9,10-KODA, an α-ketol produced by the tonoplast-localized 9-lipoxygenase ZmLOX5, plays a signaling role in maize defense against insect herbivory.

13-Lipoxygenases (LOXs) initiate the synthesis of jasmonic acid (JA), the best-understood oxylipin hormone in herbivory defense. However, the roles of 9-LOX-derived oxylipins in insect resistance remain unclear. Here, we report a novel anti-herbivory mechanism mediated by a tonoplast-localized 9-LOX, ZmLOX5, and its linolenic acid-derived product, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (9,10-KODA). Transposon-insertional disruption of ZmLOX5 resulted in the loss of resistance to insect herbivory. lox5 knockout mutants displayed greatly reduced wound-induced accumulation of multiple oxylipins and defense metabolites, including benzoxazinoids, abscisic acid (ABA), and JA-isoleucine (JA-Ile). However, exogenous JA-Ile failed to rescue insect defense in lox5 mutants, while applications of 1 µM 9,10-KODA or the JA precursor, 12-oxo-phytodienoic acid (12-OPDA), restored wild-type resistance levels. Metabolite profiling revealed that exogenous 9,10-KODA primed the plants for increased production of ABA and 12-OPDA, but not JA-Ile. While none of the 9-oxylipins were able to rescue JA-Ile induction, the lox5 mutant accumulated lower wound-induced levels of Ca2+, suggesting this as a potential explanation for lower wound-induced JA. Seedlings pretreated with 9,10-KODA exhibited rapid or more robust wound-induced defense gene expression. In addition, an artificial diet supplemented with 9,10-KODA arrested fall armyworm larvae growth. Finally, analysis of single and double lox5 and lox10 mutants showed that ZmLOX5 also contributed to insect defense by modulating ZmLOX10-mediated green leaf volatile signaling. Collectively, our study uncovered a previously unknown anti-herbivore defense and hormone-like signaling activity for a major 9-oxylipin α-ketol.

Oxylipin biosynthetic gene families of Cannabis sativa.

Cannabis sativa is a global multi-billion-dollar cash crop with numerous industrial uses, including in medicine and recreation where its value is largely owed to the production of pharmacological and psychoactive metabolites known as cannabinoids. Often underappreciated in this role, the lipoxygenase (LOX)-derived green leaf volatiles (GLVs), also known as the scent of cut grass, are the hypothetical origin of hexanoic acid, the initial substrate for cannabinoid biosynthesis. The LOX pathway is best known as the primary source of plant oxylipins, molecules analogous to the eicosanoids from mammalian systems. These molecules are a group of chemically and functionally diverse fatty acid-derived signals that govern nearly all biological processes including plant defense and development. The interaction between oxylipin and cannabinoid biosynthetic pathways remains to be explored. Despite their unique importance in this crop, there has not been a comprehensive investigation focusing on the genes responsible for oxylipin biosynthesis in any Cannabis species. This study documents the first genome-wide catalogue of the Cannabis sativa oxylipin biosynthetic genes and identified 21 LOX, five allene oxide synthases (AOS), three allene oxide cyclases (AOC), one hydroperoxide lyase (HPL), and five 12-oxo-phytodienoic acid reductases (OPR). Gene collinearity analysis found chromosomal regions containing several isoforms maintained across Cannabis, Arabidopsis, and tomato. Promoter, expression, weighted co-expression genetic network, and functional enrichment analysis provide evidence of tissue- and cultivar-specific transcription and roles for distinct isoforms in oxylipin and cannabinoid biosynthesis. This knowledge facilitates future targeted approaches towards Cannabis crop improvement and for the manipulation of cannabinoid metabolism.

Transgenic Soybeans Expressing Phosphatidylinositol-3-Phosphate-Binding Proteins Show Enhanced Resistance Against the Oomycete Pathogen Phytophthora sojae.

Oomycete and fungal pathogens cause billions of dollars of damage to crops worldwide annually. Therefore, there remains a need for broad-spectrum resistance genes, especially ones that target pathogens but do not interfere with colonization by beneficial microbes. Motivated by evidence suggesting that phosphatidylinositol-3-phosphate (PI3P) may be involved in the delivery of some oomycete and fungal virulence effector proteins, we created stable transgenic soybean plants that express and secrete two different PI3P-binding proteins, GmPH1 and VAM7, in an effort to interfere with effector delivery and confer resistance. Soybean plants expressing the two PI3P-binding proteins exhibited reduced infection by the oomycete pathogen Phytophthora sojae compared to control lines. Measurements of nodulation by nitrogen-fixing mutualistic bacterium Bradyrhizobium japonicum, which does not produce PI3P, revealed that the two lines with the highest levels of GmPH1 transcripts exhibited reductions in nodulation and in benefits from nodulation. Transcriptome and plant hormone measurements were made of soybean lines with the highest transcript levels of GmPH1 and VAM7, as well as controls, following P. sojae- or mock-inoculation. The results revealed increased levels of infection-associated transcripts in the transgenic lines, compared to controls, even prior to P. sojae infection, suggesting that the plants were primed for increased defense. The lines with reduced nodulation exhibited elevated levels of jasmonate-isoleucine and of transcripts of a JAR1 ortholog encoding jasmonate-isoleucine synthetase. However, lines expressing VAM7 transgenes exhibited normal nodulation and no increases in jasmonate-isoleucine. Overall, together with previously published data from cacao and from P. sojae transformants, the data suggest that secretion of PI3P-binding proteins may confer disease resistance through a variety of mechanisms.

Maize biochemistry in response to root herbivory was mediated by domestication, spread, and breeding.

MAIN CONCLUSION: With domestication, northward spread, and breeding, maize defence against root-herbivores relied on induced defences, decreasing levels of phytohormones involved in resistance, and increasing levels of a phytohormone involved in tolerance. We addressed whether a suite of maize (Zea mays mays) phytohormones and metabolites involved in herbivore defence were mediated by three successive processes: domestication, spread to North America, and modern breeding. With those processes, and following theoretical predictions, we expected to find: a change in defence strategy from reliance on induced defences to reliance on constitutive defences; decreasing levels of phytohormones involved in herbivore resistance, and; increasing levels of a phytohormone involved in herbivore tolerance. We tested those predictions by comparing phytohormone levels in seedlings exposed to root herbivory by Diabrotica virgifera virgifera among four plant types encompassing those processes: the maize ancestor Balsas teosinte (Zea mays parviglumis), Mexican maize landraces, USA maize landraces, and USA inbred maize cultivars. With domestication, maize transitioned from reliance on induced defences in teosinte to reliance on constitutive defences in maize, as predicted. One subset of metabolites putatively involved in herbivory defence (13-oxylipins) was suppressed with domestication, as predicted, though another was enhanced (9-oxylipins), and both were variably affected by spread and breeding. A phytohormone (indole-3-acetic acid) involved in tolerance was enhanced with domestication, and with spread and breeding, as predicted. These changes are consistent with documented changes in herbivory resistance and tolerance, and occurred coincidentally with cultivation in increasingly resource-rich environments, i.e., from wild to highly enriched agricultural environments. We concluded that herbivore defence evolution in crops may be mediated by processes spanning thousands of generations, e.g., domestication and spread, as well as by processes spanning tens of generations, e.g., breeding and agricultural intensification.

A Rapid Pipeline for Pollen- and Anther-Specific Gene Discovery Based on Transcriptome Profiling Analysis of Maize Tissues.

Recently, crop breeders have widely adopted a new biotechnology-based process, termed Seed Production Technology (SPT), to produce hybrid varieties. The SPT does not produce nuclear male-sterile lines, and instead utilizes transgenic SPT maintainer lines to pollinate male-sterile plants for propagation of nuclear-recessive male-sterile lines. A late-stage pollen-specific promoter is an essential component of the pollen-inactivating cassette used by the SPT maintainers. While a number of plant pollen-specific promoters have been reported so far, their usefulness in SPT has remained limited. To increase the repertoire of pollen-specific promoters for the maize community, we conducted a comprehensive comparative analysis of transcriptome profiles of mature pollen and mature anthers against other tissue types. We found that maize pollen has much less expressed genes (>1 FPKM) than other tissue types, but the pollen grain has a large set of distinct genes, called pollen-specific genes, which are exclusively or much higher (100 folds) expressed in pollen than other tissue types. Utilizing transcript abundance and correlation coefficient analysis, 1215 mature pollen-specific (MPS) genes and 1009 mature anther-specific (MAS) genes were identified in B73 transcriptome. These two gene sets had similar GO term and KEGG pathway enrichment patterns, indicating that their members share similar functions in the maize reproductive process. Of the genes, 623 were shared between the two sets, called mature anther- and pollen-specific (MAPS) genes, which represent the late-stage pollen-specific genes of the maize genome. Functional annotation analysis of MAPS showed that 447 MAPS genes (71.7% of MAPS) belonged to genes encoding pollen allergen protein. Their 2-kb promoters were analyzed for cis-element enrichment and six well-known pollen-specific cis-elements (AGAAA, TCCACCA, TGTGGTT, [TA]AAAG, AAATGA, and TTTCT) were found highly enriched in the promoters of MAPS. Interestingly, JA-responsive cis-element GCC box (GCCGCC) and ABA-responsive cis-element-coupling element1 (ABRE-CE1, CCACC) were also found enriched in the MAPS promoters, indicating that JA and ABA signaling likely regulate pollen-specific MAPS expression. This study describes a robust and straightforward pipeline to discover pollen-specific promotes from publicly available data while providing maize breeders and the maize industry a number of late-stage (mature) pollen-specific promoters for use in SPT for hybrid breeding and seed production.

Fusarium verticillioides Induces Maize-Derived Ethylene to Promote Virulence by Engaging Fungal G-Protein Signaling.

Seed maceration and contamination with mycotoxin fumonisin inflicted by Fusarium verticillioides is a major disease concern for maize producers worldwide. Meta-analyses of quantitative trait loci for Fusarium ear rot resistance uncovered several ethylene (ET) biosynthesis and signaling genes within them, implicating ET in maize interactions with F. verticillioides. We tested this hypothesis using maize knockout mutants of the 1-aminocyclopropane-1-carboxylate (ACC) synthases ZmACS2 and ZmACS6. Infected wild-type seed emitted five-fold higher ET levels compared with controls, whereas ET was abolished in the acs2 and acs6 single and double mutants. The mutants supported reduced fungal biomass, conidia, and fumonisin content. Normal susceptibility was restored in the acs6 mutant with exogenous treatment of ET precursor ACC. Subsequently, we showed that fungal G-protein signaling is required for virulence via induction of maize-produced ET. F. verticillioides Gß subunit and two regulators of G-protein signaling mutants displayed reduced seed colonization and decreased ET levels. These defects were rescued by exogenous application of ACC. We concluded that pathogen-induced ET facilitates F. verticillioides colonization of seed, and, in turn, host ET production is manipulated via G-protein signaling of F. verticillioides to facilitate pathogenesis.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

An updated census of the maize TIFY family.

The TIFY gene family is a plant-specific gene family encoding a group of proteins characterized by its namesake, the conservative TIFY domain and members can be organized into four subfamilies: ZML, TIFY, PPD and JAZ (Jasmonate ZIM-domain protein) by presence of additional conserved domains. The TIFY gene family is intensively explored in several model and agriculturally important crop species and here, yet the composition of the TIFY family of maize has remained unresolved. This study increases the number of maize TIFY family members known by 40%, bringing the total to 47 including 38 JAZ, 5 TIFY, and 4 ZML genes. The majority of the newly identified genes were belonging to the JAZ subfamily, six of which had aberrant TIFY domains, suggesting loss JAZ-JAZ or JAZ-NINJA interactions. Six JAZ genes were found to have truncated Jas domain or an altered degron motif, suggesting resistance to classical JAZ degradation. In addition, seven membranes were found to have an LxLxL-type EAR motif which allows them to recruit TPL/TPP co-repressors directly without association to NINJA. Expression analysis revealed that ZmJAZ14 was specifically expressed in the seeds and ZmJAZ19 and 22 in the anthers, while the majority of other ZmJAZs were generally highly expressed across diverse tissue types. Additionally, ZmJAZ genes were highly responsive to wounding and JA treatment. This study provides a comprehensive update of the maize TIFY/JAZ gene family paving the way for functional, physiological, and ecological analysis.

Amino acid-derived defense metabolites from plants: A potential source to facilitate novel antimicrobial development.

For millennia, humanity has relied on plants for its medicines, and modern pharmacology continues to reexamine and mine plant metabolites for novel compounds and to guide improvements in biological activity, bioavailability, and chemical stability. The critical problem of antibiotic resistance and increasing exposure to viral and parasitic diseases has spurred renewed interest into drug treatments for infectious diseases. In this context, an urgent revival of natural product discovery is globally underway with special attention directed toward the numerous and chemically diverse plant defensive compounds such as phytoalexins and phytoanticipins that combat herbivores, microbial pathogens, or competing plants. Moreover, advancements in "omics," chemistry, and heterologous expression systems have facilitated the purification and characterization of plant metabolites and the identification of possible therapeutic targets. In this review, we describe several important amino acid-derived classes of plant defensive compounds, including antimicrobial peptides (e.g., defensins, thionins, and knottins), alkaloids, nonproteogenic amino acids, and phenylpropanoids as potential drug leads, examining their mechanisms of action, therapeutic targets, and structure-function relationships. Given their potent antibacterial, antifungal, antiparasitic, and antiviral properties, which can be superior to existing drugs, phytoalexins and phytoanticipins are an excellent resource to facilitate the rational design and development of antimicrobial drugs.

Oxylipins are implicated as communication signals in tomato-root-knot nematode (Meloidogyne javanica) interaction.

Throughout infection, plant-parasitic nematodes activate a complex host defense response that will regulate their development and aggressiveness. Oxylipins-lipophilic signaling molecules-are part of this complex, performing a fundamental role in regulating plant development and immunity. At the same time, the sedentary root-knot nematode Meloidogyne spp. secretes numerous effectors that play key roles during invasion and migration, supporting construction and maintenance of nematodes' feeding sites. Herein, comprehensive oxylipin profiling of tomato roots, performed using LC-MS/MS, indicated strong and early responses of many oxylipins following root-knot nematode infection. To identify genes that might respond to the lipidomic defense pathway mediated through oxylipins, RNA-Seq was performed by exposing Meloidogyne javanica second-stage juveniles to tomato protoplasts and the oxylipin 9-HOT, one of the early-induced oxylipins in tomato roots upon nematode infection. A total of 7512 differentially expressed genes were identified. To target putative effectors, we sought differentially expressed genes carrying a predicted secretion signal peptide. Among these, several were homologous with known effectors in other nematode species; other unknown, potentially secreted proteins may have a role as root-knot nematode effectors that are induced by plant lipid signals. These include effectors associated with distortion of the plant immune response or manipulating signal transduction mediated by lipid signals. Other effectors are implicated in cell wall degradation or ROS detoxification at the plant-nematode interface. Being an integral part of the plant's defense response, oxylipins might be placed as important signaling molecules underlying nematode parasitism.

Differential Evolution of α-Glucan Water Dikinase (GWD) in Plants.

The alpha-glucan water dikinase (GWD) enzyme catalyzes starch phosphorylation, an integral step in transitory starch degradation. The high phosphate content in stored starch has great industrial value, due to its physio-chemical properties making it more versatile, although the phosphate content of stored starch varies depending on the botanical source. In this study, we used various computational approaches to gain insights into the evolution of the GWD protein in 48 plant species with possible roles in enzyme function and alteration of phosphate content in their stored starch. Our analyses identified deleterious mutations, particularly in the highly conserved 5 aromatic amino acid residues in the dual tandem carbohydrate binding modules (CBM-45) of GWD protein in C. zofingiensis, G. hirsutum, A. protothecoides, P. miliaceum, and C. reinhardtii. These findings will inform experimental designs for simultaneous repression of genes coding for GWD and the predicted interacting proteins to elucidate the role this enzyme plays in starch degradation. Our results reveal significant diversity in the evolution of GWD enzyme across plant species, which may be evolutionarily advantageous according to the varying needs for phosphorylated stored starch between plants and environments.

Relative contribution of LOX10, green leaf volatiles and JA to wound-induced local and systemic oxylipin and hormone signature in Zea mays (maize).

Green leaf volatiles (GLVs) and jasmonates (JAs) are the best-characterized groups of fatty acid-derived oxylipin signals that regulate wound-associated defenses. Beyond these two major groups of defense signals, plants produce an array of oxylipins in response to wounding, which possess potent signaling and/or insecticidal activities. In this study, we assessed the relative contribution of JAs and GLVs to wound-induced systemic signaling and the associated regulation of oxylipins in local and systemic tissues of maize (Zea mays). For this, we utilized GLV- and JA-deficient mutants, lox10 single and opr7opr8 double mutants, respectively, and profiled oxylipins in untreated leaves and roots, and in locally wounded and systemic leaves. In contrast to the studies in dicots, no systemic induction of JAs was observed in maize. Instead, a JA precursor, 12-OPDA, as well as ketols and C12/13 oxo-acids derived from 13-lipoxygenases (LOXs), were preferentially induced in both locally wounded and systemic unwounded leaves. Several 9-LOX-derived oxylipins (9-oxylipins) including hydroxides and ketones were also significantly induced locally. JA and JA-isoleucine (JA-Ile) were rapidly induced within 0.5 h, and were followed by a second increase in local tissue 4 h after wounding. GLV-deficient lox10 mutants displayed reduced levels of most 13-oxylipins, and elevated levels of several 9-oxylipins and the a-dioxygenase (DOX) product, 2-HOD. lox10 mutants were completely devoid of C6 volatiles and their C12 counterparts, and greatly decreased in C5 volatiles and their C13 oxo-acid counterparts. Thus, in addition to being the sole LOX isoform providing substrate for GLV synthesis, LOX10 is a major 13-LOX that provides substrate to several LOX branches that produce an array of 13-oxylipin products, including C5 volatiles. Interestingly, the rapid JA and JA-Ile increase at 0.5-2 h post-wounding was only moderately affected by the LOX10 mutation, while significantly reduced levels were observed at 4 h post-wounding. Combined with the previous findings that GLVs activate JA biosynthesis, these results suggest that both LOX10-derived substrates and/or GLVs are involved in the large second phase of JA synthesis proximal to the wound. Analyses of opr7opr8 mutants revealed that wound-induced oxylipin responses were positively regulated by JA signaling. The local and systemic accumulation of SA was not altered in the two mutants. Collectively, our results identified a subset of oxylipins strongly induced in wounded and systemic leaves, but their impact on insect defenses remain elusive. The lack of systemic induction of JAs points to substantial difference between systemic wound responses in studied dicots and maize. Our results show that GLV-deficiency and reduced JA in lox10 mutants had a greater impact on wound-induced local and systemic tissue oxylipin responses compared to the solely JA-deficient opr7opr8 double mutants. This suggests that GLVs or other LOX10-derived products heavily contribute to overall basal and wound-induced oxylipin responses. The specific roles of the GLV- and/or JA-dependent oxylipins in wound responses and defense remain to be further investigated by a combination of multiple orders of oxylipin-deficient mutants.

Green leaf volatiles and jasmonic acid enhance susceptibility to anthracnose diseases caused by Colletotrichum graminicola in maize.

Colletotrichum graminicola is a hemibiotrophic fungus that causes anthracnose leaf blight (ALB) and anthracnose stalk rot (ASR) in maize. Despite substantial economic losses caused by these diseases, the defence mechanisms against this pathogen remain poorly understood. Several hormones are suggested to aid in defence against C. graminicola, such as jasmonic acid (JA) and salicylic acid (SA), but supporting genetic evidence was not reported. Green leaf volatiles (GLVs) are a group of well-characterized volatiles that induce JA biosynthesis in maize and are known to function in defence against necrotrophic pathogens. Information regarding the role of GLVs and JA in interactions with (hemi)biotrophic pathogens remains limited. To functionally elucidate GLVs and JA in defence against a hemibiotrophic pathogen, we tested GLV- and JA-deficient mutants, lox10 and opr7 opr8, respectively, for resistance to ASR and ALB and profiled jasmonates and SA in their stalks and leaves throughout infection. Both mutants were resistant and generally displayed elevated levels of SA and low amounts of jasmonates, especially at early stages of infection. Pretreatment with GLVs restored susceptibility of lox10 mutants, but not opr7 opr8 mutants, which coincided with complete rescue of JA levels. Exogenous methyl jasmonate restored susceptibility in both mutants when applied before inoculation, whereas methyl salicylate did not induce further resistance in either of the mutants, but did induce mutant-like resistance in the wild type. Collectively, this study reveals that GLVs and JA contribute to maize susceptibility to C. graminicola due to suppression of SA-related defences.

Oxylipins Other Than Jasmonic Acid Are Xylem-Resident Signals Regulating Systemic Resistance Induced by Trichoderma virens in Maize.

Multiple long-distance signals have been identified for pathogen-induced systemic acquired resistance, but mobile signals for symbiont-induced systemic resistance (ISR) are less well understood. We used ISR-positive and -negative mutants of maize (Zea mays) and the beneficial fungus Trichoderma virens and identified 12-oxo-phytodienoic acid (12-OPDA) and α-ketol of octadecadienoic acid (KODA) as important ISR signals. We show that a maize 13-lipoxygenase mutant, lox10, colonized by the wild-type T. virens (TvWT) lacked ISR response against Colletotrichum graminicola but instead displayed induced systemic susceptibility. Oxylipin profiling of xylem sap from T. virens-treated plants revealed that 12-OPDA and KODA levels correlated with ISR. Transfusing sap supplemented with 12-OPDA or KODA increased receiver plant resistance in a dose-dependent manner, with 12-OPDA restoring ISR of lox10 plants treated with TvWT or T. virens Δsm1, a mutant unable to induce ISR. Unexpectedly, jasmonic acid (JA) was not involved, as the JA-deficient opr7 opr8 mutant plants retained the capacity for T. virens-induced ISR. Transcriptome analysis of TvWT-treated maize B73 revealed upregulation of 12-OPDA biosynthesis and OPDA-responsive genes but downregulation of JA biosynthesis and JA response genes. We propose a model that differential regulation of 12-OPDA and JA in response to T. virens colonization results in ISR induction.

Bioprospecting Plant Growth-Promoting Rhizobacteria That Mitigate Drought Stress in Grasses.

This study reports the application of a novel bioprospecting procedure designed to screen plant growth-promoting rhizobacteria (PGPR) capable of rapidly colonizing the rhizosphere and mitigating drought stress in multiple hosts. Two PGPR strains were isolated by this bioprospecting screening assay and identified as Bacillus sp. (12D6) and Enterobacter sp. (16i). When inoculated into the rhizospheres of wheat (Triticum aestivum) and maize (Zea mays) seedlings, these PGPR resulted in delays in the onset of plant drought symptoms. The plant phenotype responding to drought stress was associated with alterations in root system architecture. In wheat, both PGPR isolates significantly increased root branching, and Bacillus sp. (12D6), in particular, increased root length, when compared to the control. In maize, both PGPR isolates significantly increased root length, root surface area and number of tips when compared to the control. Enterobacter sp. (16i) exhibited greater effects in root length, diameter and branching when compared to Bacillus sp. (12D6) or the control. In vitro phytohormone profiling of PGPR pellets and filtrates using LC/MS demonstrated that both PGPR strains produced and excreted indole-3-acetic acid (IAA) and salicylic acid (SA) when compared to other phytohormones. The positive effects of PGPR inoculation occurred concurrently with the onset of water deficit, demonstrating the potential of the PGPR identified from this bioprospecting pipeline for use in crop production systems under drought stress.

CIRCADIAN CLOCK-ASSOCIATED1 Controls Resistance to Aphids by Altering Indole Glucosinolate Production.

CIRCADIAN CLOCK-ASSOCIATED1 (CCA1), a well-known central circadian clock regulator, coordinates plant responses to environmental challenges. Its daily rhythmic expression in Arabidopsis (Arabidopsis thaliana) confers host resistance to the caterpillar Trichoplusia ni However, it is unclear whether CCA1 plays a role in defense against phloem sap-feeding aphids. In this study, we showed that green peach aphid (Myzus persicae) displayed an intrinsic circadian feeding rhythm. Under constant light, wild-type Columbia-0 (Col-0) Arabidopsis plants coentrained with aphids in the same light/dark cycles exhibited greater antixenotic activity than plants preentrained in the opposite cycle from the aphids. Consistently, circadian mutants cca1-1, cca1-11, lhy-21, ztl-1, ztl-4, and lux-2 suffered more severe damage than Col-0 plants when infested by aphids, suggesting that the Arabidopsis circadian clock plays a defensive role. However, the arrhythmic CCA1 overexpression line (CCA1-OX) displayed strong antixenotic and antibiotic activities despite its loss of circadian regulation. Aphids feeding on CCA1-OX plants exhibited lower reproduction and smaller body size and weight than those on Col-0. Apparently, CCA1 regulates both clock-dependent and -independent defense responses. Systematic investigation based on bioinformatics analyses indicated that resistance to aphids in CCA1-OX plants was due primarily to heightened basal indole glucosinolate levels. Interestingly, aphid feeding induced alternatively spliced intron-retaining CCA1a/b transcripts, which are normally expressed at low levels, whereas expression of the major fully spliced CCA1 transcript remained largely unchanged. We hypothesize that posttranscriptional modulation of CCA1 expression upon aphid infestation maximizes the potential of circadian-mediated defense and stress tolerance while ensuring normal plant development.

Rosette core fungal resistance in Arabidopsis thaliana.

MAIN CONCLUSION: Unlike rosette leaves, the mature Arabidopsis rosette core can display full resistance to Botrytis cinerea revealing the importance for spatial and developmental aspects of plant fungal resistance. Arabidopsis thaliana is a model host to investigate plant defense against fungi. However, many of the reports investigating Arabidopsis fungal defense against the necrotrophic fungus, Botrytis cinerea, utilize rosette leaves as host tissue. Here we report organ-dependent differences in B. cinerea resistance of Arabidopsis. Although wild-type Arabidopsis rosette leaves mount a jasmonate-dependent defense that slows fungal growth, this defense is incapable of resisting fungal devastation. In contrast, as the fungus spreads through infected leaf petioles towards the plant center, or rosette core, there is a jasmonate- and age-dependent fungal penetration blockage into the rosette core. We report evidence for induced and preformed resistance in the rosette core, as direct rosette core inoculation can also result in resistance, but at a lower penetrance relative to infections that approach the core from infected leaf petioles. The Arabidopsis rosette core displays a distinct transcriptome relative to other plant organs, and BLADE ON PETIOLE (BOP) transcripts are abundant in the rosette core. The BOP genes, with known roles in abscission zone formation, are required for full Arabidopsis rosette core B. cinerea resistance, suggesting a possible role for BOP-dependent modifications that may help to restrict fungal susceptibility of the rosette core. Finally, we demonstrate that cabbage and cauliflower, common Brassicaceae crops, also display leaf susceptibility and rosette core resistance to B. cinerea that can involve leaf abscission. Thus, spatial and developmental aspects of plant host resistance play critical roles in resistance to necrotrophic fungal pathogens and are important to our understanding of plant defense mechanisms.

Tomato SlWRKY3 acts as a positive regulator for resistance against the root-knot nematode Meloidogyne javanica by activating lipids and hormone-mediated defense-signaling pathways.

Diseases caused by plant-parasitic nematodes in vegetables, among them Meloidogyne spp. root-knot nematodes (RKNs), lead to extensive yield decline. A molecular understanding of the mechanisms underlying plants' innate resistance may enable developing safe alternatives to harmful chemical nematicides in controlling RKNs. A tight relationship has been revealed between the WRKY transcription factors and RKN parasitism on tomato roots. We investigated the function role of tomato SlWRK3 and SlWRKY35 in regulating nematode disease development. Using promoter-GUS reporter gene fusions, we show that both SlWRKY3 and SlWRKY35 are induced within 5 days of infection and through feeding-site development and gall maturation, with a much stronger response of the former vs. the latter to nematode infection. Histological analysis of nematode-feeding sites indicated a high expression of SlWRKY3 in developing and mature feeding cells and associated vasculature cells, whereas SlWRKY35 expression was only observed in mature feeding sites. Both SlWRKY3 and SlWRKY35 promoters were induced by the defense phytohormones salicylic acid and indole-3-butyric acid, with no response to either jasmonic acid or methyl jasmonate. SlWRKY3 overexpression resulted in lower infection of the RKN Meloidogyne javanica, whereas knocking down SlWRKY3 resulted in increased infection. Phytohormone and oxylipin profiles determined by LC-MS/MS showed that the enhanced resistance in the former is coupled with an increased accumulation of defense molecules from the shikimate and oxylipin pathways. Our results pinpoint SlWRKY3 as a positive regulator of induced resistance in response to nematode invasion and infection, mostly during the early stages of nematode infection.

Oxylipins from both pathogen and host antagonize jasmonic acid-mediated defence via the 9-lipoxygenase pathway in Fusarium verticillioides infection of maize.

Oxylipins are a newly emerging group of signals that serve defence roles or promote virulence. To identify specific host and fungal genes and oxylipins governing the interactions between maize and Fusarium verticillioides, maize wild-type and lipoxygenase3 (lox3) mutant were inoculated with either F. verticillioides wild-type or linoleate-diol-synthase 1-deleted mutant (ΔFvlds1D). The results showed that lox3 mutants were more resistant to F. verticillioides. The reduced colonization on lox3 was associated with reduced fumonisin production and with a stronger and earlier induction of ZmLOX4, ZmLOX5 and ZmLOX12. In addition to the reported defence function of ZmLOX12, we showed that lox4 and lox5 mutants were more susceptible to F. verticillioides and possessed decreased jasmonate levels during infection, suggesting that these genes are essential for jasmonic acid (JA)-mediated defence. Oxylipin profiling revealed a dramatic reduction in fungal linoleate diol synthase 1 (LDS1)-derived oxylipins, especially 8-HpODE (8-hydroperoxyoctadecenoic acid), in infected lox3 kernels, indicating the importance of this molecule in virulence. Collectively, we make the following conclusions: (1) LOX3 is a major susceptibility factor induced by fungal LDS1-derived oxylipins to suppress JA-stimulating 9-LOXs; (2) LOX3-mediated signalling promotes the biosynthesis of virulence-promoting oxylipins in the fungus; and (3) both fungal LDS1- and host LOX3-produced oxylipins are essential for the normal infection and colonization processes of maize seed by F. verticillioides.

Characterization of the maize lipoxygenase gene family in relation to aflatoxin accumulation resistance.

Maize (Zea mays L.) is a globally important staple food crop prone to contamination by aflatoxin, a carcinogenic secondary metabolite produced by the fungus Aspergillus flavus. An efficient approach to reduce accumulation of aflatoxin is the development of germplasm resistant to colonization and toxin production by A. flavus. Lipoxygenases (LOXs) are a group of non-heme iron containing dioxygenase enzymes that catalyze oxygenation of polyunsaturated fatty acids (PUFAs). LOX derived oxylipins play critical roles in plant defense against pathogens including A. flavus. The objectives of this study were to summarize sequence diversity and expression patterns for all LOX genes in the maize genome, and map their effect on aflatoxin accumulation via linkage and association mapping. In total, 13 LOX genes were identified, characterized, and mapped. The sequence of one gene, ZmLOX10, is reported from 5 inbred lines. Genes ZmLOX1/2, 5, 8, 9, 10 and 12 (GRMZM2G156861, or V4 numbers ZM00001D042541 and Zm00001D042540, GRMZM2G102760, GRMZM2G104843, GRMZM2G017616, GRMZM2G015419, and GRMZM2G106748, respectively) fell under previously published QTL in one or more mapping populations and are linked to a measurable reduction of aflatoxin in maize grains. Association mapping results found 28 of the 726 SNPs tested were associated with reduced aflatoxin levels at p ≤ 9.71 x 10-4 according to association statistics. These fell within or near nine of the ZmLOX genes. This work confirms the importance of some lipoxygenases for resistance to aflatoxin accumulation and may be used to direct future genetic selection in maize.

A gene encoding maize caffeoyl-CoA O-methyltransferase confers quantitative resistance to multiple pathogens.

Alleles that confer multiple disease resistance (MDR) are valuable in crop improvement, although the molecular mechanisms underlying their functions remain largely unknown. A quantitative trait locus, qMdr9.02, associated with resistance to three important foliar maize diseases-southern leaf blight, gray leaf spot and northern leaf blight-has been identified on maize chromosome 9. Through fine-mapping, association analysis, expression analysis, insertional mutagenesis and transgenic validation, we demonstrate that ZmCCoAOMT2, which encodes a caffeoyl-CoA O-methyltransferase associated with the phenylpropanoid pathway and lignin production, is the gene within qMdr9.02 conferring quantitative resistance to both southern leaf blight and gray leaf spot. We suggest that resistance might be caused by allelic variation at the level of both gene expression and amino acid sequence, thus resulting in differences in levels of lignin and other metabolites of the phenylpropanoid pathway and regulation of programmed cell death.