Identification and Characterization of a Novel Protein Disulfide Isomerase Gene (MgPDI2) from Meloidogyne graminicola.

Protein disulfide isomerase (PDI) is a multifunctional enzyme that catalyzes rate-limiting reactions such as disulfide bond formation, isomerization, and reduction. There is some evidence that indicates that PDI is also involved in host-pathogen interactions in plants. In this study, we show that the rice root-knot nematode, Meloidogyne graminicola, has evolved a secreted effector, MgPDI2, which is expressed in the subventral esophageal glands and up-regulated during the early parasitic stage of M. graminicola. Purified recombinant MgPDI2 functions as an insulin disulfide reductase and protects plasmid DNA from nicking. As an effector, MgPDI2 contributes to nematode parasitism. Silencing of MgPDI2 by RNA interference in the pre-parasitic second-stage juveniles (J2s) reduced M. graminicola multiplication and also increased M. graminicola mortality under H2O2 stress. In addition, an Agrobacterium-mediated transient expression assay found that MgPDI2 caused noticeable cell death in Nicotiana benthamiana. An intact C-terminal region containing the first catalytic domain (a) with an active motif (Cys-Gly-His-Cys, CGHC) and the two non-active domains (b and b') is required for cell death induction in N. benthamiana. This research may provide a promising target for the development of new strategies to combat M. graminicola infections.

Chorismate mutase and isochorismatase, two potential effectors of the migratory nematode Hirschmanniella oryzae, increase host susceptibility by manipulating secondary metabolite content of rice.

Hirschmanniella oryzae is one of the most devastating nematodes on rice, leading to substantial yield losses. Effector proteins aid the nematode during the infection process by subduing plant defence responses. In this research we characterized two potential H. oryzae effector proteins, chorismate mutase (HoCM) and isochorismatase (HoICM), and investigated their enzymatic activity and their role in plant immunity. Both HoCM and HoICM proved to be enzymatically active in complementation tests in mutant Escherichia coli strains. Infection success by the migratory nematode H. oryzae was significantly higher in transgenic rice lines constitutively expressing HoCM or HoICM. Expression of HoCM, but not HoICM, increased rice susceptibility against the sedentary nematode Meloidogyne graminicola also. Transcriptome and metabolome analyses indicated reductions in secondary metabolites in the transgenic rice plants expressing the potential nematode effectors. The results presented here demonstrate that both HoCM and HoICM suppress the host immune system and that this may be accomplished by lowering secondary metabolite levels in the plant.

Real-Time Visualization of Cellulase Activity by Microorganisms on Surface.

A variety of methods to detect cellulase secretion by microorganisms has been developed over the years, none of which enables the real-time visualization of cellulase activity on a surface. This visualization is critical to study the interaction between soil-borne cellulase-secreting microorganisms and the surface of plant roots and specifically, the effect of surface features on this interaction. Here, we modified the known carboxymethyl cellulase (CMC) hydrolysis visualization method to enable the real-time tracking of cellulase activity of microorganisms on a surface. A surface was formed using pure CMC with acridine orange dye incorporated in it. The dye disassociated from the film when hydrolysis occurred, forming a halo surrounding the point of hydrolysis. This enabled real-time visualization, since the common need for post hydrolysis dyeing was negated. Using root-knot nematode (RKN) as a model organism that penetrates plant roots, we showed that it was possible to follow microorganism cellulase secretion on the surface. Furthermore, the addition of natural additives was also shown to be an option and resulted in an increased RKN response. This method will be implemented in the future, investigating different microorganisms on a root surface microstructure replica, which can open a new avenue of research in the field of plant root-microorganism interactions.

MiMIF-2 Effector of Meloidogyne incognita Exhibited Enzyme Activities and Potential Roles in Plant Salicylic Acid Synthesis.

Plant-parasitic nematodes secrete a series of effectors to promote parasitism by modulating host immunity, but the detailed molecular mechanism is ambiguous. Animal parasites secrete macrophage migration inhibitory factor (MIF)-like proteins for evasion of host immune systems, in which their biochemical activities play essential roles. Previous research demonstrated that MiMIF-2 effector was secreted by Meloidogyne incognita and modulated host immunity by interacting with annexins. In this study, we show that MiMIF-2 had tautomerase activity and protected nematodes against H2O2 damage. MiMIF-2 expression not only decreased the amount of H2O2 generation during nematode infection in Arabidopsis, but also suppressed Bax-induced cell death by inhibiting reactive oxygen species burst in Nicotiana benthamiana. Further, RNA-seq transcriptome analysis and RT-qPCR showed that the expression of some heat-shock proteins was down regulated in MiMIF-2 transgenic Arabidopsis. After treatment with flg22, RNA-seq transcriptome analysis indicated that the differentially expressed genes in MiMIF-2 expressing Arabidopsis were pointed to plant hormone signal transduction, compound metabolism and plant defense. RT-qPCR and metabolomic results confirmed that salicylic acid (SA) related marker genes and SA content were significantly decreased. Our results provide a comprehensive understanding of how MiMIF-2 modulates plant immunity and broaden knowledge of the intricate relationship between M. incognita and host plants.

Meloidogyne incognita (Nematoda: Meloidogynidae) sterol-binding protein Mi-SBP-1 as a target for its management.

Meloidogyne incognita is a polyphagous plant-parasitic nematode that causes considerable yield loss in agricultural and horticultural crops. The management options available for M. incognita are extremely limited. Here we identified and characterised a M. incognita homolog of Caenorhabditis elegans sterol-binding protein (Mi-SBP-1), a transcriptional regulator of several lipogenesis pathway genes, and used RNA interference-mediated gene silencing to establish its utility as a target for the management of M. incognita. Mi-sbp-1 is predicted to be a helix-loop-helix domain containing DNA binding transcription factor, and is present in the M. incognita genome in three copies. The RNA-Seq analysis of Mi-sbp-1 silenced second stage juveniles confirmed the key role of this gene in lipogenesis regulation in M. incognita. In vitro and host-induced gene silencing of Mi-sbp-1 in M. incognita second stage juveniles resulted in loss of nematodes' ability to utilise the stored fat reserves, slower nematode development, and reduced parasitism on adzuki bean and tobacco plants. The multiplication factor for the Mi-sbp-1 silenced nematodes on adzuki bean plants was reduced by 51% compared with the control nematodes in which Mi-sbp-1 was not silenced. Transgenic expression of the double-stranded RNA construct of the Mi-sbp-1 gene in tobacco plants caused 40-45% reduction in M. incognita multiplication, 30-43.8% reduction in the number of egg masses, and 33-54% reduction in the number of eggs per egg mass compared with the wild type control plants. Our results confirm that Mi-sbp-1 is a key regulator of lipogenesis in M. incognita and suggest that it can be used as an effective target for its management. The findings of this study can be extended to develop methods to manage other economically important parasitic nematodes.

Meloidogyne graminicola protein disulfide isomerase may be a nematode effector and is involved in protection against oxidative damage.

The rice root-knot nematode, Meloidogyne graminicola, is a serious pest in most rice-growing countries. Usually, nematodes employ antioxidants to counteract the harm of reactive oxygen species (ROS) and facilitate their infection. Here the gene encoding M. graminicola protein disulphide isomerase (MgPDI) was identified. The deduced protein is highly conserved in the putative active-site Cys-Gly-His-Cys. In situ hybridization showed that MgPDI was specifically localized within esophageal glands of pre-parasitic second stage juveniles (J2s). MgPDI was significantly up-regulated in the late parasitic J2s. Characterization of the recombinant protein showed that the purified MgPDI exhibited similar activities to other oxidases/isomerases such as the refolding of the scrambled RNase and insulin disulfide reductase and the protection of plasmid DNA and living cells from ROS damage. In addition, silencing of MgPDI by RNA interference in the pre-parasitic J2s lowered their multiplication factor. MgPDI expression was up-regulated in the presence of exogenous H2O2, whereas MgPDI silencing resulted in an increase in mortality under H2O2 stress. MgPDI is localized in the apoplast when transient expression in Nicotiana benthamiana leaves. The results indicated that MgPDI plays important roles in the reproduction and pathogenicity of M. graminicola and it also contributes to protecting nematodes from exogenous H2O2 stress.

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.

Cloning and characterization of the first serine carboxypeptidase from a plant parasitic nematode, Radopholus similis.

Radopholus similis is an important parasitic nematode of plants. Serine carboxypeptidases (SCPs) are peptidases that hydrolyse peptides and proteins and play critical roles in the development, invasion, and pathogenesis of certain parasitic nematodes and other animal pathogens. In this study, we obtained the full-length sequence of the SCP gene from R. similis (Rs-scp-1), which is 1665 bp long and includes a 1461-bp open reading frames encoding 486 amino acids with an 18-aa signal peptide. This gene is a double-copy gene in R. similis. Rs-scp-1 was expressed in the procorpus, esophageal glands and intestines of females and in the esophageal glands and intestines of juveniles. Rs-scp-1 expression levels were highest in females, followed by juveniles and males, and lowest in eggs. Rs-scp-1 expression levels were significantly suppressed after R. similis was soaked in Rs-scp-1 dsRNA for 12 h. Nematodes were then inoculated into Anthurium andraeanum after RNAi treatment. Compared with water treatment, R. similis treated with RNAi were reduced in number and pathogenicity. In summary, we obtained the first SCP gene from a plant parasitic nematode and confirmed its role in the parasitic process.

Expression and evolutionary analyses of three acetylcholinesterase genes (Mi-ace-1, Mi-ace-2, Mi-ace-3) in the root-knot nematode Meloidogyne incognita.

The full cDNA of Mi-ace-3 encoding an acetylcholinesterase (AChE) in Meloidogyne incognita was cloned and characterized. Mi-ace-3 had an open reading frame of 1875 bp encoding 624 amino acid residues. Key residues essential to AChE structure and function were conserved. The deduced Mi-ACE-3 protein sequence had 72% amino acid similarity with that of Ditylenchus destructor Dd-AChE-3. Phylogenetic analyses using 41 AChEs from 24 species showed that Mi-ACE-3 formed a cluster with 4 other nematode AChEs. Our results revealed that the Mi-ace-3 cloned in this study, which is orthologous to Caenorhabditis elegans AChE, belongs to the nematode ACE-3/4 subgroup. There was a significant reduction in the number of galls in transgenic tobacco roots when Mi-ace-1, Mi-ace-2, and Mi-ace-3 were knocked down simultaneously, whereas little or no effect were observed when only one or two of these genes were knocked down. This is an indication that the functions of these three genes are redundant.

Reported populations of Meloidogyne ethiopica in Europe identified as Meloidogyne luci.

The tropical group of root-knot nematodes (RKN) including Meloidogyne ethiopica and M. luci is a highly polyphagus and damaging group of agricultural pests. M. ethiopica has been detected in several European countries (Slovenia, Italy, Greece) and also in Turkey. However, a description of a new sister species M. luci calls for reclassification of all European and Turkish M. ethiopica populations reported up to date as M. luci. Accurate identification can be accomplished through analysis of the esterase isozyme pattern, which is the most distinguishing character between the two otherwise very similar species. Both species display a three banded esterase pattern where the upper band is slightly shifted between the two species. In addition, molecular characterization of M. ethiopica and M. luci populations revealed that the ITS, SSU, and LSU of the rDNA regions are not appropriate markers for studying relationships among the tropical group of RKNs. However, the COII/lRNA region on mtDNA proved to be very useful for analyzing the phylogenetic relationship of these very closely related species/populations. Mitochondrial sequences with low levels of heteroplasmy allowed clustering of all M. luci populations in a monophyletic clade with a clear separation of this recently described species from M. ethiopica. At the same time, a very close relationship between M. ethiopica and M. luci was confirmed again.

Tulipaline A: structure-activity aspects as a nematicide and V-ATPase inhibitor.

Carbonyl groups are known to form covalent adducts with endogenous proteins, but so far, their nematicidal mechanism of action of has been overlooked. The nematicidal activity of ten lactones was tested in vitro against the root knot nematodes Meloidogyne incognita and Meloidogynearenaria. In particular, the saturated lactones α-methylene-γ-butyrolactone or tulipaline A (1) and γ-butyrolactone (3) were active against M. incognita with an EC50/48h of 19.3±10.0 and 40.0±16.2mg/L respectively. Moreover the α, ß-unsaturated lactone 5,6-dihydro-2H-pyran-2-one (2) exhibited the strongest nematicidal activity against the two species with EC50/48h 14.5±5.3 and 21.2±9.7mg/L respectively. Here we propose that the toxic effects of lactones and aldehydes on M.incognita and M. arenaria might be a consequence of their vacuolar-type H(+)-ATPase (V-ATPase) inhibition activity; in fact α-methylene-γ-butyrolactone (1) and salicylaldehyde (12) produced an increased pH in lysosomal-like organelles on HeLa human cell line and this alteration was most likely related to a V-ATPase impairment.

Effects of catechin polyphenols and preparations from the plant-parasitic nematode Heterodera glycines on protease activity and behaviour in three nematode species.

Protease activities in preparations from the plant-parasitic nematodes Heterodera glycines and Meloidogyne incognita and the free-living nematode Panagrellus redivivus were inhibited by exposure to a series of eight catechin polyphenol analogues, (+)-catechin, (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-catechin gallate (CG), (-)-gallocatechin gallate (GCG), (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG) (1 mm each), and by a preparation from H. glycines cysts. General protease activity detected with the FRET-peptide substrate QXL520-KSAYMRF-K(5-FAM)a and proteasome chymotrypsin-like (CTL) activity detected with succinyl-LLVY-AMC were each inhibited significantly more (P < 0.05) by the gallated form of the polyphenol than by the corresponding non-gallated form. Species differences in response to inhibition across all analogues were revealed with the CTL substrate, but CG was a consistently potent inhibitor across all three species and with each substrate. A heat-stable component (CE) from H. glycines cysts inhibited M. incognita CTL activity by 92.07 ± 0.68%, significantly less (P < 0.05) in H. glycines (52.86 ± 2.77%), and by only 17.24 ± 0.55% (P < 0.05) in P. redivivus preparations. CTL activity was, however, inhibited more than 60% in all preparations by the proteasome-specific inhibitor MG-132. Hatching of M. incognita infective juveniles exposed to 1 mm CG, ECG, GCG or EGCG was reduced by 83.88 ± 4.26%, 69.98 ± 9.14%, 94.93 ± 1.71% and 87.93 ± 2.89%, respectively, while hatching of H. glycines was reduced less than 25% by each analogue. CE had no effect on nematode hatch, but did cause a 60% reduction in mobility of H. glycines infective juveniles exposed overnight to CE in vitro, which was more (P < 0.05) than the reduction of M. incognita infective juvenile mobility (20%).

Activity of chalcones derived from 2,4,5-trimethoxybenzaldehyde against Meloidogyne exigua and in silico interaction of one chalcone with a putative caffeic acid 3-O-methyltransferase from Meloidogyne incognita.

Meloidogyne exigua is a parasitic nematode of plants that causes great losses to coffee farmers. In an effort to develop parasitic controls, 154 chalcones were synthesized and screened for activity against this nematode. The best results were obtained with (2E)-1-(4'-nitrophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (6) with a 50% lethal concentration (LC50) of 171 µg/ml against M. exigua second-stage juveniles, in comparison to the commercially-available nematicide carbofuran which had an LC50 of 260 µg/ml under the same conditions. When coffee plants were used, 6 reduced the nematode population to ~50% of that observed in control plants. To investigate the mechanism of action of 6, an in silico study was carried out, which indicated that 6 may act against M. exigua through inhibition of a putative caffeic acid 3-O-methyltransferase homodimer, the amino acid sequence of which was determined by examining the genome of Meloidogyne incognita.

Molecular and biochemical characterization of the ß-1,4-endoglucanase gene Mj-eng-3 in the root-knot nematode Meloidogyne javanica.

This study describes the molecular and biochemical characterization of the ß-1,4-endoglucanase gene (Mj-eng-3) from the root knot nematode Meloidogyne javanica. A 2156-bp genomic DNA sequence of Mj-eng-3 containing six introns was obtained. Mj-eng-3 was localized in the subventral esophageal glands of M. javanica juveniles by in situ hybridization. Real-time RT-PCR assay showed that the highest transcriptional level of Mj-eng-3 occurred in pre-parasitic second-stage juveniles, and this high expression persisted in parasitic second-stage juveniles. Recombinant MJ-ENG-3 degraded carboxymethylcellulose and optimum enzyme activity at 40°C and pH 8.0. EDTA, Mg(2+), Mn(2+), Ca(2+), Co(2+), and Cu(2+) did not affect the activity of MJ-ENG-3; however, Zn(2+) and Fe(2+) inhibited MJ-ENG-3 enzyme activity. In planta Mj-eng-3 RNAi assay displayed a reduction in the number of nematodes and galls in transgenic tobacco roots. These results suggested that MJ-ENG-3 could be secreted by M. javanica to degrade the cellulose of plant cell walls to facilitate its entry and migration during the early stages of parasitism.

Post-transcriptional gene silencing of the gene encoding aldolase from soybean cyst nematode by transformed soybean roots.

Plant parasitic nematodes cause approximately 157 billion US dollars in losses worldwide annually. The soybean cyst nematode (SCN), Heterodera glycines, is responsible for an estimated one billion dollars in losses to the US farmer each year. A promising new approach for control of plant parasitic nematode control is gene silencing. We tested this approach by silencing the SCN gene HgALD, encoding fructose-1,6-diphosphate aldolase. This enzyme is important in the conversion of glucose into energy and may be especially important in actin-based motility during parasite invasion of its host. An RNAi construct targeted to silence HgALD was transformed into soybean roots of composite plants to examine its efficacy to reduce the development of females formed by SCN. The number of mature females on roots transformed with the RNAi construct designed to silence the HgALD gene was reduced by 58%. These results indicate that silencing the aldolase gene of SCN +can greatly decrease the number of female SCN reaching maturity, and it is a promising step towards broadening resistance of plants against plant-parasitic nematodes.

Knocking-down Meloidogyne incognita proteases by plant-delivered dsRNA has negative pleiotropic effect on nematode vigor.

The root-knot nematode Meloidogyne incognita causes serious damage and yield losses in numerous important crops worldwide. Analysis of the M. incognita genome revealed a vast number of proteases belonging to five different catalytic classes. Several reports indicate that M. incognita proteases could play important roles in nematode parasitism, besides their function in ordinary digestion of giant cell contents for feeding. The precise roles of these proteins during parasitism however are still unknown, making them interesting targets for gene silencing to address protein function. In this study we have knocked-down an aspartic (Mi-asp-1), a serine (Mi-ser-1) and a cysteine protease (Mi-cpl-1) by RNAi interference to get an insight into the function of these enzymes during a host/nematode interaction. Tobacco lines expressing dsRNA for Mi-ser-1 (dsSER), Mi-cpl-1 (dsCPL) and for the three genes together (dsFusion) were generated. Histological analysis of galls did not show clear differences in giant cell morphology. Interestingly, nematodes that infected plants expressing dsRNA for proteases produced a reduced number of eggs. In addition, nematode progeny matured in dsSER plants had reduced success in egg hatching, while progeny resulting from dsCPL and dsFusion plants were less successful to infect wild-type host plants. Quantitative PCR analysis confirmed a reduction in transcripts for Mi-cpl-1 and Mi-ser-1 proteases. Our results indicate that these proteases are possibly involved in different processes throughout nematode development, like nutrition, reproduction and embryogenesis. A better understanding of nematode proteases and their possible role during a plant-nematode interaction might help to develop new tools for phytonematode control.

In vitro proteolysis of nematode FMRFamide-like peptides (FLPs) by preparations from a free-living nematode (Panagrellus redivivus) and two plant-parasitic nematodes (Heterodera glycines and Meloidogyne incognita).

Proteolytic activities in extracts from three nematodes, the plant parasites Heterodera glycines and Meloidogyne incognita, and the free-living Panagrellus redivivus, were surveyed for substrate preferences using a battery of seven FRET-modified peptide substrates, all derived from members of the large FMRF-amide like peptide (FLP) family in nematodes. Overall protease activity in P. redivivus was four- to fivefold greater than in either of the parasites, a result that might reflect developmental differences. Digestion of the M. incognita FLP KHEFVRFa (substrate Abz-KHEFVRF-Y(3-NO2)a) by M. incognita extract was sevenfold greater than with H. glycines extract and twofold greater than P. redivivus, suggesting species-specific preferences. Additional species differences were revealed upon screening 12 different protease inhibitors. Two substrates were used in the screen, Abz-KHEFVRF-Y(3-NO2)a and Abz-KPSFVRF-Y(3-NO2)a), which was digested equally by all three species. The effects of various inhibitor, substrate and extract source combinations on substrate digestion suggest that M. incognita differs significantly from P. redivivus and H. glycines in its complement of cysteine proteases, particularly cathepsin L-type protease.

Data-mining of the Meloidogyne incognita degradome and comparative analysis of proteases in nematodes.

Proteases perform essential physiological functions in all living organisms. In parasitic helminths, they are of particular importance for tissue penetration, digestion of host tissues for nutrition, and evasion of host immune responses. The recent availability of the genome sequence of the nematode Meloidogyne incognita has allowed the analysis of the protease repertoire of this major crop pathogen. The M. incognita degradome consists of at least 334 proteases that are distributed into 43 families of the five known catalytic classes. Expression profiling identified protease genes with a differential transcript level between eggs and infective juveniles. Comparing the M. incognita degradome with those of five other nematodes showed discrepancies in the distribution of some protease families, including large expansion in some families, that could reflect specific aspects of the parasitic lifestyle of this organism. This comparative study should provide a framework for deciphering the diversity of protease-mediated functions in nematodes.

Molecular variability and evolution of the pectate lyase (pel-2) parasitism gene in cyst nematodes parasitizing different solanaceous plants.

While pectate lyases are major parasitism factors in plant-parasitic nematodes, there is little information on the variability of these genes within species and their utility as pathotype or host range molecular markers. We have analysed polymorphisms of pectate lyase 2 (pel-2) gene, which degrades the unesterified polygalacturonate (pectate) of the host cell-wall, in the genus Globodera. Molecular variability of the pel-2 gene and the predicted protein was evaluated in populations of G. rostochiensis, G. pallida, G. "mexicana" and G. tabacum. Seventy eight pel-2 sequences were obtained and aligned. Point mutations were observed at 373 positions, 57% of these affect the coding part of the gene and produce 129 aa replacements. The observed polymorphism does not correlate either to the pathotypes proposed in potato cyst nematodes (PCN) or the subspecies described in tobacco cyst nematodes. The trees reveal a topology different from the admitted species topology as G. rostochiensis and G. pallida sequences are more similar to each other than to G. tabacum. Species-specific sites, potentially applicable for identification, and sites distinguishing PCN from tobacco cyst nematodes, were identified. As both G. rostochiensis and G. pallida display the same host range, but distinct from G. tabacum, which cannot parasitize potato plants, it is tempting to speculate that pel-2 genes polymorphism may be implicated in this adaptation, a view supported by the fact that no active pectate lyase 2 was found in G. "mexicana", a close relative of G. pallida that is unable to develop on cultivated potato varieties.