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Ustilago esculenta is a dimorphic fungus that specifically infects Zizania latifolia, causing stem swelling and the formation of an edible fleshy stem known as jiaobai. The pathogenicity of U. esculenta is closely associated with the development of jiaobai and phenotypic differentiation. Msb2 acts as a key upstream sensor in the MAPK (mitogen-activated protein kinase) signaling pathway, playing critical roles in fungal hyphal growth, osmotic regulation, maintenance of cell wall integrity, temperature adaptation, and pathogenicity. In this study, we cloned the UeMsb2 gene from U. esculenta (GenBank No. MW768949). The open reading frame of UeMsb2 is 3015 bp in length, lacks introns, encodes a 1004-amino-acid protein with a conserved serine-rich domain, and is localized to the vacuole. Expression analysis revealed that UeMsb2 is inducibly expressed during both hyphal growth and infection processes. Deletion of UeMsb2 did not affect haploid morphology or growth rate in vitro but significantly impaired the strain's mating ability, suppressed filamentous growth, slowed host infection progression, and downregulated the expression of b signaling pathway genes associated with pathogenicity. Notably, the deletion of UeMsb2 did not influence the in vitro growth of U. esculenta under hyperosmotic, thermal, or oxidative stress conditions. These findings underscore the critical role of UeMsb2 in regulating the pathogenicity of U. esculenta. This study provides insights into the interaction between U. esculenta and Z. latifolia, particularly the mechanisms that drive host stem swelling. [ABSTRACT FROM AUTHOR]

Ustilago esculenta is a dimorphic fungus that specifically infects Zizania latifolia, causing stem swelling and the formation of an edible fleshy stem known as jiaobai. The pathogenicity of U. esculenta is closely associated with the development of jiaobai and phenotypic differentiation. Msb2 acts as a key upstream sensor in the MAPK (mitogen-activated protein kinase) signaling pathway, playing critical roles in fungal hyphal growth, osmotic regulation, maintenance of cell wall integrity, temperature adaptation, and pathogenicity. In this study, we cloned the UeMsb2 gene from U. esculenta (GenBank No. MW768949). The open reading frame of UeMsb2 is 3015 bp in length, lacks introns, encodes a 1004-amino-acid protein with a conserved serine-rich domain, and is localized to the vacuole. Expression analysis revealed that UeMsb2 is inducibly expressed during both hyphal growth and infection processes. Deletion of UeMsb2 did not affect haploid morphology or growth rate in vitro but significantly impaired the strain’s mating ability, suppressed filamentous growth, slowed host infection progression, and downregulated the expression of b signaling pathway genes associated with pathogenicity. Notably, the deletion of UeMsb2 did not influence the in vitro growth of U. esculenta under hyperosmotic, thermal, or oxidative stress conditions. These findings underscore the critical role of UeMsb2 in regulating the pathogenicity of U. esculenta. This study provides insights into the interaction between U. esculenta and Z. latifolia, particularly the mechanisms that drive host stem swelling.

The Zizania latifolia is usually infected by the obligate parasitic fungus Ustilago esculenta to form an edible fleshy stem which is an aquatic vegetable called Jiaobai in China. The infection by the teliospore (T) strain of U. esculenta induces Z. latifolia forming gray fleshy stems, while the mycelia-teliospore (MT) strain of U. esculenta induces white fleshy stems which are more suitable for edibility than gray fleshy stems. The mechanism of this phenomenon is still largely unknown. One of the possible causes is the diversity of endophytic microbial communities between these two fleshy stems. Therefore, we utilized fungal ITS1 and bacterial 16S rDNA amplicon sequencing to investigate the diversity of endophytic microbial communities in the two different fleshy stems of Z. latifolia. The results revealed that the α diversity and richness of endophytic fungi in white Z. latifolia were significantly greater than in gray Z. latifolia. The dominant fungal genus in both fleshy stems was U. esculenta, which accounted for over 90% of the endophytic fungi. The community composition of endophytic fungi in gray and white Z. latifolia was different except for U. esculenta, and a negative correlation was observed between U. esculenta and other endophytic fungi. In addition, the dominant bacterial genus in gray Z. latifolia was Alcaligenaceae which is also negatively correlated with other bacterium communities. Additionally, the co-occurrence network of white Z. latifolia was found to have a stronger scale, connectivity, and complexity compared to that of gray Z. latifolia. And the detected beneficial bacteria and pathogens in the stems of Z. latifolia potentially compete for resources. Furthermore, the function of endophytic bacteria is more abundant than endophytic fungi in Z. latifolia. This research investigated the correlation between the development of Z. latifolia fleshy stems and endophytic microbial communities. Our findings indicate that the composition of endophytic microbial communities is closely related to the type of Z. latifolia fleshy stems. This research also suggests the potential utilization of specific microbial communities to enhance the growth and development of Z. latifolia, thereby contributing to the breeding of Z. latifolia. [ABSTRACT FROM AUTHOR]

This study explored the effect of Ustilago esculenta infection on differentially expressed genes (DEGs) in male Manchurian wild rice (Zizania latifolia). We injected male Jiaobai plants at the five-leaf stage with U. esculenta suspension, and leaves at different timepoints after inoculation were collected for transcriptome sequencing. Transcriptome sequencing identified 1,226 DEGs mainly enriched in resistance-related processes. Weighted gene coexpression network analysis (WGCNA) showed that ‘plant–pathogen interaction’ and ‘MAPK signalling pathway-plant’ were the two most important upregulated pathways, and 16 candidate genes related to response to U. esculenta infection were screened. The results provide a basis for determining the molecular mechanisms of the response of male Z. latifolia to U. esculenta infection.

Smut fungi display a uniform life cycle including two phases: a saprophytic phase in vitro and a parasitic phase in host plants. Several apathogenic smut fungi are found, lacking suitable hosts in their habitat. Interestingly, MT-type Ustilago esculenta was found to maintain a parasitic life, lacking the saprophytic phase. Its long period of asexual proliferation in plant tissue results in severe defects in certain functions. In this study, the growth dynamics of U. esculenta in plant tissues were carefully observed. The mycelia of T- and MT-type U. esculenta exhibit rapid growth after karyogamy and aggregate between cells. While T-type U. esculenta successfully forms teliospores after aggregation, the aggregated mycelia of MT-type U. esculenta gradually disappeared after a short period of massive proliferation. It may be resulted by the lack of nutrition such as glucose and sucrose. After overwintering, infected Zizania latifolia plants no longer contained diploid mycelia resulting from karyogamy. This indicated that diploid mycelia failed to survive in plant tissues. It seems that diploid mycelium only serves to generate teliospores. Notably, MT-type U. esculenta keeps the normal function of karyogamy, though it is not necessary for its asexual life in plant tissue. Further investigations are required to uncover the underlying mechanism, which would improve our understanding of the life cycle of smut fungi and help the breeding of Z. latifolia. [ABSTRACT FROM AUTHOR]

The Zizania latifolia is usually infected by the obligate parasitic fungus Ustilago esculenta to form an edible fleshy stem which is an aquatic vegetable called Jiaobai in China. The infection by the teliospore (T) strain of U. esculenta induces Z. latifolia forming gray fleshy stems, while the mycelia-teliospore (MT) strain of U. esculenta induces white fleshy stems which are more suitable for edibility than gray fleshy stems. The mechanism of this phenomenon is still largely unknown. One of the possible causes is the diversity of endophytic microbial communities between these two fleshy stems. Therefore, we utilized fungal ITS1 and bacterial 16S rDNA amplicon sequencing to investigate the diversity of endophytic microbial communities in the two different fleshy stems of Z. latifolia. The results revealed that the α diversity and richness of endophytic fungi in white Z. latifolia were significantly greater than in gray Z. latifolia. The dominant fungal genus in both fleshy stems was U. esculenta, which accounted for over 90% of the endophytic fungi. The community composition of endophytic fungi in gray and white Z. latifolia was different except for U. esculenta, and a negative correlation was observed between U. esculenta and other endophytic fungi. In addition, the dominant bacterial genus in gray Z. latifolia was Alcaligenaceae which is also negatively correlated with other bacterium communities. Additionally, the co-occurrence network of white Z. latifolia was found to have a stronger scale, connectivity, and complexity compared to that of gray Z. latifolia. And the detected beneficial bacteria and pathogens in the stems of Z. latifolia potentially compete for resources. Furthermore, the function of endophytic bacteria is more abundant than endophytic fungi in Z. latifolia. This research investigated the correlation between the development of Z. latifolia fleshy stems and endophytic microbial communities. Our findings indicate that the composition of endophytic microbial communities is closely related to the type of Z. latifolia fleshy stems. This research also suggests the potential utilization of specific microbial communities to enhance the growth and development of Z. latifolia, thereby contributing to the breeding of Z. latifolia.

Ustilago esculenta is a smut fungus that obligately infects Zizania latifolia and stimulates tissue swelling to form galls. Unlike T-type, MT-type U. esculenta can only proliferate within plant tissues and infect the offspring of their host. Production of telispores, haploid life, and plant cuticle penetration are not essential for it, which may lead to the degeneration in these processes. Transcriptome changes during the mating of T- and MT-type U. esculenta were studied. The functions of several secreted proteins were further confirmed by knock-out mutants. Our results showed that MT-type U. esculenta can receive environmental signals in mating and circumstance sensing as T-type does. However, MT-type U. esculenta takes a longer time for conjunction tube formation and cytoplasmic fusion. A large number of genes encoding secreted proteins are enriched in the purple co-expression module. They are significantly up-regulated in the late stage of mating in T-type U. esculenta, indicating their relationship with infecting. The knock-out of g6161 (xylanase) resulted in an attenuated symptom. The knock-out of g943 or g4344 (function unidentified) completely blocked the infection at an early stage. This study provides a comprehensive comparison between T- and MT-type during mating and identifies two candidate effectors for further study. [ABSTRACT FROM AUTHOR]

Ustilago esculenta is a fungus of two morphological forms, among the filamentous dikaryon that can induce the plant stem to expand to form fleshy stem. In order to establish biotrophy with Zizania latifolia which belongs to the tribe Oryzeae (Poaceae), U. esculenta firstly needs to secrete a bunch of effectors, among them being cell wall degrading enzymes (CWDEs). We have isolated a gene, UeEgl1, which was differentially expressed in MT-type and T-type U. esculenta at an early stage of infection, and specifically induced in the filamentous growth of the T-type. Bioinformatics analysis and enzyme activity assay indicated that UeEgl1 functions outside the cell as a β-1,4-endoglucanase with a conserved domain of the glycosyl hydrolase family 45 (GH45) which targets the main component of the plant cell wall β-1,4 linked glycosidic bonds. The phenotype analysis of UeEgl1 deletion mutants and UeEgl1 over-expression transformants showed that UeEgl1 had no significant effect on the budding, cell fusion, and filamentous growth of U. esculenta in vitro. Further study found that over-expression of UeEgl1 promoted the proliferation of mycelia inside Z. latifolia, and raised plant defense responses. The above results show that the UeEgl1 gene may play an important role in the early stage of infection through the decomposition of the plant cell wall. [ABSTRACT FROM AUTHOR]

The smut Ustilago esculenta is an endophytic fungus that invades Zizania latifolia (Jiaobai) and plays an important role in inducing the host stem to form a swollen gall. The aims of this study were to investigate isolation, identification and inoculation methods of U. esculenta from Jiaobai. The spore suspension culturing method was the best method of isolating U. esculenta, and yeast-like fungal strains and septate hyphal strains were obtained. The universal primers ITS1 and ITS4 amplified a ribosomal DNA spacer element from all tested strains, while specific primers for Lam16A gene generated a 442 bp product only with septate hyphae strains. De novo transcriptome assembly results showed that the yeast-like fungal strains did not match Ustilago, and genomic analysis suggested the septate hyphae strains were highly similar to known smut fungi. Therefore, the septate hyphae strains were identified as U. esculenta, which indicated that the ITS primers were not species-specific but that Lam16A-specific primers can distinguish U. esculenta accurately and easily. In vitro, U. esculenta grew quickly on PDA medium at 28 °C in darkness. Furthermore, an inoculation method was established to produce swollen stems by injecting a spore suspension of U. esculenta at OD600 = 2.5 into the basal culm internode of male Jiaobai.

Ustilago esculenta is a smut fungus that obligately infects Zizania latifolia and stimulates tissue swelling to form galls. Unlike T-type, MT-type U. esculenta can only proliferate within plant tissues and infect the offspring of their host. Production of telispores, haploid life, and plant cuticle penetration are not essential for it, which may lead to the degeneration in these processes. Transcriptome changes during the mating of T- and MT-type U. esculenta were studied. The functions of several secreted proteins were further confirmed by knock-out mutants. Our results showed that MT-type U. esculenta can receive environmental signals in mating and circumstance sensing as T-type does. However, MT-type U. esculenta takes a longer time for conjunction tube formation and cytoplasmic fusion. A large number of genes encoding secreted proteins are enriched in the purple co-expression module. They are significantly up-regulated in the late stage of mating in T-type U. esculenta, indicating their relationship with infecting. The knock-out of g6161 (xylanase) resulted in an attenuated symptom. The knock-out of g943 or g4344 (function unidentified) completely blocked the infection at an early stage. This study provides a comprehensive comparison between T- and MT-type during mating and identifies two candidate effectors for further study.

Abstract Background Ustilago esculenta, a typical dimorphic fungus could infect Zizania latifolia and induce host stem swollen to form an edible vegetable called Jiaobai in China. The strains differentiation especially in the mating ability and pathogenicity is closely related to different phenotypes of Jiaobai formed in the fields. Dimorphic switching, a tightly regulated processes, is essential for the pathogenetic development of dimorphic fungi. In responses to environment cues, dimorphic switching can be activated through two conserved cell signaling pathways-PKA and MAPK pathways. Previous study indicated that exogenous arginine could induce hyphal formation in several dimorphic fungi through hydrolysis by arginase, but inhibit the dimorphic transition of U. esculenta. We conducted this study to reveal the function of arginine on dimorphic transition of U. esculenta. Results In this study, we found that arginine, but not its anabolites, could slow down the dimorphic transition of U. esculenta proportionally to the concentration of arginine. Besides, UeArginase, predicated coding arginase in U. esculenta was cloned and characterized. UeArginase mutants could actually increase the content of endogenous arginine, and slow down the dimorphic transition on either nutritious rich or poor medium. Either adding exogenous arginine or UeArginase deletion lead to down regulated expressions of UePkaC, UePrf1, mfa1.2, mfa2.1, pra1 and pra2, along with an increased content of arginine during mating process. Conclusion Results of this study indicated a direct role of arginine itself on the inhibition of dimorphic transition of U. esculenta, independent of its hydrolysis by UeArginase.

Ustilago esculenta is a fungus of two morphological forms, among the filamentous dikaryon that can induce the plant stem to expand to form fleshy stem. In order to establish biotrophy with Zizania latifolia which belongs to the tribe Oryzeae (Poaceae), U. esculenta firstly needs to secrete a bunch of effectors, among them being cell wall degrading enzymes (CWDEs). We have isolated a gene, UeEgl1, which was differentially expressed in MT-type and T-type U. esculenta at an early stage of infection, and specifically induced in the filamentous growth of the T-type. Bioinformatics analysis and enzyme activity assay indicated that UeEgl1 functions outside the cell as a β-1,4-endoglucanase with a conserved domain of the glycosyl hydrolase family 45 (GH45) which targets the main component of the plant cell wall β-1,4 linked glycosidic bonds. The phenotype analysis of UeEgl1 deletion mutants and UeEgl1 over-expression transformants showed that UeEgl1 had no significant effect on the budding, cell fusion, and filamentous growth of U. esculenta in vitro. Further study found that over-expression of UeEgl1 promoted the proliferation of mycelia inside Z. latifolia, and raised plant defense responses. The above results show that the UeEgl1 gene may play an important role in the early stage of infection through the decomposition of the plant cell wall.

Ustilago esculenta undergoes an endophytic life cycle in Zizania latifolia. It induces the stem of its host to swell, forming the edible galls called jiaobai in China, which are the second most commonly cultivated aquatic vegetable in China. Z. latifolia raised for jiaobai can only reproduce asexually because the U. esculenta infection completely inhibits flowering. The infection and proliferation in the host plants during the formation of edible gall differ from those of conventional pathogens. Previous studies have shown a close relationship between mitogen-activated protein kinase (MAPK) and fungal pathogenesis. In this study, we explored the functional properties of the MAPK UeKpp2. Cross-species complementation assays were carried out, which indicated a functional complementation between the UeKpp2 of U. esculenta and the Kpp2 of Ustilago maydis. Next, UeKpp2 mutants of the UeT14 and the UeT55 sporidia background were generated; these showed an aberrant morphology of budding cells, and attenuated mating and filamentous growth in vitro, in the context of normal pathogenicity. Interestingly, we identified another protein kinase, UeUkc1, which acted downstream of UeKpp2 and may participate in the regulation of cell shape. We also found a defect of filamentous growth in UeKpp2 mutants that was not related to a defect of the induction of mating-type genes but was directly related to a defect in UeRbf1 induction. Overall, our results indicate an important role for UeKpp2 in U. esculenta that is slightly different from those reported for other smut fungi.

Ustilago esculenta undergoes an endophytic life cycle in Zizania latifolia. It induces the stem of its host to swell, forming the edible galls called jiaobai in China, which are the second most commonly cultivated aquatic vegetable in China. Z. latifolia raised for jiaobai can only reproduce asexually because the U. esculenta infection completely inhibits flowering. The infection and proliferation in the host plants during the formation of edible gall differ from those of conventional pathogens. Previous studies have shown a close relationship between mitogen-activated protein kinase (MAPK) and fungal pathogenesis. In this study, we explored the functional properties of the MAPK UeKpp2. Cross-species complementation assays were carried out, which indicated a functional complementation between the UeKpp2 of U. esculenta and the Kpp2 of Ustilago maydis. Next, UeKpp2 mutants of the UeT14 and the UeT55 sporidia background were generated; these showed an aberrant morphology of budding cells, and attenuated mating and filamentous growth in vitro , in the context of normal pathogenicity. Interestingly, we identified another protein kinase, UeUkc1, which acted downstream of UeKpp2 and may participate in the regulation of cell shape. We also found a defect of filamentous growth in UeKpp2 mutants that was not related to a defect of the induction of mating-type genes but was directly related to a defect in UeRbf1 induction. Overall, our results indicate an important role for UeKpp2 in U. esculenta that is slightly different from those reported for other smut fungi. [ABSTRACT FROM AUTHOR]

Abstract Background Ustilago esculenta, a pathogenic basidiomycete fungus, infects Zizania latifolia to form edible galls named Jiaobai in China. The distinct growth conditions of U. esculenta induced Z. latifolia to form three different phenotypes, named male Jiaobai, grey Jiaobai and white Jiaobai. The aim of this study is to characterize the genetic and morphological differences that distinguish the two U. esculenta strains. Results In this study, sexually compatible haploid sporidia UeT14/UeT55 from grey Jiaobai (T strains) and UeMT10/UeMT46 from white Jiaobai (MT strains) were isolated. Meanwhile, we successfully established mating and inoculation assays. Great differences were observed between the T and MT strains. First, the MT strains had a defect in development, including lower teliospore formation frequency and germination rate, a slower growth rate and a lower growth mass. Second, they differed in the assimilation of nitrogen sources in that the T strains preferred urea and the MT strains preferred arginine. In addition, the MT strains were more sensitive to external signals, including pH and oxidative stress. Third, the MT strains showed an infection defect, resulting in an endophytic life in the host. This was in accordance with multiple mutated pathogenic genes discovered in the MT strains by the non-synonymous mutation analysis of the genome re-sequencing data between the MT and T strains (GenBank accession numbers of the genome re-sequencing data: JTLW00000000 for MT strains and SRR5889164 for T strains). Conclusion The MT strains appeared to have defects in growth and infection and were more sensitive to external signals compared to the T strains. They displayed an absolutely stable endophytic life in the host without an infection cycle. Accordingly, they had multiple gene mutations occurring, especially in pathogenicity. In contrast, the T strains, as phytopathogens, had a complete survival life cycle, in which the formation of teliospores is important for adaption and infection, leading to the appearance of the grey phenotype. Further studies elucidating the molecular differences between the U. esculenta strains causing differential host phenotypes will help to improve the production and formation of edible white galls.

Crop domestication is one of the essential topics in evolutionary biology. Cultivated Zizania latifolia, domesticated as the special form of a plant-fungus (the host Zizania latifolia and the endophyte Ustilago esculenta) complex, is a popular aquatic vegetable endemic in East Asia. The rapid domestication of cultivated Z. latifolia can be traced in the historical literature but still need more evidence. This study focused on deciphering the genetic relationship between wild and cultivated Z. latifolia, as well as the corresponding parasitic U. esculenta. Twelve microsatellites markers were used to study the genetic variations of 32 wild populations and 135 landraces of Z. latifolia. Model simulations based on approximate Bayesian computation (ABC) were then performed to hierarchically infer the population history. We also analyzed the ITS sequences of the smut fungus U. esculenta to reveal its genetic structure. Our results indicated a significant genetic divergence between cultivated Z. latifolia and its wild ancestors. The wild Z. latifolia populations showed significant hierarchical genetic subdivisions, which may be attributed to the joint effect of isolation by distance and hydrological unconnectedness between watersheds. Cultivated Z. latifolia was supposedly domesticated once in the low reaches of the Yangtze River. The genetic structure of U. esculenta also indicated a single domestication event, and the genetic variations in this fungus might be associated with the diversification of cultivars. These findings provided molecular evidence in accordance with the historical literature that addressed the domestication of cultivated Z. latifolia involved adaptive evolution driven by artificial selection in both the plant and fungus.

Crop domestication is one of the essential topics in evolutionary biology. Cultivated Zizania latifolia , domesticated as the special form of a plant-fungus (the host Zizania latifolia and the endophyte Ustilago esculenta) complex, is a popular aquatic vegetable endemic in East Asia. The rapid domestication of cultivated Z. latifolia can be traced in the historical literature but still need more evidence. This study focused on deciphering the genetic relationship between wild and cultivated Z. latifolia , as well as the corresponding parasitic U. esculenta. Twelve microsatellites markers were used to study the genetic variations of 32 wild populations and 135 landraces of Z. latifolia. Model simulations based on approximate Bayesian computation (ABC) were then performed to hierarchically infer the population history. We also analyzed the ITS sequences of the smut fungus U. esculenta to reveal its genetic structure. Our results indicated a significant genetic divergence between cultivated Z. latifolia and its wild ancestors. The wild Z. latifolia populations showed significant hierarchical genetic subdivisions, which may be attributed to the joint effect of isolation by distance and hydrological unconnectedness between watersheds. Cultivated Z. latifolia was supposedly domesticated once in the low reaches of the Yangtze River. The genetic structure of U. esculenta also indicated a single domestication event, and the genetic variations in this fungus might be associated with the diversification of cultivars. These findings provided molecular evidence in accordance with the historical literature that addressed the domestication of cultivated Z. latifolia involved adaptive evolution driven by artificial selection in both the plant and fungus. [ABSTRACT FROM AUTHOR]

Ustilago esculenta, infects Zizania latifolia, and induced host stem swollen to be a popular vegetable called Jiaobai in China. It is the long-standing artificial selection that maximizes the occurrence of favourable Jiaobai, and thus maintaining the plant--fungi interaction and modulating the fungus evolving from plant pathogen to entophyte. In this study, whole genome of U. esculenta was sequenced and transcriptomes of the fungi and its host were analysed. The 20.2Mb U. esculenta draft genome of 6,654 predicted genes including mating, primary metabolism, secreted proteins, shared a high similarity to related Smut fungi. But U. esculenta prefers RNA silencing not repeat-induced point in defence and has more introns per gene, indicating relatively slow evolution rate. The fungus also lacks some genes in amino acid biosynthesis pathway which were filled by up-regulated host genes and developed distinct amino acid response mechanism to balance the infection--resistance interaction. Besides, U. esculenta lost some surface sensors, important virulence factors and host range-related effectors to maintain the economic endophytic life. The elucidation of the U. esculenta genomic information as well as expression profiles can not only contribute to more comprehensive insights into the molecular mechanism underlying artificial selection but also into smut fungi--host interactions. [ABSTRACT FROM AUTHOR]

Smut fungi display a uniform life cycle including two phases: a saprophytic phase in vitro and a parasitic phase in host plants. Several apathogenic smut fungi are found, lacking suitable hosts in their habitat. Interestingly, MT-type Ustilago esculenta was found to maintain a parasitic life, lacking the saprophytic phase. Its long period of asexual proliferation in plant tissue results in severe defects in certain functions. In this study, the growth dynamics of U. esculenta in plant tissues were carefully observed. The mycelia of T- and MT-type U. esculenta exhibit rapid growth after karyogamy and aggregate between cells. While T-type U. esculenta successfully forms teliospores after aggregation, the aggregated mycelia of MT-type U. esculenta gradually disappeared after a short period of massive proliferation. It may be resulted by the lack of nutrition such as glucose and sucrose. After overwintering, infected Zizania latifolia plants no longer contained diploid mycelia resulting from karyogamy. This indicated that diploid mycelia failed to survive in plant tissues. It seems that diploid mycelium only serves to generate teliospores. Notably, MT-type U. esculenta keeps the normal function of karyogamy, though it is not necessary for its asexual life in plant tissue. Further investigations are required to uncover the underlying mechanism, which would improve our understanding of the life cycle of smut fungi and help the breeding of Z. latifolia ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Yang, Yao, Xia, Ye, Zhang, Li, Zhang and Song.)

Zizania latifolia cultivars infected by the endophytic fungus Ustilago esculenta develop an edible stem gall. Stem gall development varies among cultivars and individuals and may be affected by the strain of U. esculenta. To isolate haploids from two Z. latifolia cultivars in our paddy fields, Shirakawa and Ittenkou, we herein performed the sporadic isolation of U. esculenta strains from stem gall tissue, a PCR-based assessment of the mating type, and in vitro mating experiments. As a result, we obtained heterogametic strains of MAT-2 and MAT-3 as well as MAT-2, but not MAT-3, haploid strains. Another isolation method, in which we examined poorly growing small clusters of sporidia derived from teliospores, succeeded in isolating a MAT-3 haploid strain. We also identified the mating types of 10 U. esculenta strains collected as genetic resources from different areas in Japan. All strains, except for one MAT-1 haploid strain, were classified as MAT-2 haploid strains or heterogametic strains of MAT-2 and MAT-3. The isolated strains of MAT-1, MAT-2, and MAT-3 mated with each other to produce hyphae. Collectively, these results indicate that the mating types of U. esculenta infecting Z. latifolia cultivars in Japan are biased towards MAT-2 and MAT-3 and that U. esculenta populations in these Japanese cultivars may be characterized by the low isolation efficiency of the MAT-3 haploid.

Ustilago esculenta is a smut fungus that obligately infects Zizania latifolia and stimulates tissue swelling to form galls. Unlike T-type, MT-type U. esculenta can only proliferate within plant tissues and infect the offspring of their host. Production of telispores, haploid life, and plant cuticle penetration are not essential for it, which may lead to the degeneration in these processes. Transcriptome changes during the mating of T- and MT-type U. esculenta were studied. The functions of several secreted proteins were further confirmed by knock-out mutants. Our results showed that MT-type U. esculenta can receive environmental signals in mating and circumstance sensing as T-type does. However, MT-type U. esculenta takes a longer time for conjunction tube formation and cytoplasmic fusion. A large number of genes encoding secreted proteins are enriched in the purple co-expression module. They are significantly up-regulated in the late stage of mating in T-type U. esculenta , indicating their relationship with infecting. The knock-out of g6161 (xylanase) resulted in an attenuated symptom. The knock-out of g943 or g4344 (function unidentified) completely blocked the infection at an early stage. This study provides a comprehensive comparison between T- and MT-type during mating and identifies two candidate effectors for further study., Competing Interests: The authors declare no conflict of interest.

Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 β-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 β-glucosidase, a gene encoding a putative GH3 β-glucosidase in U. esculenta ( Uebgl3A) was cloned by PCR. Based on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 β-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and from 1,3-1,4-β-glucan and laminarin polysaccharides, indicating that UeBgl3A is a β-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin. [ABSTRACT FROM AUTHOR]

The smut fungus Ustilago esculenta infects Zizania latifolia and induces stem expansion to form a unique vegetable named Jiaobai. Although previous studies have demonstrated that hormonal control is essential for triggering stem swelling, the role of hormones synthesized by Z. latifolia and U. esculenta and the underlying molecular mechanism are not yet clear. To study the mechanism that triggers swollen stem formation, we analyzed the gene expression pattern of both interacting organisms during the initial trigger of culm gall formation, at which time the infective hyphae also propagated extensively and penetrated host stem cells. Transcriptional analysis indicated that abundant genes involving fungal pathogenicity and plant resistance were reprogrammed to maintain the subtle balance between the parasite and host. In addition, the expression of genes involved in auxin biosynthesis of U. esculenta obviously decreased during stem swelling, while a large number of genes related to the synthesis, metabolism and signal transduction of hormones of the host plant were stimulated and showed specific expression patterns, particularly, the expression of ZlYUCCA9 (a flavin monooxygenase, the key enzyme in indole-3-acetic acid (IAA) biosynthesis pathway) increased significantly. Simultaneously, the content of IAA increased significantly, while the contents of cytokinin and gibberellin showed the opposite trend. We speculated that auxin produced by the host plant, rather than the fungus, triggers stem swelling. Furthermore, from the differently expressed genes, two candidate Cys2-His2 (C2H2) zinc finger proteins, GME3058_g and GME5963_g, were identified from U. esculenta, which may conduct fungus growth and infection at the initial stage of stem-gall formation. [ABSTRACT FROM AUTHOR]

Temperature influences the physiological processes and ecology of both hosts and endophytes; however, it remains unclear how long noncoding RNAs (lncRNAs) modulate the consequences of temperature-dependent changes in host–pathogen interactions. To explore the role of lncRNAs in culm gall formation induced by the smut fungus Ustilago esculenta in Zizania latifolia, we employed RNA sequencing to identify lncRNAs and their potential cis-targets in Z. latifolia and U. esculenta under different temperatures. In Z. latifolia and U. esculenta, we identified 3194 and 173 lncRNAs as well as 126 and four potential target genes for differentially expressed lncRNAs, respectively. Further function and expression analysis revealed that lncRNA ZlMSTRG.11348 regulates amino acid metabolism in Z. latifolia and lncRNA UeMSTRG.02678 regulates amino acid transport in U. esculenta. The plant defence response was also found to be regulated by lncRNAs and suppressed in Z. latifolia infected with U. esculenta grown at 25 °C, which may result from the expression of effector genes in U. esculenta. Moreover, in Z. latifolia infected with U. esculenta, the expression of genes related to phytohormones was altered under different temperatures. Our results demonstrate that lncRNAs are important components of the regulatory networks in plant-microbe-environment interactions, and may play a part in regulating culm swelling in Z. latifolia plants. [ABSTRACT FROM AUTHOR]

The article describes the osteoclast-forming suppressive compounds from Makomatake, Zizania latifolia infected with Ustilago esculenta. According to the authors, osteoclasts play a role in bone resorption, and abnormal acceleration of osteoclast formation is one of the causes of osteoporosis. During the screening for the osteoclast-formation suppressive effects of extracts of various foodstuffs, strong activity in an extract of Makomotake was demonstrated.

Zizania latifolia is a perennial aquatic vegetable, whose symbiosis with the fungus Ustilago esculenta (member of Basidiomycota, class Ustilaginaceae ) results in the establishment of swollen gall formations. Here, we analyzed symbiotic relations of Z. latifolia and U. esculenta , using a triadimefon (TDF) treatment and transcriptome sequencing (RNA-seq). Specifically, accurately identify the whole growth cycle of Z. latifolia. Microstructure observations showed that the presence of U. esculenta could be clearly observed after gall formation but was absent after the TDF treatment. A total of 17,541 differentially expressed genes (DEGs) were identified, based on the transcriptome. According to gene ontology term and Kyoto Encyclopedia of Genes and Genomes pathway results, plant hormone signal transduction, and cell wall-loosening factors were all significantly enriched due to U. esculenta infecting Z. latifolia ; relative expression levels of hormone-related genes were identified, of which downregulation of indole 3-acetic acid (IAA)-related DEGs was most pronounced in JB_D versus JB_B. The ultra-high performance liquid chromatography analysis revealed that IAA, zeatin+trans zeatin riboside, and gibberellin 3 were increased under U. esculenta infection. Based on our results, we proposed a hormone-cell wall loosening model to study the symbiotic mechanism of gall formation after U. esculenta infects Z. latifolia . Our study thus provides a new perspective for studying the physiological and molecular mechanisms of U. esculenta infection of Z. latifolia causing swollen gall formations as well as a theoretical basis for enhancing future yields of cultivated Z. latifolia .[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.