Intracellular polyamines regulate redox homeostasis with cAMP-PKA signalling during sexual mating/filamentation and pathogenicity of Sporisorium scitamineum.

Sugarcane smut caused by Sporisorium scitamineum seriously impairs sugarcane production and quality. Sexual mating/filamentation is a critical step of S. scitamineum pathogenesis, yet the regulatory mechanisms are not fully understood. In this study, we identified the SsAGA, SsODC, and SsSAMDC genes, which are involved in polyamine biosynthesis in S. scitamineum. Deletion of SsODC led to complete loss of filamentous growth after sexual mating, and deletion of SsAGA or SsSAMDC caused reduced filamentation. Double deletion of SsODC and SsSAMDC resulted in auxotrophy for putrescine (PUT) and spermidine (SPD) when grown on minimal medium (MM), indicating that these two genes encode enzymes that are critical for PUT and SPD biosynthesis. We further showed that low PUT concentrations promoted S. scitamineum filamentation, while high PUT concentrations suppressed filamentation. Disrupted fungal polyamine biosynthesis also resulted in a loss of pathogenicity and reduced fungal biomass within infected plants at the early infection stage. SPD formed a gradient from the diseased part to nonsymptom parts of the cane stem, suggesting that SPD is probably favourable for fungal virulence. Mutants of the cAMP-PKA (SsGPA3-SsUAC1-SsADR1) signalling pathway displayed up-regulation of the SsODC gene and elevated intracellular levels of PUT. SsODC directly interacted with SsGPA3, and sporidia of the ss1uac1ΔodcΔ mutant displayed abundant pseudohyphae. Furthermore, we found that elevated PUT levels caused accumulation of intracellular reactive oxygen species (ROS), probably by suppressing transcription of ROS-scavenging enzymes, while SPD played the opposite role. Overall, our work proves that polyamines play important roles in the pathogenic development of sugarcane smut fungus, probably by collaboratively regulating intracellular redox homeostasis with the cAMP-PKA signalling pathway.

Engineered Sucrose Metabolism Improves the Smut Disease Suppression Potency of Pseudomonas sp. ST4.

Sporisorium scitamineum and Ustilago maydis are two fungal pathogens causing severe sugarcane and maize diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We showed recently that in the presence of exogenous glucose, the Pseudomonas sp. strain ST4 could block the fungal mating and display a strong disease suppression potency on S. scitamineum. With the aim of conferring strain ST4 the ability to metabolize sucrose in plants for glucose production, we identified a strong native promoter pSsrA in strain ST4 and additional promoter elements to facilitate translation and peptide translocation for the construction of a fusion gene encoding sucrose metabolism. The cscA gene encoding sucrose hydrolase from Pseudomonas protegens Pf-5 was fused to the promoter pSsrA, a translational coupler bicistronic design and a Tat signal peptide, which was then cloned into mini-Tn7 transposon. This synthetic gene cassette was integrated into the chromosome of strain ST4, and the resultant engineered strain ST4E was able to hydrolyze sucrose with high efficiency and displayed elevated inhibitory activity on the mating and virulence of S. scitamineum and U. maydis. The findings from this study provide a valuable device and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens. IMPORTANCE Sporisorium scitamineum and Ustilago maydis are typical dimorphic fungi causing severe sugarcane and maize smut diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We previously demonstrated that the biocontrol strain Pseudomonas sp. ST4 could block the fungal mating and displays a strong suppression potency on smut diseases, while it was unable to utilize the host-sourced sucrose for glucose production critical for antifungus efficiency. In this study, we constructed a high-expression gene cassette for minitransposon-mediated genome integration and sucrose hydrolysis in the bacterial periplasmic space. The resultant engineered strain ST4E was able to hydrolyze sucrose and inhibit the mating and hyphal growth of S. scitamineum and U. maydis. These findings provide a valuable tool and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens.

Aminotransferase SsAro8 Regulates Tryptophan Metabolism Essential for Filamentous Growth of Sugarcane Smut Fungus Sporisorium scitamineum.

Sugarcane smut caused by the basidiomycetous fungus Sporisorium scitamineum leads to severe economic losses globally. Sexual mating/filamentation of S. scitamineum is critical for its pathogenicity, as only the dikaryotic hyphae formed after sexual mating are capable of invading the host cane. Our comparative transcriptome analysis showed that the mitogen-activated protein kinase (MAPK) pathway and the AGC kinase Agc1 (orthologous to yeast Rim15), both governing S. scitamineum mating/filamentation, were induced by elevated tryptophol level, supporting a positive regulation of S. scitamineum mating/filamentation by tryptophol. However, the biosynthesis pathway of tryptophol remains unknown in S. scitamineum. Here, we identified an aminotransferase orthologous to the established tryptophan aminotransferase Tam1/Aro8, catalyzing the first step of tryptophan-dependent indole-3-acetic acid (IAA) production as well as that of the Ehrlich pathway for tryptophol production. We designated this S. scitamineum aminotransferase as SsAro8 and found that it was essential for mating/filamentation. Comparative metabolomics analysis revealed that SsAro8 was involved in tryptophan metabolism, likely for producing important intermediate products, including tryptophol. Exogenous addition of tryptophan or tryptophol could differentially restore mating/filamentation in the ssaro8Δ mutant, indicating that in addition to tryptophol, other product(s) of tryptophan catabolism may also be involved in S. scitamineum mating/filamentation regulation. S. scitamineum could also produce IAA, partially dependent on SsAro8 function. Surprisingly, photodestruction of IAA produced the compound(s) able to suppress S. scitamineum growth/differentiation. Lastly, we found that SsAro8 was required for proper biofilm formation, oxidative stress tolerance, and full pathogenicity in S. scitamineum. Overall, our study establishes the aminotransferase SsAro8 as an essential regulator of S. scitamineum pathogenic differentiation, as well as fungus-host interaction, and therefore of great potential as a molecular target for sugarcane smut disease control. IMPORTANCE Sugarcane smut caused by the basidiomycete fungus S. scitamineum leads to massive economic losses in sugarcane plantation globally. Dikaryotic hyphae formation (filamentous growth) and biofilm formation are two important aspects in S. scitamineum pathogenesis, yet the molecular regulation of these two processes was not as extensively investigated as that in the model pathogenic fungi, e.g., Candida albicans, Ustilago maydis, or Cryptococcus neoformans. In this study, a tryptophan aminotransferase ortholog was identified in S. scitamineum, designated SsAro8. Functional characterization showed that SsAro8 positively regulates both filamentous growth and biofilm formation, respectively, via tryptophol-dependent and -independent manners. Furthermore, SsAro8 is required for full pathogenicity and, thus, is a promising molecular target for designing anti-smut strategy.

Identification and Characterization of Auxin/IAA Biosynthesis Pathway in the Rice Blast Fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae has been known to produce the phytohormone auxin/IAA from its hyphae and conidia, but the detailed biological function and biosynthesis pathway is largely unknown. By sequence homology, we identified a complete indole-3-pyruvic acid (IPA)-based IAA biosynthesis pathway in M. oryzae, consisting of the tryptophan aminotransferase (MoTam1) and the indole-3-pyruvate decarboxylase (MoIpd1). In comparison to the wild type, IAA production was significantly reduced in the motam1Δ mutant, and further reduced in the moipd1Δ mutant. Correspondingly, mycelial growth, conidiation, and pathogenicity were defective in the motam1Δ and the moipd1Δ mutants to various degrees. Targeted metabolomics analysis further confirmed the presence of a functional IPA pathway, catalyzed by MoIpd1, which contributes to IAA/auxin production in M. oryzae. Furthermore, the well-established IAA biosynthesis inhibitor, yucasin, suppressed mycelial growth, conidiation, and pathogenicity in M. oryzae. Overall, this study identified an IPA-dependent IAA synthesis pathway crucial for M. oryzae mycelial growth and pathogenic development.

Circadian Redox Rhythm in Plant-Fungal Pathogen Interactions.

Significance: Circadian-controlled cellular growth, differentiation, and metabolism are mainly achieved by a classical transcriptional-translational feedback loop (TTFL), as revealed by investigations in animals, plants, and fungi. Recent Advances: Recently, reactive oxygen species (ROS) have been reported as part of a cellular network synchronizing nontranscriptional oscillators with established TTFL components, adding complexity to regulatory mechanisms of circadian rhythm. Both circadian rhythm and ROS homeostasis have a great impact on plant immunity as well as fungal pathogenicity, therefore interconnections of these two factors are implicit in plant-fungus interactions. Critical Issues: In this review, we aim to summarize the recent advances in circadian-controlled ROS homeostasis, or ROS-modulated circadian clock, in plant-fungus pathosystems, particularly using the rice (Oryza sativa) blast fungus (Magnaporthe oryzae) pathosystem as an example. Understanding of such bidirectional interaction between the circadian timekeeping machinery and ROS homeostasis/signaling would provide a theoretical basis for developing disease control strategies for important plants/crops. Future Directions: Questions remain unanswered about the detailed mechanisms underlying circadian regulation of redox homeostasis in M. oryzae, and the consequent fungal differentiation and death in a time-of-day manner. We believe that the rice-M. oryzae pathobiosystem would provide an excellent platform for investigating such issues in circadian-ROS interconnections in a plant-fungus interaction context. Antioxid. Redox Signal. 37, 726-738.

Tangeretin inhibits fungal ferroptosis to suppress rice blast.

Flavonoids are polyphenolic secondary metabolites that function as signaling molecules, allopathic compounds, phytoalexins, detoxifying agents and antimicrobial defensive compounds in plants. Blast caused by the fungus Magnaporthe oryzae is a serious disease affecting rice cultivation. In this study, we revealed that a natural flavonoid, tangeretin, substantially delays the formation of M. oryzae appressoria and blocks the development of blast lesions on rice plants. Our data suggest that tangeretin has antioxidant activity that interferes with conidial cell death/ferroptosis, which is critical for M. oryzae pathogenicity. Tangeretin showed a ferroptosis inhibition efficacy comparable to the well-established liproxstatin-1. Furthermore, overexpression of the NADPH oxidases NOX1 or NOX2 significantly decreased sensitivity toward tangeretin treatment, suggesting Nox-mediated lipid peroxidation as a possible target for tangeretin in regulating redox signaling and ferroptosis in M. oryzae. Our nursery and field tests showed that application of tangeretin can effectively mitigate overall disease symptoms and prevent leaf blast. Our study reveals the plant-derived fungal ferroptosis inhibitor tangeretin as a potential and novel antifungal agrochemical for the sustainable prevention of the devastating blast disease in important cereal crops.

The Histone Deacetylases MoRpd3 and MoHst4 Regulate Growth, Conidiation, and Pathogenicity in the Rice Blast Fungus Magnaporthe oryzae.

As the causal agent of the blast disease, Magnaporthe oryzae is one of the most destructive fungal pathogens of rice. Histone acetylation/deacetylation is important for remodeling of chromatin superstructure and thus altering gene expression. In this study, two genes encoding histone deacetylases, namely, MoRPD3 and MoHST4, were identified and functionally characterized in M. oryzae. MoHst4 was required for proper mycelial growth and pathogenicity, whereas overproduction of MoRpd3 led to loss of pathogenicity, likely due to a block in conidial cell death and restricted invasive growth within the host plants. Green fluorescent protein (GFP)-MoRpd3 localized to the nucleus and cytoplasm in vegetative hyphae and developing conidia. By comparative transcriptomics analysis, we identified potential target genes epigenetically regulated by histone deacetylases (HDACs) containing MoRpd3 or MoHst4, which may contribute to conidia formation and/or conidial cell death, which is a prerequisite for successful appressorium-mediated host invasion. Taken together, our results suggest that histone deacetylases MoRpd3 and MoHst4 differentially regulate mycelial growth, asexual development, and pathogenesis in M. oryzae. IMPORTANCE HDACs (histone deacetylases) regulate various aspects of growth, development, and pathogenesis in plant-pathogenic fungi. Most members of HDAC classes I to III have been functionally characterized, except for orthologous Rpd3 and Hst4, in the rice blast fungus Magnaporthe oryzae. In this study, we assessed the function of MoRpd3 and MoHst4 by reverse genetics and found that they differentially regulate M. oryzae vegetative growth, asexual development, and infection. Particularly, MoRpd3 negatively regulates M. oryzae pathogenicity, likely through suppression of conidial cell death, which we recently reported as being critical for appressorium maturation and functioning. Overall, this study broadens our understanding of fungal pathobiology and its critical regulation by histone modification(s) during cell death and in planta differentiation.

Burkholderia gladioli CGB10: A Novel Strain Biocontrolling the Sugarcane Smut Disease.

In this study, we isolated an endophytic Burkholderia gladioli strain, named CGB10, from sugarcane leaves. B. gladioli CGB10 displayed strong inhibitory activity against filamentous growth of fungal pathogens, one of which is Sporisorium scitamineum that causes sugarcane smut, a major disease affecting the quality and production of sugarcane in tropical and subtropical regions. CGB10 could effectively suppress sugarcane smut under field conditions, without itself causing any obvious damage or disease, thus underscoring a great potential as a biocontrol agent (BCA) for the management of sugarcane smut. A toxoflavin biosynthesis and transport gene cluster potentially responsible for such antifungal activity was identified in the CGB10 genome. Additionally, a quorum-sensing gene cluster was identified too and compared with two close Burkholderia species, thus supporting an overall connection to the regulation of toxoflavin synthesis therein. Overall, this work describes the in vitro and field Sporisorium scitamineum biocontrol by a new B. gladioli strain, and reports genes and molecular mechanisms potentially involved.

Ferroptosis contributes to developmental cell death in rice blast.

Ferroptosis, an iron-dependent cell death process, was found to occur in Magnaporthe oryzae, and plays a key role in infection-related development therein. Ferroptosis in the rice-blast fungus was confirmed based on five basic criteria. We confirmed the dependence of ferroptosis on ferric ions, and optimized ratio-fluorescence imaging of C11-BODIPY581/591 as a precise sensor for lipid peroxides that mediate ferroptosis in M. oryzae. We uncovered an important regulatory function for reduced glutathione and NADPH oxidases in modulating the superoxide moieties required for ferroptotic cell death. We found ferroptosis to be necessary for the developmental cell death of conidia during appressorium maturation in rice blast. Such ferroptotic cell death initiated first in the terminal cell and progressed sequentially to the entire conidium. Iron chelation or chemical inhibition of ferroptosis caused conidial cells to remain viable, and led to strong defects in host invasion by M. oryzae. Ferroptosis induction exclusively in the host severely constrained the invasive growth of M. oryzae. We found inter-reliant and independent roles for ferroptosis and autophagy in controlling such precise cell death in M. oryzae during pathogenic differentiation. Our study provides significant molecular insights into the role of developmental cell death and iron homeostasis in fungal pathogenesis.

Karyopherin MoKap119-mediated nuclear import of cyclin-dependent kinase regulator MoCks1 is essential for Magnaporthe oryzae pathogenicity.

Nuclear import of proteins relies on nuclear import receptors called importins/karyopherins (Kaps), whose functions were reported in yeasts, fungi, plants, and animal cells, including cell cycle control, morphogenesis, stress sensing/response, and also fungal pathogenecity. However, limited is known about the physiological function and regulatory mechanism of protein import in the rice-blast fungus Magnaporthe oryzae. Here, we identified an ortholog of ß-importin in M. oryzae encoded by an ortholog of KAP119 gene. Functional characterisation of this gene via reverse genetics revealed that it is required for vegetative growth, conidiation, melanin pigmentation, and pathogenicity of M. oryzae. The mokap119Δ mutant was also defective in formation of appressorium-like structure from hyphal tips. By affinity assay and liquid chromatography-tandem mass spectrometry, we identified potential MoKap119-interacting proteins and further verified that MoKap119 interacts with the cyclin-dependent kinase subunit MoCks1 and mediates its nuclear import. Transcriptional profiling indicated that MoKap119 may regulate transcription of infection-related genes via MoCks1 regulation of MoSom1. Overall, our findings provide a novel insight into the regulatory mechanism of M. oryzae pathogenesis likely by MoKap119-mediated nuclear import of the cyclin-dependent kinase subunit MoCks1.

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.

Metabolic Basis of Pathogenesis and Host Adaptation in Rice Blast.

The blast disease, caused by the ascomycete Magnaporthe oryzae, poses a great threat to rice production worldwide. Increasing use of fungicides and/or blast-resistant varieties of rice (Oryza sativa) has proved to be ineffective in long-term control of blast disease under field conditions. To develop effective and durable resistance to blast, it is important to understand the cellular mechanisms underlying pathogenic development in M. oryzae. In this review, we summarize the latest research in phototropism, autophagy, nutrient and redox signaling, and intrinsic phytohormone mimics in M. oryzae for cellular and metabolic adaptation(s) during its interactions with the host plants.

The AGC Kinase SsAgc1 Regulates Sporisorium scitamineum Mating/Filamentation and Pathogenicity.

Sporisorium scitamineum is the fungal pathogen causing severe sugarcane smut disease that leads to massive economic losses globally. S. scitamineum invades host cane by dikaryotic hyphae, formed after sexual mating of two haploid sporidia of opposite mating type. Therefore, mating/filamentation is critical for S. scitamineum pathogenicity, while its molecular mechanisms remain largely unknown. The AGC (cyclic AMP [cAMP]-dependent protein kinase 1 [protein kinase A {PKA}], cGMP-dependent protein kinase [PKG], and protein kinase C [PKC]) kinase family is a group of serine/threonine (Ser/Thr) protein kinases conserved among eukaryotic genomes, serving a variety of physiological functions, including cell growth, metabolism, differentiation, and cell death. In this study, we identified an AGC kinase, named SsAgc1 (for S. scitamineum Agc1), and characterized its function by reverse genetics. Our results showed that SsAgc1 is critical for S. scitamineum mating/filamentation and pathogenicity, and oxidative stress tolerance under some circumstances. Transcriptional profiling revealed that the SsAgc1 signaling pathway may control expression of the genes governing fungal mating/filamentation and tryptophan metabolism, especially for tryptophol production. We showed that tryptophan and tryptophol could at least partially restore ssagc1Δ mating/filamentation. Overall, our work revealed a signaling pathway mediated by AGC protein kinases to regulate fungal mating/filamentation, possibly through sensing and responding to tryptophol as signal molecules.IMPORTANCE The AGC signaling pathway represents a conserved distinct signaling pathway in regulation of fungal differentiation and virulence, while it has not been identified or characterized in the sugarcane smut fungus Sporisorium scitamineum In this study, we identified a PAS domain-containing AGC kinase, SsAgc1, in S. scitamineum Functional analysis revealed that SsAgc1 plays a regulatory role on the fungal dimorphic switch.

cAMP/PKA signalling pathway regulates redox homeostasis essential for Sporisorium scitamineum mating/filamentation and virulence.

The fungal pathogen Sporisorium scitamineum causes sugarcane smut disease. The formation and growth of dikaryotic hypha after sexual mating is critical for S. scitamineum pathogenicity, however regulation of S. scitimineum mating has not been studied in detail. We identified and characterized the core components of the conserved cAMP/PKA pathway in S. scitamineum by reverse genetics. Our results showed that cAMP/PKA signalling pathway is essential for proper mating and filamentation, and thus critical for S. scitamineum virulence. We further demonstrated that an elevated intracellular ROS (reactive oxygen species) level promotes S. scitamineum mating-filamentation, via transcriptional regulation of ROS catabolic enzymes, and is under regulation of the cAMP/PKA signalling pathway. Furthermore, we found that fungal cAMP/PKA signalling pathway is also involved in regulation of host ROS response. Overall, our work displayed a positive role of elevated intracellular ROS in fungal differentiation and virulence.

Label-Free Quantitative Proteomics of Lysine Acetylome Identifies Substrates of Gcn5 in Magnaporthe oryzae Autophagy and Epigenetic Regulation.

The rice blast fungus Magnaporthe oryzae poses a great threat to global food security. During its conidiation (asexual spore formation) and appressorium (infecting structure) formation, autophagy is induced, serving glycogen breakdown or programmed cell death function, both essential for M. oryzae pathogenicity. Recently, we identified an M. oryzae histone acetyltransferase (HAT) Gcn5 as a key regulator in phototropic induction of autophagy and asexual spore formation while serving a cellular function other than autophagy induction during M. oryzae infection. To further understand the regulatory mechanism of Gcn5 on M. oryzae pathogenicity, we set out to identify more Gcn5 substrates by comparative acetylome between the wild-type (WT) and GCN5 overexpression (OX) mutant and between OX mutant and GCN5 deletion (knockout [KO]) mutant. Our results showed that Gcn5 regulates autophagy induction and other important aspects of fungal pathogenicity, including energy metabolism, stress response, cell toxicity and death, likely via both epigenetic regulation (histone acetylation) and posttranslational modification (nonhistone protein acetylation). IMPORTANCE Gcn5 is a histone acetyltransferase that was previously shown to regulate phototropic and starvation-induced autophagy in the rice blast fungus Magnaporthe oryzae, likely via modification on autophagy protein Atg7. In this study, we identified more potential substrates of Gcn5-mediated acetylation by quantitative and comparative acetylome analyses. By epifluorescence microscopy and biochemistry experiments, we verified that Gcn5 may regulate autophagy induction at both the epigenetic and posttranslational levels and regulate autophagic degradation of a critical metabolic enzyme pyruvate kinase (Pk) likely via acetylation. Overall, our findings reveal comprehensive posttranslational modification executed by Gcn5, in response to various external stimuli, to synergistically promote cellular differentiation in a fungal pathogen.

The MAP Kinase SsKpp2 Is Required for Mating/Filamentation in Sporisorium scitamineum.

In the phytopathogenic fungus Sporisorium scitamineum, sexual mating between two compatible haploid cells and the subsequent formation of dikaryotic hyphae is essential for infection. This process was shown to be commonly regulated by a mitogen-activated protein kinase (MAPK) and a cAMP/PKA signaling pathway in the corn smut fungus Ustilago maydis but remains largely unknown in S. scitamineum. In this study, we identified a conserved putative MAP kinase Kpp2 in S. scitamineum and named it as SsKpp2. The sskpp2Δ mutant displayed significant reduction in mating/filamentation, which could be partially restored by addition of cAMP or tryptophol, a quorum-sensing molecule identified in budding yeast. Transcriptional profiling showed that genes governing S. scitamineum mating or tryptophol biosynthesis were significantly differentially regulated in the sskpp2Δ mutant compared to the WT, under mating condition. Our results demonstrate that the MAP kinase SsKpp2 is required for S. scitamineum mating/filamentation likely through regulating the conserved pheromone signal transduction pathway and tryptophol production.

Phytohormones: the chemical language in Magnaporthe oryzae-rice pathosystem.

Phytohormones (also named as plant hormones) are chemicals produced by plants in order to modulate various aspects of plant development, stress responses and defence. Recent studies revealed that fungi can also produce phytohormones or phytohormone-mimiking molecules, while it remains poorly understood about the details in the role and regulatory mechanism of such fungal produced phytohormonal molecules in plant-fungus interactions. The rice-blast fungus Magnaporthe oryzae imposes a great threat to global food security. Intensive investigation has been conducted to elucidate M. oryzae pathogenicity and rice (Oryza sativa L.) defense mechanism against blast disease, in order to provide theoretical basis and/or identify potential target(s) for developing novel disease control strategies, as well as for breeding of resistance varieties. Phytohormones have been demonstrated to play conserved and divergent roles in fine-tuning the balance of rice growth and immunity towards M. oryzae. Meanwhile, M. oryzae evolved elaborate strategy to manipulate the rice phytohormones metabolism, or even directly produce and secrete phytohormones, during their invasion process. In this review, we discuss the chemical communication in term of phytohormones in M. oryzae-rice pathosystem.

Enhanced vascular activity of a new chimeric promoter containing the full CaMV 35S promoter and the plant XYLOGEN PROTEIN 1 promoter.

To develop a new strategy that controls vascular pathogen infections in economic crops, we examined a possible enhancer of the vascular activity of XYLOGEN PROTEIN 1 promoter (Px). This protein is specifically expressed in the vascular tissues of Arabidopsis thaliana and plays an important role in xylem development. Although Px is predicted as vascular-specific, its activity is hard to detect and highly susceptible to plant and environmental conditions. The cauliflower mosaic virus 35S promoter (35S) is highly active in directing transgene expression. To test if 35S could enhance Px activity, while vascular specificity of the promoter is retained, we examined the expression of the uidA reporter gene, which encodes ß-glucuronidase (GUS), under the control of a chimeric promoter (35S-Px) or Px by generating 35S-Px-GUS and Px-GUS constructs, which were transformed into tobacco seedlings. Both 35S-Px and Px regulated gene expression in vascular tissues. However, GUS expression driven by 35S-Px was not detected in 30- and 60-day-old plants. Quantitative real-time PCR analysis showed that GUS gene expression regulated by 35S-Px was 6.2-14.9-fold higher in vascular tissues than in leaves. Histochemical GUS staining demonstrated that 35S-Px was strongly active in the xylem and phloem. Thus, fusion of 35S and Px might considerably enhance the strength of Px and increase its vascular specificity. In addition to confirming that 35S enhances the activity of a low-level tissue-specific promoter, these findings provide information for further improving the activity of such promoters, which might be useful for engineering new types of resistant genes against vascular infections.

PlMAPK10, a Mitogen-Activated Protein Kinase (MAPK) in Peronophythora litchii, Is Required for Mycelial Growth, Sporulation, Laccase Activity, and Plant Infection.

Mitogen-activated protein kinase (MAPK) pathways are ubiquitous and evolutionarily conserved signal transduction modules directing cellular respond to a diverse array of stimuli, in the eukaryotic organisms. In this study, PlMAPK10 was identified to encode a MAPK in Peronophythora litchii, the oomycete pathogen causing litchi downy blight disease. PlMAPK10, containing a specific and highly conserved dual phosphorylation lip sequence SEY (Serine-Glutamic-Tyrosine), represents a novel group of MAPKs as previously reported. Transcriptional profiling showed that PlMAPK10 expression was up-regulated in zoospore and cyst stages. To elucidate its function, the PlMAPK10 gene was silenced by stable transformation. PlMAPK10 silence did not impair oospore production, sporangium germination, zoospore encyst, or cyst germination but hindered hyphal growth, sporulation, pathogenicity, likely due to altering laccase activity. Over all, our results indicated that a MAPK encoded by PlMAPK10 gene in P. litchii is important for pathogenic development.

Phototrophy and starvation-based induction of autophagy upon removal of Gcn5-catalyzed acetylation of Atg7 in Magnaporthe oryzae.

Magnaporthe oryzae, the ascomycete fungus that causes rice blast disease, initiates conidiation in response to light when grown on Prune-Agar medium containing both carbon and nitrogen sources. Macroautophagy/autophagy was shown to be essential for M. oryzae conidiation and induced specifically upon exposure to light but is undetectable in the dark. Therefore, it is inferred that autophagy is naturally induced by light, rather than by starvation during M. oryzae conidiation. However, the signaling pathway(s) involved in such phototropic induction of autophagy remains unknown. We identified an M. oryzae ortholog of GCN5 (MGG_03677), encoding a histone acetyltransferase (HAT) that negatively regulates light- and nitrogen-starvation-induced autophagy, by acetylating the autophagy protein Atg7. Furthermore, we unveiled novel regulatory mechanisms on Gcn5 at both transcriptional and post-translational levels, governing its function associated with the unique phototropic response of autophagy in this pathogenic fungus. Thus, our study depicts a signaling network and regulatory mechanism underlying the autophagy induction by important environmental clues such as light and nutrients.