Dr. Alexander Lichius
CURRENT RESEARCH FOCUS
As University Assistant Postdoc at the Institute of Microbiology, University of Innsbruck I am now investigating pre-contact host sensing and positive chemotropic growth of the mycoparasita Trichoderma atroviride to its host fungi. I have helped to generate T. atroviride strains that express fluorescently labelled G-protein-coupled receptors (GPCRs) and downstream signalling partners of the SUR7-family. In collaboration with the Biophysics group of Prof. Gerhard Schütz of the Vienna University of Technology, we are in the process of adapting single-molecule tracking and super resolution live-cell imaging technologies for the use in filamentous fungi, in order to monitor subcellular dynamics of the labelled target proteins during mycoparasitic interactions in 4D.
In a second line of research I have implemented an improved CRIB reporter to identify individual hyphae of T. atroviride actively involved in mycoparasitism signalling at the time of observation. We are now in the process of adapting automated tip tracking software to analyse CRIB dynamics during pre-contact chemotropism towards host hyphae and their morphogenic transition to mycoparasitic attack.
With these analyses we hope to better understand how the mycoparasitic properties of T. atroviride can be used more efficiently for the biocontrol of fungal plant pathogens in agriculture
PAST AND ONGOING RESEARCH INTERESTS
As Project Assitant Postdoc at the Vienna University of Technology I have characterised the influences of nucleo-cytoplasmic transcription factor shuttling on the expression of cellulase and xylanase genes in Trichoderma reesei. I have constructed GFP-labelled fusion proteins of XYR1 and CRE1 and quantified their very distinct nuclear import and export dynamics on various cellulase and xylanase inducing and non-inducing carbon sources. I furthermore rescued the cellulase-negative T. reesei mutant strain QM9136 through the integration of a functional copy of GFP-XYR1, and reproduced the defective QM9136-phenotype in QM9414 by introducing a point-mutation in the xyr1 ORF which only generates a C-terminally truncated version of the transcription factor. Both studies together demonstrated that the C-terminal regulatory domain of XYR1 is essential for its autoregulatory-feedback mechanism required to activate high-level cellulase and xylanase gene expression upon induction. A more precise control over nuclear import of cellulase and xylanase inducing transcription factors provides means to boost industrial enzyme production, which is important to render the biotechnological application of this fungus economically profitable for the production of 2nd generation biofuels and other biorefineries.
During my Postdoc stay at CICESE I have focused on the characterisation of the architecture and spatio-temporal dynamics of the filamentous fungal polarisome. I conducted comparative live cell imaging analyses of the key polarisome components SPA-2, BUD-6 and BNI-1 during various morphogenetic processes of colony development. Another main line of research explored the application of the novel life-cell imaging markers developed during my Ph.D. to investigate molecular and structural aspects of polarised tip growth in conidial germlings and mature hyphae of Neurospora crassa, in order to formulate new models of how maintenance and reorientation of polarised apical growth is achieved in the absence or presence of the Spitzenkörper, respectively.
The main focus of my PhD research at Edinburgh University was on molecular details of eukaryotic cell-cell fusion. I have identified novel fusion mutants which suggested important roles for RhoGTPase, MAP kinase and redox signalling processes during CAT-mediated cell fusion. Using a combination of genetic, molecular biology, biochemical and live cell imaging techniques I further characterized specific roles of selected target proteins during the fusion process. These included MAP kinases and GTPases which are recruited and activated at the fusion site to induce cytoskeletal and cell wall rearrangements. I constructed novel live cell imaging markers to visualize F-actin (Lifeact-TagRP/-TagRFP-T) and activated GTPases (CRIB-sGFP/CRIB-TagRFP-T) for the first time in filamentous fungi, and used these markers to study cell polarization and directed hyphal tip growth during cell fusion in Neurospora crassa.
Supervisors: Prof. Susanne Zeilinger-Migsich, Prof. Christian P. Kubicek, Prof. Ernestina Castro-Longoria, and Prof. Nick D. Read
Phone: Tel.: +43 512 507-51256
Address: Dr. Alexander Lichius
University of Innsbruck
Institute of Microbiology
6th Floor, Room 06-04
Technikerstrasse 25d
6020 Innsbruck
Austria
As University Assistant Postdoc at the Institute of Microbiology, University of Innsbruck I am now investigating pre-contact host sensing and positive chemotropic growth of the mycoparasita Trichoderma atroviride to its host fungi. I have helped to generate T. atroviride strains that express fluorescently labelled G-protein-coupled receptors (GPCRs) and downstream signalling partners of the SUR7-family. In collaboration with the Biophysics group of Prof. Gerhard Schütz of the Vienna University of Technology, we are in the process of adapting single-molecule tracking and super resolution live-cell imaging technologies for the use in filamentous fungi, in order to monitor subcellular dynamics of the labelled target proteins during mycoparasitic interactions in 4D.
In a second line of research I have implemented an improved CRIB reporter to identify individual hyphae of T. atroviride actively involved in mycoparasitism signalling at the time of observation. We are now in the process of adapting automated tip tracking software to analyse CRIB dynamics during pre-contact chemotropism towards host hyphae and their morphogenic transition to mycoparasitic attack.
With these analyses we hope to better understand how the mycoparasitic properties of T. atroviride can be used more efficiently for the biocontrol of fungal plant pathogens in agriculture
PAST AND ONGOING RESEARCH INTERESTS
As Project Assitant Postdoc at the Vienna University of Technology I have characterised the influences of nucleo-cytoplasmic transcription factor shuttling on the expression of cellulase and xylanase genes in Trichoderma reesei. I have constructed GFP-labelled fusion proteins of XYR1 and CRE1 and quantified their very distinct nuclear import and export dynamics on various cellulase and xylanase inducing and non-inducing carbon sources. I furthermore rescued the cellulase-negative T. reesei mutant strain QM9136 through the integration of a functional copy of GFP-XYR1, and reproduced the defective QM9136-phenotype in QM9414 by introducing a point-mutation in the xyr1 ORF which only generates a C-terminally truncated version of the transcription factor. Both studies together demonstrated that the C-terminal regulatory domain of XYR1 is essential for its autoregulatory-feedback mechanism required to activate high-level cellulase and xylanase gene expression upon induction. A more precise control over nuclear import of cellulase and xylanase inducing transcription factors provides means to boost industrial enzyme production, which is important to render the biotechnological application of this fungus economically profitable for the production of 2nd generation biofuels and other biorefineries.
During my Postdoc stay at CICESE I have focused on the characterisation of the architecture and spatio-temporal dynamics of the filamentous fungal polarisome. I conducted comparative live cell imaging analyses of the key polarisome components SPA-2, BUD-6 and BNI-1 during various morphogenetic processes of colony development. Another main line of research explored the application of the novel life-cell imaging markers developed during my Ph.D. to investigate molecular and structural aspects of polarised tip growth in conidial germlings and mature hyphae of Neurospora crassa, in order to formulate new models of how maintenance and reorientation of polarised apical growth is achieved in the absence or presence of the Spitzenkörper, respectively.
The main focus of my PhD research at Edinburgh University was on molecular details of eukaryotic cell-cell fusion. I have identified novel fusion mutants which suggested important roles for RhoGTPase, MAP kinase and redox signalling processes during CAT-mediated cell fusion. Using a combination of genetic, molecular biology, biochemical and live cell imaging techniques I further characterized specific roles of selected target proteins during the fusion process. These included MAP kinases and GTPases which are recruited and activated at the fusion site to induce cytoskeletal and cell wall rearrangements. I constructed novel live cell imaging markers to visualize F-actin (Lifeact-TagRP/-TagRFP-T) and activated GTPases (CRIB-sGFP/CRIB-TagRFP-T) for the first time in filamentous fungi, and used these markers to study cell polarization and directed hyphal tip growth during cell fusion in Neurospora crassa.
Supervisors: Prof. Susanne Zeilinger-Migsich, Prof. Christian P. Kubicek, Prof. Ernestina Castro-Longoria, and Prof. Nick D. Read
Phone: Tel.: +43 512 507-51256
Address: Dr. Alexander Lichius
University of Innsbruck
Institute of Microbiology
6th Floor, Room 06-04
Technikerstrasse 25d
6020 Innsbruck
Austria
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Papers by Dr. Alexander Lichius
in sterol biosynthesis, which result in the accumulation of precursor
molecules, are commonly associated with cellular disorders and
disease. However, the effects of these sterol precursors on the
metabolism, signaling, and behavior of cells are only poorly
understood. In this study, we show that the accumulation of only
ergosterol precursors with a conjugated double bond in their
aliphatic side chain specifically disrupts cell–cell communication
and fusion in the fungus Neurospora crassa. Genetically identical
germinating spores of this fungus undergo cell–cell fusion, thereby
forming a highly interconnected supracellular network during colony
initiation. Before fusion, the cells use an unusual signaling
mechanism that involves the coordinated and alternating switching
between signal sending and receiving states of the two fusion partners.
Accumulation of only ergosterol precursors with a conjugated
double bond in their aliphatic side chain disrupts this coordinated
cell–cell communication and suppresses cell fusion. These specific
sterol precursors target a single ERK-like mitogen-activated protein
(MAP) kinase (MAK-1)-signaling cascade, whereas a secondMAP kinase
pathway (MAK-2),which is also involved in cell fusion, is unaffected.
These observations indicate that a minor specific change in sterol
structure can exert a strong detrimental effect on a key signaling
pathway of the cell, resulting in the absence of cell fusion.
hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries.
The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this
procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the
mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared
it to that of its progenitor T. reesei QM6a.
Results: In QM9136, we detected a surprisingly low number of mutagenic events in the promoter and coding regions
of genes, i.e. only eight indels and six single nucleotide variants. One of these indels led to a frame-shift in the Zn2Cys6
transcription factor XYR1, the general regulator of cellulase and xylanase expression, and resulted in its C-terminal
truncation by 140 amino acids. Retransformation of strain QM9136 with the wild-type xyr1 allele fully recovered the
ability to produce cellulases, and is thus the reason for the cellulase-negative phenotype. Introduction of an engineered
xyr1 allele containing the truncating point mutation into the moderate producer T. reesei QM9414 rendered this strain
also cellulase-negative. The correspondingly truncated XYR1 protein was still able to enter the nucleus, but failed to be
expressed over the basal constitutive level.
Conclusion: The missing 140 C-terminal amino acids of XYR1 are therefore responsible for its previously observed
auto-regulation which is essential for cellulases to be expressed. Our data present a working example of the use of
genome sequencing leading to a functional explanation of the QM9136 cellulase-negative phenotype.
Keywords: Single nucleotide polymorphism, SNP, Indel, Comparative genomics, Classical mutant, XYR1, Transcription
factor shuttling, Cellulases, Trichoderma reesei, QM9136
insights into chemoattractive mechanisms in recent years. Still, identification of specific chemoattractant molecules,
cognate receptors and downstream signaling pathways is strongly biased towards those involved in mating; probably due
to the relative ease of functional genomic comparison to the budding yeast model. The multicellular nature of filamentous
fungi, however, preserved a more complex morphology compared to unicellular fungi and revealed chemoattractive
mechanisms lost during yeast evolution. Two hallmarks of this higher complexity are the formation of an interconnected
colony network and the development of elaborate sexual reproductive organs. Morphogenesis of both structures depends
on two different modes of chemoattraction: attraction to self and attraction to nonself. Nonself chemoattraction between
genetically distinct mating partners is the basis for sexual reproduction and generally regulated through a bilateral sexpheromone/
cognate-receptor system, widely but not exclusively equivalent to that known from yeasts. In contrast, selfchemoattraction
between genetically identical cells is regulated independently of the sex-pheromone/cognate-receptor
systems, and does not exist in yeast. Although both chemoattractive modes do share a number of molecular components,
we are only beginning to understand how cell morphogenesis is regulated by means of gene expression and targeted
protein recruitment during the establishment of self-fusion. This review provides an overview on the main morphogenetic
elements involved in fungal chemoattraction, and summarizes our current understanding of the underlying molecular
mechanisms of self- and nonself-fusion during filamentous fungal development.
that occur in response to intracellular and extracellular signals.
Asexual spores (conidia) of the mold Neurospora crassa
differentiate two types of polarized cell protrusions, germ tubes
and conidial anastomosis tubes (CATs), which exhibit negative and
positive chemotropism, respectively. We provide the first evidence
that shared and separate functions of the Rho-type GTPases CDC-
42 and RAC-1 regulate these opposite chemotropisms. We
demonstrate that RAC-1 is essential for CAT formation and cell
fusion, whereas CDC-42 is necessary and sufficient for normal
germ tube development. Cdc42-Rac-interactive-binding (CRIB)
reporters were constructed to exclusively label locally activated
GTP-bound GTPases. Time course analyses showed that
repositioning of these activated GTPase clusters within germ tube
and CAT tip apices controls directional growth in the absence of a
tip-localized vesicle supply center (Spitzenkörper). We propose a
model in which the local assembly of a plasmamembrane-associated GTPase–PAK–MAPK signaling platform regulates
chemoattractant perception and secretion in order to synchronize
oscillatory cell–cell communication and directional CAT tip growth."
multiple secondary metabolite gene clusters in several fungi, and it can modify heterochromatin structure in
Aspergillus nidulans. We have recently shown that the LaeA ortholog of Trichoderma reesei (LAE1), a fungus
that is an industrial producer of cellulase and hemicellulase enzymes, regulates the expression of cellulases
and polysaccharide hydrolases. To learn more about the function of LAE1 in T. reesei, we assessed the
effect of deletion and overexpression of lae1 on genome-wide gene expression. We found that in addition
to positively regulating 7 of 17 polyketide or nonribosomal peptide synthases, genes encoding ankyrinproteins,
iron uptake, heterokaryon incompatibility proteins, PTH11-receptors, and oxidases/monoxygenases
are major gene categories also regulated by LAE1. chromatin immunoprecipitation sequencing with
antibodies against histone modifications known to be associated with transcriptionally active (H3K4me2 and
-me3) or silent (H3K9me3) chromatin detected 4089 genes bearing one or more of these methylation marks,
of which 75 exhibited a correlation between either H3K4me2 or H3K4me3 and regulation by LAE1. Transformation
of a laeA-null mutant of A. nidulans with the T. reesei lae1 gene did not rescue sterigmatocystin
formation and further impaired sexual development. LAE1 did not interact with A. nidulans VeA in yeast
two-hybrid assays, whereas it interacted with the T. reesei VeA ortholog, VEL1. LAE1 was shown to be required for
the expression of vel1, whereas the orthologs of velB and VosA are unaffected by lae1 deletion. Our data show
that the biological roles of A. nidulans LaeA and T. reesei LAE1 are much less conserved than hitherto thought. In
T. reesei, LAE1 appears predominantly to regulate genes increasing relative fitness in its environment.
fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora
crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more
complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis
in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in
gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; confirmed that all three
MAP kinase cascades are required for sexual development; and showed that the three different cascades each have
distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone
response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion.
Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal
adhesion, and envelopment during the construction of fertilizable protoperithecia.
of the two CAT plasma membranes and the formation of a fusion pore that results in cytoplasmic continuity being achieved between the fused CATs. Mutant analyses have implicated a range of other signalling pathways and processes involved in different stages of CAT fusion. These include: the Rho GTPases CDC-42 and RAC-1; the STRIPAK complex; the cell wall integrity MAP kinase pathway; redox signalling; endocytosis and five transcription factors."
of actin in filamentous fungi."
been termed conidial anastomosis tubes (CATs). Notably CAT formation, homing and fusion occurs independently of microtubules. This step-by-step protocol exploits the possibility to differentiate CATs from GTs by observing germling development in the presence of the microtubule disrupting drug Benomyl.
in sterol biosynthesis, which result in the accumulation of precursor
molecules, are commonly associated with cellular disorders and
disease. However, the effects of these sterol precursors on the
metabolism, signaling, and behavior of cells are only poorly
understood. In this study, we show that the accumulation of only
ergosterol precursors with a conjugated double bond in their
aliphatic side chain specifically disrupts cell–cell communication
and fusion in the fungus Neurospora crassa. Genetically identical
germinating spores of this fungus undergo cell–cell fusion, thereby
forming a highly interconnected supracellular network during colony
initiation. Before fusion, the cells use an unusual signaling
mechanism that involves the coordinated and alternating switching
between signal sending and receiving states of the two fusion partners.
Accumulation of only ergosterol precursors with a conjugated
double bond in their aliphatic side chain disrupts this coordinated
cell–cell communication and suppresses cell fusion. These specific
sterol precursors target a single ERK-like mitogen-activated protein
(MAP) kinase (MAK-1)-signaling cascade, whereas a secondMAP kinase
pathway (MAK-2),which is also involved in cell fusion, is unaffected.
These observations indicate that a minor specific change in sterol
structure can exert a strong detrimental effect on a key signaling
pathway of the cell, resulting in the absence of cell fusion.
hydrolysis of biomass to simple sugars, which can then be used in the production of biofuels and biorefineries.
The highly productive strains in use today were generated by classical mutagenesis. As byproducts of this
procedure, mutants were generated that turned out to be unable to produce cellulases. In order to identify the
mutations responsible for this inability, we sequenced the genome of one of these strains, QM9136, and compared
it to that of its progenitor T. reesei QM6a.
Results: In QM9136, we detected a surprisingly low number of mutagenic events in the promoter and coding regions
of genes, i.e. only eight indels and six single nucleotide variants. One of these indels led to a frame-shift in the Zn2Cys6
transcription factor XYR1, the general regulator of cellulase and xylanase expression, and resulted in its C-terminal
truncation by 140 amino acids. Retransformation of strain QM9136 with the wild-type xyr1 allele fully recovered the
ability to produce cellulases, and is thus the reason for the cellulase-negative phenotype. Introduction of an engineered
xyr1 allele containing the truncating point mutation into the moderate producer T. reesei QM9414 rendered this strain
also cellulase-negative. The correspondingly truncated XYR1 protein was still able to enter the nucleus, but failed to be
expressed over the basal constitutive level.
Conclusion: The missing 140 C-terminal amino acids of XYR1 are therefore responsible for its previously observed
auto-regulation which is essential for cellulases to be expressed. Our data present a working example of the use of
genome sequencing leading to a functional explanation of the QM9136 cellulase-negative phenotype.
Keywords: Single nucleotide polymorphism, SNP, Indel, Comparative genomics, Classical mutant, XYR1, Transcription
factor shuttling, Cellulases, Trichoderma reesei, QM9136
insights into chemoattractive mechanisms in recent years. Still, identification of specific chemoattractant molecules,
cognate receptors and downstream signaling pathways is strongly biased towards those involved in mating; probably due
to the relative ease of functional genomic comparison to the budding yeast model. The multicellular nature of filamentous
fungi, however, preserved a more complex morphology compared to unicellular fungi and revealed chemoattractive
mechanisms lost during yeast evolution. Two hallmarks of this higher complexity are the formation of an interconnected
colony network and the development of elaborate sexual reproductive organs. Morphogenesis of both structures depends
on two different modes of chemoattraction: attraction to self and attraction to nonself. Nonself chemoattraction between
genetically distinct mating partners is the basis for sexual reproduction and generally regulated through a bilateral sexpheromone/
cognate-receptor system, widely but not exclusively equivalent to that known from yeasts. In contrast, selfchemoattraction
between genetically identical cells is regulated independently of the sex-pheromone/cognate-receptor
systems, and does not exist in yeast. Although both chemoattractive modes do share a number of molecular components,
we are only beginning to understand how cell morphogenesis is regulated by means of gene expression and targeted
protein recruitment during the establishment of self-fusion. This review provides an overview on the main morphogenetic
elements involved in fungal chemoattraction, and summarizes our current understanding of the underlying molecular
mechanisms of self- and nonself-fusion during filamentous fungal development.
that occur in response to intracellular and extracellular signals.
Asexual spores (conidia) of the mold Neurospora crassa
differentiate two types of polarized cell protrusions, germ tubes
and conidial anastomosis tubes (CATs), which exhibit negative and
positive chemotropism, respectively. We provide the first evidence
that shared and separate functions of the Rho-type GTPases CDC-
42 and RAC-1 regulate these opposite chemotropisms. We
demonstrate that RAC-1 is essential for CAT formation and cell
fusion, whereas CDC-42 is necessary and sufficient for normal
germ tube development. Cdc42-Rac-interactive-binding (CRIB)
reporters were constructed to exclusively label locally activated
GTP-bound GTPases. Time course analyses showed that
repositioning of these activated GTPase clusters within germ tube
and CAT tip apices controls directional growth in the absence of a
tip-localized vesicle supply center (Spitzenkörper). We propose a
model in which the local assembly of a plasmamembrane-associated GTPase–PAK–MAPK signaling platform regulates
chemoattractant perception and secretion in order to synchronize
oscillatory cell–cell communication and directional CAT tip growth."
multiple secondary metabolite gene clusters in several fungi, and it can modify heterochromatin structure in
Aspergillus nidulans. We have recently shown that the LaeA ortholog of Trichoderma reesei (LAE1), a fungus
that is an industrial producer of cellulase and hemicellulase enzymes, regulates the expression of cellulases
and polysaccharide hydrolases. To learn more about the function of LAE1 in T. reesei, we assessed the
effect of deletion and overexpression of lae1 on genome-wide gene expression. We found that in addition
to positively regulating 7 of 17 polyketide or nonribosomal peptide synthases, genes encoding ankyrinproteins,
iron uptake, heterokaryon incompatibility proteins, PTH11-receptors, and oxidases/monoxygenases
are major gene categories also regulated by LAE1. chromatin immunoprecipitation sequencing with
antibodies against histone modifications known to be associated with transcriptionally active (H3K4me2 and
-me3) or silent (H3K9me3) chromatin detected 4089 genes bearing one or more of these methylation marks,
of which 75 exhibited a correlation between either H3K4me2 or H3K4me3 and regulation by LAE1. Transformation
of a laeA-null mutant of A. nidulans with the T. reesei lae1 gene did not rescue sterigmatocystin
formation and further impaired sexual development. LAE1 did not interact with A. nidulans VeA in yeast
two-hybrid assays, whereas it interacted with the T. reesei VeA ortholog, VEL1. LAE1 was shown to be required for
the expression of vel1, whereas the orthologs of velB and VosA are unaffected by lae1 deletion. Our data show
that the biological roles of A. nidulans LaeA and T. reesei LAE1 are much less conserved than hitherto thought. In
T. reesei, LAE1 appears predominantly to regulate genes increasing relative fitness in its environment.
fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora
crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more
complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis
in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in
gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; confirmed that all three
MAP kinase cascades are required for sexual development; and showed that the three different cascades each have
distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone
response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion.
Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal
adhesion, and envelopment during the construction of fertilizable protoperithecia.
of the two CAT plasma membranes and the formation of a fusion pore that results in cytoplasmic continuity being achieved between the fused CATs. Mutant analyses have implicated a range of other signalling pathways and processes involved in different stages of CAT fusion. These include: the Rho GTPases CDC-42 and RAC-1; the STRIPAK complex; the cell wall integrity MAP kinase pathway; redox signalling; endocytosis and five transcription factors."
of actin in filamentous fungi."
been termed conidial anastomosis tubes (CATs). Notably CAT formation, homing and fusion occurs independently of microtubules. This step-by-step protocol exploits the possibility to differentiate CATs from GTs by observing germling development in the presence of the microtubule disrupting drug Benomyl.