Genetic and chemical suppressors of mlo-mediated powdery mildew resistance

Recessively inherited mutant alleles of Mlo genes (mlo) confer broad-spectrum penetration resistance to powdery mildew pathogens in plants. Genetic analysis has identified several molecular components required for mlo-mediated resistance, such as the Ror (Required for mlo-specified resistance) genes in barley, and PEN (PENETRATION) genes in Arabidopsis. However, the exact molecular mechanism underlying mlo-mediated resistance is still a mystery. Here I utilized three complementary approaches, a forward genetic screen, reverse genetic analysis and a survey for chemical inhibitors, to identify novel suppressors of mlo resistance in barley and Arabidopsis. In the forward genetics approach, ~ 17,000 Arabidopsis plants from an ethyl methanesulfonate (EMS) mutagenized mlo2 mlo6 mutant population (generated by Sandra Noir) were screened and eight candidate suppressor mutants were identified to exhibit increased susceptibility against Golovinomyces orontii. Bulked segregant analysis in combination with a next generation sequencing (NGS)-based mapping approach detected two promising candidate mutations in two genes (CYP81F2 and AT1G03370) for two of the suppressor lines. Sequence information revealed that both candidate mutations result in amino acid changes at the protein level. In the reverse genetics approach, eight genes that are co-expressed with Arabidopsis MLO2 were investigated. Two mutants, crk19-1 (cysteine-rich receptor-like protein kinase 19) and sobir1-1 (suppressor of bir1 1), were identified to be able to suppress mlo-mediated resistance in Arabidopsis. Regarding the chemical screen, forty-one compounds were selected for the initial survey. Five chemicals were subsequently validated to be effective in suppressing mlo-mediated resistance in barley and in part in Arabidopsis. Comprehensive analysis with structural and functional analogs revealed a key role for endomembrane trafficking in mlo-mediated resistance and uncovered ribonucleosides and its derivatives as well as various polyamines as suppressors of mlo-mediated resistance. Another compound, alloxan, was identified not only to be able suppress mlo-mediated resistance in barley and Arabidopsis, but also to compromise immunity of Arabidopsis against a non-adapted powdery mildew pathogen. Histological analysis revealed that alloxan application impedes callose deposition at attempted fungal penetration sites. Further investigation indicated that alloxan interferes with the motility of cellular organelles (e.g. peroxisomes, endosomes and Endoplasmic Reticulum) and the pathogen-induced focal accumulation of a defense marker protein (PEN1) at the plasma membrane. Accumulating evidence suggests that alloxan exerts its inhibitory function through directly or indirectly disrupting the structure of actin filaments. The newly identified genetic and chemical suppressors of mlo-mediated resistance promise to deepen the mechanistic understanding of this important type of plant immunity