Journal of Integrative Agriculture2014, 13(2): 233-236 February 2014MINI REVIEW

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.doi: 10.1016/S2095-3119(13)60650-4

Messages from Powdery Mildew DNA: How the Interplay with a Host Moulds Pathogen Genomes

Pietro D Spanu

Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom

Abstract

The genomes of the barley, Arabidopsis and pea powdery mildew are significantly larger than those of related fungi. This is due to an extraordinary expansion of retro-trasposons that are evident as repetitive elements in the sequence. The protein coding genes are fewer than expected due to an overall reduction in the size of gene families, a reduction in the number of paralogs and because of the loss of certain metabolic pathways. Many of these changes have also been observed in the genomes of other taxonomically unrelated obligate biotrophic pathogens. The only group of genes that bucks the trend of gene loss, are those encoding small secreted proteins that bear the hall marks of effectors.

Key words: barley powdery mildew, genome, effectors

THE Blumeria GENOME

There are two common reasons to sequence genomes of the organisms that we are interested in. On the one hand, it provides tools for delving into its molecular genetics and biology. On the other hand, the analysis of genome structure, gene content and comparative genomics offers unique insights into the evolution and the genetic basis for the organisms’ biology. Both of these promises have been richly fulfilled by sequenc-ing the first group of powdery mildews we recently published (Spanu et al. 2010). Fortuitously, the first genome sequence and annotation of barley powdery mildew Blumeria graminis f. sp. hordei was completed almost at the same time as those of other obligate plant pathogenic fungi (Duplessis et al. 2011) and oomy-cetes (Baxter et al. 2010). The result revealed some extraordinary convergences in the evolution of obli-

gate plant diseases in basidiomycete rusts, ascomycete mildews as well as the peronosporales.

In this paper, I summarise the salient findings from this enterprise and illustrate how deciphering the B. graminis genome sequence contributes to an understanding of how the interactions between an obligate biotroph and its plant hosts have driven the evolution of the pathogen’s genome structure. This research also highlights the central importance of effectors: the players which modulate host-pathogen interactions, and drive to amplification and diversification literally moulded the mildew genomes.

A FEW SURPRISES: GENOME SIZE

Sequence, assembly and annotation of the mildew genomes challenged us with some surprising findings. The size of the B. graminis genome was the first sur-prise. As soon as the first tentative assemblies were

Received 9 July, 2013 Accepted 26 August, 2013Correspondence Pietro D Spanu, Tel: +44-207-5945384, Fax: +44-207-5842056, E-mail: p.spanu@imperial.ac.uk

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obtained, it became immediately evident that the initial coverage of the sequence was well below that expect-ed for a normal ascomycete genome (the majority of ascomycete genomes sequenced at that time were in the 30-40 Mb range). Eventually it turned out that all the powdery mildews sequenced to date (B. graminis f. sp. hordei, B. graminis f. sp. tritici (B. Keller, per-sonal communication), Golovinomyces orontii, Er-ysiphe pisi) exceed 130 Mb (Spanu et al. 2010). A detailed analysis of these genomes showed that this expansion is due to an increase in repetitive DNA that is the result of mildew specific retro-transposon activity. Filamentous fungi have a variety of mech-anisms to limit the proliferations of repetitive DNA: one pathway leads to repeat-induced point mutations (RIP) in the repeated sequences. We found that the highly conserved genes necessary for RIP are absent in all of the mildews sequenced. Moreover, a careful genome-wide analysis of the repeats showed unequiv-ocally that there was no evidence of RIP-ing in any of the repeats (Amselem, personal communication). Tak-en together these findings are consistent with the view that early in mildew evolution they lost the RIP genes and this resulted in the retro-transposon deregulation and hyper-accumulation of retro-transposon sequenc-es.

LARGE GENOMES – FEW GENES

The second surprise came when we annotated the B. graminis genome: the total number of protein cod-ing genes was <6 000, that is very few compared with that of other filamentous fungi. This constitutes an apparent paradox considering the overall increase in overall genome size (Spanu et al. 2010). Recent revi-sion of the genome annotation has somewhat increased the gene count, but it is still only <7 000 (http://www. blugen.org/). The immediate question arose as to what genes are missing from the mildew genomes. A systematic analysis showed that overall the reduced gene count can be attributed to the loss of genes in some specific functional groups and to loss of paral-ogs. Thus, for example, there are only one polyketide synthase and one non-ribosomal peptide synthase genes. Also, the overall number of genes encoding carbohydrate active enzymes (CAZy) is remarkably

low compared with that of other related ascomycetes (only two cellulase-like genes and four hemicellu-lase-like genes). In the recent publication of two Colletotrichum genomes (O’Connell et al. 2012), a comparative genome analysis showed that B. graminis has the lowest number of genes encoding CAZys of all the fungi surveyed. We also observed that there are hardly any paralogs of common primary metabolic genes. Overall, 99 normally conserved ascomycetes genes are missing in the B. graminis genome – a large portion of which are also missing in other obligate bi-otrophic plant pathogens. These genes are present in the biotrophic C. higginsianum, demonstrates that the missing genes are not per se detrimental to biotrophy but suggests that they are redundant for this special-ised lifestyle (Spanu et al. 2010). For example, the genes encoding enzymes that catalyse the reduction of inorganic nitrogen and sulphur compounds are miss-ing, reflecting the fact that nitrogen and sulphur are likely to be acquired through the assimilation of organ-ic compounds (e.g., amino acids) from host epidermal cells. Another notable example of a missing metabolic capacity is the absence of anaerobic fermentation en-zymes such as alcohol dehydrogenase. This could re-flect the fact that, uniquely amongst the fungi, the mil-dews only inhabit the aerial surfaces of plants that are permanently and fully oxygenated ecological niches. Also, B. graminis has no fungal-specific hydrophobins that are otherwise ubiquitous in filamentous ascomy-cetes and basidiomycetes; hydrophobins are necessary for the traversing of water-air interfaces. Their ab-sence in the mildews correlates with a highly unusual lifestyle that requires no liquid water for dissemination or germination of spores and conidia. Nor do mildew hyphae ever grow in a liquid aqueous medium.

EFFECTORS BUCKLE THE TREND OF

GENE REDUCTION

In the face of this generalised reduction in gene number and size of the gene families, the massive expansion of effector-like genes is thrown into an ex-ceptionally sharp relief. Originally, genes encoding candidate secreted effector protein (CSEP) genes were identified as open-reading frames in the genome pre-dicted to encode small secreted proteins that had no

Messages from Powdery Mildew DNA: How the Interplay with a Host Moulds Pathogen Genomes 235

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

obvious homologs in related fungi (Spanu et al. 2010). The observation that, unlike other mildew genes, these commonly had paralogs that could be grouped into clearly identifiable gene families led to an iterative and exhaustive “BLAST” search of the barley mil-dew genome. We have catalogued 491 CSEP genes (Pedersen et al. 2012): these represent over 7% of the barley mildew’s gene-coding capacity. There is clear experimental support for the existence and expression of the CSEP genes by gene expression studies (cDNA and RNAseq data) as well as by identification by mass spectrometry of a large proportion of the these proteins in infected plant tissues (Bindschedler et al. 2011). Overall, the expression of CSEPs and the protein accu-mulation is associated with the formation of haustoria in infected epidermis. Many of the CSEP paralogs show striking evidence of strong positive diversifying selection, particularly on aminoacids within domains predicted to be on the surface of the proteins.

Analysis of a small subset of the CSEPs has re-vealed that some of them, when knocked down by host-induced gene silencing (HIGS), result in reduced formation of haustoria and are therefore likely to en-code functional effectors (Zhang et al. 2012; Pliego et al. 2013). The prediction of the structural folds of the CSEPs revealed that many resemble secreted ribonucleases. We call these RnAse-like proteins ex-pressed in Haustoria (RALPH) effectors. Two of the functionally confirmed CSEPs are RALPH effectors (Pliego et al. 2013). We think RALPHs are not active RNAses because the aminoacids necessary for activ-ity in the RNAse are missing (Pedersen et al. 2012). This is supported by the fact that we do not detect any significant RNAse activity in BEC1054, a RALPH effector which we have expressed as a recombinant protein. However, we have experimental evidence that BEC1054 binds RNA with high affinity, suggest-ing that the effector action is due to RNA-interactions (Spanu et al., unpublished).

RALPH effectors are also found in other powdery mildews including the plantain (Podosphaera plantag-inis) (Tollenaere, personal communication), pea (Er-ysiphe pisi) and Arabidopsis (Golovinomyces orontii) (ver Loren van Themaat et al. pers. comm.) mildews. Overall a picture is emerging of that supports the idea that a proto-RALPH RNAse was co-opted early in the evolution of the powdery mildews to act as an effec-

tor. The proto-RALPH then diversified in the various mildew lineages and gave rise to at least part of the large CSEP superfamily. One distinguishing features of the CSEP genes, including RALPHs, is their close linkage to retro-transposable elements (Pedersen et al. 2012), suggesting that retro-transposon driven illegiti-mate recombination may have been the mechanism for serial duplication of these effectors. Gene duplication has two consequences: in the first place this may result in increased expression due to a simple copy-num-ber effect; in the long term, it also allows divergence through selection of mutant variants.

These processes may ultimately explain why the mildew genomes are so large and rich in retro-trans-posons: at an early point in mildew evolution, line-ages with higher retro-transposon activities were at an advantage because they had more efficient ways of generating variation in effector genes enabling them to sustain the arms race with the host recognition and defence. It is well-know that transposon insertions can disrupt protein coding genes: indeed, these events cause the phenotypes that led to the original discovery of transposons (McClintock 1950). A consequence of retro-transposon activity in the early mildew lineages was the loss of unnecessary genes described above. Disruption of gene loci is well illustrated by the de-tailed description of the usually conserved mating type locus in powdery mildews (Brewer et al. 2011). We speculate that the gene losses eventually included genes necessary for growth in non-living material, which led to the emergence of obligate biotrophy. This must have also occurred early in mildew evolu-tion because all of these fungi are, without exception, obligate pathogens (Glawe 2008).

Similar evolutionary pathways appear to have been followed by other fungal and oomycete biotrophic plant pathogens (Spanu 2012).

CONCLUSION

In summary, it appears that the structure of the ge-nome (size inflation, abundance of the repetitive DNA originating from retro-transposition) and gene loss (eventually leading to obligate biotrophy) were prices paid by the mildews to increase variation useful in their continuing struggle in an evolutionary battlefield

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represented by ever changing host immunity.

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