Mating-type loci of Ustilago esculenta are essential for mating and development.

Ustilago esculenta is closely related to the smut fungus Ustilago maydis and, in an endophytic-like life in the plant Zizania latifolia, only infects host stems and causes swollen stems to form edible galls called Jiaobai in China. In order to study its different modes of invasion and sites of symptom development from other smut fungi at the molecular level, we first characterized the a and b mating-type loci of U. esculenta. The a loci contained three a mating-type alleles, encoding two pheromones and one pheromone receptor per allele. The pheromone/receptor system controlled the conjugation formation, the initial step of mating, in which each pheromone was specific for recognition by only one mating partner. In addition, there are at least three b alleles identified in U. esculenta, encoding two subunits of heterodimeric homeodomain transcription factors bE and bW, responsible for hyphal growth and invasiveness. Hyphal formation, elongation and invasion after mating of two compatible partners occurred, only when a heterodimer complex was formed by the bE and bW proteins derived from different alleles. We also demonstrated that even with only one paired pheromone-pheromone receptor, the active b locus heterodimer triggered hyphal growth and infection.

Cloning and Characterization of Two MAPK Genes UeKpp2 and UeKpp6 in Ustilago esculenta.

Ustilago esculenta, resembling a fungal endophyte in Zizania latifolia, inhibits the host plant flowering and induces the host stems to swell and form edible galls. It is well believed that when and how the fungus infects and proliferates in the host plants during the host development is of importance in the edible gall formation. Mitogen-activated protein kinases (MAPKs) have been found to play an important role in sensing environment cues and regulating infection. Two MAPK genes UeKpp2 and UeKpp6 from U. esculenta were cloned and suggested to be involved in the Fus3/Kss1 pathway by a phylogenetic analysis with the neighbor-joining method. Quantitative RT-PCR (qRT-PCR) analyses indicated that expression of UeKpp2 and UeKpp6 were induced during mating and infection processes, and their expression patterns displayed differentially under different carbon and nitrogen sources. In addition, subcellular localization of UeKpp2 or UeKpp6 fused with the reporter green fluoresce protein was observed by confocal laser scanning microscope, and yeast two-hybrid assays were carried out. Results showed that both UeKpp2 and UeKpp6 were located in cytoplasm and interacted with UePrf1, indicating their involvement in hyphal growth and host-pathogen regulation. Only UeKpp2 but not UeKpp6 interacted with the upstream MAPK kinase UeFuz7, implying an additional MAPK pathway, in which UeKpp6 involved, existed.

Investigation on the differentiation of two Ustilago esculenta strains - implications of a relationship with the host phenotypes appearing in the fields.

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.

Functional Properties of the MAP Kinase UeKpp2 in Ustilago esculenta.

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.

Cloning and characterization of the UePrf1 gene in Ustilago esculenta.

Ustilago esculenta, an obligate parasite of Zizania latifolia, is a typical dimorphic fungus which induces host stem swelling and inhibits host inflorescence development, but is not found in host leaves. Previous studies have shown that dimorphic switching is essential for fungal pathogenicity and is regulated by protein kinase A and mitogen-activated protein kinase (MAPK) signaling pathways that are integrated by Prf1 in Ustilago maydis. In this study we identified a Prf1 homolog in U. esculenta, designated UePrf1, encoding 830 amino acids with a conserved high mobility group domain located between amino acids 124 and 195. UePrf1 was upregulated during the mating process, which induces dimorphism in U. esculenta. In vitro, UePrf1 mutants showed defects in the mating process, including cell fusion and hyphal growth. UePrf1 mutants also show reduced expression of a genes, even during the cell fusion process. Additionally, the defect in hyphal growth of the UeKpp2 and UeKpp6 mutants (MAPK signaling pathway mutants) was partially counteracted by UePrf1 overexpression, along with induced b gene expression. These results provide evidence that UePrf1 is a key factor coordinating dimorphism in U. esculenta and suggest a conserved role for UePrf1 in the regulation of the a and b genes.