A type 2C protein phosphatase FgPtc3 is involved in cell wall integrity, lipid metabolism, and virulence in Fusarium graminearum

Type 2C protein phosphatases (PP2Cs) play important roles in regulating many biological processes in eukaryotes. Currently, little is known about functions of PP2Cs in filamentous fungi. The causal agent of wheat head blight, Fusarium graminearum, contains seven putative PP2C genes, FgPTC1, -3, -5, -5R, -6, -7 and -7R. In order to investigate roles of these PP2Cs, we constructed deletion mutants for all seven PP2C genes in this study. The FgPTC3 deletion mutant (ΔFgPtc3-8) exhibited reduced aerial hyphae formation and deoxynivalenol (DON) production, but increased production of conidia. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents on potato dextrose agar plates. Pathogencity assays showed that ΔFgPtc3-8 is unable to infect flowering wheat head. All of the defects were restored when ΔFgPtc3-8 was complemented with the wild-type FgPTC3 gene. Additionally, the FgPTC3 partially rescued growth defect of a yeast PTC1 deletion mutant under various stress conditions. Ultrastructural and histochemical analyses showed that conidia of ΔFgPtc3-8 contained an unusually high number of large lipid droplets. Furthermore, the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Quantitative real-time PCR assays showed that basal expression of FgOS2, FgSLT2 and FgMKK1 in the mutant was significantly higher than that in the wild-type strain. Serial analysis of gene expression in ΔFgPtc3-8 revealed that FgPTC3 is associated with various metabolic pathways. In contrast to the FgPTC3 mutant, the deletion mutants of FgPTC1, FgPTC5, FgPTC5R, FgPTC6, FgPTC7 or FgPTC7R did not show aberrant phenotypic features when grown on PDA medium or inoculated on wheat head. These results indicate FgPtc3 is the key PP2C that plays a critical role in a variety of cellular and biological functions, including cell wall integrity, lipid and secondary metabolisms, and virulence in F. graminearum.     

Functional characterization of the three mitogen‐activated protein kinase kinases (MAP2Ks) present in the Cryphonectria parasitica genome reveals the necessity of Cpkk1 and Cpkk2, but not Cpkk3, for pathogenesis on chestnut (Castanea spp.)

The biological function(s) of the cpkk1, cpkk2 and cpkk3 genes, encoding the three mitogen‐activated protein kinase kinases (MAP2Ks) of Cryphonectria parasitica, the causal agent of chestnut blight, were examined through knockout strains. Cpkk1, the Mkk1 orthologue, acts in a phosphorylation cascade essential for cell integrity; Cpkk2 is the Ste7 orthologue involved in the pheromone response pathway; Cpkk3 is the Pbs2 orthologue, the MAP2K activated during the high‐osmolarity response. Our analysis confirmed the role of each MAP2K in its respective signalling cascade with some peculiarities: abnormal hyphae with a reduced number of septa and thinner cell walls were observed in Δcpkk1 mutants, and a strong growth defect on solid media was evident in Δcpkk2 mutants, when compared with the controls. Virulence on chestnut was affected in both the Δcpkk1 and Δcpkk2 strains, which were also unable to complete the developmental steps essential for mating. No alterations were reported in Δcpkk3, except under hyperosmotic conditions and in the presence of fludioxonil. Δcpkk2 mutants, however, showed higher sensitivity during growth in medium containing the antibiotic G418 (Geneticin).     

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.     

KRE genes are required for β‐1,6‐glucan synthesis,maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans

The polysaccharide β‐1,6‐glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in β‐1,6‐glucan synthesis. The H99 deletion mutants kre5Δ and kre6Δskn1Δ contained less cell wall β‐1,6‐glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI‐anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Δ and kre6Δskn1Δ. Our results indicate that KRE5, KRE6 and SKN1 are involved in β‐1,6‐glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.     

Septins enforce morphogenetic events during sexual reproduction and contribute to virulence of Cryptococcus neoformans

Septins are conserved, cytoskeletal GTPases that contribute to cytokinesis, exocytosis, cell surface organization and vesicle fusion by mechanisms that are poorly understood. Roles of septins in morphogenesis and virulence of a human pathogen and basidiomycetous yeast Cryptococcus neoformans were investigated. In contrast to a well‐established paradigm in S. cerevisiae, Cdc3 and Cdc12 septin homologues are dispensable for growth in C. neoformans yeast cells at 24°C but are essential at 37°C. In a bilateral cross between septin mutants, cells fuse but the resulting hyphae exhibit morphological abnormalities, including lack of properly fused specialized clamp cells and failure to produce spores. Interestingly, post‐mating hyphae of the septin mutants have a defect in nuclear distribution. Thus, septins are essential for the development of spores, clamp cell fusion and also play a specific role in nuclear dynamics in hyphae. In the post‐mating hyphae the septins localize to discrete sites in clamp connections, to the septa and the bases of the initial emerging spores. Strains lacking CDC3 or CDC12 exhibit significantly reduced virulence in a Galleria mellonella model of infection. Thus, C. neoformans septins are vital to morphology of the hyphae and contribute to virulence.     

Catecholate siderophore esterases Fes,IroD and IroE are required for salmochelins secretion following utilization,but only IroD contributes to virulence of extra‐intestinal pathogenic Escherichia coli

Salmochelins are glucosylated forms of enterobactin (enterochelin) and contribute to the virulence of Salmonella enterica and some extra‐intestinal pathogenic Escherichia coli (ExPEC). Fes, IroD and IroE esterases degrade salmochelins and enterobactin to release iron. We investigated the apparently redundant role of these esterases in virulence and in salmochelin production and utilization of the ExPEC strain χ7122. The ΔiroD, ΔfesΔiroD and ΔfesΔiroDΔiroE mutants displayed attenuated virulence phenotypes in an avian systemic infection model. Growth of ΔfesΔiroD and ΔfesΔiroDΔiroE mutants was severely reduced in the presence of conalbumin, and although enterobactin was produced, no salmochelins were detected in the culture supernatants of these mutants. Elimination of catecholate synthesis via an entA deletion in a ΔfesΔiroDΔiroE restored growth in the presence of conalbumin, but only partially restored the virulence of the strain. Salmochelin production was reestablished by reintroducing active esterases. Intracellular accumulation of cyclic mono‐glucosylated enterobactin was observed in the triple mutant ΔfesΔiroDΔiroE, and deletion of fepC, required for catecholate import into the cytoplasm, restored salmochelin detection in supernatants. These results suggest that in the absence of esterases, cyclic salmochelins are synthesized and secreted, but remain cell‐bound after internalization indicating that esterase‐mediated degradation is required for re‐secretion of catecholate siderophore molecules following their utilization.     

Identification and Genetic Division of Fusarium graminearum and Fusarium asiaticum by Species‐Specific SCAR Markers

Fusarium head blight (FHB), also called scab, is a devastating and insidious disease of cereals including wheat (Triticum spp.) and barley (Hordeum vulgare L.) worldwide. Apart from direct yield losses, the most serious concern about FHB is the contamination of the crop with mycotoxins, which pose a health risk to human and livestock. Recent research reported that phylogenetic species F. asiaticum (Fa) and F. graminearum (Fg) were the major causal agents of FHB from infected wheat heads in China. To investigate the population structure of Fusarium species in China by species‐specific as well as the chemotype‐specific markers, sequence‐related amplified polymorphism (SRAP) markers were screened on representative isolates of F. asiaticum‐NIV, F. asiaticum‐ 3ADON and F. graminearum‐15ADON to find amplification products characteristic of either species or chemotypes. Selected amplified fragments were cloned and sequenced so that sequence‐characterized amplified region (SCAR) primer pairs could be developed which permit specific detection of Fusarium species using conventional PCR. Primer pairs SCAR‐Fa1 and SCAR‐Fg1 were confirmed to be able to amplify specific products only in F. asiaticum and F. graminearum isolates, respectively. These species‐specific primers were applied to determine genetic division of F. asiaticum and F. graminearum isolates collected in Yangtze–Huaihe valley. The results indicated that F. asiaticum was the predominant species causing FHB in this wheat production area. It is the first report that SRAP markers were adapted for species characterization in Fusarium isolates.     

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.     

Mutations in the Scn8a DIIS4 voltage sensor reveal new distinctions among hypomorphic and null Nav1.6 sodium channels

Mutations in the voltage‐gated sodium channel gene SCN8A cause a broad range of human diseases, including epilepsy, intellectual disability, and ataxia. Here we describe three mouse lines on the C57BL/6J background with novel, overlapping mutations in the Scn8a DIIS4 voltage sensor: an in‐frame 9 bp deletion (Δ9), an in‐frame 3 bp insertion (?3) and a 35 bp deletion that results in a frameshift and the generation of a null allele (Δ35). Scn8a ^Δ9/+ and Scn8a ^?3/+ heterozygous mutants display subtle motor deficits, reduced acoustic startle response, and are resistant to induced seizures, suggesting that these mutations reduce activity of the Scn8a channel protein, Na_v1.6. Heterozygous Scn8a ^Δ35/+ mutants show no alterations in motor function or acoustic startle response, but are resistant to induced seizures. Homozygous mutants from each line exhibit premature lethality and severe motor impairments, ranging from uncoordinated gait with tremor (Δ9 and ?3) to loss of hindlimb control (Δ35). Scn8a ^Δ9/Δ9 and Scn8a ^?3/?3 homozygous mutants also exhibit impaired nerve conduction velocity, while normal nerve conduction was observed in Scn8a ^Δ35/Δ35 homozygous mice. Our results suggest that hypomorphic mutations that reduce Na_v1.6 activity will likely result in different clinical phenotypes compared to null alleles. These three mouse lines represent a valuable opportunity to examine the phenotypic impacts of hypomorphic and null Scn8a mutations without the confound of strain‐specific differences.     

Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae

Autophagy is a widely conserved intracellular process for degradation and recycling of proteins,organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar in...     

Nivalenol and 15‐acetyldeoxynivalenol Chemotypes of Fusarium graminearum Clade Species are Prevalent on Maize throughout China

Fusarium graminearum clade species are among the main causative agents of Gibberella ear rot (GER) in maize and responsible for the various trichothecene mycotoxins accumulated in contaminated maize grains. In this study, a total of 620 isolates from diseased maize ears collected from 59 districts in 19 provinces throughout China, previously identified morphologically as Fusarium graminearum clade, was genetically characterized at the species level based on SCAR (Sequence Characterized Amplified Region) and for their potential capability of mycotoxin production using the genetic chemotyping assay. The results showed that 359 isolates were F. asiaticum (SCAR 5), which consisted of 97% nivalenol (NIV)‐chemotypes, 0.8% 3‐acetyldeoxynivalenol (3‐ADON)‐producing isolates and 2.2% 15‐acetyldeoxynivalenol (15‐ADON) producers, whereas the remaining 261 isolates were identified as F. graminearum sensu stricto (SCAR 1), all of which produced 15‐ADON mycotoxins. This high proportion of NIV producers present in F. asiaticum is different from the chemotype patterns in F. asiaticum populations isolated from wheat and barley, where DON and its acetylated chemotypes were the predominant mycotoxins. Moreover, the majority of NIV producers (59.1%) and all the 3‐ADON‐producing strains were derived from the warmer regions in southern China, whereas most of the 15‐ADON‐producing strains (78.4%) were isolated from the colder regions in northern China. Our study is the first report of NIV chemotypes of F. asiaticum and 15‐ADON chemotypes of F. graminearum sensu stricto that were associated with the GER of maize in China.