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DNA methylation is environment-sensitive and can mediate stress responses. In trees, changes in the environment might cumulatively shape the methylome landscape over time. However, because high-resolution methylome studies usually focus on single environmental cues, the stress-specificity and long-term stability of methylation responses remain unclear. Here, we studied the methylome plasticity of a Populus nigra cv. 'Italica' clone widely distributed across Europe. Adult trees from different geographic locations were clonally propagated in a common garden experiment and exposed to cold, heat, drought, herbivory, rust infection, and salicylic acid treatments. Whole-genome bisulfite sequencing revealed stress-induced and naturally occurring DNA methylation variants. In CG/CHG contexts, the same genomic regions were often affected by multiple stresses, suggesting a generic methylome response. Moreover, these variants showed striking overlap with naturally occurring methylation variants between trees from different locations. Drought treatment triggered CHH hypermethylation of transposable elements, affecting entire superfamilies near drought-responsive genes. Thus, we revealed genomic hotspots of methylation change that are not stress-specific and that contribute to natural DNA methylation variation, and identified stress-specific hypermethylation of entire transposon superfamilies with possible functional consequences. Our results underscore the importance of studying multiple stressors in a single experiment for recognizing general versus stress-specific methylome responses. [ABSTRACT FROM AUTHOR]

The obligate biotrophic pathogen Melampsora larici‐populina, responsible for poplar foliar rust disease, causes annual epidemics and severe damages to poplar plantations worldwide. Flexible responses to external osmotic pressure changes are important for the growth and survival of pathogens. In a previous study, we suggested that the Hog1‐type MAPK MlpHog1 in M. larici‐populina may play a role in infectious growth and responses to various environmental stresses. In the present study, we analysed its biological characteristics. MlpHog1 displayed a conserved coding sequence pattern among five strains from different regions of China. MlpHog1 restored the Hog1‐orthologue mutant defects responding to hyperosmotic stress in both Saccharomyces cerevisiae and Magnaporthe oryzae. Transient expression in wheat protoplasts revealed that MlpHog1 is localized in the cytoplasm and nucleus. These results indicate that MlpHog1 plays a positive role in responding to osmotic stress in the poplar rust M. larici‐populina. [ABSTRACT FROM AUTHOR]

R2R3-MYB protein is the most abundant class of MYB transcription factor family in plants. The transcript profiles of two E4 races of Melampsora larici-populina-tolerant poplars and an intolerant poplar were investigated to characterise the role of the R2R3-MYB family genes in the poplar–E4 interaction. In this study, 217 R2R3-MYBs were identified, and 83 R2R3-MYB genes were assigned to 22 different coexpression modules by weighted gene coexpression network analysis. Most R2R3-MYB genes were unchanged in the early period of E4 infection in both tolerant and intolerant poplars. However, there were obvious increases in differentially expressed R2R3-MYB genes in tolerant poplars at 2 and 4 dpi when defence responses occurred, suggesting that differently expressed R2R3-MYB genes at these time points may play an important role in poplar resistance to E4 infection. In total, 34 R2R3-MYB genes showed differential expression at 2 and 4 dpi between tolerant and intolerant poplars. Among them, 16 differentially expressed R2R3-MYB genes were related to 43 defence-related genes that had significant differences between tolerant and intolerant poplars. There might be coregulatory relationships between R2R3-MYBs and other TFs during poplar–E4 interaction. Some differentially expressed R2R3-MYB genes were related to genes involved in flavonoid biosynthesis and IAA or free SA signal transduction and might help activate defence response during poplar–E4 interaction. MYB194 could be an important node in the convergence of IAA and SA signalling. [ABSTRACT FROM AUTHOR]

• Functional divergence likely occurred among the five OPT clades in Melampsora larici-populina. • The majority of duplicated genes exhibited up-regulated expression during the infection stage of poplar trees. • One duplicated gene, MellpOPT4g , is under balancing selection. The acquisition of nutrients from host plants by phytopathogenic fungi is critically important for their invasion success. Melampsora larici-populina , an obligate biotrophic pathogenic fungus, causes the poplar leaf rust disease and can severely damage host poplar plants. Previously, we found that oligopeptide transporters (OPTs) have undergone a convergent expansion, which might reflect adaptation to a phytoparasitic lifestyle. Here, we used various methods to evaluate this hypothesis, including conserved motif identification, positive selection signal mining, expression pattern clustering analysis, and neutral selection tests. The motif composition of the five clades in the OPT family differed, and positive selection was observed during clade differentiation. This suggests that OPTs in these five clades may be functionally differentiated, which would increase the range of transported substrates and promote the absorption of more types of nitrogen compounds from the hosts. According to clustering analysis of gene expression patterns, the expression of most genes from the two expanded clades (clade 2 and 4) was up-regulated during the infection of poplar trees, indicating that the expansion of OPTs likely occurred to promote the uptake of oligopeptides from host poplar plants. The MellpOPT4g gene was determined to be under significant balancing selection based on the neutral selection tests, suggesting that it plays a role in the pathogenic process. In conclusion, these three observations provide preliminary evidence supporting our hypothesis, as they indicate that the expansion of OPTs in M. larici-populina has aided the ability of this pathogen to acquire nutrients from host plants. [ABSTRACT FROM AUTHOR]

Tropospheric ozone and nitrogen deposition are two major environmental pollutants. A great deal of research has focused on the negative impacts of elevated O3 and the complementary effect of soil N addition on the physiological properties of trees. However, it has been overlooked how elevated O3 and N addition affect tree immunity in face of pathogen infection, as well as of the important roles of phyllosphere microbiome community in host–pathogen–environment interplay. Here, we examined the effects of elevated O3 and soil N addition on poplar leaf rust [Melampsora larici-populina] severity of two susceptible hybrid poplars [clone '107': Populus euramericana cv. '74/76'; clone '546': P. deltoides Í P. cathayana] in Free-Air-Controlled-Environment plots, in addition, the link between Mlp-susceptibility and changes in microbial community was determined using Miseq amplicon sequencing. Rust severity of clone '107' significantly increased under elevated O3 or N addition only; however, the negative impact of elevated O3 could be significantly mitigated when accompanied by N addition, likewise, this trade-off was reflected in its phyllosphere microbial α-diversity responding to elevated O3 and N addition. However, rust severity of clone '546' did not differ significantly in the cases of elevated O3 and N addition. Mlp infection altered microbial community composition and increased its sensitivity to elevated O3, as determined by the markedly different abundance of taxa. Elevated O3 and N addition reduced the complexity of microbial community, which may explain the increased severity of poplar rust. These findings suggest that poplars require a changing phyllosphere microbial associations to optimize plant immunity in response to environmental changes. [ABSTRACT FROM AUTHOR]

Populus nigra is an important autochthonous woody plant that can be grown as a renewable energy source. The possibility of its improvement through intraspecific hybridization was tested. Differences in biomass production, growth parameters, Melampsora larici-populina rust resistance and drought tolerance were evaluated among 19 intraspecific hybrids from controlled crosses, 2 clones selected from natural populations and the “MAX 4” clone (P. nigra × P. maximowiczii). These P. nigra clones from controlled crosses were chosen from more than 2000 hybrid individuals whose parents were selected from natural populations in the Czech Republic. A field trial was set up in Pruhonice, Czech Republic (320 m a.s.l., 591 mm rainfall annually, mean annual temperature of 9.5 °C). The planting density was 6061 plants ha-1, and the plants were coppiced three times at 3-year intervals. The trial was irrigated only during its establishment. Among the clones, significant differences were found in all the evaluated traits over three rotations. An average dry matter yield of the best clone “MAX 4” was 12.8 t ha-1 yr-1 over three harvests. The best black poplar clone reached up to 9.4 t ha-1 yr-1 in three harvests. Rust resistance was constant over 9 years and high for the three P. nigra clones (two from controlled crosses and one from natural populations). Moreover, the best P. nigra clones from controlled crosses showed higher drought tolerance than the “MAX 4” clone. Breeding progress was confirmed, and most of the P. nigra clones from controlled crosses performed better than the clones selected from natural populations. The trial validated the suitability of natural populations for use as gene sources for intraspecific hybridization and as sources of clones with traits comparable with those of interspecific clones. These new P. nigra clones can replace allochthonous clones in areas where autochthonous P. nigra populations are threatened by introgression.

Melampsora larici-populina is a macrocyclic rust, and the haploid stage with two nuclei and the diploid of mononuclear sequentially occur annually. During the preservation of dry urediniospores at −80°C, we found that one isolate, ΔTs06, was different from the usual wild-type isolate Ts06 at −20°C because it has mixed polykaryotic urediniospores. However, the other spores, including the 0, I, III, and IV stages of a life cycle, were the same as Ts06. After five generations of successive inoculation and harvest of urediniospores from the compatible host Populus purdomii, the isolate ΔTs06 steadily maintained more than 20% multiple nucleus spores. To test the pathogenesis variation of ΔTs06, an assay of host poplars was applied to evaluate the differences between ΔTs06 and Ts06. After ΔTs06 and Ts06 inoculation, leaves of P. purdomii were used to detect the expression of small secreted proteins (SSPs) and fungal biomasses using quantitative real-time PCR (qRT-PCR) and trypan blue staining. ΔTs06 displayed stronger expression of five SSPs and had a shorter latent period, a higher density of uredinia, and higher DNA mass. A transcriptomic comparison between ΔTs06 and Ts06 revealed that 3,224 were differentially expressed genes (DEGs), 55 of which were related to reactive oxygen species metabolism, the Mitogen-activated protein kinase (MAPK) signaling pathway, and the meiosis pathway. Ten genes in the mitotic and meiotic pathways and another two genes associated with the “response to DNA damage stimulus” all had an upward expression, which were detected by qRT-PCR in ΔTs06 during cryopreservation. Gas chromatography–mass spectrometry (GC-MS) confirmed that the amounts of hexadecanoic acid and octadecadienoic acid were much more in ΔTs06 than in Ts06. In addition, using spectrophotometry, hydrogen peroxide (H2O2) was also present in greater quantities in ΔTs06 compared with those found in Ts06. Increased fatty acids metabolism could prevent damage to urediniospores in super-low temperatures, but oxidant species that involved H2O2 may destroy tube proteins of mitosis and meiosis, which could cause abnormal nuclear division and lead to multinucleation, which has a different genotype. Therefore, the multinuclear isolate is different from the wild-type isolate in terms of phenotype and genotype; this multinucleation phenomenon in urediniospores improves the pathogenesis and environmental fitness of M. larici-populina.

Melampsora larici-populina is a macrocyclic rust, and the haploid stage with two nuclei and the diploid of mononuclear sequentially occur annually. During the preservation of dry urediniospores at −80°C, we found that one isolate, ΔTs06, was different from the usual wild-type isolate Ts06 at −20°C because it has mixed polykaryotic urediniospores. However, the other spores, including the 0, I, III, and IV stages of a life cycle, were the same as Ts06. After five generations of successive inoculation and harvest of urediniospores from the compatible host Populus purdomii , the isolate ΔTs06 steadily maintained more than 20% multiple nucleus spores. To test the pathogenesis variation of ΔTs06, an assay of host poplars was applied to evaluate the differences between ΔTs06 and Ts06. After ΔTs06 and Ts06 inoculation, leaves of P. purdomii were used to detect the expression of small secreted proteins (SSPs) and fungal biomasses using quantitative real-time PCR (qRT-PCR) and trypan blue staining. ΔTs06 displayed stronger expression of five SSPs and had a shorter latent period, a higher density of uredinia, and higher DNA mass. A transcriptomic comparison between ΔTs06 and Ts06 revealed that 3,224 were differentially expressed genes (DEGs), 55 of which were related to reactive oxygen species metabolism, the Mitogen-activated protein kinase (MAPK) signaling pathway, and the meiosis pathway. Ten genes in the mitotic and meiotic pathways and another two genes associated with the "response to DNA damage stimulus" all had an upward expression, which were detected by qRT-PCR in ΔTs06 during cryopreservation. Gas chromatography–mass spectrometry (GC-MS) confirmed that the amounts of hexadecanoic acid and octadecadienoic acid were much more in ΔTs06 than in Ts06. In addition, using spectrophotometry, hydrogen peroxide (H2O2) was also present in greater quantities in ΔTs06 compared with those found in Ts06. Increased fatty acids metabolism could prevent damage to urediniospores in super-low temperatures, but oxidant species that involved H2O2 may destroy tube proteins of mitosis and meiosis, which could cause abnormal nuclear division and lead to multinucleation, which has a different genotype. Therefore, the multinuclear isolate is different from the wild-type isolate in terms of phenotype and genotype; this multinucleation phenomenon in urediniospores improves the pathogenesis and environmental fitness of M. larici-populina. [ABSTRACT FROM AUTHOR]

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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 mega base pair genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89 mega base pair genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,841 predicted proteins of M. larici-populina to the 18,241 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic life-style include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins (SSPs), impaired nitrogen and sulfur assimilation pathways, and expanded families of amino-acid, oligopeptide and hexose membrane transporters. The dramatic upregulation of transcripts coding for SSPs, 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

Copyright of Mycosystema is the property of Mycosystema Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Melampsora larici-populina (Mlp) is a devastating pathogen of poplar trees, causing the defoliating poplar leaf rust disease. Genomic studies have revealed that Mlp possesses a repertoire of 1184 small secreted proteins (SSPs), some of them being characterized as candidate effectors. However, how they promote virulence is still unclear. This study investigates the candidate effector Mlp37347’s role during infection. We developed a stable Arabidopsis transgenic line expressing Mlp37347 tagged with the green fluorescent protein (GFP). We found that the effector accumulated exclusively at plasmodesmata (PD). Moreover, the presence of the effector at plasmodesmata favors enhanced plasmodesmatal flux and reduced callose deposition. Transcriptome profiling and a gene ontology (GO) analysis of transgenic Arabidopsis plants expressing the effector revealed that the genes involved in glucan catabolic processes are up-regulated. This effector has previously been shown to interact with glutamate decarboxylase 1 (GAD1), and in silico docking analysis supported the strong binding between Mlp37347 and GAD1 in this study. In infection assays, the effector promoted Hyalonoperospora arabidopsidis growth but not bacterial growth. Our investigation suggests that the effector Mlp37347 targets PD in host cells and promotes parasitic growth.

Rust fungi cause epidemics that threaten the production of important plant species, such as wheat and soy. Melampsora larici-populina (Mlp) causes the poplar rust and encodes at least 1184 candidate effectors (CEs) whose functions are poorly known. In this study, we sequenced the transcriptome and used mass spectrometry to analyze the metabolome of Arabidopsis plants constitutively expressing 14 Mlp CEs and of a control line to discover alterations leading to plant susceptibility. We found 2299 deregulated genes across the experiment. Genes involved in pattern-triggered immunity, such as FRK1, PR1, RBOHD, and WRKY33, as well as AUX/IAA genes were down-regulated. We further observed that 680 metabolites were deregulated in at least one CE-expressing transgenic line, with “highly unsaturated and phenolic compounds” and “peptides” enriched among down- and up-regulated metabolites. Interestingly, transgenic lines expressing unrelated CEs had correlated patterns of gene and metabolite deregulation, while expression of CEs belonging to the same family deregulated different genes and metabolites. Thus, our results uncouple effector sequence similarity and function. This supports that effector functional investigation in the context of their virulence activity and effect on plant susceptibility requires the investigation of the individual effector and precludes generalization based on sequence similarity.

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Two novel miRNAs were selected from a pre-constructed RNA library of Populus szechuanica infected with the foliar rust fungus Melampsora larici-populina in order to detect the genes regulated as targets of the miRNAs novel_mir_11 and novel_mir_357. The novel miRNAs were identified from P. szechuanica using stem-loop methods and their precursors were able to fold into a complete stem loop structure. The predicted target genes of the novel miRNAs were verified with RNA ligase-mediated 5′ rapid amplification of cDNA ends (RLM-5′RACE). The full-length sequences of target genes, RPM1 and RPS2/5, in P. szechuanica were obtained through rapid amplification of cDNA ends (RACE) and officially named PsRPM1 and PsRPS2/5. These genes contain nucleotide binding site-leucine-rich repeats (NBS-LRR) domains typical of resistance genes. The expression levels of miRNAs and their target genes in different periods post infection were analysed with quantitative real-time PCR (qRT-PCR). After infection with the foliar rust fungus, the expression levels of the novel miRNAs and their target genes were dynamic. Both novel_mir_11 and novel_mir_357 negatively regulated the expression of their target genes. In this study, the regulatory effects of two novel miRNAs through their target genes were characterized to provide further mechanistic information regarding the interaction between Populus and a foliar rust fungus. Results of this study improve our understanding of the defence response mechanisms of Populus and will stimulate future work to characterize strategies to prevent and control Populus diseases. [ABSTRACT FROM AUTHOR]

Copyright of Landbauforschung is the property of Thuenen Institut and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The TIFY domain contains approximately 36 conserved amino acids that form the core motif TIF[F/Y]XG, and they were reported to play important roles in plant growth, tissue development and defense regulation. Moreover, more and more evidence has shown that some members of the TIFY gene family perform their functions by modulating plant hormone signaling pathways. Poplar trees are found worldwide, and they comprise approximately 30 species. Benefit from the importance of poplar and its advanced platform, this tree is considered to be the model perennial plant. Here, we conducted a genome-wide identification of TIFY genes in poplar, and 24 TIFY genes were found. These 24 TIFY genes were assigned to different subfamilies according to the presence or absence of domains and motifs that they harbored. Careful analyses of their locations, structures, evolution and duplication patterns revealed an overview of this gene family in poplar. The expression profiles of these 24 TIFY genes were then analyzed in different tissues using publicly available expression data; their expression profiles following different JA/SA treatments and infection with leaf rust pathogen were also carefully examined by qRT-PCR assays. Based on their expression profiles, the functions of a number of TIFY genes could be predicted. By performing this study, we have provided valuable information for further functional characterisation of TIFY genes in poplar and candidate genes for the improvement of poplar disease resistance. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) were found to widely exist in eukaryotes and play important roles in most biological processes. Trees belonging to Populus spp. are economically important because of their fast-growing characteristics and wide use for wood, pulp, paper, and fuel. However, the prevalence of leaf rust disease caused by Melampsora spp. on some species of Populus severely affects their growth and decreases wood production. Therefore, the identification and characterization of lncRNAs involved in the defense of Melampsora spp. would not only help us to understand plant-pathogen interactions but also provide genetic elements for producing disease-resistant poplars. In the present study, an RNA-Seq transcriptome analysis was performed for poplar leaves in response to early infection by M. larici-populina. The hybrid variety 'NL895' ( P. × euramericana) was used as plant material. A total of 3994 lncRNAs were identified by mining the RNA-Seq data. The identified lncRNAs had lower abundance, fewer exons, and shorter lengths when compared to protein-coding genes. In addition, 53 lncRNAs were differentially expressed between treatments and controls. Two of these 53 lncRNAs were predicted to compete to be the target of miRNAs. Moreover, the differentially expressed lncRNAs were found to be preferentially located in close proximity to the protein-coding genes they co-expressed. In this study, we were able to show that lncRNAs may play important roles in plant-pathogen interactions in the poplar. This study also improves our understanding of how plants deploy their defense system when encountering biotic stresses. [ABSTRACT FROM AUTHOR]

The obligate biotrophic fungus Melampsora larici-populina ( Mlp) causes devastating poplar foliar rust disease. In pathogens, the Hog1-type mitogen-activated protein kinase (MAPK) gene is involved in both pathogenesis and the response to various stresses, including osmotic stress. However, there are few reports on its role in M. larici-populina. In the present study, we identified and characterized the Hog1-type MAPK gene, MlpHog1, in M. larici-populina. The MlpHog1 protein consists of 356 amino acids and contains a conserved TGY motif. The function of MlpHog1 was probed by mutant complementation studies. Expression of MlpHog1 could partially complement the osmoregulation, sexual reproduction and pathogenicity defects of the Fusarium graminearum Hog1 mutant and largely rescued the response of the mutant to cytoplasm membrane and oxidative stresses. These observations suggest that MlpHog1 may play a role in the infectious growth of M. larici-populina and the response of the fungus to various environmental stresses. [ABSTRACT FROM AUTHOR]

The rust fungi Melampsora larici-populina (Mlp) and Melampsora allii-populina (Map) are the main phytosanitary constraints for commercial poplar cultivation in Europe. Although Mlp is more aggressive and prevalent than Map, the two species may co-infect the same poplar tree or even the same poplar leaf, making the epidemiological surveys of each species difficult to achieve. In this study, a new duplex real-time PCR assay targeting each species was developed, based on single-copy genes. This test proved to be specific, inclusive and was successfully used to detect and quantify each species, starting from urediniospore samples or directly from infected poplar leaves, with or without visible symptoms. This new molecular tool was also assessed for comparative studies of time-course infection experiments on artificially inoculated poplar leaf discs. These studies showed that the growth dynamics of Map were significantly slower when the two species were co-inoculated on the same leaf disc, confirming that Map is less aggressive than Mlp. [ABSTRACT FROM AUTHOR]

Background: Rust caused by Melampsora larici-populina is one of the most damaging diseases of poplars. Rust is considered to be a model pathogen for genetic studies because both pathogen and host genomes are available. The poplar 'Robusta', whose general rust resistance is defeated by virulent rust E4, provides suitable host material for studies of the gene regulation involved in rust resistance/susceptibility. In this study, we investigated the microRNA-mediated susceptible poplar gene expression regulation associated with the infection of virulent rust. We were particularly interested in delineating the host-pathogen interactions with a specific focus on microRNAs (miRNAs). Results: To study the susceptibility of poplar to M. larici-populina, small RNA (sRNA) libraries, a degradome cDNA library and digital gene expression libraries were constructed for rust-inoculated and rust-free susceptible poplar 'Robusta' leaves through high-throughput sequencing. Altogether, 12,722 regulating interactions were identified. The results delineated the framework of post-transcriptional regulation of gene expression in the susceptible poplar, which was infected by the virulent rust. The results indicated that pathogen-associated molecular patterns (PAMPs) and PAMP-triggered immunity were induced by the infection of virulent rust E4 and that miRNAs still functioned at this stage. After this stage, miRNA-regulated R genes, such as TIR-NBS-LRR and CC-NBS-LRR, were not fully functional. Additionally, the rust-responsive miRNAs did not regulate the signaling component genes related to the salicylic acid pathway or the hypersensitive response. Conclusions: We found that the defense-related post-transcriptional regulation of the susceptible poplar 'Robusta' functions normally only at the stage of PAMPs and PAMP-triggered immunity (PTI). More importantly, the miRNA-mediated post-transcriptional regulation of defense signal pathway genes were inactivated by the infection of virulent rust at the stage of effector-triggered susceptibility and during the following stages of salicylic acid and hypersensitive responses. This inactivation was the major characteristic of 'Robusta' susceptibility. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) are small non-coding RNAs that regulate the gene expression of target mRNAs involved in plant growth, development, and abiotic stress and pathogen responses. Previous studies have reported miRNAs in Populus that respond to abiotic stresses, such as cold, heat, drought, flooding, high salt and mechanical stress. However, little is known about the regulatory roles of these molecules in the Populus response to the stress of foliar rust fungal infection. Here, we identified the miRNA profiles of Populus after inoculation with Melampsora larici-populina using high-throughput sequencing and bioinformatics analysis. Quantitative real-time PCR (qRT-PCR) was used to validate the expression levels of 10 miRNAs. Results: A total of 90 known miRNAs belonging to 42 families and 378 novel miRNAs were identified from three small-RNA libraries of Populus szechuanica infected with M. larici-populina isolates Sb052 and Th053 and a control. Comparative analysis revealed that the expression of 38 known miRNAs and 92 novel miRNAs in P. szechuanica after infection with different rust fungus isolates showed significant differences, and more miRNAs were suppressed during rust infection. Among the differentially expressed miRNAs, 7 known and 20 novel miRNAs were relevant to the rust fungus infection, and according to KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathway analysis, these miRNAs primarily regulate genes encoding disease-resistance proteins, serine/threonine protein kinases, transcription factors, and related proteins. QRT-PCR analysis indicated that most miRNAs were up-regulated in the Sb052 library and down-regulated in the Th053 library at 48 h post-inoculation (hpi). Conclusions: These results demonstrate that the expression of miRNAs was altered in poplar under stress associated with M. larici-populina infection, and different temporal dynamics were observed in incompatible and compatible libraries. These findings suggest important roles for miRNA regulation in Populus upon infection with foliar rust fungus. [ABSTRACT FROM AUTHOR]

The uredinial stage in the life cycle of Melampsora larici- populina on poplar leaves is the most important pathogenic phase. We captured partial phases of uredinial infection in the wild, aiming to reconstruct the process of uredinial ontogeny by using scanning and transmission electron microscope. At the initial infection stage, germ tubes germinated from the echinulate urediniospores. Germ tubes were frequently seen to merge with the leaf surface and cuticle breakage was observed, indicating direct hyphal penetration. Stomata penetration occurred commonly, sometimes with more than one germ tube penetrating the same stoma. Melampsora larici- populina did not form appressoria in the infection process, implying that infectious behavior of this pathogen may differ from the other rust pathogens. In general, germ tubes branched randomly, and no distinct evidence indicated that stoma could induce or orient germ tube branches. However, oriented germ tube growth has been occasionally observed in other studies. The urediniospores collapsed and finally wizened when they became nutrient stressed. At the last stage of infection, the uredinia erupted from the leaf epidermis and appeared as orange pustules on the leaf surface. [ABSTRACT FROM AUTHOR]

Most rust fungi have a complex life cycle involving up to five different spore-producing stages. The telial stage that produces melanised overwintering teliospores is one of these and plays a fundamental role for generating genetic diversity as karyogamy and meiosis occur at that stage. Despite the importance of telia for the rust life cycle, almost nothing is known about the fungal genetic programs that are activated in this overwintering structure. In the present study, the transcriptome of telia produced by the poplar rust fungus M. larici-populina has been investigated using whole genome exon oligoarrays and RT-qPCR. Comparative expression profiling at the telial and uredinial stages identifies genes specifically expressed or up-regulated in telia including osmotins/thaumatin-like proteins and aquaporins that may reflect specific adaptation to overwintering as well numerous lytic enzymes acting on plant cell wall, reflecting extensive cell wall remodelling at that stage. The temporal dynamics of karyogamy was followed using combined RT-qPCR and DAPI-staining approaches. This reveals that fusion of nuclei and induction of karyogamy-related genes occur simultaneously between the 25-39 days post inoculation time frame. Transcript profiling of conserved meiosis genes indicate a preferential induction right after karyogamy and corroborate that meiosis begins prior to overwintering and is interrupted in Meiosis I (prophase I, diplonema stage) until teliospore germination in early spring.

• Pucciniales present a reduced repertoire of TFs compared to other fungi. • TF families display different evolutionary trajectories in Pucciniales vs fungi. • The poplar rust transcriptome pinpoints TF at major life cycle stages. • Cold shock protein genes are specifically expanded in Pucciniales. • Cold Shock Proteins have a probable role in dormancy exit of the poplar rust fungus. Fungi of the order Pucciniales are obligate plant biotrophs causing rust diseases. They exhibit a complex life cycle with the production of up to five spore types, infection of two unrelated hosts and an overwintering stage. Transcription factors (TFs) are key regulators of gene expression in eukaryote cells. In order to better understand genetic programs expressed during major transitions of the rust life cycle, we surveyed the complement of TFs in fungal genomes with an emphasis on Pucciniales. We found that despite their large gene numbers, rust genomes have a reduced repertoire of TFs compared to other fungi. The proportions of C2H2 and Zinc cluster - two of the most represented TF families in fungi - indicate differences in their evolutionary relationships in Pucciniales and other fungal taxa. The regulatory gene family encoding cold shock protein (CSP) showed a striking expansion in Pucciniomycotina with specific duplications in the order Pucciniales. The survey of expression profiles collected by transcriptomics along the life cycle of the poplar rust fungus revealed TF genes related to major biological transitions, e.g. response to environmental cues and host infection. Particularly, poplar rust CSPs were strongly expressed in basidia produced after the overwintering stage suggesting a possible role in dormancy exit. Expression during transition from dormant telia to basidia confirmed the specific expression of the three poplar rust CSP genes. Their heterologous expression in yeast improved cell growth after cold stress exposure, suggesting a probable regulatory function when the poplar rust fungus exits dormancy. This study addresses for the first time TF and regulatory genes involved in developmental transition in the rust life cycle opening perspectives to further explore molecular regulation in the biology of the Pucciniales. [ABSTRACT FROM AUTHOR]

Most rust fungi have a complex life cycle involving up to five different spore-producing stages. The telial stage that produces melanized overwintering teliospores is one of these and plays a fundamental role for generating genetic diversity as karyogamy and meiosis occur at that stage. Despite the importance of telia for the rust life cycle, almost nothing is known about the fungal genetic programs that are activated in this overwintering structure. In the present study, the transcriptome of telia produced by the poplar rust fungus Melampsora larici-populina has been investigated using whole genome exon oligoarrays and RT-qPCR. Comparative expression profiling at the telial and uredinial stages identifies genes specifically expressed or up-regulated in telia including osmotins/thaumatin-like proteins (TLPs) and aquaporins that may reflect specific adaptation to overwintering as well numerous lytic enzymes acting on plant cell wall, reflecting extensive cell wall remodeling at that stage. The temporal dynamics of karyogamy was followed using combined RT-qPCR and DAPI-staining approaches. This reveals that fusion of nuclei and induction of karyogamy-related genes occur simultaneously between the 25 and 39 days post inoculation time frame. Transcript profiling of conserved meiosis genes indicates a preferential induction right after karyogamy and corroborates that meiosis begins prior to overwintering and is interrupted in Meiosis I (prophase I, diplonema stage) until teliospore germination in early spring. [ABSTRACT FROM AUTHOR]

Fungal colonization can significantly affect the secondary metabolism of the host plants. We tested the impact of a common below-ground symbiosis, i.e., ectomycorrhiza formation, on poplar leaf chemical components that are involved in the defence against a common disease, i.e., rust fungi, in N-deficient soil. A rust-susceptible poplar clone (Populus trichocarpa × deltoides ‘Beaupré’) was (a) non-associated with ectomycorrhizal fungus (EM) Hebeloma mesophaeum (Pers.) Quélet MÜN and non-infected with rust fungus Melampsora larici-populina Kleb. (isolate 98AG31), (b) associated with EM, (c) inoculated with rust fungus and (d) associated with EM and inoculated with rust fungus. Poplar leaves were analysed by photometric and mass spectrometric techniques (liquid chromatography–tandem mass spectrometry (LC-MS/MS), pyrolysis–field ionization mass spectrometry (Py-FIMS)). Both rust infection and mycorrhiza formation led to increased proportions of condensed tannins in relation to total phenolics (13% in the control, 18–19% in the fungal treatments). In contrast, salicylic acid concentration (6.8 µg g−1 in the control) was higher only in the rust treatments (17.9 and 25.4 µg g−1 with rust infection). The Py-FIMS analysis revealed that the rust-infected treatments were significantly separated from the non-rust-infected treatments on the basis of six flavonoids and one lipid. The relative abundance of these components, which have known functions in plant defence, was decreased after rust infection of non-mycorrhizal plants, but not in mycorrhizal plants. The results indicate that the ectomycorrhizal formation compensated the rust infection by a decrease in the flavonoid syntheses. The study provides new evidence for an interactive response of mycorrhizal colonization and infection with rust fungi in the metabolism of poplar. [ABSTRACT FROM AUTHOR]

Fungal plant pathogens, especially rust fungi ( Pucciniales), are well known for their complex life cycles, which include phases of sexual and asexual reproduction. The effect of asexual multiplication on population genetic diversity has been investigated in the poplar rust fungus Melampsora larici-populina using a nested hierarchical sampling scheme. Four hierarchical levels were considered: leaf, twig, tree and site. Both cultivated and wild poplar stands were sampled at two time points at the start and end of rust epidemics. A total of 641 fungal isolates was analysed using nine microsatellite markers. This study revealed that the genetic signature of asexual multiplication in the wild poplar stand was seen only at lower hierarchical levels (leaf and twig). Moreover, we observed an erosion of clonal structure through time, with an increase in both gene and genotypic diversity. New genotypes contributed to host infection over time, which demonstrates the importance of allo-infection in the epidemic process in this host-pathogen system. Compared with the wild stands, the nearly lack of detection of clonal structure in the cultivated stands reflects the higher infection level on cultivated poplars. More generally, this genetic analysis illustrates the utility of population genetics approach for elucidating the proportion of asexual reproduction in the multiplication of isolates during an epidemic, and for proper quantification of asexual dispersal in plant pathogens. [ABSTRACT FROM AUTHOR]

The advent of molecular epidemiology has greatly improved our ability to identify the population sources and track the pathogen movement. Yet the wide spatial and temporal scales usually considered are useful only to infer historical migration pathways. In this study, Bayesian genetic assignments and a landscape epidemiology approach were combined to unravel genetic origin and annual spread during a single epidemic of a plant pathogen: the poplar rust fungus Melampsora larici-populina. The study focused on a particular area-the Durance River valley-which enabled inoculum sources to be identified and channelled spread of the epidemic along a one-dimensional corridor. Spatio-temporal monitoring of disease showed that the epidemic began in the upstream part of the valley and spread out downstream. Using genetic assignment tests, individuals collected at the end of the epidemic were sorted into two genetic groups; very few hybrids were detected, although individuals from both groups coexisted locally downstream in the valley. The epidemic originated from two genetically distinct inoculum sources. Individuals of each group then dispersed southwards along the Durance River and became mixed in poplar riparian stands. These two genetic groups were found previously at a wider spatial scale and proved to result from distinct evolutionary histories on either wild or cultivated poplars. This study showed that the two groups can mix during an epidemic but do not hybridize because they then reproduce asexually. In general, the methods employed here could be useful for elucidating the genetic origin and retracing the colonization history and migration pathways of recent epidemics. [ABSTRACT FROM AUTHOR]

The effect of RUS, a major quantitative resistance (QR) factor inherited from Populus trichocarpa, was assessed in two Populus deltoides × P. trichocarpa F1 progenies against eight isolates of Melampsora larici-populina, the causal agent of Eurasian poplar rust. Six isolates were identified on which RUS had no significant effect. The first RUS-defeating isolate identified suggested a pre-existing potential to overcome RUS in the pathogen’s populations. The P. deltoides genetic background made no difference either to the ability of a given isolate to overcome RUS or to the relative and absolute aggressiveness of the isolates. This study illustrates how extreme the isolate-specificity of QR can be. It also yields insights into the relationship between size of uredinia and sporulation rate, discussing the epidemiological significance of spore production per mm2 uredinium. [ABSTRACT FROM AUTHOR]

In this study, we compared interactions of two Melampsora foliar rust species with poplar, which resulted in either limited or abundant pathogen proliferation. In the pathosystem exhibiting limited pathogen growth, a defence response was observed after invasion of poplar leaf tissues by the biotroph, with late and clear production of reactive oxygen species (ROS) and other products. Characterisation of the histological, biochemical and transcriptional events occurring in both pathosystems showed striking similarity with components of plant defence reactions observed during qualitative resistance. Key components associated with development of an active defence response, such as up-regulation of pathogenesis-related (PR) genes, were observed during infection. Moreover, the time course and strength of gene induction appear to be critical determinants for the outcome of the tree–pathogen interaction. This work provides basic biochemical characterisation and expression data for the study of so-called partial resistance in the poplar–rust pathosystem, which is also applicable to other plant–pathogen interactions resulting in quantitative disease resistance. [ABSTRACT FROM AUTHOR]

The activity of cell wall-degrading enzymes, produced in poplar cultivars infected Melampsora laricipopulina Kleb., was studied. The results show that PMG, PMTE, CX and β-glucosidase played roles during the infection. After inoculation, the activity of PMG in both susceptible and resistant cultivars had two peak values in 2 dpi and 5 dpi. The activities of PMTE and β-glucosidase had a peak value in 3 dpi, and CX in 2 dpi. Among these cell wall-degrading enzymes, the activities of PMG and PMTE were higher and the activities of CX and β-glucosidase were relatively lower. The activities of these cell wall-degrading enzymes were significantly higher in susceptible cultivars than those in resistant cultivars. All these demonstrated that these cell wall-degrading enzymes played certain roles in the infection of M. larici-populina. [ABSTRACT FROM AUTHOR]

Abstract: Dispersal has a great impact on the genetic structure of populations, but remains difficult to estimate by direct measures. In particular, gradual and stochastic dispersal are often difficult to assess and to distinguish, although they have different evolutionary consequences. Plant pathogens, especially rust fungi, are suspected to display both dispersal modes, though on different spatial scales. In this study, we inferred dispersal capacities of the poplar rust fungus Melampsora larici-populina by examining the genetic diversity and structure of 13 populations from eight European and two overseas countries in the Northern hemisphere. M. larici-populina was sampled from both cultivated hybrid poplars and on the wild host, Populus nigra. The populations were analyzed with 11 microsatellite and 8 virulence markers. Although isolates displayed different virulence profiles according to the host plant, neutral markers revealed little population differentiation with respect to the type of host. This suggests an absence of reproductive isolation between populations sampled from cultivated and wild poplars. Conversely, studying the relationship between geographic and genetic structure allowed us to distinguish between isolation by distance (IBD) patterns and long distance dispersal (LDD) events. The European populations exhibited a significant IBD pattern, suggesting a regular and gradual dispersal of the pathogen over this spatial scale. Nonetheless, the genetic differentiation between these populations was low, suggesting an important gene flow on a continental scale. The two overseas populations from Iceland and Canada were shown to result from rare LDD events, and exhibited signatures of strong founder effects. Furthermore, the high genetic differentiation between both populations suggested that these two recent introductions were independent. This study illustrated how the proper use of population genetics methods can enable contrasted dispersal modes to be revealed. [Copyright &y& Elsevier]

Qualitative resistance to Melampsora larici-populina leaf rust inherited from North American species Populus deltoides did not allow for durable control of this pathogen in interspecific hybrid cultivars. Despite significant levels of strain-specificity, quantitative resistance would exert lower selection pressures on the pathogen populations, and hence could be more durable. Previous studies restricted to a large P. × interamericana (i.e., P. deltoides × Populus trichocarpa) F1 family revealed that the presence of R1, a segregating defeated qualitative resistance gene inherited from P. deltoides, had major beneficial effects on quantitative resistance. The present study was based on 14 F1 families from a 4 × 5 P. deltoides × P. trichocarpa factorial mating design where at least four defeated qualitative resistances segregate 1:1. Even though quantitative resistance assessments were conducted in the laboratory with a M. larici-populina strain able to overcome these qualitative resistances, their presence had a significant effect on the mean level and on the genetic variability for quantitative resistance. One unprecedented result is the identification of a defeated qualitative resistance which presence is associated with lower levels of quantitative resistance. Possible inferences on the nature of the genetic relationship between both resistance types are discussed. [ABSTRACT FROM AUTHOR]

In an experiment, under controlled laboratory conditions, three species of Cladosporium were shown to be antagonistic, to different degrees, to the production of uredinia by three races of Melampsora larici-populina Kleb. on disks cut from the leaves of PopulusΧeuramericana (Dode) Guinier cv. I-488. The species of Cladosporium also colonized, to different levels, the uredinia produced by the races on the leaf disks (Sharma and Heather 1981). In a similar experiment, employing leaf disks cut from four cultivars of P.Χeuramericana, three races of M. larici-populina and five species of Cladosporium, it was demonstrated that the species of Cladosporium was more significant than the race of the pathogen, or the cultivar of the host in determining the number of uredinia induced by the races on the cultivars. Irrespective of the race or cultivar employed C. tenuissimum was the most significant antagonist to uredinial production and the most active colonizer of the uredinia formed. Furthermore the interactions of Cladosporium spp. with races and with cultivars were significant contributors to variation in the number of uredinia produced and the level of colonization of the uredinia formed (Sharma, unpublished data). [ABSTRACT FROM AUTHOR]

A study was made on the effect of a number of chemicals (benzimidazole, cycloheximide, gibberellic acid, kinetin, spermidine and spermine) and some of their combinations on the longevity of the leaf disks cut from Populus × euramericana '1-488' and P. alba cv. hickeliana. Benzimidazole and gibberellic acid maintained the leaf disks for varying periods to a miximum ol 80 days, depending on the concentration employed. In a factorial experiment of 3 chemicals × 2 concentrations × 2 cultivars × 2 races of the fungus, it was found that all these factors contributed significantly (P <0.05) to the experimental variance. However, differential effects of the chemicals or the concentrations on the cultivar × race interaction were not observed. The implications of these results in the usefulness of the detached leaf culture of are discussed. [ABSTRACT FROM AUTHOR]

We have identified AFLP markers tightly linked to the locus conferring resistance to the leaf rust Melampsora larici-populina in Populus. The study was carried out using a hybrid progeny derived from an inter-specific, controlled cross between a resistant Populus deltoides female and a susceptible P. nigra male. The segregation ratio of resistant to susceptible plants suggested that a single, dominant locus defined this resistance. This locus, which we have designated Melampsora resistance ( Mer), confers resistance against E1, E2, and E3, three different races of Melampsora larici-populina. In order to identify molecular markers linked to the Mer locus we decided to combine two different techniques: (1) the high-density marker technology, AFLP, which allows the analysis of thousands of markers in a relatively short time, and (2) the Bulked Segregant Analysis (BSA), a method which facilitates the identification of markers that are tightly linked to the locus of interest. We analyzed approximately 11,500 selectively amplified DNA fragments using 144 primer combinations and identified three markers tightly linked to the Mer locus. The markers can be useful in current breeding programs and are the basis for future cloning of the resistance gene. [ABSTRACT FROM AUTHOR]

Melampsora larici-populina (Mlp) is a devastating pathogen of poplar trees, causing the defoliating poplar leaf rust disease. Genomic studies have revealed that Mlp possesses a repertoire of 1184 small secreted proteins (SSPs), some of them being characterized as candidate effectors. However, how they promote virulence is still unclear. This study investigates the candidate effector Mlp37347's role during infection. We developed a stable Arabidopsis transgenic line expressing Mlp37347 tagged with the green fluorescent protein (GFP). We found that the effector accumulated exclusively at plasmodesmata (PD). Moreover, the presence of the effector at plasmodesmata favors enhanced plasmodesmatal flux and reduced callose deposition. Transcriptome profiling and a gene ontology (GO) analysis of transgenic Arabidopsis plants expressing the effector revealed that the genes involved in glucan catabolic processes are up-regulated. This effector has previously been shown to interact with glutamate decarboxylase 1 (GAD1), and in silico docking analysis supported the strong binding between Mlp37347 and GAD1 in this study. In infection assays, the effector promoted Hyalonoperospora arabidopsidis growth but not bacterial growth. Our investigation suggests that the effector Mlp37347 targets PD in host cells and promotes parasitic growth. [ABSTRACT FROM AUTHOR]