# On the current status of [i]Phakopsora pachyrhizi [/i] genome sequencing

Marco Loehrer, Alexander Vogel, Bruno Huettel, Richard Reinhardt, Vladimir Benes, Sébastien Duplessis, Bjoen Usadel, Ulrich Schaffrath

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Marco Loehrer, Alexander Vogel, Bruno Huettel, Richard Reinhardt, Vladimir Benes, et al. On the current status of [i]Phakopsora pachyrhizi [/i] genome sequencing. Frontiers in Plant Science, 2014, 5 (377), 5 p. 10.3389/fpls.2014.00377. hal-01268956

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# On the current status of *Phakopsora pachyrhizi* genome sequencing

Marco Loehrer<fnref n="1" />, Alexander Vogel<fnref n="2" />, Bruno Huettel<fnref n="3" />, Richard Reinhardt<fnref n="3" />, Vladimir Benes<fnref n="4" />,
Sébastien Duplessis<fnref n="5" />,<fnref n="6" />, Björn Usadel<fnref n="2" />,<fnref n="7" /> and Ulrich Schaffrath<fnref n="1" />*

**Edited by:**

David L. Joly, Université de Moncton,
Canada

**Reviewed by:**

John Fellers, United States
Department of Agriculture –
Agricultural Research Service, USA
Ralf Thomas Voegele, Universität
Hohenheim, Germany

**\*Correspondence:**

Ulrich Schaffrath, Department of Plant
Physiology, RWTH Aachen University,
Worringerweg 1, 52056 Aachen,
Germany
e-mail: schaffrath@bio3.rwth-aachen.de

Recent advances in the field of sequencing technologies and bioinformatics allow a more rapid access to genomes of non-model organisms at sinking costs. Accordingly, draft genomes of several economically important cereal rust fungi have been released in the last 3 years. Aside from the very recent flax rust and poplar rust draft assemblies there are no genomic data available for other dicot-infecting rust fungi. In this article we outline rust fungus sequencing efforts and comment on the current status of *Phakopsora pachyrhizi* (Asian soybean rust) genome sequencing.

**Keywords:** fungal genomics, rust fungi, Asian soybean rust, next-generation sequencing, herterozygosity, genome size, k-mer analysis

Sequencing of fungal genomes represented a significant milestone in the emerging era of “genomics.” In fact, the first eukaryotic genome ever sequenced was that of baker’s yeast, *Saccharomyces cerevisiae*, which consequently strengthened its position as a fun- gal model organism after the release of the 12 Mb genome with approximately 6000 genes in 1996 (Goffeau *et al.*, 1996). Some time thereafter the genomes of the fission yeast *S. pombe* (14 Mb) and the filamentous ascomycete *Neurospora crassa* (40 Mb) were released in Wood *et al.* (2002) and Galagan *et al.* (2003), respec- tively. Accelerated progress in sequencing technology from early clone-by-clone approaches through Sanger-based whole-genome shotgun sequencing (WGS) to today’s next-generation sequencing (NGS) shortened the periods between releases of novel genomes considerably (Grigoriev, 2014). This paved the way for compara- tive genomics which opened new possibilities for people working in the field of agriculture and biotechnology or combating human, animal or plant diseases (Vebø *et al.*, 2009; Manning *et al.*, 2013; Bolger *et al.*, 2014).

In the latter field, the sequencing of the genome of the ascomycete *Magnaporthe oryzae* was achieved by Dean *et al.* (2005). Along with the genome of rice (Goff *et al.*, 2002), the *M. oryzae* host plant, an understanding of the plant–pathogen interaction became possible at the genome level. Since then, sev- eral plant-pathogenic fungi were sequenced; however, a group of pathogens that exclusively feed from living plant tissue, so-called obligate biotrophs, remained recalcitrant. This was disappointing particularly because some of the most economically serious threats to human nutrition, such as powdery mildew fungi and rust fungi, are among this group.

Rust fungi have long been in the focus of plant pathologists. Already in the 19th century, Anton de Bary, who is considered as a founder of plant pathology, picked up *Puccinia graminis* with its various *formae speciales* that are specialized for para- sitism on particular cereal hosts, as subject for his groundbreaking studies. Later Harold Henry Flor developed the famous “gene- for-gene” concept based on his work on the interaction of flax rust (*Melampsora lini*) with its host plant flax (*Linum usitatissi- mum*; Flor, 1955). Despite considerable interest, sequencing of rust genomes was not achieved until most recently. Thus, the 101 Mb genome of *Melampsora larici-populina* and the 89 Mb draft genome of *Puccinia graminis* f. sp. *tritici* were sequenced in a common effort by the Joint Genome Institute and the Broad Institute, respectively, and published in Duplessis *et al.* (2011). Following, more or less advanced draft genomes of other rust fungi were sequenced and published by the community, such as several *Puccinia striiformis* f. sp. *tritici* races (56–110 Mb) and the flax rust genome *M. lini* (Cantu *et al.*, 2011, 2013; Zheng *et al.*, 2013; Nemri *et al.*, 2014; see **Table 1**). Although Puc- ciniales is an order with a lesser coverage compared to other fungi<fnref n="1" />, more genomic resources are becoming accessible. A major drawback encountered during sequencing efforts of rust genomes was their unexpected large sizes, a fact that also hampered attempts of sequencing the genome of the Asian soybean rust fungus *Phakopsora pachyrhizi*, an economically important threat to soybean cultivation. The following commentary is written to give an overview on the current status of *P. pachyrhizi* genome

<footnote n="1">¹ Department of Plant Physiology, Rheinisch-Westfälische Technische Hochschule Aachen University, Aachen, Germany</footnote>

<footnote n="2">² Institute for Botany and Molecular Genetics, Institute for Biology I, Rheinisch-Westfälische Technische Hochschule Aachen University, Aachen, Germany</footnote>

<footnote n="3">³ Max Planck Institute for Plant Breeding Research, Köln, Germany</footnote>

<footnote n="4">⁴ Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany</footnote>

<footnote n="5">⁵ Institut National de la Recherche Agronomique, Interactions Arbres/Microorganismes, UMR 1136, Champenoux, France</footnote>

<footnote n="6">⁶ Université de Lorraine, Interactions Arbres/Microorganismes, UMR 1136, Vandoeuvre-lès-Nancy, France</footnote>

<footnote n="7">⁷ Institute of Bio- and Geosciences-2 Plant Sciences, Institute for Bio- and Geosciences, Forschungszentrum Jülich, Jülich, Germany</footnote>

<footnote n="1">¹http://genome.jgi.doe.gov/programs/fungi/1000fungalgenomes.jsf</footnote>
<table><caption>Table 1 | Published rust fungi draft genomes and genome size estimations in alphabetical order.</caption><thead><tr><th>Organism</th><th>Genome size (Mb)</th><th>Estimation/sequencing method</th><th>Reference</th></tr></thead><tbody><tr><td>Cronartium quercuum f. sp. fusiforme</td><td>90</td><td>Flow cytometry</td><td>Anderson et al. (2010)</td></tr><tr><td>Hemileia vastatrix</td><td>733.5</td><td>Flow cytometry</td><td>Carvalho et al. (2013)</td></tr><tr><td>Melampsora larici-populina</td><td>101</td><td>Sanger sequencing</td><td>Duplessis et al. (2011)</td></tr><tr><td>Melampsora lini</td><td>189</td><td>Next-generation sequencing</td><td>Nemri et al. (2014)</td></tr><tr><td>Puccinia coronata</td><td>77</td><td>Flow cytometry</td><td>Eilam et al. (1994)</td></tr><tr><td>Puccinia graminis f. sp. tritici</td><td>89</td><td>Sanger sequencing</td><td>Duplessis et al. (2011)</td></tr><tr><td>Puccinia hordei</td><td>121</td><td>Flow cytometry</td><td>Eilam et al. (1994)</td></tr><tr><td>Puccinia recondita</td><td>127</td><td>Flow cytometry</td><td>Eilam et al. (1994)</td></tr><tr><td>Puccinia sorghi</td><td>102</td><td>Flow cytometry</td><td>Eilam et al. (1994)</td></tr><tr><td>Puccinia striiformis f. sp. tritici, race PST-130</td><td>65</td><td>Next-generation sequencing</td><td>Cantu et al. (2011)</td></tr><tr><td>Puccinia striiformis f. sp. tritici, races PST-21, PST-43, PST-87/7, PST-08/21</td><td>73, 71, 53, 56</td><td>Next-generation sequencing</td><td>Cantu et al. (2013)</td></tr><tr><td>Puccinia striiformis f. sp. tritici, isolate CY32</td><td>110</td><td>Next-generation sequencing, "fosmid-to-fosmid" sequencing</td><td>Zheng et al. (2013)</td></tr><tr><td>Puccinia triticiana 1-1 BBDB race 1</td><td>135</td><td>Assembly</td><td>Fellers et al. (2013)</td></tr><tr><td>Uromyces appendiculatus</td><td>418</td><td>Flow cytometry</td><td>Eilam et al. (1994)</td></tr><tr><td>Uromyces vignae</td><td>407</td><td>Flow cytometry</td><td>Eilam et al. (1994)</td></tr></tbody></table>

sequencing and is intended to initiate combined activities toward this goal.

What makes *P. pachyrhizi* so interesting? For sure it is a devastating fungal disease of the important crop plant soybean. The origin of the pathogen can be traced back to Asia and most likely it spread alongside with the propagation of soybean cultivation. *P. pachyrhizi* is able to infect more than 31 species from 17 genera of legumes, which is a rather unusual feature for rust fungi that usually are highly specialized for particular hosts (Goellner et al., 2010). *P. pachyrhizi* differs in a further important aspect from the majority of rusts: it directly penetrates leaf cells rather than entering the leaf via stomata at the uredinial stage. On the contrary, most rust fungi use stomata to get inside the host tissues at this stage and a direct penetration is only observed for some rust fungi when basidiospores infect the aecial host at later stages of the rust life cycle (Heath, 1997). Recent studies imply that generation of high turgor pressure of around 5 MPa in the non-melanized appressoria supports penetration (Loehrer et al., 2014). Penetrated epidermal cells undergo a cell death response, again an unexpected property for a biotrophic pathogen. Experiments with non-host plants such as barley and Arabidopsis showed that during penetration and concomitant epidermal cell death, marker genes associated with responses to necrotrophic pathogens are switched on and that cell death suppression had a negative influence on infection success of

*P. pachyrhizi* (Loehrer et al., 2008; Hoefle et al., 2009). Regarding its lifestyle, *P. pachyrhizi* which forms so far only a single spore type in the wild, i.e. urediospores, is a minimalist compared to, e.g., *Puccinia graminis* f. sp. tritici which has five distinct spore types and performs a host jump (Leonard and Szabo, 2005). Despite the unknown or missing sexual life cycle the genetic diversity of *P. pachyrhizi* seems not to be impaired. This may be explained by parasexual nuclear recombination occurring between different isolates after germ tube fusion or hyphal anastomosis, a feature also reported for cereal rusts (Wang and McCallum, 2009; Vittal et al., 2012).

Public information about the *P. pachyrhizi* genome sequencing project is rare. In the DoE JGI Community Sequencing Program of 2004, a project was launched to sequence the genome of *P. pachyrhizi* (isolate Taiwan 72-1) based on a fosmid shotgun sequencing approach. The genome size prediction with 50 Mb at that time was much underestimated. The sequencing project has now a "permanent draft" status at the JGI<fnref n="2" />. Besides the recently released mitochondrial genome sequence (Stone et al., 2010), information on assembly attempts of the nuclear genome have not been published. The major drawback for progress in

<footnote n="2">²http://genome.jgi.doe.gov/genome-projects/pages/project-status.jsf?projectId=16847</footnote>
P. pachyrhizi genome sequencing seems to be its huge size. An
update on this topic was given at the National Soybean Rust Sym-
posium 2005 in Nashville (TN, USA). Genome size estimations
ranged from 300 to 950 Mb depending on the analysis method used
(Posada-Buitrago et al., 2005). A similar statement was provided
by Igor Grigoriev (Head of the JGI Fungal Program) suggesting a
genome size above 850 Mb (Duplessis et al., 2012). Besides, other
general features of rust fungi genomes unraveled since then, such
as expanded multigene families and very large amount of transpos-
able elements (>45%), pose serious problems for proper genome
assembly.

We started our own efforts toward uncovering the genome size
of *P. pachyrhizi* by using our lab isolate (Brazil 05-1) and we
followed a strategy based on k-mer analysis. By breaking down
the reads obtained by Illumina sequencing into short nucleotide
sequences of defined length k (k-mers), several characteristics of
genomes, like size, heterozygosity and repeat content, can be ana-
lyzed, that would normally require a complete *de novo* assembly. As
basis for our analysis, DNA was generated from urediospores of the
*P. pachyrhizi* isolate Brazil 05-1. A total of 47 Gb Illumina whole-
genome sequencing data (100 bp paired-end reads) were then
subjected to analysis using the program JELLYFISH (Marçais and
Kingsford, 2011). In the 17-mer distribution depicted in **Figure 1**,
two peaks could be differentiated at a depth of 37 and 75. This
can be explained by the dikaryotic nature of the urediospores of
rust fungi, which means that these organisms maintain two hap-
loid nuclei separately during prolonged stages of their lifecycle.
The two peaks in the k-mer histogram point to a high degree
of heterozygosity between the two nuclei or to largely heterozy-
gotic regions within the haploid nuclei. Similar results were also
observed by Zheng et al. (2013) in the case of the wheat stripe rust
fungus.

By adding up the products of k-mer depth coverage and fre-
quency for each pair of values in **Figure 1**, divided by the depth
coverage of the first peak (=37), the size of the genome in bp was
computed, similarly as in (Li et al., 2010). Values, smaller than
the first minimum in **Figure 1**, were considered noise caused by

sequencing errors and were excluded from the calculation. Based
on this analysis, the overall size of the dikaryotic genome of *P.
pachyrhizi* is at most around 1 Gb. However, due to the unknown
degree of heterozygosity between or within the genomes of both
nuclei, this might be an overestimation (see above). The mini-
mal size of the haploid genome can be estimated to be around
500 Mb, based on the second peak in the k-mer analysis (Figure 1).
This would place the genome of the Asian soybean fungus in the
same range as published rust genomes, e.g., *Hemileia vastatrix*
(733.5 Mb) and *Uromyces* spp. (420 Mb; Table 1). It should be
noted, however, that the analysis method might considerably influ-
ence the outcome of such genome size estimations. The genome
size of *H. vastatrix*, e.g., was estimated by DNA-staining in com-
bination with flow cytometry which itself is prone to errors but
has the advantage of not being sequencing-dependent (Bainard
et al., 2010). Phenomena related to the partial heterozygosity of
the *P. pachyrhizi* genome are only detectable by assembly or k-
mer analysis as described above. Since we did not use a large
insert size sequencing approach for genome size estimation, we
obtained a N50 value of 569 bp after assembly and scaffolding
with SOAPdenovo. This allowed no prediction on gene number
or length. In future studies a combined BAC- and third generation
sequencing approach hopefully will increase the assembly qual-
ity to a point at which comprehensive gene predictions become
possible.

Working with organisms, whose genome has been sequenced
provides many advantages over working with non-sequenced
species. Besides the comprehensive prediction of all genes,
intra-genomic structural analyses or comparative genome anal-
yses between different species become possible. An alternative
to genomic-based approaches in large-scale analyses of plant-
pathogen-interactions, however, is the use of transcriptomics,
proteomics, or metabolomics (Tan et al., 2009). Up to now, only
limited information is available on *P. pachyrhizi* transcriptomics,
though very recent publications have broadened the view on par-
ticular aspects of the infection process of *P. pachyrhizi* (Tremblay
et al., 2010, 2012, 2013; Link et al., 2013). For instance, Illumina-
based transcriptome profiling at several stages of soybean leaf
infection has led to the identification of nearly 19,000 transcripts
not previously identified in other rust fungi (Tremblay et al., 2013).
This would imply a much larger gene complement in the soy-
bean rust than in other rust fungi. So far, the numbers of genes
reported in rust fungi are between 15,000 and 20,000 genes (Dup-
lessis et al., 2014). Although biases in the RNA-Seq approach can
not be excluded, it is possible that the *P. pachyrhizi* genome
has experienced a high level of gene duplication during its evo-
lution along with important transposable element activity that
could explain the huge genome size predicted for this species.
There is an urgent need for genome sequences as prerequisite
for accurate large scale expression analysis and more RNA-seq
efforts are needed. Without a genome, transcript reads have to be
assembled first and not only RNA quality and sequencing tech-
nique used will influence the resulting assembly quality but also
the algorithms used for assembly. And even if these problems
could be sufficiently solved, the resulting contigs are much smaller
than transcribed ORFs, limiting for example predictions of puta-
tively secreted proteins. Also, redundancy within gene families

<figure><img src="image_3.png" /><figcaption>**FIGURE 1 |** *K-mer analysis for P. pachyrhizi whole-genome sequencing data.* The 17-mer distribution for 47 Gb of 100 bp paired-end Illumina whole-genome sequencing data indicates two peaks at a depth of 37 and 75. These findings point to a possibly highly repetitive genome with a high degree of heterozygosity between the genomes of the two haploid nuclei.</figcaption></figure>
could be better resolved when compared to a reference genome sequence.

Hopefully in the near future, the development of novel sequencing and assembly strategies, together with dropping costs for NGS, will make the sequencing of large and complex genomes more affordable and will help to unravel the secrets of the genome of *P. pachyrhizi*.

## ACKNOWLEDGMENTS

We thank Ralph Panstruga (Biology I, RWTH Aachen) for helpful discussions. We thank Anthony Bolger (Biology I, RWTH Aachen University) for helpful advice on k-mer analysis. Sébastien Duplessis acknowledges the ANR "Investissements d'Avenir" program (ANR-11-LABX-0002-01, Lab of Excellence ARBRE).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Received: 31 May 2014; accepted: 14 July 2014; published online: 27 August 2014.

Citation: Loehrer M, Vogel A, Huettel B, Reinhardt R, Benes V, Duplessis S, Usadel B and Schaffrath U (2014) On the current status of Phakopsora pachyrhizi genome sequencing. *Front. Plant Sci.* 5:377. doi: 10.3389/fpls.2014.00377

This article was submitted to Plant-Microbe Interaction, a section of the journal
Frontiers in Plant Science.

Copyright © 2014 Loehrer, Vogel, Huettel, Reinhardt, Benes, Duplessis, Usadel and Schaffrath. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.