Document Type

Dissertation - Open Access

Award Date

2026

Degree Name

Doctor of Philosophy (PhD)

Department / School

Agronomy, Horticulture, and Plant Science

First Advisor

Gazala Ameen

Abstract

Spot blotch, caused by the fungal pathogen Bipolaris sorokiniana, causes yield losses of 30-40% in barley (Hordeum vulgare L.) globally. The rcs5 locus on the 7H chromosome of barley is the major locus and provides a recessive resistance against spot blotch disease. The rcs5 locus harbors two dominant wallassociated kinase (WAK) susceptibility genes, Sbs1 and Sbs2, within a ~234 kb genomic interval, whose pathogen-induced expression facilitates B. sorokiniana colonization during the infection. This dissertation expands our current knowledge of the mechanism of WAK-mediated immunity in plants. The project outcomes are via subcellular localization of fluorescently tagged Sbs1 and Sbs2 fusion proteins transiently expressed in Nicotiana benthamiana, confirming exclusive plasma membrane localization for both proteins, unambiguously validated by plasmolysis assays in which both signals remained associated with retracted protoplasts and Hechtian strands. Bimolecular fluorescence complementation (BiFC) in both reciprocal fluorescent-fragment orientations demonstrated direct physical interaction of Sbs1 and Sbs2 at the plasma membrane in planta, establishing that Sbs1 and Sbs2 form a heterodimeric WAK co-receptor complex, the first such interaction demonstrated between two WAK susceptibility proteins in a cereal pathosystem. Allele analysis of 142 barley cultivars and 25–28 pangenomes revealed three independently truncated Sbs1 allele classes in resistant cultivars, all lacking an intact kinase domain, demonstrating convergent loss-of-susceptibility across diverse germplasm. The Sbs2 allele produced a premature stop codon at positions 9–33 in 20 of 28 pangenomes, while all accessions encoded structurally intact full-length HvWak5 proteins. Sbs1 allele type was the principal determinant of disease outcome, accounting for 39% of phenotypic variance, with Sbs2 contributing a significant but secondary effect detectable only in the non-functional Sbs1 background; HvWak5 had no independent effect on disease severity. To characterize the genomic landscape of resistance beyond rcs5, we conducted a meta-analysis of 68 markers across 21 barley pangenome cultivars. Cumulative susceptibility allele count showed a significant positive relationship with disease severity, confirming additive genetic effects across validated loci. These findings establish Sbs1 and Sbs2 as plasma membrane co-receptors mediating WAK-based susceptibility in barley, define the molecular basis of resistance allele diversity at rcs5, and provide a genomic framework for spot blotch resistance breeding worldwide.

Publisher

South Dakota State University

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Available for download on Monday, May 15, 2028

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Rights Statement

In Copyright