The maize lipoxygenase,ZmLOX10, mediates green leaf volatile,jasmonate and herbivore‐induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro‐economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13‐LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10‐derived signaling is required for LOX8‐mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound‐induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore‐induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV‐, JA‐ and HIPV‐deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10‐dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro‐ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.     

Duplicate maize 13-lipoxygenase genes are differentially regulated by circadian rhythm, cold stress, wounding, pathogen infection, and hormonal treatments

Most plant oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and their derivatives, are produced through the lipoxygenase (LOX) pathway. Recent progress in dicots has highlighted the biological roles of oxylipins in plant defence responses to pathogens and pests. By contrast, the physiological function of LOXs and their metabolites in monocots is poorly understood. Two maize LOXs, ZmLOX10 and ZmLOX11 that share >90% amino acid sequence identity but are localized on different chromosomes, were cloned and characterized. Phylogenetic analysis revealed that ZmLOX10 and ZmLOX11 cluster together with well-characterized plastidic type 2 linoleate 13-LOXs from diverse plant species. Regio-specificity analysis of recombinant ZmLOX10 protein overexpressed in Escherichia coli proved it to be a linoleate 13-LOX with a pH optimum at approximately pH 8.0. Both predicted proteins contain putative transit peptides for chloroplast import. ZmLOX10 was preferentially expressed in leaves and was induced in response to wounding, cold stress, defence-related hormones jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA), and inoculation with an avirulent strain of Cochliobolus carbonum. These data suggested a role for this gene in maize adaptation to abiotic stresses and defence responses against pathogens and pests. ZmLOX11 was preferentially expressed in silks and was induced in leaves only by ABA, indicating its possible involvement in responses to osmotic stress. In leaves, mRNA accumulation of ZmLOX10 is strictly regulated by a circadian rhythm, with maximal expression coinciding temporally with the highest photosynthetic activity. This study reveals the evolutionary divergence of physiological roles for relatively recently duplicated genes. Possible physiological functions of these 13-LOXs are suggested.     

Disruption of a maize 9-lipoxygenase results in increased resistance to fungal pathogens and reduced levels of contamination with mycotoxin fumonisin

Plant oxylipins, produced via the lipoxygenase (LOX) pathway, function as signals in defense and development. In fungi, oxylipins are potent regulators of mycotoxin biosynthesis and sporogenesis. Previous studies showed that plant 9-LOX-derived fatty acid hydroperoxides induce conidiation and mycotoxin production. Here, we tested the hypothesis that oxylipins produced by the maize 9-LOX pathway are required by pathogens to produce spores and mycotoxins and to successfully colonize the host. Maize mutants were generated in which the function of a 9-LOX gene, ZmLOX3, was abolished by an insertion of a Mutator transposon in its coding sequence, which resulted in reduced levels of several 9-LOX-derived hydroperoxides. Supporting our hypothesis, conidiation and production of the mycotoxin fumonisin B1 by Fusarium verticillioides were drastically reduced in kernels of the lox3 mutants compared with near-isogenic wild types. Similarly, conidia production and disease severity of anthracnose leaf blight caused by Colletotrichum graminicola were significantly reduced in the lox3 mutants. Moreover, lox3 mutants displayed increased resistance to southern leaf blight caused by Cochliobolus heterostrophus and stalk rots caused by both F. verticillioides and C. graminicola. These data strongly suggest that oxylipin metabolism mediated by a specific plant 9-LOX isoform is required for fungal pathogenesis, including disease development and production of spores and mycotoxins.     

Reciprocal oxylipin-mediated cross-talk in the Aspergillus–seed pathosystem

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans , oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene ( ZmLOX3 ) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans , and into a Δ ppoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2–3 expression was decreased when infected by A. nidulans Δ ppo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus –seed pathosystem.     

Maize 9-lipoxygenase ZmLOX3 controls development, root-specific expression of defense genes, and resistance to root-knot nematodes

Root-knot nematodes (RKN) are severe pests of maize. Although lipoxygenase (LOX) pathways and their oxylipin products have been implicated in plant-nematode interactions, prior to this report there was no conclusive genetic evidence for the function of any plant LOX gene in such interactions. We showed that expression of a maize 9-LOX gene, ZmLOX3, increased steadily and peaked at 7 days after inoculation with Meloidogyne incognita RKN. Mu-insertional lox3-4 mutants displayed increased attractiveness to RKN and an increased number of juveniles and eggs. A set of jasmonic acid (JA)- and ethylene (ET)-responsive and biosynthetic genes as well as salicylic acid (SA)-dependent genes were overexpressed specifically in the roots of lox3-4 mutants. Consistent with this, levels of JA, SA, and ET were elevated in lox3-4 mutant roots, but not in leaves. Unlike wild types, in lox3-4 mutant roots, a phenylalanine ammonia lyase (PAL) gene was not RKN-inducible, suggesting a role for PAL-mediated metabolism in nematode resistance. In addition to these alterations in the defense status of roots, lox3-4 knockout mutants displayed precocious senescence and reduced root length and plant height compared with the wild type, suggesting that ZmLOX3 is required for normal plant development. Taken together, our data indicate that the ZmLOX3-mediated pathway may act as a root-specific suppressor of all three major defense signaling pathways to channel plant energy into growth processes, but is required for normal levels of resistance against nematodes.     

Fsr1, a striatin homologue,forms an endomembrane‐associated complex that regulates virulence in the maize pathogen Fusarium verticillioides

Fsr1, a homologue of mammalian striatin, containing multiple protein‐binding domains and a coiled‐coil (CC) domain, is critical for Fusarium verticillioides virulence. In mammals, striatin interacts with multiple proteins to form a STRIPAK (striatin‐interacting phosphatase and kinase) complex that regulates a variety of developmental processes and cellular mechanisms. In this study, we identified the homologue of a key mammalian STRIPAK component STRIP1/2 (striatin‐interacting proteins 1 and 2) in F. verticillioides, FvStp1, which interacts with Fsr1 in vivo. Gene deletion analysis indicates that FvStp1 is critical for F. verticillioides stalk rot virulence. In addition, we identified three proteins, designated FvCyp1, FvScp1 and FvSel1, which interact with the Fsr1 CC domain via a yeast two‐hybrid screen. Importantly, FvCyp1, FvScp1 and FvSel1 co‐localize to endomembrane structures, each having a preferred localization in the cell, and they are all required for F. verticillioides stalk rot virulence. Moreover, these proteins are necessary for the correct localization of Fsr1 to the endoplasmic reticulum (ER) and nuclear envelope. Thus, we identified several novel components in the STRIPAK complex that regulates F. verticillioides virulence, and propose that the correct organization and localization of Fsr1 are critical for STRIPAK complex function.     

Oxidation of glycerolipids by maize 9-lipoxygenase and its A562G mutant

Lipoxygenases (LOXs) are key enzymes in the biosynthesis of oxylipins, the diverse class of bioregulators involved into developmental processes, signalling and defence. This work was undertaken to better understand how LOXs control production of hydroperoxides with different positional and stereochemistry. A number of glycerolipids were tested as substrates for maize 9-LOX (ZmLOX) and its A562G mutant form. Both the wild type (WT) ZmLOX and A562G mutant were shown to dioxygenate monolinolenoylglycerol (MLG) and 2-linoleoyl-sn-glycero-3-phosphorylcholine (lysoPC). Both the WT ZmLOX and A562G mutant form oxidized the MLG predominantly into (9S)-hydroperoxide. The A562G mutation did not affect the relative yield of 13-hydroperoxide, but increased the proportion of (13R)-enantiomer. LysoPC was a poor substrate for both wild type and A562G mutant form of ZmLOX. The oxidation of lysoPC exhibited the limited regio- and stereospecificity. Nevertheless, the WT ZmLOX produced some predominance of (13S)-hydroperoxide. In contrast, the A562G mutant produced some excess of (9S)-hydroperoxide of lysoPC. The bulky polar heads of glycerolipids like MLG and lysoPC cannot penetrate into the LOX active site. Thus, the obtained data indicate that both (9S)- and (13S)-hydroperoxides can be produced when substrate is arranged within LOX active site in the “methyl end first” orientation.     

A novel plastidial lipoxygenase of maize (Zea mays) ZmLOX6 encodes for a fatty acid hydroperoxide lyase and is uniquely regulated by phytohormones and pathogen infection

Lipoxygenases (LOXs) are members of a large enzyme family that catalyze oxygenation of free polyunsaturated fatty acids into diverse hydroperoxide compounds, collectively called oxylipins. Although LOXs have been well studied in dicot species, reports of the genes encoding these enzymes are scarce for monocots, especially maize. Herein, we reported the cloning, characterization and molecular functional analysis of a novel maize LOX gene, ZmLOX6. The ZmLOX6 nucleotide sequence encodes a deduced translation product of 892 amino acids. Phylogenetic analysis showed that ZmLOX6 is distantly related to previously reported 9- or 13-LOXs from maize and other plant species, including rice and Arabidopsis. Although sequence prediction suggested cytoplasmic localization of this protein, ZmLOX6 protein has been reportedly isolated from mesophyll cell chloroplasts, emphasizing the unique features of this protein. Plastidial localization was confirmed by chloroplast uptake experiments with the in vitro translated protein. Analysis of recombinant protein revealed that ZmLOX6 has lost fatty acid hydroperoxide forming activity but 13-LOX-derived fatty acid hydroperoxides were cleaved into odd-chain ω-oxo fatty acids and as yet not identified C5-compound. In line with its reported abundance in mesophyll cells, ZmLOX6 was predominantly expressed in leaf tissue. Northern blot analysis demonstrated that ZmLOX6 was induced by jasmonic acid, but repressed by abscisic acid, salicylic acid and ethylene and was not responsive to wounding or insects. Further, this gene was strongly induced by the fungal pathogen Cochliobolus carbonum during compatible interactions, suggesting that ZmLOX6 may contribute to susceptibility to this pathogen. The potential involvement of ZmLOX6 in maize interactions with pathogens is discussed.     

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.     

Fusarium Species from the Fusarium fujikuroi Species Complex Involved in Mixed Infections of Maize in Northern Sinaloa,Mexico

Fusarium species belonging to the Fusarium fujikuroi species complex (FFSC) are associated with maize in northern Mexico and cause Fusarium ear and root rot. In order to assess the diversity of FFSC fungal species involved in this destructive disease in Sinaloa, Mexico, a collection of 108 fungal isolates was obtained from maize plants in 2007–2011. DNA sequence analysis of the calmodulin and elongation factor 1α genes identified four species: Fusarium verticillioides, F. nygamai, F. andiyazi and F. thapsinum (comprising 79, 23, 4 and 2 isolates, respectively). Differential distribution of Fusarium species in maize organs was observed, that is F. verticillioides was the most frequently isolated species from maize seeds, while F. nygamai predominated on maize roots. Mixed infections with F. verticillioides/F. thapsinum and F. verticillioides/F. nygamai were detected in maize seeds and roots, respectively. Pathogenicity assay demonstrated the ability of the four species to infect maize seedlings and induce different levels of disease severity, reflecting variation in aggressiveness, plant height and root biomass. Isolates of F. verticillioides and F. nygamai were the most aggressive. These species were able to colonize all root tissues, from the epidermis to the vascular vessels, while infection by F. andiyazi and F. thapsinum was restricted to the epidermis and adjacent cortical cells. This is the first report of F. nygamai, F. andiyazi and F. thapsinum infecting maize in Mexico and co‐infecting with F. verticillioides. Mixed infections should be taken into consideration due to the production and/or accumulation of diverse mycotoxins in maize grain.     

Fumonisin B1, a toxin produced by <Emphasis Type="Italic">Fusarium verticillioides</Emphasis>, modulates maize β-1,3-glucanase activities involved in defense response

Fusarium verticillioides is an important pathogen in maize that causes various diseases affecting all stages of plant development worldwide. The fungal pathogen could be seed borne or survive in soil and penetrate the germinating seed. Most F. verticillioides strains produce fumonisins, which are of concern because of their toxicity to animals and possibly humans, and because they enhance virulence against seedlings of some maize genotypes. In this work, we studied the action of fumonisin B1 (FB1) on the activity of maize β-1,3-glucanases involved in plant defense response. In maize embryos, FB1 induced an acidic isoform while suppressing the activity of two basic isoforms. This acidic isoform was induced also with 2,6-dichloroisonicotinic acid, an analog of salicylic acid. Repression of the basic isoforms suggested a direct interaction of the enzymes with the mycotoxin as in vitro experiments showed that pure FB1 inhibited the basic β-1,3-glucanases with an IC₅₀ of 53 μM. When germinating maize embryos were inoculated with F. verticillioides the same dual effect on β-1,3-glucanase activities that we observed with the pure toxin was reproduced. Similar levels of FB1 were recovered at 24 h germination in maize tissue when they were treated with pure FB1 or inoculated with an FB1-producing strain. These results suggest that β-1,3-glucanases are a relevant physiological target and their modulation by FB1 might contribute to F. verticillioides colonization.     

A 13‐lipoxygenase is Expressed Early in the Hypersensitive Reaction of Cotton Plants to Xanthomonas campestris pv. malvacearum

Lipoxygenases (LOXs) are enzymes responsible for lipid peroxidation processes during plant defence responses to pathogen infection. Jasmonates are lipid‐derived signals that mediate plant stress responses with chloroplastic LOXs implicated in the biosynthesis of oxylipins like jasmonic acid (JA). Hypersensitive reaction (HR) cell death of cotton to the incompatible race 18 of Xanthomonas campestris pathovar malvacearum (Xcm) is associated with 9S‐lipoxygenase activity and expression of a 9‐LOX GhLOX1. Here, we report the cloning of cotton (Gossypium hirsutum L.) LOX gene GhLOX2. Sequence analysis showed that GhLOX2 is a putative 13‐LOX with a chloroplast‐transit peptide in the amino acid terminus. GhLOX2 was found to be significantly expressed in the first hour of Xcm‐induced HR. Investigation into LOX signalization on cotyledons incubated with methyl‐jasmonate (MeJA) or infiltrated with salicylic acid (SA) or ethylene (ET) revealed that the first two treatments induced GhLOX2 gene expression. Our results show that GhLOX2 gene expression occurred at the stage of the HR prior biochemical events previously highlighted. The role that GhLOX2 may have in the defence strategy of cotton to Xcm is discussed regarding the HR.