SGT1-Specific Domain Mutations Impair Interactions with the Barley MLA6 Immune Receptor in Association with Loss of NLR Protein

Abstract

The Mla (Mildew resistance locus a) of barley (Hordeum vulgare L.) is an effective model for cereal immunity against fungal pathogens. Like many resistance proteins, variants of the MLA coiled-coil nucleotide-binding leucine-rich-repeat (CC-NLR) receptor require the HRS complex to function, which includes HSP90 (Heat Shock Protein 90), RAR1 (Required for Mla12 Resistance 1), and SGT1 (Suppressor of G-two allele of Skp1). However, functional analysis of Sgt1 has been particularly difficult as deletions are often lethal. Recently, we identified rar3 (Required for Mla6 resistance 3), an in-frame Sgt1ΔKL308-309 mutation in the SGS domain that alters resistance conferred by MLA, but without lethality. Here we use autoactive MLA6 and heterologous yeast-two-hybrid strains with stably integrated HvRar1 and HvHsp90, to determine that this mutation weakens, but doesn’t entirely disrupt, the interaction between SGT1 and MLA. This causes a concomitant reduction in MLA6 protein accumulation below the apparent threshold required for effective resistance. The ΔKL308-309 deletion had a lesser effect on intramolecular interactions than alanine or arginine substitutions, and MLA variants that display diminished interactions with SGT1 appear to be disproportionately affected by the SGT1ΔKL308-309 mutation. We hypothesize that those dimeric plant CC-NLRs that appear unaffected by Sgt1 silencing are those with the strongest intermolecular interactions with it. Combining our data with recent work in CC-NLRs, we propose a cyclical model of the MLA-HRS resistosome interactions.

Single‐cell transcriptomic analyses reveal cellular and molecular patterns of rubber tree response to early powdery mildew infection

“…Several chaperone proteins are required for NLR folding and accumulation, including HSP90 and SGT1 (Chapman et al, 2022; Shirasu, 2009). We've successfully identified the HbHSP90 and HbSGT1 gene families in rubber trees and revealed the participation of powdery mildew resistance‐related genes, HbHSP90.1 and HbSGT1b (Liu et al, 2022; Wang et al, 2023).…”

Transcriptome-based host epistasis and pathogen co-expression in barley-powdery mildew interactions

“…Lastly, Rar3 is required for Mla6 -mediated hypersensitive response and generation of H 2 O 2 . The susceptible rar3- m11526 (Mla6, Bln1, Sgt1 ΔKL308-309 ) mutant contains an in-frame Lys-Leu deletion in the SGS domain of the co-chaperone SGT1, which interacts physically with MLA (Shen et al 2003; Chapman et al 2021; Chapman et al 2022).…”

“…However, we do know that Mla6 requires Sgt1 to function properly, i.e. , express resistance to AVR a6 containing Bh isolates (Chapman et al 2021; 2022). For this second model ( Mla6, Sgt1 ), we could not separate additive from epistatic effects, but we could compare the strength of the contribution of each gene to the wild-type genotype.…”

“…To facilitate their function, host NLR immune receptors are often stabilized by additional proteins, for example, heat shock protein 90 (HSP90), required for Mla12 resistance1 (RAR1), and suppressor of the G2 allele of SKP1 (SGT1) (Shen et al 2003; Bieri et al 2004; Halterman and Wise 2004; Shirasu 2009; Chapman et al 2021; Chapman et al 2022) as well as interacting with and activating each other (Cesari et al 2014; Wróblewski et al 2018; Bentham et al 2020; Sun et al 2020; Van-Wersch et al 2020; Ngou et al 2022). On the pathogen side, coding sequences (CDS) for 534 candidate secreted effector proteins (CSEPs) have been identified in the Bh genome (Pedersen et al 2012; Menardo et al 2017; Frantzeskakis et al 2018).…”

A stripe rust fungal effector PstSIE1 targets TaSGT1 to facilitate pathogen infection

“…It has been demonstrated that Prf (Kud et al., 2013), Bs2 (Leister et al., 2005) and MLA1 (Bieri et al., 2004) interact with the SGT1 through their LRR domain. Sgt1DKL308‐309 mutation in the SGS domain alters the resistance conferred by MLA (Chapman et al., 2022). We also found that PstSIE1 interacts with the SGS domain through Y2H and BiFC assays, suggesting that PstSIE1 may affect the function of some R genes through directly interrupting the interaction between SGT1 and R proteins.…”