Early Parasitic Stages Research Articles

NRPS-like ATRR in Plant-Parasitic Nematodes Involved in Glycine Betaine Metabolism to Promote Parasitism.

Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.

A root-knot nematode effector targets the Arabidopsis cysteine protease RD21A for degradation to suppress plant defense and promote parasitism.

Meloidogyne incognita is one of the most widely distributed plant-parasitic nematodes and causes severe economic losses annually. The parasite produces effector proteins that play essential roles in successful parasitism. Here, we identified one such effector named MiCE108, which is exclusively expressed within the nematode subventral esophageal gland cells and is upregulated in the early parasitic stage of M. incognita. A yeast signal sequence trap assay showed that MiCE108 contains a functional signal peptide for secretion. Virus-induced gene silencing of MiCE108 impaired the parasitism of M. incognita in Nicotiana benthamiana. The ectopic expression of MiCE108 in Arabidopsis suppressed the deposition of callose, the generation of reactive oxygen species, and the expression of marker genes for bacterial flagellin epitope flg22-triggered immunity, resulting in increased susceptibility to M. incognita, Botrytis cinerea, and Pseudomonas syringae pv. tomato (Pst) DC3000. The MiCE108 protein physically associates with the plant defense protease RD21A and promotes its degradation via the endosomal-dependent pathway, or 26S proteasome. Consistent with this, knockout of RD21A compromises the innate immunity of Arabidopsis and increases its susceptibility to a broad range of pathogens, including M. incognita, strongly indicating a role in defense against this nematode. Together, our data suggest that M. incognita deploys the effector MiCE108 to target Arabidopsis cysteine protease RD21A and affect its stability, thereby suppressing plant innate immunity and facilitating parasitism.

First Report of Karelinia caspia (Pall.) Less as a new host of Orobanche cumana Wallr. in Xinjiang, China.

Sunflower broomrape (Orobanche cumana Wallr.) is a holoparasitic plant species which mainly parasitizes a few species of the Asteraceae in the wild and is exclusively found growing on sunflower in agricultural fields (Fernández-Martínez et al. 2015). O. cumana is a serious threat to sunflower production in Xinjiang and Inner Mongolia (Shi et al. 2015). Karelinia caspia (Pall.) Less. (Asteraceae) is an ecologically important plant species occurring across the desert ecosystems of Russia, Central Asia, and northwest China. It plays an important role in reducing wind erosion and desertification (Xu et al. 2018). During the 2018 and 2019 growing seasons, sunflower broomrape was observed parasitizing K. caspia in non-cultivated areas adjacent to sunflower fields near Beitun city (87°51'E, 47°15'N) in Xinjiang, China. Sunflower broomrape plants were identified morphologically as O. cumana according to Pujadas-Salvà and Velasco (2000). The host plants were identified morphologically as K. caspia according to Lin et al (1979). The ribosomal DNA internal transcribed spacer (ITS) and the trnL-F region of the parasite were amplified by PCR using primer pairs ITS1/ITS4 and trnL-FF/trnL-FR, respectively (Taberlet et al. 1991; Anderson et al. 2004). The ITS sequence of the parasite (Accession No. MT795725.1) showed 100% identity (675bp out of 689bp) to that of O. cernua var. cumana (KC811228.1). The trnl-F sequence of the parasite (Accession No. ON843707) showed 98% identity (675 of 689 bp) to O.cernua var. cumana (KT387722.1). Multi-locus phylogenetic analysis of the two sequences showed clustering with sunflower broomrape. The ITS region of the parasite and host was were amplified by PCR using the primer pair ITS1F/ITS4R (Taberlet et al.1991), and the ITS sequences of the host (Accession No. MT791995.1) showed 99.86% identity (728bp of 802bp) to that of K. caspia (LN607483.1). Rhizotron and pot experiments were carried out to assess the parasitic relationship between O. cumana and K. caspia. In the rhizotron experiment, 2-week-old seedlings of K. caspia were inoculated with sterilized 400 O. cumana seeds in a 15-cm petri dish filled with a sponge overlaid with glass fiber filter paper. The parasitic state of O. cumana was observed 9 days after inoculation. In another trial, seeds of K. caspia were sowed in 2-L and 4-L pots containing sand-vermiculite-compost (1:1:1 v:v:v). These pots were artificially inoculated with 50 mg of O. cumana seeds per 1 kg of substrate. After 20 and 70 days, corresponding to the early parasitic and flowering stages, respectively, of O. cumana, K. caspia plants were uprooted from the media and washed carefully. The parasitic relationship was confirmed by the attachment position of the broomrape to the K. caspia root. To our knowledge, this is the first report of O. cumana parasitizing K. caspia in Xinjiang, China. This phenomenon means that sunflower broomrape can raise up seed on a newly recognized host. Weed eradication in and near sunflower fields is a key measure to control sunflower broomrape.

A new gene family (BAPs) of Cotesia bracovirus induces apoptosis of host hemocytes

ABSTRACT Polydnaviruses (PDVs), obligatory symbionts with parasitoid wasps, function as host immune suppressors and growth and development regulator. PDVs can induce host haemocyte apoptosis, but the underlying mechanism remains largely unknown. Here, we provided evidence that, during the early stages of parasitism, the activated Cotesia vestalis bracovirus (CvBV) reduced the overall number of host haemocytes by inducing apoptosis. We found that one haemocyte-highly expressed CvBV gene, CvBV-26-4, could induce haemocyte apoptosis. Further analyses showed that CvBV-26-4 has four homologs from other Cotesia bracoviruses and BV from wasps in the genus Glyptapanteles, and all four of them possessed a similar structure containing 3 copies of a well-conserved motif (Gly-Tyr-Pro-Tyr, GYPY). Mass spectrometry analysis revealed that CvBV-26-4 was secreted into plasma by haemocytes and then degraded into peptides that induced the apoptosis of haemocytes. Moreover, ectopic expression of CvBV-26-4 caused fly haemocyte apoptosis and increased the susceptibility of flies to bacteria. Based on this research, a new family of bracovirus genes, Bracovirus apoptosis-inducing proteins (BAPs), was proposed. Furthermore, it was discovered that the development of wasp larvae was affected when the function of CvBV BAP was obstructed in the parasitized hosts. The results of our study indicate that the BAP gene family from the bracoviruses group is crucial for immunosuppression during the early stages of parasitism.

Early monitoring of parasitism by Aphidiinae parasitoids on the grain aphid Sitobion miscanthi in wheat fields using DNA barcoding.

Sitobion miscanthi is a major wheat pest at the grain-filling stage found in China. Identifying parasitoid species and understanding parasitism rates are keys to controlling the aphids via natural enemies in the wheat field. In the present study, a method based on DNA barcoding for early determination of the community composition of Aphidiinae parasitoids and parasitism on the aphid was developed. The proposed method detected Aphidius gifuensis as the predominant parasite, with parasitism rates of 40.1 ± 2.8% in 2019 and 65.7 ± 3.7% in 2022, and found that the rate varied significantly among different wheat varieties. COI primers efficiently amplified the Aphidiinae parasitoids COI fragments and amplified the aphid COI fragments derived from parasitized (mummified) S. miscanthi. Thus, the COI barcode is not sufficiently specific to unambiguously detect immature parasitoids inside their S. miscanthi hosts. However, it can be used to detect the DNA extracted from mummified aphids. In contrast, the 16S and LWRh primers effectively amplified and identified the parasitoids in parasitized aphids. The 16S primer was reliable even in the early stages of parasitism (24 h) and for DNA samples stored at -20 °C for 5 days. The three barcodes from COI, 16S, and LWRh genes could not clearly distinguish a few certain Aphidiinae species owing to relatively low intraspecific and interspecific diversity. The morphological features remain indispensable when identifying Aphidiinae species. Nonetheless, the COI and 16S primers could be used in combination for monitoring the parasitism rates on S. miscanthi in wheat fields. © 2022 Society of Chemical Industry.

Morphological characterization reveals new insights into giant cell development of Meloidogyne graminicola on rice

Main conclusionThree types of nematode-feeding sites (NFSs) caused by M. graminicola on rice were suggested, and the NFS polarized expansion stops before the full NFS maturation that occurs at adult female stage.Root-knot nematodes, Meloidogyne spp., secrete effectors and recruit host genes to establish their feeding sites giant cells, ensuring their nutrient acquisition. There is still a limited understanding of the mechanism underlying giant cell development. Here, the three-dimensional structures of M. graminicola-caused nematode-feeding sites (NFSs) on rice as well as changes in morphological features and cytoplasm density of the giant cells (GCs) during nematode parasitism were reconstructed and characterized by confocal microscopy and the Fiji software. Characterization of morphological features showed that three types of M. graminicola-caused NFSs, type I–III, were detected during parasitism at the second juvenile (J2), the third juvenile (J3), the fourth juvenile (J4) and adult female stages. Type I is the majority at all stages and type II develops into type I at J3 stage marked by its longitudinal growth. Meanwhile, NFSs underwent polarized expansion, where the lateral and longitudinal expansion ceased at later parasitic J2 stage and the non-feeding J4 stage, respectively. The investigation of giant cell cytoplasm density indicates that it reaches a peak at the midpoint of early parasitic J2 and adult female stages. Our data suggest the formation of three types of NFSs caused by M. graminicola on rice and the NFS polarized expansion stopping before full NFS maturation, which provides unprecedented spatio-temporal characterization of development of giant cells caused by a root-knot nematode.

Schistosomiasis mansoni: A new therapeutic target for ubiquinol, a natural inhibitor of neutral magnesium-dependent sphingomyelinase in murine model.

Constituting the host-parasite interface and playing a censorious role in host immune response modulation and parasite survival, tegument represents a crucial target for many antischistosomal drugs. Sphingomyelin forms a stable outer leaflet of tegumental membrane-lipid bilayer. Neutral magnesium‏-dependent sphingomyelinase (Mg2+-nSMase) is a key enzyme in sphingomyelin breakdown was identified in schistosomes. We investigated the in vivo efficacy of ubiquinol, a natural inhibitor of Mg2+-nSMase, in free and niosomes-encapsulated forms, through five-day and 15-day regimens on the early and late Schistosoma mansoni parasitic stages, respectively, compared to PZQ. Oral administration of 300 mg/kg/day ubiquinol-encapsulated niosomes (U-N) showed significant deterioration of the parasitic growth and development in the term of reduction of lung schistosomula burden (39.12%), adult worm burden (50.81%), hepatic and intestinal tissue-egg counts (80.89% and 75.54%, respectively). PZQ and free ubiquinol regimens reported reductions in lung schistosomula counts (45.36% and 22.90%, respectively) and total worm burdens of 86.28% and 24.58%, respectively. U-N therapy revealed worms de-pairing and remarkable diminution in female worms’ perimeters and fecundity. Scanning electron microscope revealed disruption of tegumental ridges with excessive longitudinal corrugation. Transmission electron microscope showed testicular and ovarian parenchymal degeneration, signs of immaturity and cell apoptosis. Indirect immunofluorescence assay approved parasite's tegumental changes. Remarkable reduction of granulomas size with amelioration of hepatic pathology and fibrosis were assumed to be attributed to the anti-inflammatory and anti-oxidant properties of ubiquinol. These findings with the drug safety profile suggest that U-N could be a promising candidate for a new antischistosomal drug development.

Characterization of Superoxide Dismutase from the Potato Cyst Nematode, Globodera pallida.

Potato cyst nematodes (PCNs), such as Globodera pallida and Globodera rostochiensis, are some of the most agriculturally and economically important pests of potato. Upon nematode infection, a principal component of plant defense is the generation of the reactive oxygen species (ROSs). ROSs are highly toxic molecules that cause damage to pathogens and host alike. To infect the plant, nematodes protect themselves from ROSs by activating their own antioxidant processes and ROS scavenging enzymes. One of these enzymes is a superoxide dismutase (SOD; EC 1.15.1.1), which prevents cellular damage by catalyzing conversion of the superoxide radical (O2-·) to hydrogen peroxide (H2O2) and molecular oxygen (O2). We have isolated a putatively secreted isoform of a Cu-Zn SOD (SOD-3) from G. pallida and localized the expression of this gene in the posterior region of the nematode. Furthermore, we studied the expression of the SOD-3 gene during early parasitic stages of infection (24 to 72 h) in the susceptible potato cultivar Desiree, the resistant potato cultivar Innovator, and an immune host, Solanum sisymbriifolium. The SOD-3 gene was significantly upregulated, regardless of the host type; however, the expression pattern differed between the susceptible and the resistant or immune hosts. This finding suggests that SOD-3 gene is responding to infection in plant roots differently depending on whether the nematode is experiencing a compatible or an incompatible interaction.

A Meloidogyne graminicola Pectate Lyase Is Involved in Virulence and Activation of Host Defense Responses.

Plant-parasitic nematodes secrete an array of cell-wall-degrading enzymes to overcome the physical barrier formed by the plant cell wall. Here, we describe a novel pectate lyase gene Mg-PEL1 from M. graminicola. Quantitative real-time PCR assay showed that the highest transcriptional expression level of Mg-PEL1 occurred in pre-parasitic second-stage juveniles, and it was still detected during the early parasitic stage. Using in situ hybridization, we showed that Mg-PEL1 was expressed exclusively within the subventral esophageal gland cells of M. graminicola. The yeast signal sequence trap system revealed that it possessed an N-terminal signal peptide with secretion function. Recombinant Mg-PEL1 exhibited hydrolytic activity toward polygalacturonic acid. Rice plants expressing RNA interference vectors targeting Mg-PEL1 showed an increased resistance to M. graminicola. In addition, using an Agrobacterium-mediated transient expression system and plant immune response assays, we demonstrated that the cell wall localization of Mg-PEL1 was required for the activation of plant defense responses, including programmed plant cell death, reactive oxygen species (ROS) accumulation and expression of defense-related genes. Taken together, our results indicated that Mg-PEL1 could enhance the pathogenicity of M. graminicola and induce plant immune responses during nematode invasion into plants or migration in plants. This provides a new insight into the function of pectate lyases in plants-nematodes interaction.

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.

Variation in intrinsic resistance of pea aphids to parasitoid wasps: A transcriptomic basis.

Evolutionary interactions between parasitoid wasps and insect hosts have been well studied at the organismal level, but little is known about the molecular mechanisms that insects use to resist wasp parasitism. Here we study the interaction between a braconid wasp (Aphidius ervi) and its pea aphid host (Acyrthosiphon pisum). We first identify variation in resistance to wasp parasitism that can be attributed to aphid genotype. We then use transcriptome sequencing to identify genes in the aphid genome that are differentially expressed at an early stage of parasitism, and we compare these patterns in highly resistant and susceptible aphid host lines. We find that resistant genotypes are upregulating genes involved in carbohydrate metabolism and several key innate immune system genes in response to parasitism, but that this response seems to be weaker in susceptible aphid genotypes. Together, our results provide a first look into the complex molecular mechanisms that underlie aphid resistance to wasp parasitism and contribute to a broader understanding of how resistance mechanisms evolve in natural populations.

Detection of Rare Objects by Flow Cytometry: Imaging, Cell Sorting, and Deep Learning Approaches.

Flow cytometry nowadays is among the main working instruments in modern biology paving the way for clinics to provide early, quick, and reliable diagnostics of many blood-related diseases. The major problem for clinical applications is the detection of rare pathogenic objects in patient blood. These objects can be circulating tumor cells, very rare during the early stages of cancer development, various microorganisms and parasites in the blood during acute blood infections. All of these rare diagnostic objects can be detected and identified very rapidly to save a patient’s life. This review outlines the main techniques of visualization of rare objects in the blood flow, methods for extraction of such objects from the blood flow for further investigations and new approaches to identify the objects automatically with the modern deep learning methods.

The Heterodera glycines effector Hg16B09 is required for nematode parasitism and suppresses plant defense response

Soybean cyst nematode (Heterodera glycines Ichinohe) is a sedentary root endoparasite that causes serious yield losses on soybean (Glycine max) worldwide. H. glycines secrets effector proteins into host cells to facilitate the success of parasitism. Nowadays, a large number of candidate effectors were identified from the genome sequence of H. glycines. However, the precise functions of these effectors in the nematode-host plant interaction are unknown. Here, an effector gene of dorsal gland protein Hg16B09 from H. glycines was cloned and functionally characterized through generating the transgenic soybean hairy roots. In situ hybridization assay and qRT-PCR analysis indicated Hg16B09 is exclusively expressed in the dorsal esophageal cells and up-regulated in the parasitic-stage juveniles. The constitutive expression of Hg16B09 in soybean hairy roots caused an enhanced susceptibility to H. glycines. In contrast, in planta silencing of Hg16B09 exhibited that nematode reproduction in hairy roots was decreased compared to the empty vector control. In addition, Hg16B09 also suppressed the expression of soybean defense-related genes induced by the pathogen-associated molecular pattern flg22. These data indicate that the effector Hg16B09 might aid H. glycines parasitism through suppressing plant basal defenses in the early parasitic stages.

Heterodera avenae GLAND5 Effector Interacts With Pyruvate Dehydrogenase Subunit of Plant to Promote Nematode Parasitism.

Heterodera avenae mainly infects cereal crops and causes severe economic losses. Many studies have shown that parasitic nematodes can secrete effector proteins to suppress plant immune responses and then promote parasitism. In this study, we showed that HaGland5, a novel effector of H. avenae, was exclusively expressed in dorsal esophageal gland cell of nematode, and up-regulated in the early parasitic stage. Transgenic Arabidopsis thaliana lines expressing HaGland5 were significantly more susceptible to H. schachtii than wild-type control plants. Conversely, silencing of HaGland5 through barley stripe mosaic virus-medicated host-induced gene silencing technique substantially reduced the infection of H. avenae in wheat. Moreover, HaGland5 could suppress the plant defense responses, including the repression of plant defense-related genes, reducing deposition of cell wall callose and the burst of reactive oxygen species. Mass spectrometry, co-immunoprecipitation, and firefly luciferase complementation imaging assays confirmed that HaGland5 interacted specifically with Arabidopsis pyruvate dehydrogenase subunit (AtEMB3003).

A Meloidogyne graminicola C‐type lectin, Mg01965, is secreted into the host apoplast to suppress plant defence and promote parasitism

SummaryC‐type lectins (CTLs), a class of multifunctional proteins, are numerous in nematodes. One CTL gene, Mg01965, shown to be expressed in the subventral glands, especially in the second‐stage juveniles of the root‐knot nematode Meloidogyne graminicola, was further analysed in this study. In vitro RNA interference targeting Mg01965 in the preparasitic juveniles significantly reduced their ability to infect host plant roots. Immunolocalizations showed that Mg01965 is secreted by M. graminicola into the roots during the early parasitic stages and accumulates in the apoplast. Transient expression of Mg01965 in Nicotiana benthamiana and targeting it to the apoplast suppressed the burst of reactive oxygen species triggered by flg22. The CTL Mg01965 suppresses plant innate immunity in the host apoplast, promoting nematode parasitism in the early infection stages.

Persistent expression of Cotesia plutellae bracovirus genes in parasitized host, Plutella xylostella.

Cotesia plutellae (= vestalis) bracovirus (CpBV) is symbiotic to an endoparasitoid wasp, C. plutellae, and plays crucial roles in parasitism against the diamondback moth, Plutella xylostella. CpBV virion genome consists of 35 circular DNAs encoding 157 putative open reading frames (ORFs). This study re-annotated 157 ORFs with update genome database and analyzed their gene expressions at early and late parasitic stages. Re-annotation has established 15 different viral gene families, to which 83 ORFs are assigned with remaining 74 hypothetical genes. Among 157 ORFs, 147 genes were expressed at early or late parasitic stages, among which 141 genes were expressed in both parasitic stages, indicating persistent nature of gene expression. Relative frequencies of different viral circles present in the ovarian lumen did not explain the expression variation of the viral ORFs. Furthermore, expression level of each viral gene was varied during parasitism along with host development. Highly up-regulated CpBV genes at early parasitic stage included BEN (BANP, E5R and NAC1), ELP (EP1-like protein), IkB (inhibitor kB), P494 (protein 494 kDa) family genes, while those at late stage were mostly hypothetical genes. Along with the viral gene expression, 362 host genes exhibited more than two fold changes in expression levels at early parasitic stage compared to nonparasitized host. At late stage, more number (1,858) of host genes was regulated. These results suggest that persistent expression of most CpBV genes may be necessary to regulate host physiological processes during C. plutellae parasitism.

A Meloidogyne incognita effector MiISE5 suppresses programmed cell death to promote parasitism in host plant

Root-knot nematodes (RKNs) are highly specialized parasites that interact with their host plants using a range of strategies. The esophageal glands are the main places where nematodes synthesize effector proteins, which play central roles in successful invasion. The Meloidogyne incognita effector MiISE5 is exclusively expressed within the subventral esophageal cells and is upregulated during early parasitic stages. In this study, we show that MiISE5 can be secreted to barley cells through infectious hyphae of Magnaporthe oryzae. Transgenic Arabidopsis plants expressing MiISE5 became significantly more susceptible to M. incognita. Inversely, the tobacco rattle virus (TRV)-mediated silence of MiISE5 decreased nematode parasitism. Moreover, transient expression of MiISE5 suppressed cell death caused by Burkholderia glumae in Nicotiana benthamiana. Based on transcriptome analysis of MiISE5 transgenic sample and the wild-type (WT) sample, we obtained 261 DEGs, and the results of GO and KEGG enrichment analysis indicate that MiISE5 can interfere with various metabolic and signaling pathways, especially the JA signaling pathway, to facilitate nematode parasitism. Results from the present study suggest that MiISE5 plays an important role during the early stages of parasitism and provides evidence to decipher the molecular mechanisms underlying the manipulation of host immune defense responses by M. incognita.

Complete Plastome Sequencing Reveals an Extremely Diminished SSC Region in HemiparasiticPedicularis ishidoyana(Orobanchaceae)

The species within Orobanchaceae cover all forms of parasitism from full autotrophy to holoparasitism. Although parasitic species have gone through dramatic genome changes, the plastomes of land plants generally contain highly conserved structures. We investigated the complete plastome sequence of hemiparasitic Pedicularis ishidoyana, belonging to the largest genus in Orobanchaceae, and compared it with the highly rearranged genomes of other taxa in that family. This plastome comprises a total of 152 571 bp, with two inverted repeats (IRs) of 35 281 bp, a large single-copy region (LSC) of 81 982 bp, and an extremely reduced small single-copy (SSC) region of 27 bp. Although IR expansion and SSC contraction have been reported in the plastomes of other hemiparasitic species in Orobanchaceae, the IR region of P. ishidoyana is extremely expanded, resulting in a very small SSC region. This genome contains 101 genes that encode 67 proteins, four rRNAs, 30 tRNAs, and 22 duplicated genes in the IR region. In contrast to other hemi- or holoparasitic members of Orobanchaceae, genes in the ndh family in P. ishidoyana (except ndhE) have lost their function through pseudogenization (six genes) or deletion (four genes). Therefore, it seems that the degradation of this gene family in P. ishidoyana has occurred independently. Phylogenetic analysis, including all parasitic types, strongly supports the conclusion that P. ishidoyana diverged early from autotrophs and is sister to holoparasitic taxa. Our results suggest that these hemiparasitic Pedicularis plants might represent an early stage of parasitism that has developed stepwise in this family from non-parasites to holoparasites.

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.

A novel Meloidogyne incognita chorismate mutase effector suppresses plant immunity by manipulating the salicylic acid pathway and functions mainly during the early stages of nematode parasitism

Meloidogyne incognita is the most economically important plant‐parasitic nematode. Meloidogyne incognita manipulates plant cell development and metabolism by injecting effectors from the oesophageal glands into the plant host. Chorismate mutase (CM) is one such effector that may contribute to successful parasitism by M. incognita. This investigation identified and functionally characterized a novel CM effector, Mi‐CM‐3, which is more similar to CMs from bacteria than from other phytoparasitic nematodes. Spatial and temporal expression assays showed Mi‐cm‐3 mRNA accumulates specifically in the subventral oesophageal glands and transcription is up‐regulated during the early parasitic stages of the nematode. In planta gene silencing of Mi‐cm‐3 attenuated nematode parasitism. In addition, Mi‐cm‐3 could fully restore the full virulence phenotype of the pathogenic bacterium Xanthomonas oryzae pv. oryzae by complementation when it was introduced into a mutant strain carrying a deletion in the CM gene. Transient expression of Mi‐CM‐3 caused a reduction in levels of salicylic acid (SA) and mRNA of gene PR1 in Nicotiana benthamiana in response to oomycete pathogen Phytophthora capsici infection, while confocal observations showed that Mi‐CM‐3 was localized within the cytoplasm and the nucleus, but not the plastids, of transfected N. benthamiana leaf cells. Constitutive expression of Mi‐CM‐3 in N. benthamiana plants inhibited root growth and increased susceptibility to M. incognita infection. These results provide direct experimental evidence to show that Mi‐CM‐3 may play an important role in suppressing plant immunity by regulating the SA pathway during the early parasitic stage of M. incognita so as to promote nematode parasitism.