Comparative analyses of responses to exogenous and endogenous antiherbivore elicitors enable a forward genetics approach to identify maize gene candidates mediating sensitivity to herbivore-associated molecular patterns.

Crop damage by herbivorous insects remains a significant contributor to annual yield reductions. Following attack, maize (Zea mays) responds to herbivore-associated molecular patterns (HAMPs) and damage-associated molecular patterns (DAMPs), activating dynamic direct and indirect antiherbivore defense responses. To define underlying signaling processes, comparative analyses between plant elicitor peptide (Pep) DAMPs and fatty acid-amino acid conjugate (FAC) HAMPs were conducted. RNA sequencing analysis of early transcriptional changes following Pep and FAC treatments revealed quantitative differences in the strength of response yet a high degree of qualitative similarity, providing evidence for shared signaling pathways. In further comparisons of FAC and Pep responses across diverse maize inbred lines, we identified Mo17 as part of a small subset of lines displaying selective FAC insensitivity. Genetic mapping for FAC sensitivity using the intermated B73 × Mo17 population identified a single locus on chromosome 4 associated with FAC sensitivity. Pursuit of multiple fine-mapping approaches further narrowed the locus to 19 candidate genes. The top candidate gene identified, termed FAC SENSITIVITY ASSOCIATED (ZmFACS), encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) that belongs to the same family as a rice (Oryza sativa) receptor gene previously associated with the activation of induced responses to diverse Lepidoptera. Consistent with reduced sensitivity, ZmFACS expression was significantly lower in Mo17 as compared to B73. Transient heterologous expression of ZmFACS in Nicotiana benthamiana resulted in a significantly increased FAC-elicited response. Together, our results provide useful resources for studying early elicitor-induced antiherbivore responses in maize and approaches to discover gene candidates underlying HAMP sensitivity in grain crops.

A sorghum genome-wide association study (GWAS) identifies a WRKY transcription factor as a candidate gene underlying sugarcane aphid (Melanaphis sacchari) resistance.

MAIN CONCLUSION: A WRKY transcription factor identified through forward genetics is associated with sorghum resistance to the sugarcane aphid and through heterologous expression reduces aphid populations in multiple plant species. Crop plant resistance to insect pests is based on genetically encoded traits which often display variability across diverse germplasm. In a comparatively recent event, a predominant sugarcane aphid (SCA: Melanaphis sacchari) biotype has become a significant agronomic pest of grain sorghum (Sorghum bicolor). To uncover candidate genes underlying SCA resistance, we used a forward genetics approach combining the genetic diversity present in the Sorghum Association Panel (SAP) and the Bioenergy Association Panel (BAP) for a genome-wide association study, employing an established SCA damage rating. One major association was found on Chromosome 9 within the WRKY transcription factor 86 (SbWRKY86). Transcripts encoding SbWRKY86 were previously identified as upregulated in SCA-resistant germplasm and the syntenic ortholog in maize accumulates following Rhopalosiphum maidis infestation. Analyses of SbWRKY86 transcripts displayed patterns of increased SCA-elicited accumulation in additional SCA-resistant sorghum lines. Heterologous expression of SbWRKY86 in both tobacco (Nicotiana benthamiana) and Arabidopsis resulted in reduced population growth of green peach aphid (Myzus persicae). Comparative RNA-Seq analyses of Arabidopsis lines expressing 35S:SbWRKY86-YFP identified changes in expression for a small network of genes associated with carbon-nitrogen metabolism and callose deposition, both contributing factors to defense against aphids. As a test of altered plant responses, 35S:SbWRKY86-YFP Arabidopsis lines were activated using the flagellin epitope elicitor, flg22, and displayed significant increases in callose deposition. Our findings indicate that both heterologous and increased native expression of the transcription factor SbWRKY86 contributes to reduced aphid levels in diverse plant models.

Differential activities of maize plant elicitor peptides as mediators of immune signaling and herbivore resistance.

Plant elicitor peptides (Peps) are conserved regulators of defense responses and models for the study of damage-associated molecular pattern-induced immunity. Although present as multigene families in most species, the functional relevance of these multigene families remains largely undefined. While Arabidopsis Peps appear largely redundant in function, previous work examining Pep-induced responses in maize (Zm) implied specificity of function. To better define the function of individual ZmPeps and their cognate receptors (ZmPEPRs), activities were examined by assessing changes in defense-associated phytohormones, specialized metabolites and global gene expression patterns, in combination with heterologous expression assays and analyses of CRISPR/Cas9-generated knockout plants. Beyond simply delineating individual ZmPep and ZmPEPR activities, these experiments led to a number of new insights into Pep signaling mechanisms. ZmPROPEP and other poaceous precursors were found to contain multiple active Peps, a phenomenon not previously observed for this family. In all, seven new ZmPeps were identified and the peptides were found to have specific activities defined by the relative magnitude of their response output rather than by uniqueness. A striking correlation was observed between individual ZmPep-elicited changes in levels of jasmonic acid and ethylene and the magnitude of induced defense responses, indicating that ZmPeps may collectively regulate immune output through rheostat-like tuning of phytohormone levels. Peptide structure-function studies and ligand-receptor modeling revealed structural features critical to the function of ZmPeps and led to the identification of ZmPep5a as a potential antagonist peptide able to competitively inhibit the activity of other ZmPeps, a regulatory mechanism not previously observed for this family.

Chemical genetic identification of a lectin receptor kinase that transduces immune responses and interferes with abscisic acid signaling.

Insight into how plants simultaneously cope with multiple stresses, for example, when challenged with biotic stress from pathogen infection and abiotic stress from drought, is important both for understanding evolutionary trade-offs and optimizing crop responses to these stresses. Mechanisms by which initial plant immune signaling antagonizes abscisic acid (ABA) signal transduction require further investigation. Using a chemical genetics approach, the small molecule [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) has previously been identified due to its ability to suppress ABA signaling via plant immune signaling components. Here, we have used forward chemical genetics screening to identify DFPM-insensitive loci by monitoring the activity of ABA-inducible pRAB18::GFP in the presence of DFPM and ABA. The ability of DFPM to attenuate ABA signaling was reduced in rda mutants (resistant to DFPM inhibition of ABA signaling). One of the mutants, rda2, was mapped and is defective in a gene encoding a lectin receptor kinase. RDA2 functions in DFPM-mediated inhibition of ABA-mediated reporter expression. RDA2 is required for DFPM-mediated activation of immune signaling, including phosphorylation of mitogen-activated protein kinase (MAPK) 3 (MPK3) and MPK6, and induction of immunity marker genes. Our study identifies a previously uncharacterized receptor kinase gene that is important for DFPM-mediated immune signaling and inhibition of ABA signaling. We demonstrate that the lectin receptor kinase RDA2 is essential for perceiving the DFPM signal and activating MAPKs, and that MKK4 and MKK5 are required for DFPM interference with ABA signal transduction.

BAK1 is involved in AtRALF1-induced inhibition of root cell expansion.

The rapid alkalinization factor (RALF) peptide negatively regulates cell expansion, and an antagonistic relationship has been demonstrated between AtRALF1, a root-specific RALF isoform in Arabidopsis, and brassinosteroids (BRs). An evaluation of the response of BR signaling mutants to AtRALF1 revealed that BRI1-associated receptor kinase1 (bak1) mutants are insensitive to AtRALF1 root growth inhibition activity. BAK1 was essential for the induction of AtRALF1-responsive genes but showed no effect on the mobilization of Ca2+ and alkalinization responses. Homozygous plants accumulating AtRALF1 and lacking the BAK1 gene did not exhibit the characteristic semi-dwarf phenotype of AtRALF1-overexpressors. Biochemical evidence indicates that AtRALF1 and BAK1 physically interact with a Kd of 4.6 µM and acridinium-labeled AtRALF1 was used to demonstrate that part of the specific binding of AtRALF1 to intact seedlings and to a microsomal fraction derived from the roots of Arabidopsis plants is BAK1-dependent. Moreover, AtRALF1 induces an increase in BAK1 phosphorylation, suggesting that the binding of AtRALF1 to BAK1 is functional. These findings show that BAK1 contains an additional AtRALF1 binding site, indicating that this protein may be part of a AtRALF1-containing complex as a co-receptor, and it is required for the negative regulation of cell expansion.

Arabidopsis thaliana rapid alkalinization factor 1-mediated root growth inhibition is dependent on calmodulin-like protein 38.

Arabidopsis thaliana rapid alkalinization factor 1 (AtRALF1) is a small secreted peptide hormone that inhibits root growth by repressing cell expansion. Although it is known that AtRALF1 binds the plasma membrane receptor FERONIA and conveys its signals via phosphorylation, the AtRALF1 signaling pathway is largely unknown. Here, using a yeast two-hybrid system to search for AtRALF1-interacting proteins in Arabidopsis, we identified calmodulin-like protein 38 (CML38) as an AtRALF1-interacting partner. We also found that CML38 and AtRALF1 are both secreted proteins that physically interact in a Ca2+- and pH-dependent manner. CML38-knockout mutants generated via T-DNA insertion were insensitive to AtRALF1, and simultaneous treatment with both AtRALF1 and CML38 proteins restored sensitivity in these mutants. Hybrid plants lacking CML38 and having high accumulation of the AtRALF1 peptide did not exhibit the characteristic short-root phenotype caused by AtRALF1 overexpression. Although CML38 was essential for AtRALF1-mediated root inhibition, it appeared not to have an effect on the AtRALF1-induced alkalinization response. Moreover, acridinium-labeling of AtRALF1 indicated that the binding of AtRALF1 to intact roots is CML38-dependent. In summary, we describe a new component of the AtRALF1 response pathway. The new component is a calmodulin-like protein that binds AtRALF1, is essential for root growth inhibition, and has no role in AtRALF1 alkalinization.

Danger-associated peptide signaling in Arabidopsis requires clathrin.

The Arabidopsis thaliana endogenous elicitor peptides (AtPeps) are released into the apoplast after cellular damage caused by pathogens or wounding to induce innate immunity by direct binding to the membrane-localized leucine-rich repeat receptor kinases, PEP RECEPTOR1 (PEPR1) and PEPR2. Although the PEPR-mediated signaling components and responses have been studied extensively, the contributions of the subcellular localization and dynamics of the active PEPRs remain largely unknown. We used live-cell imaging of the fluorescently labeled and bioactive pep1 to visualize the intracellular behavior of the PEPRs in the Arabidopsis root meristem. We found that AtPep1 decorated the plasma membrane (PM) in a receptor-dependent manner and cointernalized with PEPRs. Trafficking of the AtPep1-PEPR1 complexes to the vacuole required neither the trans-Golgi network/early endosome (TGN/EE)-localized vacuolar H(+)-ATPase activity nor the function of the brefeldin A-sensitive ADP-ribosylation factor-guanine exchange factors (ARF-GEFs). In addition, AtPep1 and different TGN/EE markers colocalized only rarely, implying that the intracellular route of this receptor-ligand pair is largely independent of the TGN/EE. Inducible overexpression of the Arabidopsis clathrin coat disassembly factor, Auxilin2, which inhibits clathrin-mediated endocytosis (CME), impaired the AtPep1-PEPR1 internalization and compromised AtPep1-mediated responses. Our results show that clathrin function at the PM is required to induce plant defense responses, likely through CME of cell surface-located signaling components.

Dynamic regulation of Pep-induced immunity through post-translational control of defence transcript splicing.

The survival of all living organisms requires the ability to detect attacks and swiftly counter them with protective immune responses. Despite considerable mechanistic advances, the interconnectivity of signalling modules often remains unclear. A newly characterized protein, IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR), negatively regulates immune responses in both maize and Arabidopsis, with disrupted function resulting in enhanced disease resistance. IRR associates with and promotes canonical splicing of transcripts encoding defence signalling proteins, including the key negative regulator of pattern-recognition receptor signalling complexes, CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28). On immune activation by Plant Elicitor Peptides (Peps), IRR is dephosphorylated, disrupting interaction with CPK28 transcripts and resulting in the accumulation of an alternative splice variant encoding a truncated CPK28 protein with impaired kinase activity and diminished function as a negative regulator. We demonstrate a new mechanism linking Pep-induced post-translational modification of IRR with post-transcriptionally mediated attenuation of CPK28 function to dynamically amplify Pep signalling and immune output.

Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity.

Specialized metabolites constitute key layers of immunity that underlie disease resistance in crops; however, challenges in resolving pathways limit our understanding of the functions and applications of these metabolites. In maize (Zea mays), the inducible accumulation of acidic terpenoids is increasingly considered to be a defence mechanism that contributes to disease resistance. Here, to understand maize antibiotic biosynthesis, we integrated association mapping, pan-genome multi-omic correlations, enzyme structure-function studies and targeted mutagenesis. We define ten genes in three zealexin (Zx) gene clusters that encode four sesquiterpene synthases and six cytochrome P450 proteins that collectively drive the production of diverse antibiotic cocktails. Quadruple mutants in which the ability to produce zealexins (ZXs) is blocked exhibit a broad-spectrum loss of disease resistance. Genetic redundancies ensuring pathway resiliency to single null mutations are combined with enzyme substrate promiscuity, creating a biosynthetic hourglass pathway that uses diverse substrates and in vivo combinatorial chemistry to yield complex antibiotic blends. The elucidated genetic basis of biochemical phenotypes that underlie disease resistance demonstrates a predominant maize defence pathway and informs innovative strategies for transferring chemical immunity between crops.

Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple.

The Rapid Alkalinization Factor (RALF) is a plant hormone peptide that inhibits proton transport causing alkalinization of the extracellular media. To detect the alkalinization response elicited by RALF peptides in root cells, Arabidopsis seedlings are carefully transferred to a gel containing the pH-sensitive indicator bromocresol purple, treated with the peptide and photographed after 30 min. Herein the protocol is optimized for evaluation of exogenous treatment, described in detail and expected results are presented.

Erratum: Correction Notice: Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple.

[This corrects the article .].