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Chasing effectors in the secretome of Melampsora larici-populina

David L. Joly1, Stéphane Hacquard2, Nicolas Feau3, Philippe Tanguay1, Francis Martin2, Richard C. Hamelin1, and Sébastien Duplessis2 1Laurentian Forestry Centre, Canadian Forest Service, Natural Resources Canada, 1055 du PEPS, PO Box 10380, Stn. Sainte-Foy, Québec, QC, Canada, G1V 4C7. 2INRA Nancy, UMR 1136, Interactions Arbres-Microorganismes, 54280 Champenoux, France. 3INRA Bordeaux-Aquitaine, UMR 1202, Biodiversité, Gènes et Communautés, 33883 Villenave d’Ornon Cedex, France.

rhamelin@nrcan.gc.ca / duplessi@nancy.inra.fr

The basidiomycete Melampsora larici-populina Kleb. (MLP), causal agent of poplar leaf rust, is an obligate macrocyclic biotroph which has its sexual stage on larch (Larix sp.) and its asexual stage on poplar (Populus species from the sections Aigeiros and Tacamahaca, and their hybrids). In the last decades, the distribution range of M. larici-populina has expanded significantly worldwide. Even though it rarely causes mortality, this rust is responsible for growth loss, premature defoliation, and predisposition to other pests and diseases. Although breeders developed several cultivars with qualitative resistance to M. larici-populina, new strains of the pathogen were able to overcome host resistances and new virulent genotypes produced by recombination were amplified by a strong selection pressure (Frey et al. 2005). Deployment of sustainable resistance requires a better understanding of the molecular mechanisms underlying Populus-Melampsora interactions (Feau et al. 2007).

Like most rust fungi, M. larici-populina urediniospore germination results in a germ tube that differentiates into a series of infection structures in the intercellular space (Rinaldi et al. 2007). The haustorium invaginates host cells and makes near-direct contact with the host plasma membrane where it plays a crucial role in nutrient acquisition (Voegele et al. 2009). This structure is also the core of cellular communication involved in the redirection of host metabolism and the suppression of host defenses (Voegele et al. 2009), which

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allows the pathogen to deliver a suite of effector proteins to modulate plant innate immunity and enable parasitic colonization (Dodds et al. 2009).

With the advent of genomics and bioinformatics, the identification of putative effectors was facilitated by tracking secreted proteins exported through the eukaryotic general secretory pathway via N-terminal amino acid sequences known as signal peptides (Torto et al. 2003). While signal peptides cannot be directly identified using molecular methods, computational tools can assign signal peptide prediction scores with a high degree of accuracy (Nielsen et al. 1997). Such bioinformatic approaches have been applied to rust fungi cDNA libraries, resulting in the identification of a number of effectors with avirulence functions (Catanzariti et al. 2006). Moreover, the availability of filamentous pathogen genome sequences enabled genome-wide survey of secreted proteins, allowing the identification of putative effectors (Soanes et al. 2007).

In the wake of the Populus genome sequencing, the ~100 Megabases genome of M. larici-populina has been sequenced by the Joint Genome Institute (http://genome.jgi-psf.org/), generating a great opportunity to identify and characterize Melampsora effectors likely involved in molecular interactions with its host plant (Duplessis et al. 2009). The analysis of this genomic sequence by an international consortium (http://mycor.nancy.inra.fr/) has revealed that this fungus is equipped with an arsenal of approximately two thousand secreted proteins, with so far 1,058 Small Secreted Proteins (<300 amino acids), herein termed SSPs. In this report, we briefly outline our current approach to identify poplar leaf rust candidate effectors in the secretome of M. larici-populina.

An integrated approach for the identification of novel rust effectors

Since effectors act during infection and outside the fungal cell, their corresponding genes can be steadily identified on the basis of in planta expression and by the presence of a signal peptide for secretion. Some of these proteins face the daunting challenge of carrying out virulence functions, while also potentially exposing the pathogen to recognition by resistance proteins, leading to an activation of host defense systems (Dodds et al. 2009). Strong selective pressures are imposed by these competing roles, and the subsequent genetic changes leave their footprints in the extant genomic variation (Ma and Guttman 2008). Such information are thus of critical importance for a comprehensive identification of candidate effectors. In order to identify effector genes in the M. larici-populina genome sequence, we

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developed a multifaceted approach broadly divided into three main themes: annotation of SSPs, which includes information regarding genomic organization, structure and homologies; gene expression profiling in infected plant tissues; and screening for positive selection.

Amino acid sequence comparisons frequently yield the first clues on possible functions of novel proteins. However, database searches with sequences of SSPs from fungi commonly do not yield recognizable homologues (Rep 2005). Indeed, among the 1,058 M. larici-populina SSP, less than 25% have homologs in public sequence databases. Surprisingly, only one third of Melampsora SSPs have identifiable homologs in the genome sequence of Puccinia graminis f. sp. tritici (PGT), the only other Pucciniales sequenced so far (http://www.broad.mit.edu). Apart from the presence of the signal peptide, the only recognizable feature of these SSPs is often a large content in cysteine residues. These cysteines are presumed to play an important role in the stability of secreted proteins, and are typical features of some fungal and oomycete effectors (Kamoun 2006; Stergiopoulos and de Wit 2009). For instance, one fourth of M. larici-populina predicted SSPs have been grouped in 30 classes according to characteristic spacings of cysteine residues, the largest family encompassing more than one hundred members. Similarities with effectors previously described in Pucciniales were uncovered, such as homologs of the Rust Transferred Protein (RTP1) described in Uromyces fabae (Kemen et al. 2005), or of the Haustorially Expressed Secreted Proteins (HESPs) from Melampsora lini (Catanzariti et al. 2006). Several of these HESPs are elicitors of the hypersensitive response in flax and co-segregate with known avirulence genes (Catanzariti et al. 2006). While only one homolog could be found for two of them (AvrL567 and AvrP123), eight and twelve homologs of AvrM and AvrP4, respectively, were recovered in the M. larici-populina genome.

Expression profiling of M. larici-populina transcripts during time-course infection of poplar leaves using exon oligonucleotide arrays (NimbleGen Systems) allowed to derive groups of genes expressed at specific stages of the interaction with the host. The number of expressed SSP genes increased all along the infection process and the expression of 56 genes peaked at 96 hpi during the biotrophic phase (Fig. 1). Pyrosequencing of transcripts (Roche 454) isolated from infected poplar leaf tissues confirmed the accumulation of several SSP transcripts at specific stages of the infection. Monitoring of candidate effector transcripts previously described in Melampsora spp. (Feau et al. 2007) by RT-qPCR also confirmed their specific temporal expression during plant colonization and biotrophic development.

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Fig. 1. Expression of Melampsora larici-populina Small Secreted Protein (SSP) transcripts during time-course infection of poplar leaves. Urediniospores of the virulent 98AG31 strain were inoculated on compatible poplar leaves as previously described (Rinaldi et al., 2007). Transcripts isolated from infected plant tissues were used to probe expression of the ~17,000 M. larici-populina predicted genes using oligonucleotide arrays. The number of SSP genes expressed above the background level is given. Number of SSP genes highly expressed is given in brackets. Microscopy pictures (© INRA, Hacquard & Duplessis) illustrate major steps of poplar leaf infection by M. larici-populina: A, spore (sp) germination and penetration through stoma (sto); B, infection hyphae (ih) and haustorium (ha) in the plant mesophyll; C, uredinia (ur) formation and release of newly formed urediniospores (sp) through the plant epidermis (pe).

Since about one third of the SSPs belong to gene families, intra- and inter-specific comparative genomics using closely related species with different host specificities appeared as a complementary strategy to target candidate effector genes. Clusters of paralogous or orthologous genes were generated to uncover the role of positive selection in the evolution of genes encoding secreted proteins. Maximum likelihood approaches were used to estimate nonsynonymous nucleotide substitution rate (dN) and synonymous substitution rate (dS) across all possible pairwise sequence comparisons within each cluster and to contrast codon substitution models (Yang et al. 2000). Our preliminary analyses suggest that most SSP-encoding genes specifically and/or highly expressed in planta with evidence of positive selection have an even number of cysteine residues, no homology in public

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databases, and a high rate of tandem duplication (Table 1). These results are consistent with other studies conducted in filamentous pathogens. Further characterization of the rust candidate effectors reported here is currently ongoing to precise their exact roles in pathogenicity.

Table 1. Examples of SSPs expressed in planta with evidence of positive selection

SSP family # of MLP # in tandem # of PGT BLAST hits # of Cys

CPG 510 4 2 0 - 4

CPG 518 3 3 0 - 8

CPG 1193+ 5 2, 2 0 M. lini HESP-C63 8

COG 747 1 - 12 - 4

Acknowledgements

The authors would like to thank Igor Grigoriev, Susan Lucas, Harris Shapiro (JGI) and Jane Grimwood (SHGC) for their outstanding efforts in sequencing, assembling and annotating the Melampsora genome. Emilie Tisserant (INRA Nancy) and Yao-Cheng Lin (VIB Ghent) are acknowledged for their support in bioinformatics. Investigations carried out in R. C. Hamelin laboratory were supported by the Canadian Forest Service Genomics Research and Development Initiative. Investigations at UMR 1136 were supported by grants from INRA (AIP Bioressources to F. Martin, innovative grant to S. Duplessis) and the Région Lorraine.

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