Igor Yakovlev

Insight into trade???off between wood decay and parasitism from the genome of a fungal forest pathogen

Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.

MicroRNAs, the epigenetic memory and climatic adaptation in Norway spruce

*Norway spruce expresses a temperature-dependent epigenetic memory from the time of embryo development, which thereafter influences the timing bud phenology. MicroRNAs (miRNAs)are endogenous small RNAs, exerting epigenetic gene regulatory impacts. We have tested for their presence and differential expression. *We prepared concatemerized small RNA libraries from seedlings of two full-sib families, originated from seeds developed in a cold and warm environment. One family expressed distinct epigenetic effects while the other not. We used available plant miRNA query sequences to search for conserved miRNAs and from the sequencing we found novel ones; the miRNAs were monitored using relative real time-PCR. *Sequencing identified 24 novel and four conserved miRNAs. Further screening of the conserved miRNAs confirmed the presence of 16 additional miRNAs. Most of the miRNAs were targeted to unknown genes. The expression of seven conserved and nine novel miRNAs showed significant differences in transcript levels in the full-sib family showing distinct epigenetic difference in bud set, but not in the nonresponding full-sib family. Putative miRNA targets were studied. *Norway spruce contains a set of conserved miRNAs as well as a large proportion of novel nonconserved miRNAs. The differentially expression of specific miRNAs indicate their putative participation in the epigenetic regulation.

Dehydrins expression related to timing of bud burst in Norway spruce

Cold deacclimation and preparation to flushing likely requires rehydration of meristems. Therefore, water stress related genes, such as dehydrins (DHN), might play an important role in providing protection during winter dormancy, deacclimation and bud burst timing processes. Here we report the sequence analysis of several Norway spruce DHN identified in late and early flushing suppressive subtraction hybridization cDNA libraries and in our Norway spruce EST database. We obtained 15 cDNAs, representing eight genes from three distinct types of DHN, and studied differential expression of these genes before and during bud burst in spring, using qRT-PCR. We found the visible reduction in transcript level of most DHN towards the bud burst, supported by a significant down-regulation of the DHN in needles during experimental induction of bud burst applied at three time points during autumn in Norway spruce grafts. For most of the DHN transcripts, their expression levels in late-flushing spruces were significantly higher than in the early flushing ones at the same calendar dates but were remarkably similar at the same bud developmental stage. From our results we may conclude that the difference between the early and the late families is in timing of the molecular processes leading to bud burst due to differences in their response to the increasing temperature in the spring. They are induced much earlier in the early flushing families.

Differential gene expression related to an epigenetic memory affecting climatic adaptation in Norway spruce

In Norway spruce, the temperature during zygotic embryogenesis appears to adjust an adaptive epigenetic memory in the progeny that may regulate bud phenology and cold acclimation. Conditions colder than normal advance the timing whilst temperatures above normal delay the onset of these processes and altered performance is long lasting in progeny with identical genetic background. As a step toward unraveling the molecular mechanism behind an epigenetic memory, transcriptional analysis was performed on seedlings from seeds of six full-sib families produced at different embryogenesis temperature-cold (CE) vs warm (WE) under long and short day conditions. We prepared two suppressive subtracted cDNA libraries, forward and reverse, representing genes predominantly expressed in plants from seeds obtained after CE and WE embryogenesis following short day treatment (inducing bud set). Sequencing and annotation revealed considerable differences in the transcriptome of WE versus CE originated plants. By using qRT-PCR we studied the expression patterns of 32 selected candidate genes chosen from subtractive cDNA libraries analysis and nine siRNA pathways genes by a direct candidate approach. Eight genes, two transposons related genes, three with no match to Databases sequences and three genes from siRNA pathways (PaDCL1 and 2, PaSGS3) showed differential expression in progeny from CE and WE correlated with the family phenotypic differences. These findings may contribute to our understanding of the epigenetic mechanisms underlying adaptive changes acquired during embryogenesis.

An adaptive epigenetic memory in conifers with important implications for seed production

Conifers are evolutionarily more ancient than their angiosperm counterparts, and thus some adaptive mechanisms and features influenced by epigenetic mechanisms appear more highly displayed in these woody gymnosperms. Conifers such as Norway spruce have very long generation times and long life spans, as well as large genome sizes. This seemingly excessive amount of genomic DNA without apparent duplications could be a rich source of sites for epigenetic regulation and modifications. In Norway spruce, an important adaptive mechanism has been identified, called epigenetic memory. This affects the growth cycle of these trees living in environments with mild summers and cold winters, allowing them to adapt rapidly to new and/or changing environments. The temperature during post-meiotic megagametogenesis and seed maturation epigenetically shifts the growth cycle programme of the embryos. This results in significant and long-lasting phenotypic change in the progeny, such as advance or delay of vital phenological processes of high adaptive value, like bud break and bud set. This phenomenon is not only of important evolutionary significance but has clear practical implications for forest seed production and conservation of forest genetic resources. The underlying molecular mechanism that causes the ‘memory’ in long-lived woody species is currently under investigation. Here we summarize the information related to epigenetic memory regulation in gymnosperms, with special emphasis on conifers. The molecular mechanism behind this is still unknown but transcriptional changes are clearly involved. Epigenetic regulation may be realized through several mechanisms, including DNA methylation, histone modification, chromatin remodelling, small non-coding RNAs and transposable element regulation, of which non-coding RNAs might be one of the most important determinants.

Substrate-specific transcription of the enigmatic GH61 family of the pathogenic white-rot fungus Heterobasidion irregulare during growth on lignocellulose

The GH61 represents the most enigmatic Glycoside Hydrolase family (GH) regarding enzymatic activity and importance in cellulose degradation. Heterobasidion irregulare is a necrotizing pathogen and white-rot fungus that causes enormous damages in conifer forests. The genome of H. irregulare allowed identification of ten HiGH61 genes. qRT-PCR analysis separate the HiGH61 members into two groups; one that show up regulation on lignocellulosic substrates (HiGH61A, HiGH61B, HiGH61D, HiGH61G, HiGH61H, and HiGH61I) and a second showing either down-regulation or constitutive expression (HiGH61C, HiGH61E, HiGH61F, and HiGH61J). HiGH61H showed up to 17,000-fold increase on spruce heartwood suggesting a pivotal role in cellulose decomposition during saprotrophic growth. Sequence analysis of these genes reveals that all GH61s except HiGH61G possess the conserved metal-binding motif essential for activity. The sequences also divide into groups having either an insert near the N terminus or an insert near the second catalytic histidine, which may represent extensions of the substrate-binding surface. Three of the HiGH61s encode cellulose-binding modules (CBM1). Interestingly, HiGH61H and HiGH61I having CBM1s are up-regulated on pure cellulose. There was a common substrate-specific induction patterns of the HiGH61s with several reference cellulolytic and hemicellulolytic GHs, this taken together with their low transcript levels on media lacking lignocellulose, reflect the concerted nature of cell wall polymer degradation.

Dehydrins in maritime pine (Pinus pinaster) and their expression related to drought stress response

Maritime pine (Pinus pinaster) is an important commercial species throughout its Atlantic distribution. With the anticipated increase in desiccation of its habitat as a result of climate change, the selection of genotypes with increased survival and growth capability under these conditions for breeding programs is of great interest for this species. We aimed to study the response to a realistic drought stress under controlled conditions, looked for a method to measure dehydration resistance, and analyzed dehydrin expression in drought-resistant and drought-sensitive clones from different ecotypes. We report here the sequence characteristics and the expression patterns of five dehydrins from P. pinaster, along with the physiological characterization of drought stress responses in different genotypes (clonally replicated plants), originating from a broad geographical distribution across France and Spain (provenances). In total, we distinguished five different dehydrin genes in silico, grouped into two types—K2 and SKn. Three of the dehydrin genes had several sequence variants, differing by multiple or single amino acid substitutions. Only two of the dehydrins (PpinDhn3 and PpinDhn4) showed an increase in transcription with increased drought stress which was dependent on provenance and genotype, suggesting their involvement in drought resistance. The other dehydrins showed decreased expression trends with increased severity of the drought stress. The lack of close association between the drought stress and expression patterns of these dehydrin genes suggest that they could have other functions and not be involved in drought resistance. Our results suggest large differences in function between different dehydrin genes.

Genes associated with lignin degradation in the polyphagous white-rot pathogen Heterobasidion irregulare show substrate-specific regulation.

The pathogenic white-rot basidiomycete Heterobasidion irregulare is able to remove lignin and hemicellulose prior to cellulose during the colonization of root and stem xylem of conifer and broadleaf trees. We identified and followed the regulation of expression of genes belonging to families encoding ligninolytic enzymes. In comparison with typical white-rot fungi, the H. irregulare genome has exclusively the short-manganese peroxidase type encoding genes (6 short-MnPs) and thereby a slight contraction in the pool of class II heme-containing peroxidases, but an expansion of the MCO laccases with 17 gene models. Furthermore, the genome shows a versatile set of other oxidoreductase genes putatively involved in lignin oxidation and conversion, including 5 glyoxal oxidases, 19 quinone-oxidoreductases and 12 aryl-alcohol oxidases. Their genetic multiplicity and gene-specific regulation patterns on cultures based on defined lignin, cellulose or Norway spruce lignocellulose substrates suggest divergent specificities and physiological roles for these enzymes. While the short-MnP encoding genes showed similar transcript levels upon fungal growth on heartwood and reaction zone (RZ), a xylem defense tissue rich in phenolic compounds unique to trees, a subset of laccases showed higher gene expression in the RZ cultures. In contrast, other oxidoreductases depending on initial MnP activity showed generally lower transcript levels on RZ than on heartwood. These data suggest that the rate of fungal oxidative conversion of xylem lignin differs between spruce RZ and heartwood. It is conceivable that in RZ part of the oxidoreductase activities of laccases are related to the detoxification of phenolic compounds involved in host-defense. Expression of the several short-MnP enzymes indicated an important role for these enzymes in effective delignification of wood by H. irregulare.

Gene expression changes during short day induced terminal bud formation in Norway spruce

The molecular basis for terminal bud formation in autumn is not well understood in conifers. By combining suppression subtractive hybridization and monitoring of gene expression by qRT-PCR analysis, we aimed to identify genes involved in photoperiodic control of growth cessation and bud set in Norway spruce. Close to 1400 ESTs were generated and their functional distribution differed between short day (SD-12 h photoperiod) and long day (LD-24 h photoperiod) libraries. Many genes with putative roles in protection against stress appeared differentially regulated under SD and LD, and also differed in transcript levels between 6 and 20 SDs. Of these, PaTFL1(TERMINAL FLOWER LIKE 1) showed strongly increased transcript levels at 6 SDs. PaCCCH(CCCH-TYPE ZINC FINGER) and PaCBF2&3(C-REPEAT BINDING FACTOR 2&3) showed a later response at 20 SDs, with increased and decreased transcript levels, respectively. For rhythmically expressed genes such as CBFs, such differences might represent a phase shift in peak expression, but might also suggest a putative role in response to SD. Multivariate analyses revealed strong differences in gene expression between LD, 6 SD and 20 SD. The robustness of the gene expression patterns was verified in 6 families differing in bud-set timing under natural light with gradually decreasing photoperiod.

EARLY BUD-BREAK1 (EBB1) defines a conserved mechanism for control of bud-break in woody perennials

Bud-break is an environmentally and economically important trait in trees, shrubs and vines from temperate latitudes. Poor synchronization of bud-break timing with local climates can lead to frost injuries, susceptibility to pests and pathogens and poor crop yields in fruit trees and vines. The rapid climate changes outpace the adaptive capacities of plants to respond through natural selection. This is particularly true for trees which have long generation cycle and thus the adaptive changes are significantly delayed. Therefore, to devise appropriate breeding and conservation strategies, it is imperative to understand the molecular underpinnings that govern dormancy mechanisms. We have recently identified and characterized the poplar EARLY BUD-BREAK 1 (EBB1) gene. EBB1 is a positive regulator of bud-break and encodes a transcription factor from the AP2/ERF family. Here, using comparative and functional genomics approaches we show that EBB1 function in regulation of bud-break is likely conserved across wide range of woody perennial species with importance to forestry and agriculture.