Two Candidate Meloidogyne javanica Effector Genes, MjShKT and MjPUT3: A Functional Investigation of Their Roles in Regulating Nematode Parasitism.

During parasitism, root-knot nematode Meloidogyne spp. inject molecules termed effectors that have multifunctional roles in construction and maintenance of nematode feeding sites. As an outcome of transcriptomic analysis of Meloidogyne javanica, we identified and characterized two differentially expressed genes encoding the predicted proteins MjShKT, carrying a Stichodactyla toxin (ShKT) domain, and MjPUT3, carrying a ground-like domain, both expressed during nematode parasitism of the tomato plant. Fluorescence in-situ hybridization revealed expression of MjShKT and MjPUT3 in the dorsal esophageal glands, suggesting their injection into host cells. MjShKT expression was upregulated during the parasitic life stages, to a maximum at the mature female stage, whereas MjPUT3 expression increased in third- to fourth-stage juveniles. Subcellular in-planta localization of MjShKT and MjPUT3 using a fused fluorescence marker indicated MjShKT co-occurrence with the endoplasmic reticulum, the perinuclear endoplasmatic reticulum, and the Golgi organelle markers, while MjPUT3 localized, to some extent, within the endoplasmatic reticulum and was clearly observed within the nucleoplasm. MjShKT inhibited programmed cell death induced by overexpression of MAPKKKα and Gpa2/RBP-1. Overexpression of MjShKT in tomato hairy roots allowed an increase in nematode reproduction, as indicated by the high number of eggs produced on roots overexpressing MjShKT. Roots overexpressing MjPUT3 were characterized by enhanced root growth, with no effect on nematode development on those roots. Investigation of the two candidate effectors suggested that MjShKT is mainly involved in manipulating the plant effector-triggered immune response toward establishment and maintenance of active feeding sites, whereas MjPUT3 might modulate roots morphology in favor of nematode fitness in the host roots. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tomato Divinyl Ether-Biosynthesis Pathway Is Implicated in Modulating of Root-Knot Nematode Meloidogyne javanica's Parasitic Ability.

The role of the 9-lipoxygenase (9-LOX)-derived oxylipins in plant defense is mainly known in solanaceous plants. In this work, we identify the functional role of the tomato divinyl ether synthase (LeDES) branch, which exclusively converts 9-hydroperoxides to the 9-divinyl ethers (DVEs) colneleic acid (CA) and colnelenic acid (CnA), during infection by the root-knot nematode Meloidogyne javanica. Analysis of LeDES expression in roots indicated a concurrent response to nematode infection, demonstrating a sharp increase in expression during the molting of third/fourth-stage juveniles, 15 days after inoculation. Spatiotemporal expression analysis using an LeDES promoter:GUS tomato line showed high GUS activity associated with the developing gall; however the GUS signal became more constricted as infection progressed to the mature nematode feeding sites, and eventually disappeared. Wounding did not activate the LeDES promoter, but auxins and methyl salicylate triggered LeDES expression, indicating a hormone-mediated function of DVEs. Heterologous expression of LeDES in Arabidopsis thaliana rendered the plants more resistant to nematode infection and resulted in a significant reduction in third/fourth-stage juveniles and adult females as compared to a vector control and the wild type. To further evaluate the nematotoxic activity of the DVEs CA and CnA, recombinant yeast that catalyzes the formation of CA and CnA from 9-hydroperoxides was generated. Transgenic yeast accumulating CnA was tested for its impact on M. javanica juveniles, indicating a decrease in second-stage juvenile motility. Taken together, our results suggest an important role for LeDES as a determinant in the defense response during M. javanica parasitism, and indicate two functional modes: directly via DVE motility inhibition effect and through signal molecule-mediated defense reactions to nematodes that depend on methyl salicylate.

Molecular cloning and characterization of high-affinity potassium transporter (AlHKT2;1) gene promoter from halophyte Aeluropus lagopoides.

HKT subfamily II functions as Na+- K+ co-transporter and prevents plants from salinity stress. A 760 bp promoter region of AlHKT2;1 was isolated, sequenced and cloned. The full length promoter D1, has many cis-regulatory elements like MYB, MBS, W box, ABRE etc. involved in abiotic stress responses. D1 and subsequent 5' deletions were cloned into pCAMBIA1301 and studied for its efficacy in stress conditions in heterologous system. Blue colour staining was observed in flower petals, anther lobe, and dehiscence slit of anther in T0 plants. The T1 seedlings showed staining in leaf veins, shoot vasculature and root except root tip. T1 seedlings were subjected to NaCl, KCl, NaCl + KCl and ABA stresses. GUS activity was quantified by 4-methylumbelliferyl glucuronide (4-MUG) assay under control and stress conditions. The smallest deletion- D4 also showed GUS expression but highest activity was observed in D2 as compared to full length promoter and other deletions. The electrophoretic mobility shift assay using stress-induced protein with different promoter deletions revealed more prominent binding in D2. These results suggest that AlHKT2;1 promoter is involved in abiotic stress response and deletion D2 might be sufficient to drive the stress-inducible expression of various genes involved in providing stress tolerance in plants.

Caenorhabditis elegans susceptibility to Daldinia cf. concentrica bioactive volatiles is coupled with expression activation of the stress-response transcription factor daf-16, a part of distinct nematicidal action.

The bionematicidal effect of a synthetic volatile mixture (SVM) of four volatile organic compounds (VOCs) emitted by the endophytic fungus Daldinia cf. concentrica against the devastating plant-parasitic root-knot nematode Meloidogyne javanica has been recently demonstrated in both in vitro and greenhouse experiments. However, the mode of action governing the observed irreversible paralysis of J2 larvae upon exposure to SVM is unknown. To unravel the mechanism underlying the anthelmintic and nematicidal activities, we used the tractable model worm Caenorhabditis elegans. C. elegans was also susceptible to both the fungal VOCs and SVM. Among compounds comprising SVM, 3-methyl-1-butanol, (±)-2-methyl-1-butanol, and 4-heptanone showed significant nematicidal activity toward L1, L4 and young adult stages. Egg hatching was only negatively affected by 4-heptanone. To determine the mechanism underlying this activity, we examined the response of C. elegans mutants for glutamate-gated chloride channel and acetylcholine transporter, targets of the nematicidal drugs ivermectin and aldicarb, respectively, to 4-heptanone and SVM. These aldicarb- and ivermectin-resistant mutants retained susceptibility upon exposure to 4-heptanone and SVM. Next, we used C. elegans TJ356 strain zIs356 (daf-16::GFP+rol-6), LD1 ldIs7 [skn-1B/C::GFP + pRF4(rol-6(su1006))], LD1171 ldIs3 [gcs-1p::gfp; rol-6(su1006))], CL2166 dvIs19 (gst-4p::GFP) and CF1553 muIs84 (sod-3p::GFP+rol-6), which have mutations in genes regulating multiple stress responses. Following exposure of L4 larvae to 4-heptanone or SVM, there was clear nuclear translocation of DAF-16::GFP, and SKN-1::GFP indicating that their susceptibility involves DAF-16 and SKN1 regulation. Application of 4-heptanone, but not SVM, induced increased expression of, gcs-1::GFP and gst-4::GFP compared to controls. In contrast, application of 4-heptanone or SVM to the sod-3::GFP line elicited a significant decline in overall fluorescence intensity compared to controls, indicating SOD-3 downregulation and therefore overall reduction in cellular redox machinery. Our data indicate that the mode of action of SVM and 4-heptanone from D. cf. concentrica differs from that of currently available nematicides, potentially offering new solutions for nematode management.

Ion homeostasis in a salt-secreting halophytic grass.

Salinity adversely affects plant growth and development, and disturbs intracellular ion homeostasis, resulting in cellular toxicity. Plants that tolerate salinity, halophytes, do so by manifesting numerous physiological and biochemical processes in coordination to alleviate cellular ionic imbalance. The present study was undertaken to analyse the salt tolerance mechanism in Aeluropus lagopoides (L.) trin. Ex Thw. (Poaceae) at both physiological and molecular levels. Plants secreted salt from glands, which eventually produced pristine salt crystals on leaves and leaf sheaths. The rate of salt secretion increased with increasing salt concentration in the growth medium. Osmotic adjustment was mainly achieved by inorganic osmolytes (Na(+)) and at 100 mM NaCl no change was observed in organic osmolytes in comparison to control plants. At 300 mM NaCl and with 150 mM NaCl + 150 mM KCl, the concentration of proline, soluble sugars and amino acids was significantly increased. Transcript profiling of transporter genes revealed differential spatial and temporal expressions in both shoot and root tissues in a manner synchronized towards maintaining ion homeostasis. In shoots, AlHKT2;1 transcript up-regulation was observed at 12 and 24 h in all the treatments, whereas in roots, maximum induction was observed at 48 h with K(+) starvation. The HAK transcript was relatively abundant in shoot tissue with all the treatments. The plasma membrane Na(+)/H(+) antiporter, SOS1, and tonoplast Na(+)/H(+) antiporter, NHX1, were found to be significantly up-regulated in shoot tissue. Our data demonstrate that AlHKT2;1, HAK, SOS1, NHX1 and V-ATPase genes play a pivotal role in regulating the ion homeostasis in A. lagopoides.

A Low-Affinity K+ Transporter AlHKT2;1 from Recretohalophyte Aeluropus lagopoides Confers Salt Tolerance in Yeast.

The high-affinity potassium transporters (HKT) are highly important for stress tolerance in plants as they uniquely maintain K(+)/Na(+) ratio for their survival and growth. In this study a novel HKT gene AlHKT2;1 was isolated and characterized from salt secreting halophyte, Aeluropus lagopoides. The AlHKT2;1 cDNA comprised of an open reading frame of 1,581 bp, encoding a protein of 526 amino acid residues. It belongs to class II HKTs and showed high homology with other HKT genes. Functional characterization of AlHKT2;1 in both K(+) uptake-deficient (WΔ6) and Na(+)-sensitive yeast mutants (G19) showed the characteristic feature of low-affinity K(+) transporter supporting the growth at >1 mM KCl concentration. The transformed yeast cells showed high sensitivity to NaCl; however, the addition of KCl along with NaCl support the growth of AlHKT2;1 expressing mutant. Ion content analysis of yeast cells with AlHKT2;1 grown in high NaCl medium supplemented with KCl revealed that salt tolerance was correlated with accumulation of K(+) during salt stress. These results suggest that AlHKT2;1 plays an important role in the K(+) uptake during salt stress and in maintaining a high K(+)/Na(+) ratio in the cytosol.

Molecular characterization and identification of target protein of an important vesicle trafficking gene AlRab7 from a salt excreting halophyte Aeluropus lagopoides.

The endomembrane system plays an important role during cellular adaptation of the plants with the extracellular environment. The small GTP-binding protein Rab7 located at the vacuolar membrane regulates the vesicle fusion with the vacuole and thereby helps in recycling of the molecules. This is the first report on isolation and characterization of AlRab7 gene from the halophyte plant, Aeluropus that extrudes NaCl through salt glands and grows luxuriantly throughout the year at the Gujarat coast, India. The AlRab7 encodes a protein with 206 amino acids, and a highly conserved effector-binding domain and four nucleotide-binding domains. The in silico analysis predicts the presence of the prenylation site for Rab geranylgeranyltransferase 2 and the Rab escort protein site. The C-terminal two cysteine residues in -XCC sequence are present for membrane attachment. Transcript expression of the AlRab7 gene was differentially regulated by different environmental stimuli such as dehydration, salinity, and hormone abscisic acid (ABA). The recombinant Escherichia coli cells showed improved growth in Luria Bertani medium supplemented with NaCl, KCl, mannitol, ABA, and indole-3-acetic acid. A novel Rab7 interacting partner AlRabring7 was identified by yeast two-hybrid screening.

Comparative molecular analysis of chemolithoautotrophic bacterial diversity and community structure from coastal saline soils, Gujarat, India.

BACKGROUND: Soils harbour high diversity of obligate as well as facultative chemolithoautotrophic bacteria that contribute significantly to CO2 dynamics in soil. In this study, we used culture dependent and independent methods to assess the community structure and diversity of chemolithoautotrophs in agricultural and coastal barren saline soils (low and high salinity). We studied the composition and distribution of chemolithoautotrophs by means of functional marker gene cbbL encoding large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and a phylogenetic marker 16S rRNA gene. The cbbL form IA and IC genes associated with carbon fixation were analyzed to gain insight into metabolic potential of chemolithoautotrophs in three soil types of coastal ecosystems which had a very different salt load and sulphur content. RESULTS: In cbbL libraries, the cbbL form IA was retrieved only from high saline soil whereas form IC was found in all three soil types. The form IC cbbL was also amplified from bacterial isolates obtained from all soil types. A number of novel monophyletic lineages affiliated with form IA and IC phylogenetic trees were found. These were distantly related to the known cbbL sequences from agroecosystem, volcanic ashes and marine environments. In 16S rRNA clone libraries, the agricultural soil was dominated by chemolithoautotrophs (Betaproteobacteria) whereas photoautotrophic Chloroflexi and sulphide oxidizers dominated saline ecosystems. Environmental specificity was apparently visible at both higher taxonomic levels (phylum) and lower taxonomic levels (genus and species). The differentiation in community structure and diversity in three soil ecosystems was supported by LIBSHUFF (P = 0.001) and UniFrac. CONCLUSION: This study may provide fundamentally new insights into the role of chemolithoautotrophic and photoautotrophic bacterial diversity in biochemical carbon cycling in barren saline soils. The bacterial communities varied greatly among the three sites, probably because of differences in salinity, carbon and sulphur contents. The cbbL form IA-containing sulphide-oxidizing chemolithotrophs were found only in high saline soil clone library, thus giving the indication of sulphide availability in this soil ecosystem. This is the first comparative study of the community structure and diversity of chemolithoautotrophic bacteria in coastal agricultural and saline barren soils using functional (cbbL) and phylogenetic (16S rDNA) marker genes.