Molecular insights into OPR gene family in Saccharum identified a ScOPR2 gene could enhance plant disease resistance.

12-Oxo-phytodienoic acid reductases (OPRs) perform vital functions in plants. However, few studies have been reported in sugarcane (Saccharum spp.), and it is of great significance to systematically investigates it in sugarcane. Here, 61 ShOPRs, 32 SsOPRs, and 36 SoOPRs were identified from R570 (Saccharum spp. hybrid cultivar R570), AP85-441 (Saccharum spontaneum), and LA-purple (Saccharum officinarum), respectively. These OPRs were phylogenetically classified into four groups, with close genes similar structures. During evolution, OPR gene family was mainly expanded via whole-genome duplications/segmental events and predominantly underwent purifying selection, while sugarcane OPR genes may function differently in response to various stresses. Further, ScOPR2, a tissue-specific OPR, which was localized in cytoplasm and cell membrane and actively response to salicylic acid (SA), methyl jasmonate, and smut pathogen (Sporisorium scitamineum) stresses, was cloned from sugarcane. In addition, both its transient overexpression and stable overexpression enhanced the resistance of transgenic plants to pathogen infection, most probably through activating pathogen-associated molecular pattern/pattern-recognition receptor-triggered immunity, producing reactive oxygen species, and initiating mitogen-activated protein kinase cascade. Subsequently, the transmission of SA and hypersensitive reaction were triggered, which stimulated the transcription of defense-related genes. These findings provide insights into the function of ScOPR2 gene for disease resistance.

Novel Secreted Effectors Conserved Among Smut Fungi Contribute to the Virulence of Ustilago maydis.

Fungal pathogens deploy a set of molecules (proteins, specialized metabolites, and sRNAs), so-called effectors, to aid the infection process. In comparison to other plant pathogens, smut fungi have small genomes and secretomes of 20 Mb and around 500 proteins, respectively. Previous comparative genomic studies have shown that many secreted effector proteins without known domains, i.e., novel, are conserved only in the Ustilaginaceae family. By analyzing the secretomes of 11 species within Ustilaginaceae, we identified 53 core homologous groups commonly present in this lineage. By collecting existing mutants and generating additional ones, we gathered 44 Ustilago maydis strains lacking single core effectors as well as 9 strains containing multiple deletions of core effector gene families. Pathogenicity assays revealed that 20 of these 53 mutant strains were affected in virulence. Among the 33 mutants that had no obvious phenotypic changes, 13 carried additional, sequence-divergent, structurally similar paralogs. We report a virulence contribution of seven previously uncharacterized single core effectors and of one effector family. Our results help to prioritize effectors for understanding U. maydis virulence and provide genetic resources for further characterization. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Exploring Potential Surrogate Systems for Studying the Early Steps of the Sporisorium scitamineum Pathogenesis.

Despite its global importance as a primary source of table sugar and bioethanol, sugarcane faces a significant threat to its production due to diseases. One of these diseases, sugarcane smut, involves the emergence of a whip-like structure from the host apical shoot. The slow onset of this pathogenesis is the most substantial challenge for researchers to investigate the molecular events leading to resistance or susceptibility. In this study, we explored the early interaction between the smut fungus Sporisorium scitamineum and foliar tissues of the model plants Arabidopsis thaliana and Nicotiana benthamiana. Upon inoculation with the fungus, A. thaliana showed a compatible reaction, producing lesions during fungus colonization, whereas N. benthamiana showed signs of nonhost resistance. In addition, we propose a sugarcane detached leaf assay using plants cultivated in vitro to reveal sugarcane smut response outcomes. We used two sugarcane genotypes with known contrasting reactions to smut in the field. Although there is no evidence of sugarcane smut fungus infecting host leaves naturally, the sugarcane detached leaf assay enabled a rapid assessment of disease outcomes. Different symptoms in the detached leaves after inoculation distinguished smut-susceptible and smut-resistant sugarcane genotypes. Microscopic observations and gene expression analysis of S. scitamineum candidate effectors confirmed the fungal growth and its restriction on the compatible and incompatible interactions, respectively. These findings offer new prospects into the disease phenotyping of S. scitamineum, which could greatly expedite the comprehension of the initial stages of the pathogenesis and predict smut resistance in sugarcane genotypes.

Characterization of a KU70-disrupted strain of the mannosylerythritol lipid-producing yeast Pseudozyma tsukubaensis constructed by a marker recycling system.

The basidiomycetous yeast Pseudozyma tsukubaensis is known as an industrial mannosylerythritol lipid producer. In this study, the PtURA5 marker gene was deleted by homologous recombination. Using the PtURA5-deleted mutant as a host strain, we obtained a derivative disrupted for the PtKU70 gene, a putative ortholog of the KU70 gene encoding a protein involved in the nonhomologous end-joining pathway of DNA repair. Subsequently, the introduced PtURA5 gene was re-deleted by marker recycling. These results demonstrated that the PtURA5 gene can be used as a recyclable marker gene. Although the frequency of homologous recombination has been shown to be increased by KU70 disruption in other fungi, the PtKU70-disrupted strain of P. tsukubaensis did not demonstrate an elevated frequency of homologous recombination. Furthermore, the PtKU70-disrupted strain did not show increased susceptibility to bleomycin. These results suggested that the function of this KU70 ortholog in P. tsukubaensis is distinct from that in other fungi.

Comparative genomics of smut fungi suggest the ability of meiosis and mating in asexual species of the genus Pseudozyma (Ustilaginales).

BACKGROUND: The Ustilaginales comprise hundreds of plant-parasitic fungi with a characteristic life cycle that directly links sexual reproduction and parasitism: One of the two mating-type loci codes for a transcription factor that not only facilitates mating, but also initiates the infection process. However, several species within the Ustilaginales have no described parasitic stage and were historically assigned to the genus Pseudozyma. Molecular studies have shown that the group is polyphyletic, with members being scattered in various lineages of the Ustilaginales. Together with recent findings of conserved fungal effectors in these non-parasitic species, this raises the question if parasitism has been lost recently and in multiple independent events or if there are hitherto undescribed parasitic stages of these fungi. RESULTS: In this study, we sequenced genomes of five Pseudozyma species together with six parasitic species from the Ustilaginales to compare their genomic capability to perform two central functions in sexual reproduction: mating and meiosis. While the loss of sexual capability is assumed in certain lineages and asexual species are common in Asco- and Basidiomycota, we were able to successfully annotate potentially functional mating and meiosis genes that are conserved throughout the whole group. CONCLUSION: Our data suggest that at least the key functions of a sexual lifestyle are maintained in the analyzed genomes, challenging the current understanding of the so-called asexual species with respect to their evolution and ecological role.

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.

Kynurenine 3-Monooxygenase Gene SsCI51640 Is Required for Sporisorium scitamineum Mating/Filamentation by Regulating cAMP Pathway and Improving Sporidia Environmental Adaptability.

Sugarcane smut is a serious disease caused by Sporisorium scitamineum, which causes significant losses to the sugar industry. It is critical to reveal the molecular pathogenic mechanism of S. scitamineum to explore a new control strategy for sugarcane smut. On the basis of transcriptome sequencing data of two S. scitamineum strains with different pathogenicity, we identified the gene, SsCI51640, which was predicted to encode kynurenine 3-monooxygenase. In this study, we obtained knockout mutants and complementary mutants of this gene and identified gene function. The results showed that the sporidial growth rate and acid production ability of knockout mutants were significantly higher and stronger than those of the wild-type and complementary mutants. The growth of knockout mutants under abiotic stress (osmotic stress and cell wall stress) was significantly inhibited. In addition, the sexual mating ability and pathogenicity of knockout mutants were significantly reduced, while this phenomenon could be restored by adding exogenous cyclic adenosine monophosphate (cAMP). It is thus speculated that the SsCI51640 gene may regulate sexual mating and pathogenicity of S. scitamineum by the cAMP signaling pathway. Moreover, the SsCI51640 gene enhanced the sporidial environmental adaptability, which promoted sexual mating and development of pathogenicity. This study provides a theoretical basis for the molecular pathogenesis of S. scitamineum.

Genetic Diversity and Pathogenic Variation of the Rice False Smut Pathogen Ustilaginoidea virens from Different Rice Cultivars.

Rice false smut, caused by Ustilaginoidea virens, has become one of the most devastating grain diseases of rice worldwide. Understanding the genetic diversity of U. virens is essential for efficient disease control and breeding for disease resistance. However, little is known about the genetic variation of U. virens from different rice cultivars. We investigated the genetic diversity and pathogenic variation of U. virens isolates from 10 rice cultivars in Zhejiang, China. A total of 260 polymorphic loci and 27 haplotypes were identified based on the 2,137-bp combined DNA fragments of all individuals; hap_4 was the most common haplotype, represented by 41 isolates. Phylogeny indicated that all isolates were divided into four genetic groups. Group I was the largest, with 98 isolates, distributed mainly in eight cultivar populations, whereas 90% of the isolates collected from a Changxiang cultivar were clustered in Group IV. Furthermore, the pairwise FST values exhibited significant genetic differentiation in 27 of the pairwise comparisons between populations, accounting for 23.21% of the total genetic variation. The genetic composition of the isolates of the CX population was distinguishable from that of the other nine populations, and genetic recombination was found in a few isolates. Finally, 27 haplotype representative isolates showed high variation in pathogenicity, and the isolates from the genetic subpopulation I were likely to be more virulent than those from genetic subpopulations II and III. Collectively, these findings suggest that differences in rice cultivars play an important role in the genetic variation of U. virens.

Kernel Smut and False Smut: The Old-Emerging Diseases of Rice-A Review.

Kernel smut, caused by Tilletia horrida, is a disease characterized by the replacement of rice grains with black sooty masses of teliospores or chlamydospores. Kernel smut differs from rice false smut, caused by Ustilaginoidea virens, in the color of chlamydospores. False smut is characterized by globose, velvety spore balls ranging from orangish yellow to greenish black in color. Both kernel smut and false smut have been persistent but are considered minor diseases in many countries since they were discovered in the late 1870s to the 1980s due to their sporadic outbreaks and limited economic impacts. In recent years, however, kernel smut and false smut have emerged as two of the most economically important diseases in rice, including organic rice, in many countries, especially in the United States. The increased use of susceptible rice cultivars, especially hybrids, excessive use of nitrogen fertilizer, and short crop rotations have resulted in an increase in kernel smut and false smut, causing significant losses in grain yield and quality. In this article, we provide a review of the distribution and economic importance of kernel smut; our current understanding of the taxonomy, biology, and epidemiology of kernel smut; and the genomics of the kernel smut fungus as compared with false smut and its causal agent. We also provide an update on the current management strategies of pathogen exclusion, cultivar resistance, fungicides, biological control, and cultural practices for kernel smut and false smut of rice.

Optimization of Ustilago nuda Inoculum Concentration for Screening Un8-Mediated Loose Smut Resistance in Barley Reveals a Resistance Reaction that Disrupts Seed Germination and Suggests a Role for Abscisic Acid in Disease Development.

Barley loose smut has been effectively controlled for decades through resistance conferred by the Un8 gene. However, evaluation of loose smut reaction using floret inoculation at the standard inoculum concentration is associated with the production of small, discolored seeds in Un8 carriers and susceptible genotypes. Interestingly, Un8 carriers also displayed significantly poorer germination than susceptible genotypes and produce short-lived seedlings following inoculation. To understand these observations, a Un8 carrier (TR11698) and susceptible non-Un8 carrier (CDC Austenson) were assessed for seed traits, Ustilago nuda biomass in the seed, infection rate, and phytohormone profile across a range of lower inoculum concentrations. At lower inoculum concentrations, seed appearance and weight improved in both genotypes, and infection rate increased in CDC Austenson. Pathogen load in the seed was similar in both genotypes and was positively correlated with the CDC Austenson infection rate. No infection was ever observed in TR11698. Significantly, germination rate improved in CDC Austenson, whereas the very low germination rate and short-lived seedlings remained associated with TR11698. It appears that poor seed appearance in both genotypes and low germination rate in the susceptible genotype can be improved by lowering the inoculum concentration. However, the very low germination rates and seedling death associated with the Un8 carrier TR11698 are indicative of Un8-mediated resistance to loose smut. Finally, profiling of 38 phytohormones revealed that larger seeds observed at some inoculum concentrations compared with mock inoculation had higher abscisic acid concentrations. This could represent a pathogen survival strategy by ensuring better growth of the host.

High-Quality Genome Resource of Ustilaginoidea virens (UV2_4G), Causal Agent of an Emerging False Smut Disease in Rice.

Ustilaginoidea virens is the fungal pathogen causing an emerging false smut disease that affects crop yield as well as deteriorates quality of the grains by producing mycotoxins. A high quality genome of U. virens isolate UV2_4G was sequenced using Nanopore and Illumina HiSeq 2,000 sequencing platforms. The total assembled genome of Indian isolate UV2_4G was 35.9 Mb, which comprised 89 scaffolds with N50 of 700,296 bp. A total of 358,697 variants were identified in the genome, out of which 355,173 were SNPs and 3,524 were INDELS. Further, 7,390 SSRs belonging to different repeat types were also identified in the genome. Out of 7,444 proteins predicted, 7,206 were functionally annotated. A total of 1,307 CAZymes, 501 signal peptides, 1,876 effectors, and 2,709 genes involved in host-pathogen interactions were identified. Comparative analysis revealed isolate UV2_4G is distinct with 31 unique clusters and placed distantly in phylogenetic analysis. Taken together, this high-quality genome assembly and sequence annotation resource can give an improved insight for characterizing the biological and pathogenic mechanisms of U. virens.

Plant Responses of Maize to Two formae speciales of Sporisorium reilianum Support Recent Fungal Host Jump.

Host jumps are a major factor for the emergence of new fungal pathogens. In the evolution of smut fungi, a putative host jump occurred in Sporisorium reilianum that today exists in two host-adapted formae speciales, the sorghum-pathogenic S. reilianum f. sp. reilianum and maize-pathogenic S. reilianum f. sp. zeae. To understand the molecular host-specific adaptation to maize, we compared the transcriptomes of maize leaves colonized by both formae speciales. We found that both varieties induce many common defense response-associated genes, indicating that both are recognized by the plant as pathogens. S. reilianum f. sp. reilianum additionally induced genes involved in systemic acquired resistance. In contrast, only S. reilianum f. sp. zeae induced expression of chorismate mutases that function in reducing the level of precursors for generation of the defense compound salicylic acid (SA), as well as oxylipin biosynthesis enzymes necessary for generation of the SA antagonist jasmonic acid (JA). In accordance, we found reduced SA levels as well as elevated JA and JA-Ile levels in maize leaves inoculated with the maize-adapted variety. These findings support a model of the emergence of the maize-pathogenic variety from a sorghum-specific ancestor following a recent host jump.

Enhanced Resistance to Fungal and Bacterial Diseases Due to Overexpression of BSR1, a Rice RLCK, in Sugarcane, Tomato, and Torenia.

Sugarcane smut caused by Sporisorium scitamineum is one of the most devastating sugarcane diseases. Furthermore, Rhizoctonia solani causes severe diseases in various crops including rice, tomato, potato, sugar beet, tobacco, and torenia. However, effective disease-resistant genes against these pathogens have not been identified in target crops. Therefore, the transgenic approach can be used since conventional cross-breeding is not applicable. Herein, the overexpression of BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice receptor-like cytoplasmic kinase, was conducted in sugarcane, tomato and torenia. BSR1-overexpressing tomatoes exhibited resistance to the bacteria Pseudomonas syringae pv. tomato DC3000 and the fungus R. solani, whereas BSR1-overexpressing torenia showed resistance to R. solani in the growth room. Additionally, BSR1 overexpression conferred resistance to sugarcane smut in the greenhouse. These three BSR1-overexpressing crops exhibited normal growth and morphologies except in the case of exceedingly high levels of overexpression. These results indicate that BSR1 overexpression is a simple and effective tool for conferring broad-spectrum disease resistance to many crops.

Transcriptomic and Metabolomic Analyses Provide Insights into the Pathogenic Mechanism of the Rice False Smut Pathogen Ustilaginoidea virens.

Rice false smut, caused by the fungal pathogen Ustilaginoidea virens, is a worldwide rice fungal disease. However, the molecular mechanism of the pathogenicity of the fungus U. virens remains unclear. To understand the molecular mechanism of pathogenesis of the fungus U. virens, we performed an integrated analysis of the transcriptome and metabolome of strongly (S) and weakly (W) virulent strains both before and after the infection of panicles. A total of 7932 differential expressed genes (DEGs) were identified using transcriptome analysis. Gene ontology (GO) and metabolic pathway enrichment analysis indicated that amino acid metabolism, autophagy-yeast, MAPK signaling pathway-yeast, and starch and sucrose metabolism were closely related to the pathogenicity of U. virens. Genes related to pathogenicity were significantly upregulated in the strongly virulent strain, and were ATG, MAPK, STE, TPS, and NTH genes. However, genes involved in the negative regulation of pathogenesis were significantly downregulated and contained TOR kinase, TORC1, and autophagy-related protein genes. Metabolome analysis identified 698 differentially accumulated metabolites (DAMs), including 13 categories of organic acids and derivatives, lipids and lipid-like molecules, organoheterocyclic compounds. The significantly enriched pathways of DAMs mainly included amino acids and carbohydrates, and they accumulated after infection by the S strain. To understand the relevance of DEGs and DAMs in the pathogenicity of U. virens, transcriptomic and metabolomic data were integrated and analyzed. These results further confirmed that the pathogenesis of U. virens was regulated by DEGs and DAMs related to these four pathways, involving arginine and proline metabolism, lysine biosynthesis, alanine, aspartate and glutamate metabolism, and starch and sugar metabolism. Therefore, we speculate that the pathogenicity of U. virens is closely related to the accumulation of amino acids and carbohydrates, and to the changes in the expression of related genes.

Autophagy-related protein UvAtg7 contributes to mycelial growth, virulence, asexual reproduction and cell stress response in rice false smut fungus Ustilaginoidea virens.

Autophagy is a conserved mechanism for nutrient and cytoplasmic components recycling in eukaryotic cell, in which E1-like enzyme Atg7 activates ubiquitin-like conjugation in the autophagy pathway. In plant pathogenic fungi Ustilaginoidea virens, UvAtg7, an ortholog of AAtg7 in baker's yeast was identified and functionally investigated. UvAtg7 was confirmed to be essential for autophagy, because the disruption of UvATG7 gene in U. virens completely blocked the fusion of autophagosome-like into vacuoles and catalytic degradation of GFP-UvAtg8 under N-starving condition. The fluorescent signal indicated UvAtg7 protein was dispersed in cytoplasma, but spatially coordinated with core autophagy protein UvAtg8 on occasion. Interestingly, disruption of UvATG7 in U. virens caused slightly reduction in mycelial growth, but resulted in a considerable decrease in virulence, conidia production in YT broth and chlamydospore formation on rice false smut balls. Moreover, the UvATG7 deletion mutants exhibited increased sensitivity to cell wall integrity stress caused by congo red and calcofluor white, meanwhile the UvATG7 deletion mutants showed decreased sensitivity to osmotic stress, cell membrane stress and reactiveoxygen stress caused by sorbitol, sodium dodecyl sulfate and H2O2, respectively. All of these defects in UvATG7 deletion mutants could be partially or completely restored by gene complementation. In general, our study indicates that UvAtg7 is essential in autophagy pathway and contributes to mycelial growth, virulence, asexual reproduction and cell stress response in U. virens.

A sugarcane smut fungus effector simulates the host endogenous elicitor peptide to suppress plant immunity.

The smut fungus Sporisorium scitamineum causes the most prevalent disease on sugarcane. The mechanism of its pathogenesis, especially the functions and host targets of its effector proteins, are unknown. In order to identify putative effectors involving in S. scitamineum infection, a weighted gene co-expression network analysis was conducted based on the transcriptome profiles of both smut fungus and sugarcane using a customized microarray. A smut effector gene, termed SsPele1, showed strong co-expression with sugarcane PLANT ELICITOR PEPTIDE RECEPTOR1 (ScPEPR1), which encodes a receptor like kinase for perception of plant elicitor peptide1 (ScPep1). The relationship between SsPele1 and ScPEPR1, and the biological function of SsPele1 were characterized in this study. The SsPele1 C-terminus contains a plant elicitor peptide-like motif, by which SsPele1 interacts strongly with ScPEPR1. Strikingly, the perception of ScPep1 on ScPEPR1 is competed by SsPele1 association, leading to the suppression of ScPEPR1-mediated immune responses. Moreover, the Ustilago maydis effector UmPele1, an ortholog of SsPele1, promotes fungal virulence using the same strategy. This study reveals a novel strategy by which a fungal effector can mimic the plant elicitor peptide to complete its perception and attenuate receptor-activated immunity.

A Bacterial Type Three Secretion-Based Delivery System for Functional Characterization of Sporisorium scitamineum Plant Immune Suppressing Effector Proteins.

The facultative biotrophic basidiomycete Sporisorium scitamineum causes smut disease in sugarcane. This study applied an assay to identify S. scitamineum candidate effectors (CEs) with plant immunity suppression activities by delivering them into Nicotiana benthamiana cells via the type-three secretion system of Pseudomonas fluorescens EtHAn. Six CEs were individually cloned into the pEDV6 vector and expressed by P. fluorescens EtHAn for translocation into the plant cells. Three CEs (g1052, g3890, and g5159) could suppress pattern-triggered immunity (PTI) responses with high reproducibility in different coinfiltration experiments with P. syringae pv. tomato DC3000. In addition, three CEs (g1052, g4549, and g5159) were also found to be AvrB-induced suppressors of effector-triggered immunity (ETI), demonstrating for the first time that S. scitamineum can defeat both PTI and ETI responses. A transcriptomic analysis at different stages of infection by the smut fungus of three sugarcane cultivars with contrasting responses to the pathogen revealed that suppressors g1052, g3890, g4549, and g5159 were induced at the early stage of infection. By contrast, the two CEs (g2666 and g6610) that did not exhibit suppression activities expressed only at the late stage of infection. Moreover, genomic structures of the CEs and searches for orthologs in other smut species suggested duplication events and further divergence in CEs evolution of S. scitamineum. Thus, the transient assay applied here demonstrated the potential of pEDV6 and P. fluorescens EtHAn as biological tools for identifying plant immune suppressors from S. scitamineum.

Uracil-auxotrophic marker recycling system for multiple gene disruption in Pseudozyma antarctica.

The basidiomycetous yeast Pseudozyma antarctica, which has multiple auxotrophic markers, was constructed, without inserting a foreign gene, as the host strain for the introduction of multiple useful genes. P. antarctica was more resistant to ultraviolet (UV) irradiation than the model yeast Saccharomyces cerevisiae, and a Paura3 mutant (C867T) was obtained after 3 min of UV exposure. A uracil-auxotrophic marker (URA3) recycling system developed in ascomycetous yeasts and fungi was applied to the P. antarctica Paura3 strain. The PaLYS12 and PaADE2 loci were disrupted via site-directed homologous recombination of PaURA3 (pop-in), followed by the removal of PaURA3 (pop-out). In the obtained double auxotrophic strain (Palys12Δ, Paura3), PaADE2 was further disrupted, and PaURA3 was removed to obtain the triple auxotrophic strain PGB800 (Paura3, Palys12Δ, Paade2Δ). The whole-genome sequence of the PGB800 strain did not contain foreign genes used for genetic manipulation and disrupted PaADE2 and PaLYS12, and removed PaURA3, as planned.

Confluenine G, a new compound from a basidiomycetous yeast Moesziomyces sp. FKI-9540 derived from the gut of a moth Acherontia lachesis (Lepidoptera, Sphingidae).

Most natural products derived from microorganisms have been sought from actinomycetes and filamentous fungi. As an attempt to develop new microbial resources in the exploratory research for natural products, we searched for new compounds from unexploited microbial taxa presumed to have biosynthetic gene clusters. A new compound confluenine G (1) and a known compound (2Z)-2-octyl-2-pentenedioic acid (2) were isolated from a cultured broth of basidiomycetous yeast, Moesziomyces sp. FKI-9540, derived from the gut of a moth Acherontia lachesis (Lepidoptera, Sphingidae). Based on the results of HR-ESI-MS and NMR analyses, the planar structure of 1 was elucidated. Confluenine G (1) was a new analog of nitrogen-oxidized isoleucine and had rare substructures with oxime and hydroxamic acid in molecule.

Establishment of an Artificial Inoculation Method of Ustilaginoidea virens Without Damaging the Rice Panicle Sheaths.

Rice false smut (RFS) is a destructive disease of rice worldwide caused by Ustilaginoidea virens. Nevertheless, there is a lack of efficient and stable artificial inoculation method to simulate the natural infection of U. virens, which is an important factor limiting further research on the pathogen. The purpose of this study was to establish an artificial inoculation method, which can simulate the natural infection process of U. virens without destroying the panicle sheath structure of rice. In this research, rice plants were inoculated by soaking roots at the seedling stage, spraying at the tillering stage, injecting at the booting stage, and again spraying at the flowering stage to determine the appropriate artificial inoculation time. Meanwhile, the panicle sheath instillation method and the injection inoculation method were compared. The results show that stages 6 to 8 of young panicle differentiation are an important period for U. virens infection. There were no significant differences in the mean rates of infected panicles, mean rates of infected grains, and maximum infected grains per panicle between the two inoculation methods. However, the frequency of RFS ball occurrence at the upper part of the panicles was significantly higher on the spikelets inoculated by the injection method than that of spikelets inoculated by natural infection and panicle sheath instillation. Therefore, panicle sheath instillation method was more similar to the natural infection of U. virens in the field. This research exhibited an innovative artificial inoculation method for identification of U. virens pathogenicity and evaluation of rice resistance against RFS.