Validation and characterization of Pyrenophora teres f. teres effector genes VR1 and VR2 conferring virulence on Rika barley

Authors: LI, JINLING - North Dakota State University; Wyatt, Nathan; NELSON, ASHLEY - North Dakota State University; BRUEGGEMAN, ROBERT - Washington State University; Friesen, Timothy

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/5/2023
Publication Date: 3/5/2023
Citation: Li, J., Wyatt, N.A., Nelson, A., Brueggeman, R., Friesen, T.L. 2023. Validation and characterization of Pyrenophora teres f. teres effector genes VR1 and VR2 conferring virulence on Rika barley. Meeting Abstract.

Interpretive Summary:

Technical Abstract: The fungal pathogen Pyrenophora teres f. teres causes net form net blotch of barley. Previously, a population developed from a cross of P. teres f. teres isolates 15A and 6A was used to identify two loci (VR1 and VR2), conferring virulence on Rika barley. Here, we identified candidate genes for VR1 and VR2, giving priority to genes encoding small, secreted proteins. Five and three genes at the VR1 and VR2 loci, respectively, were identified as the top candidates. CRISPR-Cas9-based gene disruption was used to knockout each of the genes. Disruption of a predicted carboxypeptidase at the VR1 locus and disruption of a gene encoding a hypothetical small, secreted protein at the VR2 locus changed virulent isolates to avirulent. Subsequently, we conducted CRISPR-Cas9-based gene editing (allele swaps) in an avirulent isolate, and the VR1 andVR2 edited strains became virulent on Rika. Collectively, these results validated VR1 and VR2, the genes conferring virulence on Rika barley. VR1 encodes a relatively large secreted effector (64.4 'kDa) with a serine carboxypeptidase protease domain and homologous proteins were found in various plant pathogens. In contrast, VR2 (47.6 kDa) lacks any predicted functional domains and homologues only appear in the Pyrenophora genus. Inoculation of the VR1 and VR2 edited strains onto the Rika × Kombar host mapping population showed that both VR1 and VR2 were targeting the same 6H susceptibility locus in Rika barley, however, it is not clear if these proteins are targeting the same host gene or if there are multiple host genes being targeted at the same locus. VR1 expression gradually increased from 4 hours post inoculation (hpi) until peaking at 36 hpi, while VR2 showed maximum expression at 4'hpi and rapidly decreased with the progression of the disease, indicating that VR1 and VR2 may have different functions during infection.