Engineered coumarin accumulation reduces mycotoxin-induced oxidative stress and disease susceptibility.

Coumarins can fight pathogens and are thus promising for crop protection. Their biosynthesis, however, has not yet been engineered in crops. We tailored the constitutive accumulation of coumarins in transgenic Nicotiana benthamiana, Glycine max and Arabidopsis thaliana plants, as well as in Nicotiana tabacum BY-2 suspension cells. We did so by overexpressing A. thaliana feruloyl-CoA 6-hydroxylase 1 (AtF6'H1), encoding the key enzyme of scopoletin biosynthesis. Besides scopoletin and its glucoside scopolin, esculin at low level was the only other coumarin detected in transgenic cells. Mechanical damage of scopolin-accumulating tissue led to a swift release of scopoletin, presumably from the scopolin pool. High scopolin levels in A. thaliana roots coincided with reduced susceptibility to the root-parasitic nematode Heterodera schachtii. In addition, transgenic soybean plants were more tolerant to the soil-borne pathogenic fungus Fusarium virguliforme. Because mycotoxin-induced accumulation of reactive oxygen species and cell death were reduced in the AtF6'H1-overexpressors, the weaker sensitivity to F. virguliforme may be caused by attenuated oxidative damage of coumarin-hyperaccumulating cells. Together, engineered coumarin accumulation is promising for enhanced disease resilience of crops.

The genome and lifestage-specific transcriptomes of a plant-parasitic nematode and its host reveal susceptibility genes involved in trans-kingdom synthesis of vitamin B5.

Plant-parasitic nematodes are a major threat to crop production in all agricultural systems. The scarcity of classical resistance genes highlights a pressing need to find new ways to develop nematode-resistant germplasm. Here, we sequence and assemble a high-quality phased genome of the model cyst nematode Heterodera schachtii to provide a platform for the first system-wide dual analysis of host and parasite gene expression over time, covering all major parasitism stages. Analysis of the hologenome of the plant-nematode infection site identified metabolic pathways that were incomplete in the parasite but complemented by the host. Using a combination of bioinformatic, genetic, and biochemical approaches, we show that a highly atypical completion of vitamin B5 biosynthesis by the parasitic animal, putatively enabled by a horizontal gene transfer from a bacterium, is required for full pathogenicity. Knockout of either plant-encoded or now nematode-encoded steps in the pathway significantly reduces parasitic success. Our experiments establish a reference for cyst nematodes, further our understanding of the evolution of plant-parasitism by nematodes, and show that congruent differential expression of metabolic pathways in the infection hologenome represents a new way to find nematode susceptibility genes. The approach identifies genome-editing-amenable targets for future development of nematode-resistant crops.

Mode of action of fluopyram in plant-parasitic nematodes.

Plant-parasitic nematodes (PPN) are responsible for severe yield losses in crop production. Management is challenging as effective and safe means are rare. Recently, it has been discovered that the succinate dehydrogenase (SDH) inhibitor fluopyram is highly effective against PPN while accompanying an excellent safety profile. Here we show that fluopyram is a potent inhibitor of SDH in nematodes but not in mammals, insects and earthworm, explaining the selectivity on molecular level. As a consequence of SDH inhibition, fluopyram impairs ATP generation and causes paralysis in PPN and Caenorhabditis elegans. Interestingly, efficacy differences of fluopyram amongst PPN species can be observed. Permanent exposure to micromolar to nanomolar amounts of fluopyram prevents Meloidogyne spp. and Heterodera schachtii infection and their development at the root. Preincubation of Meloidogyne incognita J2 with fluopyram followed by a recovery period effectively reduces gall formation. However, the same procedure does not inhibit H. schachtii infection and development. Sequence comparison of sites relevant for ligand binding identified amino acid differences in SDHC which likely mediate selectivity, coincidently revealing a unique amino acid difference within SDHC conserved among Heterodera spp. Docking and C. elegans mutant studies suggest that this minute difference mediates altered sensitivity of H. schachtii towards fluopyram.

Heterologous Production of ß-Caryophyllene and Evaluation of Its Activity against Plant Pathogenic Fungi.

Terpenoids constitute one of the largest and most diverse groups within the class of secondary metabolites, comprising over 80,000 compounds. They not only exhibit important functions in plant physiology but also have commercial potential in the biotechnological, pharmaceutical, and agricultural sectors due to their promising properties, including various bioactivities against pathogens, inflammations, and cancer. In this work, we therefore aimed to implement the plant sesquiterpenoid pathway leading to ß-caryophyllene in the heterologous host Rhodobacter capsulatus and achieved a maximum production of 139 ± 31 mg L-1 culture. As this sesquiterpene offers various beneficial anti-phytopathogenic activities, we evaluated the bioactivity of ß-caryophyllene and its oxygenated derivative ß-caryophyllene oxide against different phytopathogenic fungi. Here, both compounds significantly inhibited the growth of Sclerotinia sclerotiorum and Fusarium oxysporum by up to 40%, while growth of Alternaria brassicicola was only slightly affected, and Phoma lingam and Rhizoctonia solani were unaffected. At the same time, the compounds showed a promising low inhibitory profile for a variety of plant growth-promoting bacteria at suitable compound concentrations. Our observations thus give a first indication that ß-caryophyllene and ß-caryophyllene oxide are promising natural agents, which might be applicable for the management of certain plant pathogenic fungi in agricultural crop production.

The Plant Sesquiterpene Nootkatone Efficiently Reduces Heterodera schachtii Parasitism by Activating Plant Defense.

Plant parasitic nematodes, including the beet cyst nematode Heterodera schachtii, constitute a devastating problem for crops worldwide. The limited availability of sustainable management options illustrates the need for new eco-friendly control means. Plant metabolites represent an invaluable source of active compounds for the discovery of such novel antagonistic agents. Here, we evaluated the impact of eight plant terpenoids on the H. schachtii parasitism of Arabidopsis thaliana. None of the metabolites affected the plant development (5 or 10 ppm). Nootkatone decreased the number of adult nematodes on A. thaliana to 50%, with the female nematodes being smaller compared to the control. In contrast, three other terpenoids increased the parasitism and/or female size. We discovered that nootkatone considerably decreased the number of nematodes that penetrated A. thaliana roots, but neither affected the nematode viability or attraction to plant roots, nor triggered the production of plant reactive oxygen species or changed the plant's sesquiterpene profile. However, we demonstrated that nootkatone led to a significant upregulation of defense-related genes involved in salicylic and jasmonic acid pathways. Our results indicate that nootkatone is a promising candidate to be developed into a novel plant protection agent acting as a stimulator of plant immunity against parasitic nematodes.

Novel Prodiginine Derivatives Demonstrate Bioactivities on Plants, Nematodes, and Fungi.

Bacterial metabolites represent an invaluable source of bioactive molecules which can be used as such or serve as chemical frameworks for developing new antimicrobial compounds for various applications including crop protection against pathogens. Prodiginines are tripyrrolic, red-colored compounds produced by many bacterial species. Recently, due to the use of chemical-, bio-, or mutasynthesis, a novel group of prodiginines was generated. In our study, we perform different assays to evaluate the effects of prodigiosin and five derivatives on nematodes and plant pathogenic fungi as well as on plant development. Our results showed that prodigiosin and the derivatives were active against the bacterial feeding nematode Caenorhabditis elegans in a concentration- and derivative-dependent manner while a direct effect on infective juveniles of the plant parasitic nematode Heterodera schachtii was observed for prodigiosin only. All compounds were found to be active against the plant pathogenic fungi Phoma lingam and Sclerotinia sclerotiorum. Efficacy varied depending on compound concentration and chemical structure. We observed that prodigiosin (1), the 12 ring- 9, and hexenol 10 derivatives are neutral or even positive for growth of Arabidopsis thaliana depending on the applied compound concentration, whereas other derivatives appear to be suppressive. Our infection assays revealed that the total number of developed H. schachtii individuals on A. thaliana was decreased to 50% in the presence of compounds 1 or 9. Furthermore, female nematodes and their associated syncytia were smaller in size. Prodiginines seem to indirectly inhibit H. schachtii parasitism of the plant. Further research is needed to elucidate their mode of action. Our results indicate that prodiginines are promising metabolites that have the potential to be developed into novel antinematodal and antifungal agents.

Signatures of adaptation to a monocot host in the plant-parasitic cyst nematode Heterodera sacchari.

Interactions between plant-parasitic nematodes and their hosts are mediated by effectors, i.e. secreted proteins that manipulate the plant to the benefit of the pathogen. To understand the role of effectors in host adaptation in nematodes, we analysed the transcriptome of Heterodera sacchari, a cyst nematode parasite of rice (Oryza sativa) and sugarcane (Saccharum officinarum). A multi-gene phylogenetic analysis showed that H. sacchari and the cereal cyst nematode Heterodera avenae share a common evolutionary origin and that they evolved to parasitise monocot plants from a common dicot-parasitic ancestor. We compared the effector repertoires of H. sacchari with those of the dicot parasites Heterodera glycines and Globodera rostochiensis to understand the consequences of this transition. While, in general, effector repertoires are similar between the species, comparing effectors and non-effectors of H. sacchari and G. rostochiensis shows that effectors have accumulated more mutations than non-effectors. Although most effectors show conserved spatiotemporal expression profiles and likely function, some H. sacchari effectors are adapted to monocots. This is exemplified by the plant-peptide hormone mimics, the CLAVATA3/EMBRYO SURROUNDING REGION-like (CLE) effectors. Peptide hormones encoded by H. sacchari CLE effectors are more similar to those from rice than those from other plants, or those from other plant-parasitic nematodes. We experimentally validated the functional significance of these observations by demonstrating that CLE peptides encoded by H. sacchari induce a short root phenotype in rice, whereas those from a related dicot parasite do not. These data provide a functional example of effector evolution that co-occurred with the transition from a dicot-parasitic to a monocot-parasitic lifestyle.

Transcriptome and Parasitome Analysis of Beet Cyst Nematode Heterodera schachtii.

Beet cyst nematodes depend on a set of secretory proteins (effectors) for the induction and maintenance of their syncytial feeding sites in plant roots. In order to understand the relationship between the beet cyst nematode H. schachtii and its host, identification of H. schachtii effectors is crucial and to this end, we sequenced a whole animal pre-infective J2-stage transcriptome in addition to pre- and post-infective J2 gland cell transcriptome using Next Generation Sequencing (NGS) and identified a subset of sequences representing putative effectors. Comparison between the transcriptome of H. schachtii and previously reported related cyst nematodes and root-knot nematodes revealed a subset of esophageal gland related sequences and putative effectors in common across the tested species. Structural and functional annotation of H. schachtii transcriptome led to the identification of nearly 200 putative effectors. Six putative effector expressions were quantified using qPCR and three of them were functionally analyzed using RNAi. Phenotyping of the RNAi nematodes indicated that all tested genes decrease the level of nematodes pathogenicity and/or the average female size, thereby regulating cyst nematode parasitism. These discoveries contribute to further understanding of the cyst nematode parasitism.

Engineered Rhodobacter capsulatus as a Phototrophic Platform Organism for the Synthesis of Plant Sesquiterpenoids.

Sesquiterpenoids are a large class of natural compounds offering manifold properties valuable for food, cosmetics, agriculture, and pharma industry. Production in microorganisms is a sustainable approach to provide sesquiterpenoids for research and industrial use independent of their natural sources. This requires the functional transfer of the respective biocatalytic pathways in an adequate host microorganism offering a sufficient supply of precursors that is ideally adjusted to the individual demand of the recombinant biosynthesis route. The phototrophic purple bacterium Rhodobacter capsulatus offers unique physiological properties that are favorable for biosynthesis of hydrophobic terpenes. Under phototrophic conditions, it develops a large intracytoplasmic membrane suitable for hosting membrane-bound enzymes and metabolites of respective biosynthetic pathways. In addition, Rhodobacter harbors an intrinsic carotenoid biosynthesis that can be engineered toward the production of foreign terpenes. Here, we evaluate R. capsulatus as host for the production of plant sesquiterpenoids under phototrophic conditions using patchoulol and valencene as a proof of concept. The heterologous expression of patchoulol synthase PcPS from Pogostemon cablin as well as the valencene synthases CsVS from Citrus sinensis and CnVS from Callitropsis nootkatensis led to the production of the respective sesquiterpenoids in R. capsulatus. To analyze, if gradually adjustable formation of the key precursor farnesylpyrophosphate (FPP) is beneficial for sesquiterpene synthesis under phototrophic conditions, the intrinsic 1-deoxy-D-xylulose 5-phosphate (DXP) pathway genes as well as the heterologous mevalonate pathway genes were modularly expressed in various combinations. To this end, different plasmids and chromosomally integrated expression tools were developed harboring the strong and tightly controlled P nif promoter for heterologous gene expression. Notably, comparative studies identified a distinct combination of precursor biosynthetic genes as best-performing setup for each of the tested sesquiterpene synthases. In summary, we could demonstrate that R. capsulatus is a promising alternative platform organism that is suited for sustainable sesquiterpenoid formation under phototrophic cultivation conditions. A modular engineering of R. capsulatus strains via tailored co-expression of FPP biosynthetic genes further allowed adaptation of sesquiterpene precursor formation to its catalytic conversion by different plant terpene synthases.

Identification and characterization of a putative protein disulfide isomerase (HsPDI) as an alleged effector of Heterodera schachtii.

The plant-parasitic nematode Heterodera schachtii is an obligate biotroph that induces syncytial feeding sites in roots of its hosts. Nematodes produce effectors that are secreted into the host and facilitate infection process. Here we identified H. schachtii protein disulphide isomerase (HsPDI) as a putative effector that interferes with the host's redox status. In situ hybridization showed that HsPdi is specifically localized within esophageal glands of pre-parasitic second stage juveniles (J2). HsPdi is up-regulated in the early parasitic J2s. Silencing of HsPdi by RNA interference in the J2s hampers their development and leads to structural malfunctions in associated feeding sites induced in Arabidopsis roots. Expression of HsPDI in Arabidopsis increases plant's susceptibility towards H. schachtii. HsPdi expression is up-regulated in the presence of exogenous H2O2, whereas HsPdi silencing results in increased mortality under H2O2 stress. Stable expression of HsPDI in Arabidopsis plants decreases ROS burst induced by flg22. Transiently expressed HsPDI in N. benthamiana leaves is localized in the apoplast. HsPDI plays an important role in the interaction between nematode and plant, probably through inducing local changes in the redox status of infected host tissue. It also contributes to protect the nematode from exogenous H2O2 stress.

Damage-associated responses of the host contribute to defence against cyst nematodes but not root-knot nematodes.

When nematodes invade and subsequently migrate within plant roots, they generate cell wall fragments (in the form of oligogalacturonides; OGs) that can act as damage-associated molecular patterns and activate host defence responses. However, the molecular mechanisms mediating damage responses in plant-nematode interactions remain unexplored. Here, we characterized the role of a group of cell wall receptor proteins in Arabidopsis, designated as polygalacturonase-inhibiting proteins (PGIPs), during infection with the cyst nematode Heterodera schachtii and the root-knot nematode Meloidogyne incognita. PGIPs are encoded by a family of two genes in Arabidopsis, and are involved in the formation of active OG elicitors. Our results show that PGIP gene expression is strongly induced in response to cyst nematode invasion of roots. Analyses of loss-of-function mutants and overexpression lines revealed that PGIP1 expression attenuates infection of host roots by cyst nematodes, but not root-knot nematodes. The PGIP1-mediated attenuation of cyst nematode infection involves the activation of plant camalexin and indole-glucosinolate pathways. These combined results provide new insights into the molecular mechanisms underlying plant damage perception and response pathways during infection by cyst and root-knot nematodes, and establishes the function of PGIP in plant resistance to cyst nematodes.

Heterodera schachtii Tyrosinase-like protein - a novel nematode effector modulating plant hormone homeostasis.

The beet cyst nematode Heterodera schachtii causes major yield losses in sugar beet. Understanding the interaction between H. schachtii and its host plant is important for developing a sustainable management system. Nematode effectors play a crucial role in initializing and sustaining successful parasitism. In our study, we identified a gene (Hs-Tyr) encoding a tyrosinase functional domain (PF00264). We describe Hs-Tyr as a novel nematode effector. Hs-Tyr is localized in the nematode esophageal gland. Up-regulation of its expression coincided with the parasitic developmental stages of the nematode. Silencing Hs-Tyr by RNA interference made the treated nematodes less virulent. When RNAi-treated nematodes succeeded in infecting the plant, developing females and their associated syncytial nurse cells were significantly smaller than in control plants. Ectopically expressing the Hs-Tyr effector in Arabidopsis increased plant susceptibility to H. schachtii, but not to the root-knot nematode Meloidogyne incognita. Interestingly, Hs-Tyr in the plant promoted plant growth and changed the root architecture. Additionally, the expression of Hs-Tyr in Arabidopsis caused changes in the homeostasis of several plant hormones especially auxin and the ethylene precursor aminocyclopropane-carboxylic acid.

Identification of Two Meloidogyne hapla Genes and an Investigation of Their Roles in the Plant-Nematode Interaction.

Root-knot nematodes are soil-borne pathogens that invade and establish feeding sites in plant roots. They have an extremely broad host range, including most vascular plants. During infection of a susceptible host, root-knot nematodes secrete molecules called effectors that help them establish an intimate interaction with the plant and, at the same time, allow them to evade or suppress plant immune responses. Despite the fact that Meloidogyne hapla is a significant pest on several food crops, no effectors have been characterized from this root-knot nematode species thus far. Using the published genome and proteome from M. hapla, we have identified and characterized two genes, MhTTL2 and Mh265. MhTTL2 encodes a predicted secreted protein containing a transthyretin-like protein domain. The expression of MhTTL2 was up-regulated during parasitic life stages of the nematode, and in situ hybridization showed that MhTTL2 was expressed in the amphids, suggesting it has a role in the nematode nervous system during parasitism. We also studied the gene Mh265. The Mh265 transcript was localized to the subventral esophageal glands. An upregulation in Mh265 expression coincided with the pre- and early-parasitic life stages of the nematode. When Mh265 was constitutively expressed in plants, it enhanced their susceptibility to nematodes. These transgenic plants were also compromised in flg22-induced callose deposition, suggesting the Mh265 is modulating plant basal immune responses.

A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants.

Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.

Phosphonate fertilizers suppressed root knot nematodes Meloidogyne javanica and M. incognita.

The efficacy of the phosphonate fertilizers, Calphos(®) (a.i. calcium phosphonate), Magphos(®) (a.i. magnesium phosphonate and potassium phosphonate) and Phosphoros(®) (a.i. potassium phosphonate) against two species of root knot nematodes (RKN), Meloidogyne javanica and M. incognita is evaluated. Laboratory experiments showed that Calphos(®), Magphos(®) and their main components inhibited egg hatching and caused 100% mortality of the second stage juveniles (J2s) of the two RKN species; the hatching inhibition effects persisted after transferring the egg masses of both species to water. However, Phosphoros(®) (0.5%) did not suppress egg hatching or the survival of J2s of both RKN species. No hatching occurred when egg masses were treated for one week with the nematicide Vydate L(®) (2 ml/l), however, J2s hatched when the Vydate L(®) treated egg masses were moved to water. The glasshouse study indicated that Magphos(®), Calphos(®) and Phosphoros(®) reduced root galling caused by M. javanica by 98, 66 and 47%, respectively, in comparison to the untreated controls. Magphos(®) resulted in the lowest number of root galls formed by M. incognita, the reduction was 84%. In contrast, Calphos(®) and Phosphoros(®) reduced galling by 47 and 39%, respectively. The Magphos(®) treatment resulted in the lowest numbers of egg masses and the lowest reproductive factor (RF) of both nematode species. However, plants treated with Phosphoros(®) resulted in higher foliage weights compared with the application of the other two fertilizers and the untreated plants.