Roles In Fungal Pathogenicity Research Articles

Casein kinase 2 complex: a central regulator of multiple pathobiological signaling pathways in Cryptococcus neoformans.

The casein kinase 2 (CK2) complex has garnered extensive attention over the past decades as a potential therapeutic target for diverse human diseases, including cancer, diabetes, and obesity, due to its pivotal roles in eukaryotic growth, differentiation, and metabolic homeostasis. While CK2 is also considered a promising antifungal target, its role in fungal pathogens remains unexplored. In this study, we investigated the functions and regulatory mechanisms of the CK2 complex in Cryptococcus neoformans, a major cause of fungal meningitis. The cryptococcal CK2 complex consists of a single catalytic subunit, Cka1, and two regulatory subunits, Ckb1 and Ckb2. Our findings show that Cka1 plays a primary role as a protein kinase, while Ckb1 and Ckb2 have major and minor regulatory functions, respectively, in growth, cell cycle control, morphogenesis, stress response, antifungal drug resistance, and virulence factor production. Interestingly, triple mutants lacking all three subunits (cka1Δ ckb1Δ ckb2Δ) exhibited more severe phenotypic defects than the cka1Δ mutant alone, suggesting that Ckb1/2 may have Cka1-independent functions. In a murine model of systemic cryptococcosis, cka1Δ and cka1Δ ckb1Δ ckb2Δ mutants showed severely reduced virulence. Transcriptomic, proteomic, and phosphoproteomic analyses further revealed that the CK2 complex controls a wide array of effector proteins involved in transcriptional regulation, cell cycle control, nutrient metabolisms, and stress responses. Most notably, CK2 disruption led to dysregulation of key signaling cascades central to C. neoformans pathogenicity, including the Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin signaling pathways. In summary, our study provides novel insights into the multifaceted roles of the fungal CK2 complex and presents a compelling case for targeting it in the development of new antifungal drugs.IMPORTANCEThe casein kinase 2 (CK2) complex, crucial for eukaryotic growth, differentiation, and metabolic regulation, presents a promising therapeutic target for various human diseases, including cancer, diabetes, and obesity. Its potential as an antifungal target is further highlighted in this study, which explores CK2's functions in C. neoformans, a key fungal meningitis pathogen. The CK2 complex in C. neoformans, comprising the Cka1 catalytic subunit and Ckb1/2 regulatory subunits, is integral to processes like growth, cell cycle, morphogenesis, stress response, drug resistance, and virulence. Our findings of CK2's role in regulating critical signaling pathways, including Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin, underscore its importance in C. neoformans pathogenicity. This study provides valuable insights into the fungal CK2 complex, reinforcing its potential as a target for novel antifungal drug development and pointing out a promising direction for creating new antifungal agents.

Fungal small RNAs ride in extracellular vesicles to enter plant cells through clathrin-mediated endocytosis

Small RNAs (sRNAs) of the fungal pathogen Botrytis cinerea can enter plant cells and hijack host Argonaute protein 1 (AGO1) to silence host immunity genes. However, the mechanism by which these fungal sRNAs are secreted and enter host cells remains unclear. Here, we demonstrate that B. cinerea utilizes extracellular vesicles (EVs) to secrete Bc-sRNAs, which are then internalized by plant cells through clathrin-mediated endocytosis (CME). The B. cinerea tetraspanin protein, Punchless 1 (BcPLS1), serves as an EV biomarker and plays an essential role in fungal pathogenicity. We observe numerous Arabidopsis clathrin-coated vesicles (CCVs) around B. cinerea infection sites and the colocalization of B. cinerea EV marker BcPLS1 and Arabidopsis CLATHRIN LIGHT CHAIN 1, one of the core components of CCV. Meanwhile, BcPLS1 and the B. cinerea-secreted sRNAs are detected in purified CCVs after infection. Arabidopsis knockout mutants and inducible dominant-negative mutants of key components of the CME pathway exhibit increased resistance to B. cinerea infection. Furthermore, Bc-sRNA loading into Arabidopsis AGO1 and host target gene suppression are attenuated in those CME mutants. Together, our results demonstrate that fungi secrete sRNAs via EVs, which then enter host plant cells mainly through CME.

The Regulatory Hub of Siderophore Biosynthesis in the Phytopathogenic Fungus Alternaria alternata.

A GATA zinc finger-containing repressor (AaSreA) suppresses siderophore biosynthesis in the phytopathogenic fungus Alternaria alternata under iron-replete conditions. In this study, targeted gene deletion revealed two bZIP-containing transcription factors (AaHapX and AaAtf1) and three CCAAT-binding proteins (AaHapB, AaHapC, and AaHapE) that positively regulate gene expression in siderophore production. This is a novel phenotype regarding Atf1 and siderophore biosynthesis. Quantitative RT-PCR analyses revealed that only AaHapX and AaSreA were regulated by iron. AaSreA and AaHapX form a transcriptional feedback negative loop to regulate iron acquisition in response to the availability of environmental iron. Under iron-limited conditions, AaAtf1 enhanced the expression of AaNps6, thus playing a positive role in siderophore production. However, under nutrient-rich conditions, AaAtf1 plays a negative role in resistance to sugar-induced osmotic stress, and AaHapX plays a negative role in resistance to salt-induced osmotic stress. Virulence assays performed on detached citrus leaves revealed that AaHapX and AaAtf1 play no role in fungal pathogenicity. However, fungal strains carrying the AaHapB, AaHapC, or AaHapE deletion failed to incite necrotic lesions, likely due to severe growth deficiency. Our results revealed that siderophore biosynthesis and iron homeostasis are regulated by a well-organized network in A. alternata.

Tea plant (Camellia sinensis) lipid metabolism pathway modulated by tea field microbe (Colletotrichum camelliae) to promote disease.

Tea is one of the most popular healthy and non-alcoholic beverages worldwide. Tea anthracnose is a disease in tea mature leaves and ultimately affects yield and quality. Colletotrichum camelliae is a dominant fungal pathogen in the tea field that infects tea plants in China. The pathogenic factors of fungus and the susceptible factors in the tea plant are not known. In this work, we performed molecular and genetic studies to observe a cerato-platanin protein CcCp1 from C. camelliae, which played a key role in fungal pathogenicity. △CcCp1 mutants lost fungal virulence and reduced the ability to produce conidia. Transcriptome and metabolome were then performed and analysed in tea-susceptible and tea-resistant cultivars, Longjing 43 and Zhongcha 108, upon C. camelliae wild-type CCA and △CcCp1 infection, respectively. The differentially expressed genes and the differentially accumulated metabolites in tea plants were clearly overrepresented such as linolenic acid and linoleic acid metabolism, glycerophospholipid metabolism, phenylalanine biosynthesis and metabolism, biosynthesis of flavonoid, flavone and flavonol etc. In particular, the accumulation of jasmonic acid was significantly increased in the susceptible cultivar Longjing 43 upon CCA infection, in the fungal CcCp1 protein dependent manner, suggesting the compound involved in regulating fungal infection. In addition, other metabolites in the glycerophospholipid and phenylalanine pathway were observed in the resistant cultivar Zhongcha 108 upon fungal treatment, suggesting their potential role in defense response. Taken together, this work indicated C. camelliae CcCp1 affected the tea plant lipid metabolism pathway to promote disease while the lost function of CcCp1 mutants altered the fungal virulence and plant response.

Physiological race characterisation of Fusarium oxysporum strains infecting tomato employing candidate pathogenicity genes and host resistance.

Physiological races of 14 strains of Fusarium oxysporum f.sp. lycopersici were established by PCR profiling SIX gene expressions. No amplification of the SIX4 (Avr1) gene was observed in any of the 14 strains. Based on amplification of the SIX3 (Avr2) gene, 6 strains were distinguished as race 2. Race 2 strains are known to contain identical SIX3 sequences and differ from race 3 strains by single point mutations. Hence, based on polymorphic amplicons of the SIX3 gene detected by stringent PCR conditions, 8 strains were identified as race 3. The identity of the physiological races of the strains was validated by inoculating on three germplasm lines, EC-814916, FEB-2 and Pusa Rohini carrying I-2, I-3 and no I gene, respectively. The race 2 and race 3 strains were avirulent on EC-814916 and FEB-2 lines, respectively. All the 14 fungal strains were pathogenic on Pusa Rohini, the Fusarium wilt susceptible cultivar lacking R genes and exhibited different levels of virulence. In evaluating two other potential pathogenicity genes, Fow1 and Fow2 as markers for virulence, their expressions were observed among both the races of the Fol strains, and hence are not potential candidates for physiological race discrimination. However, strong expressions of the genes in the root tissues inoculated with the highly virulent strain, TOFU-IHBT in comparison to the uninoculated control indicated their roles in fungal pathogenicity. To understand the role of these pathogenicity genes in countering the host defence mechanisms, their expressions in response to ROS and phenolics, the earliest known defence mechanisms of host plants were assessed. In H2O2, the Fow2 gene expressed 1.4-fold greater than that of the control. On the contrary, in relation to the control, the expressions of Fow1 were strongly repressed exhibiting 0.7-to 0.8-fold lesser at 0.1 mM through 3 mM concentrations than that of the control indicating that the gene is modulated by the phenolic acid indicating the roles of Fow2 and Fow1 in alleviating oxidative stress and targeted by the phenolic acid, respectively.

Current progress on pathogenicity-related transcription factors in Fusarium oxysporum.

Fusarium oxysporum is a well‐known soilborne plant pathogen that causes severe vascular wilt in economically important crops worldwide. During the infection process, F. oxysporum not only secretes various virulence factors, such as cell wall‐degrading enzymes (CWDEs), effectors, and mycotoxins, that potentially play important roles in fungal pathogenicity but it must also respond to extrinsic abiotic stresses from the environment and the host. Over 700 transcription factors (TFs) have been predicted in the genome of F. oxysporum, but only 26 TFs have been functionally characterized in various formae speciales of F. oxysporum. Among these TFs, a total of 23 belonging to 10 families are required for pathogenesis through various mechanisms and pathways, and the zinc finger TF family is the largest family among these 10 families, which consists of 15 TFs that have been functionally characterized in F. oxysporum. In this review, we report current research progress on the 26 functionally analysed TFs in F. oxysporum and sort them into four groups based on their roles in F. oxysporum pathogenicity. Furthermore, we summarize and compare the biofunctions, involved pathways, putative targets, and homologs of these TFs and analyse the relationships among them. This review provides a systematic analysis of the regulation of virulence‐related genes and facilitates further mechanistic analysis of TFs important in F. oxysporum virulence.

Differential Analysis of Mycelial Proteins and Metabolites From Rigidoporus Microporus During In Vitro Interaction With Hevea Brasiliensis.

Rigidoporus microporus is the fungus accountable for the white root rot disease that is detrimental to the rubber tree, Hevea brasiliensis. The pathogenicity mechanism of R. microporus and the identity of the fungal proteins and metabolites involved during the infection process remain unclear. In this study, the protein and metabolite profiles of two R. microporus isolates, Segamat (SEG) and Ayer Molek (AM), were investigated during an in vitro interaction with H. brasiliensis. The isolates were used to inoculate H. brasiliensis clone RRIM 2025, and mycelia adhering to the roots of the plant were collected for analysis. Transmission electron microscope (TEM) images acquired confirms the hyphae attachment and colonization of the mycelia on the root of the H. brasiliensis clones after 4days of inoculation. The protein samples were subjected to 2-DE analysis and analyzed using MALDI-ToF MS/MS, while the metabolites were extracted using methanol and analyzed using LC/MS-QTOF. Based on the differential analyses, upregulation of proteins that are essential for fungal evolution such as malate dehydrogenase, fructose 1,6-biphosphate aldolase, and glyceraldehyde-3-phosphate dehydrogenase hints an indirect role in fungal pathogenicity, while metabolomic analysis suggests an increase in acidic compounds which may lead to increased cell wall degrading enzyme activity. Bioinformatics analyses revealed that the carbohydrate and amino acid metabolisms were prominently affected in response to the fungal pathogenicity. In addition to that, other pathways that were significantly affected include "Protein Ubiquitination Pathway," Unfolded Protein Response," "HIFα Signaling," and "Sirtuin Signaling Pathway." The identification of responsive proteins and metabolites from this study promotes a better understanding of mechanisms underlying R. microporus pathogenesis and provides a list of potential biological markers for early recognition of the white root rot disease.

ITRAQ-based comparative proteome analyses of different growth stages revealing the regulatory role of reactive oxygen species in the fruiting body development of Ophiocordyceps sinensis.

In this study, using an isobaric tags for relative and absolute quantitation (iTRAQ ) approach coupled with LC-MS / MS and bioinformatics, the proteomes were analyzed for the crucial three stages covering the fruiting body development of Ophiocordyceps sinensis, including sclerotium (ST), primordium (PR) and mature fruiting body (MF), with a focus on fruiting body development-related proteins and the potential mechanisms of the development. A total of 1,875 proteins were identified. Principal Component Analysis (PCA) demonstrated that the protein patterns between PR and MF were more similar than ST. Differentially accumulated proteins (DAPs) analysis showed that there were 510, 173 and 514 DAPs in the comparisons of ST vs. PR, PR vs. MF and ST vs. MF, respectively. A total of 62 shared DAPs were identified and primarily enriched in proteins related to ‘carbon transport and mechanism’, ‘the response to oxidative stress’, ‘antioxidative activity’ and ‘translation’. KEGG and GO databases showed that the DAPs were enriched in terms of ‘primary metabolisms (amino acid/fatty acid/energy metabolism)’, ‘the response to oxidative stress’ and ‘peroxidase’. Furthermore, 34 DAPs involved in reactive oxygen species (ROS) metabolism were identified and clustered across the three stages using hierarchical clustering implemented in hCluster R package . It was suggested that their roles and the underlying mechanisms may be stage-specific. ROS may play a role in fungal pathogenicity in ST, the fruit-body initiation in PR, sexual reproduction and highland adaptation in MF. Crucial ROS-related proteins were identified, such as superoxide dismutase (SOD, T5A6F1), Nor-1 (T5AFX3), electron transport protein (T5AHD1), histidine phosphotransferase (HPt, T5A9Z5) and Glutathione peroxidase (T5A9V1). Besides, the accumulation of ROS at the three stages were assayed using 2,7-dichlorofuorescin diacetate (DCFH-DA) stanning. A much stronger ROS accumulation was detected at the stage MF, compared to the stages of PR and ST. Sections of ST and fruit-body part of MF were stained by DCFH-DA and observed under the fluorescencemicroscope, showing ROS was distributed within the conidiospore and ascus. Besides, SOD activity increased across the three stages, while CAT activity has a strong increasement in MF compared to the stages of ST and PR. It was suggested that ROS may act in gradient-dependent manner to regulate the fruiting body development. The coding region sequences of six DAPs were analyzed at mRNA level by quantitative real-time PCR (qRT-PCR). The results support the result of DAPs analysis and the proteome sequencing data. Our findings offer the perspective of proteome to understand the biology of fruiting body development and highland adaptation in O. sinensis, which would inform the big industry of this valuable fungus.

Anticipatory Stress Responses and Immune Evasion in Fungal Pathogens.

In certain niches, microbes encounter environmental challenges that are temporally linked. In such cases, microbial fitness is enhanced by the evolution of anticipatory responses where the initial challenge simultaneously activates pre-emptive protection against the second impending challenge. The accumulation of anticipatory responses in domesticated yeasts, which have been termed 'adaptive prediction', has led to the emergence of 'core stress responses' that provide stress cross-protection. Protective anticipatory responses also seem to be common in fungal pathogens of humans. These responses reflect the selective pressures that these fungi have faced relatively recently in their evolutionary history. Consequently, some pathogens have evolved 'core environmental responses' which exploit host signals to trigger immune evasion strategies that protect them against imminent immune attack.

Transcriptome profiling reveals insertional mutagenesis suppressed the expression of candidate pathogenicity genes in honeybee fungal pathogen, Ascosphaera apis

Chalkbrood disease is caused by Ascosphaera apis which severely affects honeybee brood. Spore inoculation experiments shown pathogenicity varies among different strains and mutants, however, the molecular mechanism of pathogenicity is unclear. We sequenced, assembled and annotated the transcriptomes of wild type (SPE1) and three mutants (SPE2, SPE3 and SPE4) with reduced pathogenicity that were constructed in our previous study. Illumina sequencing generated a total of 394,910,604 clean reads and de novo Trinity-based assembled into 12,989 unigenes, among these, 9,598 genes were successfully annotated to known proteins in UniProt database. A total of 172, 3,996, and 650 genes were up-regulated and 4,403, 2,845, and 3,016 genes were down-regulated between SPE2-SPE1, SPE3-SPE1, and SPE4-SPE1, respectively. Overall, several genes with a potential role in fungal pathogenicity were detected down-regulated in mutants including 100 hydrolytic enzymes, 117 transcriptional factors, and 47 cell wall related genes. KEGG pathway enrichment analysis reveals 216 genes involved in nine pathways were down-regulated in mutants compared to wild type. The down-regulation of more pathways involved in pathogenicity in SPE2 and SPE4 than SPE3 supports their lower pathogenicity during in-vitro bioassay experiment. Expression of 12 down-regulated genes in mutants was validated by quantitative real time PCR. This study provides valuable information on transcriptome variation caused by mutation for further functional validation of candidate pathogenicity genes in A. apis.

The Landscape of Repetitive Elements in the Refined Genome of Chilli Anthracnose Fungus Colletotrichum truncatum.

The ascomycete fungus Colletotrichum truncatum is a major phytopathogen with a broad host range which causes anthracnose disease of chilli. The genome sequencing of this fungus led to the discovery of functional categories of genes that may play important roles in fungal pathogenicity. However, the presence of gaps in C. truncatum draft assembly prevented the accurate prediction of repetitive elements, which are the key players to determine the genome architecture and drive evolution and host adaptation. We re-sequenced its genome using single-molecule real-time (SMRT) sequencing technology to obtain a refined assembly with lesser and smaller gaps and ambiguities. This enabled us to study its genome architecture by characterising the repetitive sequences like transposable elements (TEs) and simple sequence repeats (SSRs), which constituted 4.9 and 0.38% of the assembled genome, respectively. The comparative analysis among different Colletotrichum species revealed the extensive repeat rich regions, dominated by Gypsy superfamily of long terminal repeats (LTRs), and the differential composition of SSRs in their genomes. Our study revealed a recent burst of LTR amplification in C. truncatum, C. higginsianum, and C. scovillei. TEs in C. truncatum were significantly associated with secretome, effectors and genes in secondary metabolism clusters. Some of the TE families in C. truncatum showed cytosine to thymine transitions indicative of repeat-induced point mutation (RIP). C. orbiculare and C. graminicola showed strong signatures of RIP across their genomes and “two-speed” genomes with extensive AT-rich and gene-sparse regions. Comparative genomic analyses of Colletotrichum species provided an insight into the species-specific SSR profiles. The SSRs in the coding and non-coding regions of the genome revealed the composition of trinucleotide repeat motifs in exons with potential to alter the translated protein structure through amino acid repeats. This is the first genome-wide study of TEs and SSRs in C. truncatum and their comparative analysis with six other Colletotrichum species, which would serve as a useful resource for future research to get insights into the potential role of TEs in genome expansion and evolution of Colletotrichum fungi and for development of SSR-based molecular markers for population genomic studies.

Role of fusaric acid in the development of ‘Fusarium wilt’ symptoms in tomato: Physiological, biochemical and proteomic perspectives

Fusarium wilt is one of the most prevalent and damaging diseases of tomato. Among various toxins secreted by the Fusarium oxysporum f. sp. lycopersici (causal agent of Fusarium wilt of tomato), fusaric acid (FA) is suspected to be a potent pathogenicity factor in tomato wilt disease development. With this rationale the present study was carried out with physiological, biochemical and proteomic perspectives. Treatment of FA was given to the leaves of tomato directly through infiltration to show the characteristic features of Fusarium wilt of tomato. The phytotoxic effect of FA was assessed in the form of cell death in tomato leaves which was observed by increased uptake of Evans blue stain. The measurement of electrolyte leakage was used as an indicator of the extent of cell death. The influence of FA on the leaf photosynthesis of tomato plant was investigated and it was found that FA strongly reduced the photosynthetic pigment contents of tomato leaves resulting to heavy suppression of leaf photosynthesis processes, which therefore affected leaf physiology finally leading to leaf wilting and necrosis. This cell death inducer (FA) produced an enormous oxidative burst during which large quantities of reactive oxygen species (ROS) like H2O2 was generated in the treated leaf tissues of tomato plants which was evident from enhancement in lipid peroxidation. To assess the involvement of proteolysis in the cell death cascade induced by FA treatment, total protease activity was measured in the leaf tissues and it was found that the total protease activity increased with the treatment and leading to cell death. Furthermore, proteomic study was used as a powerful tool to understand the alterations in cellular protein expression in response to FA exposure. Differential expression in several proteins was observed in the present study. Proteomic analyses, thus, clearly indicate that proteins belonging to different functional classes are significantly affected in the plant leaf tissues after FA exposure leading to deterioration of structure and metabolism of cells. Thus, it is concluded that FA plays an important role in fungal pathogenicity by decreasing cell viability.

Pex14/17, a filamentous fungus-specific peroxin, is required for the import of peroxisomal matrix proteins and full virulence of Magnaporthe oryzae.

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfil a variety of biochemical functions. The biogenesis of peroxisomes requires a variety of proteins, named peroxins, which are encoded by PEX genes. Pex14/17 is a putative recently identified peroxin, specifically present in filamentous fungal species. Its function in peroxisomal biogenesis is still obscure and its roles in fungal pathogenicity have not yet been documented. Here, we demonstrate the contributions of Pex14/17 in the rice blast fungus Magnaporthe oryzae (Mopex14/17) to peroxisomal biogenesis and fungal pathogenicity by targeting gene replacement strategies. Mopex14/17 has properties of both Pex14 and Pex17 with regard to its protein sequence. Mopex14/17 is distributed at the peroxisomal membrane and is essential for efficient peroxisomal targeting of proteins containing peroxisomal targeting signal 1. MoPEX19 deletion leads to the cytoplasmic distribution of Mopex14/17, indicating that the peroxisomal import of Pex14/17 is dependent on Pex19. The knockout mutants of MoPEX14/17 show reduced fatty acid utilization, reactive oxygen species (ROS) degradation and cell wall integrity. Moreover, Δmopex14/17 mutants show delayed conidial generation and appressorial formation, and a reduction in appressorial turgor accumulation and penetration ability in host plants. These defects result in a significant reduction in the virulence of the mutant. These data indicate that MoPEX14/17 plays a crucial role in peroxisome biogenesis and contributes to fungal development and pathogenicity.

Proteometabolomic analysis of transgenic tomato overexpressing oxalate decarboxylase uncovers novel proteins potentially involved in defense mechanism against Sclerotinia

Oxalic acid (OA) plays dual role in fungal pathogenicity in a concentration dependent manner. While at higher concentration it induces programmed cell death leading to fungal invasion, low oxalate build resistance in plant. Although OA has been identified as a virulence determinant for rot disease caused by Sclerotinia sp., our understanding of how oxalate downregulation impart host immunity is limited. We have earlier shown that ectopic expression of oxalate decarboxylase (FvOXDC) specifically degrades OA in tomato (Solanum lycopersicum). To elucidate low oxalate regulated molecular mechanism imparting immunity, a comparative proteomics approach has been applied to E8.2-OXDC tomato fruit displaying fungal resistance. Mass spectrometric analyses identified 92 OXDC-responsive immunity related protein spots (ORIRPs) presumably associated with acid metabolism, defense signaling and endoplasmic reticulum stress. Metabolome study indicated increased abundance of some of the organic acids paralleling the proteomic analysis. Further, we interrogated the proteome data using network analysis that identified modules enriched in known and novel immunity-related prognostic proteins centered around 14-3-3, translationally controlled tumor protein, annexin and chaperonin. Taken together, our data demonstrate that low oxalate may act as metabolic and immunity determinant through translational reprogramming. Biological significanceAlthough OA plays critical role as fungal elicitor, our understanding of how oxalate downregulation by decarboxylative degradation impart immunity is limited. Our study confirms the impact of oxalate down-regulation on overall cellular physiology and provides new perspectives to study plant immunity. The network representation may facilitate the prioritization of candidate proteins for patho-stress tolerance in crop plant. These findings are of great importance for future work towards functional determination and exploitation of target proteins in crop improvement program.

Fungal quorum sensing molecules: Role in fungal morphogenesis and pathogenicity.

When microorganisms live together in high numbers, they need to communicate with each other. To achieve cell-cell communication, microorganisms secrete molecules called quorum-sensing molecules (QSMs) that control their biological activities and behaviors. Fungi secrete QSMs such as farnesol, tyrosol, phenylethanol, and tryptophol. The role of QSMs in fungi has been widely studied in both yeasts and filamentous fungi, for example in Candida albicans, C. dubliniensis, Aspergillus niger, A. nidulans, and Fusarium graminearum. QSMs impact fungal morphogenesis (yeast-to-hypha formation) and also play a role in the germination of macroconidia. QSMs cause fungal cells to initiate programmed cell death, or apoptosis, and play a role in fungal pathogenicity. Several types of QSMs are produced during stages of biofilm development to control cell population or morphology in biofilm communities. This review article emphasizes the role of fungal QSMs, especially in fungal morphogenesis, biofilm formation, and pathogenicity. Information about QSMs may lead to improved measures for controlling fungal infection.

Diversity of laccase-coding genes in Fusarium oxysporum genomes.

Multiple studies confirm laccase role in fungal pathogenicity and lignocellulose degradation. In spite of broad genomic research, laccases from plant wilt pathogen Fusarium oxysporum are still not characterized. The study aimed to identify F. oxysporum genes that may encode laccases sensu stricto and to characterize the proteins in silico in order to facilitate further research on their impact on the mentioned processes. Twelve sequenced F. oxysporum genomes available on Broad Institute of Harvard and MIT (2015) website were analyzed and three genes that may encode laccases sensu stricto were found. Their amino acid sequences possess all features essential for their catalytic activity, moreover, the homology models proved the characteristic 3D laccase structures. The study shades light on F. oxysporum as a new source of multicopper oxidases, enzymes with possible high redox potential and broad perspective in biotechnological applications.

Effects of fungal root infection on the vigor of grapevines infested by root-feeding grape phylloxera

The role of fungal pathogens in damage of grapevines ( Vitis vinifera L.; cv. Chardonnay) associated with grape phylloxera ( Daktulosphaira vitifoliae FITCH) was investigated. Seven different genera of secondary fungi were isolated from surface-disinfected feeding sites of phylloxera but none from surface-disinfected root tissues undamaged by phylloxera. Damage in vines infested with phylloxera and infected with Fusarium solani (MART.) or with F. solani and Pythium ultimum TROW. was significantly greater than damage in vines infested with phylloxera only. In a greenhouse experiment, total biomass was reduced by 16% in vines infested with phylloxera and 24 to 29% in vines infested with phylloxera and infected with fungus in comparison with control vines. Chlorophyll content, average internode length, shoot biomass, and root biomass in the uninfested, uninfected vines were significantly greater than vines infested with phylloxera or vines infested with phylloxera and infected with F. solani or P. ultimum , or both. Preventative treatment with metalaxyl, benomyl or copper quinolinolate fungicides significantly decreased damage in phylloxera-infested vines in comparison with untreated vines. The implications of this research with respect to management of grape phylloxera are discussed.

Fungal Inositol Pyrophosphate IP7 Is Crucial for Metabolic Adaptation to the Host Environment and Pathogenicity.

ABSTRACTInositol pyrophosphates (PP-IPs) comprising inositol, phosphate, and pyrophosphate (PP) are essential for multiple functions in eukaryotes. Their role in fungal pathogens has never been addressed. Cryptococcus neoformans is a model pathogenic fungus causing life-threatening meningoencephalitis. We investigate the cryptococcal kinases responsible for the production of PP-IPs (IP7/IP8) and the hierarchy of PP-IP importance in pathogenicity. Using gene deletion and inositol polyphosphate profiling, we identified Kcs1 as the major IP6 kinase (producing IP7) and Asp1 as an IP7 kinase (producing IP8). We show that Kcs1-derived IP7 is the most crucial PP-IP for cryptococcal drug susceptibility and the production of virulence determinants. In particular, Kcs1 kinase activity is essential for cryptococcal infection of mouse lungs, as reduced fungal burdens were observed in the absence of Kcs1 or when Kcs1 was catalytically inactive. Transcriptome and carbon source utilization analysis suggested that compromised growth of the KCS1 deletion strain (Δkcs1 mutant) in the low-glucose environment of the host lung is due to its inability to utilize alternative carbon sources. Despite this metabolic defect, the Δkcs1 mutant established persistent, low-level asymptomatic pulmonary infection but failed to elicit a strong immune response in vivo and in vitro and was not readily phagocytosed by primary or immortalized monocytes. Reduced recognition of the Δkcs1 cells by monocytes correlated with reduced exposure of mannoproteins on the Δkcs1 mutant cell surface. We conclude that IP7 is essential for fungal metabolic adaptation to the host environment, immune recognition, and pathogenicity.

Establishment of invasive and non-invasive reporter systems to investigate American elm–Ophiostoma novo-ulmi interactions

Dutch elm disease (DED), caused by ascomycete fungi in the Ophiostoma genus, is the most devastating disease of American elm (Ulmus americana) trees. Cerato ulmin (CU), a hydrophobin secreted by the fungus, has been implicated in the development of DED, but its role in fungal pathogenicity and virulence remains uncertain and controversial. Here, we describe reporter systems based on the CU promoter and three reporter proteins (GFP, GUS and LUC), developed as research tools for quantitative and qualitative studies of DED in vitro, in vivo and in planta. A strain of the aggressive species Ophiostoma novo-ulmi was transformed with the reporter constructs using Agrobacterium-mediated transformation and the fungal transformants, namely M75-GFP, M75-GUS and M75-LUC, were examined for mitotic stability after repeated subcultures. The intensity of GFP fluorescence was strong in M75-GFP spores and hyphae, allowing microscopic investigations of spore structure, fungal morphogenesis and fungal development. The interaction of M75-GFP and U. americana callus cells was explored with scanning laser confocal microscopy facilitating qualitative studies on fungal strategies for the invasion and penetration of elm cells. M75-GUS was generated to provide an invasive, yet quantitative approach to study fungal–plant interactions in vitro and in planta. The generation of M75-LUC transformants was aimed at providing a non-destructive quantitative approach to study the role of CU in vivo. The sensitivity, low background signal and linearity of LUC assays all predict a very reliable approach to investigate and re-test previously claimed roles of this CU in fungal pathogenicity. These reporter systems provide new tools to investigate plant–pathogen interactions in this complex pathosystem and may aid in better understanding the development of DED.

FPoxDB: fungal peroxidase database for comparative genomics.

BackgroundPeroxidases are a group of oxidoreductases which mediate electron transfer from hydrogen peroxide (H2O2) and organic peroxide to various electron acceptors. They possess a broad spectrum of impact on industry and fungal biology. There are numerous industrial applications using peroxidases, such as to catalyse highly reactive pollutants and to breakdown lignin for recycling of carbon sources. Moreover, genes encoding peroxidases play important roles in fungal pathogenicity in both humans and plants. For better understanding of fungal peroxidases at the genome-level, a novel genomics platform is required. To this end, Fungal Peroxidase Database (fPoxDB; http://peroxidase.riceblast.snu.ac.kr/) has been developed to provide such a genomics platform for this important gene family.DescriptionIn order to identify and classify fungal peroxidases, 24 sequence profiles were built and applied on 331 genomes including 216 from fungi and Oomycetes. In addition, NoxR, which is known to regulate NADPH oxidases (NoxA and NoxB) in fungi, was also added to the pipeline. Collectively, 6,113 genes were predicted to encode 25 gene families, presenting well-separated distribution along the taxonomy. For instance, the genes encoding lignin peroxidase, manganese peroxidase, and versatile peroxidase were concentrated in the rot-causing basidiomycetes, reflecting their ligninolytic capability. As a genomics platform, fPoxDB provides diverse analysis resources, such as gene family predictions based on fungal sequence profiles, pre-computed results of eight bioinformatics programs, similarity search tools, a multiple sequence alignment tool, domain analysis functions, and taxonomic distribution summary, some of which are not available in the previously developed peroxidase resource. In addition, fPoxDB is interconnected with other family web systems, providing extended analysis opportunities.ConclusionsfPoxDB is a fungi-oriented genomics platform for peroxidases. The sequence-based prediction and diverse analysis toolkits with easy-to-follow web interface offer a useful workbench to study comparative and evolutionary genomics of peroxidases in fungi.