Genomic and Molecular Characterization of Pyrenphora teres f. teres

Abstract

Pyrenophora teres f. teres is the causal agent of net form net blotch of barley. P. teres f. teres is prevalent globally across all barley growing regions and globally is the most devastating foliar disease of barley. Though economically important, the molecular mechanism whereby P. teres f. teres causes disease is poorly understood and investigations into these mechanisms have been hindered by a lack of genomic resources. To set a genomic foundation for P. teres f. teres the reference isolate 0-1 was sequenced and assembled using PacBio single molecule real-time (SMRT) sequencing and scaffolded into 12 chromosomes to provide the first finished genome of P. teres f. teres. High confidence gene models were generated for the reference genome of isolate 0-1 using a combination of pure culture and in planta RNA sequencing. An additional four P. teres f. teres isolates were sequenced and assembled to the same quality as the reference isolate 0-1 and used in a comparative genomic study. Comparisons of the five P. teres f. teres isolates showed a two-speed genome architecture with the genome being partitioned into core and accessory genomic compartments. Accessory genomic compartments clustered in sub-telomeric regions of the P. teres f. teres genome with a majority of previously identified quantitative trait loci (QTL) associated with avirulence/virulence being spanned by these accessory regions. Using these genomic resources, with a bi-parental mapping population and a natural population for QTL analysis and genome wide association study (GWAS), respectively, we identified a candidate gene for the previously mapped AvrHar. QTL analysis identified a locus extending off the end of P. teres f. teres chromosome 5 and GWAS analysis identified significant associations with a gene encoding a small secreted protein. The candidate AvrHar gene was validated using CRISPR-Cas9-RNP gene disruption in parental isolates 15A and 0-1. Disruption of AvrHar in isolate 15A did not result in a phenotypic change while disruption of the 0-1 allele resulted in a complete loss of pathogenicity. This is the first identification of an effector from P. teres f. teres validated using CRISPR-Cas9-RNP gene editing.