C-terminus of serine-arginine protein kinase-like protein, SrpkF, is involved in conidiophore formation and hyphal growth under salt stress in Aspergillus aculeatus.

The serine-arginine protein kinase-like protein, SrpkF, was identified as a regulator for the cellulose-responsive induction of cellulase genes in Aspergillus aculeatus. To analyze various aspects of SrpkF function, we examined the growth of the control strain (MR12); C-terminus deletion mutant, which produced SrpkF1-327 (ΔCsrpkF); whole gene-deletion mutant of srpkF (ΔsrpkF), srpkF overexpressing strain (OEsprkF); and the complemented strain (srpkF+) under various stress conditions. All test strains grew normally on minimal medium under control, high salt (1.5 M KCl), and high osmolality (2.0 M sorbitol and 1.0 M sucrose). However, only ΔCsrpkF showed reduced conidiation on 1.0 M NaCl media. Conidiation of ΔCsrpkF on 1.0 M NaCl media was reduced to 12% compared with that of srpkF+. Further, when OEsprkF and ΔCsrpkF were pre-cultured under salt stress conditions, germination under salt stress conditions was enhanced in both strains. By contrast, deletion of srpkF did not affect hyphal growth and conidiation under the same conditions. We then quantified the transcripts of the regulators involved in the central asexual conidiation pathway in A. aculeatus. The findings revealed that the expression of brlA, abaA, wetA, and vosA was reduced in ΔCsrpkF under salt stress. These data suggest that in A. aculeatus, SrpkF regulates conidiophore development. The C-terminus of SrpkF seems to be important for regulating SrpkF function in response to culture conditions such as salt stress.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Sigesbeckia orientalis in China.

Sigesbeckia orientalis L., (St Paul's wort) is an annually grown natural herb of Asteraceae with a long therapeutic history for a wide range of inflammation-related diseases in China (Zhong et al. 2019). In June 2020, typical symptoms of powdery mildew were observed on 30% of wild S. orientalis plants grown along the roadsides and gardens in Minjiang University, Fuzhou, China. Circular to irregular white powdery fungal colonies were observed on both surfaces of the leaves and young stems, causing necrosis and premature senescence. Fungal hyphae were epigenous, flexuous to straight, branched, and septate. Appressoria on the hyphae were nipple-shaped or nearly absent. Conidiophores were straight, 30 to 210× 8 to 12 µm, and produced 3 to 7 immature conidia in chains with a crenate outline. Foot-cells were cylindrical, 45 to 75 ×10 to 12 µm, followed by 1 to 2 shorter cells. Conidia were hyaline, ellipsoid-ovoid to barrel-shaped, 25 to 38 × 18 to 23 µm with distinct fibrosin bodies. Germ tubes were produced from a lateral position on the conidia. Chasmothecia were not observed on the infected leaves. Based on anamorph characteristics, fungus was identified as Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Braun and Cook 2012). For molecular identification, total genomic DNA was extracted (Mukhtar et al. 2018) from fungal colonies on infected leaves of five collections separately. For each DNA sample, the part of LSU and ITS regions were amplified using primers LSU1/LSU2 and ITS1/ITS4 (Scholin et al. 1994; White et al. 1990), respectively. A BLAST search revealed 100 % sequences similarity with P. xanthii sequences reported on Ageratum conyzoides (KY274485), Eclipta prostrata (MT260063), Euphorbia hirta (KY388505), Sonchus asper (MN134013), and Verbena bonariensis (AB462804). Representative sequences (ITS: MZ613309; LSU: MZ614707) of an isolate were deposited in GenBank. The phylogenetic analysis also grouped the obtain sequences into P. xanthii clade. Pathogenicity was confirmed by gently pressing the infected leaves onto young leaves of five healthy one-month-old S. orientalis plants, while three non-inoculated plants were used as controls. All plants were maintained in a greenhouse at 25 ± 2°C. After, seven days, white powdery colonies were observed on inoculated plants, whereas controls remained mildew-free. On inoculated leaves, the fungus was morphologically and molecularly identical to the fungus on the original specimens. P. xanthii has been reported as a significant damaging pathogen on a wide range of plants in China (Farr and Rossman 2021). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on S. orientalis in China as well as worldwide. S. orientalis is one of the most important commercial Chinese medicinal herbs and the occurrence of powdery mildew is a threat to its production, quality, and marketability. References: Braun, U., and Cook, R. T. A. 2012. The Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series 11: CBS. Utrecht, The Netherlands. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases. Syst. Mycol. Microbiol. Lab., USDA ARS, 9 October 2021. Mukhtar, I., et al. 2018. Sydowia.70:155. Scholin, C. A., et al. 1994. J. Phycol. 30:999. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Zhong, Z., et al., 2019. Chin. Med. (U. K.) 14, 1-12. 10.1186/s13020-019-0260-y.

Evolution of asexual and sexual reproduction in the aspergilli.

Aspergillus nidulans has long-been used as a model organism to gain insights into the genetic basis of asexual and sexual developmental processes both in other members of the genus Aspergillus, and filamentous fungi in general. Paradigms have been established concerning the regulatory mechanisms of conidial development. However, recent studies have shown considerable genome divergence in the fungal kingdom, questioning the general applicability of findings from Aspergillus, and certain longstanding evolutionary theories have been questioned. The phylogenetic distribution of key regulatory elements of asexual reproduction in A. nidulans was investigated in a broad taxonomic range of fungi. This revealed that some proteins were well conserved in the Pezizomycotina (e.g. AbaA, FlbA, FluG, NsdD, MedA, and some velvet proteins), suggesting similar developmental roles. However, other elements (e.g. BrlA) had a more restricted distribution solely in the Eurotiomycetes, and it appears that the genetic control of sporulation seems to be more complex in the aspergilli than in some other taxonomic groups of the Pezizomycotina. The evolution of the velvet protein family is discussed based on the history of expansion and contraction events in the early divergent fungi. Heterologous expression of the A. nidulans abaA gene in Monascus ruber failed to induce development of complete conidiophores as seen in the aspergilli, but did result in increased conidial production. The absence of many components of the asexual developmental pathway from members of the Saccharomycotina supports the hypothesis that differences in the complexity of their spore formation is due in part to the increased diversity of the sporulation machinery evident in the Pezizomycotina. Investigations were also made into the evolution of sex and sexuality in the aspergilli. MAT loci were identified from the heterothallic Aspergillus (Emericella) heterothallicus and Aspergillus (Neosartorya) fennelliae and the homothallic Aspergillus pseudoglaucus (=Eurotium repens). A consistent architecture of the MAT locus was seen in these and other heterothallic aspergilli whereas much variation was seen in the arrangement of MAT loci in homothallic aspergilli. This suggested that it is most likely that the common ancestor of the aspergilli exhibited a heterothallic breeding system. Finally, the supposed prevalence of asexuality in the aspergilli was examined. Investigations were made using A. clavatus as a representative 'asexual' species. It was possible to induce a sexual cycle in A. clavatus given the correct MAT1-1 and MAT1-2 partners and environmental conditions, with recombination confirmed utilising molecular markers. This indicated that sexual reproduction might be possible in many supposedly asexual aspergilli and beyond, providing general insights into the nature of asexuality in fungi.

Roles of Aspergillus nidulans Cdc42/Rho GTPase regulators in hyphal morphogenesis and development.

The Rho-related family of GTPases are pivotal regulators of morphogenetic processes in diverse eukaryotic organisms. In the filamentous fungi two related members of this family, Cdc42 and Rac1, perform particularly important roles in the establishment and maintenance of hyphal polarity. The activity of these GTPases is tightly controlled by two sets of regulators: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the importance of Cdc42 and Rac1 in polarized hyphal growth, the morphogenetic functions of their cognate GEFs and GAPs have not been widely characterized in filamentous fungi outside the Saccharomycotina. Here we present a functional analysis of the Aspergillus nidulans homologs of the yeast GEF Cdc24 and the yeast GAP Rga1. We show that Cdc24 is required for the establishment of hyphal polarity and localizes to hyphal tips. We also show that Rga1 is necessary for the suppression of branching in developing conidiophores. During asexual development Rga1 appears to act primarily via Cdc42 and in doing so serves as a critical determinant of conidiophore architecture. Our results provide new insight into the roles of Cdc42 during development in A nidulans.

Cytosolic streaming in vegetative mycelium and aerial structures of Aspergillus niger.

Aspergillus niger forms aerial hyphae and conidiophores after a period of vegetative growth. The hyphae within the mycelium of A. niger are divided by septa. The central pore in these septa allows for cytoplasmic streaming. Here, we studied inter- and intra-compartmental streaming of the reporter protein GFP in A. niger. Expression of the gene encoding nuclear targeted GFP from the gpdA or glaA promoter resulted in strong fluorescence of nuclei within the vegetative hyphae and weak fluorescence in nuclei within the aerial structures. These data and nuclear run on experiments showed that gpdA and glaA are higher expressed in the vegetative mycelium when compared to aerial hyphae, conidiophores and conidia. Notably, gpdA or glaA driven expression of the gene encoding cytosolic GFP resulted in strongly fluorescent vegetative hyphae and aerial structures. Apparently, GFP streams from vegetative hyphae into aerial structures. This was confirmed by monitoring fluorescence of photo-activatable GFP (PA-GFP). In contrast, PA-GFP did not stream from aerial structures to vegetative hyphae. Streaming of PA-GFP within vegetative hyphae or within aerial structures of A. niger occurred at a rate of 10-15 µm s(-1). Taken together, these results not only show that GFP streams from the vegetative mycelium to aerial structures but it also indicates that its encoding RNA is not streaming. Absence of RNA streaming would explain why distinct RNA profiles were found in aerial structures and the vegetative mycelium by nuclear run on analysis and micro-array analysis.

Development in Aspergillus.

The genus Aspergillus represents a diverse group of fungi that are among the most abundant fungi in the world. Germination of a spore can lead to a vegetative mycelium that colonizes a substrate. The hyphae within the mycelium are highly heterogeneous with respect to gene expression, growth, and secretion. Aspergilli can reproduce both asexually and sexually. To this end, conidiophores and ascocarps are produced that form conidia and ascospores, respectively. This review describes the molecular mechanisms underlying growth and development of Aspergillus.

PdStuA Is a Key Transcription Factor Controlling Sporulation, Hydrophobicity, and Stress Tolerance in Penicillium digitatum.

Penicillium digitatum has become one of the main pathogens in citrus due to its high spore production and easy spread. In this study, the function of the APSES transcription factor StuA in P. digitatum was characterized, and the results indicated that it was involved in conidium and conidiophore development. No conidiophores were observed in the mycelium of the ∆PdStuA mutant that had grown for two days, while an abnormal conidiophore was found after another two days of incubation, and only small thin phialides as well as a very small number of spores were formed at the top of the hyphae. Moreover, it was observed that the ∆PdStuA mutant showed various defects, such as reduced hydrophobicity and decreased tolerance to cell wall inhibitors and H2O2. Compared to the original P. digitatum, the colony diameter of the ∆PdStuA mutant was not significantly affected, but the growth of aerial hyphae was obviously induced. In in vivo experiments, the spore production of the ∆PdStuA mutant grown on citrus fruit was remarkably decreased; however, there was no significant difference in the lesion diameter between the mutant and original strain. It could be inferred that less spore production might result in reduced spread in citrus, thereby reducing the green mold infection in citrus fruit during storage. This study provided a gene, PdStuA, which played key role in the sporulation of P. digitatum, and the results might provide a reference for the molecular mechanisms of sporulation in P. digitatum.

Contribution of the Tyrosinase (MoTyr) to Melanin Synthesis, Conidiogenesis, Appressorium Development, and Pathogenicity in Magnaporthe oryzae.

Dihydroxynapthalene-(DHN) and L-dihydroxyphenylalanine (L-DOPA) are two types of dominant melanin in fungi. Fungal melanins with versatile functions are frequently associated with pathogenicity and stress tolerance. In rice blast fungus, Magnaporthe oryzae, DHN melanin is essential to maintain the integrity of the infectious structure, appressoria; but the role of the tyrosinase-derived L-DOPA melanin is still unknown. Here, we have genetically and biologically characterized a tyrosinase gene (MoTyr) in M. oryzae. MoTyr encodes a protein of 719 amino acids that contains the typical CuA and CuB domains of tyrosinase. The deletion mutant of MoTyr (ΔMoTyr) was obtained by using a homologous recombination approach. Phenotypic analysis showed that conidiophore stalks and conidia formation was significantly reduced in ΔMoTyr. Under different concentrations of glycerol and PEG, more appressoria collapsed in the mutant strains than in the wild type, suggesting MoTyr is associated with the integrity of the appressorium wall. Melanin measurement confirmed that MoTyr loss resulted in a significant decrease in melanin synthesis. Accordingly, the loss of MoTyr stunted the conidia germination under stress conditions. Importantly, the MoTyr deletion affected both infection and pathogenesis stages. These results suggest that MoTyr, like DHN pigment synthase, plays a key role in conidiophore stalks formation, appressorium integrity, and pathogenesis of M. oryzae, revealing a potential drug target for blast disease control.

Contribution of the Mitochondrial Carbonic Anhydrase (MoCA1) to Conidiogenesis and Pathogenesis in Magnaporthe oryzae.

The interconversion of CO2 and HCO3 - catalyzed by carbonic anhydrases (CAs) is a fundamental biochemical process in organisms. During mammalian-pathogen interaction, both host and pathogen CAs play vital roles in resistance and pathogenesis; during planta-pathogen interaction, however, plant CAs function in host resistance but whether pathogen CAs are involved in pathogenesis is unknown. Here, we biologically characterized the Magnaporthe oryzae CA (MoCA1). Through detecting the DsRED-tagged proteins, we observed the fusion MoCA1 in the mitochondria of M. oryzae. Together with the measurement of CA activity, we confirmed that MoCA1 is a mitochondrial zinc-binding CA. MoCA1 expression, upregulated with H2O2 or NaHCO3 treatment, also showed a drastic upregulation during conidiogenesis and pathogenesis. When MoCA1 was deleted, the mutant ΔMoCA1 was defective in conidiophore development and pathogenicity. 3,3'-Diaminobenzidine (DAB) staining indicated that more H2O2 accumulated in ΔMoCA1; accordingly, ATPase genes were downregulated and ATP content decreased in ΔMoCA1. Summarily, our data proved the involvement of the mitochondrial MoCA1 in conidiogenesis and pathogenesis in the rice blast fungus. Considering the previously reported HCO3 - transporter MoAE4, we propose that MoCA1 in cooperation with MoAE4 constitutes a HCO3 - homeostasis-mediated disease pathway, in which MoCA1 and MoAE4 can be a drug target for disease control.

Characterizing the gene-environment interaction underlying natural morphological variation in Neurospora crassa conidiophores using high-throughput phenomics and transcriptomics.

Neurospora crassa propagates through dissemination of conidia, which develop through specialized structures called conidiophores. Recent work has identified striking variation in conidiophore morphology, using a wild population collection from Louisiana, United States of America to classify 3 distinct phenotypes: Wild-Type, Wrap, and Bulky. Little is known about the impact of these phenotypes on sporulation or germination later in the N. crassa life cycle, or about the genetic variation that underlies them. In this study, we show that conidiophore morphology likely affects colonization capacity of wild N. crassa isolates through both sporulation distance and germination on different carbon sources. We generated and crossed homokaryotic strains belonging to each phenotypic group to more robustly fit a model for and estimate heritability of the complex trait, conidiophore architecture. Our fitted model suggests at least 3 genes and 2 epistatic interactions contribute to conidiophore phenotype, which has an estimated heritability of 0.47. To uncover genes contributing to these phenotypes, we performed RNA-sequencing on mycelia and conidiophores of strains representing each of the 3 phenotypes. Our results show that the Bulky strain had a distinct transcriptional profile from that of Wild-Type and Wrap, exhibiting differential expression patterns in clock-controlled genes (ccgs), the conidiation-specific gene con-6, and genes implicated in metabolism and communication. Combined, these results present novel ecological impacts of and differential gene expression underlying natural conidiophore morphological variation, a complex trait that has not yet been thoroughly explored.