SlWRKY45 Interacts with Jasmonate-ZIM Domain Proteins to Negatively Regulate Defense Against Root Knot Nematode Meloidogyne incognita in Tomato

Root knot nematode (RKN), a kind of plant parasitic nematodes, leads to large reduction of crop yield, and seriously damages the agricultural production. The phytohormone jasmonates (JAs) act as important signals to regulate resistance against multiple abiotic and biotic stresses. However, little is known about the mechanism of JA-mediated defense responses against RKN in tomato. In this study, we found that the WRKY transcription factor SlWRKY45 interacts with most of the Jasmonate-ZIM domain proteins (JAZs) in yeast and plant. Overexpression of SlWRKY45 decreased plant resistance to RKN Meloidogyne incognita with increased gall index. We further generated slwrky45 mutants using the CRISPR/Cas9 technology, and discovered that the gall index and the number of nematodes and females in slwrky45 mutants are significantly reduced compared with wild type, as inoculated with RKN Meloidogyne incognita. Moreover, the contents of jasmonic acid and JA-isoleucine (JA-Ile) were highly increased in slwrky45 mutants with RKN Meloidogyne incognita infection compared with wild type. Furthermore, EMSA, and Dual-LUC assays demonstrated that SlWRKY45 directly binds and represses jasmonate biosynthesis gene ALLENE OXIDE CYCLASE ( AOC). Overall, our findings reveled that JAZ-interaction protein SlWRKY45 negatively controls plant defense against RKN Meloidogyne incognita by the regulation of JA biosynthesis in tomato.

A Laminarin-Based Formulation Protects Wheat Against Zymoseptoria tritici Via Direct Antifungal Activity and Elicitation of Host Defense-Related Genes

The present study aimed to evaluate the potential of the laminarin-based formulation Vacciplant® to protect and induce resistance in wheat against Zymoseptoria tritici, a major pathogen on this crop. Under greenhouse conditions, a single foliar spraying of the product two days before inoculation with Z. tritici reduced disease severity and pycnidium density by 42% and 45%, respectively. Vacciplant® exhibited a direct antifungal activity on Z. tritici conidial germination both in vitro and in planta. Moreover, it reduced in planta substomatal colonization as well as pycnidium formation on treated leaves. Molecular investigations revealed that Vacciplant® elicits but did not prime the expression of several wheat genes related to defense pathways, including phenylpropanoids (phenylalanine ammonia-lyase and chalcone synthase), octadecanoids (lipoxygenase and allene oxide synthase), and pathogenesis‐related proteins (β‐1,3‐endoglucanase and chitinase). By contrast, it did not modulate the expression of oxalate oxidase gene involved in the reactive oxygen species metabolism. UHPLC-MS analysis indicated limited changes in leaf metabolome after product application in both non-inoculated and inoculated conditions, suggesting a low metabolic cost associated with induction of plant resistance. This study provides evidence that the laminarin-based formulation confers protection to wheat against Z. tritici through direct antifungal activity and elicitation of plant defense-associated genes.

Developing a platform for production of the oxylipin KODA 9-hydroxy-10-oxo-octadecadienoic acid in plants

KODA (9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid) is a plant oxylipin involved in recovery from stress. As an agrichemical, KODA helps maintain crop production under various environmental stresses. In plants, KODA is synthesized from α-linolenic acids via 9-lipoxygenase (9-LOX) and allene oxide synthase (AOS), although the amount is usually low except in the free-floating aquatic plant Lemna paucicostata. To improve KODA biosynthetic yield in other plants such as Nicotiana benthamiana and Arabidopsis thaliana, we developed a system to overproduce KODA in vivo via ectopic expression of L. paucicostata 9-LOX and AOS. The transient expression in N. benthamiana showed that the expression of these two genes is sufficient to produce KODA in leaves. However, stable expression of 9-LOX and AOS (with consequent KODA production) in Arabidopsis plants succeeded only when the two proteins were localized in plastids or the endoplasmic reticulum/lipid droplets. Although only small amounts of KODA could be detected in leaf extracts of transgenic Nicotiana or Arabidopsis plants, subsequent incubation of the extracts increased KODA abundance over time. Therefore, KODA production in transgenic plants stably expressing 9-LOX and AOS requires specific subcellular localization of these two enzymes and incubation of leaf crude extracts, which liberates α-linolenic acid via breakdown of endogenous lipids.

Defense responses of arbuscular mycorrhizal fungus-colonized poplar seedlings against gypsy moth larvae: a multiomics study

Arbuscular mycorrhizal (AM) fungi may help protect plants against herbivores; however, their use for the pest control of woody plants requires further study. Here, we investigated the effect of Glomus mosseae colonization on the interactions between gypsy moth larvae and Populus alba × P. berolinensis seedlings and deciphered the regulatory mechanisms underlying the mycorrhizal-induced resistance in the leaves of mycorrhizal poplar using RNA-seq and nontargeted metabolomics. The resistance assay showed that AM fungus inoculation protected poplar seedlings against gypsy moth larvae, as evidenced by the decreased larval growth and reduced larval survival. A transcriptome analysis revealed that differentially expressed genes (DEGs) were involved in jasmonic acid biosynthesis (lipoxygenase, hydroperoxide dehydratase, and allene oxide cyclase) and signal transduction (jasmonate-ZIM domain and transcription factor MYC2) and identified the genes that were upregulated in mycorrhizal seedlings. Except for chalcone synthase and anthocyanidin synthase, which were downregulated in mycorrhizal seedlings, all DEGs related to flavonoid biosynthesis were upregulated, including 4-coumarate-CoA ligase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, and leucoanthocyanidin reductase. The metabolome analysis showed that several metabolites with insecticidal properties, including coumarin, stachydrine, artocarpin, norizalpinin, abietic acid, 6-formylumbelliferone, and vanillic acid, were significantly accumulated in the mycorrhizal seedlings. These findings suggest the potential of mycorrhiza-induced resistance for use in pest management of woody plants and demonstrate that the priming of JA-dependent responses in poplar seedlings contributes to mycorrhiza-induced resistance to insect pests.

Turnip mosaic virus P1 suppresses JA biosynthesis by degrading cpSRP54 that delivers AOCs onto the thylakoid membrane to facilitate viral infection

Jasmonic acid (JA) is a crucial hormone in plant antiviral immunity. Increasing evidence shows that viruses counter this host immune response by interfering with JA biosynthesis and signaling. However, the mechanism by which viruses affect JA biosynthesis is still largely unexplored. Here, we show that a highly conserved chloroplast protein cpSRP54 was downregulated in Nicotiana benthamiana infected by turnip mosaic virus (TuMV). Its silencing facilitated TuMV infection. Furthermore, cpSRP54 interacted with allene oxide cyclases (AOCs), key JA biosynthesis enzymes, and was responsible for delivering AOCs onto the thylakoid membrane (TM). Interestingly, TuMV P1 protein interacted with cpSRP54 and mediated its degradation via the 26S proteosome and autophagy pathways. The results suggest that TuMV has evolved a strategy, through the inhibition of cpSRP54 and its delivery of AOCs to the TM, to suppress JA biosynthesis and enhance viral infection. Interaction between cpSRP54 and AOCs was shown to be conserved in Arabidopsis and rice, while cpSRP54 also interacted with, and was degraded by, pepper mild mosaic virus (PMMoV) 126 kDa protein and potato virus X (PVX) p25 protein, indicating that suppression of cpSRP54 may be a common mechanism used by viruses to counter the antiviral JA pathway.

Poplar Allene Oxide Synthase 1 Gene Promoter Drives Rapid and Localized Expression by Wounding

Promoters play critical roles in controlling the transcription of genes and are important as tools to drive heterologous expression for biotechnological applications. In addition to core transcription factor-binding motifs that assist in the binding of RNA polymerases, there are specific nucleotide sequences in a promoter region to allow regulation of gene expression. The allene oxide synthase (AOS) gene family are cytochrome P450s that are responsive to a variety of environmental stress, making them good candidates for the discovery of inducible promoters. Populus AOS homologs separate phylogenetically into two clades. Based on the 19 promoter motifs with significant abundance differences between the two clades, Clade I AOS genes are likely more responsive to hormones, salt, and pathogen, whereas clade II homologs are likely inducible by water stress. In this study, an upstream promoter from a Clade I poplar AOS encoding gene (AOS1) was cloned and used to drive the expression of a ß-glucuronidase (GUS) gene in Arabidopsis. AOS is an essential enzyme in the lipoxygenase pathway that is responsible for the production of many non-volatile oxylipins in plants, including the jasmonates, which are regulatory phytohormones coordinating a variety of biological and stress response functions. Consistent with AOS transcript expression patterns, we found that the poplar AOS1 promoter drives rapid and localized expression by wounding. The study provides insight on the responsive elements in the poplar AOS promoters, but more importantly identifies a strong wound-inducible and localized promoter for future applications.

Transcriptomic and Metabolomic Analyses Reveals That Exogenous Methyl Jasmonate Regulates Galanthamine Biosynthesis in Lycoris longituba Seedlings

The Amaryllidaceae alkaloid galanthamine (Gal) in Lycoris longituba is a secondary metabolite that has been used to treat Alzheimer’s disease. Plant secondary metabolism is affected by methyl jasmonate (MeJA) exposure, although the regulatory mechanisms of MeJA on L. longituba seedlings remains largely unknown. In the present study, 75, 150, and 300 μM MeJA were used as treatments on L. longituba seedlings for 7, 14, 21, and 28 days, while 0 μM MeJA was used as the control (MJ-0). The effect of exogenous MeJA on Gal synthesis in L. longituba was then investigated using transcriptomic sequencing and metabolite profiling via GC-MS and LC-MS analysis. Galanthamine (Gal), lycorine (Lyc), and lycoramine (Lycm) abundances were 2. 71-, 2. 01-, and 2.85-fold higher in 75 μM MeJA (MJ-75) treatment plants compared to MJ-0 treatment plants after 7 days of cultivation. Transcriptomic analysis further showed that MJ-75 treatment significantly induced the expression of norbelladine synthase (NBS) and norbelladine 4′-O-methyltransferase (OMT), which are involved in the Gal biosynthesis pathway. In addition, increased expression was observed in MJ-75 treatment plants for genes in the JA synthesis and JA signaling pathways including those of allene oxide cyclase (AOC), 12-oxo-phytodienoic acid reductase (OPR), jasmonic acid amino acid synthase (JAR), and transcription factor MYC. The L. longituba tyrosine decarboxylase (LlTYDC) enzyme was identified and proposed to be involved in the Gal biosynthetic pathway. Metabolomics results demonstrated that the accumulation of Amaryllidaceae alkaloids, and especially alkaloids in the Gal biosynthesis pathway, could be induced by MJ-75 treatment. Interestingly, metabolites in the JA synthesis pathway were also affected by MeJA treatment. Overall, this multi-omics study suggests that both the JA synthesis/JA signaling and Gal biosynthesis pathways were affected by exogenous MeJA treatment. This comprehensive study of gene expression and metabolite contents can help us better understand the molecular mechanisms underlying MeJA-mediated Gal biosynthesis in L. longituba.

The Algal Polysaccharide Ulvan Induces Resistance in Wheat Against Zymoseptoria tritici Without Major Alteration of Leaf Metabolome

This study aimed to examine the ability of ulvan, a water-soluble polysaccharide from the green seaweed Ulva fasciata, to provide protection and induce resistance in wheat against the hemibiotrophic fungus Zymoseptoria tritici. Matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) analysis indicated that ulvan is mainly composed of unsaturated monosaccharides (rhamnose, rhamnose-3-sulfate, and xylose) and numerous uronic acid residues. In the greenhouse, foliar application of ulvan at 10 mg.ml–1 2 days before fungal inoculation reduced disease severity and pycnidium density by 45 and 50%, respectively. Ulvan did not exhibit any direct antifungal activity toward Z. tritici, neither in vitro nor in planta. However, ulvan treatment significantly reduced substomatal colonization and pycnidium formation within the mesophyll of treated leaves. Molecular assays revealed that ulvan spraying elicits, but does not prime, the expression of genes involved in several wheat defense pathways, including pathogenesis-related proteins (β-1,3-endoglucanase and chitinase), reactive oxygen species metabolism (oxalate oxidase), and the octadecanoid pathway (lipoxygenase and allene oxide synthase), while no upregulation was recorded for gene markers of the phenylpropanoid pathway (phenylalanine ammonia-lyase and chalcone synthase). Interestingly, the quantification of 83 metabolites from major chemical families using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) in both non-infectious and infectious conditions showed no substantial changes in wheat metabolome upon ulvan treatment, suggesting a low metabolic cost associated with ulvan-induced resistance. Our findings provide evidence that ulvan confers protection and triggers defense mechanisms in wheat against Z. tritici without major modification of the plant physiology.