SGT1-specific domain mutations impair interactions with the barley MLA6 immune receptor in association with loss of NLR protein

Authors: CHAPMAN, ANTONY - Iowa State University; ELMORE, J. MITCH - Iowa State University; MCREYNOLDS, MAXWELL - Iowa State University; WALLEY, JUSTIN - Iowa State University; Wise, Roger

Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2021
Publication Date: 12/9/2021
Citation: Chapman, A.V., Elmore, J., McReynolds, M., Walley, J., Wise, R.P. 2021. SGT1-specific domain mutations impair interactions with the barley MLA6 immune receptor in association with loss of NLR protein. Molecular Plant-Microbe Interactions. 35(3):274-289. https://doi.org/10.1094/MPMI-08-21-0217-R.
DOI: https://doi.org/10.1094/MPMI-08-21-0217-R

Interpretive Summary: Barley is an economically and culturally important crop that is under constant threat by fungal pathogens, such as mildews, rusts, and blights. However, it is able to combat these pathogens through the action of resistance or “R” genes. These genes encode proteins that interact with other proteins in order to function properly. One of these proteins is SGT1, which acts to stabilized the resistance protein, MLA, which provides defense to powdery mildew disease. Yet, Sgt1 has been particularly difficult to investigate in its native state as deletions are lethal. We recently identified a two amino-acid deletion mutant of SGT1, which alters resistance conferred by MLA, but without lethality. Utilizing this as a starting point, we were able to use yeast chromosome engineering and advanced protein detection methods to determine that this two amino acid domain is critical for SGT1 interactions with the MLA disease resistance protein, which is necessary for disease defense. Fundamental mechanisms of basic cell processes will allow us to discover the molecular mechanisms of how stabilizing proteins enable optimal function of disease resistance in crops. This is critical to breeders and growers that need to protect their crops, and to enhance food security in developing economies.

Technical 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 often require the HRS complex (HSP90, RAR1, and SGT1) to function. 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 Sgt1(delta)KL308-309 mutation in the SGT1-specific domain, that alters resistance conferred by MLA but without lethality. Here, we use autoactive MLA6 and recombinant yeast-two-hybrid strains with stably integrated HvRar1 and HvHsp90 to determine that this mutation weakens but does not 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 (delta)KL308-309 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 SGT1(delta)KL308-309 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.