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The Life Cycle of Ustilago maydis . In this diagram, meiosis begins soon after karyogamy, pauses at pachytene during teliospore dispersal, and meiosis resumes during teliospore germination.
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Context 1
... are also integral to fungal meiosis. Smut and rust fungi are biotrophs, meaning they derive their nutrients from living plant hosts. This interaction is very intimate, involving fungal penetration of the plant cell walls but not the plasma membranes (e.g. Snetselaar & Mims, 1992; Voegele & Mendgen, 2003). As such, most smut and rust fungi have only evolved to infect (and become meiotically competent within) one or a limited number of host species. The economic impact of these pathogens is well illustrated by considering two crops significantly damaged by them: corn and wheat. According to Capitol Commodity Services (2011), corn remains the largest valued crop in the United States, totalling $67 billion in 2010. World-wide, corn crops were estimated at $163 billion in 2010 (U.S. Grains Council, 2010). The comparable numbers for wheat were $13 billion, and $140 billion, respectively (Capitol Commodity Services, 2011; U.S. Department of Agriculture, 2011). Although mitigated by varieties with partial resistance, the maize crop loss resulting from common smut of corn, caused by Ustilago maydis , is 2% annually, equivalent to ~$1 billion (Allen et al., 2011; Martinez-Espinoza et al., 2002). Wheat crop losses due to wheat leaf rust Puccinia triticina Eriks, which is the most common and widely distributed wheat rust, results in trace to 10% crop losses in many countries around the world. In the US, from 2000 to 2004, the loss was $350 million/year, and it can be $100 million/year in Canada. The production in China is more than twice that of the US and commonly suffers 10-30% crop loss per year (Huerta- Espino et al., 2011). There is also an extreme threat from emerging races of stripe rust of wheat ( Puccinia striiformis f. sp. tritici ) and wheat stem rust ( Puccinia graminis f. sp. tritici ). The emerging stem rust races are referred to collectively as UG99 after their location of origin (Uganda), and year of detection (1999). These races are virulent on the vast majority of wheat varieties cultivated around the world. It is predicted that if resistant varieties are not developed and utilized that the UG99 epidemic in Africa will become global (Singh et al., 2011). The impact of smut and rust fungi is limited by deploying resistant crop varieties; however, the fungi overcome the resistance leading to cycles in which varieties with new resistances are released and fungi with new virulence genotypes arise. While new virulence alleles ultimately result from mutation, genotypic diversity is created through recombination. Some populations of leaf rust have a genetic structure consistent with an asexual dikaryotic population “within which stepwise mutation at avirulence or virulence loci regularly occurs” (Ordoñez & Kolmer, 2009). In contrast, greatly increased genetic diversity and epidemics of stem rust have been linked to sexual reproduction (Burdon & Reolfs, 1985; Jin, 2011) and eradicating the alternate host for stem rust, common barberry and other Berberis spp. on which sexual reproduction occurs, has provided substantial benefit in controlling wheat stem rust (Roelfs, 1982) and, inadvertently, stripe rust of wheat (Jin, 2011). Further, the corn smut pathogen U. maydis exists in predominantly out-crossing populations (Barnes et al., 2004). This suggests a key role for sexual reproduction in the emergence and maintenance of virulence genotypes. The rust fungi are obligate biotrophs and cannot be cultured outside their hosts. The wheat rusts, as typified by stem rust, have five spore stages and require two completely unrelated hosts (Schumann & D’Arcy, 2009). The primary host is wheat and the alternative host is barberry. This complex and interesting life cycle will not be discussed in detail here except to note that, in the stem rust life cycle, meiosis likely initiates in planta followed by teliospore maturation (see paragraph below on rust teliospore microscopy). The diploid teliospores are produced late in the season on the primary host, wheat. They germinate and complete meiosis yielding basidiospores that infect the alternate host. In contrast to the rust fungi, the model fungal biotrophic pathogen U. maydis (Banuett, 1995; Brefort et al., 2009) is readily cultured in the laboratory on defined media and its sexual cycle can be completed within 28 days following injection of compatible haploid cells into seedlings of the host Zea mays (corn). U. maydis is amenable to genetic analysis and molecular manipulation, including homologous gene replacement, and several vectors are available for gene expression analysis. An annotated version of the genome sequence of U. maydis was released in 2007 (Kämper et al., 2006) and the annotation continues to be improved (e.g. Donaldson & Saville, 2008; Doyle et al., 2011; Ho et al., 2007; Kronstad, 2008; Morrison et al., in preparation). This allows molecular manipulation of U. maydis outside the host, followed by molecular analysis in the host. The U. maydis life cycle (Figure 1) begins with teliospore germination and the completion of meiosis to create haploid basidiospores, which divide by budding. Compatible non- pathogenic haploids fuse to form the pathogenic filamentous dikaryon, which proliferates, branches, and penetrates the plant via the formation of specialised cells called appressoria. It grows within and between plant cells eliciting the formation of a tumour. Banuett and Herskowitz (1996) describe a series of developmental events that U. maydis undergoes in the tumour leading to teliospore formation. These events occur within the enlarged host cells and include: 1) the formation of hyphal branches at close intervals, 2) the production of a mucilaginous matrix in which the hyphae are embedded and the hyphal tips become lobed, 3) hyphae fragmentation, 4) rounding of fragmented hyphae and 5) the deposition of a pigmented thick cell wall. The pigmented teliospores enter a dormant state, the tumours disintegrate, and the teliospores are dispersed, continuing the cycle. An overview of how meiosis proceeds in U. maydis was presented by Donaldson and Saville (2008). Since the early stages of meiosis occur in planta and meiosis is temporally linked to the formation of thick walled dormant teliospores, direct microscopic observation of meiotic events has not been possible. Therefore, it is informative to review how meiosis precedes in the related homobasidiomycete, Coprinopsis cinerea . This fungus can be induced to form mushrooms (fruiting bodies) in culture and, in these fruiting bodies, meiosis proceeds in a synchronous manner with over 60% of the approximately 10 million basidia in a given cap at the same stage (Pukkila et al., 1984). Kües, (2000) reviewed meiosis in C. cinerea and noted that chromatid duplication in premeiotic S phase is followed by karyogamy, and the cytological events of prophase I precede with the condensation and alignment of chromosomes (leptotene), synapsis (zygotene), and recombination nodule appearance (pachytene). This process, from post karyogamy to pachytene, is completed in six hours (Celerin et al., 2000). It is followed by desynapsis (diplotene) and the transition to metaphase (diakinesis). The second meiotic division occurs fairly rapidly following interphase, with prophase II through telophase II being completed in ~1 hour. The second division occurs in the same plane as the first, across the longitudinal axis of the basidium. Then, chromatid separation is followed by the four nuclei migrating toward the basidium tip where basidiospores form and the nuclei migrate into them then complete a round of mitosis. This overview of basidiomycete meiosis provides a framework for U. maydis investigations. U. maydis , like other smut fungi, does not form a fruiting body. Instead, when teliospores germinate, basidia are formed in which meiosis is completed. So while a C. cinerea fruiting body has millions of basidia undergoing meiosis, in U. maydis, millions of teliospores are dispersed and each produces a basidium. What we know of the cytological events of meiosis in U. maydis is that when hyphae are enveloped in the mucilaginous matrix during teliospore formation, they contain a single nucleus, indicating karyogamy has occurred (Banuett & Herskowitz, 1996). If U. maydis meiosis follows the pathway of C. cinerea, then premeiotic S phase and the duplication of chromatids would have been completed before karyogamy occurred. The next meiotic event we are aware of in U. maydis is the germination of the teliospore when the nucleus is in late prophase I (O’Donnell & McLaughlin, 1984). Between karyogamy and germination the teliospore is dormant with extremely limited metabolic activity. This indicates that major meiotic events cannot be occurring; this leaves the possibility that, either there is a pause after karyogamy and meiosis continues with teliospore germination, or that prophase I and recombination events begin immediately after karyogamy and pause, perhaps at the pachytene checkpoint, when the teliospore becomes dormant. Following germination, the metaphase I spindles align with the longitudinal axis of the metabasidium, and a transverse septum forms, indicating the completion of telophase I and leading to the formation of two cells (O’Donnell & McLaughlin, 1984). This is rapidly followed by meiosis II, in which the nucleus in each cell migrates to a central location, divides and septa are formed, resulting in three haploid nuclei, each in a basidium cell. The fourth nucleus migrates to the base of the teliospore (Ramberg & McLaughlin, 1980). Basidospores form by budding, each basidium cell nucleus migrates into the respective basidiospore and divides, then one nucleus remains and the other migrates back into the basidium cell (Banuett, 1995). Basidiospores continue to divide by budding. Support for the idea of meiosis proceeding immediately after karyogamy and pausing at pachytene comes from microscopic analysis of a number of rust ...
Context 2
... U. maydis research, the focus has been on signals leading to pathogenesis. A look at the life cycle of this fungus (Figure 1) illustrates how closely pathogenesis is tied to the events of sexual reproduction. There are differences given U. maydis is a basidiomycete, for example, when compatible haploid cells fuse they form a filamentous dikaryon and not a diploid. ...
Context 3
... cDNA libraries were constructed from cell-types, including: germinating and dormant teliospores (Sacadura & Saville, 2003; Ho et al., 2007), filamentous diploids (Nugent et al., 2004) and dikaryons (Morrison et al., in preparation). Recall that teliospore formation and germination are temporally linked to meiosis in U. maydis (reviewed in Donaldson & Saville, 2008). Of the 319 uniESTs that did not match an annotated gene model in the U. maydis genome, 108 uniquely represented RNAs expressed in the dormant or germinating teliospores. This corresponds to 34% of the identified ncRNAs, while these two cDNA libraries account for only 17% of the total ESTs from all cell-types (Saville, unpublished). In total, ~250 NATs have been identified in U. maydis , including NATs expressed in the dormant and germinating teliospores (55 and 12, respectively). The function of the NATs and ncRNAs in U. maydis is under investigation. Ten teliospore-specific NATs, annotated during analysis of the dormant teliospore cDNA library, have been verified as teliospore-specific, using strand- specific RT-PCR (Ho et al., 2010). Unlike S. cerevisiae and S. pombe , the U. maydis NATs expressed in the dormant and germinating teliospore are not enriched for mitosis-specific genes and the NATs expressed during vegetative growth are not enriched for meiosis- specific genes. Additionally, inverse expression patterns have not been observed for sense- antisense transcript pairs; precluding transcriptional interference as the principal mechanism of action for NATs in U. maydis . Therefore, their function in the dormant and germinating teliospores appears to be unique to U. maydis . This chapter provides an overview of meiotic events in the model fungal species Saccharomyces cerevisiae , Schizosaccharomyces pombe and Coprinopsis cinerea as a means of providing context for an exploration of meiosis in the model plant pathogen Ustilago maydis . Like the yeast fungi, U. maydis has set genetic requirements for entry into meiosis; it must be diploid and contain complementary alleles at the b mating type locus. With this genetic background, the fungus is able to accept an environmental signal that triggers entry into meiosis. This signal comes from the plant host and an exploration of the stages of pathogenic development led us to hypothesize that the stage before hyphal fragmentation, and after the cells become embedded in a mucilaginous matrix, is the time it must receive a signal from the plant to trigger entry into premeiotic S phase and undergo karyogamy. We uncover similarities between the role of the MAPK and cAMP/PKA pathways in mating and meiosis initiation in yeasts and the mating and pathogenesis signal transduction pathways in U. maydis . This is very relevant because of the requirement for growth within the host for U. maydis to become meiotically competent. These comparisons emphasized that the U. maydis genes Crk1 and Prf1 , which are orthologs of the major meiosis control genes Ime2 in S. cerevisiae and Ste11 in S. pombe respectively, provide a means whereby mating type and environmental signals could be transduced to influence meiosis. This led to a model of how meiosis is triggered in U. maydis and its linkage with teliospore development. We present an overview of waves in transcription in the yeasts and present evidence for potential waves of transcription in U. maydis . The identification of U. maydis meiosis genes by bioinformatic analyses is updated with an identification of the conserved absence of core meiosis genes in plant pathogenic fungi. We also present data that identifies UmNdt80 as the first gene known to be required for meiosis completion in U. maydis . The timing of expression of six core meiosis genes in U. maydis is followed during in planta development. This uncovered support for the model that U. maydis enters meiosis very soon after karyogamy and then arrests during pachytene, when the teliospore matures and enters a dormant state. This information also identified transcriptional and posttranscriptional control of Spo11 as potential key transitions in U. maydis meiotic progression. The final portion of the chapter highlights new data on the bioinformatic discovery of ncRNAs and NATs in U. maydis and their overrepresentation among ESTs in the teliospore and germinating teliospore libraries. In the context of the emerging role for these RNAs in controlling aspects of meiosis in S. cerevisiae and S. pombe, the discovery and confirmation of these RNAs in U. maydis is compelling. This chapter identifies several areas where further research will provide tremendous insight regarding meiosis initiation and progression in U. maydis . During proofing, gametogenesis initiation (van Werven & Amon, 2011) and RNAi- independent roles for antisense transcripts in controlling meiotic genes (Chen & Neiman, 2011) in budding and fission yeasts were reviewed. We would like to acknowledge Natalie Pearson for her excellent artwork in Figure 1, thank Laura Peers for fact finding and organizational support, and thank Christine Russell for assistance in proofing this manuscript. We also acknowledge funding from NSERC of Canada, and the Ontario Ministry of Research and Innovation’s Ontario Research Fund- Research Excellence program. Abe, H. & Shimoda, C. (2000). Autoregulated expression of Schizosaccharomyces pombe meiosis-specific transcription factor Mei4 and a genome-wide search for its target genes. Genetics, Vol.154, No.4, (April 2000), pp. 1497-1508, ISSN 0016-6731 Ahmed, N.T., Bungard, D., Shin, M.E., Moore, M. & Winter, E. (2009). The Ime2 protein kinase enhances the disassociation of the Sum1 repressor from middle meiotic promoters. Molecular and cellular biology, Vol.29, No.16, (August 2009), pp. 4352- 4362, ISSN 1098-5549 Allen, A., Islamovic, E., Kaur, J., Gold, S., Shah, D. & Smith, T.J. (2011). Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut. Plant biotechnology journal, (February 2011), ISSN 1467-7652 Alvarez, B. & Moreno, S. (2006). Fission yeast Tor2 promotes cell growth and represses cell differentiation. Journal of cell science, Vol.119, No.Pt 21, (November 2006), pp. 4475- 4485, ISSN 0021-9533 Averbeck, N., Sunder, S., Sample, N., Wise, J.A. & Leatherwood, J. (2005). Negative control contributes to an extensive program of meiotic splicing in fission yeast. Molecular cell, Vol.18, No.4, (May 2005), pp. 491-498, ISSN 1097-2765 Banuett, F. (2010). Ustilago maydis and Maize: a Delightful Interaction, In: Cellular and Molecular Biology of Filamentous Fungi, K.A. Borkovich & D.J. Ebbole, (Ed.), 622- 644, ASM Press, ISSN 978-1-55581-473-1, Washington, USA Banuett, F. (2002). Pathogenic development in Ustilago maydis : A progression of morphological transitions that results in tumor formation and teliospore production, In: Molecular biology of fungal development, H.D. Osiewacz, (Ed.), 349-398, Marcel Dekker, ISSN 0824707443, New York, USA Banuett, F. & Herskowitz, I. (1996). Discrete developmental stages during teliospore formation in the corn smut fungus, Ustilago maydis. Development, Vol.122, No.10, (October 1996), pp. 2965-2976, ISSN 0950-1991 Banuett, F. (1995).Genetics of Ustilago maydis , a fungal pathogen that induces tumors in maize. Annual Review of Genetics, Vol.29, (n.d.), pp. 179-208, ISSN 0066-4197 Banuett, F. & Herskowitz, I. (1989). Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis. Proceedings of the National Academy of Sciences of the United States of America, Vol.86, No.15, (August 1989), pp. 5878-5882, ISSN 0027-8424 Barnes, C.W., Szabo, L.J., May, G. & Groth, J.V. (2004). Inbreeding levels of two Ustilago maydis populations. Mycologia, Vol.96, No.6, (November 2004), pp. 1236-1244, ISSN 0027-5514 Bauman, P., Cheng, Q.C. & Albright, C.F. (1998). The Byr2 kinase translocates to the plasma membrane in a Ras1-dependent manner. Biochemical and biophysical research communications, Vol.244, No.2, (March 1998), pp. 468-474, ISSN 0006-291X Bellani, M.A., Boateng, K.A., McLeod, D. & Camerini-Otero, R.D. (2010). The expression profile of the major mouse SPO11 isoforms indicates that SPO11beta introduces double strand breaks and suggests that SPO11alpha has an additional role in prophase in both spermatocytes and oocytes. Molecular and cellular biology, Vol.30, No.18, (September 2010), pp. 4391-4403, ISSN 1098-5549 Boehm, E.W.A., Wenstrom, J.C., McLaughlin, D.J., Szabo, L.J., Roelfs, A.P. & Bushnell, W.R. (1992). An ultrastructural pachytene karyotype for Puccinia graminis f. sp. tritici. Canadian Journal of Botany, Vol.70, (n.d.), pp. 401-4113, ISSN 0008-4026 Borkovich, K.A., Alex, L.A., Yarden, O., Freitag, M., Turner, G.E., Read, N.D., Seiler, S., Bell- Pedersen, D., Paietta, J., Plesofsky, N., Plamann, M., Goodrich-Tanrikulu, M., Schulte, U., Mannhaupt, G., Nargang, F.E., Radford, A., Selitrennikoff, C., Galagan, J.E., Dunlap, J.C., Loros, J.J., Catcheside, D., Inoue, H., Aramayo, R., Polymenis, M., Selker, E.U., Sachs, M.S., Marzluf, G.A., Paulsen, I., Davis, R., Ebbole, D.J., Zelter, A., Kalkman, E.R., O'Rourke, R., Bowring, F., Yeadon, J., Ishii, C., Suzuki, K., Sakai, W. & Pratt, R. (2004). Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiology and molecular biology reviews, Vol.68, No.1, (March 2004), pp. 1-108, ISSN 1092-2172 Brefort, T., Doehlemann, G., Mendoza-Mendoza, A., Reissmann, S., Djamei, A. & Kahmann, R. (2009). Ustilago maydis as a Pathogen. Annual Review of Phytopathology, Vol.47, (n.d.), pp. 423-445, ISSN 0066-4286 Burdon, J.J. & Roelfs, A.P. (1985). The effect of sexual and asexual reproduction on the isozyme structure of populations of Puccinia graminis. Phytopathology, Vol.75, (n.d.), pp. 1068-1073, ISSN 0031-949X Burns, C., Pukkila, P.J. & Miriam, Z.E. (2010a). Meiosis, In: Cellular and ...
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... These dikaryotic hyphae invade the plant and lead to the characteristic symptoms of the disease: chlorosis, formation of anthocyanins, the development of galls into the aerial parts of the plant (in which diploid teliospores are accumulated). Teliospores germinate outside the host and produce phragmobasidia, which give rise to budding basidiospores, reinitiating the cycle (Brefort et al., 2009;Saville et al., 2012;Snetselaar and McCann, 2017). ...
Introduction
Biological systems respond to environmental disturbances and a wide range of compounds through complex gene interaction networks. The enormous growth of experimental information obtained using large-scale genomic techniques such as microarrays and RNA sequencing led to the construction of a wide variety of gene co-expression networks in recent years. These networks allow the discovery of clusters of co-expressed genes that potentially work in the same process linking them to biological processes often of interest to industrial, medicinal, and academic research.
Methods
In this study, we built the gene co-expression network of Ustilago maydis from the gene expression data of 168 samples belonging to 19 series, which correspond to the GPL3681 platform deposited in the NCBI using WGCNA software. This network was analyzed to identify clusters of co-expressed genes, gene hubs and Gene Ontology terms. Additionally, we identified relevant modules through a hypergeometric approach based on a predicted set of transcription factors and virulence genes.
Results and Discussion
We identified 13 modules in the gene co-expression network of U. maydis. The TFs enriched in the modules of interest belong to the superfamilies of Nucleic acid-binding proteins, Winged helix DNA-binding, and Zn2/Cys6 DNA-binding. On the other hand, the modules enriched with virulence genes were classified into diseases related to corn smut, Invasive candidiasis, among others. Finally, a large number of hypothetical, a large number of hypothetical genes were identified as highly co-expressed with virulence genes, making them possible experimental targets.
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Los autores.
... Fungal biomass accumulates in tumours with the eventual formation of masses of black, sooty teliospores, which facilitate dispersal of the pathogen (Matei and Doehlemann, 2016). Smut fungi cause economically significant crop losses, particularly on cereals, and U. maydis is also a model organism for studying biotrophic plant-pathogen interactions (Saville et al., 2012). In this context, analysis of the genome sequence of U. maydis revealed clusters of genes that were expressed during maize infection and that encoded predicted, secreted proteins (K€ amper et al., 2006). ...
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 β-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 β-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. This article is protected by copyright. All rights reserved.
... During in planta growth, U. maydis cells undergo developmental transitions and it is possible that these are linked to changes in carbon source availability. For example, many fungi enter into meiosis in response to nutrient limitation (reviewed in Saville et al. 2012), so investigating the impact of carbon source shifts, including the uptake of galacturonic acid, on the initiation of teliospore production could be informative. One approach may be to determine the impact of growing U. maydis on single carbon sources such as galacturonic acid or specific sugars, on the expression of transcriptional regulators of meiosis like unh1 (Doyle et al. 2016). ...
Throughout the plant disease cycle, biotrophic fungal pathogens must obtain host-derived carbon molecules to act as building blocks and sources of energy. Gaining access to these resources requires biotrophic fungi to breach plant cell walls without eliciting substantial plant defences. The plant cell wall is composed mostly of glucose- and xylose-based polysaccharides, which can support fungal growth. Thus, fungi can acquire carbon compounds through the targeted depolymerization of specific wall components. When the plant cell wall is breached, biotrophs redirect photoassimilates, increase sink strengths, and express invertases and transporters to acquire carbon compounds. Transitions in enzyme and transporter expression during pathogenesis must be tightly controlled to ensure a continued supply of carbon compounds. This review describes carbon acquisition and metabolism, including regulation of available carbon source utilization mechanisms such as carbon catabolite repression. While carbon acquisition has been extensively studied in the ascomycetes, the mechanisms used by biotrophic fungi to acquire carbon during pathogenesis are poorly understood. Furthermore, the relationship between plant cell wall-degrading enzymes and carbon acquisition in biotrophic fungal pathogens is not well characterized. As such, this review summarizes the current knowledge of carbon source utilization by fungal pathogens, with an emphasis on research involving the corn smut pathogen Ustilago maydis, and provides a basis from which to extend our knowledge in this key area of fungal plant pathogenesis.
... The smut (Ustilaginales) and rust (Pucciniales) fungi are basidiomycete plant pathogens responsible for billions of dollars in crop losses annually ( Saville et al., 2012). Both groups of fungi produce teliospores which are dispersal agents and the protective structures for sexual reproduction. ...
The basidiomycete smut fungus Ustilago maydis causes common smut of corn. This disease is spread through the production of teliospores, which are thick-walled dormant structures characterized by low rates of respiration and metabolism. Teliospores are formed when the fungus grows within the plant, and the morphological steps involved in their formation have been described, but the molecular events leading to dormancy are not known. In U. maydis, natural antisense transcripts (NATs) can function to alter gene expression and many NATs have increased levels in the teliospore. One such NAT is as-ssm1 which is complementary to the gene for the mitochondrial seryl-tRNA synthetase (ssm1), an enzyme important to mitochondrial function. The disruption of ssm1 leads to cell lysis, indicating it is also essential for cellular viability. To assess the function of as-ssm1, it was ectopically expressed in haploid cells, where it is not normally present. This expression led to reductions in growth rate, virulence, mitochondrial membrane potential and oxygen consumption. It also resulted in the formation of as-ssm1/ssm1 double-stranded RNA and increased ssm1 transcript levels, but no change in Ssm1 protein levels was detected. Together, these findings suggest a role for as-ssm1 in facilitating teliospore dormancy through dsRNA formation and reduction of mitochondrial function. This article is protected by copyright. All rights reserved.
... Meiosis initiates during the formation of teliospores in the plant, and arrests in late prophase I when the teliospores enter dormancy (Kojic et al., 2013). Following dispersal, teliospores germinate and resume meiosis, completing meiotic divisions and generating haploid cells (Kojic et al., 2013;Saville et al., 2012). Meiosis and teliospore formation are temporally linked in U. maydis; however, up to 3% (Christensen, 1931;Kojic et al., 2002), or as high as 10% (Holliday, 1961) of the teliospores fail to complete meiosis and germinate as diploids. ...
... smuts and rusts require growth in the host plant to initiate meiosis and this initiation is linked to pathogenic development. In these fungi meiosis is paused at a time analogous to the pachytene checkpoint, or meiotic recombination checkpoint in Saccharomyces cerevisiae (Donaldson and Saville, 2008;Kojic et al., 2013;Saville et al., 2012). In S. cerevisiae this checkpoint prevents the cell from exiting prophase I and entering into meiotic divisions if recombination has not been completed (Bailis and Roeder, 2000). ...
... In S. cerevisiae this checkpoint prevents the cell from exiting prophase I and entering into meiotic divisions if recombination has not been completed (Bailis and Roeder, 2000). The progression of meiosis is consistent among sexually reproducing fungi; however, the external cues that induce entry into meiosis and the control of meiotic progression are less conserved (Saville et al., 2012). ...
In this study, Ustilago maydis Ndt80 homolog one, unh1, of the obligate sexual pathogen Ustilago maydis,is described. Unh1 is the sole Ndt80-like DNA-binding protein inU. maydis. In this model basidiomycete, Unh1 plays a role in sexual development, influencing tumor maturation, teliospore development and subsequent meiotic completion. Teliospore formation was reduced in deletion mutants, and those that did form had unpigmented, hyaline cell walls, and germinated without completing meiosis. Constitutively expressing unh1 in haploid cells resulted in abnormal pigmentation, when grown in both potato dextrose broth and minimal medium, suggesting that pigmentation may be triggered by unh1 in U. maydis. The function of Unh1 in sexual development and pigment production depends on the presence of the Ndt80-like DNA-binding domain, identified within Unh1. In the absence of this domain, or when the binding domain was altered with targeted amino acid changes, ectopic expression of Unh1 failed to complement the unh1 deletion with regards to pigment production and sexual development. An investigation of U. maydis genes with upstream motifs similar to Ndt80 recognition sequences revealed that some have altered transcript levels in Δunh1 strains. We propose that the first characterized Ndt80-like DNA-binding protein in a basidiomycete, Unh1, acts as a transcription factor that is required for teliospore maturation and the completion of meiosis in U. maydis.
... After 11 days, large masses of rounded uninucleate cells are observed and by day 14, mature teliospores with characteristic echinulate, melanin-rich cell walls appear in abundance. Given that the meiotic program initiates immediately following karyogamy in a number of fungal model systems (Rossen and Westergaard 1966;Egel and Egel-Mitani 1974;Lu and Jeng 1975;Iyengar et al. 1977;Carmi et al. 1978;Li et al. 1999), it seemed reasonable to us and others (Saville et al. 2012) to suppose that a similar process could be in place in U. maydis and that homologous recombination becomes activated during the infection stage in plants, after nuclear fusion but before teliospore maturation and not coincident with teliospore germination. ...
A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar(+) recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.
... Solopathogenic Dust1 strains were unable to induce tumors or form teliospores in infected seedlings (Garcia-Pedrajas et al., 2010). Expression of um04778 transcripts during U. maydis growth in the plant (Fig. 1a) is consistent with a role in controlling gene expression related to hyphal fragmentation, teliospore formation, teliospore maturation, or the temporally linked karyogamy and meiosis (Banuett and Herskowitz, 1996;Saville et al., 2012). Functional analysis of um04778 is underway. ...
The sustainable control of basidiomycete biotrophic plant pathogenesis requires an understanding of host responses to infection, as well as the identification and functional analysis of fungal genes involved in disease development. The creation and analysis of a suppressive subtractive hybridization (SSH) cDNA library from Ustilago maydis-infected Zea mays seedlings enabled the identification of fungal and plant genes expressed during disease development, and uncovered new insights into the interactions of this model system. Candidate U. maydis pathogenesis genes were identified by using the current SSH cDNA library analysis, and by knowledge generated from previous cDNA microarray and comparative genomic analyses. These identifications were supported by the independent determination of transcript level changes in different cell-types and during pathogenic development. The basidiomycete specific um01632, the highly in planta expressed um03046 (zig1), and the calcineurin regulatory B subunit (um10226, cnb1), were chosen for deletion experiments. um01632 and zig1 mutants showed no difference in morphology and did not have a statistically significant impact on pathogenesis. cnb1 mutants had a distinct cell division phenotype and reduced virulence in seedling assays. Infections with reciprocal wild-type × Δcnb1 haploid strain crosses revealed that the wild-type allele was unable to fully compensate for the lack of a second cnb1 allele. This haploinsufficiency was undetected in other fungal cnb1 mutational analyses. The reported data improves U. maydis genome annotation and expands on the current understanding of pathogenesis genes in this model basidiomycete.
... Second, all genes coding for the proteins involved in the mitogen-activated protein kinase and cAMP signaling cascades downstream of the G protein–coupled pheromone receptor and leading to crucial morphological and physiological modifications in pathogenic Ustilaginales were present and mostly highly conserved in P. flocculosa (see Supplemental Data Set 2 online). In U. maydis, meiosis occurs after the mating of compatible haploid mating types and in planta development of diploid teliospores (Saville et al., 2012). All genes reported to be involved in meiosis and sporulation were found in the genome of P. flocculosa (see Supplemental Data Set 3 online). ...
Pseudozyma flocculosa is related to the model plant pathogen Ustilago maydis yet is not a phytopathogen but rather a biocontrol agent of powdery mildews; this relationship makes it unique for the study of the evolution of plant pathogenicity factors. The P. flocculosa genome of ∼23 Mb includes 6877 predicted protein coding genes. Genome features, including hallmarks of pathogenicity, are very similar in P. flocculosa and U. maydis, Sporisorium reilianum, and Ustilago hordei. Furthermore, P. flocculosa, a strict anamorph, revealed conserved and seemingly intact mating-type and meiosis loci typical of Ustilaginales. By contrast, we observed the loss of a specific subset of candidate secreted effector proteins reported to influence virulence in U. maydis as the singular divergence that could explain its nonpathogenic nature. These results suggest that P. flocculosa could have once been a virulent smut fungus that lost the specific effectors necessary for host compatibility. Interestingly, the biocontrol agent appears to have acquired genes encoding secreted proteins not found in the compared Ustilaginales, including necrosis-inducing-Phytophthora-protein- and Lysin-motif- containing proteins believed to have direct relevance to its lifestyle. The genome sequence should contribute to new insights into the subtle genetic differences that can lead to drastic changes in fungal pathogen lifestyles.
... The tumours dry out and crack, leading to the dispersal of teliospores, which can remain dormant for years (Christensen, 1963 ). Teliospore germination and meiosis are temporally linked (reviewed in Saville et al., 2012), producing haploid sporidia which can initiate new rounds of infection. The numerous characteristics that have established U. maydis as the model biotrophic fungal plant pathogen have been well reviewed (Banuett, 1995; Bölker, 2001; Martinez-Espinoza et al., 2002; Basse and Steinberg, 2004; Kahmann and Kämper, 2004; Steinberg and Perez-Martin, 2008; Brefort et al., 2009; Dean et al., 2012; Djamei and Kahmann, 2012). ...
Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a two-fold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis.
