VTechWorks Repository :: Browsing by Author "Vieira, Paulo"

Browsing by Author "Vieira, Paulo"

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Cellular and Transcriptional Responses of Resistant and Susceptible Cultivars of Alfalfa to the Root Lesion Nematode, Pratylenchus penetrans
Vieira, Paulo; Mowery, Joseph; Eisenback, Jonathan D.; Shao, Jonathan; Nemchinov, Lev G. (2019-07-31)
The root lesion nematode (RLN), Pratylenchus penetrans, is a migratory species that attacks a broad range of crops, including alfalfa. High levels of infection can reduce alfalfa forage yields and lead to decreased cold tolerance. Currently, there are no commercially certified varieties with RLN resistance. Little information on molecular interactions between alfalfa and P. penetrans, that would shed light on mechanisms of alfalfa resistance to RLN, is available. To advance our understanding of the host-pathogen interactions and to gain biological insights into the genetics and genomics of host resistance to RLN, we performed a comprehensive assessment of resistant and susceptible interactions of alfalfa with P. penetrans that included root penetration studies, ultrastructural observations, and global gene expression profiling of host plants and the nematode. Several gene-candidates associated with alfalfa resistance to P. penetrans and nematode parasitism genes encoding nematode effector proteins were identified for potential use in alfalfa breeding programs or development of new nematicides. We propose that preformed or constitutive defenses, such as significant accumulation of tannin-like deposits in root cells of the resistant cultivar, could be a key to nematode resistance, at least for the specific case of alfalfa-P. penetrans interaction.
Cellular insights of beech leaf disease reveal abnormal ectopic cell division of symptomatic interveinal leaf areas
Vieira, Paulo; Kantor, Mihail R.; Jansen, Andrew; Handoo, Zafar A.; Eisenback, Jonathan D. (Public Library of Science, 2023-10-05)
The beech leaf disease nematode, Litylenchus crenatae subsp. mccannii, is recognized as a newly emergent nematode species that causes beech leaf disease (BLD) in beech trees (Fagus spp.) in North America. Changes of leaf morphology before emergence from the bud induced by BLD can provoke dramatic effects on the leaf architecture and consequently to tree performance and development. The initial symptoms of BLD appear as dark green, interveinal banding patterns of the leaf. Despite the fast progression of this disease, the cellular mechanisms leading to the formation of such aberrant leaf phenotype remains totally unknown. To understand the cellular basis of BLD, we employed several types of microscopy to provide an exhaustive characterization of nematode-infected buds and leaves. Histological sections revealed a dramatic cell change composition of these nematode-infected tissues. Diseased bud scale cells were typically hypertrophied and showed a high variability of size. Moreover, while altered cell division had no influence on leaf organogenesis, induction of cell proliferation on young leaf primordia led to a dramatic change in cell layer architecture. Hyperplasia and hypertrophy of the different leaf cell layers, coupled with an abnormal proliferation of chloroplasts especially in the mesophyll cell layers, resulted in the typical interveinal leaf banding. These discrepancies in leaf cell structure were depicted by an abnormal rate of cellular division of the leaf interveinal areas infected by the nematode, promoting significant increase of cell size and leaf thickness. The formation of symptomatic BLD leaves is therefore orchestrated by distinct cellular processes, to enhance the value of these feeding sites and to improve their nutrition status for the nematode. Our findings thus uncover relevant cellular events and provide a structural framework to understand this important disease.
Characterization and silencing of the fatty acid- and retinol-binding Pp-far-1 gene in Pratylenchus penetrans
Vieira, Paulo; Kamo, K.; Eisenback, Jonathan D. (2017-09)
Fatty acid-and retinol-binding proteins (FARs) are unique to nematodes, and are implicated in a wide range of metabolic and parasitic related functions. Along with the in silico analyses performed in this study, three different FAR members of this family were identified in Pratylenchus penetrans. The cDNA corresponding to the fatty acid-and retinol-binding Pp-far-1 gene was cloned and herein characterized at the molecular level for the first time for this genus. The translated 185 amino acid sequence of P. penetrans FAR-1 sequence, with a predicted molecular weight of 20.82 kDa and a pI of 5.49, shares highest sequence identity to FARs of other migratory nematodes of the Pratylenchidae family (90% to P. vulnus and 80% to Radopholus similis). In situ hybridization localizes Pp-far-1 transcripts in the hypodermis of the nematode. RT-qPCR detected Pp-far-1 transcripts for all nematode developmental stages, with highest expression levels found in juveniles, adult females and adult males, respectively. Pp-far-1 is also highly expressed during infection and establishment of P. penetrans in roots of different host plants, such as corn, lily and soybean. The importance of Pp-far-1 was studied by in planta RNA interference (RNAi) assays using stable soybean hairy root lines. Targeting Pp-far-1 decreased expression of the nematode Pp-far-1 and significantly reduced the reproduction of nematodes, with a total of between 44% and 70% fewer nematodes in comparison to the average number of nematodes counted for control lines. The results indicate that suppressing the expression levels of Pp-far-1 can act as an effective target gene to control P. penetrans.
Characterization of Lilium longiflorum cv. 'Nellie White' Infection with Root-lesion Nematode Pratylenchus penetrans by Bright-field and Transmission Electron Microscopy
Vieira, Paulo; Mowery, Joseph; Kilcrease, James; Eisenback, Jonathan D.; Kamo, Kathryn (2017-03)
Lilium longiflorum cv. Nellie White, commonly known as Easter lily, is an important floral crop with an annual wholesale value of over $26 million in the United States. The root-lesion nematode, Pratylenchus penetrans, is a major pest of lily due to the significant root damage it causes. In this study, we investigated the cytological aspects of this plant-nematode interaction using bright-field and transmission electron microscopy. We took advantage of an in vitro culture method to multiply lilies and follow the nematode infection over time. Phenotypic reactions of roots inoculated with P. penetrans were evaluated from 0 to 60 d after nematode infection. Symptom development progressed from initial randomly distributed discrete necrotic areas to advanced necrosis along entire roots of each inoculated plant. A major feature characterizing this susceptible host response to nematode infection was the formation of necrosis, browning, and tissue death involving both root epidermis and cortical cells. Degradation of consecutive cell walls resulted in loss of cell pressure, lack of cytoplasmic integrity, followed by cell death along the intracellular path of the nematode's migration. Pratylenchus penetrans was never seen in the vascular cylinder as the layer of collapsed endodermal cells presumably blocked the progression of nematodes into this area of the roots. This study presents the first detailed cytological characterization of P. penetrans infection of Easter lily plants.
Expanding the RNA virome of nematodes and other soil-inhabiting organisms
Vieira, Paulo; Subbotin, Sergei A.; Alkharouf, Nadim; Eisenback, Jonathan D.; Nemchinov, Lev G. (Oxford University Press, 2022-03-30)
In recent years, several newly discovered viruses infecting free-living nematodes, sedentary plant-parasitic nematodes, and migratory root lesion nematodes have been described. However, to the best of our knowledge, no comprehensive research focusing exclusively on metagenomic analysis of the soil nematode community virome has thus far been carried out. In this work, we have attempted to bridge this gap by investigating viral communities that are associated with soil-inhabiting organisms, particularly nematodes. This study demonstrates a remarkable diversity of RNA viruses in the natural soil environment. Over 150 viruses were identified in different soil-inhabiting hosts, of which more than 139 are potentially new virus species. Many of these viruses belong to the nematode virome, thereby enriching our understanding of the diversity and evolution of this complex part of the natural ecosystem.
An Expansin-Like Candidate Effector Protein from Pratylenchus penetrans Modulates Immune Responses in Nicotiana benthamiana
Vieira, Paulo; Nemchinov, Lev G. (2020-03)
The root lesion nematode (RLN) Pratylenchus penetrans is a migratory species that attacks a broad range of crops. After the RLN is initially attracted to host roots by root exudates and compounds, it releases secretions that are critical for successful parasitism. Among those secretions are nematode virulence factors or effectors that facilitate the entry and migration of nematodes through the roots and modulate plant immune defenses. The recognition of the effectors by host resistance proteins leads to effector-triggered immunity and incompatible plant- nematode interactions. Although many candidate effectors of the RLN and other plant-parasitic nematodes have been identified, the detailed mechanisms of their functions and particularly, their host targets remain largely unexplored. In this study, we sequenced and annotated genes encoding expansin-like proteins, which are major candidate effectors of P. penetrans. One of the genes, Pp-EXPB1, which was the most highly expressed during nematode infection in different plant species, was further functionally characterized via transient expression in the model plant Nicotiana benthamiana and global transcriptome profiling of gene expression changes triggered by this candidate effector in plants. As a result of this investigation, the biological roles of Pp-EXPB1 in nematode parasitism were proposed, the putative cellular targets of the proteins were identified, and the molecular mechanisms of plant responses to the nematode-secreted proteins were outlined.
First Insights into the Genetic Diversity of the Pinewood Nematode in Its Native Area Using New Polymorphic Microsatellite Loci
Mallez, Sophie; Castagnone, Chantal; Espada, Margarida; Vieira, Paulo; Eisenback, Jonathan D.; Mota, Manuel; Guillemaud, Thomas; Castagnone-Sereno, Philippe (PLOS, 2013-03-15)
The pinewood nematode, Bursaphelenchus xylophilus, native to North America, is the causative agent of pine wilt disease and among the most important invasive forest pests in the East-Asian countries, such as Japan and China. Since 1999, it has been found in Europe in the Iberian Peninsula, where it also causes significant damage. In a previous study, 94 pairs of microsatellite primers have been identified in silico in the pinewood nematode genome. In the present study, specific PCR amplifications and polymorphism tests to validate these loci were performed and 17 microsatellite loci that were suitable for routine analysis of B. xylophilus genetic diversity were selected. The polymorphism of these markers was evaluated on nematodes from four field origins and one laboratory collection strain, all originate from the native area. The number of alleles and the expected heterozygosity varied between 2 and 11 and between 0.039 and 0.777, respectively. First insights into the population genetic structure of B. xylophilus were obtained using clustering and multivariate methods on the genotypes obtained from the field samples. The results showed that the pinewood nematode genetic diversity is spatially structured at the scale of the pine tree and probably at larger scales. The role of dispersal by the insect vector versus human activities in shaping this structure is discussed.
Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans
Vicente, Cláudia S. L.; Nemchinov, Lev G.; Mota, Manuel; Eisenback, Jonathan D.; Kamo, Kathryn; Vieira, Paulo (Public Library of Science, 2019-02-22)
Similar to other plant-parasitic nematodes, root lesion nematodes possess an array of enzymes that are involved in the degradation of the plant cell wall. Here we report the identification of a gene encoding a cell wall-degrading enzyme, pectin methylesterase PME (EC 3.1.1.11), in the root lesion nematode Pratylenchus penetrans. Both genomic and coding sequences of the gene were cloned for this species, that included the presence of four introns which eliminated a possible contamination from bacteria. Expression of the Pp-pme gene was localized in the esophageal glands of P. penetrans as determined by in situ hybridization. Temporal expression of Pp-pme in planta was validated at early time points of infection. The possible function and activity of the gene were assessed by transient expression of Pp-pme in plants of Nicotiana benthamiana plants via a Potato virus X-based vector. To our knowledge, this is the first report on identification and characterization of a PME gene within the phylum Nematoda. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Meloidogyne aegracyperi n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitizing yellow and purple nutsedge in New Mexico
Eisenback, Jonathan D.; Holland, L. A.; Schroeder, Jill; Thomas, Steve H.; Beacham, Jacqueline M.; Hanson, S. F.; Paes-Takahashi, Vanessa; Vieira, Paulo (Society of Nematologists, 2019)
Meloidogyne aegracyperi n. sp. is described from roots of purple nutsedge in southern New Mexico, USA. Mature females are small (310–460 μm), pearly white, with their egg masses completely contained inside root galls. The neck is often at a 90 to 130° angleto the protruding posterior end with the perineal pattern. The distance of the dorsal esophageal gland orifice (DGO) to the base of the stylet is relatively long (4.0–6.1 μm), and the excretory pore is level with the base of the stylet. The anterior portion of the rounded lumen lining of the metacorpus contains 3 to 10 small vesicles. The perineal pattern has a rounded dorsal arch with a tail terminal area that is smooth or marked with rope-like striae. Only two males were found. The body twists 90° throughout its length. The DGO to the base of the stylet is long (3.0–3.3) μm. The cephalic framework of the second-stage juvenile is weak, and the stylet is short (10.1–11.8 μm). The DGO to the base of the stylet is long (3–5 μm). The tail is very long (64–89 μm) and the hyaline portion of the tail is very narrow, making the tail finely pointed. Eggs are typical for the genus and vary in length (85.2–99.8 μm) and width (37.1–48.1 μm), having a L/W ratio of (2.1–2.6). Maximum likelihood phylogenetic analyses of the different molecular loci (partial 18S rRNA, D2-D3 of 28S rRNA, internal transcribed spacer (ITS) rRNA, cytochrome oxidase subunit II (COII)-16S rRNA of mitochondrial DNA gene fragments and partial Hsp90 gene) placed this nematode on an independent branch in between M. graminicola and M. naasi and a cluster of species containing M. chitwoodi. M. fallax, and M. minor. Greenhouse tests showed that yellow and purple nutsedge were the best hosts, but perennial ryegrass, wheat, bentgrass, and barley were also hosts.
A new esophageal gland transcriptome reveals signatures of large scale de novo effector birth in the root lesion nematode Pratylenchus penetrans
Vieira, Paulo; Shao, Jonathan; Vijayapalani, Paramasivan; Maier, Thomas R.; Pellegrin, Clement; Eves-van den Akker, Sebastian; Baum, Thomas J.; Nemchinov, Lev G. (2020-10-23)
Background The root lesion nematode Pratylenchus penetrans is a migratory plant-parasitic nematode responsible for economically important losses in a wide number of crops. Despite the importance of P. penetrans, the molecular mechanisms employed by this nematode to promote virulence remain largely unknown. Results Here we generated a new and comprehensive esophageal glands-specific transcriptome library for P. penetrans. In-depth analysis of this transcriptome enabled a robust identification of a catalogue of 30 new candidate effector genes, which were experimentally validated in the esophageal glands by in situ hybridization. We further validated the expression of a multifaceted network of candidate effectors during the interaction with different plants. To advance our understanding of the “effectorome” of P. penetrans, we adopted a phylogenetic approach and compared the expanded effector repertoire of P. penetrans to the genome/transcriptome of other nematode species with similar or contrasting parasitism strategies. Our data allowed us to infer plausible evolutionary histories that shaped the effector repertoire of P. penetrans, as well as other close and distant plant-parasitic nematodes. Two remarkable trends were apparent: 1) large scale effector birth in the Pratylenchidae in general and P. penetrans in particular, and 2) large scale effector death in sedentary (endo) plant-parasitic nematodes. Conclusions Our study doubles the number of validated Pratylenchus penetrans effectors reported in the literature. The dramatic effector gene gain in P. penetrans could be related to the remarkable ability of this nematode to parasitize a large number of plants. Our data provide valuable insights into nematode parasitism and contribute towards basic understating of the adaptation of P. penetrans and other root lesion nematodes to specific host plants.
A novel species of RNA virus associated with root lesion nematode Pratylenchus penetrans
Vieira, Paulo; Nemchinov, Lev G. (2019-04)
The root lesion nematode Pratylenchus penetrans is a migratory species that attacks a broad range of plants. While analysing transcriptomic datasets of P. penetrans, we have identified a full-length genome of an unknown positive-sense singlestranded RNA virus, provisionally named root lesion nematode virus 1 (RLNV1). The 8614-nucleotide genome sequence encodes a single large polyprotein with conserved domains characteristic for the families Picornaviridae, Iflaviridae and Secoviridae of the order Picornavirales. Phylogenetic, BLAST and domain search analyses showed that RLNV1 is a novel species, most closely related to the recently identified sugar beet cyst nematode virus 1 and potato cyst nematode picornalike virus. In situ hybridization with a DIG-labelled DNA probe confirmed the presence of the virus within the nematodes. A negative-strand-specific RT-PCR assay detected RLNV1 RNA in nematode total RNA samples, thus indicating that viral replication occurs in P. penetrans. To the best of our knowledge, RLNV1 is the first virus identified in Pratylenchus spp.
Phytochemicals as Biopesticides against the Pinewood Nematode Bursaphelenchus xylophilus: A Review on Essential Oils and Their Volatiles
Faria, Jorge M. S.; Barbosa, Pedro; Vieira, Paulo; Vicente, Cláudia S. L.; Figueiredo, Ana Cristina; Mota, Manuel (MDPI, 2021-11-28)
The impacts of a rapidly changing environment together with the growth in global trade activities has promoted new plant pest pandemic events in forest ecosystems. The pinewood nematode (PWN), Bursaphelenchus xylophilus, causes strong worldwide economic and ecological impacts. Direct control is performed through trunk injection of powerful nematicides, however many of these (hemi)synthetic compounds have raised ecological and human health concerns for affecting non-target species and accumulating in food products. As sustainable alternatives, essential oils (EOs) have shown very promising results. In this work, available literature on the direct activity of EOs against PWN is reviewed, as a contribution to advance the search for safer and greener biopesticides to be used in sustainable PWD pest management strategies. For the first time, important parameters concerning the bioassays performed, the PWNs bioassayed, and the EOs used are summarized and comparatively analyzed. Ultimately, an overview of the chemical composition of the most active EOs allowed to uncover preliminary guidelines for anti-PWN EO efficiency. The analysis of important information on the volatile phytochemicals composing nematicidal EOs provides a solid basis to engineer sustainable biopesticides capable of controlling the PWN under an integrated pest management framework and contributes to improved forest health.
The Pratylenchus penetrans Transcriptome as a Source for the Development of Alternative Control Strategies: Mining for Putative Genes Involved in Parasitism and Evaluation of in planta RNAi
Vieira, Paulo; Eves-van den Akker, Sebastian; Verma, Ruchi; Wantoch, Sarah; Eisenback, Jonathan D.; Kamo, Kathryn (PLOS, 2015-12-14)
The root lesion nematode Pratylenchus penetrans is considered one of the most economically important species within the genus. Host range studies have shown that nearly 400 plant species can be parasitized by this species. To obtain insight into the transcriptome of this migratory plant-parasitic nematode, we used Illumina mRNA sequencing analysis of a mixed population, as well as nematode reads detected in infected soybean roots 3 and 7 days after nematode infection. Over 140 million paired end reads were obtained for this species, and de novo assembly resulted in a total of 23,715 transcripts. Homology searches showed significant hit matches to 58% of the total number of transcripts using different protein and EST databases. In general, the transcriptome of P. penetrans follows common features reported for other root lesion nematode species. We also explored the efficacy of RNAi, delivered from the host, as a strategy to control P. penetrans, by targeted knock-down of selected nematode genes. Different comparisons were performed to identify putative nematode genes with a role in parasitism, resulting in the identification of transcripts with similarities to other nematode parasitism genes. Focusing on the predicted nematode secreted proteins found in this transcriptome, we observed specific members to be up-regulated at the early time points of infection. In the present study, we observed an enrichment of predicted secreted proteins along the early time points of parasitism by this species, with a significant number being pioneer candidate genes. A representative set of genes examined using RT-PCR confirms their expression during the host infection. The expression patterns of the different candidate genes raise the possibility that they might be involved in critical steps of P. penetrans parasitism. This analysis sheds light on the transcriptional changes that accompany plant infection by P. penetrans, and will aid in identifying potential gene targets for selection and use to design effective control strategies against root lesion nematodes.
Prevalence of the root lesion nematode virus (RLNV1) in populations of Pratylenchus penetrans from North America
Vieira, Paulo; Peetz, Amy; Mimee, Benjamin; Saikai, Kanan; Mollov, Dimitre; MacGuidwin, Ann; Zasada, Inga; Nemchinov, Lev G. (2020-05-18)
Root lesion nematode virus 1 (RLNV1) was discovered in the migratory endoparasitic nematode species Pratylenchus penetrans. It was found in a P. penetrans population collected from soil samples in Beltsville, Maryland, USA. In this study, the distribution of the RLNV1 in 31 geographically distinct P. penetrans populations obtained from different crops was examined. The results demonstrate that RLNV1 is widespread in North American populations of P. penetrans and exhibits low genetic variability in the helicase and RNA-dependent RNA polymerase regions of the genome.
The Root Lesion Nematode Effector Ppen10370 Is Essential for Parasitism of Pratylenchus penetrans
Vieira, Paulo; Vicente, Cláudia S. L.; Branco, Jordana; Buchan, Gary; Mota, Manuel; Nemchinov, Lev G. (2021-06)
The root lesion nematode Pratylenchus penetrans is a migratory species that attacks a broad range of crops. Like other plant pathogens, P. penetrans deploys a battery of secreted protein effectors to manipulate plant hosts and induce disease. Although several candidate effectors of P. penetrans have been identified, detailed mechanisms of their functions and particularly their host targets remain largely unexplored. In this study, a repertoire of candidate genes encoding pioneer effectors of P. penetrans was amplified from mixed life stages of the nematode, and candidate effectors were cloned and subjected to transient expression in a heterologous host, Nicotiana benthamiana, using potato virus X-based gene vector. Among seven analyzed genes, the candidate effector designated as Ppen10370 triggered pleiotropic phenotypes substantially different from those produced by wild type infection. Transcriptome analysis of plants expressing Ppen10370 demonstrated that observed phenotypic changes were likely related to disruption of core biological processes in the plant due to effector-originated activities. Cross-species comparative analysis of Ppen10370 identified homolog gene sequences in five other Pratylenchus species, and their transcripts were found to be localized specifically in the nematode esophageal glands by in situ hybridization. RNA silencing of the Ppen10370 resulted in a significant reduction of nematode reproduction and development, demonstrating an important role of the esophageal gland effector for parasitism.
Symptom Development in Response to Combined Infection of In Vitro-grown Lilium longiflorum with Pratylenchus penetrans and Soilborne Fungi Collected from Diseased Roots of Field-grown Lilies
Lakshman, Dilip; Vieira, Paulo; Pandey, Ruchi; Slovin, Janet; Kamo, Kathryn (2017-06)
Eight fungal isolates (ELRF 1 to 8) were recovered from necrotic roots of Easter lilies, Lilium longiflorum cv. Nellie White, grown in a field in the U.S. Pacific Northwest. The eight fungal isolates were identified by sequencing and molecular phylogenetic analyses based on their ITS rDNA region. Five isolates were identified as Fusarium oxysporum, two as F. tricinctum, and one as Rhizoctonia sp. AG-I. This constitutes the first report of Rhizoctonia sp. AG-I infecting lilies worldwide and the first report of F. tricinctum infecting lilies in the United States. To study and validate their pathogenicity, pure cultures of each isolate were used to infect the roots of Easter lily plants growing in vitro. In addition, Easter lily plants growing in vitro were infected either with or without Pratylenchus penetrans, the root lesion nematode, prior to placing a culture plug of fungus 1 cm from a lily root. Pratylenchus penetrans is a nematode species commonly found in the sampled fields. The presence of both nematode and Rhizoctonia sp. AG-I isolate ELRF 3 in infected lilies was evaluated by molecular analyses, confirming the infection of roots 3 days after inoculation, prior to development of disease symptoms. Necrosis and root rot developed more rapidly with all eight fungal isolates when there had been prior infection with P. penetrans, the major nematode parasitizing Easter lily roots in the field in Oregon.

Browsing by Author "Vieira, Paulo"

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