Ctr9 promotes virulence of Candida albicans by regulating methionine metabolism.

Candida albicans, a part of normal flora, is an opportunistic fungal pathogen and causes severe health issues in immunocompromised patients. Its pathogenicity is intricately linked to the transcriptional regulation of its metabolic pathways. Paf1 complex (Paf1C) is a crucial transcriptional regulator that is highly conserved in eukaryotes. The objective of this study was to explore the role of Paf1C in the metabolic pathways and how it influences the pathogenicity of C. albicans. Paf1C knockout mutant strains of C. albicans (ctr9Δ/Δ, leo1Δ/Δ, and cdc73Δ/Δ) were generated using the CRISPR-Cas9 system. To investigate the effect of Paf1C on pathogenicity, macrophage interaction assays and mouse survival tests were conducted. The growth patterns of the Paf1C knockout mutants were analyzed through spotting assays and growth curve measurements. Transcriptome analysis was conducted under yeast conditions (30°C without serum) and hyphal conditions (37°C with 10% FBS), to further elucidate the role of Paf1C in the pathogenicity of C. albicans. CTR9 deletion resulted in the attenuation of C. albicans virulence, in macrophage and mouse models. Furthermore, we confirmed that the reduced virulence of the ctr9Δ/Δ mutant can be attributed to a decrease in C. albicans cell abundance. Moreover, transcriptome analysis revealed that metabolic processes required for cell proliferation are impaired in ctr9Δ/Δ mutant. Notably, CTR9 deletion led to the downregulation of methionine biosynthetic genes and the cAMP-PKA signaling pathway-related hypha essential genes, which are pivotal for virulence. Our results suggest that Ctr9-regulated methionine metabolism is a crucial factor for determining C. albicans pathogenicity.

PcAvh87, a virulence essential RxLR effector of Phytophthora cinnamomi suppresses host defense and induces cell death in plant nucleus.

Plants have developed intricate immune mechanisms to impede Phytophthora colonization. In response, Phytophthora secretes RxLR effector proteins that disrupt plant defense and promote infection. The specific molecular interactions through which Phytophthora RxLR effectors undermine plant immunity, however, remain inadequately defined. In this study, we delineate the role of the nuclear-localized RxLR effector PcAvh87, which is pivotal for the full virulence of Phytophthora cinnamomi. Gene expression analysis indicates that PcAvh87 expression is significantly upregulated during the initial infection stages, interacting with the immune responses triggered by the elicitin protein INF1 and pro-apoptotic protein BAX. Utilizing PEG/CaCl2-mediated protoplast transformation and CRISPR/Cas9-mediated gene editing, we generated PcAvh87 knockout mutants, which demonstrated compromised hyphal growth, sporangium development, and zoospore release, along with a marked reduction in pathogenicity. This underscores PcAvh87's crucial role as a virulence determinant. Notably, PcAvh87, conserved across the Phytophthora genus, was found to modulate the activity of plant immune protein 113, thereby attenuating plant immune responses. This implies that the PcAvh87-mediated regulatory mechanism could be a common strategy in Phytophthora species to manipulate plant immunity. Our findings highlight the multifaceted roles of PcAvh87 in promoting P. cinnamomi infection, including its involvement in sporangia production, mycelial growth, and the targeting of plant immune proteins to enhance pathogen virulence.

A highly conserved tRNA modification contributes to C. albicans filamentation and virulence.

tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker's yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis, differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits species-specific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C-the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.IMPORTANCEFungal infections are on the rise worldwide, and their global burden on human life and health is frequently underestimated. Among them, the human commensal and opportunistic pathogen, Candida albicans, is one of the major causative agents of severe infections. Its virulence is closely linked to its ability to change morphologies from yeasts to hyphae. Here, this ability is linked-to our knowledge for the first time-to modifications of tRNA and translational efficiency. One tRNA-modifying enzyme, Hma1, plays a specific role in C. albicans and its ability to invade the host. This adds a so-far unknown layer of regulation to the fungal virulence program and offers new potential therapeutic targets to fight fungal infections.

Use of the Iron-Responsive RBT5 Promoter for Regulated Expression in Candida albicans.

Engineered conditional gene expression is used in appraisal of gene function and pathway relationships. For pathogens like the fungus Candida albicans, conditional expression systems are most useful if they are active in the infection environment and if they can be utilized in multiple clinical isolates. Here, we describe such a system. It employs the RBT5 promoter and can be implemented with a few PCRs. We validated the system with RBT5 promoter fusions to two genes that promote filamentation and polarized growth, UME6 and HGC1, and with efg1Δ/Δ mutants, which are defective in an activator of filamentous growth. An RBT5 promoter fusion to either gene enabled filamentous growth of an efg1Δ/Δ mutant of strain SC5314 in iron-limited media, including RPMI with serum and yeast extract-peptone-dextrose with bathophenanthrolinedisulfonic acid. The RBT5-UME6 fusion promoted filamentation of efg1Δ/Δ mutants in RPMI with serum of four other clinical C. albicans isolates as well. In a mouse model of disseminated candidiasis, the RBT5-UME6 fusion promoted filamentation of the SC5314 efg1Δ/Δ mutant in kidney tissue, an indication that the RBT5 promoter is active in the iron-limited host environment. The RBT5 promoter expands the conditional expression toolkit for C. albicans genetics. IMPORTANCE Genetic strategies have been vital for mechanistic analysis of biological processes. Here, we describe a genetic tool for the fungal pathogen Candida albicans.

Candida albicans Filamentation Does Not Require the cAMP-PKA Pathway In Vivo.

Candida albicans is one of the most prevalent human fungal pathogens. Its ability to transition between budding yeast and filamentous morphological forms (pseudohyphae and hyphae) is tightly associated with its pathogenesis. Based on in vitro studies, the cAMP-protein kinase A (PKA) pathway is a key regulator of C. albicans morphogenesis. Using an intravital imaging approach, we investigated the role of the cAMP-PKA pathway during infection. Consistent with their roles in vitro, the downstream effectors of the cAMP-PKA pathway Efg1 and Nrg1 function, respectively, as an activator and a repressor of in vivo filamentation. Surprisingly, strains lacking the adenylyl cyclase, CYR1, showed only slightly reduced filamentation in vivo despite being completely unable to filament in RPMI + 10% serum at 37°C. Consistent with these findings, deletion of the catalytic subunits of PKA (Tpk1 and Tpk2), either singly or in combination, generated strains that also filamented in vivo but not in vitro. In vivo transcription profiling of C. albicans isolated from both ear and kidney tissue showed that the expression of a set of 184 environmentally responsive genes correlated well with in vitro filamentation (R2, 0.62 to 0.68) genes. This concordance suggests that the in vivo and in vitro transcriptional responses are similar but that the upstream regulatory mechanisms are distinct. As such, these data emphatically emphasize that C. albicans filamentation is a complex phenotype that occurs in different environments through an intricate network of distinct regulatory mechanisms. IMPORTANCE The fungus Candida albicans causes a wide range of disease in humans from common diaper rash to life-threatening infections in patients with compromised immune systems. As such, the mechanisms for its ability to cause disease are of wide interest. An intensely studied virulence property of C. albicans is its ability to switch from a round yeast form to filament-like forms (hyphae and pseudohyphae). Surprisingly, we have found that a key signaling pathway that regulates this transition in vitro, the protein kinase A pathway, is not required for filamentation during infection of the host. Our work not only demonstrates that the regulation of filamentation depends upon the specific environment C. albicans inhabits but also underscores the importance of studying these mechanisms during infection.

Bypass of Dfi1 Regulation of Candida albicans Invasive Filamentation by Iron Limitation.

Candida albicans filamentation, the ability to convert from oval yeast cells to elongated hyphal cells, is a key factor in its pathogenesis. Previous work has shown that the integral membrane protein Dfi1 is required for filamentation in cells grown in contact with a semisolid surface. Investigations into the downstream targets of the Dfi1 pathway revealed potential links to two transcription factors, Sef1 and Czf1. Sef1 regulates iron uptake and iron utilization genes under low-iron conditions, leading us to hypothesize that there exists a link between iron availability and contact-dependent invasive filamentation. In this study, we showed that Sef1 was not required for contact-dependent filamentation, but it was required for wild-type (WT) expression levels of a number of genes during growth under contact conditions. Czf1 is required for contact-dependent filamentation and for WT levels of expression of several genes. Constitutive expression and activation of either Sef1 or Czf1 individually in a dfi1 null strain resulted in a complete rescue of the dfi1 null filamentation defect. Because Sef1 is normally activated in low-iron environments, we embedded WT and dfi1 null cells in iron-free agar medium supplemented with various concentrations of ferrous ammonium sulfate (FAS). dfi1 null cells embedded in media with a low concentration of iron (20 µM FAS) showed increased filamentation in comparison to mutant cells embedded in higher concentrations of iron (50 to 500 µM). WT cells produced filamentous colonies in all concentrations. Together, the data indicate that Dfi1, Czf1, Sef1, and environmental iron regulate C. albicans contact-dependent filamentation. IMPORTANCE Candida albicans is an opportunistic pathogen responsible for a larger proportion of candidiasis and candidemia cases than any other Candida species. The ability of C. albicans cells to invade and cause disease is linked to their ability to filament. Despite this, there are gaps in our knowledge of the environmental cues and intracellular signaling that triggers the switch from commensal organism to filamentous pathogen. In this study, we identified a link between contact-dependent filamentation and iron availability. Over the course of tissue invasion, C. albicans cells encounter a number of different iron microenvironments, from the iron-rich gut to iron-poor tissues. Increased expression of Sef1-dependent iron uptake genes as a result of contact-dependent signaling will promote the adaptation of C. albicans cells to a low-iron-availability environment.

Mixed Transcriptome Analysis Revealed the Possible Interaction Mechanisms between Zizania latifolia and Ustilago esculenta Inducing Jiaobai Stem-Gall Formation.

The smut fungus Ustilago esculenta infects Zizania latifolia and induces stem expansion to form a unique vegetable named Jiaobai. Although previous studies have demonstrated that hormonal control is essential for triggering stem swelling, the role of hormones synthesized by Z. latifolia and U. esculenta and the underlying molecular mechanism are not yet clear. To study the mechanism that triggers swollen stem formation, we analyzed the gene expression pattern of both interacting organisms during the initial trigger of culm gall formation, at which time the infective hyphae also propagated extensively and penetrated host stem cells. Transcriptional analysis indicated that abundant genes involving fungal pathogenicity and plant resistance were reprogrammed to maintain the subtle balance between the parasite and host. In addition, the expression of genes involved in auxin biosynthesis of U. esculenta obviously decreased during stem swelling, while a large number of genes related to the synthesis, metabolism and signal transduction of hormones of the host plant were stimulated and showed specific expression patterns, particularly, the expression of ZlYUCCA9 (a flavin monooxygenase, the key enzyme in indole-3-acetic acid (IAA) biosynthesis pathway) increased significantly. Simultaneously, the content of IAA increased significantly, while the contents of cytokinin and gibberellin showed the opposite trend. We speculated that auxin produced by the host plant, rather than the fungus, triggers stem swelling. Furthermore, from the differently expressed genes, two candidate Cys2-His2 (C2H2) zinc finger proteins, GME3058_g and GME5963_g, were identified from U. esculenta, which may conduct fungus growth and infection at the initial stage of stem-gall formation.

Integrative multi-omics profiling reveals cAMP-independent mechanisms regulating hyphal morphogenesis in Candida albicans.

Microbial pathogens grow in a wide range of different morphologies that provide distinct advantages for virulence. In the fungal pathogen Candida albicans, adenylyl cyclase (Cyr1) is thought to be a master regulator of the switch to invasive hyphal morphogenesis and biofilm formation. However, faster growing cyr1Δ/Δ pseudorevertant (PR) mutants were identified that form hyphae in the absence of cAMP. Isolation of additional PR mutants revealed that their improved growth was due to loss of one copy of BCY1, the negative regulatory subunit of protein kinase A (PKA) from the left arm of chromosome 2. Furthermore, hyphal morphogenesis was improved in some of PR mutants by multigenic haploinsufficiency resulting from loss of large regions of the left arm of chromosome 2, including global transcriptional regulators. Interestingly, hyphal-associated genes were also induced in a manner that was independent of cAMP. This indicates that basal protein kinase A activity is an important prerequisite to induce hyphae, but activation of adenylyl cyclase is not needed. Instead, phosphoproteomic analysis indicated that the Cdc28 cyclin-dependent kinase and the casein kinase 1 family member Yck2 play key roles in promoting polarized growth. In addition, integrating transcriptomic and proteomic data reveals hyphal stimuli induce increased production of key transcription factors that contribute to polarized morphogenesis.

Rapid proliferation due to better metabolic adaptation results in full virulence of a filament-deficient Candida albicans strain.

The ability of the fungal pathogen Candida albicans to undergo a yeast-to-hypha transition is believed to be a key virulence factor, as filaments mediate tissue damage. Here, we show that virulence is not necessarily reduced in filament-deficient strains, and the results depend on the infection model used. We generate a filament-deficient strain by deletion or repression of EED1 (known to be required for maintenance of hyphal growth). Consistent with previous studies, the strain is attenuated in damaging epithelial cells and macrophages in vitro and in a mouse model of intraperitoneal infection. However, in a mouse model of systemic infection, the strain is as virulent as the wild type when mice are challenged with intermediate infectious doses, and even more virulent when using low infectious doses. Retained virulence is associated with rapid yeast proliferation, likely the result of metabolic adaptation and improved fitness, leading to high organ fungal loads. Analyses of cytokine responses in vitro and in vivo, as well as systemic infections in immunosuppressed mice, suggest that differences in immunopathology contribute to some extent to retained virulence of the filament-deficient mutant. Our findings challenge the long-standing hypothesis that hyphae are essential for pathogenesis of systemic candidiasis by C. albicans.

Candida albicans requires iron to sustain hyphal growth.

Candida albicans is an important opportunistic fungal pathogen of immunocompromised individuals. The ability to switch between yeast and hyphal growth forms is critical for its pathogenesis. Hyphal development in C. albicans requires two temporally linked regulations for initiation and maintenance. Here, we performed transcriptome sequencing (RNA-Seq) to analyze the transcriptional consequences for the two different phases of hyphal development. Genome-wide transcription profiling reveals that the sets associated with hyphal initiation were significantly enriched in genes for hyphal cell wall, biofilm matrix and actin polarization. In addition to hypha-specific genes, numerous genes involved in iron acquisition, such as FTR1 and SEF1, are highly induced specifically during sustained hyphal development even when additional free iron is supplied in the medium. Therefore, iron uptake genes are induced by signals that can support prolonged hyphal development in an iron-independent manner. The induction of iron acquisition genes during hyphal elongation was further confirmed by quantitative reverse transcription-PCR under various hypha-inducing conditions. Remarkably, preventing C. albicans from acquiring iron blocks BRG1 activation, leading to impaired hyphal maintenance, and ectopically expressed BRG1 can sustain hyphal development bypassing the requirement of iron. Our study elucidates an underlying mechanism of how multiple virulence factors are interconnected and are induced simultaneously during infection.

N-acetylglucosamine-mediated morphological transition in Candida albicans and Candida tropicalis.

Morphological transitions in Candida species are key factors in facilitating invasion and adapting to environmental changes. N-acetylglucosamine (GlcNAc) is a monosaccharide signalling molecule that can regulate morphological transitions in Candida albicans and Candida tropicalis. Interestingly, although the uptake and metabolic pathways of GlcNAc and GlcNAc-mediated white-to-opaque cell switching are similar between the two Candida species, GlcNAc induces hyphal development in C. albicans, whereas it suppresses hyphal development in C. tropicalis. These findings indicate that the characteristics of C. albicans and C. tropicalis in response to GlcNAc are remarkably different. Here, we compare the conserved and divergent GlcNAc-mediated signalling pathways and catabolism between the two Candida species. Deletion of NGT1, a GlcNAc transportation gene, inhibited hyphal formation in C. albicans but promoted hyphal development in C. tropicalis. To further understand these opposite effects on filamentous growth in response to GlcNAc in the two Candida species, the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signalling pathways in both C. albicans and C. tropicalis were compared. Interestingly, GlcNAc activated the cAMP/PKA signalling pathway of the two Candida species, suggesting that the hyphal development-regulated circuit is remarkably diverse between the two species. Indeed, the Ndt80-like gene REP1, which is critical for regulating GlcNAc catabolism, exhibits distinct roles in the hyphal development of C. albicans and C. tropicalis. These data suggest possible reasons for the divergent hyphal growth response in C. albicans and C. tropicalis upon GlcNAc induction.

Steccherinum tenuissimum and S. xanthum spp. nov. (Polyporales, Basidiomycota): New species from China.

Two new wood-inhabiting fungal species, Steccherinum tenuissimum and S. xanthum spp. nov. are described based on a combination of morphological features and molecular evidence. Steccherinum tenuissimum is characterized by an annual growth habit, resupinate basidiomata with an odontioid hymenial surface, a dimitic hyphal system with clamped generative hyphae, strongly encrusted cystidia and basidiospores measuring 3-5 × 2-3.5 µm. Steccherinum xanthum is characterized by odontioid basidiomata and a monomitic hyphal system with generative hyphae bearing clamp connections and covering by crystals, colourless, thin-walled, smooth, IKI-, CB-and has basidiospores measuring 2.7-5.5 × 1.8-4.0 µm. Sequences of the ITS and nLSU nrRNA gene regions of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony and Bayesian inference methods. The phylogenetic analyses based on molecular data of ITS + nLSU sequences showed that two new Steccherinum species felled into the residual polyporoid clade. Further investigation was obtained for more representative taxa in Steccherinum based on ITS + nLSU sequences, which demonstrated that S. tenuissimum and S. xanthum were sister to S. robustius with high support (100% BP, 100% BS and 1.00 BPP).

Roles of Dhh1 RNA helicase in yeast filamentous growth: Analysis of N-terminal phosphorylation residues and ATPase domains.

In yeast Saccharomyces cerevisiae, the Dhh1 protein, a member of the DEAD-box RNA helicase, stimulates Dcp2/Dcp1-mediated mRNA decapping and functions as a general translation repressor. Dhh1 also positively regulates translation of a selected set of mRNAs, including Ste12, a transcription factor for yeast mating and pseudohyphal growth. Given the diverse functions of Dhh1, we investigated whether the putative phosphorylation sites or the conserved motifs for the DEAD-box RNA helicases were crucial in the regulatory roles of Dhh1 during pseudohyphal growth. Mutations in the ATPase A or B motif (DHH1-K96R or DHH1-D195A) showed significant defects in pseudohyphal colony morphology and agar invasive phenotypes. The N-terminal phospho-mimetic mutation, DHH1-T16E, showed defects in pseudohyphal phenotypes. Decreased levels of Ste12 protein were also observed in these pseudohyphal-defective mutant cells under filamentous-inducing low nitrogen conditions. We suggest that the ATPase motifs and the Thr16 phosphorylation site of Dhh1 are crucial to its regulatory roles in pseudohyphal growth under low nitrogen conditions.

The msh1 gene is responsible for short life span mutant natural death and functions to maintain mitochondrial DNA integrity.

Wild-type filamentous fungus Neurospora crassa continues to grow its hyphae for a very lengthy period of time (>2 years), whereas mutations at the natural death (nd) locus shorten life span (approximately 20 days). By positional cloning based on heat augmented mutagen sensitivity of the nd strain, we identified a nonsense mutation in the msh1 gene, an eukaryotic homolog of bacterial MutS, and this mutation resulted in encoding non-functional polypeptide. By tagging with GFP, subcellular localization of the MSH1 protein in the mitochondria was observed, and knock out of the msh1 gene caused severe growth deficiency accompanying mitochondrial DNA (mtDNA) aberrations such as large-scale mtDNA deletions and rearrangements as seen in the nd strain. These results suggested that MSH1 may maintain mtDNA integrity. Thus, loss of function compromises mtDNA, leading to the acceleration of cellular aging.

Localization of NPFxD motif-containing proteins in Aspergillus nidulans.

During growth, filamentous fungi produce polarized cells called hyphae. It is generally presumed that polarization of hyphae is dependent upon secretion through the Spitzenkörper, as well as a mechanism called apical recycling, which maintains a balance between the tightly coupled processes of endocytosis and exocytosis. Endocytosis predominates in an annular domain called the sub-apical endocytic collar, which is located in the region of plasma membrane 1-5 µm distal to the Spitzenkörper. It has previously been proposed that one function of the sub-apical endocytic collar is to maintain the apical localization of polarization proteins. These proteins mark areas of polarization at the apices of hyphae. However, as hyphae grow, these proteins are displaced along the membrane and some must then be removed at the sub-apical endocytic collar in order to maintain the hyphoid shape. While endocytosis is fairly well characterized in yeast, comparatively little is known about the process in filamentous fungi. Here, a bioinformatics approach was utilized to identify 39 Aspergillus nidulans proteins that are predicted to be cargo of endocytosis based on the presence of an NPFxD peptide motif. This motif is a necessary endocytic signal sequence first established in Saccharomyces cerevisiae, where it marks proteins for endocytosis through an interaction with the adapter protein Sla1p. It is hypothesized that some proteins that contain this NPFxD peptide sequence in A. nidulans will be potential targets for endocytosis, and therefore will localize either to the endocytic collar or to more proximal polarized regions of the cell, e.g. the apical dome or the Spitzenkörper. To test this, a subset of the motif-containing proteins in A. nidulans was tagged with GFP and the dynamic localization was evaluated. The documented localization patterns support the hypothesis that the motif marks proteins for localization to the polarized cell apex in growing hyphae.

Phylogeny and taxonomy of Ceriporia and other related taxa and description of three new species.

Species of Ceriporia (Irpicaceae, Basidiomycota) are saprotrophs or endophytes in forest ecosystems. To evaluate the taxonomy and generic relationships of Ceriporia and other related taxa, we used morphology and multigene phylogenetic analyses based on sequence data from nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) region, nuc 28S rDNA (28S), and RNA polymerase II largest subunit (rpb1). Our results show that Ceriporia sensu lato is polyphyletic and distributed across multiple clades in the Irpicaceae, Phanerochaetaceae, and Meruliaceae. Some species previously considered in Ceriporia are now recovered in Meruliopsis, resulting in four new combinations: M. albomellea, M. crassitunicata, M. nanlingensis, and M. pseudocystidiata. Two new species of Meruliopsis are described: M. leptocystidiata from northeast China and South Korea and M. parvispora from Taiwan. Ceriporia arbuscula is described as a new species from Taiwan. Ceriporia mellita and Meruliopsis nanlingensis are newly recorded from Japan and Taiwan, and M. taxicola is recorded from Taiwan for the first time.

A mycorrhiza-specific H+ -ATPase is essential for arbuscule development and symbiotic phosphate and nitrogen uptake.

Most land plants can form symbiosis with arbuscular mycorrhizal (AM) fungi to enhance uptake of mineral nutrients, particularly phosphate (Pi) and nitrogen (N), from the soil. It is established that transport of Pi from interfacial apoplast into plant cells depends on the H+ gradient generated by the H+ -ATPase located on the periarbuscular membrane (PAM); however, little evidence regarding the potential link between mycorrhizal N transport and H+ -ATPase activity is available to date. Here, we report that a PAM-localized tomato H+ -ATPase, SlHA8, is indispensable for arbuscule development and mycorrhizal P and N uptake. Knockout of SlHA8 resulted in truncated arbuscule morphology, reduced shoot P and N accumulation, and decreased H+ -ATPase activity and acidification of apoplastic spaces in arbusculated cells. Overexpression of SlHA8 in tomato promoted both P and N uptake, and increased total colonization level, but did not affect arbuscule morphology. Heterogeneous expression of SlHA8 in the rice osha1 mutant could fully complement its defects in arbuscule development and mycorrhizal P and N uptake. Our results propose a pivotal role of the SlHA8 in energizing both the symbiotic P and N transport, and highlight the evolutionary conservation of the AM-specific H+ -ATPase orthologs in maintaining AM symbiosis across different mycorrhizal plant species.

Virulence and pathogenicity of a Candida albicans mutant with reduced filamentation.

Deletion of DNA polymerase eta (Rad30/Polη) in pathogenic yeast Candida albicans is known to reduce filamentation induced by serum, ultraviolet, and cisplatin. Because nonfilamentous C. albicans is widely accepted as avirulent form, here we explored the virulence and pathogenicity of a rad30Δ strain of C. albicans in cell-based and animal systems. Flow cytometry of cocultured fungal and differentiated macrophage cells revealed that comparatively higher percentage of macrophages was associated with the wild-type than rad30Δ cells. In contrast, higher number of Polη-deficient C. albicans adhered per macrophage membrane. Imaging flow cytometry showed that the wild-type C. albicans developed hyphae after phagocytosis that caused necrotic death of macrophages to evade their clearance. Conversely, phagosomes kill the fungal cells as estimated by increased metacaspase activity in wild-type C. albicans. Despite the morphological differences, both wild-type and rad30∆ C. albicans were virulent with a varying degree of pathogenicity in mice models. Notably, mice with Th1 immunity were comparatively less susceptible to systemic fungal infection than Th2 type. Thus, our study clearly suggests that the modes of interaction of morphologically different C. albicans strains with the host immune cells are diverged, and host genetic background and several other attributing factors of the fungus could additionally determine their virulence.

Ochroconis globalis infecting Atlantic salmon (Salmo salar), with a review of Ochroconis species in cold-blooded animals.

Necropsy examination of an adult Atlantic salmon (Salmo salar) from the Dalälven River in Sweden revealed numerous large, white nodules, with spherical cysts and granulomata in kidney and liver. Histopathology showed dark, septate, thin-walled hyphae. The aetiologic agent was found to be an Ochroconis species (Venturiales) that differed from known fish-associated species of the genus. Molecular phylogenetic studies of the culture (strain UIII09 = CBS 135766) demonstrated that Ochroconis globalis was concerned. The isolate proved to be susceptible to all investigated antifungals, as it is known for another Ochroconis species. The role of Ochroconis in opportunism of cold-blooded animals was discussed, and the diagnostic methods using DNA sequences for routine identification of the fungus were proposed.

Mitochondrial proline catabolism activates Ras1/cAMP/PKA-induced filamentation in Candida albicans.

Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence.