SignificanceHow pathogens manipulate host cellular machinery to enable infection is a major question in biology. The ability of Cochliobolus carbonum race 1 to infect susceptible corn plants relies on production of HC-toxin (HCT). While it is known that HC-toxin is a histone deacetylase inhibitor, knowledge of how HCT actually promotes virulence has remained elusive. Here, we use mass spectrometry to quantify protein abundance and levels of protein acetylation in HCT-treated or pathogen-infected plants. These analyses revealed that the activity of plant-encoded enzymes can be modulated to alter both histone and nonhistone protein acetylation during a susceptible interaction and suggest that virulent C. carbonum utilizes HCT to reprogram the transcriptional response to infection, resulting in an ineffective defense response.

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.

Peptide mass spectrometry is an invaluable technique to globally quantify the proteome. Central to proteome profiling are efficient methods to extract proteins, digest proteins into peptides, and enrich for posttranslationally modified peptides prior to mass spectrometry. In this chapter, we describe methods to extract proteins, process them into peptides, and optionally enrich for phospho- and acetyl-peptides prior to analysis by mass spectrometry.

Lysine acetylation is a key post-translational modification that regulates diverse proteins involved in a range of biological processes. The role of histone acetylation in plant defense is well established and it is known that pathogen effector proteins encoding acetyltransferses can directly acetylate host proteins to alter immunity. However, it is unclear whether endogenous plant enzymes can modulate protein acetylation during an immune response. Here we investigate how the effector molecule HC-toxin, a histone deacetylase inhibitor, produced by Cochliobolus carbonum race 1 promotes pathogen virulence in maize through altering protein acetylation. Using mass spectrometry we globally quantified the abundance of 3,636 proteins and the levels of acetylation at 2,791 sites in maize plants treated with HC-toxin as well as HC-toxin deficient or producing strains of C. carbonum. Analyses of these data demonstrate that acetylation is a widespread post-translational modification impacting proteins encoded by many intensively studied maize genes. Furthermore, the application of exogenous HC-toxin enabled us to show that the activity of plant-encoded enzymes can be modulated to alter acetylation of non-histone proteins during an immune response. Collectively, these results provide a resource for further mechanistic studies examining the regulation of protein function and offer insight into the complex immune response triggered by virulent C. carbonum.

Auxin is a key regulator of root morphogenesis across angiosperms. To better understand auxin-regulated networks underlying maize root development, we have characterized auxin-responsive transcription across two time points (30 and 120 min) and four regions of the primary root: the meristematic zone, elongation zone, cortex and stele. Hundreds of auxin-regulated genes involved in diverse biological processes were quantified in these different root regions. In general, most auxin-regulated genes are region unique and are predominantly observed in differentiated tissues compared with the root meristem. Auxin gene regulatory networks were reconstructed with these data to identify key transcription factors that may underlie auxin responses in maize roots. Additionally, Auxin-Response Factor subnetworks were generated to identify target genes that exhibit tissue or temporal specificity in response to auxin. These networks describe novel molecular connections underlying maize root development and provide a foundation for functional genomic studies in a key crop.