Plant-nematode Interactions Research Articles

RNAi of esophageal gland-specific gene Mi-msp-1 alters early stage infection behaviour of root-knot nematode, Meloidogyne incognita

Root-knot nematodes are economically important plant pathogens that cause considerable damage to agricultural crops worldwide. They secrete a range of effector proteins from their specialized gland cells to modify host cell physiology to favour parasitism of the host tissue. A deeper understanding of the roles of effectors in nematode parasitism is necessary to unravel the mysteries of plant–nematode interactions. Herein, mRNAs of Mi-msp-1 (or Mi-vap-1), a gene encoding venom allergen-like effector protein, was localized to the subventral esophageal glands of Meloidogyne incognita. Mi-msp-1 was highly transcribed in the pre- and post-parasitic juveniles of M. incognita, suggesting the association of this effector gene during early stage infection behaviour of nematode. The targeted knockdown of Mi-msp-1 using in vitro RNAi rendered the nematode less successful in penetrating the tomato root. Silencing of Mi-msp-1 also interfered with the development and reproduction of M. incognita in adzuki bean. Intriguingly, induced suppression of Mi-msp-1 significantly altered the transcription of cell-wall-modifying enzymes (CWMEs, effectors involved in early parasitic process) and vice versa in M. incognita, indicating a possible interaction among msp-1 and CWMEs during the early stage of nematode–plant interaction. Our results showed that Mi-msp-1 could be a potential target/choke point for developing anti-nematode strategies.

Rice bean (Vigna umbellata) as an alternative to Adzuki bean (Vigna angularis) for culturing Meloidogyne incognita in soilless CYG germination pouches

Root-knot nematode, Meloidogyne incognita is a key global pest of agricultural and horticultural crops. The soil dwelling second-stage juveniles of the nematode species enter the plant roots and develop specialized feeding cells by modifying host plant physiology. Being obligate in nature the nematode cannot be multiplied in artificial media, and the traditional culturing is usually on soil-based tritrophic microcosm. The alternative use of CYG germination pouches provides a clean and contamination free culturing of the nematode species, where adzuki bean is used as a susceptible host. The present study introduces rice bean as an alternative to adzuki bean for maintenance of M. incognita in CYG germination pouches. The rice bean plants were found to harbour comparatively higher number of females leading to higher eggmass production than the adzuki plants. The reproductive fitness of M. incognita was not compromised in rice bean, and the maintenance of plants was easier due to determinate plant type with sturdy stems. This provides additional advantage of using rice bean in the germination pouches. Lastly, the rice bean plants offer clean and contamination free nematode culture which is highly suitable for downstream basic research to understand compatible/incompatible plant nematode interactions.

Functions of Flavonoids in Plant⁻Nematode Interactions.

Most land plants can become infected by plant parasitic nematodes in the field. Plant parasitic nematodes can be free-living or endoparasitic, and they usually infect plant roots. Most damaging are endoparasites, which form feeding sites inside plant roots that damage the root system and redirect nutrients towards the parasite. This process involves developmental changes to the root in parallel with the induction of defense responses. Plant flavonoids are secondary metabolites that have roles in both root development and plant defense responses against a range of microorganisms. Here, we review our current knowledge of the roles of flavonoids in the interactions between plants and plant parasitic nematodes. Flavonoids are induced during nematode infection in plant roots, and more highly so in resistant compared with susceptible plant cultivars, but many of their functions remain unclear. Flavonoids have been shown to alter feeding site development to some extent, but so far have not been found to be essential for root–parasite interactions. However, they likely contribute to chemotactic attraction or repulsion of nematodes towards or away from roots and might help in the general plant defense against nematodes. Certain flavonoids have also been associated with functions in nematode reproduction, although the mechanism remains unknown. Much remains to be examined in this area, especially under field conditions.

Molecular and Physiological Detection of Cyst Nematodes on Potato During Plant Nematode Interactions

Molecular and Physiological Detection of Cyst Nematodes on Potato During Plant Nematode Interactions

Imaging Mass Spectrometry of Endogenous Polypeptides and Secondary Metabolites from Galls Induced by Root-Knot Nematodes in Tomato Roots.

Nematodes are devastating pests that infect most cultivated plant species and cause considerable agricultural losses worldwide. The understanding of metabolic adjustments induced during plant-nematode interaction is crucial to generate resistant plants or to select more efficient molecules to fight against this pest. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used herein for in situ detection and mapping endogenous polypeptides and secondary metabolites from nematode-induced gall tissue. One of the major critical features of this technique is sample preparation; mainly, the generation of intact sections of plant cells with their rigid cell walls and vacuolated cytoplasm. Our experimental settings allowed us to obtain sections without contamination of exogenous ions or diffusion of molecules and to map the differential presence of low and high molecular weight ions in uninfected roots compared with nematode-induced galls. We predict the presence of lipids in both uninfected roots and galls, which was validated by MALDI time-of-flight tandem mass spectrometry and high-resolution mass spectrometry analysis of lipid extracts. Based on the isotopic ion distribution profile, both esters and glycerophospholipids were predicted compounds and may be playing an important role in gall development. Our results indicate that the MALDI-MSI technology is a promising tool to identify secondary metabolites as well as peptides and proteins in complex plant tissues like galls to decipher molecular processes responsible for infection and maintenance of these feeding sites during nematode parasitism.

Canonical and noncanonical ethylene signaling pathways that regulate Arabidopsis susceptibility to the cyst nematode Heterodera schachtii.

Plant-parasitic cyst nematodes successfully exploit various phytohormone signaling pathways to establish a new hormonal equilibrium that facilitates nematode parasitism. Although it is largely accepted that ethylene regulates plant responses to nematode infection, a mechanistic understanding of how ethylene shapes plant-nematode interactions remains largely unknown. In this study, we examined the involvement of various components regulating ethylene perception and signaling in establishing Arabidopsis susceptibility to the cyst nematode Heterodera schachtii using a large set of well-characterized single and higher order mutants. Our analyses revealed the existence of two pathways that separately engage ethylene with salicylic acid (SA) and cytokinin signaling during plant response to nematode infection. One pathway involves the canonical ethylene signaling pathway in which activation of ethylene signaling results in suppression of SA-based immunity. The second pathway involves the ethylene receptor ETR1, which signals independently of SA acid to affect immunity, instead altering cytokinin-mediated regulation of downstream components. Our results reveal important mechanisms through which cyst nematodes exploit components of ethylene perception and signaling to affect the balance of hormonal signaling through ethylene interaction with SA and cytokinin networks. This hormonal interaction overcomes plant defense and provokes a susceptible response.

Plant-Nematode Interactions Assisted by Microbes in the Rhizosphere.

Plant health is strongly influenced by the interactions between parasites/pathogens and beneficial microorganisms. In this chapter we will summarize the up-to date knowledge on soil suppressiveness as a biological tool against phytonematodes and explore the nature of monoculture versus crop rotation in this regard. Since nematodes are successfully antagonized by different microbiological agents, we highlighted this phenomenon with respect to the most important antagonists, and a nature of these interactions. The focus is on the hyperparasitic microbes of phytonematodes such as Pasteuria sp. and egg parasites. Furthermore, we comprised the studies on the defence system expressions in plants triggered by nematode-associated microbes. The attachment of bacteria and fungi to phytonematodes and putative effects of the attachment on the induced systemic resistance in plants are discussed. Finally, our chapter is rounded up with the importance of incorporating the knowledge on plant-nematode-microbe interactions in the integrated pest management.

Effect of homeopathic nematicide pellets on plant-nematode interaction under controlled conditions

Effect of homeopathic nematicide pellets on plant-nematode interaction under controlled conditions

A role for ALF4 during gall and giant cell development in the biotic interaction between Arabidopsis and Meloidogyne spp.

Root-knot nematodes (RKNs; Meloidogyne spp.) are a major pest for the agriculture worldwide. RKNs induce specialized feeding cells (giant cells, GCs) inside galls which are de novo formed pseudo-organs in the roots that share similarities with other developmental processes as lateral root (LR) and callus formation or grafting involving new vascular development or pericycle proliferation. Hence, it is pertinent to study the molecular mechanisms directing the plant-nematode interaction. In this respect, ALF4 is a key gene during LR formation, vascular vessels reconnection in grafting, hormone-induced callus formation or de novo root organogenesis from leaf explants. Our results show that ALF4 is also induced in galls at early infection stages in an auxin-independent way. Furthermore, ALF4 activity is necessary for the formation of proper galls and GCs, as the mutant alf4-1 presents aberrant galls and GCs with severe structural abnormalities leading to a dramatic reduction in the nematode egg production. However, a low-reproduction rate is maintained, that might be explained by the local auxin maximum build by the nematodes in galls, partially rescuing alf4-1 phenotype. This would be similar to the partial rescue described for LR formation with exogenous auxins and also agrees with the LR emergence from alf4-1 galls but not from uninfected roots. In addition, ALF4 is also induced in syncytia formed by cyst nematodes. All these data support a pivotal role for ALF4 during de novo organogenesis processes induced by endoparasitic nematodes, in addition to its role in LR formation, callus development or vessel reconnection during grafting.

Signal Transduction in Plant⁻Nematode Interactions.

To successfully invade and infect their host plants, plant parasitic nematodes (PPNs) need to evolve molecular mechanisms to overcome the defense responses from the plants. Nematode-associated molecular patterns (NAMPs), including ascarosides and certain proteins, while instrumental in enabling the infection, can be perceived by the host plants, which then initiate a signaling cascade leading to the induction of basal defense responses. To combat host resistance, some nematodes can inject effectors into the cells of susceptible hosts to reprogram the basal resistance signaling and also modulate the hosts’ gene expression patterns to facilitate the establishment of nematode feeding sites (NFSs). In this review, we summarized all the known signaling pathways involved in plant–nematode interactions. Specifically, we placed particular focus on the effector proteins from PPNs that mimic the signaling of the defense responses in host plants. Furthermore, we gave an updated overview of the regulation by PPNs of different host defense pathways such as salicylic acid (SA)/jasmonic acid (JA), auxin, and cytokinin and reactive oxygen species (ROS) signaling to facilitate their parasitic successes in plants. This review will enhance the understanding of the molecular signaling pathways involved in both compatible and incompatible plant–nematode interactions.

The Novel Secreted Meloidogyne incognita Effector MiISE6 Targets the Host Nucleus and Facilitates Parasitism in Arabidopsis.

Meloidogyne incognita is highly specialized parasite that interacts with host plants using a range of strategies. The effectors are synthesized in the esophageal glands and secreted into plant cells through a needle-like stylet during parasitism. In this study, based on RNA-seq and bioinformatics analysis, we predicted 110 putative Meloidogyne incognita effectors that contain nuclear localization signals (NLSs). Combining the Burkholderia glumae–pEDV based screening system with subcellular localization, from 20 randomly selected NLS effector candidates, we identified an effector MiISE6 that can effectively suppress B. glumae-induced cell death in Nicotiana benthamiana, targets to the nuclei of plant cells, and is highly expressed in early parasitic J2 stage. Sequence analysis showed that MiISE6 is a 157-amino acid peptide, with an OGFr_N domain and two NLS motifs. Hybridization in situ verified that MiISE6 is expressed in the subventral esophageal glands. Yeast invertase secretion assay validated the function of the signal peptide harbored in MiISE6. Transgenic Arabidopsis thaliana plants expressing MiISE6 become more susceptible to M. incognita. Inversely, the host-derived RNAi of MiISE6 of the nematode can decrease its parasitism on host. Based on transcriptome analysis of the MiISE6 transgenic Arabidopsis samples and the wild-type samples, we obtained 852 differentially expressed genes (DEGs). Integrating Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, we found that expression of MiISE6 in Arabidopsis can suppress jasmonate signaling pathway. In addition, the expression of genes related to cell wall modification and the ubiquitination proteasome pathway also have detectable changes in the transgenic plants. Results from the present study suggest that MiISE6 is involved in interaction between nematode-plant, and plays an important role during the early stages of parasitism by interfering multiple signaling pathways of plant. Moreover, we found homologs of MiISE6 in other sedentary nematodes, Meloidogyne hapla and Globodera pallida. Our experimental results provide evidence to decipher the molecular mechanisms underlying the manipulation of host immune defense responses by plant parasitic nematodes, and transcriptome data also provide useful information for further study nematode–plant interactions.

The Role of Programmed Cell Death Regulator LSD1 in Nematode-Induced Syncytium Formation.

Cyst-forming plant-parasitic nematodes are common pests of many crops. They inject secretions into host cells to induce the developmental and metabolic reprogramming that leads to the formation of a syncytium, which is the sole food source for growing nematodes. As in other host-parasite models, avirulence leads to rapid and local programmed cell death (PCD) known as the hypersensitive response (HR), whereas in the case of virulence, PCD is still observed but is limited to only some cells. Several regulators of PCD were analyzed to understand the role of PCD in compatible plant–nematode interactions. Thus, Arabidopsis plants carrying recessive mutations in LESION SIMULATING DISEASE1 (LSD1) family genes were subjected to nematode infection assays with juveniles of Heterodera schachtii. LSD1 is a negative and conditional regulator of PCD, and fewer and smaller syncytia were induced in the roots of lsd1 mutants than in wild-type Col-0 plants. Mutation in LSD ONE LIKE2 (LOL2) revealed a pattern of susceptibility to H. schachtii antagonistic to lsd1. Syncytia induced on lsd1 roots compared to Col0 showed significantly retarded growth, modified cell wall structure, increased vesiculation, and some myelin-like bodies present at 7 and 12 days post-infection. To place these data in a wider context, RNA-sequencing analysis of infected and uninfected roots was conducted. During nematode infection, the number of transcripts with changed expression in lsd1 was approximately three times smaller than in wild-type plants (1440 vs. 4206 differentially expressed genes, respectively). LSD1-dependent PCD in roots is thus a highly regulated process in compatible plant–nematode interactions. Two genes identified in this analysis, coding for AUTOPHAGY-RELATED PROTEIN 8F and 8H were down-regulated in syncytia in the presence of LSD1 and showed an increased susceptibility to nematode infection contrasting with lsd1 phenotype. Our data indicate that molecular regulators belonging to the LSD1 family play an important role in precise balancing of diverse PCD players during syncytium development required for successful nematode parasitism.

SRNAs involved in the regulation of plant developmental processes are altered during the root-knot nematode interaction for feeding site formation

Plant parasitic root- knot nematodes (RKNs; Meloidogyne spp.) are a significant threat for the agriculture as they infect multiple crops, causing severe economic losses worldwide. Most of the effective chemical nematicides have been or are in the process of being banned on the basis of their harmful effect to the environment and human health. These nematodes penetrate the root, migrate intercellularly and establish in the vascular cylinder. Then, nematode effectors delivered into the plant cells alter the plant development to induce their feeding cells, called giant cells (GCs) that serve to nourish them till life cycle completion. The GCs are embedded into a swelling in the root, called gall, formed by the proliferation and/or hypertrophy of the surrounding tissues. Further studies on the development of the nematode-feeding sites, i.e. to deepen into the molecular bases of the plant-nematode interaction, are required to identify alternative approaches for nematode control. Here, we summarize recent advances specifically in the role of the small RNAs during the compatible interactions between RKNs and Arabidopsis and tomato hosts. Most of the studies describe the deregulation in the galls and/or GCs of gene regulatory modules composed by miRNAs and transcription factors which have already an assigned function during development and/or organogenesis processes in the plant. This may point to a role for these miRNAs as molecular hubs of pathways triggered either by developmental plant cues or by a biotic stress as the nematode infection.

Gibberellin antagonizes jasmonate-induced defense against Meloidogyne graminicola in rice.

Gibberellin (GA) regulates various plant growth and developmental processes, but its role in pathogen attack, and especially nematode-plant interactions, still remains to be elucidated. An in-depth characterization of the role of GA in nematode infection was conducted using mutant lines of rice, chemical inhibitors, and phytohormone measurements. Our results showed that GA influences rice-Meloidogyne graminicola interactions in a concentration-dependent manner. Foliar spray of plants with a low concentration of gibberellic acid enhanced nematode infection. Biosynthetic and signaling mutants confirmed the importance of gibberellin for rice susceptibility to M.graminicola infection. Our study also demonstrates that GA signaling suppresses jasmonate (JA)-mediated defense against M.graminicola, and likewise the JA-induced defense against M.graminicola requires SLENDER RICE1 (SLR1)-mediated repression of the GA pathway. In contrast to observations from other plant-pathogen interactions, GA plays a dominant role over JA in determining susceptibility to M.graminicola in rice. This GA-induced nematode susceptibility was largely independent of auxin biosynthesis, but relied on auxin transport. In conclusion, we showed that GA-JA antagonistic crosstalk is at the forefront of the interaction between rice and M.graminicola, and SLR1 plays a central role in the JA-mediated defense response in rice against this nematode.

A Phenotyping Method of Giant Cells from Root-Knot Nematode Feeding Sites by Confocal Microscopy Highlights a Role for CHITINASE-LIKE 1 in Arabidopsis.

Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells’ area in an Arabidopsis line overexpressing CHITINASE-LIKE-1 (CTL1) from optical sections by confocal microscopy, assigning a role for CTL1 and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species (Arabidopsis, cucumber or Medicago). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions.

New live screening of plant-nematode interactions in the rhizosphere

Free living nematodes (FLN) are microscopic worms found in all soils. While many FLN species are beneficial to crops, some species cause significant damage by feeding on roots and vectoring viruses. With the planned legislative removal of traditionally used chemical treatments, identification of new ways to manage FLN populations has become a high priority. For this, more powerful screening systems are required to rapidly assess threats to crops and identify treatments efficiently. Here, we have developed new live assays for testing nematode responses to treatment by combining transparent soil microcosms, a new light sheet imaging technique termed Biospeckle Selective Plane Illumination Microscopy (BSPIM) for fast nematode detection, and Confocal Laser Scanning Microscopy for high resolution imaging. We show that BSPIM increased signal to noise ratios by up to 60 fold and allowed the automatic detection of FLN in transparent soil samples of 1.5 mL. Growing plant root systems were rapidly scanned for nematode abundance and activity, and FLN feeding behaviour and responses to chemical compounds observed in soil-like conditions. This approach could be used for direct monitoring of FLN activity either to develop new compounds that target economically damaging herbivorous nematodes or ensuring that beneficial species are not negatively impacted.

Chemotaxis responses of Meloidogyne incognita towards tomato roots depends on concentration gradient of root exudates in vitro assay

Nematodes respond differently to various host-derived odorants which can be either an attractant or a repellent. However, nematodes require suitable concentration gradient of the host cues to respond. In the present study, we have systematically assessed the distance and optimum incubation time at which Meloidogyne incognita second stage juveniles (J2s) respond to tomato root exudates and invasion via an in vitro assay using PF-127 medium. In vitro assay is highly suitable to investigate the nematode's host finding and invasion behaviour under stringent laboratory conditions. Tomato roots incubated for 2 hrs in pluoronic gel followed by exposure to M. incognita J2s gave significant attraction with directional movement and penetration after 8 hrs as compared to the roots incubated for 0 hr, ½ hr, 1 hr and 1½ hrs. In addition, response of J2s to host root signals was confirmed in terms of stylet movement or thrusting which was significantly increased by exposure to tomato root exudates and neurotransmitter resorcinol that is known to induce stylet thrusting along with accumulation of esophageal gland secretions. Similar stylet thrusting was not seen in water. Thus, the present study indicates that nematodes require proper gradient of host signals during the host recognition. These findings can be utilized for understanding the intricate plant nematode interactions which in turn beneficial for the development of novel nematode control strategies.

A multi-seasonal model of plant-nematode interactions and its use to identify durable plant resistance deployment strategies

• Root-knot nematodes (RKNs) are soil-borne polyphagous pests with major impact on crop yield worlwide. Resistant crops efficiently control avirulent RKNs, but favour the emergence of virulent forms. Virulence being associated with fitness costs, susceptible crops counter-select virulent RKNs. In this study we identify optimal rotation strategies between susceptible and resistant crops to control RKNs and maximize crop yield. • We developed an epidemiological model describing the within-season dynamics of avirulent and virulent RKNs on susceptible or resistant plant root-systems, and their between-season survival. The model was fitted to experimental data and used to predict yield-maximizing rotation strategies, with special attention to the impact of epidemic and genetic parameters. • Crop rotations were found to be efficient under realistic parameter ranges. They were characterised by low ratios of resistant plants, and were robust to parameter uncertainty. Rotations provide significant gain over resistant-only strategies, especially under intermediate fitness costs and severe epidemic contexts. • Switching from the current general deployment of resistant crops to custom rotation strategies could not only maintain or increase crop yield, but also preserve the few and valuable R-genes available to us. 1 All rights reserved. No reuse allowed without permission. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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.

A root-knot nematode small glycine and cysteine-rich secreted effector, MiSGCR1, is involved in plant parasitism.

Root-knot nematodes, Meloidogyne spp., are obligate endoparasites that maintain a biotrophic relationship with their hosts. They infect roots as microscopic vermiform second-stage juveniles, and establish specialized feeding structures called 'giant-cells', from which they withdraw water and nutrients. The nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we compared Illumina RNA-seq transcriptomes for M.incognita obtained at various points in the lifecycle, and identified 31 genes more strongly expressed in parasitic stages than in preparasitic juveniles. We then selected candidate effectors for functional characterization. Quantitative real-time PCR and insitu hybridizations showed that the validated differentially expressed genes are predominantly specifically expressed in oesophageal glands of the nematode. We also soaked the nematodes in siRNA to silence these genes and to determine their role in pathogenicity. The silencing of the dorsal gland specific-Minc18876 and its paralogues resulted in a significant, reproducible decrease in the number of mature females with egg masses, demonstrating a potentially important role for the small glycine- and cysteine-rich effector MiSGCR1 in early stages of plant-nematode interaction. Finally, we report that MiSGCR1 suppresses plant cell death induced by bacterial or oomycete triggers of plant defense.