bioRxiv preprint doi: https://doi.org/10.1101/475574; this version posted January 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Cellular Dynamics and Genomic Identity of Centromeres in the Cereal Blast Fungus 2 3 Vikas Yadav1¶,#a, Fan Yang2¶, Md. Hashim Reza1, Sanzhen Liu3, Barbara Valent3, Kaustuv 1* 2* 4 Sanyal , Naweed I. Naqvi 5 6 1Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific 7 Research, Jakkur, Bangalore, India 8 2Temasek Life Sciences Laboratory, and Department of Biological Sciences, 1 Research Link, 9 Singapore 10 3Department of Plant Pathology, Kansas State University, Manhattan, KS, USA 11 #aCurrent address: Department of Molecular Genetics and Microbiology, Duke University 12 Medical Center, Durham, NC, USA 13 14 * Corresponding authors: 15 E-mail: [email protected] (NIN); [email protected] (KS) 16 17 ¶These authors contributed equally to this work. 18 19 Running title: Centromeres in M. oryzae. 1 bioRxiv preprint doi: https://doi.org/10.1101/475574; this version posted January 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 20 Abstract 21 A series of well-synchronized events mediated by kinetochore-microtubule interactions ensure 22 faithful chromosome segregation in eukaryotes. Centromeres scaffold kinetochore assembly and 23 are among the fastest evolving chromosomal loci in terms of the DNA sequence, length, and 24 organization of intrinsic elements. Neither the centromere structure nor the kinetochore dynamics 25 is well studied in plant pathogenic fungi. Here, we sought to understand the process of 26 chromosome segregation in the rice blast fungus, Magnaporthe oryzae. High-resolution confocal 27 imaging of GFP-tagged inner kinetochore proteins, CenpA and CenpC, revealed an unusual 28 albeit transient declustering of centromeres just before anaphase separation in M. oryzae. 29 Strikingly, the declustered centromeres positioned randomly at the spindle midzone without an 30 apparent metaphase plate per se. Using chromatin immunoprecipitation followed by deep 31 sequencing, all seven centromeres were identified as CenpA-rich regions in the wild-type Guy11 32 strain of M. oryzae. The centromeres in M. oryzae are regional and span 57 to 109 kb 33 transcriptionally poor regions. No centromere-specific DNA sequence motif or repetitive 34 elements could be identified in these regions suggesting an epigenetic specification of 35 centromere function in M. oryzae. Highly AT-rich and heavily methylated DNA sequences were 36 the only common defining features of all the centromeres in rice blast fungus. PacBio genome 37 assemblies and synteny analyses facilitated comparison of the centromere regions in distinct 38 isolate(s) of rice blast, wheat blast, and in M. poae. Overall, this study identified unusual 39 centromere dynamics and precisely mapped the centromere DNA sequences in the top model 40 fungal pathogens that belong to the Magnaporthales and cause severe losses to global production 41 of food crops and turf grasses. 42 2 bioRxiv preprint doi: https://doi.org/10.1101/475574; this version posted January 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 43 Author summary 44 Magnaporthe oryzae is an important fungal pathogen that causes an annual loss of 10- 45 30% of the rice crop due to the devastating blast disease. In most organisms, kinetochores are 46 arranged either in the metaphase plate or are clustered together to facilitate synchronized 47 anaphase separation of chromosomes. In this study, we show that the initially clustered 48 kinetochores separate and position randomly prior to anaphase in M. oryzae. Centromeres, 49 identified as the site of kinetochore assembly, are regional type without any shared sequence 50 motifs in M. oryzae. Together, this study reveals atypical kinetochore dynamics and identifies 51 functional centromeres in M. oryzae, thus paving the way to define heterochromatin boundaries 52 and understand the process of kinetochore assembly on epigenetically specified centromere loci 53 in the economically important cereal blast and summer patch pathogens. This study paves the 54 way for understanding the contribution of heterochromatin in genome stability and virulence of 55 the blast fungus. 56 3 bioRxiv preprint doi: https://doi.org/10.1101/475574; this version posted January 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 57 Introduction 58 Faithful chromosome segregation is one of the essential processes required for 59 maintaining genome integrity in dividing cells. This process is successfully carried out by the 60 attachment of microtubules, emanating from opposite spindle poles, to the proteinaceous multi- 61 subunit structure, the kinetochore, that is pre-assembled onto centromeres [1, 2]. The centromere 62 forms a crucial part of this machinery and yet, it is one of the most rapidly evolving loci in 63 eukaryotic genomes [3, 4]. On the contrary, the proteins that bind to centromere DNA are 64 evolutionary conserved [2]. Centromere DNA shows a wide diversity in the length and 65 composition of the underlying DNA sequence. A few fungal species, like Saccharomyces 66 cerevisiae, harbor centromeres that are less than 400 bp comprising of conserved DNA sequence 67 elements to form point centromeres [5]. Most others possess regional centromeres that span from 68 a few kilobases to several megabases. Unlike point centromeres, regional centromeres in an 69 organism can span hundred of kbs and are often are defined by epigenetic factors. For example, 70 the regional centromeres in Schizosaccharomyces pombe and Candida tropicalis have a 71 homogenized central core flanked by inverted repeats [6, 7]. Likewise, the regional centromeres 72 in Cryptococcus neoformans possess specific retrotransposons that are present randomly therein 73 [8]. In contrast, centromeres in Candida albicans, Candida lusitaniae, and Candida dubliniensis 74 differ between all chromosomes and lack a conserved DNA sequence element [9-11]. 75 Centromeres in filamentous fungi like Neurospora crassa, on the other hand, span long stretches 76 of repetitive DNA but lack a consensus sequence or pattern [12, 13]. Metazoans and plants also 77 have regional centromeres that are up to few megabases long, and mostly consist of repetitive 78 DNA or transposons [14-16]. 4 bioRxiv preprint doi: https://doi.org/10.1101/475574; this version posted January 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 79 Despite this sequence divergence, centromeres in most studied organisms are bound by a 80 centromere-specific histone H3 variant CENP-A /CenH3/Cse4, also known as the hallmark of 81 centromere identity [4, 17]. CENP-A forms the foundation of the kinetochore assembly and is 82 essential for cell viability in all organisms studied until date. Evolutionary conservation of 83 CENP-A along with other kinetochore proteins also provides an efficient tool to identify 84 centromeres. Additionally, studies with fluorescently-labeled inner kinetochore proteins such as 85 CENP-A or CENP-C/Cen-C/Mif2 has led to an understanding of spatial dynamics of the 86 kinetochore within the nucleus [18-22]. These studies established that kinetochores in most yeast 87 species are clustered throughout the nuclear division, and unlike metazoan CEN, do not align on 88 a metaphase plate. However, more recently, some variations to the metaphase plate or 89 kinetochore clustering have been reported revealing the diversity in this phenomenon. 90 Kinetochores remain clustered throughout the cell cycle in two well-studied ascomycetes, S. 91 cerevisiae, and C. albicans [23, 24]. In S. pombe, kinetochores undergo a brief declustering 92 during mitosis but remain clustered otherwise [18, 25]. Another ascomycete, Zymoseptoria 93 tritici, shows multiple kinetochore foci instead of a single cluster during interphase although 94 their localization dynamics during mitosis remains unexplored [26]. On the other hand, the cells 95 of a basidiomycete C. neoformans display multiple foci of kinetochores in interphase, but 96 kinetochores gradually cluster during mitosis [19, 22]. Even the phenomenon of 97 centromere/kinetochore clustering is observed in Drosophila that depends on centric chromatin 98 rather than specific DNA sequences [27]. 99 Besides CENP-A, several other chromatin features are known to be associated with 100 centromeres. For example, centromeres are devoid of genes/ORFs and exhibit a significantly low 101 level of polyA transcription as compared to the rest of the genome [8, 28]. Furthermore, 5 bioRxiv preprint doi: https://doi.org/10.1101/475574; this version posted January 20, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 102 centromeres in many organisms are heterochromatic in nature and harbor the heterochromatic 103 marks like H3K9di/trimethylation and DNA methylation [8, 13, 29]. A preference