Deletion of a Rare Fungal PKS CgPKS11 Promotes Chaetoglobosin A Biosynthesis, Yet Defers the Growth and Development of Chaetomium globosum

We previously reported that chaetoglobosin A (ChA) exhibits a great potential in the biocontrol of nematodes and pathogenic fungi. To improve the production of ChA, a CRISPR-Cas9 system was created and applied for eliminating potential competitive polyketide products. One of the polyketide synthase encoding genes, Cgpks11, which is putatively involved in the biosynthesis of chaetoglocin A, was disrupted. Cgpks11 deletion led to the overexpression of the CgcheA gene cluster, which is responsible for ChA biosynthesis, and a 1.6-fold increase of ChA. Transcription of pks-1, a melanin PKS, was simultaneously upregulated. Conversely, the transcription of genes for chaetoglocin A biosynthesis, e.g., CHGG_10646 and CHGG_10649, were significantly downregulated. The deletion also led to growth retardation and seriously impaired ascospore development. This study found a novel regulatory means on the biosynthesis of ChA by CgPKS11. CgPKS11 affects chaetoglobosin A biosynthesis, growth, and development in Chaetomium globosum.

The FgCYP51B Y123H mutation confers reduced sensitivity to prochloraz and is important for conidiation and ascospore development in Fusarium graminearum

Fusarium graminearum is one of the most important causal agent of Fusarium Head Blight disease and now were controlled mainly by chemicals such as DMI fungicides. FgCYP51B is one of the DMI targets in F. graminearum and Tyrosine123 is an important amino acid in Fusarium graminearum CYP51B, located in one of the predicted substrate binding pockets based on the binding mode between demethylation inhibitors (DMIs) and CYP51B. Previous study suggests that resistance to DMI fungicides is primarily attributed to point mutations in the CYP51 gene and that the Y123H mutation in F. verticillioides CYP51 confers prochloraz resistance in the laboratory. To investigate the function of FgCYP51B Y123 residue in the growth and development, pathogenicity, and DMI-resistance, the FgCYP51B Y123H mutant was generated and analyzed. Results revealed that Y123H mutation led to reduced conidial sporulation and affected ascospore development and moreover, the mutation conferred reduced sensitivity to prochloraz. The qPCR and molecular docking were performed to investigate the resistance mechanism. Results indicated that Y123H mutation changed the target gene expression and decreased the binding affinity of FgCYP51 to prochloraz. These results will attract more attention to the potential DMI-resistant mutation of F. graminearum and further deepen our understanding of the DMI resistance mechanism.

The Notorious Soilborne Pathogenic Fungus Sclerotinia sclerotiorum: An Update on Genes Studied with Mutant Analysis

Ascomycete Sclerotinia sclerotiorum (Lib.) de Bary is one of the most damaging soilborne fungal pathogens affecting hundreds of plant hosts, including many economically important crops. Its genomic sequence has been available for less than a decade, and it was recently updated with higher completion and better gene annotation. Here, we review key molecular findings on the unique biology and pathogenesis process of S. sclerotiorum, focusing on genes that have been studied in depth using mutant analysis. Analyses of these genes have revealed critical players in the basic biological processes of this unique pathogen, including mycelial growth, appressorium establishment, sclerotial formation, apothecial and ascospore development, and virulence. Additionally, the synthesis has uncovered gaps in the current knowledge regarding this fungus. We hope that this review will serve to build a better current understanding of the biology of this under-studied notorious soilborne pathogenic fungus.

Influence of moisture on maturation rate of the Venturia inaequalis (Cooke) Wint. ascospores in central Poland

Influence of moisture on maturation rate of the airborne Venturia inaequalis (Cooke) Wint. ascospores was numerically examined using field data collected from spore traps in Skierniewice, Poland, during 2005–2008 and 2010–2014. In order to avoid a bias resulting from different abundance of airborne ascospores in different seasons, the cumulative proportions of mature ascospores observed in the season were analyzed. The maturation rate was expressed with the probit of cumulative proportion which linearized its sigmoidal shape and made the effect of the weather during the entire season of the same importance. The significant, positive association between the probit slope and the moisture indicators related to precipitation, leaf wetness, and high relative humidity were observed. Additionally, the significant, negative relationships with the average and maximal air temperature were found. The results suggested that rain should be treated as the major factor driving the ascospore development and leaf wetness as the second factor. In conclusion, the main sources of moisture should be taken into account in statistical models of ascospore development, but it seems reasonable to take into account different strength of their association with the development rate.

Mid1, a Mechanosensitive Calcium Ion Channel, Affects Growth, Development, and Ascospore Discharge in the Filamentous Fungus Gibberella zeae

ABSTRACT The role of Mid1, a stretch-activated ion channel capable of being permeated by calcium, in ascospore development and forcible discharge from asci was examined in the pathogenic fungus Gibberella zeae (anamorph Fusarium graminearum ). The Δ mid1 mutants exhibited a >12-fold reduction in ascospore discharge activity and produced predominately abnormal two-celled ascospores with constricted and fragile septae. The vegetative growth rate of the mutants was ∼50% of the wild-type rate, and production of macroconidia was >10-fold lower than in the wild type. To better understand the role of calcium flux, Δ mid1 Δ cch1 double mutants were also examined, as Cch1, an L-type calcium ion channel, is associated with Mid1 in Saccharomyces cerevisiae . The phenotype of the Δ mid1 Δ cch1 double mutants was similar to but more severe than the phenotype of the Δ mid1 mutants for all categories. Potential and current-voltage measurements were taken in the vegetative hyphae of the Δ mid1 and Δ cch1 mutants and the wild type, and the measurements for all three strains were remarkably similar, indicating that neither protein contributes significantly to the overall electrical properties of the plasma membrane. Pathogenicity of the Δ mid1 and Δ mid1 Δ cch1 mutants on the host (wheat) was not affected by the mutations. Exogenous calcium supplementation partially restored the ascospore discharge and vegetative growth defects for all mutants, but abnormal ascospores were still produced. These results extend the known roles of Mid1 to ascospore development and forcible discharge. However, Neurospora crassa Δ mid1 mutants were also examined and did not exhibit defects in ascospore development or in ascospore discharge. In comparison to ion channels in other ascomycetes, Mid1 shows remarkable adaptability of roles, particularly with regard to niche-specific adaptation.

The L-Type Calcium Ion Channel Cch1 Affects Ascospore Discharge and Mycelial Growth in the Filamentous Fungus Gibberella zeae (Anamorph Fusarium graminearum)

ABSTRACT Cch1, a putative voltage-gated calcium ion channel, was investigated for its role in ascus development in Gibberella zeae. Gene replacement mutants of CCH1 were generated and found to have asci which did not forcibly discharge spores, although morphologically ascus and ascospore development in the majority of asci appeared normal. Additionally, mycelial growth was significantly slower, and sexual development was slightly delayed in the mutant; mutant mycelia showed a distinctive fluffy morphology, and no cirrhi were produced. Wheat infected with Δcch1 mutants developed symptoms comparable to wheat infected with the wild type; however, the mutants showed a reduced ability to protect the infected stalk from colonization by saprobic fungi. Transcriptional analysis of gene expression in mutants using the Affymetrix Fusarium microarray showed 2,449 genes with significant, twofold or greater, changes in transcript abundance across a developmental series. This work extends the role of CCH1 to forcible spore discharge in G. zeae and suggests that this channel has subtle effects on growth and development.

Intracellular Siderophores Are Essential for Ascomycete Sexual Development in Heterothallic Cochliobolus heterostrophus and Homothallic Gibberella zeae

ABSTRACT Connections between fungal development and secondary metabolism have been reported previously, but as yet, no comprehensive analysis of a family of secondary metabolites and their possible role in fungal development has been reported. In the present study, mutant strains of the heterothallic ascomycete Cochliobolus heterostrophus, each lacking one of 12 genes (NPS1 to NPS12) encoding a nonribosomal peptide synthetase (NRPS), were examined for a role in sexual development. One type of strain (Δnps2) was defective in ascus/ascospore development in homozygous Δnps2 crosses. Homozygous crosses of the remaining 11 Δnps strains showed wild-type (WT) fertility. Phylogenetic, expression, and biochemical analyses demonstrated that the NRPS encoded by NPS2 is responsible for the biosynthesis of ferricrocin, the intracellular siderophore of C. heterostrophus. Functional conservation of NPS2 in both heterothallic C. heterostrophus and the unrelated homothallic ascomycete Gibberella zeae was demonstrated. G. zeae Δnps2 strains are concomitantly defective in intracellular siderophore (ferricrocin) biosynthesis and sexual development. Exogenous application of iron partially restored fertility to C. heterostrophus and G. zeae Δnps2 strains, demonstrating that abnormal sexual development of Δnps2 strains is at least partly due to their iron deficiency. Exogenous application of the natural siderophore ferricrocin to C. heterostrophus and G. zeae Δnps2 strains restored WT fertility. NPS1, a G. zeae NPS gene that groups phylogenetically with NPS2, does not play a role in sexual development. Overall, these data demonstrate that iron and intracellular siderophores are essential for successful sexual development of the heterothallic ascomycete C. heterostrophus and the homothallic ascomycete G. zeae.