Genomic and Genetic Insights Into a Cosmopolitan Fungus, Paecilomyces variotii (Eurotiales)

Abstract

Species in the genus Paecilomyces, a member of the fungal order Eurotiales, are ubiquitous in nature and impact a variety of human endeavors. Here, the biology of one common species, Paecilomyces variotii, was explored using genomics and functional genetics. Sequencing the genome of two isolates revealed key genome and gene features in this species. A striking feature of the genome was the two-part nature, featuring large stretches of DNA with normal GC content separated by AT-rich regions, a hallmark of many plant-pathogenic fungal genomes. These AT-rich regions appeared to have been mutated by repeat-induced point (RIP) mutations. We developed methods for genetic transformation of P. variotii, including forward and reverse genetics as well as crossing techniques. Using transformation and crossing, RIP activity was identified, demonstrating for the first time that RIP is an active process within the order Eurotiales. A consequence of RIP is likely reflected by a reduction in numbers of genes within gene families, such as in cell wall degradation, and reflected by growth limitations on P. variotii on diverse carbon sources. Furthermore, using these transformation tools we characterized a conserved protein containing a domain of unknown function (DUF1212) and discovered it is involved in pigmentation.

Full text

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ORIGINAL RESEARCH
published: 13 December 2018
doi: 10.3389/fmicb.2018.03058
Edited by:
Monika Schmoll,
Austrian Institute of Technology (AIT),
Austria
Reviewed by:
Roberto Silva,
University of São Paulo, Brazil
Martin Münsterkötter,
University of Sopron, Hungary
*Correspondence:
Alexander Idnurm
alexander.idnurm@unimelb.edu.au
Specialty section:
This article was submitted to
Fungi and Their Interactions,
a section of the journal
Frontiers in Microbiology
Received: 15 August 2018
Accepted: 27 November 2018
Published: 13 December 2018
Citation:
Urquhart AS, Mondo SJ,
Mäkelä MR, Hane JK, Wiebenga A,
He G, Mihaltcheva S, Pangilinan J,
Lipzen A, Barry K, de Vries RP,
Grigoriev IV and Idnurm A (2018)
Genomic and Genetic Insights Into
a Cosmopolitan Fungus,
Paecilomyces variotii (Eurotiales).
Front. Microbiol. 9:3058.
doi: 10.3389/fmicb.2018.03058
Genomic and Genetic Insights Into a
Cosmopolitan Fungus, Paecilomyces
variotii (Eurotiales)
Andrew S. Urquhart1, Stephen J. Mondo2, Miia R. Mäkelä3, James K. Hane4,5 ,
Ad Wiebenga6, Guifen He2, Sirma Mihaltcheva2, Jasmyn Pangilinan2, Anna Lipzen2,
Kerrie Barry2, Ronald P. de Vries6, Igor V. Grigoriev2and Alexander Idnurm1*
1School of BioSciences, University of Melbourne, Melbourne, VIC, Australia, 2U.S. Department of Energy Joint Genome
Institute, Walnut Creek, CA, United States, 3Department of Microbiology, Faculty of Agriculture and Forestry, Viikki
Biocenter 1, University of Helsinki, Helsinki, Finland, 4CCDM Bioinformatics, Centre for Crop and Disease Management,
Curtin University, Bentley, WA, Australia, 5Curtin Institute for Computation, Curtin University, Bentley, WA, Australia, 6Fungal
Physiology, Westerdijk Fungal Biodiversity Institute and Fungal Molecular Physiology, Utrecht University, Utrecht, Netherlands
Species in the genus Paecilomyces, a member of the fungal order Eurotiales, are
ubiquitous in nature and impact a variety of human endeavors. Here, the biology of one
common species, Paecilomyces variotii, was explored using genomics and functional
genetics. Sequencing the genome of two isolates revealed key genome and gene
features in this species. A striking feature of the genome was the two-part nature,
featuring large stretches of DNA with normal GC content separated by AT-rich regions,
a hallmark of many plant-pathogenic fungal genomes. These AT-rich regions appeared
to have been mutated by repeat-induced point (RIP) mutations. We developed methods
for genetic transformation of P. variotii, including forward and reverse genetics as well
as crossing techniques. Using transformation and crossing, RIP activity was identified,
demonstrating for the first time that RIP is an active process within the order Eurotiales.
A consequence of RIP is likely reflected by a reduction in numbers of genes within
gene families, such as in cell wall degradation, and reflected by growth limitations on
P. variotii on diverse carbon sources. Furthermore, using these transformation tools we
characterized a conserved protein containing a domain of unknown function (DUF1212)
and discovered it is involved in pigmentation.
Keywords: Agrobacterium tumefaciens-mediated transformation, Byssochlamys spectabilis, DUF1212,
Eurotiales, genome defense, leuA, mitochondrial membrane carrier
INTRODUCTION
Species in the order Eurotiales are amongst some of the best characterized fungi. They include
the source of life-saving penicillin Penicillium rubens, the model filamentous fungus Aspergillus
nidulans, the industrial species and source of citric acid Aspergillus niger, and the human pathogen
Aspergillus fumigatus (Galagan et al., 2005;Max et al., 2010;de Vries et al., 2017). While a handful
of these species have been extensively studied most have not received a high level of investigation,
yet might provide similar benefits or risks to people.
Paecilomyces variotii is a ubiquitous thermo-tolerant species that is encountered in food
products, soil, indoor environments and clinical samples (Houbraken et al., 2010). Its thermo-
tolerance and ability to grow at low oxygen levels allows it to survive heat treatment and it has
been widely isolated as a contaminant of products such as heat-treated fruit juices (Houbraken
et al., 2006). Furthermore, it is emerging as an opportunistic human pathogen (Steiner et al., 2013),
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Urquhart et al. Genome Analysis of Paecilomyces variotii
with cases of P. variotii and the closely related species
Paecilomyces formosus infection in immuno-compromised
individuals (Torres et al., 2014;Polat et al., 2015;Feldman et al.,
2016;Kuboi et al., 2016;Swami et al., 2016;Bellanger et al., 2017;
Uzunoglu and Sahin, 2017) and plant disease (Heidarian et al.,
2018). While this organism can be detrimental to human health,
it also lends itself to diverse industrial applications. P. variotii has
been explored as a source of industrial tannase, as its tannase has
beneficial characteristics including a high optimum temperature
(Battestin and Macedo, 2007a,b). Among its other enzymes with
favorable properties for industry are a thermostable glucoamylase
(Michelin et al., 2008), a glucose-tolerant β-glucosidase (Job
et al., 2010) and an alcohol oxidase that displays stability at high
temperature (50◦C) and over a wide pH range (from 5 to 10)
(Kondo et al., 2008).
Despite the relevance of Paecilomyces species to human
activities across the world, no well-annotated genome sequence
is currently available for any members in the Paecilomyces
genus except for draft genomes of P. formosus (Oka et al.,
2014) and P. niveus (Biango-Daniels et al., 2018). Furthermore,
methods for genetic manipulation or classical genetics have not
been described for Paecilomyces, further limiting our ability to
understand gene functions in the genus.
Here, we sequenced and annotated the genome of P. variotii
[Byssochlamys spectabilis] CBS 101075, which is the type strain
of the teleomorphic state (Houbraken et al., 2008), and strain
CBS 144490 that was isolated in this study. The genomes have
a bi-modal pattern of overall DNA G:C content with alternating
stretches of G:C-equilibrated or A:T rich DNA, reminiscent of
those found in the genomes of many plant pathogens as a
consequence of repeat induced point mutation (RIP) (Testa et al.,
2016). RIP is a fungal process in which repetitive sequences are
recognized during the sexual cycle and targeted for mutation
(Hane et al., 2015). Experimental evidence of RIP – that is a
mutagenic process targeted to duplicated DNA sequences that
occurs during mating – is limited to fungi of the fungal classes
Dothideomycetes [L. maculans (Idnurm and Howlett, 2003;Van
de Wouw et al., 2019)] and Sordariomycetes [Fusarium spp.,
Magnaporthe oryzae,Neurospora crassa,Podospora anserina, and
Trichoderma reesei (Selker and Garrett, 1988;Nakayashiki et al.,
1999;Graïa et al., 2001;Cuomo et al., 2007;Coleman et al.,
2009;Li et al., 2017), reviewed by (Hane et al., 2015)]. In silico
sequence analysis suggests that RIP occurs extensively in the
fungi [for example a potential activity in the Basidiomycota
(Horns et al., 2012)], including species in the Eurotiales like
A. niger (Braumann et al., 2008), A. nidulans (Nielsen et al.,
2001;Clutterbuck, 2004), Aspergillus oryzae (Montiel et al., 2006),
Penicillium chrysogenum (Braumann et al., 2008) and Penicillium
roqueforti (Ropars et al., 2012). However, in these species
whether those patterns of mutation represent RIP, the natural
accumulation of mutations over time, or another mechanism
of DNA mutation such as the spontaneous deamination of
methylated cytosines (Lindahl, 1993;Lutsenko and Bhagwat,
1999), remains unknown. This point is well illustrated in the case
of A. nidulans, a genetic model for many decades yet in which
RIP has not been observed despite the in silico evidence (Nielsen
et al., 2001;Clutterbuck, 2004). Second, we developed methods
for the genetic transformation of P. variotii, including an efficient
next-generation-sequencing-based method to identify genes that
are mutated in forward genetic screens, and classical genetics
in which parents are crossed and their progeny used in genetic
segregation analysis. Using these new tools, we characterized two
genes of previously unknown function.
By combining these methods, we demonstrate RIP activity
experimentally for the first time in the Eurotiales, vastly
expanding the phylogenetic breadth of the fungi experimentally
verified to undergo RIP and thereby suggesting this is indeed
a fundamental force that shapes fungal genome evolution. In
addition, we compared the plant biomass degrading ability of
P. variotii to other Eurotiales, hypothesizing that the active RIP
mechanism in this species might reduce gene duplication events
and thus limit the expansion of gene families in this species.
Consistent with this hypothesis, our analysis revealed the poorest
CAZy genome content in P. variotii among the fungal species
used for comparison. This, and the identification of a phenotype
associated with mutating a gene encoding a protein with a
DUF1212 domain, which is at present an enigmatic yet widely
conserved domain, highlights how research on P. variotii offers
new perspectives to understand the biology of Eurotiales fungi,
and fungi more broadly.
MATERIALS AND METHODS
Wild-Type Strains and Preparation of
Growth Media
The ex-type strain of Paecilomyces variotii, i.e., strain CBS 101075,
was obtained from the Commonwealth Scientific and Industrial
Research Organisation culture collection (FRR5219). A second
strain was isolated as a contaminant after water damage to the
laboratory, having attracted attention because of its ability to
inhibit the growth of a plant pathogenic fungus Leptosphaeria
maculans. This strain has been deposited at the Westerdijk
Institute as CBS 144490. As described below, CBS 10105 (MAT1-
1) and CBS 144490 (MAT1-2) are of opposite mating type. An
Aspergillus niger strain was isolated from an onion (identification
including ITS sequencing, as GenBank MH605508), and used
as source of DNA in molecular biology experiments. The strain
was deposited to the Westerdijk Institute as CBS 144491. The
strains of Eurotiales species used for carbon utilization profiling
are given in Table 1.
Cleared and uncleared V8 juice was adjusted to pH 6 with
NaOH, and used at 10% v/v for media. Potato dextrose agar
(PDA) and potato dextrose broth were obtained commercially
(Difco). Minimal medium was prepared using an adaptation
of Sutter (1975), and consisted of per liter: 20 g glucose, 2 g
asparagine, 5 g KH2PO4, 500 mg MgSO4, 28 mg CaCl2, 2 mg
citric acid·H2O, 1.5 mg ferric sulfate, 1 mg ZnSO4·7H2O, 300 µg
MnSO4·H2O, 50 µg CuSO4·5H2O, 50 µg molybdic acid. 1 mg/ml
leucine was added when needed.
Genome Sequencing of P. variotii Strains
Genomic DNA of the two strains was isolated as described
previously (Pitkin et al., 1996). The genome of P. variotii
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TABLE 1 | Additional fungal species and strains used in this study.
Species Strain Reference
CAZy gene comparison
Talaromyces marneffei ATCC 18224 Nierman et al., 2015
Penicillium rubens Wisconsin 54-1255 van den Berg et al.,
2008
Penicillium subrubescens FBCC1632, CBS 132785 Peng et al., 2017
Aspergillus wentii CBS 141173 de Vries et al., 2017
Aspergillus glaucus CBS 516.65 de Vries et al., 2017
Aspergillus clavatus NRRL1 Sherlock et al., 2012
Aspergillus fumigatus Af293 Nierman et al., 2005
Aspergillus terreus NIH 2624 Sherlock et al., 2012
Aspergillus oryzae RIB40 Machida et al., 2005
Aspergillus nidulans FGSC A4 Galagan et al., 2005
Aspergillus niger ATCC 1015 Andersen et al., 2011
Secondary metabolite cluster comparison
Aspergillus aculeatinus CBS 121060 Vesth et al., 2018
Aspergillus bombycis NRRL 26010 Moore et al., 2016
Aspergillus calidoustus SF006504 Horn et al., 2016
Aspergillus fijiensis CBS 313.89 Vesth et al., 2018
Aspergillus homomorphus CBS 101889 Vesth et al., 2018
Aspergillus ibericus CBS 121593 Vesth et al., 2018
Aspergillus nidulans FGSC A4 Galagan et al., 2005
Aspergillus uvarum CBS 121591 Vesth et al., 2018
Penicillium griseofulvum PG3 Banani et al., 2016
Penicillium steckii IBT 24891 Nielsen et al., 2017
Penicillium subrubescens FBCC1632, CBS132785 Peng et al., 2017
Thermoascus aurantiacus ATCC 26904
strain CBS 101075 was sequenced using the Pacific Biosciences
platform. Unamplified libraries were generated using Pacific
Biosciences standard template preparation protocol for
creating >10 kb libraries. Five µg of gDNA was used to
generate each library and the DNA was sheared using Covaris
g-TUBEs to generate sheared fragments of >10 kb in length.
The sheared DNA fragments were then prepared using Pacific
Biosciences SMRTbell template preparation kit, where the
fragments were treated with DNA damage repair, had their ends
repaired so that they were blunt-ended, and 50phosphorylated.
Pacific Biosciences hairpin adapters were ligated to the fragments
to create the SMRTbell template for sequencing. The SMRTbell
templates were then purified using exonuclease treatments
and size-selected using AMPure PB beads. PacBio Sequencing
primer was then annealed to the SMRTbell template library
and sequencing polymerase was bound to them using Sequel
Binding kit 2.0. The prepared SMRTbell template libraries were
then sequenced on a Pacific Biosystem’s Sequel sequencer using
v3 sequencing primer, 1M v2 SMRT cells, and Version 2.0
sequencing chemistry with 1 ×360 sequencing movie run times.
The filtered PacBio sub-read data were assembled together
with Falcon version 1.8.81, improved with FinisherSC version 2.0
(Lam et al., 2015), and polished with Arrow version SMRTLink
v.5.0.0.6792.2
1https://github.com/PacificBiosciences/FALCON
2https://github.com/PacificBiosciences/GenomicConsensus
To aid in gene predictions and annotation, the P. variotii
transcriptome was sequenced with Illumina. To generate a
diversity of transcripts, mycelia were cultured under four
conditions for 4 days without shaking: at two temperatures (30◦C
and 37◦C) and in 10% cleared V8 juice pH 6 and potato dextrose
broth. RNA was isolated from mycelium using TRIzol reagent
(Invitrogen) following the manufacturer’s recommendations, and
equal quantities of RNA isolated from each mycelium were
pooled. Stranded cDNA libraries were generated using the
Illumina Truseq Stranded RNA LT kit. mRNA was purified
from 1 µg of total RNA using magnetic beads containing poly-
T oligos. mRNA was fragmented and reverse transcribed using
random hexamers and Superscript II (Invitrogen) followed by
second strand synthesis. The fragmented cDNA was treated with
end-pair, A-tailing, adapter ligation, and 8 cycles of PCR. The
prepared library was then quantified using KAPA Biosystem’s
next-generation sequencing library qPCR kit and run on a
Roche LightCycler 480 real-time PCR instrument. The quantified
library was then multiplexed with other libraries, and the pool
of libraries was prepared for sequencing on the Illumina HiSeq
sequencing platform utilizing a TruSeq paired-end cluster kit,
v4, and Illumina’s cBot instrument to generate a clustered flow
cell. Sequencing of the flow cell was performed on the Illumina
HiSeq 2500 sequencer using HiSeq TruSeq SBS sequencing kits,
v4, following a 2 ×150 indexed run recipe. Illumina reads
were filtered for quality and artifacts, RNA spike-in, PhiX, and
N-containing reads, trimmed and assembled into consensus
sequences using Trinity version 2.3.2 (Grabherr et al., 2011).
The genome was annotated using the JGI Annotation Pipeline,
and made publicly available via JGI fungal genome portal
MycoCosm (Grigoriev et al., 2014).
Strain CBS 144490 HYG1 is a transformant of strain CBS
144490, with a T-DNA inserted into its genome from plasmid
pCSB1. This strain was sequenced to represent the genome of
CBS 144490. Illumina sequencing of strain CBS 144490 HYG1
was conducted at the Australian Genome Research Facility
(AGRF), with 100 bp paired-end reads on an Illumina HiSeq 2500
instrument. The nuclear genome was assembled using Velvet
with the k-mer setting at 67 and auto detect for low coverage cut
off (Zerbino and Birney, 2008). The mitochondrial genome was
assembled using the inbuilt assembler in Geneious version 10.1.3
and annotated along with the mitochondrial genome of strain
CBS 101075 using MFannot (Supplementary Figure S1).
Phylogenetic Analysis of Strains of
Paecilomyces
Three gene regions, encoding calmodulin and β-tubulin and
the internal transcribed spacers (ITS), were used to build
phylogenetic trees between strains. Sequences were those used
previously (Samson et al., 2009), with the addition of the
corresponding regions of the “P. variotii” number 5 CBS
144490 and CBS 101075 obtained via BLAST searches of
the whole genome sequences. Sequences were aligned using
MUSCLE (Edgar, 2004) and phylogenetic relationships were
inferred using MrBayes (Huelsenbeck and Ronquist, 2001)
implemented through Geneious version 11.0.4 using the
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HKY85 substitution model and 1,100,000 iterations with the
sequences from Paecilomyces divaricatus CBS 284.48 set as the
outgroups.
In addition, a species tree was generated using 3,374 single
copy gene orthologs, identified using mcl (Enright et al., 2002).
Genes were individually aligned using mafft (Katoh and Standley,
2013), trimmed using Gblocks (Castresana, 2000) using the
following parameters: −t = p, −e = .gb, and −b4 = 5, then
the phylogeny was reconstructed using RAxML (Stamatakis,
2014) under the PROTGAMMAWAGF substitution model. 100
bootstrap replicates were performed (all branches were fully
supported).
Generation of Plasmids for Fungal
Transformation Using Agrobacterium
tumefaciens
Plasmids were constructed for the transformation of P. variotii
using A. tumefaciens for differing purposes. These plasmids are
described in the following eight subsections.
(i) Mitochondrial GFP barcode series. The nucleotide
sequence corresponding to the first 76 amino acids of the
L. maculans citrate synthase gene (Lema_T101280.1) was
amplified using primers AU268 and AU269 (Supplementary
Table S1) off the genomic DNA of strain M1. Suelmann and
Fischer (2000) showed that the corresponding sequence from
A. nidulans was sufficient to direct GFP localization to the
mitochondria. The coding region of the GFP gene was amplified
using primers AU108 and AU68 off plasmid PLAU17 (Idnurm
et al., 2017). These two fragments were then cloned into plasmid
PLAU2 (Idnurm et al., 2017) using Gibson assembly (New
England Biolabs). The resultant plasmid was linearized with PmeI
and a 20 nucleotide “barcode” was inserted into this site using
Gibson assembly. The barcode contained 20 semi-randomized
nucleotides (NMNMNMNMNMNMNMNMNMNM; where N
is any nucleotide and M is either A or C, as based on Hensel
et al. (1995), and appropriate flanking sequence for Gibson
assembly was included as a single stranded oligonucleotide
AU257 that was made double-stranded via a PCR reaction with
primers AU258 and AU259. The pool of resulting fragments
was cloned into the PmeI site resulting in a series of plasmids
with different barcodes. The sequences of clones in individual
plasmids were determined by Sanger sequencing with primer
ai076 (Supplementary Table S2).
(ii) H2B-CFP. A fusion protein of A. nidulans histone H2B
and GFP has previously been shown to be nuclear-localized
(Maruyama et al., 2002). The coding region of the histone H2B
gene of A. niger strain CBS 144491 was amplified using primers
AU492 and AU493 off genomic DNA. The coding region of
CFP was amplified using primers AU494 and AU495 off plasmid
PLAU41 (a PLAU2-based expression plasmid for CFP, analogous
to PLAU17) and cloned into the BglII site of PLAU2 using Gibson
assembly.
(iii) dspA complementation construct. The dspA gene region
was amplified using primers AU463 and AU464 and cloned into
the XbaI site of plasmid pMAI2 (Idnurm et al., 2017) using
Gibson assembly.
(iv) prmJ complementation construct. Two fragments
corresponding to the gene were amplified with primers AU461
and AU438, and AU437 and AU462 and cloned into the XbaI site
of plasmid pMAI2 (Idnurm et al., 2017).
(v) A. niger prmJ cross-species complementation construct.
The coding region of the A. niger prmJ gene was amplified in two
parts; the first using primers FD1212AFPLAU2 and FD1212ER,
and FD1212DF and FD1212FRPLAU2 and then combined into
the BglII site of PLAU53 (Idnurm et al., 2017) using Gibson
assembly.
(vi) mCherry-tagged DspA. The coding region of the dspA
gene was amplified by PCR using the dspA complementation
construct as a template with primers AU516 and AU473. The
mCherry coding sequence was amplified using primers AU474
and AU517. These two fragments were cloned into the BglII site
of plasmid PLAU2 using Gibson assembly.
(vii) Mitochondrial GFP in a plasmid conferring resistance to
G418. A plasmid expressing mitochondrially localized GFP and
G418 resistance was created for co-localization experiments. The
coding region of the citrate synthase-GFP fusion was amplified
from plasmid CSB1 using primers AU268 and AU68 and cloned
into the BglII site of plasmid PLAU53 (Idnurm et al., 2017) using
Gibson assembly.
(viii) leuA gene knockout and complementation. A genomic
fragment (1,449 bp) corresponding to the 50flank of the
leuA homolog was amplified from strain CBS 101075 using
primers MAI0442 and MAI0443. The 30flank of the gene
(1,439 bp) was amplified with primers MAI0444 and MAI0445.
The hygromycin expression cassette of plasmid pMAI17 was
amplified using primers MAI0440 and MAI0441. The three
fragments were cloned, using Gibson assembly, into plasmid
pPZP-201BK (Covert et al., 2001) that had been linearized with
EcoRI and HindIII restriction enzymes. P. variotii transformants
were generated with this plasmid, as described below, and assayed
for their ability to grow on minimal media without leucine. PCR
analysis to confirm the successful integration of the knockout
construct into the leuA gene was conducted using primer pairs:
MAI0023 +MAI0446 and MAI0022 +MAI0447 that amplify
from the hph gene into the 50or 30flank of the leuA locus,
respectively.
As a complementation control, the wild type copy of leuA was
amplified with primers MAI0442 and MAI0445 and cloned into
pPZP-201BK linearized with EcoRI and HindIII. The plasmid
and the empty pPZP-201BK were electroporated separately into
A. tumefaciens strain EHA105. These two A. tumefaciens strains
were co-cultured with two leuA1strains of P. variotii for 3 days,
then overlaid with minimal medium and cefotaxime.
Confirmation of T-DNA Insertion Sites
and Verification of Complementation by
PCR
The T-DNA insertion sites of two mutant strains were confirmed
by PCR, i.e., strains AU2_33 and AU1_63. Primers used for
AU2_33 were AU446 and ai076 for the intragenic T-DNA
and Match2F and Match2R for the intergenic T-DNA. Primers
used for 1_63 were AU437 and ai076. The integration of the
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constructs into the genome, for the complementation of strains,
was confirmed by PCR using primers AU446 and AU448 for
AU2_33 (dspA) and AU437 and AU439 for AU1_63 (prmJ).
Transformation of P. variotii by
A. tumefaciens
Agrobacterium tumefaciens strain EHA105 was transformed with
plasmids by electroporation, as described previously (Urquhart
and Idnurm, 2017), with selection on LB agar +50 µg/ml
kanamycin. An amount of Agrobacterium cells equivalent to a rice
grain was scraped directly off the Agrobacterium transformation
plate and suspended in 1 ml of SOC media. P. variotii spores
were harvested off V8 agar cultures and suspended in dH2O
at approximately 106spores per ml. Five hundred µl of fungal
spores and 100 µl of Agrobacterium suspensions were pipetted
onto the center of a 145 mm petri dish containing 25 ml of
solidified induction media (Gardiner and Howlett, 2004). The
mixture was spread around the plate and incubated at 22◦C for
3 days and then overlaid with 25 ml of molten CV8 containing
200 µg/ml cefotaxime and either 100 µg/ml hygromycin or
200 µg/ml G418 as appropriate for selection of transformants.
Leucine (10 mg/ml) was added to the overlay in the case of
the transformation aiming at gene replacement of leuA. Fungal
transformants appeared after 5 days and were transferred onto
fresh V8 plates containing half the antibiotic concentrations used
in the overlay.
Barcoding Mutagenesis and NGS to
Locate DNA Inserts
DNA was extracted from a number of P. variotii transformants
that showed growth phenotypes, using a buffer containing
CTAB as described previously (Pitkin et al., 1996). The genomic
DNA was pooled and sequenced at the AGRF with Illumina
sequencing using the same instrument and parameters as strain
CBS 144490. Analysis of the next generation sequencing data was
conducted in Geneious version 10.1.2. To identify the positions
of T-DNA insertions in the genome of a given strain the NGS
reads containing the “barcode” from the construct with which
that strain was transformed were pulled out (Supplementary
Figure S2). Many of these reads extended out from the T-DNA
into the sequence adjacent to the T-DNA and this section of the
P. variotii genome was then identified using BLAST against the
genome sequence.
Microscopy
A Leica M205 stereomicroscope was used for the examination
of mating cultures on agar plates. Fluorescence microscopy
was performed using a Leica DM6000 microscope. Cell wall
staining was conducted using calcofluor white M2R (0.0004%)
and emission was detected using a DAPI filter cube. Images were
overlaid using ImageJ software.
Genetic Crosses
Crossing was conducted as described by Houbraken et al. (2008).
Recombination in the progeny was confirmed using genetic
markers that were based on PCR amplification of genomic
fragments followed by digestion with restriction enzymes. An
exception was for the mating type locus where a multiplex
PCR resulting in different product sizes was employed. These
markers and primers used for amplification are summarized in
Supplementary Table S3.
Amplification and Sequencing of the hph
Gene Conferring Hygromycin Resistance
From Progeny of a AU2_33 ×CBS
101075 Cross
A region of each of the T-DNAs was amplified using
primer MAI0022 located at the start of the hygromycin
phosphotransferase (hph) open reading frame and a primer
specific to the genomic region flanking each of the T-DNA
insertion sites (primer Match2R or primer AU439). The resulting
PCR product was then used as the template from which to
amplify the hph coding region by PCR using primers MAI0022
and MAI0023. This PCR product was sequenced using Sanger
chemistry at the AGRF.
Southern Blot Analysis of T-DNA Insert
Copy Number
Approximately 10 µg of genomic DNA was digested with HindIII
and separated on a 1% agarose gel by electrophoresis. DNA
was blotted onto Hybond-N+membrane (GE Healthcare) using
standard methods. A fragment of the hph gene was labeled
with the PCR DIG Probe Synthesis Kit (Roche), as per the
manufacturer’s directions, hybridized to the blot overnight, and
the probe was detected using the DIG wash and block buffer set
(Roche) and the DIG Luminescent Detection Kit following the
manufacturer’s directions. An image of the blot was captured with
a ChemiDoc MP (Bio-Rad) using the High Sensitivity Chemi
setting.
Profiling Fungal Growth on Different
Carbon Sources
Fungi were grown on Aspergillus minimal medium containing
25 mM monosaccharide or 1% polysaccharide for 2–5 days
(depending on the species; Table 1), after which pictures were
taken. Growth was compared using D-glucose as an internal
reference, so that growth on a specific carbon source relative to
growth on glucose was compared between the species.
Analysis of Gene Content
Gene numbers in different functional categories for the two
P. variotii strains were obtained using the “cluster” option from
the MycoCosm portal (Grigoriev et al., 2014), comparing the two
strains with other species in the Eurotiales as well as Neurospora
crassa where RIP is most extensively characterized. As a focused
case study, the putative Carbohydrate-Active enzymes (CAZys)
were filtered for families known to be involved in plant biomass
degradation (de Vries et al., 2017). It should be noted that for
families the genes could not always be split by the predicted
activity, resulting in some cases in an over-prediction of the
number of genes encoding enzymes for the utilization of a certain
polysaccharide.
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RESULTS
Genome Sequence Characteristics of
P. variotii Strains CBS 101075 and CBS
144490
The genome of ex-type strain P. variotii CBS 101075 was
sequenced using long reads of Pacific Biosciences technology,
and genes were annotated using the JGI annotation pipeline
(Grigoriev et al., 2014). The mitochondrial genome was
annotated separately with MFannot software (Supplementary
Figure S1). The P. variotii CBS 101075 genome is approximately
30.1 Mb in total size, 4.53% of which is comprised of repetitive
DNA of simple repeats and putative transposable elements
(Supplementary Table S4). The genome appears to represent
gene-encoding regions completely, as estimated by the presence
of 100% of CEGMA genes [the Core Eukaryotic Genes Mapping
Approach (Parra et al., 2007)]. Analysis using Benchmarking
Universal Single-Copy Orthologs (BUSCO V3; Waterhouse et al.,
2018) also indicated a high level of completeness to the genome,
with 99.6 and 99.0% of BUSCO genes being present using the
Fungi or Eukaryota settings, respectively. Assembly statistics
are comparable to other related, recently published genomes
(Supplementary Table S4). We identified 9,270 genes in the
P. variotii genome (Supplementary Table S4), most of which are
complete by having both start and stop codons (98.76%) and have
well supported matches in various genomic databases, including
NCBI (95.2% of genes) and Pfam (75.02%) (Finn et al., 2016).
MCL-based ortholog clustering (Enright et al., 2002) using the
genomes in Supplementary Table S4 reveals 8,808 P. variotii
genes in orthologous gene clusters, and 462 unique genes. The
genome assembly and related data for P. variotii CBS 101075 is
available from https://genome.jgi.doe.gov/Paevar1, and the whole
genome shotgun project deposited to GenBank as accession
RCNU00000000.
The genome of a second isolate, CBS 144490 HYG1, was
generated using short read technology. A total of 15,229,380
100 bp paired end reads were generated and were assembled into
126 contigs (N50 = 642,740) totaling 32,365,222 bp. This genome
was annotated based on that of CBS 101075, and is available
from https://genome.jgi.doe.gov/Paevar_HGY_1, and deposited
in GenBank under accession RHLL00000000 and in the short
read archive as PRJNA497137.
Phylogenetic Resolution of Sequenced
Strains Within the Paecilomyces Genus
A phylogenetic analysis was conducted to confirm the species-
level taxonomy of strain CBS 144490, and “P. variotii” strain
number 5 whose genome was previously sequenced (Oka
et al., 2014). The calmodulin and β-tubulin gene regions and
ITS separates P. formosus and P. variotii into separate clades
(Supplementary Figure S3), in agreement with previous studies
(Samson et al., 2009). The regions obtained from the genome
sequence of CBS 101075 were identical to those deposited
previously for this isolate in GenBank. Strain CBS 144490,
isolated in this study, also clearly groups with the other P. variotii
strains. However, strain “P. variotii” number 5 (Oka et al.,
2014) groups within the P. formosus clade, and not with
P. variotii.
Agrobacterium tumefaciens Can Be
Used for the Efficient Transformation of
P. variotii
Although the genomes of P. variotii contain a number of
interesting genes and other features, testing their function
requires methods for genetic manipulation. In the first step for
this process, transformation with exogenous DNA was tested
using delivery of T-DNA molecules from Agrobacterium
tumefaciens. The T-DNA used expressed hygromycin
phosphotransferase, GFP with an N-terminal mitochondrial
targeting sequence, and contained a “barcode” sequence inward
of the right border. Following selection on hygromycin, colonies
were examined for GFP fluorescence: the hyphae of all strains
(n= 100) had fluorescent tubules consistent with mitochondria,
indicating that when using this transformation system 100%
of the resultant colonies have integrated the T-DNA construct,
including the DNA for expression of GFP, into their genome
(Figure 1).
Targeted Gene Disruption in P. variotii Is
Possible Despite the Multinucleate
Nature of Its Conidiospores
Many fungi produce multinucleate spores, meaning that after
transformation several passaging steps are required to isolate a
homokaryotic mycelium. A histone H2B-CFP fusion construct,
causing the localization of CFP to the nucleus, was transformed
into P. variotii to allow the number of nuclei in the conidiospores
to be counted. Most of the spores contained two or more nuclei
and some spores containing up to four nuclei (Figure 2).
The experiments above indicated that P. variotii could
be transformed with DNA. However, whether targeted gene
mutations were possible and if mutants could be easily
isolated from a population containing multinucleate spores
were unknown. To address this, the feasibility of targeted gene
disruption via homologous recombination in this species was
tested. The leuA gene, encoding α-isopropylmalate synthase
required for leucine biosynthesis, was chosen as mutation
of homologs of the gene results in leucine auxotrophy
in ascomycetes, basidiomycetes and Mucoromycota species
(Kohlhaw, 2003;Larson and Idnurm, 2010;Ianiri et al., 2011)
that are easy to identify by their inability to growth on media
without leucine. Of 25 hygromycin-resistant strains transformed
with the leuA knockout construct, which contains approximately
1.5 kb of homologous sequence on either side of the construct for
hygromycin resistance, four showed reduced growth on minimal
media without amino acids and 21 showed wild type growth rate
(Figure 3A). The growth of the four strains was restored by the
addition of leucine to the medium (Figure 3A). PCR analysis
confirmed the correct integration of the hygromycin resistance
cassette into the leuA locus in the four leucine auxotrophs
(Figure 3B).
To confirm that the leucine auxotrophy was due to the gene
deletion, two deletion strains were complemented with the wild
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FIGURE 1 | Transformation of P. variotii using Agrobacterium-mediated delivery of the exogenous DNA. The construct encodes an enzyme conferring hygromycin
resistance for selection and a hybrid protein with a mitochondrial-targeted sequence fused to GFP. (A) Mitochondrial GFP fluorescence of was observed in 100 out
of 100 hygromycin-resistant strains obtained after transformation; one representative strain is shown. (B) Cell walls fluoresce blue from staining with calcofluor white.
(C) The overlay of the GFP and calcofluor white signals. Scale bar = 20 µm.
type copy of leuA. The full length gene was amplified from wild
type DNA and cloned into plasmid pPZP-201BK. The pPZP-
201BK-leuA and pPZP-201BK empty plasmids were used to
transform the two strains using Agrobacterium-mediated delivery
of their T-DNAs, with selection on minimal medium without
leucine. Colonies were obtained when using the leuA plasmid, but
not empty plasmid (data not shown).
Rapid Identification of T-DNA Insertion
Sites in Barcoded Mutants by
Next-Generation Sequencing
To assess the potential for forward genetics using insertional
mutagenesis of T-DNA molecules delivered from Agrobacterium
in P. variotii, approximately 500 transformants were screened
for growth or development phenotypes on V8 juice medium
and minimal medium. Transformants with such phenotypes were
obtained, and seven were further investigated toward identifying
the genes disrupted within them.
A NGS approach was used in which a pool of DNA from
the seven strains carrying a barcode near the right border of
the T-DNA was sequenced, to identify the location of T-DNA
insertion sites (Supplementary Table S5). Three of the strains
were found to each contain at least two T-DNA insertions. No
reads containing barcode number 4 could be found and thus the
location of the T-DNA is strain AU4_W could not be determined.
Three of the strains contained the same barcode sequence
(barcode 1). Only two T-DNAs corresponding to barcode 1 were
found. However, reads were present which contained barcode 1
and vector sequence extending beyond the right border, so it is
likely that one of these strains contains an abnormally integrated
T-DNA. Two of the strains in which the T-DNA had clearly
inserted within the open reading frame of genes were further
studied, namely strains AU1_63 and AU2_33.
Three of the strains whose DNA were pooled for sequencing
were derived from transformation with the same plasmid so
therefore contained the same barcode sequence (#1). PCR was
employed to distinguish the insertion events between them, to
reveal the presence of the mutation in a gene with a domain
of unknown function (DUF1212) in strain AU1_63 (Figure 4).
The insert is located approximately in the center of the single
exon of the gene, upstream of the region encoding the conserved
DUF1212 domain (Figure 4C). We named this gene prmJ after
the Saccharomyces cerevisiae homolog PRM10, employing the
gene nomenclature used for A. nidulans and other Eurotiales
species to P. variotii.
The strain AU2_33 contains two insertion sites, one in the
coding region of a mitochondrial membrane carrier (delayed
sporulation A, dspA) and one that was intergenic. The genes
near the intergenic insertion were not further characterized. The
intragenic T-DNA insert was located 64 bp into the first exon of
the dspA gene (Figure 5C).
Strains AU1_63 and AU2_33 were analyzed by Southern
blotting to confirm the number of T-DNA inserts as indicated
by the genome sequencing data (Supplementary Figure S4). The
single T-DNA insertion in strain AU1_63 was supported by the
hph gene fragment hybridizing to a single HindIII fragment of
approximately 3.9 kb, while two T-DNAs in strain AU2_33 were
indicated as hybridization to two HindIII restriction fragments
of ∼6.4 kb and ∼8.3 kb. These sizes are consistent with size
predictions based on HindIII sites in the genome sequence data
adjacent to the T-DNA insertion sites.
Strain AU1_63 Has a Media-Dependent
Impairment in Spore Pigmentation, Due
to Mutation of a Gene With an
Uncharacterized Domain
Strain AU1_63 has a pale phenotype on cleared V8 juice (CV8)
agar medium because it produces conidiospores that lacked the
characteristic yellow pigmentation of P. variotii (Figure 4A). The
phenotype was not observed when the strain was cultured instead
on potato dextrose medium. A wild type copy of the prmJ gene
was amplified and cloned into a plasmid containing a construct
conferring G418 resistance, and then transformed into strain
AU1_63. Of three strains transformed with the complementation
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FIGURE 2 | Paecilomyces variotii produces a mix of uni- and multinucleate spores. Nucleus copy numbers in strain CBS 101075 conidiospores were visualized
through the expression of a CFP-Histone H2B fusion construct. (A) CFP fluorescence, (B) DIC image, and (C) overlay, scale bar = 25 µm. (D) Histogram of the
distribution of the number of nuclei per spore (n= 138); two nuclei per spore is the most common, and 32.6% of spores are uninucleate.
construct, two had a phenotype similar to wild type and one
resembled the AU1_63 mutant. However, PCR analysis showed
that this non-complementing transformant has not integrated the
wild-type copy of the gene into its genome whereas the two other
strains with the wild type spore pigmentation had (Figure 4B).
Paecilomyces variotii is heterothallic, and comparison of
strains CBS 144490 (MAT1-1) and CBS 101075 (MAT1-2)
revealed that each has a distinct gene complement at its MAT
locus (Supplementary Figure S5). The pair therefore allows
the potential for crossing. The 32 progeny of a cross between
mutant AU1_63 and CBS 144490 showed prefect co-segregation
of hygromycin resistance with the pale colony pigmentation, as
shown in Table S6. Two additional genetic markers, 123A and
123B (Supplementary Table S3), located 1,069,000 bp apart on
contig 123 of CBS 144490 were examined in these progeny.
These markers demonstrated that recombination events take
place during crossing, consistent with meiotic reduction events
rather than parasexual reduction in chromosome numbers as can
occur in some Eurotiales species.
The Mutation in prmJ in Strain AU1_63
Can Be Cross-Species Complemented
by the Aspergillus niger prmJ Homolog
The DUF1212-containing protein (PrmJ) identified in P. variotii
shows strong sequence similarity to homologs from the genus
Aspergillus. As a representative example, the alignment of the
A. niger homolog (GenBank: EHA28452.1) has 66% identical
amino acids with PrmJ of P. variotii.
To test the hypothesis that the PrmJ proteins have a conserved
function, the coding region of the homologous gene from
A. niger was cloned into the constitutive expression plasmid
PLAU2 and transformed into the AU1_63 mutant. Five putative
transformants were obtained; all showed an increase in colony
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FIGURE 3 | Targeted gene disruption, through homologous recombination, of
the leuA gene in P. variotii.(A) Growth of two representative transformants, 15
that is a putative leuA deletion strain and 11 that is an ectopic insertion of the
deletion construct, on minimal medium with (+) or without (–) leucine. (B) PCR
amplification of the 50and 30regions adjacent to leuA into the hph selectable
marker gene illustrate correct integration of the knockout construct by
amplifying sequences unique to a correct integrant (3,440 and 2,850 bp) in
transformants 15 and 20, but not in ectopic insertion strains 4 and 11.
pigmentation, and one of these transformants was further
analyzed (Figure 6A). PCR confirmed that the transformant
contained both the mutated copy of the prmJ allele and the
introduced A. niger transgene (Figure 6B). Thus, the A. niger
homolog can complement the functions lost in the P. variotii
prmJ gene mutant.
The DUF1212 Domain Protein Is Not
Essential for Mating in P. variotii
There is little information about DUF1212 proteins in
fungi, other than that the PRM10 gene of S. cerevisiae is
transcriptionally induced in response to sexual pheromones
(Heiman and Walter, 2000). Of the progeny of the
AU1_63 ×CBS 101075 cross, eight contained the disrupted prmJ
allele and were of the opposite mating type (MAT1-2) to strain
AU1_63 (MAT1-1) (Supplementary Table S6). One of these
isolates was back-crossed to strain AU1_63, and this combination
of strains was able to produce the sexual cleistothecia structures
and viable progeny from ascospores (Supplementary Figure S6).
Hence, the DUF1212 domain protein is not essential for sexual
crossing in P. variotii.
Strain AU2_33 Has Delayed Sporulation
and Growth Defect Phenotypes Due to
Mutation of the Mitochondrial Membrane
Carrier DspA
Strain AU2_33 showed delayed sporulation on CV8 medium,
with spore production beginning at around 3–4 days, in contrast
to the wild type that produces spores as soon as the colony
begins to expand (Figure 5A). Even after 14 days, the amount
of sporulation was reduced. On this medium the radial growth
rate was not noticeably reduced. In contrast, on defined minimal
medium, the radial growth rate of the AU2_33 mutant was highly
reduced as it showed close to no growth. A complementation
construct was produced with a wild type copy of the gene, and
when transformed into strain AU2_33, the transformants showed
a phenotype resembling that of the wild type. As expected, PCR
analysis of the two complemented isolates indicated that they
contain both a mutant and a wild-type allele in their genomes
(Figure 5B).
Transformants of CBS 101075 expressing a DspA-mCherry
fusion protein displayed red fluorescence. This co-localized with
the green fluorescence of a mitochondrially localized GFP-citrate
synthase fusion protein, indicating that this putative carrier
protein also localizes to the mitochondrion (Figure 5D).
The T-DNA insertion in strain AU2_33 co-segregated with
the delayed sporulation phenotype in 18 out of 20 progeny as
assessed by PCR (Supplementary Table S7). Two progeny, 17
and 19, contained the mutant dspA allele yet did not display
the mutant phenotype, which might be due to the effect of
other genetic rearrangements taking place during crossing. The
two T-DNA inserts of mutant AU2_33 displayed genetic linkage
co-segregating in 19 of 20 progeny. There was recombination
between the T-DNAs and mating type locus, demonstrating the
progeny were the result of meiotic events. Intriguingly, all of the
progeny from this cross were sensitive to hygromycin.
The hph Gene, Conferring Hygromycin
Resistance, Is Mutated by
Repeat-Induced Point Mutation (RIP) in
the Progeny of a Cross Between AU2_33
and CBS 101075
None of the 20 progeny resulting from a cross between
mutant AU2_33 and CBS 101075 showed a hygromycin
resistance phenotype, despite the T-DNA construct being present
in 10 of these progeny as demonstrated by PCR analysis
(Figures 7A,B). Therefore the coding region of the hph
gene, which confers resistance to hygromycin, in one of the
progeny (progeny 3) was sequenced. The open reading frame
of the hph gene amplified from both T-DNA insertion copies
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FIGURE 4 | (A) Mutation of the prmJ gene, in strain AU1_63, results in a pale phenotype on cleared V8 juice (CV8) agar but not on potato dextrose agar (PDA). Two
of the three strains transformed with the complementation construct have a wild type phenotype while one resembles the prmJ mutant. (B) PCR analysis of the
genotypes of the AU1_63 mutant, wild type CBS 101075, and the three strains after transformation with the complementation construct. The AU1_63CompB
transformant, in which the phenotype was not complemented, has not integrated a wild-type copy of the prmJ gene. (C) Location of the T-DNA insert in the prmJ
gene. Green represents sequence of the T-DNA and red represents nucleotides lost when the T-DNA integrated into the genome in the mutant strain AU1_63.
revealed substitution mutations characteristic of RIP (Figure 7C).
A 780 bp region was sequenced and 141 (18.1%) and 156 (20%)
nucleotides were mutated. The mutations were all C to T or
G to A. RIPCAL analysis revealed bias toward CpA to TpA
dinucleotides and the complementary TpG to TpA mutations
that are characteristic of RIP in other fungal species such as
N. crassa (Figure 7D).
The P. variotii Genome Features
Evidence of RIP
The genome sequences of P. variotii have a bimodal GC content,
containing long stretches of approximately 50% G:C interspersed
by relatively shorter regions of approximately 20% G:C. The
example of the first 450,000 bp of CBS 144490 contig 49 is
given in Figure 8A. Overall, these AT rich regions constitute
approximately 8.49% of the CBS 101075 assembly and 13.8%
of CBS 144490 assembly (Figure 8B). It should be noted that
because of the different sequencing strategies – long reads
from Pacific Bioscience vs. 100 nucleotide reads from Illumina
technologies – these proportions can only be compared broadly.
One mechanism by which AT-rich regions can be created is RIP
(Testa et al., 2016), which has been shown to defend the N. crassa
genome against transposons (Kinsey et al., 1994). We hypothesize
that the AT-rich regions identified in the P. variotii genome are
due to RIP.
In support of this hypothesis, a putative Tf2-type
retrotransposon, Tn123, on contig 123 (nucleotide position
231,587–238,677) of CBS 144490 was identified via BLASTx
searches (Altschul et al., 1990). BLASTn comparison of this
sequence against the two P. variotii genomes revealed sequence
similarity between this transposon and a number of the AT-rich
regions in both genomes. Furthermore, there was a clear pattern
of C →T and G →A mutations that are characteristic of RIP.
RIPCAL analysis showed that most of the RIP-like mutations
targeted CpA dinucleotides, which is also highly characteristic of
RIP [(Hane et al., 2015); Figure 8C]. This strongly suggested that
RIP mutation of retrotransposons including, but not limited to
Tn123, is responsible for the formation of at least some of these
AT-rich regions.
P. variotii Has a Reduced Expansion of
Gene Families, Including Polysaccharide
Degradation Related CAZy Genes,
Relative to Other Eurotiales Species
Given the genomic and experimental evidence for the active
occurrence of RIP in P. variotii we assessed whether a
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FIGURE 5 | Strain AU2_33 has a growth and sporulation defect due to mutation of the dspA gene. (A) Sporulation on CV8 was delayed in the mutant AU2_33 at
both 3 and 14 days after growth on clear V8 juice medium compared to the wild type CBS 144490 and two complemented strains. The AU2_33 mutant also had
impaired growth on minimal medium (MM). (B) PCR analysis of the genotypes of the AU2_33 mutant, wild type and two complemented isolates. (C) The T-DNA
insertion is located in the first exon of the dspA gene. Green represents sequence of the T-DNA and red represents sequence lost from the genome in the mutant.
(D) Co-localization of mCherry-tagged DspA protein and mitochondrially localized GFP: (i) red fluorescence from the DspA-mCherry fusion, (ii) green fluorescence of
citrate synthase-GFP, (iii) blue fluorescence due to calcofluor white staining of the cell wall, and (iv) the merged image. Scale bar = 10 µm.
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FIGURE 6 | Cross-species complementation of the P. variotii AU1_63 strain
with the Aspergillus niger prmJ homolog restores the colony pigmentation to
wild type levels of the AU1_63 mutant of CV8 media. (A) Growth of the wild
type (CBS 101075), T-DNA insertion mutant (AU1_63), and insertion mutant
transformed with the A. niger prmJ gene (AU1_63+AnComp) on cleared V8
juice medium. (B) A PCR analysis for the P. variotii mutated allele and the
introduced A. niger alleles in the three strains, with wild type A. niger as a
control.
consequence is the limited expansion of gene families in this
species. A comparison of P. variotii with other Eurotiales species
shows that these strains have the fewest genes (Figure 9).
We compared the bi-directional similarity of P. variotii
genes against the second closest BLAST match in its own
genome. Consistent with RIP P. variotii has fewer genes with
close similarity than the comparison species in the genera
Talaromyces,Penicillium,Saccharomyces,Schizosaccharomyces
and most Aspergilli. However, several Aspergillus species
including A. clavatus also had few similar genes, possibly
indicating past or current RIP in these species (Figure 10A).
Comparative cluster data obtained through the MycoCosm
portal (Grigoriev et al., 2014) show that P. variotii along with
two other species in the family Thermoascaceae (P. formosus
and Thermoascus aurantiacus) contains considerably fewer
genes in the 100 most populous gene clusters (Figure 10B
and Supplementary Table S8). For example, examination of
secondary metabolite gene clusters shows that the three species
in the Thermoascaceae contain fewer secondary metabolite
clusters than other species in the Eurotiales (Figure 10C).
P. variotii is particularly depleted in genes encoding polyketide
synthases, with strain CBS 101075 containing only six such
genes compared to as many as 28 in some of the Aspergillus
species examined. Two other striking reductions in gene family
numbers were seen for amino acid permeases (cluster 12)
and major facilitator superfamilies (clusters 10 and 13). The
one exception to the reduction in gene numbers in the
Thermoascaceae species examined was an expansion in genes
encoding methyltransferases (cluster 11).
Comparison of the CBS 144490 genome with that of CBS
101075 revealed a high level of similarity; however, CBS 144490
contains an additional 1.2 Mb of sequence, some of which is
made up of repetitive elements, while estimated to have 40 fewer
genes overall (Supplementary Table S4). A comparison between
genomes revealed that the CBS 144490 strain has 372 genes and
the CBS 101075 strain has 450 genes that are unique to each
strain and not found in the other. No examples of recent DNA
duplications were observed in either genome. In many cases,
genes unique to one or the other strain were found as clusters
of varying size of such unique genes. The most striking example
is the presence of scaffold 108 (151 kb) in CBS 144490 that is
absent from CBS 101075. This region includes 52 predicted genes,
including a putative non-ribosomal peptide synthase. However,
despite these differences to date no in vitro growth differences
have been observed for the two strains.
Evidence for the lack of expansion of gene families in
P. variotii can be seen in the genes encoding CAZys (for
plant polysaccharide degradation), as P. variotii had the fewest
number of such genes (74 genes) of all tested Eurotiales species
(Figure 10D and Supplementary Dataset S1). In total, this
number is most similar to Aspergillus glaucus (92 genes), while
significantly higher CAZy gene numbers in all of the other species
suggests a better capability for plant polysaccharide degradation.
Assimilation Capabilities Are Reduced in
P. variotii for Some Carbohydrate
Sources
To assess if the reduction in gene family numbers has a
consequence on biology, the growth of P. variotii on different
carbon sources was compared with other Eurotiales species.
Overall, P. variotii is less able to use these plant-derived
compounds as a carbon source than most other species
(Figure 11). The growth profile of P. variotii is also most similar
to A. glaucus, consistent with the genome content of CAZys.
Growth of P. variotii is particularly poor on cellulose, xylan
and inulin, which correlates with the very low number of
genes encoding cellulolytic (8 genes), xylanolytic (30 genes) and
inulinolytic (2 genes) enzymes compared to other species in the
Eurotiales. Talaromyces marneffei has no inulinolytic genes and
A. nidulans has the same number as P. variotii: these species also
grow very poorly on inulin, as do several others (Supplementary
Dataset S1 and Figure 11). Interestingly, P. variotii can produce
high levels of invertase when cultivated on agricultural and
industrial residues (Job et al., 2010). Growth on commercial
cellulose (Avicel) is challenging for most fungi, so it is hard to
use it to draw comparative conclusions about species differences,
although reasonable radial growth was observed for P. variotii.
However, P. variotii has been shown to produce a glucose-
tolerant β-glucosidase (Job et al., 2010) indicating its ability to
release glucose molecules from short oligosaccharides. A clear
difference in growth of the species is seen on xylan, but this does
not fully correlate with the number of xylanolytic genes. Growth
is poor for P. variotii and A. glaucus that have a low number of
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FIGURE 7 | Repeat induced point mutation is active in the Eurotiales. (A) PCR analysis indicates that four progeny (P2, P3, P4, and P10) of a cross between the wild
type (WT) and AU2_33 contain the hygromycin phosphotransferase gene, despite (B) these progeny being sensitive to hygromycin (HYG). (C) Nucleotide sequence
alignment of 780 bp of the hph coding region of both T-DNAs in progeny number 3 showed a pattern of C to T and A to G mutations, consistent with the RIP
process. Dots represent identical nucleotides. (D) RIPCAL analysis of the sequencing information in (C) revealed a basis towards mutation of CpA dinucleotides, also
consistent with RIP mutation.
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FIGURE 8 | (A) Paecilomyces variotii isolates show a bipartite genome structure that is characteristic of a consequence of repeat induced point mutation acting in
the organism. (B) The genome assembly of strain CBS 144490 contains a greater proportion of AT-rich regions than does that of strain CBS 101075 (C). A putative
transposon was identified on contig 123 of CBS 144490 with similarity to some of the AT-rich regions present in both genomes: RIPCAL analysis revealed that most
of the putative mutations were CpA to TpA (TpG to TpA in the reverse strand), which is a feature of DNA that has undergone RIP.
xylanolytic genes, but also for A. wentii that has a similar number
(66 genes) to species that grow well on this substrate. In contrast,
good growth was observed for T. marneffei (47 genes), which has
a similar number of genes as A. glaucus (41 genes). However,
aP. variotii strain showing high xylanase production has been
described (de Laguna et al., 2015), suggesting that even with a
few genes those enzymatic activities may reach high levels.
Paecilomyces variotii grows relatively well on guar gum
(galactomannan) even though it has a low number of
mannanolytic genes in its genome (10 genes), similar to
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FIGURE 9 | Paecilomyces variotii has fewer genes than many other species in the Eurotiales. Phylogenetic relationships between the Eurotiales species, with two
yeast species as outgroups, were defined from a comparison of 3,374 single copy gene orthologs. The graph shows total numbers of genes in each species and the
distribution of the homologs.
A. glaucus. Both species grow better on guar gum than on xylan,
suggesting that their limited enzyme system is sufficient for
degradation of galactomannan. Neither species contains a known
endomannanase, but both contain exo-enzymes, β-mannosidases
and α-galactosidases that can release the monomeric sugars from
galactomannan. A. niger and P. subrubescens, which both have
a much more extensive mannanolytic gene system, including
several endomannanase encoding genes, grow much better on
guar gum.
Similarly to guar gum, P. variotii showed good growth on
apple pectin despite having the lowest number of pectinolytic
genes (25 genes) from the tested species. Exo-polygalacturonases
have also been purified from P. variotii cultures further
demonstrating its pectinolytic capability (de Lima Damásio et al.,
2010;Patil et al., 2012). This is in contrast to poor growth of
A. clavatus, which has a reduced number of pectinolytic genes (43
genes) compared to most other Aspergilli, but still almost twice
as many as P. variotii. The better growth of P. variotii on apple
pectin could be explained by a higher number of GH28 pectin
hydrolases (6 genes) compared to A. clavatus (3 genes). This may
also explain the poorer growth of A. glaucus on this substrate, as
while it has a similar number of pectinolytic genes as P. variotii,
it only contains two genes encoding GH28 enzymes.
Paecilomyces variotii has been shown to produce thermostable
glucoamylase and α-amylase with potential in industrial
applications (Michelin et al., 2008;Michelin et al., 2010). Growth
of P. variotii on starch was similar to most other species.
Despite a somewhat reduced amylolytic gene set (16 genes),
P. variotii has all the enzymatic activities for degradation
of starch, which likely explains the growth observed on this
polysaccharide.
DISCUSSION
The genus Paecilomyces has received limited attention for
functional genomics, despite its role in industry, human disease,
and as a commonly encountered saprobe found around the
world. This research has generated high quality genome sequence
resources, demonstrates that genetic segregation analysis is
possible, and shows that gene disruption by either targeted or
reverse genetics is highly feasible for gene discovery. Several key
points arising from this work are described in the following
sections.
Using P. variotii, new properties associated with fungal
genes of unknown function have been defined. For example,
proteins with a DUF1212 are widely conserved in fungi and
include Prm10 in S. cerevisiae and NCU00717 in N. crassa.
In S. cerevisiae, the gene was found to be up-regulated three-
fold in response to pheromone and predicted to contain five
transmembrane segments (Heiman and Walter, 2000). However,
the biological function of these proteins has not been elucidated
and no phenotypes found in gene disruption strains. This study
reports a pigmentation phenotype associated with disruption
of the DUF1212 homolog in P. variotii (Figure 4). Given that
the PRM10 gene is up-regulated in response to pheromone in
S. cerevisiae (Heiman and Walter, 2000), we assessed whether
the protein is required for mating in P. variotii. Crosses between
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Urquhart et al. Genome Analysis of Paecilomyces variotii
FIGURE 10 | Paecilomyces variotii and related Thermoascaceae have a reduced expansion in gene families. (A) A limited number of highly similar gene duplicates
are observed in P. variotii compared to other Eurotiales. For each genome, a self BLASTp was conducted to identify orthologs by reciprocal best hit via BLAST, then
the fraction of orthologs at various identity levels were plotted. x-axis: percent identity, y-axis: lineage, z-axis: fraction of all orthologs at a given % identity. Lineages
are colored at the genus level, green: Paecilomyces, purple: Talaromyces, blue: Penicillium, dark red: Aspergillus, red: Saccharomyces, yellow:
Schizosaccharomyces.(B) Three Thermoascaceae species have relatively fewer genes in the 100 most populous gene clusters in the comparative cluster analysis
obtained through MycoCosm. Similarly, these species showed a more restricted set of (C) secondary metabolite genes and (D) genes encoding
Carbohydrate-Active enZYmes (CAZys).
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FIGURE 11 | Growth of P. variotii compared to other Eurotiales species on different plant polysaccharides as the sole carbon source, compared to glucose. Petri
dishes containing minimal medium and differing carbon sources were inoculated with different species of Eurotiales and growth photographed after 2–5 days
depending on the species.
two isolates carrying the DUF1212 mutant allele produced
cleistothecia, sexual spores and viable progeny (Supplementary
Figure S6). This suggests that the PRM10 homolog (prmJ) is
not essential for the sexual cycle of P. variotii. The ability of
the A. niger prmJ homolog to complement the pigmentation
phenotype of the P. variotii mutant strain implies that the
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Urquhart et al. Genome Analysis of Paecilomyces variotii
function of this protein is conserved between the two genera
(Figure 6). Identification of a phenotype associated with a
Domain of Unknown Function (DUF) protein that has a
conserved function in a related species suggests that P. variotii
is a species in which to study this protein family in greater
detail.
A second insertional mutant investigated in detail contained
a T-DNA in the dspA gene encoding a mitochondrial carrier
family protein. DspA is localized to the mitochondria and
mutation of the dspA gene delays sporulation in a manner
dependent on the medium composition (Figure 5). As in the
case of the AU1_63 strain, the phenotype of the AU2_33
(dspA mutant) strain is influenced by the composition of the
media. On a minimal medium, growth on the strain was highly
restricted (Figure 5). This provides a possible direction for
future studies into the function of this putative mitochondrial
carrier protein. That is, if a compound can be found that
when supplemented into the media restores the phenotype
of this mutant, that compound might represent the substrate
of the carrier. Despite their annotation, not all mitochondrial
carrier family proteins are localized to the mitochondria. For
example, proteins in this family have been found localized
to chloroplasts (Palmieri et al., 2009) and peroxisomes (Jank
et al., 1993). Thus, the localization of mitochondrial carrier
family proteins cannot be predicted, so must be determined
experimentally. We demonstrate, through co-localization with a
known mitochondrial protein, that the DspA protein in P. variotii
has a mitochondrial localization.
An unexpected finding from the genetic segregation analysis
of mutant AU2_33, which contains two T-DNAs, was that all
of the progeny were hygromycin sensitive despite half of the
progeny encoding the hygromycin resistance gene when they
were tested by PCR (Figure 7). We traced this loss of resistance to
mutation of the hph gene by RIP. Thus, this analysis in P. variotii
provides important evidence that Eurotiales fungi have active RIP
mechanisms.
Analysis of the P. variotii genome sequence shows evidence
for past RIP activity, both in its bi-modal G:C content and more
conclusively the presence of a putative retrotransposon, Tn123,
some copies of which strongly appear to have been affected by
RIP (Figure 8). In the majority of genomes analyzed for past RIP
activity there is a dinucleotide profile that is biased toward RIP-
like CpA mutations (Hane et al., 2015). Analysis of the Tn123
sequences also indicated a strong CpA bias, strengthening our
conclusion that the mutated copies of this transposon sequence
have been created through RIP mutation (Figure 8).
The predicted consequences of RIP are limitations in the
expansion of genes by gene duplication. Evidence for this
comes from the analysis of gene families when compared with
other ascomycete species. As illustrated in Figures 9,10 and
Supplementary Table S8,P. variotii consistently has the lowest
number of genes other than N. crassa, where RIP has been
demonstrated to occur, and Thermoascus aurantiacus, where little
is know about its genetics. While one predicted consequence of
RIP should be limitation in the expansion of gene families, this
is not always the case: in this analysis the species with the largest
number of families, Nectria haematococca, also has an active RIP
process (Coleman et al., 2009). Analysis of the ability of P. variotii
to degrade polysaccharides suggests that it has much smaller gene
set related to the degradation of plant polysaccharides compared
to most of the other tested Eurotiales species (Figure 10D). One
interpretation of this finding is that RIP mutation has reduced
gene duplication and thus the expansion of these gene families.
This may represent evidence of the hypothesized evolutionary
cost associated with the genome protection afforded by RIP
(Galagan and Selker, 2004). A flipside of the evolutionary cost of
RIP has also been hypothesized that certain loci within repeat rich
compartments may undergo accelerated evolution; this remains
to be experimentally validated.
Despite recent advances, not least in the rapid rate of genome
sequencing (Grigoriev et al., 2014), only a minute fraction of
the millions of fungal species believed to exist (Blackwell, 2011)
have been studied at the genetic level. This is because the
necessary combination of tools required for functional biology,
i.e., a genome sequence, transformation protocols, targeted
gene mutations and genetic crosses, have been developed in
relatively few species. However, research conducted beyond the
current model organisms is vital to gain a more comprehensive
understanding of fungal biology.
Paecilomyces variotii is one of the vast number of fungal
species for which techniques for genetic manipulation have
not previously been reported, despite its relevance to human
activities. In this study, we have produced genome assemblies
for two strains as well as developing transformation, efficient
targeted gene disruption using Agrobacterium and convenient
genetic crosses. Considering that PEG-mediated protoplast
transformation is commonly used in several species of the
Eurotiales (Nara et al., 1993;de Bekker et al., 2009;Arentshorst
et al., 2012;Oakley et al., 2012;Weyda et al., 2017), it is likely that
this protocol could also be adapted to P. variotii. Taken together,
P. variotii could now be considered as a convenient model for
studying aspects of the diverse biology of the Eurotiales (de
Vries et al., 2017), and in particular the family Thermoascaceae,
including studying RIP. Future work will undoubtedly uncover
more novelties or shared features in this ubiquitous organism.
AUTHOR CONTRIBUTIONS
AU, SM, MM, AW, and AI performed the experiments. AU,
SM, GH, SM, JP, AL, KB, IG, and AI were involved in genome
sequencing, assembly and annotation. AU, SM, JH, IG, and AI
analyzed the data. AU, MM, SM, RdV, IG, and AI designed the
experiments, discussed the results, and wrote the manuscript. All
authors provided final approval for the manuscript.
FUNDING
This work was supported by the Australian Research Council
(AI), a scholarship from the Grains Research and Development
Corporation, an RTP scholarship from the Australian
Government and the Australasian Mycological Society (AU). The
Joint Genome Institute, a DOE Office of Science User Facility,
Frontiers in Microbiology | www.frontiersin.org 18 December 2018 | Volume 9 | Article 3058

fmicb-09-03058 December 11, 2018 Time: 17:41 # 19
Urquhart et al. Genome Analysis of Paecilomyces variotii
is supported by the Office of Science of the U.S. Department of
Energy (Contract No. DE-AC02-05CH11231).
ACKNOWLEDGMENTS
We thank Allison van de Meene (University of Melbourne)
for assistance with microscopy, Mark Wilson (CSIRO FRR
culture collection) for providing strains, and Ciara Redmond
for technical assistance in crossing experiments. The U.S.
Department of Energy Joint Genome Institute, a DOE Office of
Science User Facility, is supported by the Office of Science of the
U.S. Department of Energy.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2018.03058/full#supplementary-material
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