Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora laricipopulina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.comparative genomics | plant pathogen | basidiomycete | evolution | rust disease

In western North America, the current outbreak of the mountain pine beetle (MPB) and its microbial associates has destroyed wide areas of lodgepole pine forest, including more than 16 million hectares in British Columbia. Grosmannia clavigera ( Gc ), a critical component of the outbreak, is a symbiont of the MPB and a pathogen of pine trees. To better understand the interactions between Gc , MPB, and lodgepole pine hosts, we sequenced the ∼30-Mb Gc genome and assembled it into 18 supercontigs. We predict 8,314 protein-coding genes, and support the gene models with proteome, expressed sequence tag, and RNA-seq data. We establish that Gc is heterothallic, and report evidence for repeat-induced point mutation. We report insights, from genome and transcriptome analyses, into how Gc tolerates conifer-defense chemicals, including oleoresin terpenoids, as they colonize a host tree. RNA-seq data indicate that terpenoids induce a substantial antimicrobial stress in Gc , and suggest that the fungus may detoxify these chemicals by using them as a carbon source. Terpenoid treatment strongly activated a ∼100-kb region of the Gc genome that contains a set of genes that may be important for detoxification of these host-defense chemicals. This work is a major step toward understanding the biological interactions between the tripartite MPB/fungus/forest system.

The obligate biotrophic rust fungus Melampsora larici-populina is the most devastating and widespread pathogen of poplars. Studies over recent years have identified various small secreted proteins (SSP) from plant biotrophic filamentous pathogens and have highlighted their role as effectors in host-pathogen interactions. The recent analysis of the M. larici-populina genome sequence has revealed the presence of 1,184 SSP-encoding genes in this rust fungus. In the present study, the expression and evolutionary dynamics of these SSP were investigated to pinpoint the arsenal of putative effectors that could be involved in the interaction between the rust fungus and poplar. Similarity with effectors previously described in Melampsora spp., richness in cysteines, and organization in large families were extensively detailed and discussed. Positive selection analyses conducted over clusters of paralogous genes revealed fast-evolving candidate effectors. Transcript profiling of selected M. laricipopulina SSP showed a timely coordinated expression during leaf infection, and the accumulation of four candidate effectors in distinct rust infection structures was demonstrated by immunolocalization. This integrated and multifaceted approach helps to prioritize candidate effector genes for functional studies.

Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulosedegrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems. poplar pathogen | tree disease | fungal genomics | Septoria canker

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BackgroundObligate biotrophs such as rust fungi are believed to establish long-term relationships by modulating plant defenses through a plethora of effector proteins, whose most recognizable feature is the presence of a signal peptide for secretion. Since the phenotypes of these effectors extend to host cells, their genes are expected to be under accelerated evolution stimulated by host-pathogen coevolutionary arms races. Recently, whole genome sequence data has allowed the prediction of secretomes, facilitating the identification of putative effectors.ResultsWe generated cDNA libraries from four poplar leaf rust pathogens (Melampsora spp.) and used computational approaches to identify and annotate putative secreted proteins with the aim of uncovering new knowledge about the nature and evolution of the rust secretome. While more than half of the predicted secretome members encoded lineage-specific proteins, similarities with experimentally characterized fungal effectors were also identified. A SAGE analysis indicated a strong stage-specific regulation of transcripts encoding secreted proteins. The average sequence identity of putative secreted proteins to their closest orthologs in the wheat stem rust Puccinia graminis f. sp. tritici was dramatically reduced compared with non-secreted ones. A comparative genomics approach based on homologous gene groups unravelled positive selection in putative members of the secretome.ConclusionWe uncovered robust evidence that different evolutionary constraints are acting on the rust secretome when compared to the rest of the genome. These results are consistent with the view that these genes are more likely to exhibit an effector activity and be involved in coevolutionary arms races with host factors.

Hybridization and introgression are pervasive evolutionary phenomena that provide insight into the selective forces that maintain species boundaries, permit gene flow, and control the direction of evolutionary change. Poplar trees (Populus L.) are well known for their ability to form viable hybrids and maintain their distinct species boundaries despite this interspecific gene flow. We sought to quantify the hybridization dynamics and postzygotic fitness within a hybrid stand of balsam poplar (Populus balsamifera L.), eastern cottonwood (P. deltoides Marsh.), and their natural hybrids to gain insight into the barriers maintaining this stable hybrid zone. We observed asymmetrical hybrid formation with P. deltoides acting as the seed parent, but with subsequent introgression biased toward P. balsamifera. Native hybrids expressed fitness traits intermediate to the parental species and were not universally unfit. That said, native hybrid seedlings were absent from the seedling population, which may indicate additional selective pressures controlling their recruitment. It is imperative that we understand the selective forces maintaining this native hybrid zone in order to quantify the impact of exotic poplar hybrids on this native system.

Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada.