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Plant Molecular Biology

, Volume 62, Issue 4–5, pp 735–752 | Cite as

A novel approach for developing resistance in rice against phloem limited viruses by antagonizing the phloem feeding hemipteran vectors

  • Prasenjit Saha
  • Indranil Dasgupta
  • Sampa DasEmail author
Article

Abstract

Rice production is known to be severely affected by virus transmitting rice pests, brown planthopper (BPH) and green leafhopper (GLH) of the order hemiptera, feeding by phloem abstraction. ASAL, a novel lectin from leaves of garlic (Allium sativum) was previously demonstrated to be toxic towards hemipteran pests when administered in artificial diet as well as in ASAL expressing transgenic plants. In this report ASAL was targeted under the control of phloem-specific Agrobacterium rolC and rice sucrose synthase-1 (RSs1) promoters at the insect feeding site into popular rice cultivar, susceptible to hemipteran pests. PCR, Southern blot and C-PRINS analyses of transgenic plants have confirmed stable T-DNA integration and the transgenes were co-segregated among self-fertilized progenies. The T0 and T1 plants, harbouring single copy of intact T-DNA expression cassette, exhibit stable expression of ASAL in northern and western blot analyses. ELISA showed that the level of expressed ASAL was as high as 1.01% of total soluble protein. Immunohistofluorescence localization of ASAL depicted the expected expression patterns regulated by each promoter type. In-planta bioassay studies revealed that transgenic ASAL adversely affect survival, growth and population of BPH and GLH. GLH resistant T1 plants were further evaluated for the incidence of tungro disease, caused by co-infection of GLH vectored Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV), which appeared to be dramatically reduced. The result presented here is the first report of such GLH mediated resistance to infection by RTBV/RTSV in ASAL expressing transgenic rice plant.

Keywords

ASAL transgenic rice GLH Immunohistofluorescence RTBV resistance 

Abbreviations

ASAL

Allium sativum agglutinin from leaf

BAP

benzylamino purine

BPH

brown planthopper

BSA

bovine serum albumin

cv.

cultivar

C-PRINS

cycling-primed in situ labelling

dpi

days post inoculation

ELISA

enzyme linked immunosorbent assay

FITC

fluorescein isothiocyanate

GLH

green leafhopper

Hyg

hygromycin

MS

Murashige and Skoog

NAA

α-napthaline acetic acid

PBS

phosphate buffered saline

RSs1

rice sucrose synthase-1 promoter

RTBV

Rice tungro bacilliform virus

RTSV

Rice tungro spherical virus

SDS-PAGE

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

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Notes

Acknowledgements

Authors are grateful to Council of Scientific and Industrial Research; Government of India for providing fellowships to PS. We are grateful to Prof. S. C. Roy, Centre of Advanced Study (CAS), Cell and Chromosome Research, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, India for his help to carry out C-PRINS study in his laboratory. Authors thank the Programme Coordinator, CAS, Dept. Bot. CU for providing above technical opportunities. We are thankful to Regional Rice Research Station, Chinsurah, West Bengal, India for providing nuclear stock seed of Pusa Basmati 1 rice cultivar. For back up service of Mr. Arup Kumar Dey, BI is sincerely acknowledged. Authors are also thankful to Gautam Basu for critically reading the manuscript. The expert technical help of Anand Singh Rana in viral assays and Trilok Singh Rawat in inoculations of DU(SC) are acknowledged.

Supplementary material

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  1. 1.Plant Molecular and Cellular GeneticsBose InstituteKolkataIndia
  2. 2.Department of Plant Molecular BiologyUniversity Delhi South CampusNew DelhiIndia

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