American Journal of Potato Research

, Volume 89, Issue 5, pp 374–383

Generation of PVY Coat Protein siRNAs in Transgenic Potatoes Resistant to PVY

  • Kent F. McCue
  • Grisel Ponciano
  • David R. Rockhold
  • Jonathan L. Whitworth
  • Stewart M. Gray
  • Yuriy Fofanov
  • William R. Belknap
Article

DOI: 10.1007/s12230-012-9257-0

Cite this article as:
McCue, K.F., Ponciano, G., Rockhold, D.R. et al. Am. J. Pot Res (2012) 89: 374. doi:10.1007/s12230-012-9257-0
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Abstract

Global climate change has the potential to quickly alter the distribution and profile of crop pests and pathogens. Ready tools for mobilization of resistance into elite crops will be part of an integrated strategy to maintain agricultural productivity. Engineered virus resistance through expression of coat protein is well established. The mechanisms of virus suppression by small RNAs (sRNAs) have only recently been elucidated. Understanding the role of sRNAs in gene regulation and development is rapidly evolving. High throughput sequencing (HTS) can generate large data sets of sRNA sequences which can be analyzed to recognize the abundant sRNAs that result from transcript degradation and to identify the regulatory micro RNAs (miRNA) involved in gene regulation and small inhibitory RNAs (siRNA) that confer virus resistance. Transgenic Solanum tuberosum that are glass house or field grown and that express the potato virus Y coat protein (PVY-CP) inverted hairpin RNA (ihRNA) from the 409s promoter with GBSS6 intron as a spacer exhibited resistance to PVY. Southern blots of genomic DNA indicated one or two copies of the transgene and analysis of HTS data of the small RNA population indicated high levels of siRNA production from the transgenic hairpin construct. Up to 47 of the top 200 most frequent sRNA reads were attributable to the PVY-CP transgenic construct in Ranger Russet line #5. This combination of transgene and transcription strength is functional for constructing efficient resistance cassettes for crop protection. Up to 95 of the top 200 sRNA sequences were 21 nucleotides in length with as many as 19 sequences corresponding to known miRNA transcripts. Additional endogenous sRNAs with homology to resistance gene analogs may be indicative of additional or complimentary resistance mechanisms produced in concert with the PVY siRNAs arising from PVY-CP RNA transcription.

Keywords

sRNA High throughput sequencing 409s pBINPLUS/ARS ihRNA miRNA 

Abbreviations

PVY

Potato virus Y

CP

Coat protein

sRNA

Small RNA

miRNA

Micro RNA

siRNA

Small inhibitory RNA

ihRNA

Inverted hairpin RNA

PTGS

Post transcriptional gene silencing

RISC

RNA induced silencing complex

ELISA

Enzyme linked immunosorbent assay

AT

Atlantic

RR

Ranger Russet

Resumen

El cambio climático global tiene el potencial de alterar rápidamente la distribución y el perfil de plagas y patógenos de los cultivos. Las herramientas listas para movilización de la resistencia a cultivos elite será parte de una estrategia integrada para mantener la productividad agrícola. Esta bien establecida la ingeniería de la resistencia a virus a través de la expresión de la cubierta proteica. Recientemente se han elucidado los mecanismos de la supresión del virus mediante pequeños ARNs (sRNAs). Esta evolucionando rápidamente el entendimiento del papel de los sRNAs en la regulación y desarrollo de genes. La obtención de alta secuenciación (HTS) puede generar grandes grupos de datos de secuencias de sRNA que se pueden analizar para reconocer la abundancia de sRNAs que resultan de la degradación de la transcripción e identificar los micro RNAs regulatorios (miRNA) involucrados en la regulación de genes y pequeños ARN inhibitorios (siRNA) que confieren resistencia a virus. Solanum tuberosum transgenicas que se cultivan en invernadero o campo y que expresan la horquilla invertida de RNA (ihRNA) de la cubierta proteica del virus Y de la papa (PVY-CP) del promotor 409s con el intron GBSS6 como un espaciador, exhibieron resistencia al PVY. Las técnicas de manchas sureñas (southern blots) de ADN genómico indicaron una o dos copias del transgene y el análisis de datos de HTS de una población de pequeño ARN indicó altos niveles de producción de siRNA de la construcción transgénica de la horquilla. Hasta 47 de las 200 mas frecuentes lecturas de sRNA fueron atribuibles a la construcción transgénica de PVY-CP en Ranger Russet línea #5. Esta combinación de transgene y fuerza de transcripción es funcional en la construcción de cartuchos de resistencia eficientes para la protección de los cultivos. Hasta 95 de las mejores 200 secuencias de sRNA fueron de una longitud de 21 nucleótidos con tantas como 19 secuencias correspondientes a transcripciones miRNA conocidas. sRNAs endógenos adicionales con homología a genes de resistencia análogos, pudieran ser indicativos de mecanismos de resistencia adicionales o complementarios producidos en concordancia con los PVY siRNAs surgidos de la transcripción de ARN PVY-CP.

Supplementary material

12230_2012_9257_MOESM1_ESM.docx (37 kb)
Supplemental Table 1Most abundant sRNA of Ranger Russet potato leaf. The top 192 sRNA from the potato leaf sorted by relative abundance (number of reads). This file can be used to create a fastA file for comparing to other sRNA libraries. (DOCX 37 kb)
12230_2012_9257_MOESM2_ESM.docx (38 kb)
Supplemental Table 2Abundant sRNA overlapping sets of RR5 potato leaf. Sequences are sorted alphabetically and overlapping sets are boxed. Longest or consensus BLAST identity is indicated in the annotation. SNPs are highlighted in bold. Sequences with identity in the tomato (SOLly) sRNA database are indicated. (DOCX 38 kb)
12230_2012_9257_MOESM3_ESM.docx (355 kb)
Supplemental Figure 1Southern blot analysis of transgenic Atlantic potato lines containing the PVY-CP construct. Transgene integration patterns in individual transgenic lines of Atlantic containing the PVY-CP ihRNA transgene. DNA was digested with EcoRI and probed with a randomly primed 32P-labeled PVY-CP sequence. (DOCX 354 kb)

Copyright information

© Potato Association of America 2012

Authors and Affiliations

  • Kent F. McCue
    • 1
  • Grisel Ponciano
    • 1
  • David R. Rockhold
    • 1
  • Jonathan L. Whitworth
    • 2
  • Stewart M. Gray
    • 3
    • 4
  • Yuriy Fofanov
    • 5
    • 6
  • William R. Belknap
    • 1
  1. 1.Crop Improvement and Utilization Research Unit, USDA Agricultural Research ServiceAlbanyUSA
  2. 2.Small Grains and Potato Germplasm Research Unit, USDA Agricultural Research ServiceAberdeenUSA
  3. 3.Biological Integrated Pest Management Unit, USDA Agricultural Research ServiceIthacaUSA
  4. 4.Department of Plant Pathology & Plant-Microbe BiologyCornell UniversityIthacaUSA
  5. 5.Department of Computer ScienceUniversity of HoustonHoustonUSA
  6. 6.Department of Biology and BiochemistryUniversity of HoustonHoustonUSA

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