Gervais Pelletier

Regeneration of transgenic Picea glauca, P. mariana, and P. abies after cocultivation of embryogenic tissue with Agrobacterium tumefaciens

SummaryTransgenic plants of three Picea species were produced after coculture of embryogenic tissue with the disarmed strain of Agrobacterium tumefaciens C58/pMP90/pBIV10 and selection on medium containing kanamycin. In addition to the nptII selectable gene (conferring resistance to kanamycin), the vector carried the uidA (β-glucuronidase) marker gene. Transformation frequencies were dependent on the species, genotype, and post-cocultivation procedure. Of the three species tested, P. mariana was transformed at the highest frequency, followed by P. glauca and P. abies. The transgenic state of the embryogenic tissue was initially, confirmed by histochemical β-glucuronidase (GUS) assay followed by Southern hybridization. One to over five copies of T-DNA were detected in various transgenic lines analyzed. Transgenic plants were regenerated for all species using modified protocols for maturation and germination of somatic embryos.

Large-scale screening of transcription factor–promoter interactions in spruce reveals a transcriptional network involved in vascular development

This research aimed to investigate the role of diverse transcription factors (TFs) and to delineate gene regulatory networks directly in conifers at a relatively high-throughput level. The approach integrated sequence analyses, transcript profiling, and development of a conifer-specific activation assay. Transcript accumulation profiles of 102 TFs and potential target genes were clustered to identify groups of coordinately expressed genes. Several different patterns of transcript accumulation were observed by profiling in nine different organs and tissues: 27 genes were preferential to secondary xylem both in stems and roots, and other genes were preferential to phelloderm and periderm or were more ubiquitous. A robust system has been established as a screening approach to define which TFs have the ability to regulate a given promoter in planta. Trans-activation or repression effects were observed in 30% of TF–candidate gene promoter combinations. As a proof of concept, phylogenetic analysis and expression and trans-activation data were used to demonstrate that two spruce NAC-domain proteins most likely play key roles in secondary vascular growth as observed in other plant species. This study tested many TFs from diverse families in a conifer tree species, which broadens the knowledge of promoter–TF interactions in wood development and enables comparisons of gene regulatory networks found in angiosperms and gymnosperms.

Stress-Responsive Mitogen-Activated Protein Kinases Interact with the EAR Motif of a Poplar Zinc Finger Protein and Mediate Its Degradation through the 26S Proteasome

Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors.

Hormonally regulated overexpression of Arabidopsis WUS and conifer LEC1 ( CHAP3A ) in transgenic white spruce: implications for somatic embryo development and somatic seedling growth

Adult conifers are still recalcitrant in clonal propagation despite significant advances in forest tree biotechnology. Plant regeneration through somatic embryogenesis from explants older than mature zygotic embryos is either difficult or impossible to achieve. To investigate if ectopic expression of transcription factors involved in the induction of the embryogenic process would induce somatic embryogenesis in Picea glauca (white spruce) somatic plants, we used the LEAFY-COTYLEDON1 homolog cloned from Picea mariana, CHAP3A, and Arabidopsis thaliana WUS to transform embryonal mass of P. glauca. Ectopic gene expression was induced by 17-β-estradiol during stages of somatic embryogenesis (early embryogenesis and late embryogenesis) and somatic seedling growth in the transgenics. Of the two transcription factors, only WUS produced severe phenotypes by disrupting the development of somatic embryos on the maturation medium and inhibiting germination. However, none of the transgenes induced ectopic somatic embryogenesis even in the presence of plant growth regulators. Absolute quantitative PCR confirmed the expression of both CHAP3A and WUS in transgenic embryonal mass and in all parts of somatic seedlings. A high expression of the transgenes did not influence expression profiles of any of the ten other transcription factors tested, some of which have been known to be involved in the process of embryogenesis. Implications of these results for further work are discussed.

Impact of endochitinase-transformed white spruce on soil fungal biomass and ectendomycorrhizal symbiosis.

ABSTRACT The impact of transgenic white spruce [Picea glauca (Moench) Voss] containing the endochitinase gene (ech42) on soil fungal biomass and on the ectendomycorrhizal fungi Wilcoxina spp. was tested using a greenhouse trial. The measured level of endochitinase in roots of transgenic white spruce was up to 10 times higher than that in roots of nontransformed white spruce. The level of endochitinase in root exudates of three of four ech42-transformed lines was significantly greater than that in controls. Analysis soil ergosterol showed that the amount of fungal biomass in soil samples from control white spruce was slightly larger than that in soil samples from ech42-transformed white spruce. Nevertheless, the difference was not statistically significant. The rates of mycorrhizal colonization of transformed lines and controls were similar. Sequencing the internal transcribed spacer rRNA region revealed that the root tips were colonized by the ectendomycorrhizal fungi Wilcoxina spp. and the dark septate endophyte Phialocephala fortinii. Colonization of root tips by Wilcoxina spp. was monitored by real-time PCR to quantify the fungus present during the development of ectendomycorrhizal symbiosis in ech42-transformed and control lines. The numbers of Wilcoxina molecules in the transformed lines and the controls were not significantly different (P > 0.05, as determined by analysis of covariance), indicating that in spite of higher levels of endochitinase expression, mycorrhization was not inhibited. Our results indicate that the higher levels of chitinolytic activity in root exudates and root tissues from ech42-transformed lines did not alter the soil fungal biomass or the development of ectendomycorrhizal symbiosis involving Wilcoxina spp.

Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR

Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada.

Multiplex real-time polymerase chain reaction (PCR) for detection of Phytophthora ramorum, the causal agent of sudden oak death

Since 1995, Phytophthora ramorum has been causing sudden oak death in California and Oregon, affecting tens of thousands of oak trees over large areas and infecting more than 100 other plant species. Quarantine measures are in effect, and regulatory agencies need to detect this organism in a timely and efficient manner. Various molecular assays have been developed over the past few years, and redundancy using multiple gene regions of P. ramorum was shown to increase the reliability in detecting the pathogen. However, such multi-gene assays require different polymerase chain reactions (PCRs) to test a single sample. To improve P. ramorum detection, three different TaqMan assays were multiplexed with a fourth TaqMan specific to the Phytophthora genus in a single reaction. A second multiplex TaqMan PCR assay to detect oomycetes and give a positive PCR reaction in the presence of plant DNA was also designed and tested in conjunction with the P. ramorum internal transcribed spacer (ITS) and Phytophthora genus TaqMan assays. These assays were tested on different Phytophthora species and were verified on two different sets of field samples previously assayed by other laboratories. These were obtained from multiple field hosts infected by various Phytophthora species, and the DNA from one set was extracted from ELISA lysates. All known P. ramorum samples from pure cultures or field samples were detected using these multiplex real-time PCR assays. In general, TaqMan multiplex assays showed lower detection sensitivity than single separated reactions. However, the multiplex assays still detected P. ramorum accurately while decreasing the cost and increasing throughput.

Transcriptome Analysis of Poplar during Leaf Spot Infection with Sphaerulina spp.

Diseases of poplar caused by the native fungal pathogen Sphaerulina musiva and related species are of growing concern, particularly with the increasing interest in intensive poplar plantations to meet growing energy demands. Sphaerulina musiva is able to cause infection on leaves, resulting in defoliation and canker formation on stems. To gain a greater understanding of the different responses of poplar species to infection caused by the naturally co-evolved Sphaerulina species, RNA-seq was conducted on leaves of Populus deltoides, P. balsamifera and P. tremuloides infected with S. musiva, S. populicola and a new undescribed species, Ston1, respectively. The experiment was designed to contain the pathogen in a laboratory environment, while replicating disease development in commercial plantations. Following inoculation, trees were monitored for disease symptoms, pathogen growth and host responses. Genes involved in phenylpropanoid, terpenoid and flavonoid biosynthesis were generally upregulated in P. balsamifera and P. tremuloides, while cell wall modification appears to play an important role in the defense of P. deltoides. Poplar defensive genes were expressed early in P. balsamifera and P. tremuloides, but their expression was delayed in P. deltoides, which correlated with the rate of disease symptoms development. Also, severe infection in P. balsamifera led to leaf abscission. This data gives an insight into the large differences in timing and expression of genes between poplar species being attacked by their associated Sphaerulina pathogen.

The Poplar Rust-Induced Secreted Protein (RISP) Inhibits the Growth of the Leaf Rust Pathogen Melampsora larici-populina and Triggers Cell Culture Alkalinisation

Plant cells secrete a wide range of proteins in extracellular spaces in response to pathogen attack. The poplar rust-induced secreted protein (RISP) is a small cationic protein of unknown function that was identified as the most induced gene in poplar leaves during immune responses to the leaf rust pathogen Melampsora larici-populina, an obligate biotrophic parasite. Here, we combined in planta and in vitro molecular biology approaches to tackle the function of RISP. Using a RISP-mCherry fusion transiently expressed in Nicotiana benthamiana leaves, we demonstrated that RISP is secreted into the apoplast. A recombinant RISP specifically binds to M. larici-populina urediniospores and inhibits their germination. It also arrests the growth of the fungus in vitro and on poplar leaves. Interestingly, RISP also triggers poplar cell culture alkalinisation and is cleaved at the C-terminus by a plant-encoded mechanism. Altogether our results indicate that RISP is an antifungal protein that has the ability to trigger cellular responses.