Poplar Rust Fungus Melampsora Larici-populina Research Articles

Host-Specific and Homologous Pairs of Melampsora larici-populina Effectors Unveil Novel Nicotiana benthamiana Stromule Induction Factors.

The poplar rust fungus Melampsora larici-populina is part of one of the most devastating group of fungi (Pucciniales) and causes important economic losses to the poplar industry. Because M. larici-populina is a heteroecious obligate biotroph, its spread depends on its ability to carry out its reproductive cycle through larch and then poplar parasitism. Genomic approaches have identified more than 1,000 candidate secreted effector proteins (CSEPs) from the predicted secretome of M. larici-populina that are potentially implicated in the infection process. In this study, we selected CSEP pairs (and one triplet) among CSEP gene families that share high sequence homology but display specific gene expression profiles among the two distinct hosts. We determined their subcellular localization by confocal microscopy through expression in the heterologous plant system Nicotiana benthamiana. Five out of nine showed partial or complete chloroplastic localization. We also screened for potential protein interactors from larch and poplar by yeast two-hybrid assays. One pair of CSEPs and the triplet shared common interactors, whereas the members of the two other pairs did not have common targets from either host. Finally, stromule induction quantification revealed that two pairs and the triplet of CSEPs induced stromules when transiently expressed in N.benthamiana. The use of N.benthamiana eds1 and nrg1 knockout lines showed that CSEPs can induce stromules through an eds1-independent mechanism. However, CSEP homologs shared the same impact on stromule induction and contributed to discovering a new stromule induction cascade that can be partially and/or fully independent of eds1. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Issue Information

Molecular EcologyVolume 32, Issue 10 p. i-ii ISSUE INFORMATIONFree Access Issue Information First published: 09 May 2023 https://doi.org/10.1111/mec.16516AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Cover Illustrations: (Left to Right): (Image 1): N/A; (Image 2): Sourdough photography taken on a SEM; (Image 3): Soybean pod with symptoms of anthracnose; (Image 4): Population Genetics provides a link across the three scientific communities interested in improving the durability of plant resistances; (Image 5): Uredinia of the poplar rust fungus Melampsora larici-populina on a poplar leaf; (Image 6): Coffee berries infected with coffee berry disease caused by the fungal pathogen Colletotrichum kahawae which infects the young developing berries causing black sunken lessions on the outer skin of the berries. Eventually the infection covers the whole berries and the berries will be mummified (current stage) and drop from the shrub; (Image 7): The association relationship analysis between the average deoxynivalenol accumulation and Fusarium OTU diversity for different sampling sites; (Image 8): Paddy fields before harvest, Yuanyang Terraces, Yunnan, China; (Image 9): Active sites of multidrug efflux pumps in prominent gut bacterial symbionts of humans contain polymorphisms indicative of a history of balancing selection; (Image 10): Each city subway is distinguishable by a particular microbiome signature. The subway microbiome may be a proxy of the city microbiome itself; (Image 11): We used a combination of sampling, sequencing and culturing to analyse variation of microbial community composition among different locations in Switzerland and different types of shops; (Image 12): Field collection of rhizosphere soil of the model legume Medicago truncatula to culture symbiotic nitrogen-fixing rhizobia in the south of France; (Image 13): Network representing the sharing of plasmid genes among the Lysobacteraceae bacterial family. Photo Credit: (Image 1): J. P. Sampaio; (Image 2): Bernard Onno; (Image 3): Flavia Rogerio; (Image 4): M. Saubin; (Image 5): Benjamin PETRE; (Image 6): Beyene Zewdie; (Image 7): Qiang Wang, Xiaoping Hu; (Image 8): E. FOURNIER, INRAE; (Image 9): Andrew Moeller; (Image 10): Mariana Peimbert; (Image 11): Helena Klein (illuzation-hk.com); (Image 12): Michael Grillo; (Image 13): Damien Richard. Volume32, Issue10Special Issue: EVOLUTIONARY ECOLOGY OF HUMAN‐ASSOCIATED MICROBESMay 2023Pages i-ii RelatedInformation

Genomic Signatures of a Major Adaptive Event in the Pathogenic Fungus Melampsora larici-populina.

The recent availability of genome-wide sequencing techniques has allowed systematic screening for molecular signatures of adaptation, including in nonmodel organisms. Host–pathogen interactions constitute good models due to the strong selective pressures that they entail. We focused on an adaptive event which affected the poplar rust fungus Melampsora larici-populina when it overcame a resistance gene borne by its host, cultivated poplar. Based on 76 virulent and avirulent isolates framing narrowly the estimated date of the adaptive event, we examined the molecular signatures of selection. Using an array of genome scan methods based on different features of nucleotide diversity, we detected a single locus exhibiting a consistent pattern suggestive of a selective sweep in virulent individuals (excess of differentiation between virulent and avirulent samples, linkage disequilibrium, genotype–phenotype statistical association, and long-range haplotypes). Our study pinpoints a single gene and further a single amino acid replacement which may have allowed the adaptive event. Although our samples are nearly contemporary to the selective sweep, it does not seem to have affected genome diversity further than the immediate vicinity of the causal locus, which can be explained by a soft selective sweep (where selection acts on standing variation) and by the impact of recombination in mitigating the impact of selection. Therefore, it seems that properties of the life cycle of M. larici-populina, which entails both high genetic diversity and outbreeding, has facilitated its adaptation.

A point mutation and large deletion at the candidate avirulence locus AvrMlp7 in the poplar rust fungus correlate with poplar RMlp7 resistance breakdown.

The deployment of plant varieties carrying resistance genes (R) exerts strong selection pressure on pathogen populations. Rapidly evolving avirulence genes (Avr) allow pathogens to escape R-mediated plant immunity through a variety of mechanisms, leading to virulence. The poplar rust fungus Melampsora larici-populina is a damaging pathogen of poplars in Europe. It underwent a major adaptive event in 1994, with the breakdown of the poplar RMlp7 resistance gene. Population genomics studies identified a locus in the genome of M. larici-populina that probably corresponds to the candidate avirulence gene AvrMlp7. Here, to further characterize this effector, we used a population genetics approach on a comprehensive set of 281 individuals recovered throughout a 28-year period encompassing the resistance breakdown event. Using two dedicated molecular tools, genotyping at the candidate locus highlighted two different alterations of a predominant allele found mainly before the resistance breakdown: a nonsynonymous mutation and a complete deletion of this locus. This results in six diploid genotypes: three genotypes related to the avirulent phenotype and three related to the virulent phenotype. The temporal survey of the candidate locus revealed that both alterations were found in association during the resistance breakdown event. They pre-existed before the breakdown in a heterozygous state with the predominant allele cited above. Altogether, these results suggest that the association of both alterations at the candidate locus AvrMlp7 drove the poplar rust adaptation to RMlp7-mediated immunity. This study demonstrates for the first time a case of adaptation from standing genetic variation in rust fungi during a qualitative resistance breakdown.

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.

Patterns of genomic variation in the poplar rust fungus Melampsora larici-populina identify pathogenesis-related factors.

Melampsora larici-populina is a fungal pathogen responsible for foliar rust disease on poplar trees, which causes damage to forest plantations worldwide, particularly in Northern Europe. The reference genome of the isolate 98AG31 was previously sequenced using a whole genome shotgun strategy, revealing a large genome of 101 megabases containing 16,399 predicted genes, which included secreted protein genes representing poplar rust candidate effectors. In the present study, the genomes of 15 isolates collected over the past 20 years throughout the French territory, representing distinct virulence profiles, were characterized by massively parallel sequencing to assess genetic variation in the poplar rust fungus. Comparison to the reference genome revealed striking structural variations. Analysis of coverage and sequencing depth identified large missing regions between isolates related to the mating type loci. More than 611,824 single-nucleotide polymorphism (SNP) positions were uncovered overall, indicating a remarkable level of polymorphism. Based on the accumulation of non-synonymous substitutions in coding sequences and the relative frequencies of synonymous and non-synonymous polymorphisms (i.e., PN/PS), we identify candidate genes that may be involved in fungal pathogenesis. Correlation between non-synonymous SNPs in genes encoding secreted proteins (SPs) and pathotypes of the studied isolates revealed candidate genes potentially related to virulences 1, 6, and 8 of the poplar rust fungus.

Genome-wide patterns of segregation and linkage disequilibrium: the construction of a linkage genetic map of the poplar rust fungus Melampsora larici-populina.

The poplar rust fungus Melampsora larici-populina causes significant yield reduction and severe economic losses in commercial poplar plantations. After several decades of breeding for qualitative resistance and subsequent breakdown of the released resistance genes, breeders now focus on quantitative resistance, perceived to be more durable. But quantitative resistance also can be challenged by an increase of aggressiveness in the pathogen. Thus, it is of primary importance to better understand the genetic architecture of aggressiveness traits. To this aim, our goal is to build a genetic linkage map for M. larici-populina in order to map quantitative trait loci related to aggressiveness. First, a large progeny of M. larici-populina was generated through selfing of the reference strain 98AG31 (which genome sequence is available) on larch plants, the alternate host of the poplar rust fungus. The progeny's meiotic origin was validated through a segregation analysis of 115 offspring with 14 polymorphic microsatellite markers, of which 12 segregated in the expected 1:2:1 Mendelian ratio. A microsatellite-based linkage disequilibrium analysis allowed us to identify one potential linkage group comprising two scaffolds. The whole genome of a subset of 47 offspring was resequenced using the Illumina HiSeq 2000 technology at a mean sequencing depth of 6X. The reads were mapped onto the reference genome of the parental strain and 144,566 SNPs were identified across the genome. Analysis of distribution and polymorphism of the SNPs along the genome led to the identification of 2580 recombination blocks. A second linkage disequilibrium analysis, using the recombination blocks as markers, allowed us to group 81 scaffolds into 23 potential linkage groups. These preliminary results showed that a high-density linkage map could be constructed by using high-quality SNPs based on low-coverage resequencing of a larger number of M. larici-populina offspring.

Transcriptome analysis of poplar rust telia reveals overwintering adaptation and tightly coordinated karyogamy and meiosis processes

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.

Exploring the role of asexual multiplication in poplar rust epidemics: impact on diversity and genetic structure

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.

Disentangling the genetic origins of a plant pathogen during disease spread using an original molecular epidemiology approach

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.

Characterization of 41 microsatellite loci developed from the genome sequence of the poplar rust fungus, Melampsora larici-populina

Forty-one microsatellite markers were developed for the poplar rust fungus Melampsora larici-populina. The genome sequence of M. larici-populina strain 98AG31 was screened in silico for microsatellite loci using Magellan software. Thirty-nine loci, evaluated on a sexual population of 35 M. larici-populina isolates sampled on larch trees in the French Alps, were identified to be highly polymorphic. These markers revealed two to 34 alleles, with an average of 7.23 alleles per locus. The estimated heterozygosity ranged from 0.029 to 0.958 across the 41 microsatellite loci. These markers will be highly useful for population genetics studies of M. larici-populina.

Genetic structure of the poplar rust fungus Melampsora larici-populina: Evidence for isolation by distance in Europe and recent founder effects overseas

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.