward both Vd and Fov. RESULTS: The population size of endophytic bacteria in root was significantly larger than that in leaf and stem. The populations at seedling stage were generally lower than those at the flowering/maturing stage in root, the populations in stem and leaf were fluctuant at different development stages, but variation law was not observed obviously. Furthermore, although no significant differences of the population densities in root were found among 6 cotton cultivars, the population densities in stem and leaf showed cultivar differences. The proportion of endophytic bacteria antagonizing Vd (V107, V396) and Fov (F108) in root was higher than that in stem/leaf, moreover, the amount of endophytic bacteria antagonizing toward V107 was less than that toward V396/F108. Based on 16S rDNA sequence analysis, all 44 DAEB isolates consisted of two phyla, i.e., Bacteroidetes (1 out of 44) and Proteobacteria (43 out of 44), and fell into 8 genera. The genus enterobacter (18 out of 44) and Pantoea (15 out of 44) were predominant. Notably, ten DAEB isolates demonstrated <97% sequence similarity with the most similar sequences of strain deposited in the Ribosomal Database, these DAEB isolates might be potential novel species. CONCLUSION: This article suggested that plant genotype, development stage, and tissue influenced the population of endophytic bacteria. We discovered that DAEB with predominant and various genus existed in cotton. Endophytic bacteria in cotton could serve as a pool for discovering biocontrol agent toward cotton pathogens.