An Efficient Strategy for Obtaining Mutants by Targeted Gene Deletion in Ophiostoma novo-ulmi

Abstract

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.

Comparative Analysis of Transcriptomes of Ophiostoma novo-ulmi ssp. americana Colonizing Resistant or Sensitive Genotypes of American Elm

“…Since genetic transformation systems for O. novo-ulmi were developed [ 105 ], the contribution of 13 genes to pathogenicity (including eight genes analyzed in this work) has been investigated by producing and phenotyping knockdown- and knockout mutants [ 21 , 22 , 30 ]. Genes OnuG6779 ( ade7 ) and OnuG7434 ( ade1 ), which code for two enzymes involved in the adenine biosynthesis pathway, were shown to be required for pathogenicity [ 30 ], thereby confirming previous reports of altered pathogenicity phenotype in adenine auxotrophs from several plant pathogenic fungi (e.g., [ 106 ]). Mutants for the other candidate genes were all highly virulent.…”

“…We also unveiled networks of fungal genes that were co-expressed during the infection of S elms. Targeted knockdown or deletion mutants obtained for the eight genes we assessed initially did not show significant changes in virulence but we expect that the combination of transcriptomic data and tools for efficient production of mutants [ 30 ] will facilitate the formal identification of genes controlling pathogenicity and virulence in O. novo-ulmi . In turn, this may help breeders identify elm genes encoding molecules and receptors that interact with fungal factors in order to select and breed American elms that are more resistant to DED.…”

“…Knockout mutants for genes aox1 (OnuG5955), opf2 (OnuG1642) and bct2 (OnuG2340) were produced using Agrobacterium tumefaciens -mediated transformation (ATMT) of an O. novo-ulmi ∆ mus52 strain deficient in non-homologous end joining (NHEJ), followed by counterselection of ectopic integration of the T-DNA using binary vector pOSCAR-HSVtk [ 30 , 47 ]. Deletion plasmids were prepared as described previously [ 30 , 48 ]. Primers designed to amplify the region flanking the ORF of the target genes contained one of four different att B recombination sites in their 5′ extension ( Table S3 ).…”

“…In addition to mutants for candidate pathogenicity genes, treatments included WT strain H327, deletion mutant Δmus52 (OnuG0757), and a negative control consisting in sterile distilled water. Each treatment was applied to 10 individual saplings in a randomized block design, using a procedure described previously [ 30 ]. Defoliation was recorded for each sapling two and three weeks after inoculation and the mean defoliation was calculated for each treatment.…”

“…The impact of this obstacle was lowered by using RNA interference [ 27 ] to produce knockdown mutants containing a carbon source regulated promoter to drive cassette expression and downregulate specific genes or gene families identified through sequence analysis [ 28 , 29 ]. More recently, targeted knockout mutants were recovered at high frequency after subjecting a Δ mus52 derivative of strain H327 defective for non-homologous end-joining of DNA (NHEJ) to a dual selection system in OSCAR plasmids [ 30 ]. The next development in this area will likely be the development of protocols based on CRISPR/Cas (Clustered regularly interspersed short palindromic repeats/CRISPR associated; [ 31 ]) for precise genome editing in O. novo-ulmi [ 32 ].…”