The dimorphic fungus Ustilago maydis is a pathogen of maize and it was used for decades to understand the molecular basis of plant pathogenicity aspects. Recently, much effort went into understanding the cell biology that underlies the virulence of U. maydis. It was shown previously that early endosomes (EEs) move bidirectionally within fungal hyphal cells. Although it was shown that the motility of EEs facilitates growth of the infectious hypha and mutants defective for kinesin-3 (Kin3), the major EE transporter, exhibit impaired polarized growth, the importance of EEs and their motility in plant colonization is not known. The first part of this thesis is focused on the role of EE motility during plant infection. In collaboration with Natalie Steinberg, who performed the plant infection assays, I used a synthetic molecular anchor, K1rPX, to block the motility of EEs at early and late stages during the host plant infection and I found that EE motility is essential during the first two days of pathogenic development, when infectious hyphae exhibit most prominent elongation, whereas blockage of EE motility after 3 days post infection does not inhibit plant colonization. Moreover, I documented that the blockage of EE motility during early stages of the infection causes high plant defence response, which means that the pathogen becomes recognized by the host plant defence system. These results indicate that EE motility is crucial during initial stages of the plant host infection and enables colonization by U. maydis and additionally suggests involvement of EEs in some defence response machinery. The second part of the thesis addresses the relationship between Kin3, the major motor for EE motility, and the microtubule (MT) array. I demonstrate here that Kin3 uses all MT tracks available in the cell, which is in contrast to published results in other systems. In the third part I focused on the interaction between Kin3 and the EEs. I found that the pleckstrin homology (PH) domain localized at the distal part of the Kin3 tail is of minor importance for EE association. This conclusion is supported by in vivo experiments, showing that truncated Kin3PH, which lacks the PH domain, was still able to bind to the organelles. By systematic truncation of parts of the Kin3 tail I found two adjacent regions, a DUF3694 domain and a "linker" region, that are important for binding of Kin3 to EEs. By using a synthetic anchor composed of Kin1 rigor domain and selected Kin3 domains I proved that both domains anchor the EEs to MTs and inhibit EE motility. I also showed that the PH domain is not able to block EE motility. In collaboration with Dr. Nicholas Harmer, who performed structural modelling of selected PH domains, I demonstrated that the PH domain is likely to interact with the motor domain of Kin3. This result was confirmed by using a yeast-two hybrid approach and a protein affinity assay. This indicates a globular organization of the Kin3 motor, which was confirmed by a split-YFP assay in living cells. Deletion of the PH domain and most probably lack of intramolecular interaction between the tail and motor domain reduces Kin3 motility parameters like velocity, frequency and run length indicating that the interaction of the PH domain with the motor domain has a role in the control of Kin3 motility.

Striatin family proteins contain a caveolin binding domain, a coiled-coil motif, a calmodulin binding domain, and a WD-repeat domain. Homologs of striatin protein have been However, our knowledge of the function and the molecular mechanism of fungal striatin homologs is limited. Based on the conserved sequences of functional domains, I hypothesized that the fungal striatin orthologs also act as scaffolding proteins that are functionally conserved among fungal species and involved in multiple types of development in the diverse kingdom Mycota. I used reverse genetic strategies to study the function of the Aspergillus nidulans striatin ortholog (strA) and the Colletotrichum graminicola striatin ortholog (str1). In assays of sexual development, the strA deletion strain (ΔstrA) produces fewer ascospores with smaller cleistothecia, while the str1 deletion strain (Δstr1) is defective in perithecia development. The ΔstrA phenotypes indicate that StrA is associated with ascosporogenesis in cleistothecia. Both ΔstrA and Δstr1 are reduced in radial growth and in conidia production. The Δstr1 strain is also altered in its spiral growth pattern and morphology of conidia and hyphopodia, but it produces appressoria similar to wild type. The pairing of nitrate non-utilizing mutants demonstrates that Str1 is required for hyphal fusion. In pathogenicity, Δstr1 is less virulent in maize anthracnose leaf blight and stalk rot. The phenotypes of Δstr1 are complemented by the Fusarium verticillioides striatin ortholog (fsr1), indicating that Fsr1 and Str1 are functionally conserved. Over-expression of StrA reveals its positive role in conidiation and the sexual production. StrA::eGFP localizes mainly to the endoplasmic reticulum. After comparing the results from these two species and other studied fungal species, I suggest that fungal striatins are involved in five types of development including hyphal growth, hyphal fusion, conidiation, sexual development, and virulence, and propose a model of fungal striatin protein interactions to account for these diverse phenotypes.

Invasive growth through a substrate requires a massive amount of penetrative force, and this is generated in the space of a few microns in a growing tip. This process is known to be critical in the root hair, pollen tube, rhizoids, and the topic of this thesis, hyphal growth. However defining the mechanisms underlying the tip growth remains a contentious issue. Shortcomings in control of direction and regulation of growth began to undermine early turgor-based theories, and the cytoskeletal protein actin, ubiquitous in nature and with crucial roles in structure and motility became a target for investigation. A major breakthrough came with the discovery that a characteristic actin depleted zone (ADZ) occurs at the growing tip of hyphae during invasive but not non-invasive hyphal growth. The ADZ is likely to have an important role in generating the greater protrusive force required for invasive growth. However, since its discovery, little has been determined about the characteristics of the ADZ. Uncertainty in the description of the physical environment the hyphae face adds a layer of complexity to interpretation of results. This thesis aims to address this issue, studying the impact of increasing agarose substrate concentration on the presence and dimensions of the ADZ in the oomycete A. bisexualis. Furthermore, agarose is examined by compression and imaging to compare the physical characteristics of the agar samples over the range of concentrations, and determine whether increasing agarose concentration influences agarose gel structure. Results suggest a difference in the number of ADZ observed in non-invasive compared with invasive samples, however no significant differences in the number or dimensions of ADZ were found amongst the 1-4% w/v agarose concentrations. The 0% sample showed 20.7 percent of hyphae exhibited depleted zones, while 1, 2, 3 and 4% samples showed 56.9%, 48.8%, 40.9% and 54.2% respectively. ADZ dimensions did not correlate with agarose concentration. The average ADZ area:hyphal diameter ratio was 0.634, 0.526, 0.430, 1.09, and 0.65 for 0-4% agarose concentrations respectively. Additionally, investigation of gel compression forces revealed gel strength increases with agarose concentration. The force required to compress the agarose increased from 1.85 Psi in 1% agarose to 4.85, 7.09 and 12.22 Psi in 2, 3 and 4% agarose concentrations respectively. SEM imaging, however, suggests heterogeneity of the fibrous interconnected network of agarose gels at a microscopic scale with variable porous structure at all agarose concentrations. This scale is relevant to hyphal tip growth. In combination, these results suggest F-actin depletion may be a response mechanism to provide greater force for invasive growth. Additionally, this response is not dependent on the concentration of the agarose media, possibly due to the variability encountered within the media. These results contribute another important step forward in unraveling the elusive mechanism of tip growth.

Invasive growth through a substrate requires a massive amount of penetrative force, and this is generated in the space of a few microns in a growing tip. This process is known to be critical in the root hair, pollen tube, rhizoids, and the topic of this thesis, hyphal growth. However defining the mechanisms underlying the tip growth remains a contentious issue. Shortcomings in control of direction and regulation of growth began to undermine early turgor-based theories, and the cytoskeletal protein actin, ubiquitous in nature and with crucial roles in structure and motility became a target for investigation. A major breakthrough came with the discovery that a characteristic actin depleted zone (ADZ) occurs at the growing tip of hyphae during invasive but not non-invasive hyphal growth. The ADZ is likely to have an important role in generating the greater protrusive force required for invasive growth. However, since its discovery, little has been determined about the characteristics of the ADZ. Uncertainty in the description of the physical environment the hyphae face adds a layer of complexity to interpretation of results. This thesis aims to address this issue, studying the impact of increasing agarose substrate concentration on the presence and dimensions of the ADZ in the oomycete A. bisexualis. Furthermore, agarose is examined by compression and imaging to compare the physical characteristics of the agar samples over the range of concentrations, and determine whether increasing agarose concentration influences agarose gel structure. Results suggest a difference in the number of ADZ observed in non-invasive compared with invasive samples, however no significant differences in the number or dimensions of ADZ were found amongst the 1-4% w/v agarose concentrations. The 0% sample showed 20.7 percent of hyphae exhibited depleted zones, while 1, 2, 3 and 4% samples showed 56.9%, 48.8%, 40.9% and 54.2% respectively. ADZ dimensions did not correlate with agarose concentration. The average ADZ area:hyphal diameter ratio was 0.634, 0.526, 0.430, 1.09, and 0.65 for 0-4% agarose concentrations respectively. Additionally, investigation of gel compression forces revealed gel strength increases with agarose concentration. The force required to compress the agarose increased from 1.85 Psi in 1% agarose to 4.85, 7.09 and 12.22 Psi in 2, 3 and 4% agarose concentrations respectively. SEM imaging, however, suggests heterogeneity of the fibrous interconnected network of agarose gels at a microscopic scale with variable porous structure at all agarose concentrations. This scale is relevant to hyphal tip growth. In combination, these results suggest F-actin depletion may be a response mechanism to provide greater force for invasive growth. Additionally, this response is not dependent on the concentration of the agarose media, possibly due to the variability encountered within the media. These results contribute another important step forward in unraveling the elusive mechanism of tip growth.

La polarité cellulaire est un phénomène biologique essentiel du monde vivant. Chez la levure Candida albicans, sa capacité à croître sous une forme hyperpolarisée semble être un élément déterminant de sa pathogénicité. Nous avons entrepris d’identifier les éléments moléculaires d’une structure essentielle à cette croissance hyphale, le Spitzenkörper, afin de mieux comprendre le rôle de ce corps apical dans la croissance polarisée. Nous nous sommes également intéressés à la régulation des protéines Rho3 et Rho4 impliquées dans la croissance polarisée de C. albicans, à travers l’identification et l’étude de la protéine RhoGAP commune à ces deux protéines Rhos, la protéine Rgd1.Chez la levure Saccharomyces cerevisiæ, les protéines Rho3 et Rho4 sont également impliquées dans le contrôle de la croissance polarisée, et sont régulées par la protéine Rgd1. Le laboratoire, à l’origine de la découverte de ce régulateur commun, étudiait des aspects de croissance polarisée contrôlée par les protéines Rho3 et Rho4, à travers l’étude de la régulation de la protéine Rgd1. Nous avons notamment mis en évidence que Rgd1p est modifiée au niveau post-traductionnel par des phosphorylations. La kinase Ipl1 de la famille des kinases Aurora est un des acteurs de cette modification. Différents éléments indiquent que le complexe phosphatase Glc7-Bud14 serait également impliqué dans le contrôle de l’état de phosphorylation de Rgd1p, de façon antagoniste à la kinase Ipl1 / Cell polarity is an essential process for living organisms. In the yeast Candida albicans, its ability of hyperpolarized growth seems to be a decisive element for its pathogenicity. We undertook to identify molecular elements of an essential structure for hyphal growth, named Spitzenkörper, to better understand the role of this apical body in polarised growth. We also studied regulation of Rho3 and Rho4 proteins implicated in C. albicans polarised growth, through identification and study of a shared RhoGAP protein, named Rgd1.In the yeast Saccharomyces cerevisiæ, Rho3 and Rho4 proteins are also implicated in polarised growth control, and are regulated by Rgd1 protein. The laboratory, which identified this shared regulator, studied polarised growth aspects controlled by Rho3 and Rho4 proteins, through study of Rgd1p regulation. We showed that Rgd1p is post-translationally modified, by phosphorylations. The Ipl1 kinase, an Aurora family member, is implicated in this modification. Several elements indicate that the Glc7-Bud14 phosphatase complex would be also implicated in the control of Rgd1 phosphorylation state, antagonistically to Ipl1p

Candida albicans est une levure pathogène qui, à l’état commensal, colonise les muqueuses de la cavité orale et du tractus gastro-intestinal. De nature opportuniste, C. albicans cause de nombreuses infections, allant des candidoses superficielles (muguet buccal, vulvo-vaginite) aux candidoses systémiques sévères. C. albicans a la capacité de se développer sous diverses morphologies, telles que les formes levures, pseudohyphes et hyphes. Des stimuli environnementaux mimant les conditions retrouvées chez l’hôte (température de 37°C, pH neutre, présence de sérum) induisent la transition levure-à-hyphe (i.e. morphogenèse ou filamentation). Cette transition morphologique contribue à la pathogénicité de C. albicans, du fait que des souches présentant un défaut de filamentation sont avirulentes. Non seulement la morphogenèse est un facteur de virulence, mais elle constituerait aussi une cible pour le développement d’antifongiques. En effet, il a déjà été démontré que l’inhibition de la transition levure-à-hyphe atténuait la virulence de C. albicans lors d’infections systémiques. Par ailleurs, des études ont démontré que de nombreuses molécules pouvaient moduler la morphogenèse. Parmi ces molécules, certains acides gras, dont l’acide linoléique conjugué (CLA), inhibent la formation d’hyphes. Ainsi, le CLA posséderait des propriétés thérapeutiques, du fait qu’il interfère avec un déterminant de pathogénicité de C. albicans. Par contre, avant d’évaluer son potentiel thérapeutique dans un contexte clinique, il est essentiel d’étudier son mode d’action. Ce projet vise à caractériser l’activité anti-filamentation des acides gras et du CLA et à déterminer le mécanisme par lequel ces molécules inhibent la morphogenèse chez C. albicans. Des analyses transcriptomiques globales ont été effectuées afin d’obtenir le profil transcriptionnel de la réponse de C. albicans au CLA. L’acide gras a entraîné une baisse des niveaux d’expression de gènes encodant des protéines hyphes-spécifiques et des régulateurs de morphogenèse, dont RAS1. Ce gène code pour la GTPase Ras1p, une protéine membranaire de signalisation qui joue un rôle important dans la transition levure-à-hyphe. Des analyses de PCR quantitatif ont confirmé que le CLA inhibait l’induction de RAS1. De plus, le CLA a non seulement causé une baisse des niveaux cellulaires de Ras1p, mais a aussi entraîné sa délocalisation de la membrane plasmique. En affectant les niveaux et la localisation cellulaire de Ras1p, le CLA nuit à l’activation de la voie de signalisation Ras1p-dépendante, inhibant ainsi la morphogenèse. Il est possible que le CLA altère la structure de la membrane plasmique et affecte indirectement la localisation membranaire de Ras1p. Ces travaux ont permis de mettre en évidence le mode d’action du CLA. Le potentiel thérapeutique du CLA pourrait maintenant être évalué dans un contexte d’infection, permettant ainsi de vérifier qu’une telle approche constitue véritablement une stratégie pour le traitement des candidoses. / The yeast Candida albicans is an inhabitant of the oral cavity, the gastrointestinal and genitourinary tracts of humans. Generally encountered as a commensal, it is also an opportunistic pathogen that causes a spectrum of infections, ranging from superficial mycoses (thrush, vulvovaginitis) to severe and life-threatening systemic infections. A striking feature of C. albicans is its ability to grow in different morphological forms, including budding yeasts, pseudohyphae, and hyphae. Environmental cues that mimic host conditions (elevated temperature, neutral or alkaline pH, and serum) induce the yeast-to-hypha transition. Morphogenesis is considered to be an attribute of pathogenesis, as mutants locked as yeasts or filamentous forms are avirulent. Given that the yeast-to-hypha transition is a virulence factor, it may also constitute a target for the development of antifungal drugs. Indeed, evidence has shown that impairing morphogenesis is a means to treat systemic candidiasis. Concurrently, a number of molecules have been reported to modulate morphogenesis in C. albicans. For instance, several fatty acids, including conjugated linoleic acid (CLA), inhibited the yeast-to-hypha transition. By interfering with an important attribute of C. albicans pathogenesis, CLA may harbor antifungal properties. However, before assessing its therapeutic potential in a clinical context, it is mandatory to address CLA’s mode of action. The present study aims to further characterize the hypha-inhibiting properties of fatty acids and CLA and to elucidate the mechanism by which these molecules inhibit the yeast-to-hypha transition in C. albicans. Gene expression analyses were performed to gain insight into the transcriptional response of cells to CLA on a genome-wide scale and to probe the fatty acid’s mode of action. CLA downregulated the expression of hypha-specific genes and blocked the induction of genes encoding regulators of hyphal growth, including that of RAS1, which encodes the small GTPase Ras1p. A membrane-associated signaling protein, Ras1p plays a major role in morphogenesis. Quantitative PCR analyses showed that CLA prevented the increase in RAS1 mRNA levels which occurred at the onset of the yeast-to-hypha transition. Unexpectedly, CLA reduced the steady-state levels of Ras1p. Additionally, CLA caused the delocalization of GFP-Ras1p from the plasma membrane. These findings indicate that CLA treatment results in suboptimal Ras1p cellular concentrations and localization, which impedes Ras1p signaling and inhibits the yeast-to-hypha transition. CLA may indirectly affect Ras1p localization by altering the structure of the plasma membrane. These studies have provided the mechanism underlying CLA’s hypha-inhibiting properties and may serve as the rationale to examine CLA’s therapeutic potential in the context of a Candida infection. There is a general lack of clinical evidence demonstrating that impairing morphogenesis is a sound approach to treat candidiasis. To remedy this situation, the therapeutic potential of molecules that modulate morphogenesis, such as CLA, should be clinically assessed.

Candida albicans est une levure pathogène qui, à l’état commensal, colonise les muqueuses de la cavité orale et du tractus gastro-intestinal. De nature opportuniste, C. albicans cause de nombreuses infections, allant des candidoses superficielles (muguet buccal, vulvo-vaginite) aux candidoses systémiques sévères. C. albicans a la capacité de se développer sous diverses morphologies, telles que les formes levures, pseudohyphes et hyphes. Des stimuli environnementaux mimant les conditions retrouvées chez l’hôte (température de 37°C, pH neutre, présence de sérum) induisent la transition levure-à-hyphe (i.e. morphogenèse ou filamentation). Cette transition morphologique contribue à la pathogénicité de C. albicans, du fait que des souches présentant un défaut de filamentation sont avirulentes. Non seulement la morphogenèse est un facteur de virulence, mais elle constituerait aussi une cible pour le développement d’antifongiques. En effet, il a déjà été démontré que l’inhibition de la transition levure-à-hyphe atténuait la virulence de C. albicans lors d’infections systémiques. Par ailleurs, des études ont démontré que de nombreuses molécules pouvaient moduler la morphogenèse. Parmi ces molécules, certains acides gras, dont l’acide linoléique conjugué (CLA), inhibent la formation d’hyphes. Ainsi, le CLA posséderait des propriétés thérapeutiques, du fait qu’il interfère avec un déterminant de pathogénicité de C. albicans. Par contre, avant d’évaluer son potentiel thérapeutique dans un contexte clinique, il est essentiel d’étudier son mode d’action. Ce projet vise à caractériser l’activité anti-filamentation des acides gras et du CLA et à déterminer le mécanisme par lequel ces molécules inhibent la morphogenèse chez C. albicans. Des analyses transcriptomiques globales ont été effectuées afin d’obtenir le profil transcriptionnel de la réponse de C. albicans au CLA. L’acide gras a entraîné une baisse des niveaux d’expression de gènes encodant des protéines hyphes-spécifiques et des régulateurs de morphogenèse, dont RAS1. Ce gène code pour la GTPase Ras1p, une protéine membranaire de signalisation qui joue un rôle important dans la transition levure-à-hyphe. Des analyses de PCR quantitatif ont confirmé que le CLA inhibait l’induction de RAS1. De plus, le CLA a non seulement causé une baisse des niveaux cellulaires de Ras1p, mais a aussi entraîné sa délocalisation de la membrane plasmique. En affectant les niveaux et la localisation cellulaire de Ras1p, le CLA nuit à l’activation de la voie de signalisation Ras1p-dépendante, inhibant ainsi la morphogenèse. Il est possible que le CLA altère la structure de la membrane plasmique et affecte indirectement la localisation membranaire de Ras1p. Ces travaux ont permis de mettre en évidence le mode d’action du CLA. Le potentiel thérapeutique du CLA pourrait maintenant être évalué dans un contexte d’infection, permettant ainsi de vérifier qu’une telle approche constitue véritablement une stratégie pour le traitement des candidoses. / The yeast Candida albicans is an inhabitant of the oral cavity, the gastrointestinal and genitourinary tracts of humans. Generally encountered as a commensal, it is also an opportunistic pathogen that causes a spectrum of infections, ranging from superficial mycoses (thrush, vulvovaginitis) to severe and life-threatening systemic infections. A striking feature of C. albicans is its ability to grow in different morphological forms, including budding yeasts, pseudohyphae, and hyphae. Environmental cues that mimic host conditions (elevated temperature, neutral or alkaline pH, and serum) induce the yeast-to-hypha transition. Morphogenesis is considered to be an attribute of pathogenesis, as mutants locked as yeasts or filamentous forms are avirulent. Given that the yeast-to-hypha transition is a virulence factor, it may also constitute a target for the development of antifungal drugs. Indeed, evidence has shown that impairing morphogenesis is a means to treat systemic candidiasis. Concurrently, a number of molecules have been reported to modulate morphogenesis in C. albicans. For instance, several fatty acids, including conjugated linoleic acid (CLA), inhibited the yeast-to-hypha transition. By interfering with an important attribute of C. albicans pathogenesis, CLA may harbor antifungal properties. However, before assessing its therapeutic potential in a clinical context, it is mandatory to address CLA’s mode of action. The present study aims to further characterize the hypha-inhibiting properties of fatty acids and CLA and to elucidate the mechanism by which these molecules inhibit the yeast-to-hypha transition in C. albicans. Gene expression analyses were performed to gain insight into the transcriptional response of cells to CLA on a genome-wide scale and to probe the fatty acid’s mode of action. CLA downregulated the expression of hypha-specific genes and blocked the induction of genes encoding regulators of hyphal growth, including that of RAS1, which encodes the small GTPase Ras1p. A membrane-associated signaling protein, Ras1p plays a major role in morphogenesis. Quantitative PCR analyses showed that CLA prevented the increase in RAS1 mRNA levels which occurred at the onset of the yeast-to-hypha transition. Unexpectedly, CLA reduced the steady-state levels of Ras1p. Additionally, CLA caused the delocalization of GFP-Ras1p from the plasma membrane. These findings indicate that CLA treatment results in suboptimal Ras1p cellular concentrations and localization, which impedes Ras1p signaling and inhibits the yeast-to-hypha transition. CLA may indirectly affect Ras1p localization by altering the structure of the plasma membrane. These studies have provided the mechanism underlying CLA’s hypha-inhibiting properties and may serve as the rationale to examine CLA’s therapeutic potential in the context of a Candida infection. There is a general lack of clinical evidence demonstrating that impairing morphogenesis is a sound approach to treat candidiasis. To remedy this situation, the therapeutic potential of molecules that modulate morphogenesis, such as CLA, should be clinically assessed.