The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase,Is Involved in Cell Wall Architecture and Virulence

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity.     

Unraveling the molecular determinants of the anti-phagocytic protein cloak of plague bacteria

The pathogenic bacterium Yersina pestis is protected from macrophage engulfment by a capsule like antigen, F1, formed of long polymers of the monomer protein, Caf1. However, despite the importance of this pathogen, the mechanism of protection was not understood. Here we demonstrate how F1 protects the bacteria from phagocytosis. First, we show that Escherichia coli expressing F1 showed greatly reduced adherence to macrophages. Furthermore, the few cells that did adhere remained on the macrophage surface and were not engulfed. We then inserted, by mutation, an “RGDS” integrin binding motif into Caf1. This did not change the number of cells adhering to macrophages but increased the fraction of adherent cells that were engulfed. Therefore, F1 protects in two separate ways, reducing cell adhesion, possibly by acting as a polymer brush, and hiding innate receptor binding sites needed for engulfment. F1 is very robust and we show that E. coli expressing weakened mutant polymers are engulfed like the RGDS mutant. This suggests that innate attachment sites on the native cell surface are exposed if F1 is weakened. Single-molecule force spectroscopy (SMFS) experiments revealed that wild-type F1 displays a very high mechanical stability of 400 pN. However, the mechanical resistance of the destabilised mutants, that were fully engulfed, was only 20% weaker. By only marginally exceeding the mechanical force applied to the Caf1 polymer during phagocytosis it may be that the exceptional tensile strength evolved to resist the forces applied at this stage of engulfment.     

Tim4- and MerTK-Mediated Engulfment of Apoptotic Cells by Mouse Resident Peritoneal Macrophages

Apoptotic cells are swiftly engulfed by macrophages to prevent the release of noxious materials from dying cells. Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, and macrophages engulf them by recognizing PtdSer using specific receptors and opsonins. Here, we found that mouse resident peritoneal macrophages expressing Tim4 and MerTK are highly efficient at engulfing apoptotic cells. Neutralizing antibodies against either Tim4 or MerTK inhibited the macrophage engulfment of apoptotic cells. Tim4-null macrophages exhibited reduced binding and engulfment of apoptotic cells, whereas MerTK-null macrophages retained the ability to bind apoptotic cells but failed to engulf them. The incubation of wild-type peritoneal macrophages with apoptotic cells induced the rapid tyrosine phosphorylation of MerTK, which was not observed with Tim4-null macrophages. When mouse Ba/F3 cells were transformed with Tim4, apoptotic cells bound to the transformants but were not engulfed. Transformation of Ba/F3 cells with MerTK had no effect on the binding or engulfment of apoptotic cells; however, Tim4/MerTK transformants exhibited strong engulfment activity. Taken together, these results indicate that the engulfment of apoptotic cells by resident peritoneal macrophages proceeds in two steps: binding to Tim4, a PtdSer receptor, followed by MerTK-mediated cell engulfment.     

Stage Specific Assessment of Candida albicans Phagocytosis by Macrophages Identifies Cell Wall Composition and Morphogenesis as Key Determinants

Candida albicans is a major life-threatening human fungal pathogen. Host defence against systemic Candida infection relies mainly on phagocytosis of fungal cells by cells of the innate immune system. In this study, we have employed video microscopy, coupled with sophisticated image analysis tools, to assess the contribution of distinct C. albicans cell wall components and yeast-hypha morphogenesis to specific stages of phagocytosis by macrophages. We show that macrophage migration towards C. albicans was dependent on the glycosylation status of the fungal cell wall, but not cell viability or morphogenic switching from yeast to hyphal forms. This was not a consequence of differences in maximal macrophage track velocity, but stems from a greater percentage of macrophages pursuing glycosylation deficient C. albicans during the first hour of the phagocytosis assay. The rate of engulfment of C. albicans attached to the macrophage surface was significantly delayed for glycosylation and yeast-locked morphogenetic mutant strains, but enhanced for non-viable cells. Hyphal cells were engulfed at a slower rate than yeast cells, especially those with hyphae in excess of 20 µm, but there was no correlation between hyphal length and the rate of engulfment below this threshold. We show that spatial orientation of the hypha and whether hyphal C. albicans attached to the macrophage via the yeast or hyphal end were also important determinants of the rate of engulfment. Breaking down the overall phagocytic process into its individual components revealed novel insights into what determines the speed and effectiveness of C. albicans phagocytosis by macrophages.     

Dynamics of the Establishment of Multinucleate Compartments in Fusarium oxysporum

Nuclear dynamics can vary widely between fungal species and between stages of development of fungal colonies. Here we compared nuclear dynamics and mitotic patterns between germlings and mature hyphae in Fusarium oxysporum. Using fluorescently labeled nuclei and live-cell imaging, we show that F. oxysporum is subject to a developmental transition from a uninucleate to a multinucleate state after completion of colony initiation. We observed a special type of hypha that exhibits a higher growth rate, possibly acting as a nutrient scout. The higher growth rate is associated with a higher nuclear count and mitotic waves involving 2 to 6 nuclei in the apical compartment. Further, we found that dormant nuclei of intercalary compartments can reenter the mitotic cycle, resulting in multinucleate compartments with up to 18 nuclei in a single compartment.     

Molecular Characterization of Fusarium oxysporum f. melongenae by ISSR and RAPD Markers on Eggplant

Fusarium oxysporum f. melongenae is a major soil-borne pathogen of eggplant (Solanum melongena). ISSR and RAPD markers were used to characterize Fusarium oxysporum f. melongenae isolates collected from eggplant fields in southern Turkey. Those isolates were not pathogenic to tomato. Pathogens were identified by their morphology, and their identity was confirmed by PCR amplification using the specific primer PF02-3. The isolates were classified into groups on the basis of ISSR and RAPD fingerprints, which showed a level of genetic specificity and diversity not previously identified in Fusarium oxysporum f. melongenae, suggesting that genetic differences are related to the pathogen in the Mediterranean region. The primers selected to characterize Fusarium oxysporum f. melongenae may be used to determine genetic differences and pathogen virulence. This study is the first to characterize eggplant F. oxysporum species using ISSR and RAPD.     

FoEG1, a secreted glycoside hydrolase family 12 protein from Fusarium oxysporum,triggers cell death and modulates plant immunity

Fusarium oxysporum is an important soilborne fungal pathogen with many different formae speciales that can colonize the plant vascular system and cause serious crop wilt disease worldwide. We found a glycoside hydrolase family 12 protein FoEG1, secreted by F. oxysporum, that acted as a pathogen-associated molecular pattern (PAMP) targeting the apoplast of plants to induce cell death. Purified FoEG1 protein triggered cell death in different plants and induced the plant defence response to enhance the disease resistance of plants. The ability of FoEG1 to induce cell death was mediated by leucine-rich repeat (LRR) receptor-like kinases BAK1 and SOBIR1, and this ability was independent of its hydrolase activity. The mutants of cysteine residues did not affect the ability of FoEG1 to induce cell death, and an 86 amino acid fragment from amino acid positions 144 to 229 of FoEG1 was sufficient to induce cell death in Nicotiana benthamiana. In addition, the expression of FoEG1 was strongly induced in the early stage of F. oxysporum infection of host plants, and FoEG1 deletion or loss of enzyme activity reduced the virulence of F. oxysporum. Therefore, our results suggest that FoEG1 can contribute to the virulence of F. oxysporum depending on its enzyme activity and can also act as a PAMP to induce plant defence responses.     

The secreted ribonuclease T2 protein FoRnt2 contributes to Fusarium oxysporum virulence

Secreted RNase proteins have been reported from only a few pathogens, and relatively little is known about their biological functions. Fusarium oxysporum is a soilborne fungal pathogen that causes Fusarium wilt, one of the most important diseases on tomato. During the infection of F. oxysporum, some proteins are secreted that modulate host plant immunity and promote pathogen invasion. In this study, we identify an RNase, FoRnt2, from the F. oxysporum secretome that belongs to the ribonuclease T2 family. FoRnt2 possesses an N-terminal signal peptide and can be secreted from F. oxysporum. FoRnt2 exhibited ribonuclease activity and was able to degrade the host plant total RNA in vitro dependent on the active site residues H80 and H142. Deletion of the FoRnt2 gene reduced fungal virulence but had no obvious effect on mycelial growth and conidial production. The expression of FoRnt2 in tomato significantly enhanced plant susceptibility to pathogens. These data indicate that FoRnt2 is an important contributor to the virulence of F. oxysporum, possibly through the degradation of plant RNA.     

Differential responses of molecular mechanisms and physiochemical characters in wild and cultivated soybeans against invasion by the pathogenic Fusarium oxysporum Schltdl

Cultivated soybean (Glycine max) was derived from the wild soybean (Glycine soja), which has genetic resources that can be critically important for improving plant stress resistance. However, little information is available pertaining to the molecular and physiochemical comparison between the cultivated and wild soybeans in response to the pathogenic Fusarium oxysporum Schltdl. In this study, we first used comparative phenotypic and paraffin section analyses to indicate that wild soybean is indeed more resistant to F. oxysporum than cultivated soybean. Genome‐wide RNA‐sequencing approach was then used to elucidate the genetic mechanisms underlying the differential physiological and biochemical responses of the cultivated soybean, and its relative, to F. oxysporum. A greater number of genes related to cell wall synthesis and hormone metabolism were significantly altered in wild soybean than in cultivated soybean under F. oxysporum infection. Accordingly, a higher accumulation of lignins was observed in wild soybean than cultivated soybean under F. oxysporum infection. Collectively, these results indicated that secondary metabolites and plant hormones may play a vital role in differentiating the response between cultivated and wild soybeans against the pathogen. These important findings may provide future direction to breeding programs to improve resistance to F. oxysporum in the elite soybean cultivars by taking advantage of the genetic resources within wild soybean germplasm.     

Analysis of interspecific relationships between Funneliformis mosseae and Fusarium oxysporum in the continuous cropping of soybean rhizosphere soil during the branching period

This study analysed the interspecific relationships between the dominant arbuscular mycorrhizal (AM) fungus, Funneliformis mosseae, and the major soybean root rot pathogen, Fusarium oxysporum, in the rhizosphere soil of continuous cropped soybean. Our aim was to provide theoretical evidence on the AM fungi to overcome the obstacles of soybean continuous cropping. We selected soybean cultivars, including Kenfeng 16 (an intermediate cultivar), Heinong 44 (a high-fat cultivar) and Heinong 48 (a high-protein cultivar), and sowed in the soybean continuous cropping soil under different treatments. The infection status of the soybean roots during the branching period by Fu. mosseae and F. oxysporum was estimated using the standard polymerase chain reaction method, as well as their colonisation status in rhizosphere soil. The AM fungal colonisation rates and F. oxysporum disease incidence of soybean roots were determined, respectively. Quantitative polymerase chain reaction was applied to analyse the DNA content of Fu. mosseae and F. oxysporum to investigate the relationship between Fu. mosseae and F. oxysporum. The results show that both Fu. mosseae and F. oxysporum can infect the soybean roots during the branching period and colonise the rhizosphere. However, the DNA content of F. oxysporum clearly decreased in soybean root and rhizosphere samples after the inoculation with Fu. mosseae. In addition, the disease incidence of F. oxysporum significantly decreased after inoculation with Fu. mosseae, which might indicate inhibitive effects of Fu. mosseae over F. oxysporum.     

Effect of Co-infection by Fusarium oxysporum and Cowpea Mosaic Virus on the Growth and Colonization of Cowpea Seedlings (Vigna unguiculata [L.] Walp.)

Combined infection of cowpea seedlings (c. v. ‘California Blackeye”) by cowpea mosaic virus (CPMV) and Fusarium oxysporum induced greater losses in leaf area, fresh and dry weights than infection by either pathogen alone. The growth of seedlings infected by F. oxysporum f. sp. tracheiphilum was less than that of comparable seedlings infected by F. oxysporum f. sp. phaseoli. The virus infectivity of extracts of the trifoliate leaves of dual-infected plants was significantly higher than that of comparable extracts from the leaves of plants singly infected with CPMV. The nature of the effects of multiple infection in cowpea is discussed.     

The changes in pectin metabolism in flax infected with Fusarium

Fusarium culmorum and Fusarium oxysporum are the most common fungal pathogens of flax (Linum usitatissimum L.), thus leading to the greatest losses in crop yield. A subtractive cDNA library was constructed from flax seedlings exposed for two days to F. oxysporum. This revealed a set of genes that are potentially involved in the flax defense responses. Two of those genes directly participate in cell wall sugar polymer metabolism: UDP-d-glucuronate 4-epimerase (GAE; EC 5.1.3.6) and formate dehydrogenase (FDH; EC 1.2.1.2). GAE delivers the main substrate for pectin biosynthesis, and decreases were detected in its mRNA level after Fusarium infection. FDH participates in the metabolism of formic acid, and the expression level of its gene increased after Fusarium infection. However, metabolite profiling analysis disclosed that the pectin content in the infected plants remained unchanged, but that there were reductions in both the levels of the soluble sugars that serve as pectin precursors, and in the level of formic acid. Since formic acid is the product of pectin demethylesterification, the level of mRNAs coding for pectin methylesterase (EC 3.1.1.11) in the infected flax was measured, revealing a decrease in its expression upon plant infection. Transgenic flax plants overexpressing fungal polygalacturonase (EC 3.2.1.15) and rhamnogalacturonase (EC 3.2.1.-) showed a decrease in the pectin content and an elevated level of formic acid, but the level of expression of the FDH gene remained unchanged. It is suspected that the expression of the formate dehydrogenase gene is directly controlled by the pathogen in the early stage of infection, and additionally by pectin degradation in the later stages.     

Genetic Variation of Strawberry Fusarium Wilt Pathogen Population in Korea

Strawberries are a popular economic crop, and one of the major plantations and exporting countries is Korea in the world. The Fusarium oxysporum species complex (FOSC) is a soil-borne pathogen with genetic diversity, resulting in wilt disease in various crops. In Korea, strawberries wilt disease was first reported in the 1980s due to the infection of FOSC, causing significant economic damage every year. The causal agent, F. oxysporum f. sp. fragariae, is a soil-borne pathogen with a characteristic of FOSC that is difficult to control chemically and mutates easily. This study obtained genetic polymorphism information that was based on AFLP, of F. oxysporum f. sp. fragariae 91 strains, which were isolated from strawberry cultivation sites in Gyeongsangnam-do and Chungcheongnam-do, and compared strains information, which was the isolated location, host variety, response to chemical fungicide, and antagonistic bacteria, and mycelium phenotype. As a result, AFLP phylogeny found that two groups were mainly present, and group B was present at a high frequency in Gyeongsangnam-do. Group B proved less sensitive to tebuconazole than group A through Student’s t-test. In addition, the fractions pattern of AFLP was calculated by comparing the strain information using PCA and PERMANOVA, and the main criteria were separated localization and strawberry varieties (PERMANOVA; p < 0.05). And tebuconazole was different with weak confidence (PERMANOVA; p < 0.10). This study suggests that the F. oxysporum f. sp. fragariae should be continuously monitored and managed, including group B, which is less chemically effective.     

Host perception of jasmonates promotes infection by Fusarium oxysporum formae speciales that produce isoleucine‐ and leucine‐conjugated jasmonates

Three pathogenic forms, or formae speciales (f. spp.), of Fusarium oxysporum infect the roots of Arabidopsis thaliana below ground, instigating symptoms of wilt disease in leaves above ground. In previous reports, Arabidopsis mutants that are deficient in the biosynthesis of abscisic acid or salicylic acid or insensitive to ethylene or jasmonates exhibited either more or less wilt disease, than the wild‐type, implicating the involvement of hormones in the normal host response to F. oxysporum. Our analysis of hormone‐related mutants finds no evidence that endogenous hormones contribute to infection in roots. Mutants that are deficient in abscisic acid and insensitive to ethylene show no less infection than the wild‐type, although they exhibit less disease. Whether a mutant that is insensitive to jasmonates affects infection depends on which forma specialis (f. sp.) is infecting the roots. Insensitivity to jasmonates suppresses infection by F. oxysporum f. sp. conglutinans and F. oxysporum f. sp. matthioli, which produce isoleucine‐ and leucine‐conjugated jasmonate (JA‐Ile/Leu), respectively, in culture filtrates, whereas insensitivity to jasmonates has no effect on infection by F. oxysporum f. sp. raphani, which produces no detectable JA‐Ile/Leu. Furthermore, insensitivity to jasmonates has no effect on wilt disease of tomato, and the tomato pathogen F. oxysporum f. sp. lycopersici produces no detectable jasmonates. Thus, some, but not all, F. oxysporum pathogens appear to utilize jasmonates as effectors, promoting infection in roots and/or the development of symptoms in shoots. Only when the infection of roots is promoted by jasmonates is wilt disease enhanced in a mutant deficient in salicylic acid biosynthesis.     

Modeling competition for infection sites on roots by nonpathogenic strains of Fusarium oxysporum

By use of plane and solid geometry and probability models, efficiencies of infection and competition for nutrients and infection sites by a nonpathogenic strain of Fusarium oxysporum (C14) with F. oxysporum f. sp. cucumerinum on the rhizoplane of cucumber were calculated. The model is derived from previously published data. Efficiencies for successful infection were 0.04 chlamydospores per infection site for both pathogen and nonpathogen. Observed successful infections by the pathogen in competition with the nonpathogen were close in values to the competition ratio (CR) calculated as the number of chlamydospores on the infection court of the pathogen divided by the total number of both pathogen and nonpathogen at relatively low densities. When total chlamydospores were, on average, closer than 175 μm apart, however, competition for nutrients/mutual inhibition occurred. At such densities there was an overestimation of the effect of competition for infection sites. These relationships were modeled at inoculum densities of pathogen and/or nonpathogen of 5000 chlamydospores per g soil and above, however, in the field, maximum densities of 1000 colony forming units/g (cfu) were observed. Most likely models of competition for infection sites at this density of the pathogen revealed that infection efficiency was only approximately halved, even when 0.98 of the possible 30 infection sites were occupied by the nonpathogen. It is conclude that competition for nutrients and/or infection sites is an insignificant factor in biocontrol of Fusarium wilt diseases by nonpathogenic fusaria.     

Biochemical and genetic analysis of a unique poly(ADP-ribosyl) glycohydrolase (PARG) of the pathogenic fungus <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

The genome sequence of the plant pathogen Fusarium oxysporum f. sp. lycopersici contains a single gene encoding a predicted poly(ADP-ribose) glycohydrolase (FOXG_05947.2, PARG). Here, we assessed whether this gene has a role as a global regulator of DNA repair or in virulence as an ADP ribosylating toxin homologue of bacteria. The PARG protein was purified after expressing its encoding gene in Escherichia coli. Its inhibition by 6,9-diamino-2-ethoxyacridine lactate monohydrate and tannins was similar to its human orthologue that is involved in DNA repair. A deletion strain of F. oxysporum f. sp. lycopersici showed no growth defects and was not affected in pathogenicity. Together, our results indicate that the PARG protein of F. oxysporum f. sp. lycopersici is involved in DNA repair and does not act in pathogenicity as an effector.     

Repression of Inflammasome by Francisella tularensis during Early Stages of Infection

Francisella tularensis is an important human pathogen responsible for causing tularemia. F. tularensis has long been developed as a biological weapon and is now classified as a category A agent by the Centers for Disease Control because of its possible use as a bioterror agent. F. tularensis represses inflammasome; a cytosolic multi-protein complex that activates caspase-1 to produce proinflammatory cytokines IL-1β and IL-18. However, the Francisella factors and the mechanisms through which F. tularensis mediates these suppressive effects remain relatively unknown. Utilizing a mutant of F. tularensis in FTL_0325 gene, this study investigated the mechanisms of inflammasome repression by F. tularensis. We demonstrate that muted IL-1β and IL-18 responses generated in macrophages infected with F. tularensis live vaccine strain (LVS) or the virulent SchuS4 strain are due to a predominant suppressive effect on TLR2-dependent signal 1. Our results also demonstrate that FTL_0325 of F. tularensis impacts proIL-1β expression as early as 2 h post-infection and delays activation of AIM2 and NLRP3-inflammasomes in a TLR2-dependent fashion. An enhanced activation of caspase-1 and IL-1β observed in FTL_0325 mutant-infected macrophages at 24 h post-infection was independent of both AIM2 and NLRP3. Furthermore, F. tularensis LVS delayed pyroptotic cell death of the infected macrophages in an FTL_0325-dependent manner during the early stages of infection. In vivo studies in mice revealed that suppression of IL-1β by FTL_0325 early during infection facilitates the establishment of a fulminate infection by F. tularensis. Collectively, this study provides evidence that F. tularensis LVS represses inflammasome activation and that F. tularensis-encoded FTL_0325 mediates this effect.