Ecto- and endo-mycorrhizal colonization of Populus roots have a positive impact on the overall tree health and growth. A complete molecular understanding of these interactions will have important implications for increasing agricultural or forestry sustainability using plant:microbe-based strategies. These beneficial associations entail extensive morphological changes orchestrated by the genetic reprogramming in both organisms. In this study, we performed a comparative analysis of two Populus species (Populus deltoides and P. trichocarpa) that were colonized by either an arbuscular mycorrhizal fungus (AmF), Rhizophagus irregularis or an ectomycorrhizal fungus (EmF), Laccaria bicolor, to describe the small RNA (sRNA) landscape including small open reading frames (sORFs) and micro RNAs (miRNAs) involved in these mutualistic interactions. We identified differential expression of sRNAs that were, to a large extent, 1) within the genomic regions lacking annotated genes in the Populus genome and 2) distinct for each fungal interaction. These sRNAs may be a source of novel sORFs within a genome, and in this regard, we identified potential sORFs encoded by the sRNAs. We predicted a higher number of differentially-expressed miRNAs in P. trichocarpa (4 times more) than in P. deltoides (conserved and novel). In addition, 44 miRNAs were common in P. trichocarpa between the EmF and AmF treatments, and only 4 miRNAs were common in P. deltoides between the treatments. Root colonization by either fungus was more effective in P. trichocarpa than in P. deltoides, thus the relatively few differentially-expressed miRNAs predicted in P. deltoides might reflect the extent of the symbiosis. Finally, we predicted several genes targets for the plant miRNAs identified here, including potential fungal gene targets. Our findings shed light on additional molecular tiers with a role in Populus-fungal mutualistic associations and provides a set of potential molecular targets for future enhancement.

Cenococcum geophilum is an ectomycorrhizal fungus with global distribution in numerous habitats and associates with a large range of host species including gymnosperm and angiosperm trees. Moreover, C. geophilum is the unique ectomycorrhizal species within the clade Dothideomycetes, the largest class of Ascomycetes containing predominantly saprotrophic and many devastating phytopathogenic fungi. Recent studies highlight that mycorrhizal fungi, as pathogenic ones, use effectors in form of Small Secreted Proteins (SSPs) as molecular keys to promote symbiosis. In order to better understand the biotic interaction of C. geophilum with its host plants, the goal of this work was to characterize mycorrhiza-induced small-secreted proteins (MiSSPs) that potentially play a role in the ectomycorrhiza formation and functioning of this ecologically very important species. We combined different approaches such as gene expression profiling, genome localization and conservation of MiSSP genes in different C. geophilum strains and closely related species as well as protein subcellular localization studies of potential targets of MiSSPs in interacting plants using in tobacco leaf cells. Gene expression analyses of C. geophilum interacting with Pinus sylvestris (pine) and Populus tremula × Populus alba (poplar) showed that similar sets of genes coding for secreted proteins were up-regulated and only few were specific to each host. Whereas pine induced more carbohydrate active enzymes (CAZymes), the interaction with poplar induced the expression of specific SSPs. We identified a set of 22 MiSSPs, which are located in both, gene-rich, repeat-poor or gene-sparse, repeat-rich regions of the C. geophilum genome, a genome showing a bipartite architecture as seen for some pathogens but not yet for an ectomycorrhizal fungus. Genome re-sequencing data of 15 C. geophilum strains and two close relatives Glonium stellatum and Lepidopterella palustris were used to study sequence conservation of MiSSP-encoding genes. The 22 MiSSPs showed a high presence-absence polymorphism among the studied C. geophilum strains suggesting an evolution through gene gain/gene loss. Finally, we showed that six CgMiSSPs target four distinct sub-cellular compartments such as endoplasmic reticulum, plasma membrane, cytosol and tonoplast. Overall, this work presents a comprehensive analysis of secreted proteins and MiSSPs in different genetic level of C. geophilum opening a valuable resource to future functional analysis.

Project Goals: The goal of the PMI SFA is to understand the genome-dependent molecular and cellular events involved in establishing and maintaining beneficial interactions between plants and microbes. Populus and its associated microbial community serves as the experimental system for understanding how these molecular events manifest themselves within the spatially, structurally, and temporally complex scales of natural systems. To achieve this goal, we focus on 1) characterizing host and environmental drivers for diversity and function in the Populus microbiome, 2) utilizing microbial model system studies to elucidate Populus-microbial interactions at the molecular level and dissecting the signals and pathways responsible for initiating and maintaining microbial relationships and 3) develop metabolic and genomic modeling of these interactions to aid in interpreting the molecular mechanisms shaping the Populus-microbial interface.

A Populus deltoides × Populus trichocarpa F1 pedigree was analyzed for quantitative trait loci (QTLs) affecting ectomycorrhizal development and for microarray characterization of gene networks involved in this symbiosis. A 300 genotype progeny set was evaluated for its ability to form ectomycorrhiza with the basidiomycete Laccaria bicolor. The percentage of mycorrhizal root tips was determined on the root systems of all 300 progeny and their two parents. QTL analysis identified four significant QTLs, one on the P. deltoides and three on the P. trichocarpa genetic maps. These QTLs were aligned to the P. trichocarpa genome and each contained several megabases and encompass numerous genes. NimbleGen whole-genome microarray, using cDNA from RNA extracts of ectomycorrhizal root tips from the parental genotypes P. trichocarpa and P. deltoides, was used to narrow the candidate gene list. Among the 1,543 differentially expressed genes (p value ≤ 0.05; ≥5.0-fold change in transcript level) having different transcript levels in mycorrhiza of the two parents, 41 transcripts were located in the QTL intervals: 20 in Myc_d1, 14 in Myc_t1, and seven in Myc_t2, while no significant differences among transcripts were found in Myc_t3. Among these 41 transcripts, 25 were overrepresented in P. deltoides relative to P. trichocarpa; 16 were overrepresented in P. trichocarpa. The transcript showing the highest overrepresentation in P. trichocarpa mycorrhiza libraries compared to P. deltoides mycorrhiza codes for an ethylene-sensitive EREBP-4 protein which may repress defense mechanisms in P. trichocarpa while the highest overrepresented transcripts in P. deltoides code for proteins/genes typically associated with pathogen resistance.

The Mycorrhiza Helper Bacterium (MHB) Pseudomonas fluorescens BBc6R8 promotes the ectomycorrhizal symbiosis between Douglas fir roots and Laccaria bicolor. In this study, we identified a non-flagellar type III secretion system (T3SS) in the draft genome of BBc6R8 similar to that described in the biocontrol strain P. fluorescens SBW25. We examined whether this T3SS plays a role in the BBc6R8 mycorrhizal helper effect by creating a deletion in the rscRST genes encoding the central channel of the injectisome. The in vitro effect of BBc6R8 T3SS mutants on the radial growth rate of L. bicolor was unchanged compared with the parental strain. In contrast, T3SS mutants were unable to promote mycorrhization, suggesting that type III secretion plays an important role in the mycorrhizal helper effect of P. fluorescens BBc6R8 independent of the promotion of hyphal growth that BBc6R8 exhibits in vitro.

The aim of this study was to determine the existence of a genetic basis for the ability to form ectomycorrhiza on a model angiosperm tree (Populus, poplar). Parental clones and 18 progeny from a controlled interspecific cross between Populus deltoides and Po- pulus trichocarpa were grown in a glasshouse and inoculated with mycelium of the ectomycorrhizal fungus Laccaria bicolor. Three months after inoculation, the percentage of mycorrhizal root tips was determined for each inoculated plant. The data indicate variability in the ability to form ectomycorrhizas among the F1 progeny, including individual progeny which are different to either parent. This suggests a genetic basis for mycorrhiza formation.