The Genome of Xylona Heveae Provides a Window Into Fungal Endophytism
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fungal biology 120 (2016) 26e42 journal homepage: www.elsevier.com/locate/funbio The genome of Xylona heveae provides a window into fungal endophytism Romina GAZISa,*, Alan KUOb, Robert RILEYb, Kurt LABUTTIb, Anna LIPZENb, Junyan LINb, Mojgan AMIREBRAHIMIb, Cedar N. HESSEc,d, Joseph W. SPATAFORAc, Bernard HENRISSATe,f,g, Matthieu HAINAUTe, Igor V. GRIGORIEVb, David S. HIBBETTa aClark University, Biology Department, 950 Main Street, Worcester, MA 01610, USA bUS Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA cOregon State University, Department of Botany and Plant Pathology, Corvallis, OR 97331, USA dLos Alamos National Laboratory, Bioscience Division, Los Alamos, NM, USA eAix-Marseille Universite, CNRS, UMR 7257, Marseille, France fAix-Marseille Universite, Architecture et Fonction des Macromolecules Biologiques, 13288 Marseille cedex 9, France gKing Abdulaziz University, Department of Biological Sciences, Jeddah 21589, Saudi Arabia article info abstract Article history: Xylona heveae has only been isolated as an endophyte of rubber trees. In an effort to under- Received 12 August 2015 stand the genetic basis of endophytism, we compared the genome contents of X. heveae Received in revised form and 36 other Ascomycota with diverse lifestyles and nutritional modes. We focused on 18 September 2015 genes that are known to be important in the hostefungus interaction interface and that Accepted 5 October 2015 presumably have a role in determining the lifestyle of a fungus. We used phylogenomic Available online 22 October 2015 data to infer the higher-level phylogenetic position of the Xylonomycetes, and mined ITS Corresponding Editor: sequences to explore its taxonomic and ecological diversity. The X. heveae genome contains Teun Boekhout a low number of enzymes needed for plant cell wall degradation, suggesting that Xylona is a highly adapted specialist and likely dependent on its host for survival. The reduced rep- Keywords: ertoire of carbohydrate active enzymes could reflect an adaptation to intercellulary growth CAZymes and to the avoidance of the host’s immune system, suggesting that Xylona has a strictly en- Glycoside hydrolases dophytic lifestyle. Phylogenomic data resolved the position of Xylonomycetes as sister to Horizontally transmitted endophytes Lecanoromycetes and Eurotiomycetes and placed the beetle-endosymbiont Symbiotaphrina Sapwood endophytes as a member of this class. ITS data revealed that Trinosporium is also part of the Xylonomy- Symbiotaphrina cetes, extending the taxonomic and ecological diversity of this group. Trinosporium ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction Rodriguez et al. 2009). HTE have also been the target of several bioprospecting studies, which have reported their potential as Horizontally transmitted endophytes (HTE) are known for sources of novel antibiotics, enzymes with applications in the their ubiquitous presence, broad taxonomic host associations bio-fuel industry and drugs (e.g., Strobel & Daisy 2003; and high alpha and beta diversity (Arnold & Lutzoni 2007; Suryanarayanan et al. 2009; Aly et al. 2013). Despite the * Corresponding author. Tel.: þ1 (508) 793 7332; fax: þ1 (508) 793 7174. E-mail address: [email protected] (R. Gazis). http://dx.doi.org/10.1016/j.funbio.2015.10.002 1878-6146/ª 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. The genome of Xylona heveae 27 research interest towards this ecologically and taxonomically ITSeLSU dataset that addresses the taxonomic composition diverse group, much of their biology remains to be under- of the Xylonomycetes. stood. For instance, it is not known if all HTE are metabolically active while living intercellularly within their hosts or what is Materials and methods the extent of their colonization. The degree of specificity that HTE have with their host is also unclear, with some studies Fungal strains suggesting evidence of host specificity (Chaverri et al. 2011; Quilliam & Jones 2012; Unterseher et al. 2013) and others sup- Xylona heveae strain TC161 (type strain) was selected for porting a host generalist habit (Cannon & Simmons 2002; whole-genome sequencing. TC161 was isolated as a sapwood Higgins et al. 2011). endophyte from cultivated Hevea brasiliensis in San Martin, The mechanisms that gave rise to the evolution of endo- Peru. The strain is deposited in the Centraalbureau voor phytism and the genetic basis of the endophytic habit are Schimmelcultures (CBS), Utrecht, The Netherlands (CBS also poorly understood. It has been speculated that endo- 132557). phytic lineages have evolved several times from plant patho- genic ancestors, but it has also been suggested that plant pathogenic species have arisen from endophytic lineages Genomic DNA and RNA extraction (Delaye et al. 2013; Xu et al. 2014). While the mechanisms un- derlying evolutionary changes between endophytic and para- Xylona heveae was grown in 500 ml Potato Dextrose Broth me- sitic lifestyles are expected to be lineage-specific, the genetic dia (PDB, Difco) at 25 C for one month. Fungal mass was col- background of the fungal lineage should influence the transi- lected, dried and frozen in liquid nitrogen. DNA was extracted w tion potential. from 10 g of frozen mycelium using the Qiagen Blood and The main reason why the ecological role of HTE cannot be Cell Culture DNA Kit Maxi (Catalog # 13362) according to the generalized is that they are polyphyletic (Rodriguez et al. 2009). manufacturer’s instructions. A total concentration of À1 Most classes of Ascomycota have been reported as having en- 72 ng ul was used in the genome sequencing. Total RNA dophytic representatives (Arnold & Lutzoni 2007), suggesting was extracted from a 2 week-old PDB culture mycelial mat us- that different species of endophytes probably interact with ing the Qiagen RNeasy Midi Kit (Catalog # 75144), following their substrate or host in a distinct manner. The ‘endophytic manufacturer’s instructions. continuum’ (Schulz & Boyle 2005) model suggests that the out- come of the plantefungus interaction, which can range from Genome sequencing assembly and annotation mutualism to parasitism, depends on the fungal species, the host genetic background and the environment (Kogel et al. The genome of Xylona heveae was sequenced using two Illu- 2006). mina libraries, standard with 270 bp insert size and 4 Kbp Gazis et al. (2012) described the Ascomycota class Xylono- long insert paired end (LFPE) and supplemented with a single mycetes based on the species Xylona heveae. Isolates of X. transcriptomics Illumina library, procedures were similar as heveae were found mainly as sapwood endophytes of Hevea the ones described in Floudas et al. (2015). Reads were QC fil- brasiliensis, the main source of natural rubber (Chengalroyen tered for artifact/process contamination and subsequently as- & Dabbs 2013). DNA sequences from X. heveae isolates (ITS, In- sembled together with AllPathsLG version R46652 (Gnerre et al. ternal Transcribed Spacer and LSU, Large Subunit of the ribo- 2011) and de novo transcript contigs were assembled using somal RNA) were compared to an extensive database Rnnotator (Martin et al. 2010). General aspects of library con- comprising saprotrophs, plant pathogens, endophytes and se- struction and sequencing can be found at the JGI website quences obtained from environmental surveys. No close rela- http://www.jgi.doe.gov/. tives were found, suggesting that X. heveae might represent The genome of X. heveae was annotated using the JGI anno- a strict endophyte, in the sense that it cannot be associated tation pipeline, which combines multiple tools for gene pre- with any other lifestyle. The same study also reported that, diction, annotation and analysis (Grigoriev et al. 2014; Kuo in contrast to what has been reported for many leaf endo- et al. 2014). The assembled genomic scaffolds were masked us- phytes (Oses et al. 2006; Promputtha et al. 2010), X. heveae ing RepeatMasker ver. 3 (Smit et al. 2004). Multiple sets of gene was not able to degrade cellulose or lignin under laboratory models were predicted using different gene predictors and au- conditions. tomatically filtered based on similarity to proteins from other In this study, we analysed the genome of X. heveae in a com- organisms and gene expression support to produce a final parative framework to address the genetic basis of the endo- non-redundant catalog of genes representing the best gene phytic habit and shifts along the ‘endophytic continuum’. model found at each locus site. Multi-gene families were pre- We focused on genes that are thought to play a role in the dicted using the Markov clustering algorithm (MCL, Enright hostefungus interaction, with emphasis on carbohydrate ac- et al. 2002), a part of the JGI annotation pipeline, and annotated tive enzymes (CAZymes). Recent comparative studies have using PFAM domains detected in cluster member sequences. demonstrated a strong relationship between the suite of All other genomes included in the analysis were annotated CAZymes in fungal genomes and their lifestyle and nutritional as above, except the Epichloe€ festucae genome, which was an- mode (Parrent et al. 2009; Eastwood et al. 2011; Ohm et al. 2012; notated by the working group based at the University of Ken- Kohler et al. 2015). We also performed a phylogenomic analy- tucky (http://csbio-l.csr.uky.edu/ef2011/). To assess the sis to infer the placement of the Xylonomycetes within the genome coverage