Molecular cloning and functional characterization of UBC13 and MMS2 from Candida albicans.

To maintain genome stability, eukaryotes have evolved a powerful DNA damage response system called DNA-damage tolerance (DDT) to deal with replication-blocking lesions. In the budding yeast Saccharomyces cerevisiae, K63-linked polyubiquitination of proliferating cell nuclear antigen (PCNA) is mediated by a Ubc13-Mms2 heterodimer, leading to error-free DDT. Candida albicans is one of the most studied fungal pathogens and to date no data regarding K63-linked ubiquitination or error-free DDT has been available. Here we report the identification and functional characterization of UBC13 and MMS2 genes from C. albicans. Both genes are highly conserved between S. cerevisiae and C. albicans. However, CaUbc13 differs from all other eukaryotes in that it contains a 21-amino acid tail that appears to attenuate its interaction with CaMms2, suggesting a possible regulatory mechanism in C. albicans. Both CaUBC13 and CaMMS2 genes can functionally rescue the corresponding budding yeast mutants from increased spontaneous mutagenesis and killing by DNA-damaging agents, indicating an error-free DDT pathway in C. albicans. Indeed Caubc13Δ/Δ and Camms2Δ/Δ null mutants were constructed and displayed characteristic sensitivity to DNA-damaging agents.

Ricin B lectin-like proteins of the microsporidian Encephalitozoon cuniculi and Anncaliia algerae are involved in host-cell invasion.

Microsporidia are obligate intracellular pathogens capable of infecting a wide variety of hosts ranging from invertebrates to vertebrates. The infection process requires a step of prior adherence of Microsporidia to the surface of host cells. A few studies demonstrated the involvement of proteins containing a ricin-B lectin (RBL) domain in parasite infection. In this study Anncalia algerae and Encephalitozoon cuniculi genomes were screened by bioinformatic analysis to identify proteins with an extracellular prediction and possessing RBL-type carbohydrate-binding domains, being both potentially relevant factors contributing to host cell adherence. Three proteins named AaRBLL-1 and AaRBLL-2 from A. algerae and EcRBLL-1 from E. cuniculi, were selected and comparative analysis of sequences suggested their belonging to a multigenic family, with a conserved structural RBL domain despite a significant amino acid sequence divergence. The production of recombinant proteins and antibodies against the three proteins allowed their subcellular localization on the spore wall and/or the polar tube. Adherence inhibition assays based on pre-treatments with recombinant proteins or antibodies highlighted the significant decrease of the proliferation of both E. cuniculi and A. algerae, strongly suggesting that these proteins are involved in the infection process.

Fungal effector SIB1 of Colletotrichum orbiculare has unique structural features and can suppress plant immunity in Nicotiana benthamiana.

Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five ß-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel ß-barrel structure. However, the ß-strands were found to display a unique topology, one pair of these ß-strands formed a parallel ß-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern-triggered immunity in N. benthamiana.

Candida albicans ENT2 Contributes to Efficient Endocytosis, Cell Wall Integrity, Filamentation, and Virulence.

Epsins play a pivotal role in the formation of endocytic vesicles and potentially provide a linkage between endocytic and other trafficking pathways. We identified a Candida albicans epsin, ENT2, that bears homology to the Saccharomyces cerevisiae early endocytosis genes ENT1 and ENT2 and studied its functions by a reverse genetic approach utilizing CRISPR-Cas9-mediated gene deletion. The C. albicans ent2Δ/Δ null mutant displayed cell wall defects and altered antifungal drug sensitivity. To define the role of C. albicans ENT2 in endocytosis, we performed assays with the lipophilic dye FM4-64 that revealed greatly reduced uptake in the ent2Δ/Δ mutant. Next, we showed that the C. albicans ent2Δ/Δ mutant was unable to form hyphae and biofilms. Assays for virulence properties in an in vitro keratinocyte infection model demonstrated reduced damage of mammalian adhesion zippers and host cell death from the ent2Δ/Δ mutant. We conclude that C. albicans ENT2 has a role in efficient endocytosis, a process that is required for maintaining cell wall integrity, hyphal formation, and virulence-defining traits. IMPORTANCE The opportunistic fungal pathogen Candida albicans is an important cause of invasive infections in hospitalized patients and a source of considerable morbidity and mortality. Despite its clinical importance, we still need to improve our ability to diagnose and treat this common pathogen. In order to support these advancements, a greater understanding of the biology of C. albicans is needed. In these studies, we are focused on the fundamental biological process of endocytosis, of which little is directly known in C. albicans. In addition to studying the function of a key gene in this process, we are examining the role of endocytosis in the virulence-related processes of filamentation, biofilm formation, and tissue invasion. These studies will provide greater insight into the role of endocytosis in causing invasive fungal infections.

Functional Roles of α1-, α2-, ß1-, and ß2-Tubulins in Vegetative Growth, Microtubule Assembly, and Sexual Reproduction of Fusarium graminearum.

The plant pathogen Fusarium graminearum contains two α-tubulin isotypes (α1 and α2) and two ß-tubulin isotypes (ß1 and ß2). The functional roles of these tubulins in microtubule assembly are not clear. Previous studies reported that α1- and ß2-tubulin deletion mutants showed severe growth defects and hypersensitivity to carbendazim, which have not been well explained. Here, we investigated the interaction between α- and ß-tubulin of F. graminearum. Colocalization experiments demonstrated that ß1- and ß2-tubulin are colocalized. Coimmunoprecipitation experiments suggested that ß1-tubulin binds to both α1- and α2-tubulin and that ß2-tubulin can also bind to α1- or α2-tubulin. Interestingly, deletion of α1-tubulin increased the interaction between ß2-tubulin and α2-tubulin. Microtubule observation assays showed that deletion of α1-tubulin completely disrupted ß1-tubulin-containing microtubules and significantly decreased ß2-tubulin-containing microtubules. Deletion of α2-, ß1-, or ß2-tubulin had no obvious effect on the microtubule cytoskeleton. However, microtubules in α1- and ß2-tubulin deletion mutants were easily depolymerized in the presence of carbendazim. The sexual reproduction assay indicates that α1- and ß1-tubulin deletion mutants could not produce asci and ascospores. These results implied that α1-tubulin may be essential for the microtubule cytoskeleton. However, our Δα1-2×α2 mutant (α1-tubulin deletion mutant containing two copies of α2-tubulin) exhibited normal microtubule network, growth, and sexual reproduction. Interestingly, the Δα1-2×α2 mutant was still hypersensitive to carbendazim. In addition, both ß1-tubulin and ß2-tubulin were found to bind the mitochondrial outer membrane voltage-dependent anion channel (VDAC), indicating that they could regulate the function of VDAC. IMPORTANCE In this study, we found that F. graminearum contains four different α-/ß-tubulin heterodimers (α1-/ß1-, α1-/ß2-, α2-/ß1-, and α2-/ß2-tubulin heterodimers), and they assemble together into a single microtubule. Moreover, α1- and α2-tubulins are functionally interchangeable in microtubule assembly, vegetative growth, and sexual reproduction. These results provide more insights into the functional roles of different tubulins of F. graminearum, which could be helpful for purification of tubulin heterodimers and development of new tubulin-binding agents.

Mapping yeast mitotic 5' resection at base resolution reveals the sequence and positional dependence of nucleases in vivo.

Resection of the 5'-terminated strand at DNA double-strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Recent studies have described a multi-step model of DSB resection where endonucleolytic cleavage mediated by Mre11 and Sae2 leads to further degradation mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with high-throughput sequencing using molecular identifiers, allowing exact counting of cleaved 5' ends at base resolution. Mutant strains, including exo1Δ, mre11-H125N and exo1Δ sgs1Δ, revealed a major Mre11-dependent cleavage position 60-70 bp from the DSB end whose exact position depended on local sequence. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1Δ sgs1Δ yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB. These results provide detailed in vivo views of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.

Electrophysiological characterization of a diverse group of sugar transporters from Trichoderma reesei.

Trichoderma reesei is an ascomycete fungus known for its capability to secrete high amounts of extracellular cellulose- and hemicellulose-degrading enzymes. These enzymes are utilized in the production of second-generation biofuels and T. reesei is a well-established host for their production. Although this species has gained considerable interest in the scientific literature, the sugar transportome of T. reesei remains poorly characterized. Better understanding of the proteins involved in the transport of different sugars could be utilized for engineering better enzyme production strains. In this study we aimed to shed light on this matter by characterizing multiple T. reesei transporters capable of transporting various types of sugars. We used phylogenetics to select transporters for expression in Xenopus laevis oocytes to screen for transport activities. Of the 18 tested transporters, 8 were found to be functional in oocytes. 10 transporters in total were investigated in oocytes and in yeast, and for 3 of them no transport function had been described in literature. This comprehensive analysis provides a large body of new knowledge about T. reesei sugar transporters, and further establishes X. laevis oocytes as a valuable tool for studying fungal sugar transporters.

A Novel Hexose Transporter ChHxt6 Is Required for Hexose Uptake and Virulence in Colletotrichum higginsianum.

Colletotrichum higginsianum is an important hemibiotrophic plant pathogen that causes crucifer anthracnose worldwide. To date, some hexose transporters have been identified in fungi. However, the functions of hexose transporters in virulence are not clear in hemibiotrophic phytopathogens. In this study, we identified and characterized a new hexose transporter gene named ChHxt6 from a T-DNA insertion pathogenicity-deficient mutant G256 in C. higginsianum. Expression profiling analysis revealed that six ChHxt genes, ChHxt1 to ChHxt6, exhibited specific expression patterns in different infection phases of C. higginsianum. The ChHxt1 to ChHxt6 were separately deleted using the principle of homologous recombination. ChHxt1 to ChHxt6 deletion mutants grew normally on PDA plates, but only the virulence of ChHxt4 and ChHxt6 deletion mutants was reduced. ChHxt4 was required for fungal infection in both biotrophic and necrotrophic stages, while ChHxt6 was important for formation of necrotrophic hyphae during infection. In addition, ChHxts were functional in uptake of different hexoses, but only ChHxt6-expressing cells could grow on all five hexoses, indicating that the ChHxt6 was a central hexose transporter and crucial for hexose uptake. Site-directed mutation of T169S and P221L positions revealed that these two positions were necessary for hexose transport, whereas only the mutation Thr169 caused reduced virulence and defect in formation of necrotrophic hyphae. Taken together, ChHxt6 might regulate fungal virulence by modulating the utilization of hexose.

Structure prediction and function characterization of WC-2 proteins in Blakeslea trispora.

Blakeslea trispora is known for its potential to produce an excess of carotenoids in mixed cultures of strains of opposite sex. The biosynthesis of ß-carotene in B. trispora is activated not only by sex hormone trisporic acid but also by light, especially blue light. In fungi, the most intensively investigated blue-light reception proteins are WC-1 and WC-2, and the two proteins form a transcription factor complex which is called WCC by their PAS domains. Notably, multiple genes similar to wc-1 and wc-2 have been identified and characterized in Phycomyces, Mucor, and Rhizopus. Here we report that there are four members of wc-2-like gene family in B. trispora genome: Btwc-2a, Btwc-2b, Btwc-2c, and Btwc-2d. When the mycelia were exposed to blue light, their transcription levels are regulated differentially. Except for BtWC-2b, which only has a PAS domain, the other three proteins contain both a PAS domain and a ZnF domain. BtWC-2a interacts with either BtWC-1a or BtWC-1c to form different photoreceptor complexes in yeast two-hybrid assays, which is the unique situation not yet described in other fungi. In addition, the protein-protein docking analysis by the predicted 3D structures showed that the two complexes are structurally different. These results suggested that WC proteins of B. trispora are still involved in light regulation by forming WCC and the regulation mechanism of the photobiology appears to be more complex.

Important role of melanin for fertility in the fungus Podospora anserina.

Melanins are pigments used by fungi to withstand various stresses and to strengthen vegetative and reproductive structures. In Sordariales fungi, their biosynthesis starts with a condensation step catalyzed by an evolutionary-conserved polyketide synthase. Here we show that complete inactivation of this enzyme in the model ascomycete Podospora anserina through targeted deletion of the PaPks1 gene results in reduced female fertility, in contrast to a previously analyzed nonsense mutation in the same gene that retains full fertility. We also show the utility of PaPks1 mutants for detecting rare genetic events in P. anserina, such as parasexuality and possible fertilization and/or apomixis of nuclei devoid of mating-type gene.

Dynamical analysis of the fission yeast cell cycle via Markov chain.

The cell cycle is a complex network involved in the regulation of cell growth and proliferation. Intrinsic molecular noise in gene expression in the cell cycle network can generate fluctuations in protein concentration. How the cell cycle network maintains its robust transitions between cell cycle phases in the presence of these fluctuations remains unclear. To understand the complex and robust behavior of the cell cycle system in the presence of intrinsic noise, we developed a Markov model for the fission yeast cell cycle system. We quantified the effect of noise on gene and protein activity and on the probability of transition between different phases of the cell cycle. Our analysis shows how network perturbations decide the fate of the cell. Our model predicts that the cell cycle pathway (subsequent transitions from [Formula: see text]) is the most robust and probable pathway among all possible trajectories in the cell cycle network. We performed a sensitivity analysis to find correlations between protein interaction weights and transition probabilities between cell cycle phases. The sensitivity analysis predicts how network perturbations affect the transition probability between different cell cycle phases and, consequently, affect different cell fates, thus, forming testable in vitro/in vivo hypotheses. Our simulation results agree with published experimental findings and reveal how noise in the cell cycle regulatory network can affect cell cycle progression.

Recording of DNA-binding events reveals the importance of a repurposed Candida albicans regulatory network for gut commensalism.

Candida albicans is a fungal component of the human gut microbiota and an opportunistic pathogen. C. albicans transcription factors (TFs), Wor1 and Efg1, are master regulators of an epigenetic switch required for fungal mating that also control colonization of the mammalian gut. We show that additional mating regulators, WOR2, WOR3, WOR4, AHR1, CZF1, and SSN6, also influence gut commensalism. Using Calling Card-seq to record Candida TF DNA-binding events in the host, we examine the role and relationships of these regulators during murine gut colonization. By comparing in-host transcriptomes of regulatory mutants with enhanced versus diminished commensal fitness, we also identify a set of candidate commensalism effectors. These include Cht2, a GPI-linked chitinase whose gene is bound by Wor1, Czf1, and Efg1 in vivo, that we show promotes commensalism. Thus, the network required for a C. albicans sexual switch is biochemically active in the host intestine and repurposed to direct commensalism.

Fungal X-Intrinsic Protein Aquaporin from Trichoderma atroviride: Structural and Functional Considerations.

The major intrinsic protein (MIP) superfamily is a key part of the fungal transmembrane transport network. It facilitates the transport of water and low molecular weight solutes across biomembranes. The fungal uncharacterized X-Intrinsic Protein (XIP) subfamily includes the full protein diversity of MIP. Their biological functions still remain fully hypothetical. The aim of this study is still to deepen the diversity and the structure of the XIP subfamily in light of the MIP counterparts-the aquaporins (AQPs) and aquaglyceroporins (AQGPs)-and to describe for the first time their function in the development, biomass accumulation, and mycoparasitic aptitudes of the fungal bioagent Trichoderma atroviride. The fungus-XIP clade, with one member (TriatXIP), is one of the three clades of MIPs that make up the diversity of T. atroviride MIPs, along with the AQPs (three members) and the AQGPs (three members). TriatXIP resembles those of strict aquaporins, predicting water diffusion and possibly other small polar solutes due to particularly wider ar/R constriction with a Lysine substitution at the LE2 position. The XIP loss of function in ∆TriatXIP mutants slightly delays biomass accumulation but does not impact mycoparasitic activities. ∆TriatMIP forms colonies similar to wild type; however, the hyphae are slightly thinner and colonies produce rare chlamydospores in PDA and specific media, most of which are relatively small and exhibit abnormal morphologies. To better understand the molecular causes of these deviant phenotypes, a wide-metabolic survey of the ∆TriatXIPs demonstrates that the delayed growth kinetic, correlated to a decrease in respiration rate, is caused by perturbations in the pentose phosphate pathway. Furthermore, the null expression of the XIP gene strongly impacts the expression of four expressed MIP-encoding genes of T. atroviride, a plausible compensating effect which safeguards the physiological integrity and life cycle of the fungus. This paper offers an overview of the fungal XIP family in the biocontrol agent T. atroviride which will be useful for further functional analysis of this particular MIP subfamily in vegetative growth and the environmental stress response in fungi. Ultimately, these findings have implications for the ecophysiology of Trichoderma spp. in natural, agronomic, and industrial systems.

The Golgin Protein RUD3 Regulates Fusarium graminearum Growth and Virulence.

Golgins are coiled-coil proteins that play prominent roles in maintaining the structure and function of the Golgi complex. However, the role of golgin proteins in phytopathogenic fungi remains poorly understood. In this study, we functionally characterized the Fusarium graminearum golgin protein RUD3, a homolog of ScRUD3/GMAP-210 in Saccharomyces cerevisiae and mammalian cells. Cellular localization observation revealed that RUD3 is located in the cis-Golgi. Deletion of RUD3 caused defects in vegetative growth, ascospore discharge, deoxynivalenol (DON) production, and virulence. Moreover, the Δrud3 mutant showed reduced expression of tri genes and impairment of the formation of toxisomes, both of which play essential roles in DON biosynthesis. We further used green fluorescent protein (GFP)-tagged SNARE protein SEC22 (SEC22-GFP) as a tool to study the transport between the endoplasmic reticulum (ER) and Golgi and observed that SEC22-GFP was retained in the cis-Golgi in the Δrud3 mutant. RUD3 contains the coiled coil (CC), GRAB-associated 2 (GA2), GRIP-related Arf binding (GRAB), and GRAB-associated 1 (GA1) domains, which except for GA1, are indispensable for normal localization and function of RUD3, whereas only CC is essential for normal RUD3-RUD3 interaction. Together, these results demonstrate how the golgin protein RUD3 mediates retrograde trafficking in the ER-to-Golgi pathway and is necessary for growth, ascospore discharge, DON biosynthesis, and pathogenicity in F. graminearumIMPORTANCEFusarium head blight (FHB) caused by the fungal pathogen Fusarium graminearum is an economically important disease of wheat and other small grain cereal crops worldwide, and limited effective control strategies are available. A better understanding of the regulation mechanisms of F. graminearum development, deoxynivalenol (DON) biosynthesis, and pathogenicity is therefore important for the development of effective control management of this disease. Golgins are attached via their extreme carboxy terminus to the Golgi membrane and are involved in vesicle trafficking and organelle maintenance in eukaryotic cells. In this study, we systematically characterized a highly conserved Golgin protein, RUD3, and found that it is required for vegetative growth, ascospore discharge, DON production, and pathogenicity in F. graminearum Our findings provide a comprehensive characterization of the golgin family protein RUD3 in plant-pathogenic fungus, which could help to identify a new potential target for effective control of this devastating disease.

An improved staining method of cell cycle analysis with Sybr Green I for fungi: Cryptococcus neoformans and Saccharomyces cerevisiae.

Cryptococcus neoformans is a pathogenic fungus which causes millions of deaths and infections, especially threatening immunocompromised individuals. During the development of new drugs, the ubiquitination has been found to play an important role in the regulation of the virulence and cell cycle of this fungus. Based on this mechanism, ubiquitination-related mutant strains exhibiting cell cycle arrest have been established for drug development for the fungus. However, flow cytometry detection of the cell cycle in fungi is generally difficult because the thick cell wall and capsule of fungi generally contribute to a nonspecific signal of cytometry. In this study, an improved method, derived from Saccharomyces cerevisiae assays, is developed to specifically stain C. neoformans, in whose cell cycle the G1 and G2 peaks are separated enough to be allowed for cell cycle analysis. As a result, the improved method facilitates the detection of the alterations in the cell cycle of C. neoformans with a mutation that results in cell cycle arrest, which distinctly delays the cell division of C. neoformans. Thus, the improved method reported here provides detailed technical information regarding assays on C. neoformans and, more importantly, offers a solution for assessing the cell cycle in other fungi in the future. Abbreviation: PI: propidium iodide.

Expression of L-amino acid oxidase of Trichoderma harzianum in tobacco confers resistance to Sclerotinia sclerotiorum and Botrytis cinerea.

L-amino acid oxidase (ThLAAO) secreted by Trichoderma harzianum ETS323 is a flavoenzyme with antimicrobial characteristics. In this study, we transformed the ThLAAO gene into tobacco to elucidate whether ThLAAO can activate defense mechanisms and confer resistance against phytopathogens. Transgenic tobacco overexpressing ThLAAO showed enhanced resistance against Sclerotinia sclerotiorum and Botrytis cinerea and activated the expression of defense-related genes and the genes involved in salicylic acid, jasmonic acid, and ethylene biosynthesis accompanied by substantial accumulation of H2O2 in chloroplasts, cytosol around chloroplasts, and cell membranes of transgenic tobacco. Scavenge of H2O2 with ascorbic acid abolished disease resistance against B. cinerea infection and decreased the expression of defense-related genes. ThLAAO-FITC application on tobacco protoplast or overexpression of ThLAAO-GFP in tobacco revealed the localization of ThLAAO in chloroplasts. Chlorophyll a/b binding protein (CAB) was isolated through ThLAAO-ConA affinity chromatography. The pull down assay results confirmed ThLAAO-CAB binding. Application of ThLAAO-Cy5.5 on cabbage roots promptly translocated to the leaves. Treatment of ThLAAO on cabbage roots induces systemic resistance against B. cinerea. Overall, these results demonstrate that ThLAAO may target chloroplast and activate defense mechanisms via H2O2 signaling to confer resistance against S. sclerotiorum and B. cinerea.

Cell cycle-dependent and independent mating blocks ensure fungal zygote survival and ploidy maintenance.

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins postfusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 not only imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least 2 mechanisms where the zygotic fate imposed by Mei2 and the cell cycle reentry triggered by Mei3 synergize to prevent zygotic mating.

Plasmopara viticola effector PvRXLR111 stabilizes VvWRKY40 to promote virulence.

Plasmopara viticola, the causal organism of grapevine downy mildew, secretes a vast array of effectors to manipulate host immunity. Previously, several cell death-inducing PvRXLR effectors have been identified, but their functions and host targets are poorly understood. Here, we investigated the role of PvRXLR111, a cell death-inducing RXLR effector, in manipulating plant immunity. When coexpressed with other PvRXLR effectors, PvRXLR111-induced cell death was prevented. Transient expression of PvRXLR111 in Nicotiana benthamiana suppressed bacterial flagellin peptide flg22-elicited immune responses and enhanced Phytophthora capsici infection. PvRXLR111 induction in Arabidopsis increased susceptibility to Hyaloperonospora arabidopsidis. PvRXLR111 expression in Pseudomonas syringae promoted bacterial colonization. By immunoprecipitation-mass spectrometry analysis, yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays, it was shown that PvRXLR111 interacted with Vitis vinifera putative WRKY transcription factor 40 (VvWRKY40), which increased VvWRKY40 stability. Transient expression of VvWRKY40 in N. benthamiana inhibited flg22-induced reactive oxygen species burst and enhanced P. capsici infection and silencing NbWRKY40 attenuated P. capsici colonization. These results suggest VvWRKY40 functions as a negative regulator in plant immunity and that PvRXLR111 suppresses host immunity by stabilizing VvWRKY40.

Paracoccin Overexpression in Paracoccidioides brasiliensis Enhances Fungal Virulence by Remodeling Chitin Properties of the Cell Wall.

BACKGROUND: The thermodimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis. Although poorly studied, paracoccin (PCN) from Paracoccidioides brasiliensis has been shown to harbor lectinic, enzymatic, and immunomodulatory properties that affect disease development. METHODS: Mutants of P. brasiliensis overexpressing PCN (ov-PCN) were constructed by Agrobacterium tumefaciens-mediated transformation. ov-PCN strains were analyzed and inoculated intranasally or intravenously to mice. Fungal burden, lung pathology, and survival were monitored to evaluate virulence. Electron microscopy was used to evaluate the size of chito-oligomer particles released by ov-PCN or wild-type strains to growth media. RESULTS: ov-PCN strains revealed no differences in cell growth and viability, although PCN overexpression favored cell separation, chitin processing that results in the release of smaller chito-oligomer particles, and enhanced virulence. Our data show that PCN triggers a critical effect in the cell wall biogenesis through the chitinase activity resulting from overexpression of PCN. As such, PCN overexpression aggravates the disease caused by P. brasiliensis. CONCLUSIONS: Our data are consistent with a model in which PCN modulates the cell wall architecture via its chitinase activity. These findings highlight the potential for exploiting PCN function in future therapeutic approaches.

HOG-Independent Osmoprotection by Erythritol in Yeast Yarrowia lipolytica.

Erythritol is a polyol produced by Yarrowia lipolytica under hyperosmotic stress. In this study, the osmo-sensitive strain Y. lipolytica yl-hog1Δ was subjected to stress, triggered by a high concentration of carbon sources. The strain thrived on 0.75 M erythritol medium, while the same concentrations of glucose and glycerol proved to be lethal. The addition of 0.1 M erythritol to the medium containing 0.75 M glucose or glycerol allowed the growth of yl-hog1Δ. Supplementation with other potential osmolytes such as mannitol or L-proline did not have a similar effect. To examine whether the osmoprotective effect might be related to erythritol accumulation, we deleted two genes involved in erythritol utilization, the transcription factor Euf1 and the enzyme erythritol dehydrogenase Eyd1. The strain eyd1Δ yl hog1Δ, which lacked the erythritol utilization enzyme, reacted to the erythritol supplementation significantly better than yl-hog1Δ. On the other hand, the strain euf1Δ yl-hog1Δ became insensitive to supplementation, and the addition of erythritol could no longer improve the growth of this strain in hyperosmotic conditions. This indicates that Euf1 regulates additional, still unknown genes involved in erythritol metabolism.