Appressorium Formation Research Articles

Ornithine decarboxylase of the fungal pathogen Colletotrichum higginsianum plays an important role in regulating global metabolic pathways and virulence.

Colletotrichum higginsianum is an important fungal pathogen causing anthracnose disease of cruciferous plants. In this study, we characterized a putative orthologue of yeast SPE1 in C. higginsianum, named ChODC. Deletion mutants of ChODC were defective in hyphal and conidial development. Importantly, deletion of ChODC significantly affected appressorium-mediated penetration in C. higginsianum. However, polyamines partially restore appressorium function and virulence indicating that loss of ChODC caused significantly decreased virulence by the crosstalk between polyamines and other metabolic pathways. Subsequently, transcriptomic and metabolomic analyses demonstrated that ChODC played an important role in metabolism of various carbon and nitrogen compounds including amino acids, carbohydrates and lipids. Along with these clues, we found deletion of ChODC affected glycogen and lipid metabolism, which were important for conidial storage utilization and functional appressorium formation. Loss of ChODC affected the mTOR signalling pathway via modulation of autophagy. Interestingly, cAMP treatment restored functional appressoria to the ΔChODC mutant, and rapamycin treatment also stimulated formation of functional appressoria in the ΔChODC mutant. Overall, ChODC was associated with the polyamine biosynthesis pathway, as a mediator of cAMP and mTOR signalling pathways to regulate appressorium function. Our study provides evidence of a link between ChODC and the cAMP signalling pathway and defines a novel mechanism by which ChODC regulates infection-associated autophagy and plant infection by fungi.

PoRal2 Is Involved in Appressorium Formation and Virulence via Pmk1 MAPK Pathways in the Rice Blast Fungus Pyricularia oryzae.

Pyricularia oryzae is an important plant pathogenic fungus that can severely damage rice and wheat crops, leading to significant reductions in crop productivity. To penetrate into and invade tissues of its plant host, this fungus relies on an invasive structure known as an appressorium. Appressorium formation is rigorously regulated by the cAMP-PKA and Pmk1 MAPK pathways. Here, we identified PoRal2, a homologous protein of Schizosaccharomyces pombe Ral2, and characterized its roles in fungal development and virulence in P. oryzae. PoRal2 contains N-terminal kelch repeats and C-terminal BTB domains. PoRal2 is involved in sporulation, aerial hypha and conidiophore differentiation, appressorium formation, plant penetration, and virulence. During appressorium formation, ∆Poral2 mutants generate appressoria with long germ tubes on hydrophobic surfaces. ∆Poral2 mutants exhibited a defective response to exogenous cAMP and the activated RAS2G18V on a hydrophilic surface, indicating impairment in the cAMP-PKA or Pmk1 MAPK signaling pathways. Deletion of PoRAL2 leads to lowered Pmk1 phosphorylation level in the mutant. Moreover, PoRal2 is found to interact with Scd1, Smo1, and Mst50, which are involved in activation of Pmk1. In addition, the expression levels of MPG1, WISH, and PDEH in the cAMP-PKA pathway, RAS2 in both the cAMP-PKA and Pmk1 MAPK pathways, and melanin biosynthesis genes (ALB1, BUF1, and RSY1) were significantly down-regulated in the ∆Poral2. Therefore, PoRal2 is involved in fungal development and virulence by its crosstalk in the cAMP-PKA and Pmk1 MAPK signaling pathways.

Transcriptional Landscapes of Long Non-coding RNAs and Alternative Splicing in Pyricularia oryzae Revealed by RNA-Seq

Pyricularia oryzae causes the rice blast, which is one of the most devastating crop diseases worldwide, and is a model fungal pathogen widely used for dissecting the molecular mechanisms underlying fungal virulence/pathogenicity. Although the whole genome sequence of P. oryzae is publicly available, its current transcriptomes remain incomplete, lacking the information on non-protein coding genes and alternative splicing. Here, we performed and analyzed RNA-Seq of conidia and hyphae, resulting in the identification of 3,374 novel genes. Interestingly, the vast majority of these novel genes likely transcribed long non-coding RNAs (lncRNAs), and most of them were localized in the intergenic regions. Notably, their expressions were concomitant with the transcription of neighboring genes thereof in conidia and hyphae. In addition, 2,358 genes were found to undergo alternative splicing events. Furthermore, we exemplified that a lncRNA was important for hyphal growth likely by regulating the neighboring protein-coding gene and that alternative splicing of the transcription factor gene CON7 was required for appressorium formation. In summary, results from this study indicate that lncRNA transcripts and alternative splicing events are two important mechanisms for regulating the expression of genes important for conidiation, hyphal growth, and pathogenesis, and provide new insights into transcriptomes and gene regulation in the rice blast fungus.

A double‐edged sword: reactive oxygen species (ROS) during the rice blast fungus and host interaction

Magnaporthe oryzae is a hemibiotrophic fungus that also needs host nutrients for propagation during infection. During its interaction with rice, reactive oxygen species (ROS) mediate important signaling reactions impacting both the pathogen and the host. In M. oryzae, the accumulation of ROS is important for the formation and maturation of the infectious structure appressorium. On the other hand, upon M. oryzae infection, rice generates further ROS to restrict invasive hyphae (IH) spreading. Despite ROS receptors remaining to be identified, M. oryzae recruits several strategies to respond and suppress ROS accumulation through the secretion of various effector molecules. These findings suggest that the balance between the generation and scavenging of ROS is sophisticatedly controlled during M. oryzae-rice interaction. In this review, we discuss advances to understand the regulation mechanisms for the generation, accumulation, and transduction of ROS.

Fungal Jasmonate as a Novel Morphogenetic Signal for Pathogenesis.

A key question that has remained unanswered is how pathogenic fungi switch from vegetative growth to infection-related morphogenesis during a disease cycle. Here, we identify a fungal oxylipin analogous to the phytohormone jasmonic acid (JA), as the principal regulator of such a developmental switch to isotropic growth and pathogenicity in the rice-blast fungus Magnaporthe oryzae. Using specific inhibitors and mutant analyses, we determined the molecular function of intrinsic jasmonates during M. oryzae pathogenesis. Loss of 12-Oxo-phytodienoic Acid (OPDA) Reductase and/or consequent reduction of jasmonate biosynthesis, prolonged germ tube growth and caused delayed initiation and improper development of infection structures in M. oryzae, reminiscent of phenotypic defects upon impaired cyclic AMP (cAMP) signaling. Chemical- or genetic-complementation completely restored proper vegetative growth and appressoria in opr1Δ. Mass spectrometry-based quantification revealed increased OPDA accumulation and significantly decreased jasmonate levels in opr1Δ. Most interestingly, exogenous jasmonate restored proper appressorium formation in pth11Δ that lacks G protein/cAMP signaling; but failed to do so in the Mitogen-activated protein (MAP) kinase mutants. Epistasis analysis placed jasmonate upstream of the cAMP pathway in rice blast. Mechanistically, intrinsic jasmonate orchestrates timely cessation of the vegetative phase and induces pathogenic development via a complex regulatory interaction with the cAMP-PKA cascade and redox signaling in rice blast.

Mucin Msb2 cooperates with the transmembrane protein Sho1 in various plant surface signal sensing and pathogenic processes in the poplar anthracnose fungus Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides is a hemibiotrophic ascomycete fungus that causes anthracnose on numerous plants worldwide and forms a specialized infection structure known as an appressorium in response to various plant surface signals. However, the associated mechanism of host surface signal recognition remains unclear. In the present study, three putative sensors, namely the mucin Msb2, the membrane sensor protein Sho1, and the G‐protein‐coupled receptor Pth11, were identified and characterized. The results showed that CgMsb2 plays a major role in the recognition of various host surface signals; deletion of CgMsb2 resulted in significant defects in appressorium formation, appressorium penetration, cellophane membrane penetration, and pathogenicity. CgSho1 plays a minor role and together with CgMsb2 cooperatively regulates host signal recognition, cellophane membrane penetration, and pathogenicity; deletion of CgSho1 resulted in an expansion defect of infection hyphae. Deletion of CgPth11 in wildtype, ΔCgMsb2, and ΔCgSho1 strains only resulted in a slight defect in appressorium formation at the early stage, and CgPth11 was dispensable for penetration and pathogenicity. However, exogenous cAMP failed to restore the defect of appressorium formation in ΔCgPth11 at the early stage. CgMsb2 contributed to the phosphorylation of the mitogen‐activated protein kinase CgMk1, which is essential for infection‐associated functions, while CgSho1 was unable to activate CgMk1 alone but rather cooperated with CgMsb2 to activate CgMk1. These data suggest that CgMsb2 contributes to the activation of CgMk1 and has overlapping functions with CgSho1 in plant surface sensing, appressorium formation, and pathogenicity.

Functional Characterization of the Novel Laminaripentaose-Producing β-1,3-Glucanase MoGluB and Its Biocontrol of Magnaporthe oryzae

Fungal cell wall synthesizing enzymes or remodeling enzymes represent key factors for the interaction of plant pathogen and antifungal agents, which are regarded as potential biocontrol agents. In this study, a novel endo-β-1,3-glucanase from Magnaporthe oryzae was expressed and characterized. The expression of MoGluB was significantly upregulated after 2 days of liquid culture and 48 h after infection, indicating that it may be involved in cell wall reconstitution. Purified MoGluB exhibited high activity on insoluble β-glucans, with a specific activity of 8.18 U/mg toward yeast glucan at pH 9.0 and 50 °C. MoGluB hydrolyzed pachymaran and yeast glucan into oligosaccharides dominated by laminaripentaose, suggesting that it is an endo-β-1,3-glucanase. Incubation of 8 μg of MoGluB with 106 spores/mL resulted in the inhibition of conidial germination and appressorium formation of M. oryzae, illustrating effective biocontrol activity. Hydrolysates of pachymaran induced the expression of defense genes restricting M. oryzae infection in rice plants, indicating an immunostimulatory effect of MoGluB hydrolysates.

An insight of anopheline larvicidal mechanism of Trichoderma asperellum (TaspSKGN2)

Anopheline larvicidal property of T. asperellum has been found recently in medical science. The mechanism of actions exhibited by T. asperellum to infect mosquito larvae is the pivotal context of our present study. To infect an insect, entomopathogens must undergo some events of pathogenesis. We performed some experiments to find out the mechanisms of action of T. asperellum against anopheline larvae and compared its actions with other two well recognized entomopathogens like Metarhizium anisopliae and Beauveria bassiana. The methodology adopted for this includes Compound light and SE Microscopic study of host–pathogen interaction, detection of fungal spore adhesion on larval surface (Mucilage assay), detection of cuticle degrading enzymes (Spore bound pr1, chitinase and protease) by spectro-photometric method, Quantitative estimation of chitinase and protease enzymes, and determination of nuclear degeneration of hemocyte cells of ME (methanolic extract) treated larvae by T. asperellum under fluorescence microscope. Compound light microscopic studies showed spore attachment, appressorium and germ tube formation, invasion and proliferated hyphal growth of T. asperellum on epicuticle and inside of dead larvae. SEM study also supported them. After 3 h of interaction, spores were found to be attached on larval surface exhibiting pink colored outer layer at the site of attachment indicating the presence of mucilage surrounding the attached spores. The enzymatic cleavage of the 4-nitroanilide substrate yields 4-nitroaniline which indicates the presence of spore-bound PR1 protein (Pathogenecity Related 1 Protein) and it was highest (absorbance 1.298 ± 0.002) for T. asperellum in comparison with control and other two entomopathogens. T. asperellum exhibited highest enzymatic index values for both chitinase (5.20) and protease (2.77) among three entomopathogens. Quantitative experiment showed that chitinase enzyme concentration of T. asperellum (245 µg mL−1) was better than other two M. anisopliae (134.59 µg mL−1) and B. bassiana (128.65 µg mL−1). Similarly protease enzyme concentration of this fungus was best (298.652 µg mL−1) among three entomopathogens. Here we have detected and estimated fragmentized nuclei of hemocyte cells by fluorescence microscopy in treated larvae with different ME doses of T. asperellum, and also observed that mosquito larvae exposed to 0.1 mg mL−1 dose of ME showed maximum (100%) nuclear fragmentations of hemocytes and while 20, 45, 70 and 85% of nuclear deformities were recorded at 0.02, 0.04, 0.06 and 0.08 mg mL−1 concentrations of ME. The knowledge of this work certainly will help in understanding of mechanism of action of T. asperellum for anopheline larval killing and consequently in eradication of malaria vector.

O-mannosyltransferase MaPmt2 contributes to stress tolerance, cell wall integrity and virulence in Metarhizium acridum

As a conserved post-translational modification, O-mannosyltransferase families play important roles in many cellular processes. Three subfamilies (MaPmt1, MaPmt2 and MaPmt4) are grouped in Metarhizium acridum according to sequence homology. The functions of MaPmt1 and MaPmt4 have been characterized in M. acridum previously. In this study, the functions of another member belonging to the Pmt2 subfamily, MaPmt2, were identified through RNAi strategy. The three MaPmt2 knockdown mutants showed dramatically decreased expression of MaPmt2. Phenotypic analyses showed that the mutants exhibited decreased tolerances to wet-heat, UV-B irradiation and cell wall perturbing chemicals. Further studies revealed that the mutants presented thinner cell walls observed by transmission electron microscope combined with changed cell wall components. Besides, knockdown of MaPmt2 decelerated conidial germination and decreased conidial yield. Compared with the wild-type strain, the MaPmt2 knockdown mutants caused impaired virulence only by topical inoculation. Results illustrated that the decreased virulence by inoculation could result from the delayed conidial germination on locust wings, reduced appressorium formation, as well as reduced turgor pressure in MaPmt2 knockdown mutants.

Effect of Osmotic Stress on the Growth, Development and Pathogenicity of Setosphaeria turcica.

Osmotic stress is a severe condition frequently encountered by microorganisms; however, there is limited knowledge on the influence of hyperosmotic stress on the growth, development and pathogenicity of phytopathogenic fungi. Here, three osmotic conditions (0.4 M NaCl, 0.4 M KCl, and 0.6 M sorbitol supplemented in potato dextrose agar medium) were used to identify the effect of osmotic stress on the growth, development and pathogenicity of Setosphaeria turcica which is a plant pathogenic fungus and causes northern corn leaf blight disease in maize, sorghum, and related grasses. In osmotic stress, the growth rate of mycelium was decreased, and the number of vesicular structures and flocculent secretion outside the hypha cell wall were significantly increased. The qRT-PCR results showed that the osmotic stress quickly activated the HOG-MAPK pathway, up-regulated the expression of the downstream genes, and these genes were most highly expressed within 30 min of exposure to osmotic stress. Furthermore, the germination rate and the yield of conidia were significantly higher under osmotic stress than in the control. A pathogenicity analysis confirmed that pathogenicity of the conidia which were cultured under osmotic stress was significantly enhanced. By analyzing the knock-out mutants of an osmotic stress responsed gene StFPS1, an aquaglyceroporin downstream of the HOG-MAPK pathway, we found that StFPS1 was involved in the formation of appressorium and penetration peg, which affected the penetration ability of S. turcica. In summary, our work explained the correlation between osmotic stress and growth, development, and pathogenicity in S. turcica.

Regulation of conidiation, polarity growth, and pathogenicity by MrSte12 transcription factor in entomopathogenic fungus, Metarhizium rileyi

Metarhizium rileyi, a well-known filamentous biocontrol fungus, is the main pathogen of numerous field pests, especially noctuid pests. To explore the potential factors involved in the fungal pathogenicity, MrSte12, an important and conserved functional transcription factor in mitogen-activated protein kinase pathway was carried out by functional analysis. Homologous recombination was used to disrupt the MrSte12 gene in M. rileyi. The deletant fungal strain exhibited malformed hyphae and impaired conidiogenesis, and conidia could not be collected from △MrSte12 in vitro towards SMAY medium. Although conidia could be collected again supplemented with KCl within SMAY medium, the conidial germination, growth and stress tolerance were much weaker compared with that in WT. Additionally, △MrSte12 showed a dramatic reduction in virulence in intra-hemolymph injections and no pathogenicity in topical inoculations against noctuid pests, which is due to the failure of appressorium formation. Moreover, the content of chitin and β-1, 3-glucan in cell wall significantly reduced in mutant conidia. These results indicate that the MrSte12 gene markedly contributes to invasive growth and conidiation, as well as the major pathogenicity in M. rileyi.

MoCpa1-mediated arginine biosynthesis is crucial for fungal growth, conidiation, and plant infection of Magnaporthe oryzae.

Arginine is an important amino acid involved in processes such as cell signal transduction, protein synthesis, and sexual reproduction. To understand the biological roles of arginine biosynthesis in pathogenic fungi, we used Cpa1, the carbamoyl phosphate synthase arginine-specific small chain subunit in Saccharomyces cerevisiae as a query to identify its ortholog in the Magnaporthe oryzae genome and named it MoCpa1. MoCpa1 is a 471-amino acid protein containing a CPSase_sm_chain domain and a GATase domain. MoCpa1 transcripts were highly expressed at the conidiation, early-infection, and late-infection stages of the fungus. Targeted deletion of the MoCPA1 gene resulted in a ΔMocpa1 mutant exhibiting arginine auxotrophy on minimum culture medium (MM), confirming its role in de novo arginine biosynthesis. The ΔMocpa1 mutant presented significantly decreased sporulation with some of its conidia being defective in morphology. Furthermore, the ΔMocpa1 mutant was nonpathogenic on rice and barley leaves, which was a result of defects in appressorium-mediated penetration and restricted invasive hyphal growth within host cells. Addition of exogenous arginine partially rescued conidiation and pathogenicity defects on the barley and rice leaves, while introduction of the MoCPA1 gene into the ΔMocpa1 mutant fully complemented the lost phenotype. Further confocal microscopy examination revealed that MoCpa1 is localized in the mitochondria. In summary, our results demonstrate that MoCpa1-mediated arginine biosynthesis is crucial for fungal development, conidiation, appressorium formation, and infection-related morphogenesis in M. oryzae, thus serving as an attractive target for mitigating obstinate fungal plant pathogens. KEY POINTS: • MoCpa1 is important for aerial hyphal growth and arginine biosynthesis. • MoCpa1 is pivotal for conidial morphogenesis and appressorium formation. • MoCpa1 is crucial for full virulence in M. oryzae.

Genome-Wide Analysis of Nutrient Signaling Pathways Conserved in Arbuscular Mycorrhizal Fungi.

Arbuscular mycorrhizal (AM) fungi form a mutualistic symbiosis with a majority of terrestrial vascular plants. To achieve an efficient nutrient trade with their hosts, AM fungi sense external and internal nutrients, and integrate different hierarchic regulations to optimize nutrient acquisition and homeostasis during mycorrhization. However, the underlying molecular networks in AM fungi orchestrating the nutrient sensing and signaling remain elusive. Based on homology search, we here found that at least 72 gene components involved in four nutrient sensing and signaling pathways, including cAMP-dependent protein kinase A (cAMP-PKA), sucrose non-fermenting 1 (SNF1) protein kinase, target of rapamycin kinase (TOR) and phosphate (PHO) signaling cascades, are well conserved in AM fungi. Based on the knowledge known in model yeast and filamentous fungi, we outlined the possible gene networks functioning in AM fungi. These pathways may regulate the expression of downstream genes involved in nutrient transport, lipid metabolism, trehalase activity, stress resistance and autophagy. The RNA-seq analysis and qRT-PCR results of some core genes further indicate that these pathways may play important roles in spore germination, appressorium formation, arbuscule longevity and sporulation of AM fungi. We hope to inspire further studies on the roles of these candidate genes involved in these nutrient sensing and signaling pathways in AM fungi and AM symbiosis.

The Msn2 Transcription Factor Regulates Acaricidal Virulence in the Fungal Pathogen Beauveria bassiana.

Beauveria bassiana holds promise as a feasible biological control agent for tick control. The B. bassiana stress–response transcription factor Msn2 is known to contribute to fungal growth, conidiogenesis, stress–response and virulence towards insects; however, little is known concerning whether Msn2 is involved in infection across Arthropoda classes. We evaluated the effects of Msn2 on B. bassiana virulence against Rhipicephalus microplus (Acari, Ixodidae) using wild-type, targeted gene knockout (ΔBbmsn2) and complemented mutant (ΔBbmsn2/Bbmsn2) strains. Reproductive parameters of R. microplus engorged females treated topically or by an intra-hemocoel injection of conidial suspensions were assessed. Treated cuticles of engorged females were analyzed by microscopy, and proteolytic activity of B. bassiana on cuticles was assessed. Topically treated engorged females showed high mean larval hatching (>84%) in control and ΔBbmsn2 treatments, whereas treatment with the wild-type or ΔBbmsn2/Bbmsn2 strains resulted in significantly decreased (lowered egg viability) larval hatching. Percent control of R. microplus topically treated with ΔBbmsn2 was lower than in the groups treated with wild-type (56.1%) or ΔBbmsn2/Bbmsn2 strains. However, no differences on reproductive parameters were detected when R. microplus were treated by intra-hemocoel injection using low (800 conidia/tick) doses for all strains tested; R. microplus injected with high doses of wild-type or mutant strains (106 conidia/tick) died before laying eggs (~48 h after treatment). SEM analyses of B. bassiana infection showed similar conidial germination and formation of pseudo-appressoria on tick cuticle. Histological sections of ticks treated with the wild-type or ΔBbmsn2/Bbmsn2 strains showed fungal penetration through the cuticle, and into the tick interior. Hyphae of ΔBbmsn2, however, did not appear to penetrate or breach the tick exocuticle 120 h after treatment. Protease activity was lower on tick cuticles treated with ΔBbmsn2 than those treated with the wild-type or ΔBbmsn2/Bbmsn2 strains. These data show that loss of the Msn2 transcription factor reduced B. bassiana virulence against R. microplus, but did not interfere with conidial germination, appressoria formation or sporulation on tick cadavers, and plays only a minimal role once the cuticle is breached. Our results indicate that the BbMsn2 transcription factor acts mainly during the fungal penetration process and that decreased protease production may be one mechanism that contributes to the inability of the mutant strain to breach the tick cuticle.

Alternative Splicing of MoPTEN Is Important for Growth and Pathogenesis in Magnaporthe oryzae.

Human PTEN, a dual-phosphatase tumor suppressor, is frequently dysregulated by alternative splicing. Fungi harbor PTEN homologs, but alternative splicing of fungal PTENs has not been reported as far as we know. Here, we described an alternative splicing case in the PTEN homolog of Magnaporthe oryzae (MoPTEN). Two splice variants of MoPTEN were detected and identified, which are resulted from an intron retention and exclusion (MoPTEN-1/2). Both proteins were different in lipid and protein phosphatase activity and in expression patterns. The MoPTEN deletion mutant (ΔMoPTEN) showed the defects in conidiation, appressorium formation, and pathogenesis. ΔMoPTEN could be completely restored by MoPTEN, but rescued partially by MoPTEN-1 in the defect of conidium and appressorium formation, and by MoPTEN-2 in the defect of invasive development. Assays to assess sensitivity to oxidative stress reveal the involvement of MoPTEN-2 in scavenging exogenous and host-derived H2O2. Taken together, MoPTEN undergoes alternative splicing, and both variants cooperatively contribute to conidium and appressorium development, and invasive hyphae growth in plant cells, revealing a novel disease development pathway in M. oryzae.

A slicing mechanism facilitates host entry by plant-pathogenic Phytophthora.

Phytophthora species, classified as oomycetes, are among the most destructive plant pathogens worldwide and pose a substantial threat to food security. Plant pathogens have developed various methods to breach the cuticle and walls of plant cells. For example, plant-pathogenic fungi use a 'brute-force' approach by producing a specialized and fortified invasion organ to generate invasive pressures. Unlike in fungi, the biomechanics of host invasion in oomycetes remains poorly understood. Here, using a combination of surface-deformation imaging, molecular-fracture sensors and modelling, we find that Phytophthora infestans, Phytophthora palmivora and Phytophthora capsici slice through the plant surface to gain entry into host tissues. To distinguish this mode of entry from the brute-force approach of fungi that use appressoria, we name this oomycete entry without appressorium formation 'naifu' invasion. Naifu invasion relies on polarized, non-concentric, force generation onto the surface at an oblique angle, which concentrates stresses at the site of invasion to enable surface breaching. Measurements of surface deformations during invasion of artificial substrates reveal a polarized mechanical geometry that we describe using a mathematical model. We confirm that the same mode of entry is used on real hosts. Naifu invasion uses actin-mediated polarity, surface adherence and turgor generation to enable Phytophthora to invade hosts without requiring specialized organs or vast turgor generation.

MaAreB, a GATA Transcription Factor, Is Involved in Nitrogen Source Utilization, Stress Tolerances and Virulence in Metarhizium acridum.

The nitrogen catabolite repression (NCR) pathway is involved in nitrogen utilization, in which the global GATA transcription factor AreA plays an indispensable role and has been reported in many fungi. However, relatively few studies are focused on AreB, another GATA transcription factor in the NCR pathway and the functions of AreB are largely unknown in entomopathogenic fungi. Here, we characterized MaAreB in the model entomopathogenic fungus Metarhizium acridum. Sequence arrangement found that MaAreB had a conserved GATA zinc finger DNA binding domain and a leucine zipper domain. Disruption of MaAreB affected the nitrogen utilization and led to decelerated conidial germination and hyphal growth, decreased conidial yield, and lower tolerances to UV-B irradiation and heat-shock. Furthermore, the MaAreB mutant (ΔMaAreB) exhibited increased sensitivity to CFW (Calcofluor white), decreased cell wall contents (chitin and β-1,3-glucan) and reduced expression levels of some genes related to cell wall integrity, indicating that disruption of MaAreB affected the cell wall integrity. Bioassays showed that the virulence of the ΔMaAreB strain was decreased in topical inoculation but not in intra-hemocoel injection. Consistently, deletion of MaAreB severely impaired the appressorium formation and reduced the turgor pressure of appressorium. These results revealed that MaAreB regulated fungal nitrogen utilization, cell wall integrity and biological control potential, which would contribute to the functional characterization of AreB homologous proteins in other insect fungal pathogens, and even filamentous fungi.

The molecular mechanism underlying pathogenicity inhibition by sanguinarine in Magnaporthe oryzae.

Sanguinarine (SAN) is a benzophenanthridine alkaloid that broadly targets a range of pathways in mammalian and fungal cells. In this study we set out to explore the molecular mechanism of sanguinarine inhibition of the fungal development and pathogenicity of Magnaporthe oryzae with the hope that sanguinarine will bolster the development of antiblast agents. We found that the fungus exhibited a significant reduction in vegetative growth and hyphal melanization while the spores produced long germ tubes on the artificial hydrophobic surface characteristic of a defect in thigmotropic sensing when exposed to 4, 8 and 0.5μm sanguinarine, respectively. Consistent with these findings, we observed that the genes involved in melanin biosynthesis and the fungal hydrophobin MoMPG1 were remarkably suppressed in mycelia treated with 8μm sanguinarine. Additionally, sanguinarine inhibited appressorium formation at a dose of 1.0μm and this defect was restored by supplementing 5 mM of exogenous cAMP. By qRT-PCR assay we found cAMP pathway signalling genes such as MoCAP1 and MoCpkA were significantly repressed whereas MoCDTF1 and MoSOM1 were upregulated in sanguinarine-treated strains. Furthermore, we showed that sanguinarine does not selectively inhibit vegetative growth and appressorium formation of Guy11 but also other strains of M. oryzae. Finally, treatment of sanguinarine impaired the appressorium-mediated penetration and pathogenicity of M. oryzae in a dose-dependent manner. Based on our results we concluded that sanguinarine is an attractive antimicrobial candidate for fungicide development in the control of rice blast disease. © 2021 Society of Chemical Industry.

The rice blast fungus MoRgs1 functioning in cAMP signaling and pathogenicity is regulated by casein kinase MoCk2 phosphorylation and modulated by membrane protein MoEmc2.

GTP-binding protein (G-protein) and regulator of G-protein signaling (RGS) mediated signal transduction are critical in the growth and virulence of the rice blast pathogen Magnaporthe oryzae. We have previously reported that there are eight RGS and RGS-like proteins named MoRgs1 to MoRgs8 playing distinct and shared regulatory functions in M. oryzae and that MoRgs1 has a more prominent role compared to others in the fungus. To further explore the unique regulatory mechanism of MoRgs1, we screened a M. oryzae cDNA library for genes encoding MoRgs1-interacting proteins and identified MoCkb2, one of the two regulatory subunits of the casein kinase (CK) 2 MoCk2. We found that MoCkb2 and the sole catalytic subunit MoCka1 are required for the phosphorylation of MoRgs1 at the plasma membrane (PM) and late endosome (LE). We further found that an endoplasmic reticulum (ER) membrane protein complex (EMC) subunit, MoEmc2, modulates the phosphorylation of MoRgs1 by MoCk2. Interestingly, this phosphorylation is also essential for the GTPase-activating protein (GAP) function of MoRgs1. The balance among MoRgs1, MoCk2, and MoEmc2 ensures normal operation of the G-protein MoMagA-cAMP signaling required for appressorium formation and pathogenicity of the fungus. This has been the first report that an EMC subunit is directly linked to G-protein signaling through modulation of an RGS-casein kinase interaction.

MoSep3 and MoExo70 are needed for MoCK2 ring assembly essential for appressorium function in the rice blast fungus, Magnaporthe oryzae.

Polar growth during appressorium formation is vital for the penetration peg formation in the rice blast fungus, Magnaporthe oryzae. Previous research has shown that the Sln1‐septin‐exocyst complex, localized at the base of the appressorium in contact with the leaf surface, forms a ring structure that influences growth polarity and affects penetration peg formation, and is necessary for pathogenicity. Our previous research showed CK2 proteins assemble another ring structure positioned perpendicular to the Sln1‐septin‐exocyst complex. Our research showed that the CK2 ring needs to become correctly assembled for penetration peg function and subsequent plant infection. In the present study, we found that the ring structures of CK2 are absent in the appressorium of ΔMoSep3 septin deletion mutants lacking the septin ring of the Sln1‐septin‐exocyst complex. Sln1 affects the septin proteins that recruit the exocyst complex that localizes as another ring at the appressorium's bottom. Destruction of the exocyst complex by mutation also causes incorrect localization of the CK2 ring structure. In conclusion, CK2 probably takes part in reestablishing the appressorium' spolarity growth necessary for penetration peg formation. We can also conclude that the correct localization and assembly of one or more CK2 ring structures in the appressorium depend on the initial assembly of the Sln1‐septin‐exocyst complex two rings at the base of the appressorium.