Project description:Five years old Ulmus minor plants from three different genotypes, two tolerant and one sensitive to Dutch Elm disease, were inoculated with an aggressive local strain of Ophiostoma novo-ulmi (Z-BU1) while the other half were inoculated with sterile and distilled water as control treatment. following the procedure described by Solla et al. (2005). A healthy 3-year-old branch located around 2 meters tall, from both inoculated and control plants, were collected at 1, 3, 7, 14 and 21 days after inoculation. The stem from the branch was individualized and RNA isolated to hybridize two colors microarrays.
Project description:6-year-old ramets of U. minor (Atinian elm clone) were inoculated with O. novo-ulmi in order to evaluate molecular responses activated during plant colonization. To elucidate the different genes involved in the U. minor immune system and the molecular changes suffered after inoculation, oligomicroarrays were constructed using the data from the transcriptome available in the Dryad database (Perdiguero et al., 2015; http://dx.doi.org/10.5061/dryad.ps837), and hybridized with cDNA obtained from the ramets over a time course following inoculation. Three biological replicates for control and inoculated plants for each sampling point (1, 3, 7, 14 and 21 days post-inoculation) were hybridised using two colors (inoculated vs control).
Project description:The emergence of new microbial pathogens can result in destructive outbreaks, since their hosts have limited resistance and pathogens may be excessively aggressive. Described as the major ecological incident of the twentieth century, Dutch elm disease, caused by ascomycete fungi from the Ophiostoma genus, has caused a significant decline in elm tree populations (Ulmus sp.) in North America and Europe. Genome sequencing of the two main causative agents of Dutch elm disease (Ophiostoma ulmi and Ophiostoma novo-ulmi), along with closely related species with different lifestyles, allows for unique comparisons to be made to identify how pathogens and virulence determinants have emerged. Among several established virulence determinants, secondary metabolites (SMs) have been suggested to play significant roles during phytopathogen infection. Interestingly, the secondary metabolism of Dutch elm pathogens remains almost unexplored, and little is known about how SM biosynthetic genes are organized in these species. To better understand the metabolic potential of O. ulmi and O. novo-ulmi, we performed a deep survey and description of SM biosynthetic gene clusters (BGCs) in these species and assessed their conservation among eight species from the Ophiostomataceae family. Among 19 identified BGCs, a fujikurin-like gene cluster (OpPKS8) was unique to Dutch elm pathogens. Phylogenetic analysis revealed that orthologs for this gene cluster are widespread among phytopathogens and plant-associated fungi, suggesting that OpPKS8 may have been horizontally acquired by the Ophiostoma genus. Moreover, the detailed identification of several BGCs paves the way for future in-depth research and supports the potential impact of secondary metabolism on Ophiostoma genus' lifestyle.
Project description:Some fungal endophytes of forest trees are recognized as beneficial symbionts against stresses. In previous works, two elm endophytes from the classes Cystobasidiomycetes and Eurotiomycetes promoted host resistance to abiotic stress, and another elm endophyte from Dothideomycetes enhanced host resistance to Dutch elm disease (DED). Here, we hypothesize that the combined effect of these endophytes activate the plant immune and/or antioxidant system, leading to a defense priming and/or increased oxidative protection when exposed to the DED pathogen Ophiostoma novo-ulmi. To test this hypothesis, the short-term defense gene activation and antioxidant response were evaluated in DED-susceptible (MDV1) and DED-resistant (VAD2 and MDV2.3) Ulmus minor genotypes inoculated with O. novo-ulmi, as well as two weeks earlier with a mixture of the above-mentioned endophytes. Endophyte inoculation induced a generalized transient defense activation mediated primarily by salicylic acid (SA). Subsequent pathogen inoculation resulted in a primed defense response of variable intensity among genotypes. Genotypes MDV1 and VAD2 displayed a defense priming driven by SA, jasmonic acid (JA), and ethylene (ET), causing a reduced pathogen spread in MDV1. Meanwhile, the genotype MDV2.3 showed lower defense priming but a stronger and earlier antioxidant response. The defense priming stimulated by elm fungal endophytes broadens our current knowledge of the ecological functions of endophytic fungi in forest trees and opens new prospects for their use in the biocontrol of plant diseases.
Project description:The dimorphic fungus Ophiostoma novo-ulmi is the highly aggressive pathogen responsible for the current, highly destructive, pandemic of Dutch elm disease (DED). Genome and transcriptome analyses of this pathogen previously revealed that a large set of genes expressed during dimorphic transition were also potentially related to plant infection processes, which seem to be regulated by molecular mechanisms different from those described in other dimorphic pathogens. Then, O. novo-ulmi can be used as a representative species to study the lifestyle of dimorphic pathogenic fungi that are not shared by the "model species" Candida albicans and Ustilago maydis. In order to gain better knowledge of molecular aspects underlying infection process and symptom induction by dimorphic fungi that cause vascular wilt disease, we developed a high-throughput gene deletion protocol for O. novo-ulmi. The protocol is based on transforming a Δmus52 O. novo-ulmi mutant impaired for non-homologous end joining (NHEJ) as the recipient strain, and transforming this strain with the latest version of OSCAR plasmids. The latter are used for generating deletion constructs containing the toxin-coding Herpes simplex virus thymidine kinase (HSVtk) gene which prevents ectopic integration of the T-DNA in Ophiostoma DNA. The frequency of gene deletion by homologous recombination (HR) at the ade1 locus associated with purine nucleotide biosynthesis was up to 77.8% in the Δmus52 mutant compared to 2% in the wild-type (WT). To validate the high efficiency of our deletion gene methodology we deleted ade7, which also belongs to the purine nucleotide pathway, as well as bct2, ogf1, and opf2 which encode fungal binuclear transcription factors (TFs). The frequency of gene replacement by HR for these genes reached up to 94%. We expect that our methodology combining the use of NHEJ deficient strains and OSCAR plasmids will function with similar high efficiencies for other O. novo-ulmi genes and other filamentous fungi.