Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to NRP
TARGETING ESSENTIAL METABOLIC AND PARASITISM GENES OF THE ROOT-LESION NEMATODE TO DEVELOP NEMATODE-RESISTANT PLANTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1005202
Grant No.
2015-67012-22834
Cumulative Award Amt.
$150,000.00
Proposal No.
2014-02142
Multistate No.
(N/A)
Project Start Date
Dec 1, 2014
Project End Date
Nov 30, 2017
Grant Year
2015
Program Code
[A7201]- AFRI Post Doctoral Fellowships
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Plant Path, Phys, & Weed Sci
Non Technical Summary
Worldwide crop losses due to plant-parasitic nematodes have been estimated at $118 billion annually, with Pratylenchus spp. (commonly known as root-lesion nematodes, RLN) ranking third in terms of economic losses. The range of available nematicide products is limited, and some of these are expected to be banned in the future. Most of the research for controlling nematodes has been done on sedentary nematodes such as root-knot and cyst nematodes, rather than migratory nematodes such as RLN. The goal of this project is to produce genetically enhanced crops with resistance against RLN. Comparative transcriptome analyses (Illumina mRNA-Seq) of soybean roots infected with Pratylenchus penetrans have been performed, providing basic knowledge about the molecular mechanisms involved in the RLN-host interaction. Engineered soybean roots will be employed using two different strategies against RLN: 1) Silencing of essential metabolic and parasitism candidate genes of RLN through RNA- mediated interference technology; 2) Blocking RLN development by overexpression of proteins with nematicidal effect, such as cysteine proteinase inhibitors and Bacillus thuringiensis crystal proteins, delivery during nematode feeding in planta. The identification and experimental validation of these approaches genes would represent a break-through for novel management practices against such resilient plant-parasitic nematodes.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21221211120100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
2121 - Cut flowers, foliage, and greens;

Field Of Science
1120 - Nematology;
Goals / Objectives
The main goal of this project is to produce genetically enhanced crops with resistance against root lesion nematodes by the development of novel, environmentally friendly approaches based on molecular strategies.Objective 1: Determine the specific Pratylenchus penetrans and Glycine max/Lilium longiflorum genes involved during the initial stages of root infection.1.1. Elucidate the molecular mechanisms by which RLN interact with host plants1.2. Identify and select essential metabolic and parasitism genes of RLNObjective 2: Characterize two biotechnological strategies, RNAi and overexpression of dietary proteins, against root-lesion nematodes2.1. Study the potential of inducing an effective host resistance against RLN through RNAi2.2. Study the potential of dietary protein uptake from plants against RLN, by overexpressing cysteine proteinase inhibitors (cystatins) and Bacillus thuringiensis crystal proteins
Project Methods
To achieve our obejctives the following methodologies will be implemented:1: Determine the specific Pratylenchus penetrans and Glycine max/Lilium longiflorumgenes involved during the initial stages of root infection.1.1: Elucidate the molecular mechanisms by which RLN interact with host plantsThe transcriptome data will be managed and analyzed using the reference genome (Gmax189) and the abundance of reads estimated using the Tophat and Cufflinks suite. The abundance will be evaluated at the isoform level in Fragments Per Kilobase of transcript per Million mapped reads (FPKM) as defined in the Cufflinks program. Integration and interpretation of the datasets will be conducted by using the soybean database (phytozome.net/soybean) of metabolic pathways and enzymes, as well as the KEGG pathways database. Pair-wise comparison will be performed and transcript isoforms showing significant difference (q-value < 0.05) will be chosen for GO and KEGG pathway enrichment analysis using DAVID. A set of selected genes will be validated by qRT-PCR with the same RNA samples that were used for the RNAseq experiments. This objective is expected to yield unique insights into the molecular mechanisms and novel gene networks responsible for the induction of the disease by RLN.1.2. Identify and select essential metabolic and parasitism genes of RLNAvailable EST sequences for Pratylenchus in http://www.wormbase.org and http://www.nematode.net, and recently published transcriptomes for other species of Pratylenchus will be use as a dataset. De novo assembly of P. penetrans contigs generated from mRNA-Seq samples will be aligned against the C. elegans genome. Following alignment, multiple-sequence alignment will identify sequences conserved across Pratylenchus species and C. elegans. Mapping genomic coordinates of known RNAi lethal phenotypes (WormBase: "lethal", "embryonic lethal", and "larval lethal") onto such conserved sequences will generate candidate sequences that produce. For the identification of putative related-parasitism genes BLAST search analysis will be also conducted against public genome datasets of plant-parasitic nematodes (e.g. Meloidogyne incognita as a sedentary species, and Bursaphelenchus xylophilus as a migratory species of nematodes). Specific research directions will be evaluated and prioritized as they are encountered, i.e. high priority will be given to candidate genes that will most likely contribute to essential metabolic pathways of the nematode's biology.Objective 2: Characterize two biotechnological strategies, RNAi and overexpression of dietary proteins, against root-lesion nematodes2.1. Study the potential of inducing an effective host resistance against RLN through RNAiThis task will be started with the validation of the transcript levels of P. penetrans candidate genes by qRT-PCR. A set of genes (10 to 15) will be evaluated. To test this hypothesis, RNAi constructs will be prepared from the list of candidate genes, and transform hairy root cultures of soybean to demonstrate suppression of the RLN gene. PCR products of the candidate targets will be initially cloned in pENTR, and then subcloned as an inverted repeat separated by a linker fragment into a T-DNA vector suitable for A. rhizogenes-mediated plant transformation. A control construct will also be prepared that includes inverted repeats of the E. coli GUS gene. For nematode infection tests, a mixed-stage population of P. penetranswill be placed in the Petri plate with the roots, and incubated in the dark at 30°C, for two months. As a control two different options will be used: 1) non-transformed hairy roots and 2) hairy roots with inverted repeats of the E. coli GUS gene. The number of nematodes developing in each root will be used to determine infection levels. In addition, a microscopic screening of the extracted nematodes will be performed to identify any possible nematode phenotype as supporting evidence for the RNAi suppression of a specific gene. Primer pairs outside the gene fragments targeted for RNAi will be used to monitor the suppression of the candidate genes by qRT-PCR. Candidate genes showinggood levels of resistance in soybean hairy roots, the gene will be then subcloned and used to transformLilium'Nellie White'.2.2. Study the potential of dietary protein uptake from plants against RLN, by overexpressing cysteine proteinase inhibitors (cystatins) and Bacillus thuringiensis crystal proteins.Soybean hairy roots will be transformed with A. rhizogenes containing Bt candidate genes. Genes cloned in a T-DNA vector under the CaMV 35S promoter, containing nptII as the selectable marker gene. Transformed hairy roots will be selected in Petri plates of MS medium with 50 mg/L kanamycin in the dark at 25°C. Putatively- transformed soybean hairy roots will be analyzed by PCR using transgene-specific primer sequences to determine if they have the gene of interest. Levels of candidate genesexpression will be compared using RT-PCR, and genomic DNA from selected lines will be used for Southern hybridization to confirm integration of the transgene. A set of 5 lines with the relatively highest level of RNA for the transgene will be used for challenge inoculation with P. penetrans. The number of nematodes developing in each root will determine infection levels.If either Bt gene shows good levels of resistance in soybean hairy roots, the gene will be then subcloned into a pUC-based vector under control of the CaMV-35S promoter and used to transform Lilium 'Nellie White'.

Progress 12/01/16 to 11/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 2.2: We have completed in situ hybridization for 70 gene candidates, and we are currently conducting semi-quantitative RT-PCR analyses for nematode genes found specifically in the esophageal glands in different developmental stages of P. penetrans. In additional, temporal expression analyses of these genes were initiatedby qPCR in infected roots at 1, 3 and 7 days after nematode infection.

Publications


    Progress 12/01/14 to 11/30/17

    Outputs
    Target Audience:The new results obtained during this project were disseminated among researchers working in different areas, such as nematology, plant-pathology, students, as well as growers dealing with root lesion nematodes. Our research results have been shared frequently in national and international conferences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Information about the final results of this research project has been presented on the 56th Annual Meeting of the Society of Nematology, to the APS Annual Meeting and to an international conference on Plant Protection. The three papers submitted in 2016 were accepted and published during the year of 2017. One additional paper has been submitted to a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Objective 1.2: In this final year we combined spatially and temporally resolved next generation sequencing datasets of P. penetrans to select a list of candidate genes aimed at the identification of a panel of effector genes for this species. We determined the spatial expression of transcripts of 22 candidate effector genes within the esophageal glands of P. penetrans by in situ hybridization, out of a total of 100 nematode genes selected. These comprised homologues of known effectors of other plant-parasitic nematodes with diverse putative functions (e.g. cell wall-degrading enzymes), as well as eight novel pioneer effectors specific to RLN. We then combined in situ localization of effectors with available genomic data to identify a non-coding motif that is enriched promoter regions of a subset of P. penetrans effectors, and thus a putative hallmark of spatial expression. A selected subset of candidate effectors was shown to be actively expressed during the early steps of plant infection using RT-qPCR analyses. Our current results provide the most comprehensive panel of effector genes found for RLN. Considering the damage caused by P. penetrans, this information provides valuable data to elucidate the pathogenicity basis of this broad-host-nematode and offers useful suggestions regarding the potential use of RLN-specific target effector genes to control this important pathogen.

    Publications

    • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Vieira, P., Mayer, T., Eves-van den Akker, S., Howe, D.K., Zasada, I., Baum, T., Eisenback, J.D., Kamo, K. 2017. Identification of candidate effector genes of Pratylenchus penetrans. Submitted
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Lakshman, D.K., Kamo, K., Vieira, P., and Pandey, R. Exploring the causal agents of lily bulb- and root-rot disease in the U.S. Pacific Northwest. APS Annual meeting, 5-9 August, San Antonio, Texas, US.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Vieira, P., Maier, T., Eves-van den Akker, S., Zasada, I., Baum, T., Eisenback, J.D. and Kamo, K. Identification of a panel of effector genes for Pratylenchus penetrans. 56th Annual meeting of the Society of Nematologists, 13-16 August, Williamsburg, Virginia, US.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Branco, J., Vicente, C., Mota, M. Eisenback, J.D., Kamo, K. and Vieira, P. Characterization of a set of cell wall-degrading enzymes of the root lesion nematode Pratylenchus penetrans. 2 Simp�sio SCAP de Protec��o de Plantas; 8 Congresso da Sociedade Portuguesa de Fitopatologia, and 11 Encontro Nacional de Protec��o Integrada. 26-27 October, Santar�m, Portugal.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Vieira, P. Multi-strategy biotechnological application towards the root lesion nematode control. USDA NIFA Fellowship Project Directors Meeting, 22-23 August, Washington, DC, US.


    Progress 12/01/16 to 11/30/16

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Objective 2.2: We have completed in situ hybridization for 70 gene candidates, and we are currently conducting semi-quantitative RT-PCR analyses for nematode genes found specifically in the esophageal glands in different developmental stages of P. penetrans. In additional, temporal expression analyses of these genes were initiatedby qPCR in infected roots at 1, 3 and 7 days after nematode infection.

    Publications


      Progress 12/01/15 to 11/30/16

      Outputs
      Target Audience:The new results of this project provided relevant information for researchers working in different areas, such as nematology, plant-pathology, students, and growers dealing with root lesion nematodes. Our research results have been shared frequently with Dr. Kathryn Kamo (Floral and Nursery Plants Research Unit, Beltsville, USDA) in joint collaboration meetings, which has a close collaboration with lily growers in California. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Research training for 1 research assistant. How have the results been disseminated to communities of interest?Information about the progress and results of this research project has been presented on 27th Annual USDA-Beltsville Poster Day, the 55th Annual Meeting of the Society of Nematology and to the 32nd European Society Symposium of Nematology. A seminar was also given to the graduate students in Virginia Tech. One paper has been published, and three others have been submitted to peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?Over the next 12 months we will finalize our studies on the plant molecular pathways involved upon P. penetrans infection. We will also finalize our screen analyses for the identification of new nematode parasitism genes in P. penetrans, and respective silencing using the RNAi setup established during this project. Finally, we will gather all data generated and submit the results to peer-reviewed journals.

      Impacts
      What was accomplished under these goals? Objective 1.1: Characterize transcriptional expression profiles of plant molecular pathways upon root lesion nematode infection is important to have an overview of the molecular actors of the plant to this nematode. Since the last reporting period we have validated by qPCR analyses gene expression profiles belonging to the main defense pathways of two economic important plants (soybean and lilies) against P. penetrans, using a series of time points. We are currently working on a publication dealing with the results obtained from these analyses.To complement our molecular analyses, we also investigated the cytological aspects of the interaction of this nematode in lilies using bright-field light 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 days after nematode infection. Symptom development progressed from initial randomly distributed discrete necrotic areas to advanced necrosis along entire roots of each inoculated plant. The induction and severity of symptoms could be correlated with the number of nematodes found parasitizing roots. 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. This study presents the first detailed cytological characterization of P. penetrans infection of Easter lily plants. In addition, as P. penetrans is often associated with other soil borne pathogens, we studied the symptom development responses of Lilium longiflorum cv. Nellie White to a combination of infections with P. penetrans and soilborne fungi collected from diseased roots of field-grown lilies. Along this study eight fungal isolates were isolated from necrotic roots of L. longiflorum, 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. The isolates were identified within different species of Fusarium and Rhizoctonia. To study and validate their pathogenicity, pure cultures of each isolate were used to infect the roots of Easter lily plants growing in vitro. Lily plants were then infected in a combination of fungal and nematode assays. Observations of necrosis and root rot showed more rapid development of disease symptoms when both nematode and fungal isolates where co-infecting the lily roots. Objective 1.2:The reference transcriptome assembly generated for P. penetrans during the first year of this project constitutes a relevant output for this objective (Vieira et al. 2015). In the second year, we conducted more detailed analyses for the identification and characterization of parasitism genes of P. penetrans. The predicted transcripts containing a signal peptide and no transmembrane domain were ranked according their normalized expression. This allowed us to look at the highly expressed nematode secreted candidate genes, and to increase the likelihood of identifying genes relevant for parasitism, we selected a set of genes for which there is evidence for expression in planta base on our recent transcriptome analyses (Vieira et al. 2015). We conducted in situ hybridization for a set of 50 genes, and studied their localization within the nematode tissues. We were able to validate and confirm the specific localization of transcripts encoding for orthologues of known parasitism genes from other plant-parasitic nematodes (e.g. cell wall degrading enzymes), as well as new pioneer genes in the esophageal glands of P. penetrans. We are currently conducting qPCR analyses to evaluate the expression levels of those genes found in the esophageal glands of P. penetrans, in different nematode developmental stages (eggs, juveniles and adults), and at different time points of plant infection. A manuscript dealing with these results is under preparation. Objective 2.1:RNAi interference represents a powerful technique for the analysis of gene function, and has shown promising results in the control of plant pathogens, including plant-parasitic nematodes. We have validated this proof of concept and conducted RNAi experiments against metabolic and parasitism related genes of P. penetrans. Two genes related to locomotion and muscle architecture (Pp-pat-10 and Pp-unc-87), which were highly abundant among the nematode transcripts identified from infected roots, provide significant nematode reduction after plant-mediated RNAi silencing. In addition, other parasitism-related genes involved in different molecular pathways (unpublished data) have been analyzed, with some of them showing a significant reduction of nematodes (unpublished and submitted data for publication). Objective 2.2:After validation the efficiency of overexpression two cystatin genes (OC-I and OC-II), and two Bt genes (Cry5B and Cry6A) against P. penetrans using soybean hairy roots, the reductions on the nematode development reach up to 15% reduction only, and we prioritized our studies using an RNAi assay approach instead.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2015 Citation: Vieira P, Eves-van den Akker S, Verma R, Wantoch S, Eisenback JD and Kamo K. 2015. 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. PlosOne 10: e0144674.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Identification of nematode target genes for root lesion nematode (Pratylenchus penetrans) control. Paulo Vieira, Sarah Wantoch, Jonathan D. Eisenback, and Kathryn Kamo. 27thAnnual Beltsville USDA Poster Day, 27 April, Beltsville, US.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Cytological changes of easter lily (Lilium longiflorum) upon root lesion nematode (Pratylenchus penetrans) infection. Paulo Vieira, Joseph Mowery, James Kilcrease, Jonathan D. Eisenback, and Kathryn Kamo. 55th Annual meeting of the Society of Nematologists, 17-22 July, Montreal, Canada.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Data mining of the root lesion nematode (Pratylenchus penetrans) transcriptome for identification of candidate effector genes. Vieira, Paulo, T. Maier, I. A. Zasada, T. Baum, K. Kamo, and J. D. Eisenback. 55th Annual meeting of the Society of Nematologists, 17-22 July, Montreal, Canada.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Identification of parasitism-related genes in Pratylenchus penetrans. Paulo Vieira, Thomas Maier, Inga A. Zasada, Thomas Baum, Kathryn Kamo, and Jonathan D. Eisenback. 32nd Symposium of the European Society of Nematologists, 28 August-1 September, Braga, Portugal.
      • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Lakshman D, Vieira P, Pandey R, Slovin J and Kamo K. 2016. Symptom development in response to combined infection of in vitro grown Lilium longiflorum with the root lesion nematode Pratylenchus penetrans and soilborne fungi collected from diseased roots of field-grown lilies.
      • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Vieira P, Mowery J, Kilcrease J, Eisenback JD and Kamo K. 2016. Characterization of Lilium longiflorum cv. Nellie White infection with root lesion nematode Pratylenchus penetrans by bright-field and transmission electron microscopy.
      • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Vieira P, Kamo K and Eisenback JD. 2016. Plant-mediated silencing of a fatty acid- and retinoid-binding gene can reduce development of the root lesion nematode, Pratylenchus penetrans.


      Progress 12/01/14 to 11/30/15

      Outputs
      Target Audience:This project aims to expanded knowledge about the root lesion nematode (Pratylenchus penetrans) molecular biology and its interaction with plants. In addition we attempt to develop new biotechnology tools aimed at the control of such pathogens. The results obtained during this period were shared with other researchers and students with oral and poster presentations, during national and international research meetings. In addition, we are sharing our research results frequently with Dr. Kathryn Kamo (Floral and Nursery Plants Research Unit, Beltsville, USDA) in joint collaboration meetings, as Dr. Kamo's lab is working with root lesion nematodes and interacts directly with Oregon lily growers. Our recently submitted manuscript will target other researchers working directly with root lesion nematodes and nematode parasitism related genes. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PI of this project has increased his skills of bioinformatic tools to understand the molecular mechanisms related to this nematode-plant interaction. A technician has participated in this project and has been trained in nematology and molecular biology. Other researchers gained additional knowledge about plant-nematode molecular interaction of hosts affected by this economic important species. How have the results been disseminated to communities of interest?Information about the progress and results of this research project has been presented on the 26th Annual Beltsville Poster Day, on the 54th Annual Meeting of the Society of Nematologists, and on the Annual Meeting of the American Phytopathological Society. Through the established collaboration with Dr. Kathryn Kamo (Beltsville, USDA), the results of this project has been shared with her team and with lily growers. Growers became aware of the importance of implementing future biotechnology approaches for controlling this group of nematodes (e.g. lily growers). What do you plan to do during the next reporting period to accomplish the goals?Over the next year of the project we will finalize the analysis of the mRNA-seq results generated for the nematode infected soybean roots, which will enable a better understanding of the molecular actors of this nematode-plant interaction. A second replication of the current resistant tests will be performed for the most promising nematode silencing genes, as well as for the lines overexpressing both Bacillus thuringiensis and cystatin genes. We anticipate adding additional nematode target genes for RNAi silencing (mainly putative parasitism genes), and test their efficacy for nematode control.

      Impacts
      What was accomplished under these goals? Objective 1: The specific characterization of the molecular mechanism/genes involved during the initial stages of root lesion infection is relevant to understand how this plant-nematode is able to induce root lesion disease. The root lesion nematode Pratylenchus penetrans is an important plant parasitic species causing significant yield decrease of several crops. Objective 1.1: During this first year of the project we have achieved and finalized the mRNA-seq of different soybean samples infected with the nematode (3 replicates x two time points after nematode infection, plus respective controls) using Illumina technology. Considering the total number of biological replicates (9 samples), RNA representing the entire transcriptome of infected soybean roots with P. penetrans was sequenced and a total of 732,066,470 reads were generated. In total 424,998,906 reads were acquired from infected and 307,067,564 from uninfected root tissues from both time points. All reads have been mapped against the soybean reference genome, and the remaining in silico analyses are still under evaluation. In addition RNA of nematode infected lilies samples have collected and prepared for subsequent mRNA-seq analysis. Objective 1.2: To obtain insight into the transcriptome of this migratory plant-parasitic nematode, we used Illumina mRNA sequencing analysis of a mixed population (two samples), in order to generate a reference transcriptome for this species. Over 140 million paired end reads were obtained for this species, and de novo assembly resulted in a total of 23,175 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. 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 (e.g. cell wall degrading enzymes). In addition, nematode reads detected in infected soybean roots 3 and 7 days after nematode infection were also characterized. Focusing on the predicted nematode secreted proteins found in this transcriptome; we have found nematode specific members to be up-regulated at the early time points of host infection. A representative set of these genes was examined using RT-PCR to confirm their expression during the host infection. The expression patterns of the different candidate genes raises the possibility that they might be involve in critical steps of P. penetrans parasitism, which may constitute appropriated targets for nematode control. This work has been submitted for publication. Objective 2.1: We initiated the efficacy of RNAi, delivered from the host, as a strategy to control P. penetrans, by targeted knock-down of selected metabolic (five candidates) and putative-parasitism related nematode genes (five candidates) using dsRNA constructs. After validation of the transcript levels of each P. penetrans candidate gene target by qPCR, PCR products of each nematode candidate (ranging size from 350 bp to 750 bp) have been cloned in a Gateway system (pENTR vector) using P. penetrans cDNA, and sub-cloned as an inverted repeat separated by a linker fragment into a T-DNA vector suitable (pRAP17). The pRAP17 constructs were then individually transferred to competent Agrobacterium rhizogenes (K559), and used to generate stable transgenic soybean hairy root lines. All transformations were confirmed by PCR, and expression levels of each dsRNA construct in planta were validated using the expression of the intron that separates each fragment by semi-quantitative RT-PCR. For each nematode candidate gene a minimum of 10 lines have been established. We are currently performing nematode resistant tests for 10 nematode candidate genes using a minimum of tree to five independent lines for each nematode gene. Objective 2.1: To evaluate this strategy against P. penetrans we have generated transgenic soybean hairy roots overexpressing two Bacillus thuringiensis genes. For comparison transgenic soybean hairy roots have been also generated overexpressing two previous isolated rice cystatin genes (OC-I and OC-II) (Samac and Smigocki, 2003), while control hairy root lines were generated using A. rhizogenes harboring an empty vector (pBIN-JIT) that provides kanamycin resistance in both bacteria and plants (Ferrandiz et al. 2000). Specific primers were designed for genomic detection of each gene in soybean hairy roots, as well as level of transcript expression of each gene using a semi-quantitative RT-PCR amplification. As controls the Gm-UBQ3 (Polyubiquitin gene) gene-specific primers have been used. For each nematode candidate gene a minimum of 5 lines have been established. We are currently performing nematode resistant tests for these lines, using a minimum of tree to five independent lines for each gene.

      Publications

      • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Vieira, P., Wantoch, S., Eisenback, J.D., Kamo, K. 2015. Multi-strategy biotechnological application towards root lesion nematode control. 26th Annual Beltsville Poster Day. May 20th, National Agricultural Library, Beltsville, Maryland.
      • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Vieira, P., Wantoch, S., Eisenback, J.D., Kamo, K. 2015. Insight into the soybean transcriptional profiling upon infection by root lesion nematode. 54th Annual Meeting of the Society of Nematologists. July 19-24, East Lansing, Michigan.
      • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Vieira, P., Wantoch, S., Eisenback, J.D., Kamo, K. 2015. Characterization of the transcriptional profiling of the migratory root lesion nematode infection on important crop an floral species. 2015 Annual Meeting of The American Phytopathological Society. August 1-5, Pasadena, California.
      • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Vieira, P., Eves-van den Akker, S., Verma, R., Wantoch, S., Eisenback, J.D., Kamo, K. 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. Under Review.