citrus greening pip spinach defensin sod2 and sod7 … 2016... · citrus greening pip spinach...
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Citrus Greening PIP Spinach
Defensin SoD2 and SoD7 Dossier
SCRA Regulatory Assistance Workshop II
Milutin S. Djurickovic, BiologistU.S. EPA, Office of Pesticide Programs
Biopesticides and Pollution Prevention Division
September 20, 2016
Product Characterization
Data for EUP• Molecular characterization: Description of
Disease Resistant Citrus: Plasmid Maps and
Transformation Method
• Molecular characterization of Disease
Resistance Events
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Product Characterization
• SoD2 and SoD7 derived from Spinach
• Expressed in sweet orange (Hamlin),
grapefruit (Ruby Red), and lemon (Frost
Lisbon)
• Disarmed Agrobacterium tumefaciens
• Constructs: • pBINplus/34SGUS/SoD2-NoSp
• pBINplus/34SGUS/SoD7-NoSP
• pBINplus/34SGUS/SoD2+7 with SP
Toxicity Allergenicity Data
• Acute Oral Toxicity
• Thermolability
• Stability in Gastric Fluid
• Stability in Fresh and Pasteurized Juices
• Physiochemical Characterization of SoD
proteins
• Bioinformatics of Potential Allergenicity
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Confirmation •Successful transformation after grafting confirmed by Southern and Northern blotting with selectable markers (NPTII) and GUS.
•Northern blotting – RNA transcript 800 to 900 base pairs long
•Comparison of amounts of transcript in each event
•Southern blot – predicted single fragment of expected size for each event – indicating a single insertion
•Western blot – confirms low expression levels of SoD proteins
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Toxicity and Allergenicity
• Acute oral rats – no toxicity
• Remaining toxicity studies waived – lack of
exposure via those routes (ex. Dermal,
Injection, Eye, Pulmonary, Hypersensitivity,
Cell culture)
Con’t
• SoD2 and SoD7 - 5 minute pepsin half life and
30 second pancreatin half life – simulated
gastric – microbially produced – LC/MS
• Proteolyzed to amino acids and small peptide
fragments
• No Allergens – FASTA3, NCBI, Literature
• Cross reactivity ? – Specific serum screening
tests
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Degradation of Proteins
• Thermolability – Microbially produced SoD2
and SoD7 proteins
• Incubated at 100 °C for 30 min
• Bioassay – SoD2 activity is compromised
• LC/MS screening, SoD2 45% loss of activity
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Con’t
• Assay SoD2 and SoD7 degraded in fresh orange and
grapefruit juice, but stable in pasteurized juice
• SoD2 stable in fresh and pasteurized lemon juice
• SoD7 degraded in fresh lemon juice, but stable in
pasteurized lemon juice
• Could not extract adequate amount defensin proteins
from citrus for comparison between microbially
produced and citrus produced
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Simplot W8-Potato PIP Rpi-vnt1
SCRA Regulatory Assistance Workshop II
Milutin S. Djurickovic, BiologistU.S. EPA, Office of Pesticide Programs
Biopesticides and Pollution Prevention Division
September 20, 2016
Product Characterization
• W8 – Russet Burbank – Rpi-Vnt1 from
Solanum venturii producing VNT1 resistance
protein
• VNT1 – 891 amino acid homolog of Tm-
22 tomato mosaic virus
• Resistance to Phytophthora infestans (late
blight)
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Con’t
• Phytophthora infestans infection – AVR-VNT1
effector protein
• Recognized by VNT1 – localized plant cell death for
protection
• Limits spread of Phytophthora infestans
• Phytophthora – hemibiotroph
• Can switch between biotrophic and necrotic phases
rapidly
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Experimental Use Permit
• ~95 acres across 10 states
• All data submitted sufficient for EUP but
more needed for a Section 3 registration
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Needed information
• Bioinformatics Review of genes homologous to Rpi-
Vnt1
• Majority matched with wild Solanum species and
TMV
• More information needed…
• Need natural history of wild Solanum species
• How wild Solanum has been used as a genetic
resource for currently consumed potatoes
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Bioassay data
• Submitted data on assay to measure Rpi-vnt1
in W8 potato using RT-qPCR
• Need total RNA of all test samples
• Need results from all replicates for
comparison
• Differences in expression and how this
relates to efficacy
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Con’t
• Bioassay for detection of VNT1
• Protein expressed below detectable
levels
• Collection of efficacy data strongly
encouraged
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Experimental Program
• Information on how tubers collected
from experimental plots are stored and
kept out of the food supply is needed
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Acute Toxicity
• Data waivers requested for EUP, since
VNT1 protein is below detectable levels
and crop destruction for EUP
• Address endpoints for pepsin stability
and toxicity testing due to lack of
available protein
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Bioinformatics Allergens
and Toxins• Bioinformatics done
• Should conduct in silico analysis of putative
Open Reading Frame sequences at site of
insertion for possible allergens and toxins
• Evaluate newly created ORF’s that span 5’ and 3’
junction of transgenic insert or the two internal
junctions created when T-DNA inserts during
transformation
• Looking for newly created chimeric ORF’s20
