Canadian Journal of Microbiology The mitochondrial genome of Ophiostoma himal-ulmi and comparison with other Dutch elm disease causing fungi. Journal: Canadian Journal of Microbiology Manuscript ID cjm-2020-0589.R1 Manuscript Type: Article Date Submitted by the 04-Feb-2021 Author: Complete List of Authors: Wai, Alvan; University of Manitoba, Hausner, Georg; University of Manitoba, Buller Building 213 Keyword: Introns, Ophiostomatales, homing endonucleases, Dutch Elm Disease Is the invited manuscript for Draft consideration in a Special Not applicable (regular submission) Issue? : © The Author(s) or their Institution(s) Page 1 of 42 Canadian Journal of Microbiology 1 The mitochondrial genome of Ophiostoma himal-ulmi and comparison with other Dutch 2 elm disease causing fungi. 3 4 Alvan Wai and Georg Hausner# 5 Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, R3T 2N2 6 #Corresponding author: [email protected] 7 8 Draft 1 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 2 of 42 9 Abstract 10 The mitochondrial genome of Ophiostoma himal-ulmi, a species endemic to the Western 11 Himalayas and a member of the Dutch elm disease-causing fungi, has been sequenced and 12 characterized. The mitochondrial genome was compared with other available genomes for 13 members of the Ophiostomatales, including other agents of Dutch elm disease (Ophiostoma ulmi, 14 Ophiostoma novo-ulmi subspecies novo-ulmi and Ophiostoma novo-ulmi subspecies americana) 15 and it was observed that gene synteny is highly conserved and variability among members of the 16 Dutch-elm disease-causing fungi is primarily due to the number of intron insertions. Among the 17 Dutch elm disease-causing fungi examined, O. himal-ulmi has the largest mitochondrial genomes 18 ranging from 94 934 bp to 111 712 bp due to the expansion of the number of introns. 19 Draft 20 Keywords: introns, Ophiostomatales, homing endonucleases, Dutch elm disease 21 2 © The Author(s) or their Institution(s) Page 3 of 42 Canadian Journal of Microbiology 22 Introduction 23 Brasier and Mehrotra (1995) conducted a mycological survey in northern Himachal 24 Pradesh (Western Himalayas) with the focus on Ophiostoma species isolated from breeding 25 galleries of scolytid beetles present within the bark of Ulmus wallichiana. They isolated an 26 Ophiostoma species that resembled Ophiostoma ulmi but had a set of physiological and 27 morphological features that set it apart from O. ulmi and Ophiostoma novo-ulmi. In addition, 28 interfertility tests demonstrated reproductive isolation, therefore this new species was designated 29 as Ophiostoma himal-ulmi. Brasier and Mehrotra hoped that the discovery of this potential 30 “sibling species” may contribute towards a better understanding of the origins of Dutch elm 31 disease. Historically Ophiostoma ulmi (Buisman) Melin & Nannf. (1934) was viewed to be the 32 causative agent of Dutch elm disease (DED).Draft However, more recently it has been recognized that 33 O. ulmi has been replaced by more aggressive forms assigned to Ophiostoma novo-ulmi (Brasier 34 1991). Members of the O. ulmi species complex are vectored by bark beetles and are agents of a 35 devastating wilt disease that had and continues to have significant impact on urban forests. 36 Ophiostoma novo-ulmi based on cultural and molecular characters has been separated into two 37 subspecies: O. novo-ulmi subsp. novo-ulmi and O. novo-ulmi subsp. americana (Brasier and Kirk 38 2001; Harrington et al. 2001). Both subspecies are well established in Europe but so far O. novo- 39 ulmi subsp. novo-ulmi has only been reported from Europe and Eurasia (Brasier 2001; Brasier 40 and Buck 2001). 41 Most fungi are obligate aerobes so functional mitochondria are essential for their survival 42 and fitness. With regards to structure, mitochondrial DNA can be circular, linear, and in a few 43 cases segmented (i.e., multi-chromosomal) (Valach et al. 2011; Lang 2018). For most fungi, 44 mitochondrial genomes exist as multiple copies and are represented as circular molecules, and 3 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 4 of 42 45 are compacted into mitochondrial nucleoids possibly composed of multimeric (concatemers) 46 molecules and DNA-binding complexes (Miyakawa 2017). The mitochondrial DNA 47 concatemers might be due to DNA replication mechanisms that involve rolling circles (Bendich 48 1996; Hausner et al. 2006, Chen and Clark-Walker 2018). Fungal mitochondrial genomes can 49 vary greatly in size from 12.055 kbp to > 500 kbp (James et al. 2013; Liu et al. 2020). The size 50 variation can be explained, in part, by the presence of introns, intergenic spacers, duplications, 51 proliferation of repeats, and insertions of plasmid components (Hausner 2003; Himmelstrand et 52 al. 2014; Freel et al. 2015; Deng et al. 2018; 2020; Lang 2018; Sandor et al. 2018; Medina et al. 53 2020). 54 Although fungal mitochondrial genomes show great variation in size, filamentous 55 Ascomycetes fungi encode a similar geneDraft complement: genes involved in translation [small and 56 large ribosomal subunit RNAs (rns and rnl)] plus a set of tRNAs; genes coding for components 57 of the respiratory chain such as subunits for Complexes III and IV (cob, cox1, cox2, and cox3), 58 subunits of NADH dehydrogenase (nad1 to nad6 and nad4L), and subunits for the ATP synthase 59 (atp6, atp8, and usually atp9) (Lang 2018; Zardoya 2020). In addition, most members of the 60 Ascomycota encode the ribosomal protein RPS3 (rps3; Hausner 2003; Freel et al. 2015), where it 61 can be encoded within an intron or positioned as a free-standing gene (Wai et al. 2019). 62 Fungal mtDNA introns are unique as they are potential mobile elements that encode so 63 called intron-encoded proteins (IEPs) that catalyze their mobility to cognate intron-less alleles 64 and the IEPs, in some instances, enhance (maturase activity) the intron RNAs ability to self- 65 splice from the transcripts of host genes they have invaded (Belfort 2003; Lang et al. 2007; 66 Hausner 2012). Based on the splicing mechanism and secondary structures mitochondrial introns 67 can be assigned to either group I or group II introns (Belfort et al. 2002). Homing endonucleases 4 © The Author(s) or their Institution(s) Page 5 of 42 Canadian Journal of Microbiology 68 (HEs) or maturases, which are DNA-cutting enzymes or proteins that facilitate splicing and 69 reverse transcriptases are IEPs associated usually with group I and group II introns, respectively. 70 Although, there are examples of group II introns that encode HEs that have the potential to 71 catalyze the mobility of their host group II introns (Toor and Zimmerly 2002; Mullineux et al. 72 2010). In fungal mtDNAs, two families of HEs are noted to be encoded by homing endonuclease 73 genes (HEGs), named by the presence of conserved amino acid motifs: the LAGLIDADG and 74 the GIY-YIG families of HEs (Stoddard 2014). Although it has been noted that HEGs can move 75 independently of their ribozyme partners (Mota and Collins 1988), more frequently they appear 76 to have co-evolved with their intron counterparts that encode them (Megarioti and Kouvelis 77 2020). The gain and loss of introns and intron mobility appears to promote mitochondrial DNA 78 size polymorphisms and rearrangementsDraft by promoting intra- and inter mitochondrial genome 79 recombination events (Kanzi et al. 2016; Franco et al. 2017; Wu and Hao 2019; Deng et al. 80 2020). 81 Previously, we characterized the mitogenome for Ophiostoma novo-ulmi subsp. novo- 82 ulmi (Abboud et al. 2018). Here, we report the complete mitogenome of Ophiostoma himal-ulmi 83 and compare it with those of other members of the DED-causing fungal species complex (Brasier 84 and Buck 2001; Hessenauer et al. 2020). The data may provide some insight in understanding the 85 origin of O. novo-ulmi and provide a resource for developing markers that allow for 86 distinguishing the various species (or subspecies) that can cause Dutch elm disease. 87 88 Material and Methods 89 Source of culture and culturing methods 5 © The Author(s) or their Institution(s) Canadian Journal of Microbiology Page 6 of 42 90 Ophiostoma himal-ulmi Brasier & M.D. Mehrotra (CBS 374.67; Mycobank: 363234, 91 isolated from an Ulmus wallichiana twig, India, Kashmir, Baba Reshi) was obtained from The 92 Westerdijk Fungal Biodiversity Institute (Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands). 93 An approximately 5 mm3 block of stock culture maintained on malt extract agar (MEA; 94 supplemented with yeast extract) slant (30 g/L malt extract, 1 g/L yeast extract, 2 g/L agar) was 95 transferred to a malt extract agar plate (100 mm diameter Petri plate containing approximately 40 96 mL MEA) and incubated in the dark at 20 °C for up to four weeks. Five 250 mL Erlenmeyer 97 flask containing 100 mL PYG broth (1 g/L peptone, 1 g/L yeast extract, 3 g/L glucose) were 98 each inoculated with ten agar blocks (cut from the edge of the mycelium; approximate 99 dimensions = 2 mm x 2 mm x 1 mm) and incubated in the dark at 20 °C for up to two weeks. Draft 100 101 Mitochondria and mitochondrial DNA extraction 102 Fungal mass was collected by vacuum filtration. Briefly, the liquid culture was filtered 103 through a Whatman® Grade 1 qualitative filter paper placed in a Büchner funnel under vacuum 104 until most of the liquid broth was removed. The fungal mass was transferred to a prechilled (-20 105 °C) mortar and mixed, per 1 g fungal mass, with 1.5 g acid-washed, autoclaved sand and 2 mL 106 isolation buffer [10 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 440 mM sucrose].