Redox signalling in plant-nematode interactions: Insights into molecular crosstalk and defense mechanisms.

Plant-parasitic nematodes, specifically cyst nematodes (CNs) and root-knot nematodes (RKNs), pose significant threats to global agriculture, leading to substantial crop losses. Both CNs and RKNs induce permanent feeding sites in the root of their host plants, which then serve as their only source of nutrients throughout their lifecycle. Plants deploy reactive oxygen species (ROS) as a primary defense mechanism against nematode invasion. Notably, both CNs and RKNs have evolved sophisticated strategies to manipulate the host's redox environment to their advantage, with each employing distinct tactics to combat ROS. In this review, we have focused on the role of ROS and its scavenging network in interactions between host plants and CNs and RKNs. Overall, this review emphasizes the complex interplay between plant defense mechanism, redox signalling and nematode survival tactics, suggesting potential avenues for developing innovative nematode management strategies in agriculture.

ROS Detection and Quantification in Plant-Nematode Interactions.

Reactive oxygen species (ROS) accumulation is one of the earliest hallmarks upon successful pathogen recognition in plants. H2O2 is considered the most important ROS in plant defense considering its relatively high stability and capacity to cross long distances in the plant. However, ROS also play roles in cell development and could hence facilitate nematode feeding site development. Several methods to analyze the cellular redox state exist, among which ROS detection and quantification and the evaluation of ROS scavenging enzyme activity (peroxidase activity, catalase activity, etc.). Here, we describe DAB staining, which is used to detect and localize ROS in planta upon an external trigger. Furthermore, ROS quantification using the FOX assay is described. Both methods have been used extensively in research and yield repeatable results in various plants.

Molecular analysis of broad-spectrum induced resistance in rice by the green leaf volatile Z-3-hexenyl acetate.

Green leaf volatiles (GLVs), volatile organic compounds released by plants upon tissue damage, are key signaling molecules in plant immunity. The ability of exogenous GLV application to trigger an induced resistance (IR) phenotype against arthropod pests has been widely reported, but its effectiveness against plant pathogens is less well understood. In this study, we combined mRNA sequencing-based transcriptomics and phytohormone measurements with multispectral imaging-based precision phenotyping to gain insights into the molecular basis of Z-3-hexenyl acetate-induced resistance (Z-3-HAC-IR) in rice. Furthermore, we evaluated the efficacy of Z-3-HAC-IR against a panel of economically significant rice pathogens: Pyricularia oryzae, Rhizoctonia solani, Xanthomonas oryzae pv. oryzae, Cochliobolus miyabeanus, and Meloidogyne graminicola. Our data revealed rapid induction of jasmonate metabolism and systemic induction of plant immune responses upon Z-3-HAC exposure, as well as a transient allocation cost due to accelerated chlorophyll degradation and nutrient remobilization. Z-3-HAC-IR proved effective against all tested pathogens except for C. miyabeanus, including against the (hemi)biotrophs M. graminicola, X. oryzae pv. oryzae, and P. oryzae. The Z-3-HAC-IR phenotype was lost in the jasmonate (JA)-deficient hebiba mutant, which confirms the causal role of JA in Z-3-HAC-IR. Together, our results show that GLV exposure in rice induces broad-spectrum, JA-mediated disease resistance with limited allocation costs, and may thus be a promising alternative crop protection approach.

Potential of Exogenous Treatment with Dehydroascorbate to Control Root-knot Nematode Infection in Rice.

Induced resistance (IR) is a unique physiological state characterized by reduced plant susceptibility to (a)biotic stress. Our previous studies showed that exogenous foliar application of dehydroascorbate (DHA), the oxidized form of ascorbic acid, induces systemic resistance against root-knot nematode Meloidogyne graminicola in rice. In the present study, the potential of DHA in protecting rice plants against M. graminicola was evaluated in lab, pot, and field studies. In an experiment where the interval between foliar treatment and inoculation was varied, 20 mM DHA was found to protect rice plants from M. graminicola for at least 14 days. Pot and field studies confirmed that 10 or 20 mM DHA are highly effective in reducing gall formation and led to a significant increase in rice seed yield. A half dose of DHA (10 mM) combined with another IR-stimulus - piperonylic acid (PA) 300 µM - was at par with DHA 20 mM, leading to reductions in gall formation of more than 80%. In in vitro bioassays, DHA was found to be highly nematicidal to the second-stage juveniles of M. graminicola, with more than 90% mortality within 3 h of exposure to 10 or 20 mM concentrations. While seed treatment had no effect, root drenching or root dipping was also effective in reducing rice susceptibility to M. graminicola, next to foliar treatment. As a dual-action compound with extended protection and ease of application, DHA has great potential for effective nematode management in rice.

Dicer-like 3a mediates intergenerational resistance against root-knot nematodes in rice via hormone responses.

In a continuously changing and challenging environment, passing down the memory of encountered stress factors to offspring could provide an evolutionary advantage. In this study, we demonstrate the existence of "intergenerational acquired resistance" in the progeny of rice (Oryza sativa) plants attacked by the belowground parasitic nematode Meloidogyne graminicola. Transcriptome analyses revealed that genes involved in defense pathways are generally downregulated in progeny of nematode-infected plants under uninfected conditions but show a stronger induction upon nematode infection. This phenomenon was termed "spring loading" and depends on initial downregulation by the 24-nucleotide (nt) siRNA biogenesis gene dicer-like 3a (dcl3a) involved in the RNA-directed DNA methylation pathway. Knockdown of dcl3a led to increased nematode susceptibility and abolished intergenerational acquired resistance, as well as jasmonic acid/ethylene spring loading in the offspring of infected plants. The importance of ethylene signaling in intergenerational resistance was confirmed by experiments on a knockdown line of ethylene insensitive 2 (ein2b), which lacks intergenerational acquired resistance. Taken together, these data indicate a role for DCL3a in regulating plant defense pathways during both within-generation and intergenerational resistance against nematodes in rice.

Diproline-induced resistance to parasitic nematodes in the same and subsequent rice generations: Roles of iron, nitric oxide and ethylene.

Induced resistance (IR) is a plant phenotype characterized by lower susceptibility to biotic challenges upon elicitation by so-called IR stimuli. Earlier, we identified diproline (cyclo(l-Pro-l-Pro)) as IR stimulus that protects rice (Oryza sativa) against the root-knot nematode Meloidogyne graminicola (Mg). In the current study, detailed transcriptome analyses at different time points, and under uninfected and nematode-infected conditions revealed that this rice IR phenotype is correlated with induction of genes related to iron (Fe), ethylene (ET) and reactive oxygen species (ROS)/reactive nitrogen species (RNS) metabolism. An infection experiment under Fe limiting conditions confirmed that diproline-IR is only effective under optimal Fe supply. Although total root Fe levels were not affected in diproline-treated plants, phytosiderophore secretion was found to be induced by this treatment. Experiments on mutant and transgenic rice lines impaired in ET or ROS/RNS metabolism confirmed that these metabolites are involved in diproline-IR. Finally, we provide evidence for transgenerational inheritance of diproline-IR (diproline-TIR), as two successive generations of diproline-treated ancestors exhibited an IR phenotype while themselves never being exposed to diproline. Transcriptome analyses on the offspring plants revealed extensive overlap between the pathways underpinning diproline-IR and diproline-TIR. Although diproline induces significant systemic changes in global DNA methylation levels early after treatment, such changes in DNA methylation were not detected in the descendants of these plants. To our knowledge, this is the first report of TIR in rice and the first transcriptional assessment of TIR in monocots.

ß-Aminobutyric acid induced phytotoxicity and effectiveness against nematode is stereomer-specific and dose-dependent in tomato.

ß-Aminobutyric acid (BABA) induces resistance to a/biotic stress but is associated with phytotoxicity in some plant species. There are two enantiomers of BABA, the R and S enantiomers. We evaluated the phytotoxicity caused by the RS BABA (racemic mixture of R and S BABA), evaluating the dose-response effect and different modes of application on tomato. Results show that RS BABA-induced phytotoxicity in tomato is dose-dependent and stronger with foliar applications than with soil drench. We further evaluated the phytotoxicity of the two enantiomers separately and observed that BABA-induced phytotoxicity is stereomer-specific. In comparison with less phytotoxic effects induced by S BABA, R BABA induces dose-dependent and systemic phytotoxic symptoms. To investigate the possible physiological causes of this phytotoxicity, we measured levels of oxidative stress and anthocyanins and validated the findings with gene expression analyses. Our results show that high doses of RS and R BABA induce hydrogen peroxide, lipid peroxidation, and anthocyanin accumulation in tomato leaves, while this response is milder and more transient upon S BABA application. Next, we evaluated BABA induced resistance against root-knot nematode Meloidogyne incognita in tomato. BABA-induced resistance was found to be stereomer-specific and dependent on dose and mode of application. R or RS BABA multiple soil drench application at low doses induces resistance to nematodes with less phytotoxic effects. Taken together, our data provide useful knowledge on how BABA can be applied in crop production by enhancing stress tolerance and limiting phytotoxicity.

Opposing effects of trans- and cis-cinnamic acid during rice coleoptile elongation.

The phenylpropanoid cinnamic acid (CA) is a plant metabolite that can occur under a trans- or cis-form. In contrast to the proven bioactivity of the cis-form (c-CA), the activity of trans-CA (t-CA) is still a matter of debate. We tested both compounds using a submerged rice coleoptile assay and demonstrated that they have opposite effects on cell elongation. Notably, in the tip of rice coleoptile t-CA showed an inhibiting and c-CA a stimulating activity. By combining transcriptomics and (untargeted) metabolomics with activity assays and genetic and pharmacological experiments, we aimed to explain the underlying mechanistic processes. We propose a model in which c-CA treatment activates proton pumps and stimulates acidification of the apoplast, which in turn leads to the loosening of the cell wall, necessary for elongation. We hypothesize that c-CA also inactivates auxin efflux transporters, which might cause a local auxin accumulation in the tip of the coleoptile. For t-CA, the phenotype can partially be explained by a stimulation of cell wall polysaccharide feruloylation, leading to a more rigid cell wall. Metabolite profiling also demonstrated that salicylic acid (SA) derivatives are increased upon t-CA treatment. As SA is a known antagonist of auxin, the shift in SA homeostasis provides an additional explanation of the observed t-CA-mediated restriction on cell growth.

Distinct chemical resistance-inducing stimuli result in common transcriptional, metabolic, and nematode community signatures in rice root and rhizosphere.

Induced resistance (IR), a phenotypic state induced by an exogenous stimulus and characterized by enhanced resistance to future (a)biotic challenge, is an important component of plant immunity. Numerous IR-inducing stimuli have been described in various plant species, but relatively little is known about 'core' systemic responses shared by these distinct IR stimuli and the effects of IR on plant-associated microbiota. In this study, rice (Oryza sativa) leaves were treated with four distinct IR stimuli (ß-aminobutyric acid, acibenzolar-S-methyl, dehydroascorbic acid, and piperonylic acid) capable of inducing systemic IR against the root-knot nematode Meloidogyne graminicola and evaluated their effect on the root transcriptome and exudome, and root-associated nematode communities. Our results reveal shared transcriptional responses-notably induction of jasmonic acid and phenylpropanoid metabolism-and shared alterations to the exudome that include increased amino acid, benzoate, and fatty acid exudation. In rice plants grown in soil from a rice field, IR stimuli significantly affected the composition of rhizosphere nematode communities 3 d after treatment, but by 14 d after treatment these changes had largely reverted. Notably, IR stimuli did not reduce nematode diversity, which suggests that IR might offer a sustainable option for managing plant-parasitic nematodes.

Genetic disruption of Arabidopsis secondary metabolite synthesis leads to microbiome-mediated modulation of nematode invasion.

In-depth understanding of metabolite-mediated plant-nematode interactions can guide us towards novel nematode management strategies. To improve our understanding of the effects of secondary metabolites on soil nematode communities, we grew Arabidopsis thaliana genetically altered in glucosinolate, camalexin, or flavonoid synthesis pathways, and analyzed their root-associated nematode communities using metabarcoding. To test for any modulating effects of the associated microbiota on the nematode responses, we characterized the bacterial and fungal communities. Finally, as a proxy of microbiome-modulating effects on nematode invasion, we isolated the root-associated microbiomes from the mutants and tested their effect on the ability of the plant parasitic nematode Meloidogyne incognita to penetrate tomato roots. Most mutants had altered relative abundances of several nematode taxa with stronger effects on the plant parasitic Meloidogyne hapla than on other root feeding taxa. This probably reflects that M. hapla invades and remains embedded within root tissues and is thus intimately associated with the host. When transferred to tomato, microbiomes from the flavonoid over-producing pap1-D enhanced M. incognita root-invasion, whereas microbiomes from flavonoid-deficient mutants reduced invasion. This suggests microbiome-mediated effect of flavonoids on Meloidogyne infectivity plausibly mediated by the alteration of the abundances of specific microbial taxa in the transferred microbiomes, although we could not conclusively pinpoint such causative microbial taxa.

Dehydroascorbate induces plant resistance in rice against root-knot nematode Meloidogyne graminicola.

Ascorbic acid (AsA) is an important antioxidant in plants and regulates various physiological processes. In this study, we show that exogenous treatments with the oxidized form of AsA, that is, dehydroascorbate (DHA), activates induced systemic resistance in rice against the root-knot nematode Meloidogyne graminicola, and investigate the molecular and biochemical mechanisms underlying this phenotype. Detailed transcriptome analysis on roots of rice plants showed an early and robust transcriptional response on foliar DHA treatment, with induction of several genes related to plant stress responses, immunity, antioxidant activity, and secondary metabolism already at 1 day after treatment. Quantitative and qualitative evaluation of H2 O2 levels confirmed the appearance of a reactive oxygen species (ROS) burst on DHA treatment, both at the site of treatment and systemically. Experiments using chemical ROS inhibitors or scavengers confirmed that H2 O2 accumulation contributes to DHA-based induced resistance. Furthermore, hormone measurements in DHA-treated plants showed a significant systemic accumulation of the defence hormone salicylic acid (SA). The role of the SA pathway in DHA-based induced resistance was confirmed by nematode infection experiments using an SA-signalling deficient WRKY45-RNAi line and reverse transcription-quantitative PCR on SA marker genes. Our results collectively reveal that DHA activates induced systemic resistance in rice against the root-knot nematode M. graminicola, mediated through the production of ROS and activation of the SA pathway.

Rice diterpenoid phytoalexins are involved in defence against parasitic nematodes and shape rhizosphere nematode communities.

Rice diterpenoid phytoalexins (DPs) are secondary metabolites with a well known role in resistance to foliar pathogens. As DPs are also known to be produced and exuded by rice roots, we hypothesised that they might play an important role in plant-nematode interactions, and particularly in defence against phytoparasitic nematodes. We used transcriptome analysis on rice roots to analyse the effect of infection by the root-knot nematode Meloidogyne graminicola or treatment with resistance-inducing chemical stimuli on DP biosynthesis genes, and assessed the susceptibility of mutant rice lines impaired in DP biosynthesis to M. graminicola. Moreover, we grew these mutants and their wild-type in field soil and used metabarcoding to assess the effect of impairment in DP biosynthesis on rhizosphere and root nematode communities. We show that M. graminicola suppresses DP biosynthesis genes early in its invasion process and, conversely, that resistance-inducing stimuli transiently induce the biosynthesis of DPs. Moreover, we show that loss of DPs increases susceptibility to M. graminicola. Metabarcoding on wild-type and DP-deficient plants grown in field soil reveals that DPs significantly alter the composition of rhizosphere and root nematode communities. Diterpenoid phytoalexins are important players in basal and inducible defence against nematode pathogens of rice and help shape rice-associated nematode communities.

Spatiotemporal expression profile of novel and known small RNAs throughout rice plant development focussing on seed tissues.

BACKGROUND: Small RNAs (sRNAs) regulate numerous plant processes directly related to yield, such as disease resistance and plant growth. To exploit this yield-regulating potential of sRNAs, the sRNA profile of one of the world's most important staple crops - rice - was investigated throughout plant development using next-generation sequencing. RESULTS: Root and leaves were investigated at both the vegetative and generative phase, and early-life sRNA expression was characterized in the embryo and endosperm. This led to the identification of 49,505 novel sRNAs and 5581 tRNA-derived sRNAs (tsRNAs). In all tissues, 24 nt small interfering RNAs (siRNAs) were highly expressed and associated with euchromatic, but not heterochromatic transposable elements. Twenty-one nt siRNAs deriving from genic regions in the endosperm were exceptionally highly expressed, mimicking previously reported expression levels of 24 nt siRNAs in younger endosperm samples. In rice embryos, sRNA content was highly diverse while tsRNAs were underrepresented, possibly due to snoRNA activity. Publicly available mRNA expression and DNA methylation profiles were used to identify putative siRNA targets in embryo and endosperm. These include multiple genes related to the plant hormones gibberellic acid and ethylene, and to seed phytoalexin and iron content. CONCLUSIONS: This work introduces multiple sRNAs as potential regulators of rice yield and quality, identifying them as possible targets for the continuous search to optimize rice production.

Beneficial worm allies warn plants of parasite attack below-ground and reduce above-ground herbivore preference and performance.

Antagonistic interactions among different functional guilds of nematodes have been recognized for quite some time, but the underlying explanatory mechanisms are unclear. We investigated responses of tomato (Solanum lycopersicum) to two functional guilds of nematodes-plant parasite (Meloidogyne javanica) and entomopathogens (Heterorhabditis bacteriophora, Steinernema feltiae below-ground, and S. carpocapsae)-as well as a leaf mining insect (Tuta absoluta) above-ground. Our results indicate that entomopathogenic nematodes (EPNs): (1) reduced root knot nematode (RKN) infestation below-ground, (2) reduced herbivore (T. absoluta) host preference and performance above-ground, and (3) induced overlapping plant defence responses by rapidly activating polyphenol oxidase and guaiacol peroxidase activity in roots, but simultaneously suppressing this activity in above-ground tissues. Concurrently, we investigated potential plant signalling mechanisms underlying these interactions using transcriptome analyses. We found that both entomopathogens and plant parasites triggered immune responses in plant roots with shared gene expression. Secondary metabolite transcripts induced in response to the two nematode functional guilds were generally overlapping and showed an analogous profile of regulation. Likewise, we show that EPNs modulate plant defence against RKN invasion, in part, by suppressing active expression of antioxidant enzymes. Inoculations of roots with EPN triggered an immune response in tomato via upregulated phenylpropanoid metabolism and synthesis of protease inhibitors in plant tissues, which may explain decreased egg laying and developmental performance exhibited by herbivores on EPN-inoculated plants. Furthermore, changes induced in the volatile organic compound-related transcriptome indicated that M. javanica and/or S. carpocapsae inoculation of plants triggered both direct and indirect defences. Our results support the hypothesis that plants "mistake" subterranean EPNs for parasites, and these otherwise beneficial worms activate a battery of plant defences associated with systemic acquired resistance and/or induced systemic resistance with concomitant antagonistic effects on temporally co-occurring subterranean plant pathogenic nematodes and terrestrial herbivores.

Short-term effects of cadmium on leaf growth and nutrient transport in rice plants.

Consumption of rice grains contaminated with high concentrations of cadmium (Cd) can cause serious long-term health problems. Moreover, even low Cd concentrations present in the soil can result in the abatement of plant performance, leading to lower grain yield. Studies examining the molecular basis of plant defense against Cd-induced oxidative stress could pave the way in creating superior rice varieties that display an optimal antioxidative defense system to cope with Cd toxicity. In this study, we showed that after one day of Cd exposure, hydroponically grown rice plants exhibited adverse shoot biomass and leaf growth effects. Cadmium accumulates especially in the roots and the leaf meristematic region, leading to a disturbance of manganese homeostasis in both the roots and leaves. The leaf growth zone showed an increased amount of lipid peroxidation indicating that Cd exposure disturbed the oxidative balance. We propose that an increased expression of genes related to the glutathione metabolism such as glutathione synthetase 2, glutathione reductase and phytochelatin synthase 2, rather than genes encoding for antioxidant enzymes, is important in combating early Cd toxicity within the leaves of rice plants. Furthermore, the upregulation of two RESPIRATORY BURST OXIDASE HOMOLOG genes together with a Cd concentration-dependent increase of abscisic acid might cause stomatal closure or cell wall modification, potentially leading to the observed leaf growth reduction. Whereas abscisic acid was also elevated at long term exposure, a decrease of the growth hormone auxin might further contribute to growth inhibition and concomitantly, an increase in salicylic acid might stimulate the activity of antioxidative enzymes after a longer period of Cd exposure. In conclusion, a clear interplay between phytohormones and the oxidative challenge affect plant growth and acclimation during exposure to Cd stress.

Biochar-Enhanced Resistance to Botrytis cinerea in Strawberry Fruits (But Not Leaves) Is Associated With Changes in the Rhizosphere Microbiome.

Biochar has been reported to play a positive role in disease suppression against airborne pathogens in plants. The mechanisms behind this positive trait are not well-understood. In this study, we hypothesized that the attraction of plant growth-promoting rhizobacteria (PGPR) or fungi (PGPF) underlies the mechanism of biochar in plant protection. The attraction of PGPR and PGPF may either activate the innate immune system of plants or help the plants with nutrient uptake. We studied the effect of biochar in peat substrate (PS) on the susceptibility of strawberry, both on leaves and fruits, against the airborne fungal pathogen Botrytis cinerea. Biochar had a positive impact on the resistance of strawberry fruits but not the plant leaves. On leaves, the infection was more severe compared with plants without biochar in the PS. The different effects on fruits and plant leaves may indicate a trade-off between plant parts. Future studies should focus on monitoring gene expression and metabolites of strawberry fruits to investigate this potential trade-off effect. A change in the microbial community in the rhizosphere was also observed, with increased fungal diversity and higher abundances of amplicon sequence variants classified into Granulicella, Mucilaginibacter, and Byssochlamys surrounding the plant root, where the latter two were reported as biocontrol agents. The change in the microbial community was not correlated with a change in nutrient uptake by the plant in either the leaves or the fruits. A decrease in the defense gene expression in the leaves was observed. In conclusion, the decreased infection of B. cinerea in strawberry fruits mediated by the addition of biochar in the PS is most likely regulated by the changes in the microbial community.