Assaying Effector Cell-to-Cell Mobility in Plant Tissues Identifies Hypermobility and Indirect Manipulation of Plasmodesmata.

In plants, plasmodesmata establish cytoplasmic continuity between cells to allow for communication and resource exchange across the cell wall. While plant pathogens use plasmodesmata as a pathway for both molecular and physical invasion, the benefits of molecular invasion (cell-to-cell movement of pathogen effectors) are poorly understood. To establish a methodology for identification and characterization of the cell-to-cell mobility of effectors, we performed a quantitative live imaging-based screen of candidate effectors of the fungal pathogen Colletotrichum higginsianum. We predicted C. higginsianum effectors by their expression profiles, the presence of a secretion signal, and their predicted and in planta localization when fused to green fluorescent protein. We assayed for cell-to-cell mobility of nucleocytosolic effectors and identified 14 that are cell-to-cell mobile. We identified that three of these effectors are "hypermobile," showing cell-to-cell mobility greater than expected for a protein of that size. To explore the mechanism of hypermobility, we chose two hypermobile effectors and measured their impact on plasmodesmata function and found that even though they show no direct association with plasmodesmata, each increases the transport capacity of plasmodesmata. Thus, our methods for quantitative analysis of cell-to-cell mobility of candidate microbe-derived effectors, or any suite of host proteins, can identify cell-to-cell hypermobility and offer greater understanding of how proteins affect plasmodesmal function and intercellular connectivity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Comparative phyloproteomics identifies conserved plasmodesmal proteins.

Plasmodesmata are cytosolic bridges, lined by the plasma membrane and traversed by endoplasmic reticulum; plasmodesmata connect cells and tissues, and are critical for many aspects of plant biology. While plasmodesmata are notoriously difficult to extract, tissue fractionation and proteomic analyses can yield valuable knowledge of their composition. Here we have generated two novel proteomes to expand tissue and taxonomic representation of plasmodesmata: one from mature Arabidopsis leaves and one from the moss Physcomitrium patens, and leveraged these and existing data to perform a comparative analysis to identify evolutionarily conserved protein families that are associated with plasmodesmata. Thus, we identified ß-1,3-glucanases, C2 lipid-binding proteins, and tetraspanins as core plasmodesmal components that probably serve as essential structural or functional components. Our approach has not only identified elements of a conserved plasmodesmal proteome, but also demonstrated the added power offered by comparative analysis for recalcitrant samples. Conserved plasmodesmal proteins establish a basis upon which ancient plasmodesmal function can be further investigated to determine the essential roles these structures play in multicellular organism physiology in the green lineages.

Diffusion and bulk flow of amino acids mediate calcium waves in plants.

In plants, a variety of stimuli trigger long-range calcium signals that travel rapidly along the vasculature to distal tissues via poorly understood mechanisms. Here, we use quantitative imaging and analysis to demonstrate that traveling calcium waves are mediated by diffusion and bulk flow of amino acid chemical messengers. We propose that wounding triggers release of amino acids that diffuse locally through the apoplast, activating the calcium-permeable channel GLUTAMATE RECEPTOR-LIKE 3.3 as they pass. Over long distances through the vasculature, the wound-triggered dynamics of a fluorescent tracer show that calcium waves are likely driven by bulk flow of a channel-activating chemical. We observed that multiple stimuli trigger calcium waves with similar dynamics, but calcium waves alone cannot initiate all systemic defense responses, suggesting that mobile chemical messengers are a core component of complex systemic signaling in plants.

Effectors from a Bacterial Vector-Borne Pathogen Exhibit Diverse Subcellular Localization, Expression Profiles, and Manipulation of Plant Defense.

Climate change is predicted to increase the prevalence of vector-borne disease due to expansion of insect populations. 'Candidatus Liberibacter solanacearum' is a phloem-limited pathogen associated with multiple economically important diseases in solanaceous crops. Little is known about the strategies and pathogenicity factors 'Ca. L. solanacearum' uses to colonize its vector and host. We determined the 'Ca. L. solanacearum' effector repertoire by predicting proteins secreted by the general secretory pathway across four different 'Ca. L. solanacearum' haplotypes, investigated effector localization in planta, and profiled effector expression in the vector and host. The localization of 'Ca. L. solanacearum' effectors in Nicotiana spp. revealed diverse eukaryotic subcellular targets. The majority of tested effectors were unable to suppress plant immune responses, indicating they possess unique activities. Expression profiling in tomato and the psyllid Bactericera cockerelli indicated 'Ca. L. solanacearum' differentially interacts with its host and vector and can switch effector expression in response to these environments. This study reveals 'Ca. L. solanacearum' effectors possess complex expression patterns, target diverse host organelles and the majority are unable to suppress host immune responses. A mechanistic understanding of 'Ca. L. solanacearum' effector function will reveal novel targets and provide insight into phloem biology. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Three Common Symbiotic ABC Subfamily B Transporters in Medicago truncatula Are Regulated by a NIN-Independent Branch of the Symbiosis Signaling Pathway.

Several ATP-binding cassette (ABC) transporters involved in the arbuscular mycorrhizal symbiosis and nodulation have been identified. We describe three previously unreported ABC subfamily B transporters, named AMN1, AMN2, and AMN3 (ABCB for mycorrhization and nodulation), that are expressed early during infection by rhizobia and arbuscular mycorrhizal fungi. These ABCB transporters are strongly expressed in symbiotically infected tissues, including in root-hair cells with rhizobial infection threads and arbusculated cells. During nodulation, the expression of these genes is highly induced by rhizobia and purified Nod factors and is dependent on DMI3 but is not dependent on other known major regulators of infection, such as NIN, NSP1, or NSP2. During mycorrhization their expression is dependent on DMI3 and RAM1 but not on NSP1 and NSP2. Therefore, they may be commonly regulated through a distinct branch of the common symbiotic pathway. Mutants with exonic Tnt1-transposon insertions were isolated for all three genes. None of the single or double mutants showed any differences in colonization by either rhizobia or mycorrhizal fungi, but the triple amn1 amn2 amn3 mutant showed an increase in nodule number. Further studies are needed to identify potential substrates of these transporters and understand their roles in these beneficial symbioses.[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.

Chitin perception in plasmodesmata characterizes submembrane immune-signaling specificity in plants.

The plasma membrane (PM) is composed of heterogeneous subdomains, characterized by differences in protein and lipid composition. PM receptors can be dynamically sorted into membrane domains to underpin signaling in response to extracellular stimuli. In plants, the plasmodesmal PM is a discrete microdomain that hosts specific receptors and responses. We exploited the independence of this PM domain to investigate how membrane domains can independently integrate a signal that triggers responses across the cell. Focusing on chitin signaling, we found that responses in the plasmodesmal PM require the LysM receptor kinases LYK4 and LYK5 in addition to LYM2. Chitin induces dynamic changes in the localization, association, or mobility of these receptors, but only LYM2 and LYK4 are detected in the plasmodesmal PM. We further uncovered that chitin-induced production of reactive oxygen species and callose depends on specific signaling events that lead to plasmodesmata closure. Our results demonstrate that distinct membrane domains can integrate a common signal with specific machinery that initiates discrete signaling cascades to produce a localized response.

A protein complex required for polar growth of rhizobial infection threads.

During root nodule symbiosis, intracellular accommodation of rhizobia by legumes is a prerequisite for nitrogen fixation. For many legumes, rhizobial colonization initiates in root hairs through transcellular infection threads. In Medicago truncatula, VAPYRIN (VPY) and a putative E3 ligase LUMPY INFECTIONS (LIN) are required for infection thread development but their cellular and molecular roles are obscure. Here we show that LIN and its homolog LIN-LIKE interact with VPY and VPY-LIKE in a subcellular complex localized to puncta both at the tip of the growing infection thread and at the nuclear periphery in root hairs and that the punctate accumulation of VPY is positively regulated by LIN. We also show that an otherwise nuclear and cytoplasmic exocyst subunit, EXO70H4, systematically co-localizes with VPY and LIN during rhizobial infection. Genetic analysis shows that defective rhizobial infection in exo70h4 is similar to that in vpy and lin. Our results indicate that VPY, LIN and EXO70H4 are part of the symbiosis-specific machinery required for polar growth of infection threads.

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

Plant synthetic biology and cereal engineering depend on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well-established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimization, intron-mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small-scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multigenic traits where the expression of each transgene needs to be precisely regulated. Here, we present data on the characterization of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimization and intron-mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimization and intron-mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use and also propose a general set of principles and guidelines for those engineering cereal species.

NIN Acts as a Network Hub Controlling a Growth Module Required for Rhizobial Infection.

The symbiotic infection of root cells by nitrogen-fixing rhizobia during nodulation requires the transcription factor Nodule Inception (NIN). Our root hair transcriptomic study extends NIN's regulon to include Rhizobium Polar Growth and genes involved in cell wall modification, gibberellin biosynthesis, and a comprehensive group of nutrient (N, P, and S) uptake and assimilation genes, suggesting that NIN's recruitment to nodulation was based on its role as a growth module, a role shared with other NIN-Like Proteins. The expression of jasmonic acid genes in nin suggests the involvement of NIN in the resolution of growth versus defense outcomes. We find that the regulation of the growth module component Nodulation Pectate Lyase by NIN, and its function in rhizobial infection, are conserved in hologalegina legumes, highlighting its recruitment as a major event in the evolution of nodulation. We find that Nodulation Pectate Lyase is secreted to the infection chamber and the lumen of the infection thread. Gene network analysis using the transcription factor mutants for ERF Required for Nodulation1 and Nuclear Factor-Y Subunit A1 confirms hierarchical control of NIN over Nuclear Factor-Y Subunit A1 and shows that ERF Required for Nodulation1 acts independently to control infection. We conclude that while NIN shares functions with other NIN-Like Proteins, the conscription of key infection genes to NIN's control has made it a central regulatory hub for rhizobial infection.

Fatty acids in arbuscular mycorrhizal fungi are synthesized by the host plant.

Plants form beneficial associations with arbuscular mycorrhizal fungi, which facilitate nutrient acquisition from the soil. In return, the fungi receive organic carbon from the plants. The transcription factor RAM1 (REQUIRED FOR ARBUSCULAR MYCORRHIZATION 1) is crucial for this symbiosis, and we demonstrate that it is required and sufficient for the induction of a lipid biosynthetic pathway that is expressed in plant cells accommodating fungal arbuscules. Lipids are transferred from the plant to mycorrhizal fungi, which are fatty acid auxotrophs, and this lipid export requires the glycerol-3-phosphate acyltransferase RAM2, a direct target of RAM1. Our work shows that in addition to sugars, lipids are a major source of organic carbon delivered to the fungus, and this is necessary for the production of fungal lipids.

A Medicago truncatula Cystathionine-ß-Synthase-like Domain-Containing Protein Is Required for Rhizobial Infection and Symbiotic Nitrogen Fixation.

The symbiosis between leguminous plants and soil rhizobia culminates in the formation of nitrogen-fixing organs called nodules that support plant growth. Two Medicago truncatula Tnt1-insertion mutants were identified that produced small nodules, which were unable to fix nitrogen effectively due to ineffective rhizobial colonization. The gene underlying this phenotype was found to encode a protein containing a putative membrane-localized domain of unknown function (DUF21) and a cystathionine-ß-synthase domain. The cbs1 mutants had defective infection threads that were sometimes devoid of rhizobia and formed small nodules with greatly reduced numbers of symbiosomes. We studied the expression of the gene, designated M truncatula Cystathionine-ß-Synthase-like1 (MtCBS1), using a promoter-ß-glucuronidase gene fusion, which revealed expression in infected root hair cells, developing nodules, and in the invasion zone of mature nodules. An MtCBS1-GFP fusion protein localized itself to the infection thread and symbiosomes. Nodulation factor-induced Ca(2+) responses were observed in the cbs1 mutant, indicating that MtCBS1 acts downstream of nodulation factor signaling. MtCBS1 expression occurred exclusively during Medicago-rhizobium symbiosis. Induction of MtCBS1 expression during symbiosis was found to be dependent on Nodule Inception (NIN), a key transcription factor that controls both rhizobial infection and nodule organogenesis. Interestingly, the closest homolog of MtCBS1, MtCBS2, was specifically induced in mycorrhizal roots, suggesting common infection mechanisms in nodulation and mycorrhization. Related proteins in Arabidopsis have been implicated in cell wall maturation, suggesting a potential role for CBS1 in the formation of the infection thread wall.

Conservation and divergence of the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway in two plant-pathogenic fungi: Fusarium graminearum and F. verticillioides.

The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway is a central signalling cascade that transmits extracellular stimuli and governs cell responses through the second messenger cAMP. The importance of cAMP signalling in fungal biology has been well documented and the key conserved components, adenylate cyclase (AC) and the catalytic subunit of PKA (CPKA), have been functionally characterized. However, other genes involved in this signalling pathway and their regulation are not well understood in filamentous fungi. Here, we performed a comparative transcriptomics analysis of AC and CPKA mutants in two closely related fungi: Fusarium graminearum (Fg) and F. verticillioides (Fv). Combining available Fg transcriptomics and phenomics data, we reconstructed the Fg cAMP signalling pathway. We developed a computational program that combines sequence conservation and patterns of orthologous gene expression to facilitate global transcriptomics comparisons between different organisms. We observed highly correlated expression patterns for most orthologues (80%) between Fg and Fv. We also identified a subset of 482 (6%) diverged orthologues, whose expression under all conditions was at least 50% higher in one genome than in the other. This enabled us to dissect the conserved and unique portions of the cAMP-PKA pathway. Although the conserved portions controlled essential functions, such as metabolism, the cell cycle, chromatin remodelling and the oxidative stress response, the diverged portions had species-specific roles, such as the production and detoxification of secondary metabolites unique to each species. The evolution of the cAMP-PKA signalling pathway seems to have contributed directly to fungal divergence and niche adaptation.

Cytokinin responses counterpoint auxin signaling during rhizobial infection.

The transcriptomics approach to study gene expression in root hairs from M. truncatula has shed light on the developmental events during rhizobial infection and the underlying hormone responses. This approach revealed the induction of several cyclins and an aurora kinase which suggests that the cell-division machinery plays a role in rhizobial infection. Changes in the cell cycle in plants are governed by hormones, in particular auxin and cytokinin. Through gene expression and genetic analyses, we have shown auxin plays a role during rhizobial infection. Here we provide further analysis of the data showing the induction of a set of cytokinin signaling components. These include genes encoding 2 cytokinin-activating enzymes, the cytokinin receptor CRE1, and 5 type-A cytokinin response regulators. We discuss the possible interactions between auxin and cytokinin signaling during the infection process. We also consider a potential role for cytokinin signaling in rhizobial attachment.

The root hair "infectome" of Medicago truncatula uncovers changes in cell cycle genes and reveals a requirement for Auxin signaling in rhizobial infection.

Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads.

Metabolome and transcriptome of the interaction between Ustilago maydis and Fusarium verticillioides in vitro.

The metabolome and transcriptome of the maize-infecting fungi Ustilago maydis and Fusarium verticillioides were analyzed as the two fungi interact. Both fungi were grown for 7 days in liquid medium alone or together in order to study how this interaction changes their metabolomic and transcriptomic profiles. When grown together, decreased biomass accumulation occurs for both fungi after an initial acceleration of growth compared to the biomass changes that occur when grown alone. The biomass of U. maydis declined most severely over time and may be attributed to the action of F. verticillioides, which secretes toxic secondary metabolites and expresses genes encoding adhesive and cell wall-degrading proteins at higher levels than when grown alone. U. maydis responds to cocultivation by expressing siderophore biosynthetic genes and more highly expresses genes potentially involved in toxin biosynthesis. Also, higher expression was noted for clustered genes encoding secreted proteins that are unique to U. maydis and that may play a role during colonization of maize. Conversely, decreased gene expression was seen for U. maydis genes encoding the synthesis of ustilagic acid, mannosylerythritol D, and another uncharacterized metabolite. Ultimately, U. maydis is unable to react efficiently to the toxic response of F. verticillioides and proportionally loses more biomass. This in vitro study clarifies potential mechanisms of antagonism between these two fungi that also may occur in the soil or in maize, niches for both fungi where they likely interact in nature.

Npc1 is involved in sterol trafficking in the filamentous fungus Fusarium graminearum.

The ortholog of the human gene NPC1 was identified in the plant pathogenic, filamentous fungus Fusarium graminearum by shared amino acid sequence, protein domain structure and cellular localization of the mature fungal protein. The FusariumNpc1 gene shares 34% amino acid sequence identity and 51% similarity to the human gene, has similar domain structure and is constitutively expressed, although up-regulated in ungerminated macroconidia and ascospores. GFP-tagged Npc1p localizes to the fungal vacuolar membrane. Cultures derived from a Δnpc1 mutant strain contain significantly more ergosterol than cultures of the wildtype. Staining with the fluorescent, sterol binding dye filipin, shows that ergosterol accumulates in vacuoles of the Δnpc1 mutant but not the wildtype strain. The Δnpc1 mutant has a temperature dependent reduction in growth and greater sensitivity to the ergosterol synthesis inhibiting fungicide tebuconazole compared with the wildtype strain or the mutant complemented with wildtype Npc1. The mutant also is significantly reduced in pathogenicity to wheat. Our results are consistent with the interpretation that Npc1p is important for normal transport of ergosterol from the vacuole and is essential for proper membrane function under particular environmental conditions.

The transcription factor FgStuAp influences spore development, pathogenicity, and secondary metabolism in Fusarium graminearum.

Fusarium graminearum is an important plant-pathogenic fungus and the major cause of cereal head blight. Here, we report the functional analysis of FgStuA, the gene for a transcription factor with homology to key developmental regulators in fungi. The deletion mutant was greatly reduced in pathogenicity on wheat heads and in production of secondary metabolites. Spore production was significantly impaired in ΔFgStuA, which did not develop perithecia and sexual ascospores, and lacked conidiophores and phialides, leading to delayed production of aberrant macroconidia. FgStuAp appears to act as a global regulator that may affect many diverse aspects of the life cycle of F. graminearum. Transcriptome analysis shows that thousands of genes are differentially expressed in the mutant during asexual sporulation and infection of wheat heads and under conditions that induce secondary metabolites, including many that could account for the mutant phenotypes observed. The primary regulatory targets of FgStuAp are likely genes involved in cell-cycle control, and the predicted FgStuAp sequence has an APSES domain, with homology to helix-loop-helix proteins involved in cell-cycle regulation. The Aspergillus StuAp response element (A/TCGCGT/ANA/C) was found highly enriched in the promoter sequences of cell-cycle genes, which was upregulated in the ΔFgStuA deletion mutant.

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Systematic discovery of regulatory motifs in Fusarium graminearum by comparing four Fusarium genomes.

BACKGROUND: Fusarium graminearum (Fg), a major fungal pathogen of cultivated cereals, is responsible for billions of dollars in agriculture losses. There is a growing interest in understanding the transcriptional regulation of this organism, especially the regulation of genes underlying its pathogenicity. The generation of whole genome sequence assemblies for Fg and three closely related Fusarium species provides a unique opportunity for such a study. RESULTS: Applying comparative genomics approaches, we developed a computational pipeline to systematically discover evolutionarily conserved regulatory motifs in the promoter, downstream and the intronic regions of Fg genes, based on the multiple alignments of sequenced Fusarium genomes. Using this method, we discovered 73 candidate regulatory motifs in the promoter regions. Nearly 30% of these motifs are highly enriched in promoter regions of Fg genes that are associated with a specific functional category. Through comparison to Saccharomyces cerevisiae (Sc) and Schizosaccharomyces pombe (Sp), we observed conservation of transcription factors (TFs), their binding sites and the target genes regulated by these TFs related to pathways known to respond to stress conditions or phosphate metabolism. In addition, this study revealed 69 and 39 conserved motifs in the downstream regions and the intronic regions, respectively, of Fg genes. The top intronic motif is the splice donor site. For the downstream regions, we noticed an intriguing absence of the mammalian and Sc poly-adenylation signals among the list of conserved motifs. CONCLUSION: This study provides the first comprehensive list of candidate regulatory motifs in Fg, and underscores the power of comparative genomics in revealing functional elements among related genomes. The conservation of regulatory pathways among the Fusarium genomes and the two yeast species reveals their functional significance, and provides new insights in their evolutionary importance among Ascomycete fungi.

CopA:GFP localizes to putative Golgi equivalents in Aspergillus nidulans.

The Golgi complex is a main component of the eukaryotic secretory system and functions to modify nascent proteins sent from the endoplasmic reticulum. Ultrastructural studies of filamentous fungi have shown Golgi to be individual smooth membrane cisternae that are referred to as Golgi equivalents or dictyosomes. The Aspergillus nidulans copA gene encodes a homolog of mammalian coat protein (alpha-COP), a constituent of the Golgi-localized COPI vesicle coat. Here, the localization of A. nidulansalpha-COP was examined in live cells using the reporter green fluorescent protein (GFP). CopA:GFP localized to putative Golgi equivalents that were concentrated at hyphal tips. The localization was disrupted by the fungal metabolite brefeldin A. To investigate the significance of the microtubule cytoskeleton in the localization of putative Golgi equivalents, the copA:gfp fusion was expressed in a temperature-sensitive dynein mutant. In addition, a wild-type strain expressing copA:gfp was treated with the microtubule-disrupting drug nocodazole. The results suggest that the microtubule cytoskeleton is not the primary mechanism of localizing putative Golgi equivalents in A. nidulans.