Sylvie Herrmann

Manipulation of the onset of ectomycorrhiza formation by indole-3- acetic acid, activated charcoal or relative humidity in the association between oak microcuttings and Piloderma croceum: influence on plant development and photosynthesis

A Petri dish system in which development of oak (Quercus robur L.) microcuttings is stimulated by the late stage ectomycorrhizal (EM) fungus Piloderma croceum J. Erikss. & Hjortst. in a long pre-symbiotic stage was optimised to allow synchronous, rhythmic plant growth. Addition of indole-3-acetic acid or activated charcoal to the medium caused an early and more intensive EM formation coupled with suppression of most developmental effects of P. croceum. Leaf area, chlorophyll fluorescence, and content were compared in inoculated and uninoculated plants grown at two relative humidity levels (45 and 95%) and under consideration of three possible answers to inoculation, that is, no or EM formation after the 1st or the 2nd shoot flush. The culture conditions for uninoculated plants were suboptimal, leading toward photochemical stress reflected by a non photochemical quenching (qE) increase and a reduced Chl content at the end of the assay. Prior to EM formation, inoculation itself enhanced the optimal (Fv/Fm) and effective (phiPSII) quantum yield in leaves of the 1st shoot flush under reduced relative humidity. It also fully protected the plants against stress during the complete assays. The results indicate that inoculated plants only form EM once they have acquired a sufficient development level and C-providing capacity. However, the fungus actively improves the development and photosynthesis of plants up to the pre-mycorrhizal stage, helping them to reach this capacity.

OakContigDF159.1, a reference library for studying differential gene expression in Quercus robur during controlled biotic interactions: use for quantitative transcriptomic profiling of oak roots in ectomycorrhizal symbiosis

Oaks (Quercus spp.), which are major forest trees in the northern hemisphere, host many biotic interactions, but molecular investigation of these interactions is limited by fragmentary genome data. To date, only 75 oak expressed sequence tags (ESTs) have been characterized in ectomycorrhizal (EM) symbioses. We synthesized seven beneficial and detrimental biotic interactions between microorganisms and animals and a clone (DF159) of Quercus robur. Sixteen 454 and eight Illumina cDNA libraries from leaves and roots were prepared and merged to establish a reference for RNA-Seq transcriptomic analysis of oak EMs with Piloderma croceum. Using the Mimicking Intelligent Read Assembly (MIRA) and Trinity assembler, the OakContigDF159.1 hybrid assembly, containing 65 712 contigs with a mean length of 1003 bp, was constructed, giving broad coverage of metabolic pathways. This allowed us to identify 3018 oak contigs that were differentially expressed in EMs, with genes encoding proline-rich cell wall proteins and ethylene signalling-related transcription factors showing up-regulation while auxin and defence-related genes were down-regulated. In addition to the first report of remorin expression in EMs, the extensive coverage provided by the study permitted detection of differential regulation within large gene families (nitrogen, phosphorus and sugar transporters, aquaporins). This might indicate specific mechanisms of genome regulation in oak EMs compared with other trees.

Streptomyces-Induced Resistance Against Oak Powdery Mildew Involves Host Plant Responses in Defense, Photosynthesis, and Secondary Metabolism Pathways

Rhizobacteria are known to induce defense responses in plants without causing disease symptoms, resulting in increased resistance to plant pathogens. This study investigated how Streptomyces sp. strain AcH 505 suppressed oak powdery mildew infection in pedunculate oak, by analyzing RNA-Seq data from singly- and co-inoculated oaks. We found that this Streptomyces strain elicited a systemic defense response in oak that was, in part, enhanced upon pathogen challenge. In addition to induction of the jasmonic acid/ethylene-dependent pathway, the RNA-Seq data suggests the participation of the salicylic acid-dependent pathway. Transcripts related to tryptophan, phenylalanine, and phenylpropanoid biosynthesis were enriched and phenylalanine ammonia lyase activity increased, indicating that priming by Streptomyces spp. in pedunculate oak shares some determinants with the Pseudomonas-Arabidopsis system. Photosynthesis-related transcripts were depleted in response to powdery mildew infection, but AcH 505 alleviated this inhibition, which suggested there is a fitness benefit for primed plants upon pathogen challenge. This study offers novel insights into the mechanisms of priming by actinobacteria and highlights their capacity to activate plant defense responses in the absence of pathogen challenge.

Transcriptional changes in two types of pre-mycorrhizal roots and in ectomycorrhizas of oak microcuttings inoculated with Piloderma croceum

The formation of the ectomycorrhiza implies an alteration in gene expression of both the plant and fungal partners, a process which starts before the formation of any symbiotic interface. However, little is known on the regulation pattern occurring in different parts of the root system. Our experimental system consisting of a micropropagated oak with a hierarchical root system was shown to exhibit symbiosis functional traits prior to any mycorrhizal tissue differentiation after the inoculation with the basidiomycete Piloderma croceum. Using a cDNA array, the plant gene regulation was analyzed in the pre-mycorrhizal phase. Seventy-five transcripts showed differential expression in pre-mycorrhizal lateral and principal roots, and both root types exhibited different sets of responsive genes. For transcripts selected according to a statistical analysis, the alteration in gene expression was confirmed by RT-PCR and quantitative real-time PCR. Genes regulated in pre-mycorrhizal lateral roots displayed an almost identical expression in mycorrhizas. In contrast, genes regulated in pre-mycorrhizal principal roots were often regulated differently in ectomycorrhizas. Down-regulation affected most of the regulated genes involved in metabolism, whereas most of the regulated genes related to cell rescue functions, water regulation and defence response were up-regulated. Regulation of such genes could explain the increase of global resistance observed in mycorrhizal plants.

Differential expression of two class III chitinases in two types of roots of Quercus robur during pre-mycorrhizal interactions with Piloderma croceum

Expression of two plant chitinase genes, representing members of class III chitinases, was studied in Quercus robur roots during interactions in a pre-mycorrhizal stage with the ectomycorrhizal fungus Piloderma croceum. Chitinase gene expression was compared in lateral roots destined to form ectomycorrhiza, and in principal roots that are not directly involved in mycorrhizal interactions. The transcript level of the first chitinase (QrchitIII-1) was upregulated in lateral roots, whereas no significant differential expression was observed in principal roots. The second chitinase (QrchitIII-2) was regulated neither in lateral nor in principal roots in presence of the fungus. Because P. croceum did not induce significant chitinase responses in principal roots, the enhanced expression of QrchitIII-1 in lateral roots after inoculation may be related to some steps in symbiosis ontogenesis.

Detection and quantification of a mycorrhization helper bacterium and a mycorrhizal fungus in plant-soil microcosms at different levels of complexity

BackgroundHost plant roots, mycorrhizal mycelium and microbes are important and potentially interacting factors shaping the performance of mycorrhization helper bacteria (MHB). We investigated the impact of a soil microbial community on the interaction between the extraradical mycelium of the ectomycorrhizal fungus Piloderma croceum and the MHB Streptomyces sp. AcH 505 in both the presence and the absence of pedunculate oak microcuttings.ResultsSpecific primers were designed to target the internal transcribed spacer of the rDNA and an intergenic region between two protein encoding genes of P. croceum and the intergenic region between the gyrA and gyrB genes of AcH 505. These primers were used to perform real-time PCR with DNA extracted from soil samples. With a sensitivity of 10 genome copies and a linear range of 6 orders of magnitude, these real-time PCR assays enabled the quantification of purified DNA from P. croceum and AcH 505, respectively. In soil microcosms, the fungal PCR signal was not affected by AcH 505 in the absence of the host plant. However, the fungal signal became weaker in the presence of the plant. This decrease was only observed in microbial filtrate amended microcosms. In contrast, the PCR signal of AcH 505 increased in the presence of P. croceum. The increase was not significant in sterile microcosms that contained plant roots.ConclusionsReal-time quantitative PCR assays provide a method for directly detecting and quantifying MHB and mycorrhizal fungi in plant microcosms. Our study indicates that the presence of microorganisms and plant roots can both affect the nature of MHB-fungus interactions, and that mycorrhizal fungi may enhance MHB growth.

Endogenous rhythmic growth in oak trees is regulated by internal clocks rather than resource availability

Highlight Increased resource availability has no impact on endogenous rhythmic growth and related allocation shifts in oak trees; an internal clock regulates core genes during shoot and root growth cessation phases.

Large scale transcriptome analysis reveals interplay between development of forest trees and a beneficial mycorrhiza helper bacterium

BackgroundPedunculate oak, Quercus robur is an abundant forest tree species that hosts a large and diverse community of beneficial ectomycorrhizal fungi (EMFs), whereby ectomycorrhiza (EM) formation is stimulated by mycorrhiza helper bacteria such as Streptomyces sp. AcH 505. Oaks typically grow rhythmically, with alternating root flushes (RFs) and shoot flushes (SFs). We explored the poorly understood mechanisms by which oaks integrate signals induced by their beneficial microbes and endogenous rhythmic growth at the level of gene expression. To this end, we compared transcript profiles of oak microcuttings at RF and SF during interactions with AcH 505 alone and in combination with the basidiomycetous EMF Piloderma croceum.ResultsThe local root and distal leaf responses to the microorganisms differed substantially. More genes involved in the recognition of bacteria and fungi, defence and cell wall remodelling related transcription factors (TFs) were differentially expressed in the roots than in the leaves of oaks. In addition, interaction with AcH 505 and P. croceum affected the expression of a higher number of genes during SF than during RF, including AcH 505 elicited defence response, which was attenuated by co-inoculation with P. croceum in the roots during SF. Genes encoding leucine-rich receptor-like kinases (LRR-RLKs) and proteins (LRR-RLPs), LRR containing defence response regulators, TFs from bZIP, ERF and WRKY families, xyloglucan cell wall transglycolases/hydrolases and exordium proteins were differentially expressed in both roots and leaves of plants treated with AcH 505. Only few genes, including specific RLKs and TFs, were induced in both AcH 505 and co-inoculation treatments.ConclusionTreatment with AcH 505 induces and maintains the expression levels of signalling genes encoding candidate receptor protein kinases and TFs and leads to differential expression of cell wall modification related genes in pedunculate oak microcuttings. Local gene expression response to AcH 505 alone and in combination with P. croceum are more pronounced when roots are in resting stages, possibly due to the fact that non growing roots re-direct their activity towards plant defence rather than growth.

Oak genome reveals facets of long lifespan

Oaks are an important part of our natural and cultural heritage. Not only are they ubiquitous in our most common landscapes1 but they have also supplied human societies with invaluable services, including food and shelter, since prehistoric times2. With 450 species spread throughout Asia, Europe and America3, oaks constitute a critical global renewable resource. The longevity of oaks (several hundred years) probably underlies their emblematic cultural and historical importance. Such long-lived sessile organisms must persist in the face of a wide range of abiotic and biotic threats over their lifespans. We investigated the genomic features associated with such a long lifespan by sequencing, assembling and annotating the oak genome. We then used the growing number of whole-genome sequences for plants (including tree and herbaceous species) to investigate the parallel evolution of genomic characteristics potentially underpinning tree longevity. A further consequence of the long lifespan of trees is their accumulation of somatic mutations during mitotic divisions of stem cells present in the shoot apical meristems. Empirical4 and modelling5 approaches have shown that intra-organismal genetic heterogeneity can be selected for6 and provides direct fitness benefits in the arms race with short-lived pests and pathogens through a patchwork of intra-organismal phenotypes7. However, there is no clear proof that large-statured trees consist of a genetic mosaic of clonally distinct cell lineages within and between branches. Through this case study of oak, we demonstrate the accumulation and transmission of somatic mutations and the expansion of disease-resistance gene families in trees.Oaks can live hundreds of years. Comparative genomics using a high-quality genome sequence provides new insights that may explain tree longevity. Samples from branches and corresponding acorns also help quantify heritable somatic mutations.

Transcriptome analysis in oak uncovers a strong impact of endogenous rhythmic growth on the interaction with plant-parasitic nematodes.

BackgroundPedunculate oak (Quercus robur L.), an important forest tree in temperate ecosystems, displays an endogenous rhythmic growth pattern, characterized by alternating shoot and root growth flushes paralleled by oscillations in carbon allocation to below- and aboveground tissues. However, these common plant traits so far have largely been neglected as a determining factor for the outcome of plant biotic interactions. This study investigates the response of oak to migratory root-parasitic nematodes in relation to rhythmic growth, and how this plant-nematode interaction is modulated by an ectomycorrhizal symbiont. Oaks roots were inoculated with the nematode Pratylenchus penetrans solely and in combination with the fungus Piloderma croceum, and the systemic impact on oak plants was assessed by RNA transcriptomic profiles in leaves.ResultsThe response of oaks to the plant-parasitic nematode was strongest during shoot flush, with a 16-fold increase in the number of differentially expressed genes as compared to root flush. Multi-layered defence mechanisms were induced at shoot flush, comprising upregulation of reactive oxygen species formation, hormone signalling (e.g. jasmonic acid synthesis), and proteins involved in the shikimate pathway. In contrast during root flush production of glycerolipids involved in signalling cascades was repressed, suggesting that P. penetrans actively suppressed host defence. With the presence of the mycorrhizal symbiont, the gene expression pattern was vice versa with a distinctly stronger effect of P. penetrans at root flush, including attenuated defence, cell and carbon metabolism, likely a response to the enhanced carbon sink strength in roots induced by the presence of both, nematode and fungus. Meanwhile at shoot flush, when nutrients are retained in aboveground tissue, oak defence reactions, such as altered photosynthesis and sugar pathways, diminished.ConclusionsThe results highlight that gene response patterns of plants to biotic interactions, both negative (i.e. plant-parasitic nematodes) and beneficial (i.e. mycorrhiza), are largely modulated by endogenous rhythmic growth, and that such plant traits should be considered as an important driver of these relationships in future studies.