ZmPILS6 is an auxin efflux carrier required for maize root morphogenesis.

Plant root systems play a pivotal role in plant physiology and exhibit diverse phenotypic traits. Understanding the genetic mechanisms governing root growth and development in model plants like maize is crucial for enhancing crop resilience to drought and nutrient limitations. This study focused on identifying and characterizing ZmPILS6, an annotated auxin efflux carrier, as a key regulator of various crown root traits in maize. ZmPILS6-modified roots displayed reduced network area and suppressed lateral root formation, which are desirable traits for the "steep, cheap, and deep" ideotype. The research revealed that ZmPILS6 localizes to the endoplasmic reticulum and plays a vital role in controlling the spatial distribution of indole-3-acetic acid (IAA or "auxin") in primary roots. The study also demonstrated that ZmPILS6 can actively efflux IAA when expressed in yeast. Furthermore, the loss of ZmPILS6 resulted in significant proteome remodeling in maize roots, particularly affecting hormone signaling pathways. To identify potential interacting partners of ZmPILS6, a weighted gene coexpression analysis was performed. Altogether, this research contributes to the growing knowledge of essential genetic determinants governing maize root morphogenesis, which is crucial for guiding agricultural improvement strategies.

Temporal and spatial auxin responsive networks in maize primary roots.

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.

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

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 Sgt1ΔKL308-309 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 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.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.

Fungal-induced protein hyperacetylation in maize identified by acetylome profiling.

Lysine acetylation is a key posttranslational 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 acetyltransferases 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 (HCT), a histone deacetylase inhibitor produced by the fungal pathogen Cochliobolus carbonum race 1, promotes 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 HCT as well as HCT-deficient or HCT-producing strains of C. carbonum Analyses of these data demonstrate that acetylation is a widespread posttranslational modification impacting proteins encoded by many intensively studied maize genes. Furthermore, the application of exogenous HCT enabled us to show that the activity of plant-encoded enzymes (histone deacetylases) can be modulated to alter acetylation of nonhistone proteins during an immune response. Collectively, these results provide a resource for further mechanistic studies examining the regulation of protein function by reversible acetylation and offer insight into the complex immune response triggered by virulent C. carbonum.

Sample Preparation Protocols for Protein Abundance, Acetylome, and Phosphoproteome Profiling of Plant Tissues.

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.