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.

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.

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.