Canadian Forest Service Publications
Agrobacterium tumefaciens-mediated transformation for targeted disruption and over expression of genes in the poplar pathogen Sphaerulina musiva. 2014. Foster, A.J.; Morency, M.-J.; Séguin, A.; Tanguay, P. For. Pathol. 44:233-241.
Issued by: Laurentian Forestry Centre
Catalog ID: 35555
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Sphaerulina musiva causes both leaf spots and cankers on poplar. Leaf spots can lead to defoliation and cankers on branches and primary stems can lead to stem breakage and tree mortality. The recent availability of both the S. musiva and Populus trichocarpa genomes offers a great opportunity to study host–pathogen interactions. To better understand the factors involved in S. musiva pathology, we present a strategy for the transformation of this species using Agrobacterium tumefaciens. Binary plasmids were generated with hygromycin B phosphotransferase (hph) flanked by upstream and downstream sequences of polyketide synthase-like (PKS-L1) gene to generate targeted gene disruptants by homologous recombination. Plasmids were also constructed for constitutive expression reporter genes eGFP and mCherry to help with histological characterization of the pathogen during infection. Gene knockouts were identified by PCR and confirmed by sequencing and Southern blotting. No significant differences were observed in melanin production between PKS-L1 disruptants and wild type isolates. Colonies expressing reporter genes were identified by fluorescent stereomicroscopy. This method is a promising tool for the characterization of pathogen genes through reverse and forward genetics and for introducing markers for histopathological study.
Plain Language Summary
The Sphaerulina musiva fungus causes leaf spots and cankers on the twigs and trunks of poplars. Leaf spots can lead to defoliation while cankers can cause branch breakage and, ultimately, lead to tree mortality.
Recently acquired knowledge on the genomes of the S. musiva fungus and black cottonwood has given researchers a better understanding of the genetic processes involved between this fungus and its host. Using these processes as a basis, researchers have developed genetic tools to validate the results obtained from genomic analysis. They can thus study in fine detail the attack mechanisms of the pathogen and the methods of resistance employed by its host tree. The methods developed in this study will contribute to the development of molecular detection tools.