Invasive Hyphae Research Articles

OsWRKY70 Plays Opposite Roles in Blast Resistance and Cold Stress Tolerance in Rice

The transcription factor WRKYs play pivotal roles in the adapting to adverse environments in plants. Prior research has demonstrated the involvement of OsWRKY70 in resistance against herbivores and its response to abiotic stress. Here, we reported the functional analysis of OsWRKY70 in immunity against fungal diseases and cold tolerance. The results revealed that OsWRKY70 was induced by various Magnaporthe oryzae strains. Knock out mutants of OsWRKY70, which were generated by the CRISPR/Cas9 system, exhibited enhanced resistance to M. oryzae. This was consistent with fortifying the reactive oxygen species (ROS) burst after inoculation in the mutants, elevated transcript levels of defense-responsive genes (OsPR1b, OsPBZ1, OsPOX8.1 and OsPOX22.3) and the observation of the sluggish growth of invasive hyphae under fluorescence microscope. RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) validations demonstrated that differentially expressed genes were related to plant-pathogen interactions, hormone transduction and MAPK cascades. Notably, OsbHLH6, a key component of the JA signaling pathway, was down-regulated in the mutants compared to wild type plants. Further investigation confirmed that OsWRKY70 bound to the promoter of OsbHLH6 by semi-in vivo chromatin immunoprecipitation (ChIP). Additionally, the loss-function of OsWRKY70 impaired cold tolerance in rice. The enhanced susceptibility in the mutants characterized by excessive ROS production, elevated ion leakage rate and increased malondialdehyde content, as well as decreased activity of catalase (CAT) and peroxidase (POD) under low temperature stress was, which might be attributed to down-regulation of cold-responsive genes (OsLti6b and OsICE1). In conclusion, our findings indicate that OsWRKY70 negatively contributes to blast resistance but positively regulates cold tolerance in rice, providing a strategy for crop breeding with tolerance to stress.

The actin motor protein OsMYA1 associates with OsExo70H1 and contributes to rice secretory defense by modulating OsSyp121 distribution.

Magnaporthe oryzae (M. oryzae) is a devastating hemibiotrophic pathogen. Its biotrophic invasive hyphae (IH) are enclosed in the extrainvasive hyphal membrane produced by plant cells, thus generating a front line of the battlefield between the pathogen and the host plants. In plants, defense-related complexes such as proteins, callose-rich materials and vesicles, are directionally secreted to this interface to confer defense responses, but the underlying molecular mechanism is poorly understood. In this study, we found that a Myosin gene, Myosin A1 (OsMYA1), contributed to rice defense. The OsMYA1 knockout mutant exhibited decreased resistance to M. oryzae infection. OsMYA1 localizes to the actin cytoskeleton and surrounds the IH of M. oryzae. OsMYA1 interacts with an exocyst subunit, OsExo70H1, and regulates its accumulation at the plasma membrane (PM) and pathogen-plant interface. Furthermore, OsExo70H1 interacted with the rice syntaxin of the plants121 protein (OsSyp121), and the distribution of OsSyp121 to the PM or the pathogen-plant interface was disrupted in both the OsMYA1 and OsExo70H1 mutants. Overall, these results not only reveal a new function of OsMYA1 in rice blast resistance, but also uncover a molecular mechanism by which plants regulate defense against M. oryzae by OsMYA1-initiated vesicle secretory pathway, which originates from the actin cytoskeleton to the PM.

Analysis of the antagonistic effect of Stenotrophomonas geniculata WXY53 on Magnaporthe oryzae through bioassays and Whole-Genome sequencing

Magnaporthe oryzae causes rice blast, which poses a significant threat to agriculture. The use of environmentally friendly biological control agents is one of the methods used to reduce disease incidence. Our study identified the isolate WXY53 as Stenotrophomonas geniculata using 16S rDNA and phylogenetic tree analysis. WXY53 exhibited a 53.39 % inhibition rate on the growth diameter of M. oryzae hyphae in the dual culture assay. The WXY53 fermentation broth treatment inhibited 97.89 % of appressoria formation and delayed the colonization of plant cells by invasive hyphae. Applying WXY53 fermentation broth had a significant impact on preventing rice blast in the greenhouse experiment. It suppressed the size of necrotic lesions and reduced the number of lesions in rice and barley by 66.25 % and 72.0 %, respectively. The whole-genome sequencing of WXY53 revealed a genome size of 4,504,923 bp, with 66.51 % GC content and 4038 protein-coding genes. The study utilized multiple databases, including the Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups of proteins, Gene Ontology, Pathogen-Host Interactions, and Carbohydrate-Active enZYmes to annotate gene functions. The analysis revealed genes related to environmental information processing, biological processes, cell wall/membrane/envelope biogenesis, glycoside hydrolases, and metabolism. Antibiotics & Secondary Metabolite Analysis Shell analysis predicted that WXY53 has the potential to produce aryl polymers, entolysin, and 2,3-dihydroxybenzoyl serine, in addition to the three gene clusters with unknown functions. Based on these findings, strain WXY53 could be a promising novel source of biological agents for managing rice blast. This study provides a theoretical basis for further investigation into microbial resources and metabolic gene clusters for agricultural biological control.

MoSey1 regulates the unfolded protein response, appressorium development, and pathogenicity of Magnaporthe oryzae

Endoplasmic reticulum (ER) is an enclosed three-dimensional eukaryotic membrane network composed of flattened sacs. Fusion of homologous membranes to the ER membrane is essential for the maintenance of this network structure. In yeast, ER membrane fusion is mediated by Sey1p, whose paralogues function distinctly in different species. In this study, we investigated the biological functions of MoSEY1 in the devastating rice blast fungus Magnaporthe oryzae by functional genomic approach. Compared to wild type, deletion of MoSEY1 considerably decreased the growth and conidia production of M. oryzae. Additionally, the absence of MoSEY1 delayed appressorium formation and invasive hyphae growth. The appressorium function was also impaired in ΔMosey1 mutant. Subcellular localization analysis revealed that MoSey1 is localized at the ER. The ΔMosey1 mutant showed augmented sensitivity to ER stress. Additionally, we found that MoSey1 regulated the unfolded protein response, autophagy, and protein secretion in M. oryzae. In conclusion, our study unveiled the involvement of MoSey1 in the development, pathogenesis, and ER functions in M. oryzae.

Cytological and ultrastructural investigation of pathogen infection pathway and host responses in asparagus stem infected by Phomopsis asparagi

Asparagus stem blight, a highly destructive disease in global asparagus cultivation, is caused by the fungus Phomopsis asparagi. However, the underlying mechanisms of the infectious process and pathogenesis of P. asparagi remain poorly understood. This study aims to elucidate the infection event of P. asparagi at the cytological and ultrastructural levels in asparagus stem through a microscopic observation. The host responses were also examined by microscopic observation and fluorescent probe. It revealed that P. asparagi germinated at either the tip or the middle of the conidia to produce short germ tubes on the surfaces of the asparagus stem at 20 h post-inoculation (hpi). The germ tubes penetrated the host cell wall with appressorium-like structures or narrow pegs at 1 day post-inoculation (dpi). At 3 − 5 dpi, a large number of P. asparagi hyphae colonized the epidermal cells. The hyphae were found to grow both intracellularly and intercellularly. The movement of hyphae between cells was facilitated by constricted invasive hyphae pegs. The hyphae exhibited bidirectional intracellular growth, extending and branching along the inner side of the cell wall within the stem cortex and towards the central cylinder. The fungal colonization resulted in cellular damage in plants, which is characterized by plasmolysis, rupture of the cell wall, and disruption of the cytoplasm. At 11 dpi, the fungi penetrated the parenchyma cells, and the fungal pycnidia were formed. At 13 dpi, the fungi penetrated the stem center parenchyma cell, where the conidia were released. In addition, the host defense response was investigated, which revealed a notably reduced germination rate of conidium, the formation of callose analogs, and the reactive oxygen burst. These findings provide unexpected perspectives on the infection process and host response in P. asparagi-plant interaction.

Streptomyces pratensis S10 inhibits spread of Fusarium graminearum invasive hyphae and toxisome formation in wheat plants.

Fusarium head blight (FHB) of wheat, mainly caused by Fusarium graminearum, leads to severe economic losses worldwide. Effective management measures for controlling FHB are not available, due to a lack of resistant cultivars. Currently, the utilization of biological control is a promising approach that can be used to help manage FHB. Previous studies have confirmed that Streptomyces pratensis S10 harbors excellent inhibitory effects on F. graminearum. However, there is no information regarding invasive hyphae of F. graminearum are inhibited by S10. Thus, we investigated the effects of S10 on F. graminearum strain PH-1 hyphae extension, toxisome formation, and TRI5 gene expression on wheat plants via microscopic observation. The results showed that S10 effectively inhibited spread of F. graminearum hyphae along the rachis, restricting the infection of neighboring florets via the phloem. In the presence of S10, the hyphal growth is impeded by formation of dense cell wall thickenings in the rachis internode surrounding the F. graminearum infection site, avoiding cell plasmolysis and collapse. We further demonstrated that S10 largely prevented cell-to-cell invasion of fungal hyphae inside wheat coleoptiles using a constitutively green fluorescence protein-expressing F. graminearum strain, PH-1. Importantly, S. pratensis S10 inhibited toxisome formation and TRI5 gene expression in wheat plants during infection. Collectively, these findings indicated that S. pratensis S10 prevents spread of F. graminearum invasive hyphae via the rachis.

In Vitro and Ex Vivo Antifungal Activities of Metconazole against the Rice Blast Fungus Pyricularia oryzae.

Rice blast, caused by the filamentous fungus Pyricularia oryzae, has long been one of the major threats to almost all rice-growing areas worldwide. Metconazole, 5-(4-chlorobenzyl)-2, 2-dimethyl-1-(1H-1, 2, 4-triazol-1-ylmethyl) cyclopentanol, is a lipophilic, highly active triazole fungicide that has been applied in the control of various fungal pathogens of crops (cereals, barley, wheat), such as the Fusarium and Alternaria species. However, the antifungal activity of metconazole against P. oryzae is unknown. In this study, metconazole exhibited broad spectrum antifungal activities against seven P. oryzae strains collected from rice paddy fields and the wild type strain P131. Scanning electron microscopic analysis and fluorescein diacetate staining assays revealed that metconazole treatment damaged the cell wall integrity, cell membrane permeability and even cell viability of P. oryzae, resulting in deformed and shrunken hyphae. The supplementation of metconazole in vitro increased fungal sensitivity to different stresses, such as sodium dodecyl sulfate, congo red, sodium chloride, sorbitol and oxidative stress (H2O2). Metconazole could inhibit key virulence processes of P. oryzae, including conidial germination, germ tube elongation and appressorium formation. Furthermore, this chemical prevented P. oryzae from infecting barley epidermal cells by disturbing appressorium penetration and subsequent invasive hyphae development. Pathogenicity assays indicated a reduction of over 75% in the length of blast lesions in both barley and rice leaves when 10 μg/mL of metconazole was applied. This study provides evidence to understand the antifungal effects of metconazole against P. oryzae and demonstrates its potential in rice blast management.

MoAti1 mediates mitophagy by facilitating recruitment of MoAtg8 to promote invasive growth in Magnaporthe oryzae.

Mitophagy is a selective autophagy for the degradation of damaged or excessive mitochondria to maintain intracellular homeostasis. In Magnaporthe oryzae, a filamentous ascomycetous fungus that causes rice blast, the most devastating disease of rice, mitophagy occurs in the invasive hyphae to promote infection. To date, only a few proteins are known to participate in mitophagy and the mechanisms of mitophagy are largely unknown in pathogenic fungi. Here, by a yeast two-hybrid screen with the core autophagy-related protein MoAtg8 as a bait, we obtained a MoAtg8 interactor MoAti1 (MoAtg8-interacting protein 1). Fluorescent observations and protease digestion analyses revealed that MoAti1 is primarily localized to the peripheral mitochondrialouter membrane and is responsible for recruiting MoAtg8 to mitochondria under mitophagy induction conditions. MoAti1 is specifically required for mitophagy, but not for macroautophagy and pexophagy. Infection assays suggested that MoAti1 is required for mitophagy in invasive hyphae during pathogenesis. Notably, no homologues of MoAti1 were found in rice and human protein databases, indicating that MoAti1 may be used as a potential target to control rice blast. By the host-induced gene silencing (HIGS) strategy, transgenic rice plants targeted to silencing MoATI1 showed enhanced resistance against M. oryzae with unchanged agronomic traits. Our results suggest that MoATI1 is required for mitophagy and pathogenicity in M. oryzae and can be used as a target for reducing rice blast.

A protein kinase coordinates cycles of autophagy and glutaminolysis in invasive hyphae of the fungus Magnaporthe oryzae within rice cells

The blast fungus Magnaporthe oryzae produces invasive hyphae in living rice cells during early infection, separated from the host cytoplasm by plant-derived interfacial membranes. However, the mechanisms underpinning this intracellular biotrophic growth phase are poorly understood. Here, we show that the M. oryzae serine/threonine protein kinase Rim15 promotes biotrophic growth by coordinating cycles of autophagy and glutaminolysis in invasive hyphae. Alongside inducing autophagy, Rim15 phosphorylates NAD-dependent glutamate dehydrogenase, resulting in increased levels of α-ketoglutarate that reactivate target-of-rapamycin (TOR) kinase signaling, which inhibits autophagy. Deleting RIM15 attenuates invasive hyphal growth and triggers plant immunity; exogenous addition of α-ketoglutarate prevents these effects, while glucose addition only suppresses host defenses. Our results indicate that Rim15-dependent cycles of autophagic flux liberate α-ketoglutarate – via glutaminolysis – to reactivate TOR signaling and fuel biotrophic growth while conserving glucose for antioxidation-mediated host innate immunity suppression.

Evaluation of the Antifungal Activity of Polysubstituted Cyclic 1,2-Diketones against Colletotrichum gloeosporioides

(1) Anthracnose caused by Colletotrichum damages crops, ornamentals, and forest trees severely, resulting in enormous economic losses to agricultural and forestry systems. Overusing traditional chemical fungicides leads to fungicide resistance, environmental pollution, and potential risks to public health. Therefore, priorities should be given to developing efficient and environmentally friendly approaches to pest management, including anthracnose. (2) In this study, the antifungal activity of botanical derivative polysubstituted cyclic 1,2-diketones (FPL001) against C. gloeosporioides was examined. (3) FPL001 significantly inhibited the vegetative growth of C. gloeosporioides with an EC50 of 160.23 µg/mL. When the concentration of FPL001 reached 30 µg/mL, the conidial germination and appressorium formation of C. gloeosporioides were completely inhibited. FPL001 also significantly suppressed the invasive hyphae development and plant infection of C. gloeosporioides. FPL001 did not exhibit toxicity to model organisms such as alfalfa and silkworm larvae. (4) These results indicate that compound FPL001 is a potential and efficient agent for green control of C. gloeosporioides.

Cutaneous Rhizopus Infection Complicating Left Submammary Intertrigo

Abstract Introduction Mucormycosis is a rare, opportunistic infection commonly associated with diabetes mellitus, acidosis, neutropenia, organ transplantation, high-dose glucocorticoid therapy, iron overload syndromes, and hematologic malignancy. Case Invasive infection usually presents with rhino-orbital-cerebral (34%), cutaneous (22%), and pulmonary (20%) manifestations. Cutaneous cases are almost always precipitated by trauma or wounds. Only 6 previous cases of cutaneous mucormycosis (Mucor, Rhizopus, Lichtheimia, and others) infection of the breast have been reported in the literature. We herein report a woman with fatal submammary mucormycosis. Report A 45-year-old diabetic woman with alcohol use disorder was admitted from her basement living quarters with septic shock and lactic acidosis. During her intensive care unit stay, she developed worsening eschar of left submammary region. Cultures revealed Rhizopus species and surgical resection revealed invasive hyphae classic for mucormycosis. Despite treatment with liposomal amphotericin and complete resection, the patient died. Discussion Rhizopus infection complicated sepsis-related lactic acidosis and preexisting submammary intertrigoseveral days after admission. This case emphasizes the importance of a continued thorough physical examination that includes visualization of intertriginous areas, including those in submammary areas in septic patients in the critical care unit.

The Cell Wall Integrity MAP Kinase Signaling Pathway Is Required for Development, Pathogenicity, and Stress Adaption of the Pepper Anthracnose Fungus Colletotrichum scovillei

The cell wall integrity (CWI) signaling pathway plays important roles in the dissemination and infection of several plant pathogenic fungi. However, its roles in the pepper fruit anthracnose fungus Colletotrichum scovillei remain uninvestigated. In this study, the major components of the CWI signaling pathway—CsMCK1 (MAPKKK), CsMKK1 (MAPKK), and CsMPS1 (MAPK)—were functionally characterized in C. scovillei via homology-dependent gene replacement. The ΔCsmck1, ΔCsmkk1, and ΔCsmps1 mutants showed impairments in fungal growth, conidiation, and tolerance to CWI and salt stresses. Moreover, ΔCsmck1, ΔCsmkk1, and ΔCsmps1 failed to develop anthracnose disease on pepper fruits due to defects in appressorium formation and invasive hyphae growth. These results suggest that CsMCK1, CsMKK1, and CsMPS1 play important roles in mycelial growth, conidiation, appressorium formation, plant infection, and stress adaption of C. scovillei. These findings will contribute to a better understanding of the roles of the CWI signaling pathway in the development of pepper fruit anthracnose disease.

60-Year-Old Patient with Invasive Mold Infection Consequent to an Encounter with a Barnyard Animal

Abstract Introduction/Objective Lichtheimia and Scedosporium species are ubiquitous fungi associated with a range of clinical presentations. We report a complex soft tissue coinfection caused by Lichtheimia spp. and Scedosporium spp. following a puncture wound by a rooster spur. Methods/Case Report A 60-year-old immunosuppressed man with a history of heart transplant presented with a non-healing wound 2-months after sustaining a lower leg puncture secondary to a rooster spur. The patient was previously treated with Augmentin for presumed cellulitis without improvement. To characterize the ongoing disease process, the wound was sampled with tissue portions routed for both histopathologic review and culture. In short order, the biopsy demonstrated invasive hyaline pauciseptate fungal hyphae consistent with organisms of the Mucorales order. In congruence, culture rapidly revealed a mucoraceous mold, subsequently identified as a Lichtheimia spp. Thus, the patient was treated with liposomal amphotericin B. However, days later, a second fungus - a Scedosporium spp. - was isolated from the original specimen. Consequently, isavuconazonium sulfate therapy was added. Re-review of the initial biopsy enabled distinction of two populations of fungal hyphae plausibly representing both molds. Despite numerous debridements and optimal medical management, the patient was ultimately referred for amputation. Results (if a Case Study enter NA) NA. Conclusion When encountered in an immunocompromised host, providers understandably respond to an invasive mucoraceous mold with alarm followed by quick, decisive action. However, septate molds, such as the Scedosporium spp., can also be aggressive. Further, the presence of more than one invasive mold presents a treatment challenge as the mucormycetes may require surgery coupled with amphotericin B therapy; whereas, the a Scedosporium spp. may be wholly resistant to amphotericin B. This case illustrates the critical role that an alert pathologist plays in guiding management through integration of data from different diagnostic modalities. It also constitutes an emphatic reminder that an illness may be caused by more than one actionable, invasive fungus.

The histone deacetylase Cfhos2 is a key epigenetic factor regulating appressorium development and pathogenesis in apple Glomerella leaf spot fungus Colletotrichum fructicola

Glomerella leaf spot (GLS) is a devastating fungal disease that damages the leaves and fruits and reduces tree vigor of apple (Malus domestica). The pathogen infection mechanism, however, remains elusive. Histone-modifying enzymes, which regulate eukaryotic chromatin conformation and gene expression, are key epigenetic factors controlling fungal development, virulence, and secondary metabolism. To dissect the epigenetic regulation of GLS pathogenesis, we characterized a histone deacetylase gene Cfhos2 in Colletotrichum fructicola, the causing agent of GLS. Cfhos2 deletion mutants were mildly reduced in vegetative growth rate, but almost lost pathogenicity on apple leaves. Cfhos2 deletion mutants induced strong plant defense responses manifested by epidermal cell browning, granulation, and distortion of pathogen invasive hyphae. The mutants also showed defect in appressorial development on cellophane, but not on parafilm or on apple leaf surface, suggesting that the defect in appressorial development is surface-dependent. RNA-seq based transcriptome analysis highlighted that Cfhos2 regulates secondary metabolism-related virulence genes during infection. Moreover, the expression of an apple defense-related F-box protein was strongly induced by infection with Cfhos2 deletion mutants. Taken together, we demonstrate that Cfhos2 is a key epigenetic factor regulating appressorium development, virulence gene expression, and GLS pathogenesis in C. fructicola. The results provide important information for understanding the virulence mechanisms of C. fructicola.

Endoplasmic reticulum membrane protein MoScs2 is important for asexual development and pathogenesis of Magnaporthe oryzae.

Most secretory proteins are folded and modified in the endoplasmic reticulum (ER). In Saccharomyces cerevisiae, the absence of Scs2 protein will lead to the separation of the endoplasmic reticulum and plasma membrane, resulting in endoplasmic reticulum dysfunction, but its function is not clear in rice blast fungus or even filamentous fungus. In this study, we report the identification and characterization of MoSCS2 in the pathogenesis of the rice blast fungus Magnaporthe oryzae. Protein subcellular localization showed that MoSCS2 is mainly localized in the endoplasmic reticulum. Compared to the wild-type strain Guy11, the deletion mutant ΔMoscs2 showed a significant reduction in growth and conidiation. MoSCS2 deficiency also resulted in abnormal conidial morphology and septum formation. The ΔMoscs2 mutant shows delayed appressorium formation, and the appressorium of ΔMoscs2 mutant could not form huge turgor pressure to penetrate the host epidermal cell wall. Pathogenicity and plant leave infection assays showed that knockout of MoSCS2 significantly inhibited the expansion of the invasive hyphae in host cells, ultimately leading to the decline of pathogenicity. Moreover, MoSCS2 gene is also involved in the regulation of cell wall and endoplasmic reticulum stress response. In conclusion, MoSCS2 plays an important role in the growth, asexual production, conidia morphogenesis, infection-related morphogenesis and pathogenicity of M. oryzae.

MoWhi2 Mediates Mitophagy to Regulate Conidiation and Pathogenesis in Magnaporthe oryzae.

Mitophagy refers to the specific process of degrading mitochondria, which is an important physiological process to maintain the balance of mitochondrial quantity and quality in cells. At present, the mechanisms of mitophagy in pathogenic fungi remain unclear. Magnaporthe oryzae (Syn. Pyricularia oryzae), the causal agent of rice blast disease, is responsible for the most serious disease of rice. In M. oryzae, mitophagy occurs in the foot cells and invasive hyphae to promote conidiation and infection. In this study, fluorescent observations and immunoblot analyses showed that general stress response protein MoWhi2 is required for mitophagy in M. oryzae. In addition, the activation of the autophagy, pexophagy and cytoplasm-to-vacuole targeting (CVT) pathway upon nitrogen starvation was determined using the GFP-MoATG8, GFP-SRL and MoAPE1-GFP strains and the ΔMowhi2 mutant in these backgrounds. The results indicated that MoWhi2 is specifically required for mitophagy in M. oryzae. Further studies showed that mitophagy in the foot cells and invasive hyphae of the ΔMowhi2 was interrupted, leading to reduced conidiation and virulence in the ΔMowhi2 mutant. Taken together, we found that MoWhi2 contributes to conidiation and invasive growth by regulating mitophagy in M. oryzae.

Two Magnaporthe appressoria-specific (MAS) proteins, MoMas3 and MoMas5, are required for suppressing host innate immunity and promoting biotrophic growth in rice cells.

In the devastating rice blast fungus Magnaporthe oryzae, six Magnaporthe appressoria‐specific (MAS) proteins are encoded by MoGAS1, MoGAS2 and MoMAS3–MoMAS6. MoGAS1 and MoGAS2 were previously characterized as M. oryzae virulence factors; however, the roles of the other four genes are unknown. Here, we found that, although the loss of any MAS gene did not affect appressorial formation or vegetative growth, ∆Momas3 and ∆Momas5 mutant strains (but not the others) were reduced in virulence on susceptible CO‐39 rice seedlings. Focusing on ∆Momas3 and ∆Momas5 mutant strains, we found that they could penetrate host leaf surfaces and fill the first infected rice cell but did not spread readily to neighbouring cells, suggesting they were impaired for biotrophic growth. Live‐cell imaging of fluorescently labelled MoMas3 and MoMas5 proteins showed that during biotrophy, MoMas3 localized to the apoplastic compartment formed between fungal invasive hyphae and the plant‐derived extra‐invasive hyphal membrane while MoMas5 localized to the appressoria and the penetration peg. The loss of either MoMAS3 or MoMAS5 resulted in the accumulation of reactive oxygen species (ROS) in infected rice cells, resulting in the triggering of plant defences that inhibited mutant growth in planta. ∆Momas3 and ∆Momas5 biotrophic growth could be remediated by inhibiting host NADPH oxidases and suppressing ROS accumulation. Thus, MoMas3 and MoMas5 are novel virulence factors involved in suppressing host plant innate immunity to promote biotrophic growth.

Aspartate Transaminase AST2 Involved in Sporulation and Necrotrophic Pathogenesis in the Hemibiotrophs Magnaporthe oryzae and Colletotrichum graminicola.

Aspartate family includes five additional amino acids other than aspartate, among which most except aspartate have been reported for their action in pathogenesis by amino acid biosynthesis. However, how aspartate, the initial substrate of this family metabolic pathway, is involved in pathogenesis remains unknown. Here, we focused on aspartate transaminase (AST) that catalyzes transamination reaction between glutamate-aspartate in Magnaporthe oryzae. Three MoAST genes were bioinformatically analyzed, of which MoAST2 was uniquely upregulated when invasive hyphae switched to necrotrophic pathogenesis. MoAST2 deletion (ΔMoast2) caused a drastic reduction in conidiogenesis and appressorium formation. Particularly, ΔMoast2 was observed to be proliferated at the biotrophic phase but inhibited at the necrotrophic stage, and with invisible symptoms detected, suggesting a critical role in necrotrophic phase. Glutamate family restored the ΔMoast2 defects but aspartate family did not, inferring that transamination occurs from aspartate to glutamine. MoAST2 is cytosolic and possessed H2O2 stress tolerance. In parallel, Colletotrichum graminicola AST2, CgAST2 was proven to be a player in necrotrophic anthracnose development. Therefore, conserved AST2 is qualified to be a drug target for disease control.

Invasive aspergillosis-on-chip: A quantitative treatment study of human Aspergillus fumigatus infection

Invasive pulmonary aspergillosis is associated with a high mortality rate and poses a direct threat to immunocompromised patients. Here, we present the invasive aspergillosis-on-chip (IAC) model to investigate Aspergillus fumigatus infection in vitro. The model allows the study of the lateral growth and the invasive behaviour of fungal hyphae from the epithelium into the endothelial cell layer in an alveolus-on-chip model. We established an algorithm-based analysis pipeline for three-dimensional confocal microscopy images to visualize and quantify fungal morphology, including hyphal growth and branching. Human macrophages in the IAC model partially inhibited the growth of the fungus, contributed to the release of proinflammatory cytokines (IL-1, IL-6, TNF) and chemokines (IL-8 and MCP-1) associated with an increased number of invasive hyphae. Similar to in vivo, the application of the fungistatic drug caspofungin limited the fungal growth and resulted in morphological changes of the hyphal tree previously described in other studies. The IAC infection model allows the identification and characterization of cellular infection targets and in vitro testing of antifungal drugs in clinically relevant concentrations. It thus represents a promising tool to broaden the understanding of pathogenicity and pathophysiology of invasive aspergillosis.

Clumping Morphology Influences Virulence Uncoupled from Echinocandin Resistance in Candida glabrata.

ABSTRACTHere, we report two paired sets of an index wild-type Candida glabrata bloodstream isolate and subsequent echinocandin-resistant FKS mutant. One paired set exhibited a higher proportion of clumping cells and was more virulent in the invertebrate host Galleria mellonella than the other paired set. No virulence difference between the paired index and FKS strains was observed. These findings imply a potential link of clumping morphology with virulence in C. glabrata that is uncoupled from FKS-mediated echinocandin resistance.IMPORTANCE Candida glabrata is a leading cause of invasive candidiasis. In contrast to other species, it has a high propensity for developing resistance to echinocandins, which are the first-line treatment. Unlike the dimorphic Candida albicans which can grow invasive filamentous hyphae, C. glabrata lacks this ability. Here, we report a link between virulence and clumping cell morphology in two different sets of clinical C. glabrata strains obtained from patients failing echinocandin therapy. One set of paired strains (echinocandin-susceptible and subsequent resistant mutant) had a high proportion of clumping cells in the population and were significantly more virulent than another set which had fewer clumping cells. Additionally, we corroborate that echinocandin resistance does not impart a significant fitness cost. Our findings suggest that clumping morphology may be an important but previously underestimated virulence factor for C. glabrata and also aid our understand for the high prevalence of resistance observed in this species.