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.

Plant surface cues prime Ustilago maydis for biotrophic development.

Infection-related development of phytopathogenic fungi is initiated by sensing and responding to plant surface cues. This response can result in the formation of specialized infection structures, so-called appressoria. To unravel the program inducing filaments and appressoria in the biotrophic smut fungus Ustilago maydis, we exposed cells to a hydrophobic surface and the cutin monomer 16-hydroxy hexadecanoic acid. Genome-wide transcriptional profiling at the pre-penetration stage documented dramatic transcriptional changes in almost 20% of the genes. Comparisons with the U. maydis sho1 msb2 double mutant, lacking two putative sensors for plant surface cues, revealed that these plasma membrane receptors regulate a small subset of the surface cue-induced genes comprising mainly secreted proteins including potential plant cell wall degrading enzymes. Targeted gene deletion analysis ascribed a role to up-regulated GH51 and GH62 arabinofuranosidases during plant penetration. Among the sho1/msb2-dependently expressed genes were several secreted effectors that are essential for virulence. Our data also demonstrate specific effects on two transcription factors that redirect the transcriptional regulatory network towards appressorium formation and plant penetration. This shows that plant surface cues prime U. maydis for biotrophic development.

Taz protects hematopoietic stem cells from an aging-dependent decrease in PU.1 activity.

Specific functions of the immune system are essential to protect us from infections caused by pathogens such as viruses and bacteria. However, as we age, the immune system shows a functional decline that can be attributed in large part to age-associated defects in hematopoietic stem cells (HSCs)-the cells at the apex of the immune cell hierarchy. Here, we find that the Hippo pathway coactivator TAZ is potently induced in old HSCs and protects these cells from functional decline. We identify Clca3a1 as a TAZ-induced gene that allows us to trace TAZ activity in vivo. Using CLCA3A1 as a marker, we can isolate "young-like" HSCs from old mice. Mechanistically, Taz acts as coactivator of PU.1 and to some extent counteracts the gradual loss of PU.1 expression during HSC aging. Our work thus uncovers an essential role for Taz in a previously undescribed fail-safe mechanism in aging HSCs.

Symbiosis-related pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices.

The arbuscular mycorrhiza association results from a successful interaction between genomes of the plant and fungal symbiotic partners. In this study, we analyzed the effect of inactivation of late-stage symbiosis-related pea genes on symbiosis-associated fungal and plant molecular responses in order to gain insight into their role in the functional mycorrhizal association. The expression of a subset of ten fungal and eight plant genes, previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated wild-type and isogenic genotypes of pea mutated for the PsSym36, PsSym33, and PsSym40 genes where arbuscule formation is inhibited or fungal turnover modulated, respectively. Microdissection was used to corroborate arbuscule-related fungal gene expression. Molecular responses varied between pea genotypes and with fungal development. Most of the fungal genes were downregulated when arbuscule formation was defective, and several were upregulated with more rapid fungal development. Some of the plant genes were also affected by inactivation of the PsSym36, PsSym33, and PsSym40 loci, but in a more time-dependent way during root colonization by G. intraradices. Results indicate a role of the late-stage symbiosis-related pea genes not only in mycorrhiza development but also in the symbiotic functioning of arbuscule-containing cells.

An STE12 gene identified in the mycorrhizal fungus Glomus intraradices restores infectivity of a hemibiotrophic plant pathogen.

Mechanisms of root penetration by arbuscular mycorrhizal (AM) fungi are unknown and investigations are hampered by the lack of transformation systems for these unculturable obligate biotrophs. Early steps of host infection by hemibiotrophic fungal phytopathogens, sharing common features with those of AM fungal colonization, depend on the transcription factor STE12. Using degenerated primers and rapid amplification of cDNA ends, we isolated the full-length cDNA of an STE12-like gene, GintSTE, from Glomus intraradices and profiled GintSTE expression by real-time and in situ RT-PCR. GintSTE activity and function were investigated by heterologous complementation of a yeast ste12Delta mutant and a Colletotrichum lindemuthianum clste12Delta mutant. * Sequence data indicate that GintSTE is similar to STE12 from hemibiotrophic plant pathogens, especially Colletotrichum spp. Introduction of GintSTE into a noninvasive mutant of C. lindemuthianum restored fungal infectivity of plant tissues. GintSTE expression was specifically localized in extraradicular fungal structures and was up-regulated when G. intraradices penetrated roots of wild-type Medicago truncatula as compared with an incompatible mutant. Results suggest a possible role for GintSTE in early steps of root penetration by AM fungi, and that pathogenic and symbiotic fungi may share common regulatory mechanisms for invasion of plant tissues.

Development and activity of Glomus intraradices as affected by co-existence with Glomus claroideum in one root system.

The co-existence of two arbuscular mycorrhizal fungal (AMF) species, Glomus intraradices and Glomus claroideum, in the root systems of plants was investigated in a greenhouse experiment aimed at reconstructing interactions during an early stage of primary succession on a coal-mine spoil bank in Central Europe. Two plant species, Tripleurospermum inodorum and Calamagrostis epigejos, were inoculated either with one or both AMF species. Fungal development, determined by trypan blue and alkaline phosphatase staining as well as by PCR amplification of rRNA genes with species-specific primers, and the expression of five genes with different metabolic functions in the intraradical structures of G. intraradices were followed after 6 and 9 weeks of cultivation. The two AMF closely co-existed in the root systems of both plants possibly through similar colonisation rates and competitivity. Inoculation with the two fungi, however, did not bring any additional benefit to the host plants in comparison with single inoculation; moreover, plant growth depression observed after inoculation with G. claroideum persisted also in mixed inoculation. The expression of all the assayed G. intraradices genes was affected either by host plant or by co-inoculation with G. claroideum. The effects of both factors depended on the time of sampling, which underlines the importance of addressing this topic in time-course studies.

Spatial monitoring of gene activity in extraradical and intraradical developmental stages of arbuscular mycorrhizal fungi by direct fluorescent in situ RT-PCR.

Gene expression profiling based on tissue extracts gives only limited information about genes associated with complex developmental processes such as those implicated in fungal interactions with plant roots during arbuscular mycorrhiza development and function. To overcome this drawback, a direct fluorescent in situ RT-PCR methodology was developed for spatial mapping of gene expression in different presymbiotic and symbiotic structures of an arbuscular mycorrhizal fungus. Transcript detection was optimized by targeting the LSU rRNA gene of Glomus intraradices and monitoring expression of a stearoyl-CoA-desaturase gene that is consistently expressed at high levels in spores, hyphae, arbuscules and vesicles. This method was further validated by localizing expression of fungal peptidylprolyl isomerase and superoxide dismutase genes, which are expressed to different extents in fungal structures. Direct fluorescent in situ RT-PCR offers new perspectives for the sensitive analysis of fungal developmental processes that occur during functional differentiation in symbiotic arbuscular mycorrhiza interactions.

Author Correction: TRPS1 shapes YAP/TEAD-dependent transcription in breast cancer cells.

In the original version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include the following: "This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001144), the UK Medical Research Council (FC001144), and the Wellcome Trust (FC001144)." https://doi.org/10.1038/s41467-018-05370-7 .

TRPS1 shapes YAP/TEAD-dependent transcription in breast cancer cells.

Yes-associated protein (YAP), the downstream transducer of the Hippo pathway, is a key regulator of organ size, differentiation and tumorigenesis. To uncover Hippo-independent YAP regulators, we performed a genome-wide CRISPR screen that identifies the transcriptional repressor protein Trichorhinophalangeal Syndrome 1 (TRPS1) as a potent repressor of YAP-dependent transactivation. We show that TRPS1 globally regulates YAP-dependent transcription by binding to a large set of joint genomic sites, mainly enhancers. TRPS1 represses YAP-dependent function by recruiting a spectrum of corepressor complexes to joint sites. Loss of TRPS1 leads to activation of enhancers due to increased H3K27 acetylation and an altered promoter-enhancer interaction landscape. TRPS1 is commonly amplified in breast cancer, which suggests that restrained YAP activity favours tumour growth. High TRPS1 activity is associated with decreased YAP activity and leads to decreased frequency of tumour-infiltrating immune cells. Our study uncovers TRPS1 as an epigenetic regulator of YAP activity in breast cancer.

Ustilago maydis effectors and their impact on virulence.

Biotrophic fungal plant pathogens establish an intimate relationship with their host to support the infection process. Central to this strategy is the secretion of a range of protein effectors that enable the pathogen to evade plant immune defences and modulate host metabolism to meet its needs. In this Review, using the smut fungus Ustilago maydis as an example, we discuss new insights into the effector repertoire of smut fungi that have been gained from comparative genomics and discuss the molecular mechanisms by which U. maydis effectors change processes in the plant host. Finally, we examine how the expression of effector genes and effector secretion are coordinated with fungal development in the host.

Fungal effectors and plant susceptibility.

Plants can be colonized by fungi that have adopted highly diverse lifestyles, ranging from symbiotic to necrotrophic. Colonization is governed in all systems by hundreds of secreted fungal effector molecules. These effectors suppress plant defense responses and modulate plant physiology to accommodate fungal invaders and provide them with nutrients. Fungal effectors either function in the interaction zone between the fungal hyphae and host or are transferred to plant cells. This review describes the effector repertoires of 84 plant-colonizing fungi. We focus on the mechanisms that allow these fungal effectors to promote virulence or compatibility, discuss common plant nodes that are targeted by effectors, and provide recent insights into effector evolution. In addition, we address the issue of effector uptake in plant cells and highlight open questions and future challenges.

Expression profiling of fungal genes during arbuscular mycorrhiza symbiosis establishment using direct fluorescent in situ RT-PCR.

Expression profiling of fungal genes in the arbuscular mycorrhiza (AM) symbiosis has been based on studies of RNA extracted from fungal tissue or mycorrhizal roots, giving only a general picture of overall transcript levels in the targeted tissues. Information about the spatial distribution of transcripts within AM fungal structures during different developmental stages is essential to a better understanding of fungal activity in symbiotic interactions with host roots and to determine molecular events involved in establishment and functioning of the AM symbiosis. The obligate biotrophic nature of AM fungi is a challenge for developing new molecular methods to identify and localize their activity in situ. The direct fluorescent in situ (DIFIS) RT-PCR procedure described here represents a novel tool for spatial mapping of AM fungal gene expression simultaneously prior to root penetration, within fungal tissues in the host root and in the extraradical stage of fungal development.In order to enhance detection sensitivity of the in situ RT-PCR technique and enable localization of low abundance mRNA, we have adopted direct fluorescent labeling of primers for the amplification step to overcome the problem of low detection associated with digoxigenin or biotin-labeled primers and to avoid the multiplicity of steps associated with immunological detection. Signal detection has also been greatly improved by eliminating autofluorescence of AM fungal and root tissues using confocal microscopy.

Changes in membrane lipid composition in ethanol- and acid-adapted Oenococcus oeni cells: characterization of the cfa gene by heterologous complementation.

Cyclopropane fatty acid (CFA) synthesis was investigated in Oenococcus oeni. The data obtained demonstrated that acid-grown cells or cells harvested in the stationary growth phase showed changes in fatty acid composition similar to those of ethanol-grown cells. An increase of the CFA content and a decrease of the oleic acid content were observed. The biosynthesis of CFAs from unsaturated fatty acid phospholipids is catalysed by CFA synthases. Quantitative real-time-PCR experiments were performed on the cfa gene of O. oeni, which encodes a putative CFA synthase. The level of cfa transcripts increased when cells were harvested in stationary phase and when cells were grown in the presence of ethanol or at low pH, suggesting transcriptional regulation of the cfa gene under different stress conditions. In contrast to Escherichia coli, only one functional promoter was identified upstream of the cfa gene of O. oeni. The function of the cfa gene was confirmed by complementation of a cfa-deficient E. coli strain. Nevertheless, the complementation remained partial because the conversion percentage of unsaturated fatty acids into CFA of the complemented strain was much lower than that of the wild-type strain. Moreover, a prevalence of cycC19 : 0 was observed in the membrane of the complemented strain. This could be due to a specific affinity of the CFA synthase from O. oeni. In spite of this partial complementation, the complemented strain of E. coli totally recovered its viability after ethanol shock (10 %, v/v) whereas its viability was only partly recovered after an acid shock at pH 3.0.