Appressorium-mediated Penetration Research Articles

Transcription Factor CgSte12 Regulates Pathogenicity by Affecting Appressorium Structural Development in the Anthracnose-Causing Fungus Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides is the causal agent of poplar anthracnose, which induces major economic losses and adversely affects the ecosystem services of poplar forests. The appressorium serves as a penetration structure for many pathogenic fungi, including C. gloeosporioides. The production of mucilage and the formation of penetration pegs are critically important for the appressorium-mediated penetration of host tissues. We previously found that CgPmk1 is a key protein involved in appressorium formation, penetration, and pathogenicity. Although CgSte12, which is a transcription factor that functions downstream of CgPmk1, regulates the formation of penetration pegs, its role in C. gloeosporioides appressorium development and pathogenicity has not been elucidated. Here, we developed C. gloeosporioides CgSTE12 mutants and characterized the molecular and cellular functions of CgSTE12. The results showed that mycelial growth and morphology were not affected in the CgSTE12 knockout mutants, which produced normal melanized appressoria. However, these mutants had less mucilage secreted around the appressoria, impaired appressorial cone formation, and the inability to form penetration pores and pegs, which ultimately led to a significant loss of pathogenicity. Our comparative transcriptome analysis revealed that CgSte12 controls the expression of genes involved in appressorium development and function, including genes encoding cutinases, NADPH oxidase, spermine biosynthesis-related proteins, ceramide biosynthesis-related proteins, fatty acid metabolism-related proteins, and glycerophospholipid metabolism-related proteins. Overall, our findings indicate that CgSte12 is a critical regulator of appressorium development and affects C. gloeosporioides pathogenicity by modulating the structural integrity of appressoria.

The bZIP transcription factor BIP1 of the rice blast fungus is essential for infection and regulates a specific set of appressorium genes.

The rice blast fungus Magnaporthe oryzae differentiates specialized cells called appressoria that are required for fungal penetration into host leaves. In this study, we identified the novel basic leucine zipper (bZIP) transcription factor BIP1 (B-ZIP Involved in Pathogenesis-1) that is essential for pathogenicity. BIP1 is required for the infection of plant leaves, even if they are wounded, but not for appressorium-mediated penetration of artificial cellophane membranes. This phenotype suggests that BIP1 is not implicated in the differentiation of the penetration peg but is necessary for the initial establishment of the fungus within plant cells. BIP1 expression was restricted to the appressorium by both transcriptional and post-transcriptional control. Genome-wide transcriptome analysis showed that 40 genes were down regulated in a BIP1 deletion mutant. Most of these genes were specifically expressed in the appressorium. They encode proteins with pathogenesis-related functions such as enzymes involved in secondary metabolism including those encoded by the ACE1 gene cluster, small secreted proteins such as SLP2, BAS2, BAS3, and AVR-Pi9 effectors, as well as plant cuticle and cell wall degrading enzymes. Interestingly, this BIP1 network is different from other known infection-related regulatory networks, highlighting the complexity of gene expression control during plant-fungal interactions. Promoters of BIP1-regulated genes shared a GCN4/bZIP-binding DNA motif (TGACTC) binding in vitro to BIP1. Mutation of this motif in the promoter of MGG_08381.7 from the ACE1 gene cluster abolished its appressorium-specific expression, showing that BIP1 behaves as a transcriptional activator. In summary, our findings demonstrate that BIP1 is critical for the expression of early invasion-related genes in appressoria. These genes are likely needed for biotrophic invasion of the first infected host cell, but not for the penetration process itself. Through these mechanisms, the blast fungus strategically anticipates the host plant environment and responses during appressorium-mediated penetration.

Phosphocholine cytidylyltransferase MoPct1 is crucial for vegetative growth, conidiation, and appressorium-mediated plant infection by Magnaporthe oryzae.

Phosphatidylcholine (PC) plays crucial biological roles in eukaryotic cells. In Saccharomyces cerevisiae, apart from phosphatidylethanolamine (PE) methylation pathway, PC is also synthesized via CDP-choline pathway. Phosphocholine cytidylyltransferase Pct1 is the rate-limiting enzyme to catalyze the conversion from phosphocholine to CDP-choline in this pathway. Here, we report the identification and functional characterization of an ortholog of the budding yeast PCT1 in Magnaporthe oryzae, named MoPCT1. Targeted gene deletion mutants of MoPCT1 were impaired in vegetative growth, conidiation, appressorium turgor accumulation and cell wall integrity. Also, the mutants were severely compromised in appressorium-mediated penetration, infectious growth and pathogenicity. Western blot analysis revealed that cell autophagy was activated by the deletion of MoPCT1 under nutrient-rich conditions. Moreover, we found several key genes in PE methylation pathway, such as MoCHO2, MoOPI3, and MoPSD2, were significantly up-regulated in the ΔMopct1 mutants, indicating that a pronounced compensation effect exists between the two PC biosynthesis pathways in M. oryzae. Interestingly, in the ΔMopct1 mutants, histone H3 was hypermethylated and expression levels of several methionine cycling-related genes were significantly up-regulated, suggesting that MoPCT1 is involved in histone H3 methylation and methionine metabolism. Taken together, we conclude that the phosphocholine cytidylyltransferase coding gene MoPCT1 plays important roles in vegetative growth, conidiation and appressorium-mediated plant infection by M. oryzae.

NADPH Oxidases Are Required for Appressorium-Mediated Penetration in Colletotrichum scovillei-Pepper Fruit Pathosystem.

NADPH oxidase (Nox) complexes are known to play essential roles in differentiation and proliferation of many filamentous fungi. However, the functions of Noxs have not been elucidated in Colletotrichum species. Therefore, we set out to characterize the roles of Nox enzymes and their regulators in Colletotrichum scovillei, which causes serious anthracnose disease on pepper fruits in temperate and subtropical and temperate region. In this study, we generated targeted deletion mutants for CsNox1, CsNox2, CsNoxR, and CsNoxD via homologous recombination. All deletion mutants were normal in mycelial growth, conidiation, conidial germination, and appressorium formation, suggesting that CsNox1, CsNox2, CsNoxR, and CsNoxD are not involved in those developmental processes. Notably, conidia of ΔCsnox2 and ΔCsnoxr, other than ΔCsnox1 and ΔCsnoxd, failed to cause anthracnose on intact pepper fruits. However, they still caused normal disease on wounded pepper fruits, suggesting that Csnox2 and CsnoxR are essential for penetration-related morphogenesis in C. scovillei. Further observation proved that ΔCsnox2 and ΔCsnoxr were unable to form penetration peg, while they fully developed appressoria, revealing that defect of anthracnose development by ΔCsnox2 and ΔCsnoxr resulted from failure in penetration peg formation. Our results suggest that CsNox2 and CsNoxR are critical for appressorium-mediated penetration in C. scovillei-pepper fruit pathosystem, which provides insight into understanding roles of Nox genes in anthracnose disease development.

Distinct Transcriptional Programs Underlie Differences in Virulence of Isolates on Host Plants in a Fungal Pathogen, Colletotrichum gloeosporioides.

Susceptible host plants challenged by fungal pathogens can display different types of lesions, which can be attributed to environmental factors affecting the nature of interactions between the host and pathogen. During our survey of apple anthracnose in Korea, two distinct types of disease symptoms, designated as progressive (PS) and static symptoms (SS), were recognized. PS is a typical, rapidly enlarging symptom of apple anthracnose, while SS is a small, dark speck that does not expand further until the harvesting season. Isolation and genotyping of pathogens from disease lesions suggested that all of them belong to Colletotrichum gloeosporioides, a well-known causal agent of apple anthracnose. Two types of isolates were comparable in growth on media, spore germination and appressorium formation, virulence test on fruits at various temperature conditions. Furthermore, they were analyzed at the molecular level by a phylogenetic tree, RNA-seq, and expression of virulence gene. However, the SS isolates were defective in appressorium-mediated penetration into the underlying substratum. RNA-seq analysis of PS and SS isolates showed that distinct transcriptional programs underlie the development of different types of anthracnose symptoms in host plants. One downregulated gene in SS encoded isocitrate lyase is essential for disease development via its involvement in the glyoxylate cycle. It partly explains why SS is less virulent than PS on host plants. Overall, our work challenges the traditional view on the development of different lesion types and provides valuable insights into variations that exist in the pathogen population.

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.

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.

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.

Transcription Factor CfSte12 of Colletotrichum fructicola Is a Key Regulator of Early Apple Glomerella Leaf Spot Pathogenesis.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola, is a rapidly emerging disease leading to defoliation, fruit spot, and storage fruit rot on apple in China. Little is known about the mechanisms of GLS pathogenesis. Early transcriptome analysis revealed that expression of the zinc finger transcription factor Ste12 gene in C. fructicola (CfSte12) was upregulated in appressoria and leaf infection. To investigate functions of CfSte12 during pathogenesis, we constructed gene deletion mutants (ΔCfSte12) by homologous recombination. Phenotypic analysis revealed that CfSte12 was involved in pathogenesis of nonwounded apple fruit and leaf, as well as wounded apple fruit. Subsequent histological studies revealed that loss of pathogenicity by ΔCfSte12 on apple leaf was expressed as defects of conidium germination, appressorium development, and appressorium-mediated penetration. Further RNA sequencing-based transcriptome comparison revealed that CfSte12 modulates the expression of genes related to appressorium function (e.g., genes for the tetraspanin PLS1, Gas1-like proteins, cutinases, and melanin biosynthesis) and candidate effectors likely involved in plant interaction. In sum, our results demonstrated that CfSte12 is a key regulator of early apple GLS pathogenesis in C. fructicola In addition, CfSte12 is also needed for sexual development of perithecia and ascospores.IMPORTANCE Glomerella leaf spot (GLS) is an emerging fungal disease of apple that causes huge economic losses in Asia, North America, and South America. The damage inflicted by GLS manifests in rapid necrosis of leaves, severe defoliation, and necrotic spot on the fruit surface. However, few studies have addressed mechanisms of GLS pathogenesis. In this study, we identified and characterized a key pathogenicity-related transcription factor, CfSte12, of Colletotrichum fructicola that contributes to GLS pathogenesis. We provide evidence that the CfSte12 protein regulates many important pathogenic processes of GLS, including conidium germination, appressorium formation, appressorium-mediated penetration, and colonization. CfSte12 also impacts development of structures needed for sexual reproduction which are vital for the GLS disease cycle. These results reveal a key pathogenicity-related transcription factor, CfSte12, in C. fructicola that causes GLS.

A Histone Deacetylase, MoHDA1 Regulates Asexual Development and Virulence in the Rice Blast Fungus.

Interplay between histone acetylation and deacetylation is one of the key components in epigenetic regulation of transcription. Here we report the requirement of Mo-HDA1-mediated histone deacetylation during asexual development and pathogenesis for the rice blast fungus, Magnaporthe oryzae. Structural similarity and phylogenetic analysis suggested that MoHDA1 is an ortholog of Saccharomyces cerevisiae Hda1, which is a representative member of class II histone deacetylases. Targeted deletion of MoHDA1 caused a little decrease in radial growth and large reduction in asexual sporulation. Comparison of acetylation levels for H3K9 and H3K14 showed that lack of MoHDA1 gene led to significant increase in H3K9 and H3K14 acetylation level, compared to the wild-type and complementation strain, confirming that it is a bona fide histone deacetylase. Expression analysis on some of the key genes involved in asexual reproduction under sporulation-promoting condition showed almost no differences among strains, except for MoCON6 gene, which was up-regulated more than 6-fold in the mutant than wild-type. Although the deletion mutant displayed little defects in germination and subsequent appressorium formation, the mutant was compromised in its ability to cause disease. Woundinoculation showed that the mutant is impaired in invasive growth as well. We found that the mutant was defective in appressorium-mediated penetration of host, but did not lose the ability to grow on the media containing H2O2. Taken together, our data suggest that MoHDA1-dependent histone deacetylation is important for efficient asexual development and infection of host plants in M. oryzae.

Biomimetic Mineralization-Based CRISPR/Cas9 Ribonucleoprotein Nanoparticles for Gene Editing

Delivery of the CRISPR/Cas9 ribonucleoprotein (RNP) complex has provided an alternative strategy for the regulation of CRISPR functions, offering a transient and DNA-free means for genomic editing. Chemical methods of delivering the RNPs via nanocomplexes have the potential to address these delivery problems for efficiency, safety, and packaging capacity. Here, we developed a biomimetic mineralization-mediated strategy for efficient DNA-free genome editing by using CRISPR/Cas9 RNPs. We found that the RNPs have the ability to form the biomimetic mineralized RNP nanoparticles (Bm-RNP NPs) quickly in situ and can be effectively delivered into the fungal protoplast cells. Biomimetic mineralization can maintain the natural function of Cas9 protein and protect the sgRNA activity. At the same time, the encapsulated RNPs can be effectively released into the cytoplasm, and the Sytalone dehydratase (SDH) gene can be edited in a targeted manner. Except for phenotypic defects, molecular detections indicated that the delivery of Bm-RNP NPs achieved 20% genomic editing for Magnaporthe oryzae compared to RNPs alone. Moreover, the Bm-RNP NP-mediated editing of the SDH gene significantly affects the appressorium-mediated penetration and invasive growth in M. oryzae. Our system has the advantages of being cheap, fast, and effective, without the traditional transformation process, suggesting the potential application of this DNA-free gene-editing strategy in different organisms.

MoGT2 Is Essential for Morphogenesis and Pathogenicity of Magnaporthe oryzae.

Magnaporthe oryzae causes the rice blast disease, which is one of the most serious diseases of cultivated rice worldwide. Glycosylation is an important posttranslational modification of secretory and membrane proteins in all eukaryotes, catalyzed by glycosyltransferases (GTs). In this study, we identified and characterized a type 2 glycosyltransferase, MoGt2, in M. oryzae Targeted gene deletion mutants of MoGT2 (mogt2Δ strains) were nonpathogenic and were impaired in vegetative growth, conidiation, and appressorium formation at hyphal tips. Moreover, MoGT2 plays an important role in stress tolerance and hydrophobin function of M. oryzae Site-directed mutagenesis analysis showed that conserved glycosyltransferase domains (DxD and QxxRW) are critical for biological functions of MoGt2. MoGT2 deletion led to altered glycoproteins during M. oryzae conidiation. By liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified several candidate proteins as potential substrates of MoGt2, including several heat shock proteins, two coiled-coil domain-containing proteins, aminopeptidase 2, and nuclease domain-containing protein 1. On the other hand, we found that a conidiation-related gene, genes involved in various metabolism pathways, and genes involved in cell wall integrity and/or osmotic response were differentially regulated in the mogt2Δ mutant, which may potentially contribute to its condiation defects. Taken together, our results show that MoGt2 is important for infection-related morphogenesis and pathogenesis in M. oryzaeIMPORTANCE The ascomycete fungus Magnapothe oryzae is the causal agent of rice blast disease, leading to severe loss in cultivated rice production worldwide. In this study, we identified a conserved type 2 glycosyltransferase named MoGt2 in M. oryzae The mogt2Δ targeted gene deletion mutants exhibited pleiotropic defects in vegetative growth, conidiation, stress response, hyphal appressorium-mediated penetration, and pathogenicity. Furthermore, conserved glycosyltransferase domains are critical for MoGt2 function. The comparative transcriptome analysis revealed potential target genes under MoGt2 regulation in M. oryzae conidiation. Identification of potential glycoproteins modified by MoGt2 provided information on its regulatory mechanism of gene expression and biological functions. Overall, our study represents the first report of type 2 glycosyltransferase function in M. oryzae infection-related morphogenesis and pathogenesis.

Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.

Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity.

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection. Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers invitro with high affinity and specificity and that both proteins suppress the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases in the host Arabidopsis. Using RNAi-mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium-mediated penetration of both Arabidopsis epidermal cells and cellophane membranes invitro. The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin-triggered immunity and as proteins required for appressorium function.

Peltaster fructicola genome reveals evolution from an invasive phytopathogen to an ectophytic parasite.

Sooty blotch and flyspeck (SBFS) fungi are unconventional plant pathogens that cause economic losses by blemishing the surface appearance of infected fruit. Here, we introduce the 18.14-Mb genome of Peltaster fructicola, one of the most prevalent SBFS species on apple. This undersized assembly contains only 8,334 predicted protein-coding genes and a very small repertoire of repetitive elements. Phylogenomics and comparative genomics revealed that P. fructicola had undergone a reductive evolution, during which the numbers of orphan genes and genes involved in plant cell wall degradation, secondary metabolism, and secreted peptidases and effectors were drastically reduced. In contrast, the genes controlling 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis and appressorium-mediated penetration were retained substantially. Additionally, microscopic examination of the surfaces of infected apple indicated for the first time that P. fructicola can not only dissolve epicuticular waxes but also partially penetrate the cuticle proper. Our findings indicate that genome contraction, characterized mainly by the massive loss of pathogenicity-related genes, has played an important role in the evolution of P. fructicola (and by implication other SBFS species) from a plant-penetrating ancestor to a non-invasive ectophyte, displaying a novel form of trophic interaction between plants and fungi.

MoARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae

Arginine is one of the most versatile amino acids in eukaryote cells, which plays important roles in a multitude of processes such as protein synthesis, nitrogen metabolism, nitric oxide (NO) and urea biosynthesis. The de novo arginine biosynthesis pathway is conserved among fungal kingdom, but poorly understood in plant pathogenic fungi. Here, we characterized the functions of three synthetic enzyme-encoding genes MoARG1, MoARG5,6, and MoARG7, which involved the seventh step, second–third step and fifth step of arginine biosynthesis in Magnaporthe oryzae, respectively. Deletion of MoARG1 or MoARG5,6, resulted in arginine auxotrophic mutants, which had a strict requirement for arginine on minimal medium (MM). Both ΔMoarg1 and ΔMoarg5,6 severely reduced in aerial hyphal growth, pigmentation, conidiogenesis, sexual reproduction and pathogenicity. Interestingly, like Saccharomyces cerevisiae, deletion of MoARG7 caused a leaky arginine auxotrophy, and attenuated pathogenicity. Limited appressorium-mediated penetration and restricted invasive hyphae growth in host cells are responsible for the severely attenuated pathogenicity of the Arg− mutants. Additionally, we monitored the NO generation during conidial germination and appressorial formation in both Arg− mutants and wild type, and demonstrated that NO generation may not occur via arginine-dependent pathway in M. oryzae. In summary, MoARG1, MoARG5,6, and MoARG7 are required for growth, conidiogenesis, sexual reproduction, and pathogenicity in M. oryzae.

Appressorium-mediated penetration of Magnaporthe oryzae and Colletotrichum orbiculare into surface-cross-linked agar media

Many phytopathogenic fungi form appressoria on some artificial substances. However, it is difficult to induce appressorium-mediated penetration into artificial substances. In the present study, novel artificial agar media were developed to investigate the in vitro penetration process of phytopathogenic fungi. The media contained sodium carboxymethyl cellulose or sodium alginate, and the surfaces were subjected to ionic cross-linking using trivalent metal ions. The hemibiotrophic phytopathogenic fungi, rice blast fungus and cucurbit anthracnose fungus, formed appressoria and penetrated into the surface cross-linked artificial agar media from the base of appressoria. These artificial media appeared to induce fungal infection behaviour that occurred on host plants.

Methionine biosynthesis is essential for infection in the rice blast fungus Magnaporthe oryzae.

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.

Appressorium formation in the corn smut fungus Ustilago maydis requires a G2 cell cycle arrest

Many of the most important plant diseases are caused by fungal pathogens that form specialized cell structures to breach the leaf surface as well as to proliferate inside the plant. To initiate pathogenic development, the fungus responds to a set of inductive cues. Some of them are of extracellular nature (environmental signals) while others respond to intracellular conditions (developmental signals). These signals have to be integrated into a single response that has as a major outcome changes in the morphogenesis of the fungus. The cell cycle regulation is pivotal during these cellular differentiations, and we hypothesized that cell cycle regulation would be likely to provide control points for infection development by fungal pathogens. Although efforts have been done in various fungal systems, there is still limited information available regarding the relationship of these processes with the induction of the virulence programs. Hence, the role of fungal cell cycle regulators –which are wide conserved elements– as true virulence factors, has yet to be defined. Here we discuss the recent finding that the formation of the appressorium, a structure required for plant penetration, in the corn smut fungus Ustilago maydis seems to be incompatible with an active cell cycle and, therefore genetic circuits evolved in this fungus to arrest the cell cycle during the growth of this fungus on plant surface, before the appressorium-mediated penetration into the plant tissue.

Genome-wide analyses of DNA-binding proteins harboring AT-hook motifs and their functional roles in the rice blast pathogen, Magnaporthe oryzae

Transcriptional regulation is a complex process mediated by coordinated assembly complexes to ensure temporal and spatial gene expression. The AT-hook is a DNA-binding motif originally described in the high mobility group A of non-histone chromatin components. The AT-hook proteins bind to the minor groove of adenine–thymine (AT) rich regions of DNA and act as transcriptional cofactors coordinating nucleoproteins during transcriptional regulation. In this study, a genome-wide in silico analysis of AT-hook proteins was performed on the ascomycete plant pathogenic fungus, Magnaporthe oryzae. Quantitative real-time RT-PCR analysis revealed differential expression patterns of MoATH genes during development and plant infection. To increase our understanding of the functional role of AT-hook proteins in M. oryzae development and pathogenicity, a deletion mutant of MoATH10 was functionally characterized. Targeted deletion of MoATH10 significantly increased pigmentation and conidiation, indicating that MoATH10 is negatively involved in the regulation of pigmentation and conidiation in M. oryzae. Pathogenicity assays revealed that the ΔMoath10 mutant was less virulent. The reduced disease development of the ΔMoath10 mutant was due to a partial defect in invasive growth inside plant cells, but not appressorium-mediated penetration. These results suggest that MoATH10 is important for growth, development, and virulence in M. oryzae.