Melampsora Research Articles

A Poplar Rust Effector Protein Associates with Protein Disulfide Isomerase and Enhances Plant Susceptibility.

Melampsora larici-populina (Mlp), the causal agent of Populus leaf rust, secretes an array of effectors into the host through the haustorium to gain nutrients and suppress immunity. The precise mechanisms by which these effectors promote virulence remain unclear. To address this question, we developed a transgenic Arabidopsis line expressing a candidate effector, Mlp124357. Constitutive expression of the effector increased plant susceptibility to pathogens. A GxxxG motif present in Mlp124357 is required for its subcellular localization at the vacuolar membrane of the plant cell, as replacement of the glycine residues with alanines led to the delocalization of Mlp124357 to the nucleus and cytoplasm. We used immunoprecipitation and mass spectrometry (MS) to identify Mlp124357 interaction partners. Only one of the putative interaction partners knock-out line caused delocalization of the effector, indicating that Arabidopsis protein disulfide isomerase-11 (AtPDI-11) is required for the effector localization. This interaction was further confirmed by a complementation test, a yeast-two hybrid assay and a molecular modeling experiment. Moreover, localization results and infection assays suggest that AtPDI-11 act as a helper for Mlp124357. In summary, our findings established that one of Mlp effectors resides at the vacuole surface and modulates plant susceptibility.

Evolution of isoprenyl diphosphate synthase-like terpene synthases in fungi

Terpene synthases (TPSs) and trans-isoprenyl diphosphate synthases (IDSs) are among the core enzymes for creating the enormous diversity of terpenoids. Despite having no sequence homology, TPSs and IDSs share a conserved “α terpenoid synthase fold” and a trinuclear metal cluster for catalysis, implying a common ancestry with TPSs hypothesized to evolve from IDSs anciently. Here we report on the identification and functional characterization of novel IDS-like TPSs (ILTPSs) in fungi that evolved from IDS relatively recently, indicating recurrent evolution of TPSs from IDSs. Through large-scale bioinformatic analyses of fungal IDSs, putative ILTPSs that belong to the geranylgeranyl diphosphate synthase (GGDPS) family of IDSs were identified in three species of Melampsora. Among the GGDPS family of the two Melampsora species experimentally characterized, one enzyme was verified to be bona fide GGDPS and all others were demonstrated to function as TPSs. Melampsora ILTPSs displayed kinetic parameters similar to those of classic TPSs. Key residues underlying the determination of GGDPS versus ILTPS activity and functional divergence of ILTPSs were identified. Phylogenetic analysis implies a recent origination of these ILTPSs from a GGDPS progenitor in fungi, after the split of Melampsora from other genera within the class of Pucciniomycetes. For the poplar leaf rust fungus Melampsora larici-populina, the transcripts of its ILTPS genes were detected in infected poplar leaves, suggesting possible involvement of these recently evolved ILTPS genes in the infection process. This study reveals the recurrent evolution of TPSs from IDSs since their ancient occurrence and points to the possibility of a wide distribution of ILTPS genes in three domains of life.

Verification of new Populus nigra L. clone improvement based on their performance over three rotations

Abstract: 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.

Identification of Genes Underlying the Resistance to Melampsora larici-populina in an R Gene Supercluster of the Populus deltoides Genome.

Identification of the particular genes in an R genes supercluster underlying resistance to the rust fungus Melampsora larici-populina in poplar genome remains challenging. Based on the de novo assembly of the Populus deltoides genome, all of the detected major genetic loci conferring resistance to M. larici-populina were confined to a 3.5-Mb region on chromosome 19. The transcriptomes of the resistant and susceptible genotypes were sequenced for a timespan from 0 to 168 hours postinoculation. By mapping the differentially expressed genes to the target genomic region, we identified two constitutive expression R genes and one inducible expression R gene that might confer resistance to M. larici-populina. Nucleotide variations were predicted based on the reconstructed haplotypes for each allele of the candidate genes. We also confirmed that salicylic acid was the phytohormone mediating signal transduction pathways, and PR-1 was identified as a key gene inhibiting rust reproduction. Finally, quantitative reverse transcription PCR assay revealed consistent expressions with the RNA-sequencing data for the detected key genes. This study presents an efficient approach for the identification of particular genes underlying phenotype of interest by the combination of genetic mapping, transcriptome profiling, and candidate gene sequences dissection. The identified key genes would be useful for host resistance diagnosis and for molecular breeding of elite poplar cultivars exhibiting resistance to M. larici-populina infection. The detected R genes are also valuable for testing whether the combination of individual R genes can induce durable quantitative resistance.

Insight into plant cell wall degradation and pathogenesis of Ganoderma boninense via comparative genome analysis.

BackgroundG. boninense is a hemibiotrophic fungus that infects oil palms (Elaeis guineensis Jacq.) causing basal stem rot (BSR) disease and consequent massive economic losses to the oil palm industry. The pathogenicity of this white-rot fungus has been associated with cell wall degrading enzymes (CWDEs) released during saprophytic and necrotrophic stage of infection of the oil palm host. However, there is a lack of information available on the essentiality of CWDEs in wood-decaying process and pathogenesis of this oil palm pathogen especially at molecular and genome levels.MethodsIn this study, comparative genome analysis was carried out using the G. boninense NJ3 genome to identify and characterize carbohydrate-active enzyme (CAZymes) including CWDE in the fungal genome. Augustus pipeline was employed for gene identification in G. boninense NJ3 and the produced protein sequences were analyzed via dbCAN pipeline and PhiBase 4.5 database annotation for CAZymes and plant-host interaction (PHI) gene analysis, respectively. Comparison of CAZymes from G. boninense NJ3 was made against G. lucidum, a well-studied model Ganoderma sp. and five selected pathogenic fungi for CAZymes characterization. Functional annotation of PHI genes was carried out using Web Gene Ontology Annotation Plot (WEGO) and was used for selecting candidate PHI genes related to cell wall degradation of G. boninense NJ3.ResultsG. boninense was enriched with CAZymes and CWDEs in a similar fashion to G. lucidum that corroborate with the lignocellulolytic abilities of both closely-related fungal strains. The role of polysaccharide and cell wall degrading enzymes in the hemibiotrophic mode of infection of G. boninense was investigated by analyzing the fungal CAZymes with necrotrophic Armillaria solidipes, A. mellea, biotrophic Ustilago maydis, Melampsora larici-populina and hemibiotrophic Moniliophthora perniciosa. Profiles of the selected pathogenic fungi demonstrated that necrotizing pathogens including G. boninense NJ3 exhibited an extensive set of CAZymes as compared to the more CAZymes-limited biotrophic pathogens. Following PHI analysis, several candidate genes including polygalacturonase, endo β-1,3-xylanase, β-glucanase and laccase were identified as potential CWDEs that contribute to the plant host interaction and pathogenesis.DiscussionThis study employed bioinformatics tools for providing a greater understanding of the biological mechanisms underlying the production of CAZymes in G. boninense NJ3. Identification and profiling of the fungal polysaccharide- and lignocellulosic-degrading enzymes would further facilitate in elucidating the infection mechanisms through the production of CWDEs by G. boninense. Identification of CAZymes and CWDE-related PHI genes in G. boninense would serve as the basis for functional studies of genes associated with the fungal virulence and pathogenicity using systems biology and genetic engineering approaches.

Structural genomics applied to the rust fungus Melampsora larici-populina reveals two candidate effector proteins adopting cystine knot and NTF2-like protein folds

Rust fungi are plant pathogens that secrete an arsenal of effector proteins interfering with plant functions and promoting parasitic infection. Effectors are often species-specific, evolve rapidly, and display low sequence similarities with known proteins. How rust fungal effectors function in host cells remains elusive, and biochemical and structural approaches have been scarcely used to tackle this question. In this study, we produced recombinant proteins of eleven candidate effectors of the leaf rust fungus Melampsora larici-populina in Escherichia coli. We successfully purified and solved the three-dimensional structure of two proteins, MLP124266 and MLP124017, using NMR spectroscopy. Although both MLP124266 and MLP124017 show no sequence similarity with known proteins, they exhibit structural similarities to knottins, which are disulfide-rich small proteins characterized by intricate disulfide bridges, and to nuclear transport factor 2-like proteins, which are molecular containers involved in a wide range of functions, respectively. Interestingly, such structural folds have not been reported so far in pathogen effectors, indicating that MLP124266 and MLP124017 may bear novel functions related to pathogenicity. Our findings show that sequence-unrelated effectors can adopt folds similar to known proteins, and encourage the use of biochemical and structural approaches to functionally characterize effector candidates.

Leaf Cuticle Can Contribute to Non-Host Resistance to Poplar Leaf Rust

The plant leaf cuticle is a chemically complex but largely waxy outer shell that limits water loss and also prevents some pathogens from gaining access to internal mesophyll. Rust fungi are obligate parasites, and most bypass the cuticle by thigmotropically locating stomata, growing through the stomatal openings, and then parasitizing mesophyll cells with haustoria. It is thought that even non-hosts of a given rust fungus do not resist until their mesophyll cells are contacted in this way. In other words, it is thought that the cuticle plays no role in non-host resistance. Here, we tested the hypothesis that poplar leaf cuticles might contribute to non-host resistance to rust fungi by chemically impeding the germination and growth of urediniosporelings of Melampsora larici-populina. Following an initial survey in China of the resistance of 36 genotypes of various species and interspecific hybrids of Populus to M. larici-populina, we selected three genotypes for the initial test of hypothesis: (1) A Populus purdomii genotype that is fully susceptible; (2) a Populus deltoides cv. ‘I-69′ that is incompletely resistant (i.e., a resistant host); and (3) a Populus tomentosa genotype that is a non-host to M. larici-populina. Urediniospores were assayed for germination in extracts of the cuticles of the three genotypes. Germination was most reduced by the P. tomentosa non-host cuticular extracts that also reduced the growth of germ tubes to 36 times less than that in controls or in the extract of the susceptible P. purdomii. Four cuticular components were identified as putative defense compounds given greater concentrations in P. tomentosa than in P. purdomii: Aucubin, hexakis(trimethylsilyl) ether, catechol, 7,9-Di-tert-buty l-1-oxaspiro (4,5) deca-6, 9-diene-2,8-dione and trifluoroacetamide. These four compounds were then tested, and they reduced urediniospore germination and uredinial density in inoculations of normally susceptible P. purdomii with Melampsora larici-populina. Thus, the cuticle of P. tomentosa can contribute to pre-haustorial, non-host resistance to M. larici-populina.

Molecular Characterization of the Transcription Factors in Susceptible Poplar Infected with Virulent Melampsora larici-populina.

Transcription factors (TFs) have been shown to play important roles in determining poplar susceptibility. In this study, the transcript profiles of five resistance-related TF groups at different time points were investigated to study the roles of TFs in the compatible interaction between ‘Robusta’ (Populus nigra × P. deltoides) and the virulent E4 race of Melampsora larici-populina. The susceptibility test indicated that the parasitic process of E4 could be divided into two representative time periods: the infection phase and the production phase. Bioinformatics analysis showed that in these two phases, E4 infection induced a network of TFs in ‘Robusta’. Although some TFs responded rapidly and positively, most TFs did not respond to E4, especially during the infection phase. The ethylene, jasmonic acid, and auxin pathways were downregulated, while a calcium-binding protein was upregulated. No other significantly changed phytohormone-related genes were found, which was consistent with the pathological process in the absence of an immune response, suggesting that the lack of response of most TFs during the infection phase of E4 is related to the susceptibility of ‘Robusta’.

Molecular characterization of the ERF family in susceptible poplar infected by virulent Melampsora larici-populina

Melampsora larici-populina causes serious poplar foliar diseases called rust worldwide. The ethylene response factor (ERF) family, which is one of the largest groups of transcription factors (TFs) families, is considered to play a crucial role in regulating networks related to plant disease resistance. To investigate the transcript profiles of ERF genes involved in poplar rust resistance, Populus nigra × P. deltoides (Pnd), which is susceptible to the E4 race of M. larici-populina, was selected for this study. Digital gene expression (DGE) libraries of mixed time points of E4-inoculated (rust+) or E4-free (rust-) Pnd leaves were examined, yielding 143 Pnd-ERF genes of which 41 Pnd-ERF genes had altered transcript levels. RT-qPCR results indicated that transcript levels of these Pnd-ERF genes were mostly reduced or unchanged when plants were at 4 dpi. Then transcriptome sequencing for rust + or rust- Pnd leaves at 4 dpi was performed and interaction network of Pnd-ERFs and their target genes were predicted and analyzed. Based on the results of the transcriptome sequencing, transcript levels of 93 Pnd-ERF genes in rust + Pnd leaves were significantly different from those in rust- Pnd leaves, including 21 upregulated and 72 downregulated genes. In addition, 199 target genes were predicted to be regulated by 24 Pnd-ERFs in 216 interactions. Among them, only 31 target genes were activated, whereas 70 target genes were unchanged and the remaining 98 target genes were repressed. GO enrichment analysis indicated that most disease resistance-related target genes of Pnd-ERFs from rust + Pnd leaves were downregulated compared with their transcript levels in rust- Pnd leaves. Nevertheless, a negative regulator of plant innate immunity, the histone deacetylase 1 gene, which was targeted by a transcriptional repressor, Pnd-ERF40, was dramatically upregulated at 4 dpi. The inactivation of most Pnd-ERF genes and their disease resistance-related target genes, along with the activation of the negative regulator of plant innate immunity, at 4 dpi might account for the susceptibility of Pnd. Therefore, the inactivation of the Pnd-ERF gene network at the crucial time point should work on the breaking down of the resistance of Pnd.

Identification of novel miRNAs and their target genes from Populus szechuanica infected with Melampsora larici-populina.

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.

Integrated Transcriptome Analysis Reveals Plant Hormones Jasmonic Acid and Salicylic Acid Coordinate Growth and Defense Responses upon Fungal Infection in Poplar.

Plants have evolved a sophisticated system to respond to various stresses. Fungal attack or infection is one of the most important biotic stresses for most plants. During the defense response to fungal infection, the plant hormones jasmonic acid (JA) and salicylic acid (SA) play critical roles. Here, gene expression data on JA/SA treatments and Melampsora larici-populina (MLP) infection were generated. Integrated transcriptome analyses of these data were performed, and 943 genes in total were identified as common responsive genes (CRG). Gene ontology (GO) term analysis revealed that the genes from CRG are generally involved in the processes of stress responses, metabolism, and growth and development. The further cluster analysis of the CRG identified a set of core genes that are involved in the JA/SA-mediated response to fungal defense with distinct gene expression profiles upon JA/SA treatment, which highlighted the different effects of these two hormones on plant fungal defenses. The modifications of several pathways relative to metabolism, biotic stress, and plant hormone signal pathways suggest the possible roles of JA/SA on the regulation of growth and defense responses. Co-expression modules (CMs) were also constructed using the poplar expression data on JA, SA, M. larici-populina, Septoria musiva, and Marssonina brunnea treatment or infection. A total of 23 CMs were constructed, and different CMs clearly exhibited distinct biological functions, which conformably regulated the concerted processes in response to fungal defense. Furthermore, the GO term analysis of different CMs confirmed the roles of JA and SA in regulating growth and defense responses, and their expression profiles suggested that the growth ability was reduced when poplar deployed defense responses. Several transcription factors (TFs) among the CRG in the co-expression network were proposed as hub genes in regulating these processes. According to this study, our data finely uncovered the possible roles of JA/SA in regulating the balance between growth and defense responses by integrating multiple hormone signaling pathways. We were also able to provide more knowledge on how the plant hormones JA/SA are involved in the regulation of the balance between growth and plant defense.

Rust Infection of Black Poplar Trees Reduces Photosynthesis but Does Not Affect Isoprene Biosynthesis or Emission.

Poplar (Populus spp.) trees are widely distributed and play an important role in ecological communities and in forestry. Moreover, by releasing high amounts of isoprene, these trees impact global atmospheric chemistry. One of the most devastating diseases for poplar is leaf rust, caused by fungi of the genus Melampsora. Despite the wide distribution of these biotrophic pathogens, very little is known about their effects on isoprene biosynthesis and emission. We therefore infected black poplar (P. nigra) trees with the rust fungus M. larici-populina and monitored isoprene emission and other physiological parameters over the course of infection to determine the underlying mechanisms. We found an immediate and persistent decrease in photosynthesis during infection, presumably caused by decreased stomatal conductance mediated by increased ABA levels. At the same time, isoprene emission remained stable during the time course of infection, consistent with the stability of its biosynthesis. There was no detectable change in the levels of intermediates or gene transcripts of the methylerythritol 4-phosphate (MEP) pathway in infected compared to control leaves. Rust infection thus does not affect isoprene emission, but may still influence the atmosphere via decreased fixation of CO2.

A rust fungal effector binds plant DNA and modulates transcription

The basidiomycete Melampsora larici-populina causes poplar rust disease by invading leaf tissues and secreting effector proteins through specialized infection structures known as haustoria. The mechanisms by which rust effectors promote pathogen virulence are poorly understood. The present study characterized Mlp124478, a candidate effector of M. larici-populina. We used the models Arabidopsis thaliana and Nicotiana benthamiana to investigate the function of Mlp124478 in plant cells. We established that Mlp124478 accumulates in the nucleus and nucleolus, however its nucleolar accumulation is not required to promote growth of the oomycete pathogen Hyaloperonospora arabidopsidis. Stable constitutive expression of Mlp124478 in A. thaliana repressed the expression of genes involved in immune responses, and also altered leaf morphology by increasing the waviness of rosette leaves. Chip-PCR experiments showed that Mlp124478 associats'e with the TGA1a-binding DNA sequence. Our results suggest that Mlp124478 exerts a virulence activity and binds the TGA1a promoter to suppress genes induced in response to pathogen infection.

Defense Compounds Rather Than Nutrient Availability Shape Aggressiveness Trait Variation Along a Leaf Maturity Gradient in a Biotrophic Plant Pathogen.

Foliar pathogens face heterogeneous environments depending on the maturity of leaves they interact with. In particular, nutrient availability as well as defense levels may vary significantly, with opposing effects on the success of infection. The present study tested which of these factors have a dominant effect on the pathogen’s development. Poplar leaf disks of eight maturity levels were inoculated with the poplar rust fungus Melampsora larici-populina using an innovative single-spore inoculation procedure. A set of quantitative fungal traits (infection efficiency, latent period, uredinia size, mycelium quantity, sporulation rate, sporulation capacity, and spore volume) was measured on each infected leaf disk. Uninfected parts of the leaves were analyzed for their nutrient (sugars, total C and N) and defense compounds (phenolics) content. We found that M. larici-populina is more aggressive on more mature leaves as indicated by wider uredinia and a higher sporulation rate. Other traits varied independently from each other without a consistent pattern. None of the pathogen traits correlated with leaf sugar, total C, or total N content. In contrast, phenolic contents (flavonols, hydroxycinnamic acid esters, and salicinoids) were negatively correlated with uredinia size and sporulation rate. The pathogen’s fitness appeared to be more constrained by the constitutive plant defense level than limited by nutrient availability, as evident in the decrease in sporulation.

Identification of Virulences of the Rust Fungus Melampsora larici-populina Occurring in Chile.

Melampsora larici-populina Kleb. is a Eurasian species that causes leaf rust of many species of Populus from sections Tacamahaca and Aigeiros. This rust has been inadvertently introduced to many parts around the world affecting poplar species that grow naturally, which may be susceptible to this fungus. In Chile, early European settlers introduced poplars; rust, attributed to M. larici-populina, has been reported in these trees since 1918. However, a modern confirmation has been lacking, and pathogenic variation of the Chilean population of M. larici-populina has not been investigated. Using a morphological analysis of urediniospores and sequencing of internal transcribed spacer (ITS) regions, we confirmed the presence of M. larici-populina in Chile. ITS regions exhibited 100% homology with M. larici-populina. Scanning electron microscopy showed that spores were 26 to 47 µm in length, 13 to 16 µm in width, and echinulate except for apices, which are smooth, characteristics described for this species of rust. The variability of M. larici-populina is characterized by the presence of pathotypes, which allows the fungus to infect despite the resistance of certain poplar hybrids. We concluded that the identified spores belong to M. larici-populina, with virulences 1, 2, 3, 4, 5, and 6. These results describe variation in virulence of M. larici-populina, which suggests the presence of a sexual stage in Chile.

New insight into bulb dynamics in the vacuolar lumen ofArabidopsiscells

Plant vacuoles are multifunctional organelles with dynamic and transient membranous structures, such as trans-vacuolar strands and bulbs. Bulbs are highly mobile structures that travel along trans-vacuolar strands. A candidate effector protein from Melampsora larici-populina (Mlp124357) fused with the enhanced green fluorescent protein (eGFP) was used to investigate the properties of central vacuoles and their bulbs. We discovered the coexistence of two bulb populations in Arabidopsis cells. In addition to previously-described bulbs, which present even marker protein distribution on the bulb surface, we discerned bulbs displaying irregular fusion protein distribution. Using fluorescence recovery after photobleaching (FRAP), we also demonstrated that bulbs do not exchange proteins with the tonoplast once they are formed. These results show that more than one type of bulb may co-exist in the same cell and provide evidence of micro-domains on the bulb surface. They also reveal that proteins do not flow freely from the tonoplast to the bulb membrane, giving new insight into the biology of tonoplast-derived substructures.

Induction of Resistance in Poplar to Melampsora larici-populina Using L-form Bacteria

Induction of Resistance in Poplar to Melampsora larici-populina Using L-form Bacteria

The Rust Fungus Melampsora larici-populina Expresses a Conserved Genetic Program and Distinct Sets of Secreted Protein Genes During Infection of Its Two Host Plants, Larch and Poplar.

Mechanisms required for broad-spectrum or specific host colonization of plant parasites are poorly understood. As a perfect illustration, heteroecious rust fungi require two alternate host plants to complete their life cycles. Melampsora larici-populina infects two taxonomically unrelated plants, larch, on which sexual reproduction is achieved, and poplar, on which clonal multiplication occurs, leading to severe epidemics in plantations. We applied deep RNA sequencing to three key developmental stages of M. larici-populina infection on larch: basidia, pycnia, and aecia, and we performed comparative transcriptomics of infection on poplar and larch hosts, using available expression data. Secreted protein was the only significantly overrepresented category among differentially expressed M. larici-populina genes between the basidial, the pycnial, and the aecial stages, highlighting their probable involvement in the infection process. Comparison of fungal transcriptomes in larch and poplar revealed a majority of rust genes were commonly expressed on the two hosts and a fraction exhibited host-specific expression. More particularly, gene families encoding small secreted proteins presented striking expression profiles that highlight probable candidate effectors specialized on each host. Our results bring valuable new information about the biological cycle of rust fungi and identify genes that may contribute to host specificity.

Flavan-3-ols Are an Effective Chemical Defense against Rust Infection.

Phenolic secondary metabolites are often thought to protect plants against attack by microbes, but their role in defense against pathogen infection in woody plants has not been investigated comprehensively. We studied the biosynthesis, occurrence, and antifungal activity of flavan-3-ols in black poplar (Populus nigra), which include both monomers, such as catechin, and oligomers, known as proanthocyanidins (PAs). We identified and biochemically characterized three leucoanthocyanidin reductases and two anthocyanidin reductases from P. nigra involved in catalyzing the last steps of flavan-3-ol biosynthesis, leading to the formation of catechin [2,3-trans-(+)-flavan-3-ol] and epicatechin [2,3-cis-(-)-flavan-3-ol], respectively. Poplar trees that were inoculated with the biotrophic rust fungus (Melampsora larici-populina) accumulated higher amounts of catechin and PAs than uninfected trees. The de novo-synthesized catechin and PAs in the rust-infected poplar leaves accumulated significantly at the site of fungal infection in the lower epidermis. In planta concentrations of these compounds strongly inhibited rust spore germination and reduced hyphal growth. Poplar genotypes with constitutively higher levels of catechin and PAs as well as hybrid aspen (Populus tremula × Populus alba) overexpressing the MYB134 transcription factor were more resistant to rust infection. Silencing PnMYB134, on the other hand, decreased flavan-3-ol biosynthesis and increased susceptibility to rust infection. Taken together, our data indicate that catechin and PAs are effective antifungal defenses in poplar against foliar rust infection.

FIRST REPORT OF LEAF RUST CAUSED BY MELAMPSORA MAGNUSIANA ON POPULUS ALBA IN ITALY

In October 2016, Melampsora spp. hypophyllous uredinia (0.3-0.5 mm in diameter) were observed on leaves of Populus alba L. clones cultivated in Casale Monferrato at CREA-PLF (Piedmont, northern Italy). Morphological observations and DNA sequencing of the Internal Transcribed Spacer (ITS) region were performed to identify the fungus at the species level. Urediniospores were yellowish, obovate, verrucose and on average 23.3 x 18.8 µm (wall 1.22-2.44 µm thick). DNA was extracted from urediniospores with the Quiagen Plant DNeasy Mini-kit following manufacturer’s instructions. The ITS region was amplified and sequenced with taxon selective primers ITS1-F and ITS4-B (Gardes and Bruns, 1993). Sequences were deposited in GenBank (accession Nos. KY364897, KY364898) and compared with those of known Melampsora species using the NCBI GenBank nucleotide BLAST search. Based on morphological features of uredinia and urediniospores and DNA sequences the fungus was identified, according to Vialle et al. (2011), as Melampsora magnusiana G.H. Wagner, belonging to M. populnea complex and associated with P. alba, P. tremula L. and P. x canescens (Aiton) Sm. telial hosts. The identification of this Melampsora species was also supported by the presence of its known aecial hosts Chelidonium majus L. and Corydalis spp. in the neighborhood of the sampling site (Picco, 2010). Telia were not observed. Disease symptoms consisting of chlorotic spots were reproduced on the abaxial surface of ten leaves cut from five P. x canescens, belonging to the same clone, artificially inoculated by spraying a spore suspension of the fungus (105 spores ml-1 ). Uredinia were observed ten days after inoculation. Non-inoculated leaves remained symptomless. To our knowledge, this is the first report of M. magnusiana on P. alba in Italy, although it was observed in some European countries (Pei and McCraken, 2005) on both Populus spp. and aecial hosts.