RHO-3 plays a significant role in hyphal extension rate, conidiation, and the integrity of the Spitzenkörper in Neurospora crassa.

The Rho family of monomeric GTPases act as signaling proteins to establish and maintain cell polarity and other essential cellular processes. Rho3 is a GTPase of the Rho family that is exclusive of fungi that regulate cell polarity in yeast. However, studies have yet to explore its function in filamentous fungi. In this work, we investigated the role of RHO-3 in the model organism Neurospora crassa. Confocal microscopy analysis revealed that RHO-3 localizes in the outer region of the Spitzenkörper (Spk), in the plasma membrane from region II to the beginning of region III, and in the septa of mature hyphae. The phenotypic effect of the rho-3 deletion was analyzed. The results revealed that the rho-3 null strain showed severe defects in growth rate, aerial hyphae length, and conidia production. The organization of the Spk is also affected in the absence of RHO-3. Co-expression analysis of GFP-RHO-3 with glucan synthase 1 (GS-1-mChFP) and chitin synthase 1 (CHS-1-mChFP) revealed that RHO-3 localizes in the external region of the Spk in the macrovesicles zone. In summary, our results suggest that RHO-3 is not essential for the polarized growth of hyphae but plays a significant role in hyphal extension rate, conidiation, sexual reproduction and the integrity of the Spk, possibly regulating the delivery of macrovesicles to the apical dome.

The small Ras-like GTPase BUD-1 modulates conidial germination and hyphal growth guidance in the filamentous fungus Neurospora crassa.

In filamentous fungi, the hypha orientation is essential for polarized growth and morphogenesis. The ability to re-orient tip growth in response to environmental cues is critical for the colony survival. Therefore, hyphal tip orientation and tip extension are distinct mechanisms that operate in parallel during filamentous growth. In yeast, the axial growth orientation requires a pathway regulated by Rsr1p/Bud1p, a Ras-like GTPase protein, which determines the axial budding pattern. However, in filamentous fungi the function of the Rsr1/Bud1p gene (krev-1 homolog) has not been completely characterized. In this work, we characterized the phenotype of a homokaryon mutant Bud1p orthologous in Neurospora crassa (△bud-1) and tagged BUD-1 with the green fluorescent protein (GFP) to determine its localization and cell dynamics under confocal microscopy. During spore germination BUD-1 was localized at specific points along the plasma membrane and during germ tube emergence it was located at the tip of the germ tubes. In mature hyphae BUD-1 continued to be located at the cell tip and was also present at sites of branch emergence and at the time of septum formation. The △bud-1 mutant showed a delayed germination, and the orientation of hyphae was somewhat disrupted. Also, the hypha diameter was reduced approximately 37 % with respect to the wild type. The lack of BUD-1 affected the Spitzenkörper (Spk) formation, trajectory, the localization of polarisome components BNI-1 and SPA-2, and the actin cytoskeleton polarization. The results presented here suggest that BUD-1 participates in the establishment of a new polarity axis. It may also mediate the delivery of secretory vesicles for the efficient construction of new plasma membrane and cell wall.

The cytoplasmic microtubule array in Neurospora crassa depends on microtubule-organizing centers at spindle pole bodies and microtubule +end-depending pseudo-MTOCs at septa.

γ-Tubulin ring complexes (γ-TuRC) mediate nucleation and anchorage of microtubules (MTs) to microtubule organizing centers (MTOCs). In fungi, the spindle pole body (SPB) is the functional equivalent of the centrosome, which is the main MTOC. In addition, non-centrosomal MTOCs (ncMTOCs) contribute to MT formation in some fungi like Schizosaccharomyces pombe and Aspergillus nidulans. In A. nidulans, MTOCs are anchored at septa (sMTOC) and share components of the outer plaque of the SPB. Here we show that the Neurospora crassa SPB is embedded in the nuclear envelope, with the γ-TuRC targeting proteins PCP-1Pcp1/PcpA located at the inner plaque and APS-2Mto1/ApsB located at the outer plaque of the SPB. PCP-1 was a specific component of nuclear MTOCs, while APS-2 was also present at the septal pore. Although γ-tubulin was only detected at the nucleus, spontaneous MT nucleation occurred in the apical and subapical cytoplasm during recovery from benomyl-induced MT depolymerization experiments. There was no evidence for MT nucleation at septa. However, without benomyl treatment MT plus-ends were organized in the septal pore through MTB-3EB1. Those septal MT plus ends polymerized MTs from septa in interphase cells Thus we conclude that the SPB is the only MT nucleation site in N. crassa, but the septal pore aids the MT network arrangement through the anchorage of the MT plus-ends through a pseudo-MTOC.

F-actin dynamics following mechanical injury of Trichoderma atroviride and Neurospora crassa hyphae.

We investigated hyphae regeneration in Trichoderma atroviride and Neurospora crassa, with particular focus on determining the role of the actin cytoskeleton after mechanical injury. Filamentous actin (F-actin) dynamics was observed by live-cell confocal microscopy in both T. atroviride and N. crassa strains expressing Lifeact-GFP. In growing hyphae of both fungi, F-actin localized in three different structural forms: patches, cables and actomyosin rings. Most patches were conspicuously arranged in a collar in the hyphal subapex. A strong F-actin signal, likely actin filaments, colocalized with the core of the Spitzenkörper. Filaments and cables of F-actin were observed along the cortex throughout hyphae. Following mechanical damage at the margin of growing mycelia of T. atroviride and N. crassa, the severed hyphae lost their cytoplasmic contents, but plugging of the septal pore by a Woronin body occured, and the rest of the hyphal tube remained whole. In both fungi, patches of F-actin began accumulating next to the plugged septum. Regeneration was attained by the emergence of a new hyphal tube as an extension of the plugged septum wall. The septum wall was gradually remodeled into the apical wall of the emerging hypha. Whereas in T. atroviride the re-initiation of polarized growth took  âˆ¼ 1 h, in N. crassa, actin patch accumulation began almost immediately, and new growing hyphae were observed âˆ¼ 30 min after injury. By confocal microscopy, we found that chitin synthase 1 (CHS-1), a microvesicle (chitosome) component, accumulated next to the plugged septum in regenerating hyphae of N. crassa. We concluded that the actin cytoskeleton plays a key role in hyphal regeneration by supporting membrane remodeling, helping to facilitate transport of vesicles responsible for new wall growth and organization of the new tip-growth apparatus.

The actin motor MYO-5 effect in the intracellular organization of Neurospora crassa.

In filamentous fungi, polarized growth is the result of vesicle secretion at the hyphal apex. Motor proteins mediate vesicle transport to target destinations on the plasma membrane via actin and microtubule cytoskeletons. Myosins are motor proteins associated with actin filaments. Specifically, class V myosins are responsible for cargo transport in eukaryotes. We studied the dynamics and localization of myosin V in wild type hyphae of Neurospora crassa and in hyphae that lacked MYO-5. In wild type hyphae, MYO-5-GFP was localized concentrated in the hyphal apex and colocalized with Spitzenkörper. Photobleaching studies showed that MYO-5-GFP was transported to the apex from subapical hyphal regions. The deletion of the class V myosin resulted in a reduced rate of hyphal growth, apical hyperbranching, and intermittent loss of hyphal polarity. MYO-5 did not participate in breaking the symmetrical growth during germination but contributed in the apical organization upon establishment of polarized growth. In the Δmyo-5 mutant, actin was organized into thick cables in the apical and subapical hyphal regions, and the number of endocytic patches was reduced. The microvesicles-chitosomes observed with CHS-1-GFP were distributed as a cloud occupying the apical dome and not in the Spitzenkörper as the WT strain. The mitochondrial movement was not associated with MYO-5, but tubular vacuole position is MYO-5-dependent. These results suggest that MYO-5 plays a role in maintaining apical organization and the integrity of the Spitzenkörper and is required for normal hyphal growth, polarity, septation, conidiation, and proper conidial germination.

Characterization of a Polymicrobial Dermal Infection in a Peninsular Pronghorn ( Antilocapra americana peninsularis) in Baja California, Mexico.

In situ conservation efforts are assisting the recovery of free-ranging populations of the endangered peninsular pronghorns ( Antilocapra americana peninsularis) at the Vizcaino Biosphere Reserve, Baja California Sur, Mexico. We detected a polymicrobial dermal infection. Etiologic agents were identified as a keratinophilic dermatomycete and opportunistic pathogenic bacteria.

Candida species diversity and antifungal susceptibility patterns in oral samples of HIV/AIDS patients in Baja California, Mexico.

Candidiasis is the most common opportunistic fungal infection in HIV patients. The aims of this study were to identify the prevalence of carriers of Candida, Candida species diversity, and in vitro susceptibility to antifungal drugs. In 297 HIV/AIDS patients in Baja California, Mexico, Candida strains were identified by molecular methods (PCR-RFLP) from isolates of oral rinses of patients in Tijuana, Mexicali, and Ensenada. 56.3% of patients were colonized or infected with Candida. In Tijuana, there was a significantly higher percentage of carriers (75.5%). Out of the 181 strains that were isolated, 71.8% were Candida albicans and 28.2% were non-albicans species. The most common non-albicans species was Candida tropicalis (12.2%), followed by Candida glabrata (8.3%), Candida parapsilosis (2.2%), Candida krusei (1.7%), and Candida guilliermondii (1.1%). Candida dubliniensis was not isolated. Two associated species were found in 11 patients. In Mexicali and Ensenada, there was a lower proportion of Candida carriers compared to other regions in Mexico and worldwide, however, in Tijuana, a border town with many peculiarities, a higher carrier rate was found. In this population, only a high viral load was associated with oral Candida carriers. Other factors such as gender, use of antiretroviral therapy, CD4+ T-lymphocyte levels, time since diagnosis, and alcohol/ tobacco consumption, were not associated with Candida carriers.

Two microtubule-plus-end binding proteins LIS1-1 and LIS1-2, homologues of human LIS1 in Neurospora crassa.

LIS1 is a microtubule (Mt) plus-end binding protein that interacts with the dynein/dynactin complex. In humans, LIS1 is required for proper nuclear and organelle migration during cell growth. Although gene duplication is absent from Neurospora crassa, we found two paralogues of human LIS1. We named them LIS1-1 and LIS1-2 and studied their dynamics and function by fluorescent tagging. At the protein level, LIS1-1 and LIS1-2 were very similar. Although, the characteristic coiled-coil motif was not present in LIS1-2. LIS1-1-GFP and LIS1-2-GFP showed the same cellular distribution and dynamics, but LIS1-2-GFP was less abundant. Both LIS1 proteins were found in the subapical region as single fluorescent particles traveling toward the cell apex, they accumulated in the apical dome forming prominent short filament-like structures, some of which traversed the Spitzenkörper (Spk). The fluorescent structures moved exclusively in anterograde fashion along straight paths suggesting they traveled on Mts. There was no effect in the filament behavior of LIS1-1-GFP in the Δlis1-2 mutant but the dynamics of LIS1-2-GFP was affected in the Δlis1-1 mutant. Microtubular integrity and the dynein-dynactin complex were necessary for the formation of filament-like structures of LIS1-1-GFP in the subapical and apical regions; however, conventional kinesin (KIN-1) was not. Deletion mutants showed that the lack of lis1-1 decreased cell growth by ∼75%; however, the lack of lis1-2 had no effect on growth. A Δlis1-1;Δlis1-2 double mutant showed slower growth than either single mutant. Conidia production was reduced but branching rate increased in Δlis1-1 and the Δlis1-1;Δlis1-2 double mutants. The absence of LIS1-1 had a strong effect on Mt organization and dynamics and indirectly affected nuclear and mitochondrial distribution. The absence of LIS1-1 filaments in dynein mutants (ropy mutants) or in benomyl treated hyphae indicates the strong association between this protein and the regulation of the dynein-dynactin complex and Mt organization. LIS1-1 and LIS1-2 had a high amino acid homology, nevertheless, the absence of the coiled-coil motif in LIS1-2 suggests that its function or regulation may be distinct from that of LIS1-1.

MTB-3, a microtubule plus-end tracking protein (+TIP) of Neurospora crassa.

The microtubule (MT) "plus end" constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called "MT plus-end tracking proteins" (+TIPs). +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassaMicroTubule Binding protein-3 (MTB-3) is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4× faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Δmtb-3 mutants but there were fewer MTs in Δmtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Δmtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics.

Coronin is a component of the endocytic collar of hyphae of Neurospora crassa and is necessary for normal growth and morphogenesis.

Coronin plays a major role in the organization and dynamics of actin in yeast. To investigate the role of coronin in a filamentous fungus (Neurospora crassa), we examined its subcellular localization using fluorescent proteins and the phenotypic consequences of coronin gene (crn-1) deletion in hyphal morphogenesis, Spitzenkörper behavior and endocytosis. Coronin-GFP was localized in patches, forming a subapical collar near the hyphal apex; significantly, it was absent from the apex. The subapical patches of coronin colocalized with fimbrin, Arp2/3 complex, and actin, altogether comprising the endocytic collar. Deletion of crn-1 resulted in reduced hyphal growth rates, distorted hyphal morphology, uneven wall thickness, and delayed establishment of polarity during germination; it also affected growth directionality and increased branching. The Spitzenkörper of Δcrn-1 mutant was unstable; it appeared and disappeared intermittently giving rise to periods of hyphoid-like and isotropic growth respectively. Uptake of FM4-64 in Δcrn-1 mutant indicated a partial disruption in endocytosis. These observations underscore coronin as an important component of F-actin remodeling in N. crassa. Although coronin is not essential in this fungus, its deletion influenced negatively the operation of the actin cytoskeleton involved in the orderly deployment of the apical growth apparatus, thus preventing normal hyphal growth and morphogenesis.

Visualization of F-actin localization and dynamics with live cell markers in Neurospora crassa.

Filamentous actin (F-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. We visualized F-actin organization and dynamics in living Neurospora crassa cells via confocal microscopy of growing hyphae expressing GFP fusions with homologues of the actin-binding proteins fimbrin (FIM) and tropomyosin (TPM-1), a subunit of the Arp2/3 complex (ARP-3) and a recently developed live cell F-actin marker, Lifeact (ABP140 of Saccharomyces cerevisiae). FIM-GFP, ARP-3-GFP, and Lifeact-GFP associated with small patches in the cortical cytoplasm that were concentrated in a subapical ring, which appeared similar for all three markers but was broadest in hyphae expressing Lifeact-GFP. These cortical patches were short-lived, and a subset was mobile throughout the hypha, exhibiting both anterograde and retrograde motility. TPM-1-GFP and Lifeact-GFP co-localized within the Spitzenkörper (Spk) core at the hyphal apex, and were also observed in actin cables throughout the hypha. All GFP fusion proteins studied were also transiently localized at septa: Lifeact-GFP first appeared as a broad ring during early stages of contractile ring formation and later coalesced into a sharper ring, TPM-1-GFP was observed in maturing septa, and FIM-GFP/ARP3-GFP-labeled cortical patches formed a double ring flanking the septa. Our observations suggest that each of the N. crassa F-actin-binding proteins analyzed associates with a different subset of F-actin structures, presumably reflecting distinct roles in F-actin organization and dynamics. Moreover, Lifeact-GFP marked the broadest spectrum of F-actin structures; it may serve as a global live cell marker for F-actin in filamentous fungi.