A kiwellin disarms the metabolic activity of a secreted fungal virulence factor.

Fungi-induced plant diseases affect global food security and plant ecology. The biotrophic fungus Ustilago maydis causes smut disease in maize (Zea mays) plants by secreting numerous virulence effectors that reprogram plant metabolism and immune responses1,2. The secreted fungal chorismate mutase Cmu1 presumably affects biosynthesis of the plant immune signal salicylic acid by channelling chorismate into the phenylpropanoid pathway3. Here we show that one of the 20 maize-encoded kiwellins (ZmKWL1) specifically blocks the catalytic activity of Cmu1. ZmKWL1 hinders substrate access to the active site of Cmu1 through intimate interactions involving structural features that are specific to fungal Cmu1 orthologues. Phylogenetic analysis suggests that plant kiwellins have a versatile scaffold that can specifically counteract pathogen effectors such as Cmu1. We reveal the biological activity of a member of the kiwellin family, a widely conserved group of proteins that have previously been recognized only as important human allergens.

Pathogenesis-Related 1 (PR1) Protein Family Genes Involved in Sugarcane Responses to Ustilago scitaminea Stress.

Plant resistance against biotic stressors is significantly influenced by pathogenesis-related 1 (PR1) proteins. This study examines the systematic identification and characterization of PR1 family genes in sugarcane (Saccharum spontaneum Np-X) and the transcript expression of selected genes in two sugarcane cultivars (ROC22 and Zhongtang3) in response to Ustilago scitaminea pathogen infection. A total of 18 SsnpPR1 genes were identified at the whole-genome level and further categorized into four groups. Notably, tandem and segmental duplication occurrences were detected in one and five SsnpPR1 gene pairs, respectively. The SsnpPR1 genes exhibited diverse physio-chemical attributes and variations in introns/exons and conserved motifs. Notably, four SsnpPR1 (SsnpPR1.02/05/09/19) proteins displayed a strong protein-protein interaction network. The transcript expression of three SsnpPR1 (SsnpPR1.04/06/09) genes was upregulated by 1.2-2.6 folds in the resistant cultivar (Zhongtang3) but downregulated in the susceptible cultivar (ROC22) across different time points as compared to the control in response to pathogen infection. Additionally, SsnpPR1.11 was specifically upregulated by 1.2-3.5 folds at 24-72 h post inoculation (hpi) in ROC22, suggesting that this gene may play an important negative regulatory role in defense responses to pathogen infection. The genetic improvement of sugarcane can be facilitated by our results, which also establish the basis for additional functional characterization of SsnpPR1 genes in response to pathogenic stress.

N-glycosylation of the protein disulfide isomerase Pdi1 ensures full Ustilago maydis virulence.

Fungal pathogenesis depends on accurate secretion and location of virulence factors which drive host colonization. Protein glycosylation is a common posttranslational modification of cell wall components and other secreted factors, typically required for correct protein localization, secretion and function. Thus, the absence of glycosylation is associated with animal and plant pathogen avirulence. While the relevance of protein glycosylation for pathogenesis has been well established, the main glycoproteins responsible for the loss of virulence observed in glycosylation-defective fungi have not been identified. Here, we devise a proteomics approach to identify such proteins and use it to demonstrate a role for the highly conserved protein disulfide isomerase Pdi1 in virulence. We show that efficient Pdi1 N-glycosylation, which promotes folding into the correct protein conformation, is required for full pathogenic development of the corn smut fungus Ustilago maydis. Remarkably, the observed virulence defects are reminiscent of those seen in glycosylation-defective cells suggesting that the N-glycosylation of Pdi1 is necessary for the full secretion of virulence factors. All these observations, together with the fact that Pdi1 protein and RNA expression levels rise upon virulence program induction, suggest that Pdi1 glycosylation is important for normal pathogenic development in U. maydis. Our results provide new insights into the role of glycosylation in fungal pathogenesis.

Nuclear status and leaf tumor formation in the Ustilago maydis-maize pathosystem.

Ustilago maydis is a biotrophic fungus causing smut disease in corn. The infectious forms are dikaryotic hyphae. Here we analyze mutants lacking the nlt1 transcription factor and investigate why these mutants are unable to induce leaf tumors. The study involved reverse genetics, complementation, epistasis analysis, microscopy, gene expression analysis by quantitative reverse transcriptase PCR and virulence assays. We show that nlt1 mutants colonize maize leaves efficiently but fail to undergo karyogamy and are attenuated in late proliferation. Nlt1 activates transcription of ros1, a transcription factor controlling karyogamy, and represses see1, an effector previously shown to contribute to leaf tumor induction. In mononuclate solopathogenic strains, nlt1 mutants cause attenuated leaf tumor formation. In actively dividing maize organs, nlt1 mutants undergo karyogamy and induce tumor formation. Sporisorium reilianum, a smut fungus unable to induce leaf tumors, possesses an ortholog of nlt1 that controls the fusion of dikaryotic nuclei late in infection during cob colonization. Our results have established a regulatory connection between nlt1, ros1 and see1 and suggest the existence of two stages contributing to leaf tumor formation, one before nuclear fusion and involving nlt1 and one after karyogamy that is nlt1 independent.

The Biotrophic Development of Ustilago maydis Studied by RNA-Seq Analysis.

The maize smut fungus Ustilago maydis is a model organism for elucidating host colonization strategies of biotrophic fungi. Here, we performed an in depth transcriptional profiling of the entire plant-associated development of U. maydis wild-type strains. In our analysis, we focused on fungal metabolism, nutritional strategies, secreted effectors, and regulatory networks. Secreted proteins were enriched in three distinct expression modules corresponding to stages on the plant surface, establishment of biotrophy, and induction of tumors. These modules are likely the key determinants for U. maydis virulence. With respect to nutrient utilization, we observed that expression of several nutrient transporters was tied to these virulence modules rather than being controlled by nutrient availability. We show that oligopeptide transporters likely involved in nitrogen assimilation are important virulence factors. By measuring the intramodular connectivity of transcription factors, we identified the potential drivers for the virulence modules. While known components of the b-mating type cascade emerged as inducers for the plant surface and biotrophy module, we identified a set of yet uncharacterized transcription factors as likely responsible for expression of the tumor module. We demonstrate a crucial role for leaf tumor formation and effector gene expression for one of these transcription factors.

In vivo insertion pool sequencing identifies virulence factors in a complex fungal-host interaction.

Large-scale insertional mutagenesis screens can be powerful genome-wide tools if they are streamlined with efficient downstream analysis, which is a serious bottleneck in complex biological systems. A major impediment to the success of next-generation sequencing (NGS)-based screens for virulence factors is that the genetic material of pathogens is often underrepresented within the eukaryotic host, making detection extremely challenging. We therefore established insertion Pool-Sequencing (iPool-Seq) on maize infected with the biotrophic fungus U. maydis. iPool-Seq features tagmentation, unique molecular barcodes, and affinity purification of pathogen insertion mutant DNA from in vivo-infected tissues. In a proof of concept using iPool-Seq, we identified 28 virulence factors, including 23 that were previously uncharacterized, from an initial pool of 195 candidate effector mutants. Because of its sensitivity and quantitative nature, iPool-Seq can be applied to any insertional mutagenesis library and is especially suitable for genetically complex setups like pooled infections of eukaryotic hosts.

Role of Long Noncoding RNAs ZlMSTRG.11348 and UeMSTRG.02678 in Temperature-Dependent Culm Swelling in Zizania latifolia.

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.

Two Is Better Than One: Studying Ustilago bromivora-Brachypodium Compatibility by Using a Hybrid Pathogen.

Pathogenic fungi can have devastating effects on agriculture and health. One potential challenge in dealing with pathogens is the possibility of a host jump (i.e., when a pathogen infects a new host species). This can lead to the emergence of new diseases or complicate the management of existing threats. We studied host specificity by using a hybrid fungus formed by mating two closely related fungi: Ustilago bromivora, which normally infects Brachypodium spp., and U. hordei, which normally infects barley. Although U. hordei was unable to infect Brachypodium spp., the hybrid could. These hybrids also displayed the same mating-type bias that had been observed in U. bromivora and provide evidence of a dominant spore-killer-like system on the sex chromosome of U. bromivora. By analyzing the genomic composition of 109 hybrid strains, backcrossed with U. hordei over four generations, we identified three regions associated with infection on Brachypodium spp. and 75 potential virulence candidates. The most strongly associated region was located on chromosome 8, where seven genes encoding predicted secreted proteins were identified. The fact that we identified several regions relevant for pathogenicity on Brachypodium spp. but that none were essential suggests that host specificity, in the case of U. bromivora, is a multifactorial trait which can be achieved through different subsets of virulence factors.

Acetate provokes mitochondrial stress and cell death in Ustilago maydis.

The fungal pathogen Ustilago maydis causes disease on maize by mating to establish an infectious filamentous cell type that invades the host and induces tumours. We previously found that ß-oxidation mutants were defective in virulence and did not grow on acetate. Here, we demonstrate that acetate inhibits filamentation during mating and in response to oleic acid. We therefore examined the influence of different carbon sources by comparing the transcriptomes of cells grown on acetate, oleic acid or glucose, with expression changes for the fungus during tumour formation in planta. Guided by the transcriptional profiling, we found that acetate negatively influenced resistance to stress, promoted the formation of reactive oxygen species, triggered cell death in stationary phase and impaired virulence on maize. We also found that acetate induced mitochondrial stress by interfering with mitochondrial functions. Notably, the disruption of oxygen perception or inhibition of the electron transport chain also influenced filamentation and mating. Finally, we made use of the connections between acetate and ß-oxidation to test metabolic inhibitors for an influence on growth and virulence. These experiments identified diclofenac as a potential inhibitor of virulence. Overall, these findings support the possibility of targeting mitochondrial metabolic functions to control fungal pathogens.

Chromatin modification factors in plant pathogenic fungi: Insights from Ustilago maydis.

Adaptation to the environment is a requirement for the survival of every organism. For pathogenic fungi this also implies coping with the different conditions that occur during the infection cycle. After detecting changes to external media, organisms must modify their gene expression patterns in order to accommodate the new circumstances. Control of gene expression is a complex process that involves the coordinated action of multiple regulatory elements. Chromatin modification is a well-known mechanism for controlling gene expression in response to environmental changes in all eukaryotes. In pathogenic fungi, chromatin modifications are known to play crucial roles in controlling host interactions and their virulence capacity, yet little is known about the specific genes they directly target and to which signals they respond. The smut fungus Ustilago maydis is an excellent model system in which multiple molecular and cellular approaches are available to study biotrophic interactions. Many target genes regulated during the infection process have been well studied, however, how they are controlled and specifically how chromatin modifications affect gene regulation in the context of infection is not well known in this organism. Here, we analyse the presence of chromatin modifying enzymes and complexes in U. maydis and discuss their putative roles in this plant pathogen in the context of findings from other organisms, including other plant pathogens such as Magnaporthe oryzae and Fusarium graminearum. We propose U. maydis as a remarkable organism with interesting chromatin features, which would allow finding new functions of chromatin modifications during plant pathogenesis.

Cellular and proteomic events associated with the localized formation of smut-gall during Zizania latifolia-Ustilago esculenta interaction.

The perennial wild rice Zizania latifolia is confined in the swampy habitat and wetland of the Indo-Burma biodiversity hotspot of India and infection by the biotrophic fungus Ustilago esculenta is hallmarked by swellings that develop to form localized smut-gall at the topmost internodal region. The cellular and proteomic events involved in the non-systemic colonization of Z. latifolia by U. esculenta leading to smut-gall formation is poorly understood. Proteins were extracted from the smut-gall region at the topmost internodal region below the apical meristematic tissue from the infected and uninfected parts of Z. latifolia. By combining transmission electron microscopy (TEM) and fluorescent microscopy (FM), we showed that U. esculenta hyphal morphological transitions and movement occurred both intercellularly and intracellularly while sporulation occurred intracellularly in selective cells. Following proteome profiling using two dimensional SDS-PAGE at different phenological phases of smut-gall development and U. esculenta infection, differentially expressed proteins bands and their relative abundance were detected and subjected to liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Importantly, the fungus explores at least 7 metabolic pathways and 5 major biological processes to subdue the host defense and thrive successfully on Z. latifolia. The fungus U. esculenta produces proteases and energy acquisition proteins those enhance it's defensive and survival mode in the host. The identified differentially regulated proteins shed-light into why inflorescence is being replaced by bulbous smut-gall at late stages of the disease, as well as the development of resistance in some Z. latifolia plants against U. esculenta infection.

New Insights of Ustilago maydis as Yeast Model for Genetic and Biotechnological Research: A Review.

The basidiomycete Ustilago maydis is a biotrophic organism responsible for corn smut disease. In recent years, it has become one of the most promising models for biochemical and biotechnological research due to advantages, such as rapid growth, and easy genetic manipulation. In some aspects, this yeast is more similar to complex eukaryotes, such as humans, compared to standard laboratory yeast models. U. maydis can be employed as a tool to explore physiological processes with more versatility than other fungi. Previously, U. maydis was only considered as a phytopathogenic fungus, but different studies have shown its potential as a research model. Therefore, numerous promising studies have focused on deepening our understanding of the natural interactions, enzyme production, and biotechnological capacity. In this review, we explore general characteristics of U. maydis, both as pathogenic and "innocuous" basidiomycete. Additionally, a comparison with other yeast models focusing on genetic, biochemical, and biotechnological research are analyzed, to emphasize the versatility, dynamism, and novelty that U. maydis has as a research model. In this review, we highlight the applications of the yeast form of the fungus; however, since the filamentous form is also of relevance, it is addressed in the present work, as well.

The WOPR Protein Ros1 Is a Master Regulator of Sporogenesis and Late Effector Gene Expression in the Maize Pathogen Ustilago maydis.

The biotrophic basidiomycete fungus Ustilago maydis causes smut disease in maize. Hallmarks of the disease are large tumors that develop on all aerial parts of the host in which dark pigmented teliospores are formed. We have identified a member of the WOPR family of transcription factors, Ros1, as major regulator of spore formation in U. maydis. ros1 expression is induced only late during infection and hence Ros1 is neither involved in plant colonization of dikaryotic fungal hyphae nor in plant tumor formation. However, during late stages of infection Ros1 is essential for fungal karyogamy, massive proliferation of diploid fungal cells and spore formation. Premature expression of ros1 revealed that Ros1 counteracts the b-dependent filamentation program and induces morphological alterations resembling the early steps of sporogenesis. Transcriptional profiling and ChIP-seq analyses uncovered that Ros1 remodels expression of about 30% of all U. maydis genes with 40% of these being direct targets. In total the expression of 80 transcription factor genes is controlled by Ros1. Four of the upregulated transcription factor genes were deleted and two of the mutants were affected in spore development. A large number of b-dependent genes were differentially regulated by Ros1, suggesting substantial changes in this regulatory cascade that controls filamentation and pathogenic development. Interestingly, 128 genes encoding secreted effectors involved in the establishment of biotrophic development were downregulated by Ros1 while a set of 70 "late effectors" was upregulated. These results indicate that Ros1 is a master regulator of late development in U. maydis and show that the biotrophic interaction during sporogenesis involves a drastic shift in expression of the fungal effectome including the downregulation of effectors that are essential during early stages of infection.

Virulence function of the Ustilago maydis sterol carrier protein 2.

The peroxisomal sterol carrier protein 2 (Scp2) of the biotrophic maize pathogen Ustilago maydis was detected in apoplastic fluid, suggesting that it might function as a secreted effector protein. Here we analyze the role of the scp2 gene during plant colonization. We used reverse genetics approaches to delete the scp2 gene, determined stress sensitivity and fatty acid utilization of mutants, demonstrated secretion of Scp2, used quantitative reverse transcription polymerase chain reaction for expression analysis and expressed GFP-Scp2 fusion proteins for protein localization. scp2 mutants were strongly attenuated in virulence and this defect manifested itself during penetration. Scp2 localized to peroxisomes and peroxisomal targeting was necessary for its virulence function. Deletion of scp2 in U. maydis interfered neither with growth nor with peroxisomal ß-oxidation. Conventionally secreted Scp2 protein could not rescue the virulence defect. scp2 mutants displayed an altered localization of peroxisomes. Our results show a virulence function for Scp2 during penetration that is probably carried out by Scp2 in peroxisomes. We speculate that Scp2 affects the lipid composition of membranes and in this way ensures the even cellular distribution of peroxisomes.

Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi.

Peroxisomes are eukaryotic organelles important for the metabolism of long-chain fatty acids. Here we show that in numerous fungal species, several core enzymes of glycolysis, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK), reside in both the cytoplasm and peroxisomes. We detected in these enzymes cryptic type 1 peroxisomal targeting signals (PTS1), which are activated by post-transcriptional processes. Notably, the molecular mechanisms that generate the peroxisomal isoforms vary considerably among different species. In the basidiomycete plant pathogen Ustilago maydis, peroxisomal targeting of Pgk1 results from ribosomal read-through, whereas alternative splicing generates the PTS1 of Gapdh. In the filamentous ascomycete Aspergillus nidulans, peroxisomal targeting of these enzymes is achieved by exactly the opposite mechanisms. We also detected PTS1 motifs in the glycolytic enzymes triose-phosphate isomerase and fructose-bisphosphate aldolase. U. maydis mutants lacking the peroxisomal isoforms of Gapdh or Pgk1 showed reduced virulence. In addition, mutational analysis suggests that GAPDH, together with other peroxisomal NADH-dependent dehydrogenases, has a role in redox homeostasis. Owing to its hidden nature, partial peroxisomal targeting of well-studied cytoplasmic enzymes has remained undetected. Thus, we anticipate that further bona fide cytoplasmic proteins exhibit similar dual targeting.

Programmed cell cycle arrest is required for infection of corn plants by the fungus Ustilago maydis.

Ustilago maydis is a plant pathogen that requires a specific structure called infective filament to penetrate the plant tissue. Although able to grow, this filament is cell cycle arrested on the plant surface. This cell cycle arrest is released once the filament penetrates the plant tissue. The reasons and mechanisms for this cell cycle arrest are unknown. Here, we have tried to address these questions. We reached three conclusions from our studies. First, the observed cell cycle arrest is the result of the cooperation of at least two distinct mechanisms: one involving the activation of the DNA damage response (DDR) cascade; and the other relying on the transcriptional downregulation of Hsl1, a kinase that modulates the G2/M transition. Second, a sustained cell cycle arrest during the infective filament step is necessary for the virulence in U. maydis, as a strain unable to arrest the cell cycle was severely impaired in its ability to infect corn plants. Third, production of the appressorium, a structure required for plant penetration, is incompatible with an active cell cycle. The inability to infect plants by strains defective in cell cycle arrest seems to be caused by their failure to induce the appressorium formation process. In summary, our findings uncover genetic circuits to arrest the cell cycle during the growth of this fungus on the plant surface, thus allowing the penetration into plant tissue.

Transcriptomic analysis of basidiocarp development in Ustilago maydis (DC) Cda.

Previously, we demonstrated that when Ustilago maydis (DC) Cda., a phytopathogenic basidiomycete and the causal agent of corn smut, is grown in the vicinity of maize embryogenic calli in a medium supplemented with the herbicide Dicamba, it developed gastroid-like basidiocarps. To elucidate the molecular mechanisms involved in the basidiocarp development by the fungus, we proceeded to analyze the transcriptome of the process, identifying a total of 2002 and 1064 differentially expressed genes at two developmental stages, young and mature basidiocarps, respectively. Function of these genes was analyzed with the use of different databases. MIPS analysis revealed that in the stage of young basidiocarp, among the ca. two thousand differentially expressed genes, there were some previously described for basidiocarp development in other fungal species. Additional elements that operated at this stage included, among others, genes encoding the transcription factors FOXO3, MIG3, PRO1, TEC1, copper and MFS transporters, and cytochromes P450. During mature basidiocarp development, important up-regulated genes included those encoding hydrophobins, laccases, and ferric reductase (FRE/NOX). The demonstration that a mapkk mutant was unable to form basidiocarps, indicated the importance of the MAPK signaling pathway in this developmental process.

Functional analysis of oxidative burst in sugarcane smut-resistant and -susceptible genotypes.

MAIN CONCLUSION: Smut pathogen induced an early modulation of the production and scavenging of reactive oxygen species during defence responses in resistant sugarcane that coincided with the developmental stages of fungal growth. Sporisorium scitamineum is the causal agent of sugarcane smut disease. In this study, we characterized sugarcane reactive oxygen species (ROS) metabolism in response to the pathogen in smut-resistant and -susceptible genotypes. Sporisorium scitamineum teliospore germination and appressorium formation coincided with H2O2 accumulation in resistant plants. The superoxide dismutase (SOD) activity was not responsive in any of the genotypes; however, a higher number of isoenzymes were detected in resistant plants. In addition, related to resistance were lipid peroxidation, a decrease in catalase (CAT), and an increase in glutathione S-transferase (GST) activities and an earlier transcript accumulation of ROS marker genes (CAT3, CATA, CATB, GST31, GSTt3, and peroxidase 5-like). Furthermore, based on proteomic data, we suggested that the source of the increased hydrogen peroxide (H2O2) may be due to a protein of the class III peroxidase, which was inhibited in the susceptible genotype. H2O2 is sensed and probably transduced through overlapping systems related to ascorbate-glutathione and thioredoxin to influence signalling pathways, as revealed by the presence of thioredoxin h-type, ascorbate peroxidase, and guanine nucleotide-binding proteins in the infected resistant plants. Altogether, our data depicted the balance of the oxidative burst and antioxidant enzyme activity in the outcome of this interaction.

The Hos2 Histone Deacetylase Controls Ustilago maydis Virulence through Direct Regulation of Mating-Type Genes.

Morphological changes are critical for host colonisation in plant pathogenic fungi. These changes occur at specific stages of their pathogenic cycle in response to environmental signals and are mediated by transcription factors, which act as master regulators. Histone deacetylases (HDACs) play crucial roles in regulating gene expression, for example by locally modulating the accessibility of chromatin to transcriptional regulators. It has been reported that HDACs play important roles in the virulence of plant fungi. However, the specific environment-sensing pathways that control fungal virulence via HDACs remain poorly characterised. Here we address this question using the maize pathogen Ustilago maydis. We find that the HDAC Hos2 is required for the dimorphic switch and pathogenic development in U. maydis. The deletion of hos2 abolishes the cAMP-dependent expression of mating type genes. Moreover, ChIP experiments detect Hos2 binding to the gene bodies of mating-type genes, which increases in proportion to their expression level following cAMP addition. These observations suggest that Hos2 acts as a downstream component of the cAMP-PKA pathway to control the expression of mating-type genes. Interestingly, we found that Clr3, another HDAC present in U. maydis, also contributes to the cAMP-dependent regulation of mating-type gene expression, demonstrating that Hos2 is not the only HDAC involved in this control system. Overall, our results provide new insights into the role of HDACs in fungal phytopathogenesis.

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.