Page 158 - PC2019 Program & Proceedings

PLANT CANADA 2019

S101. Targeted mutation of multiple putative effectors in Fusarium graminearum utilizing
CRISPR/Cas9
Foster, A; R. Subramaniam
Agriculture and Agri-Food Canada

Fusarium graminearum, a causal agent of Fusarium head blight (FHB) in wheat, barley and oats, utilizes
secreted cysteine-rich effector proteins to modulate host immunity. One method to characterize fungal
effectors is gene disruption through homologous recombination, but this approach is limited as only one
gene or gene cluster can be targeted per transformation and many effector proteins are suspected of
having functional redundancy. CRISPR-Cas9 recently emerged as a powerful new tool for gene editing in
molecular biology. Among the different uses of the CRISPR/Cas9 system is the ability to target multiple
genes with a single transformation event. The main objective of this work was to develop a streamlined
method to disrupt multiple effectors. Expression vectors were constructed using a dual ribozyme
expression system for sgRNA with GFP as a marker and codon optimized Cas9. We targeted a group of
three putative effectors by protoplast transformation that were not clustered together in the genome with
five different sgRNAs. This approach successfully induced deletions from 200 bp to over 2000 bp in one
or more target genes in approximately 20% of transformed isolates. Additional transformations were also
performed to determine if the putative effectors are secreted during infection, by tagging genes with GFP.
Pathology tests with the CRISPR edited lines on wheat and potato are ongoing and results will be
discussed.

Adam Foster (adam.foster2@canada.ca)

S102. Development of Simplicillium lamellicola as a biocontrol agent against the wheat pathogen
Fusarium graminearum
Abaya, A.*; T. Hsiang
University of Guelph

The endophytic fungus Simplicillium lamellicola was isolated from tissues of moderately resistant
cultivars (winter wheat ‘AC Morley’ and ‘25R34’, Triticum aestivum). This isolate was assayed for the
ability to suppress disease caused by Fusarium graminearum under growth room and field conditions.
Agar inoculum of S. lamellicola applied 3 days before an agar plug of F. graminearum on wheat leaves
reduced disease by more than 80% in growth room tests. Spore suspensions of S. lamellicola which were
applied at anthesis of lab grown plants at three hours before the pathogen inoculation, reduced disease by
41-58% for susceptible cultivar ‘Wilkin’ and moderately resistant ‘Glenn’. In field tests, the biocontrol
agent reduced disease for three cultivars, susceptible ‘Wilkin’ (65%), moderately resistant ‘AAC Scotia’
(72%), and moderately resistant ‘Glenn’ (77%). The endophytic fungus S. lamellicola isolate AA2016
was effective against F. graminearum for reducing disease in lab and field tests and should be further
developed as a biological control agent.

Abdurraouf Abaya (abayaa@uoguelph.ca)

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