Coccinia grandis is an interesting model system to understand dioecy in Cucurbitaceae family. Recent transcriptomics and proteomics studies carried out to understand the sex expression in C. grandis have resulted in identification of many candidate sex-biased genes. In absence of an efficient genetic transformation protocol for C. grandis, virus-induced gene silencing (VIGS) would be a powerful tool to enable gene functional analysis. In current study, we explored the apple latent spherical virus (ALSV) for gene knockdown in C. grandis. The viral infection was achieved through mechanical inoculation of ALSV-infected Chenopodium quinoa leaf extract onto the cotyledons of C. grandis. ALSV-VIGS mediated knockdown of CgPDS gene was successfully achieved in C. grandis by mechanical inoculation method resulting in characteristic photobleaching. Subsequently, we developed agroinfiltration compatible vectors for direct infection of C. grandis and shortened the time-frame by skipping viral propagation in C. quinoa. Typical yellow-leaf phenotype was observed in C. grandis plants agroinfiltrated with ALSV-CgSU constructs, indicating robust silencing of CgSU gene. In addition, we improved the infection efficiency of ALSV by co-infiltration of P19 viral silencing suppressor. These results suggest that ALSV-VIGS is suitable for characterization of gene function in dioecious C. grandis and it can help us understand the mechanism of sex expression.
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We thank Prof. Yoshikawa, Iwate University, Japan for kindly providing us pEALSR1 and pEALSR2L5R5 vectors. We acknowledge Dr. P. V. Shivaprasad, NCBS, Bangalore for kindly providing us the pDGB3alpha2_35S:P19:Tnos vector. We thank Mr. Nitish Lahigude for maintaining C. grandis plants in the green house as well as in the field. We would also like to thank Mr. Shailendra Kushwaha, Ms. Shruti Kulshrestha and Ms. Diksha Kumari for their help with some of the vector construction in the current study.
R.S.D. acknowledges research fellowship obtained from CSIR, HRDG, New Delhi. Financial support from DBT, Govt. of India (Grant No-BT/PR16399/NER/95/125/2015), and Director, IISER Pune are thankfully acknowledged. Funding body was not involved in the design of the study; collection, analysis, interpretation of data and in writing the manuscript.
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Supplementary file1 Online Resource 1: Fig. S1 Chenopodium quinoa plants infected with ALSV infectious cDNA for viral propagation. Infected C. quinoa leaves are used as inocula for C. grandis infection. White arrows indicate the chlorotic spots characteristic of ALSV infection in C. quinoa leaves (TIF 1846 kb)
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Devani, R.S., Kute, A., John, S. et al. Development of a Virus‐Induced Gene Silencing System for Dioecious Coccinia grandis. Mol Biotechnol (2020). https://doi.org/10.1007/s12033-020-00259-7
- Apple latent spherical virus
- Virus-induced gene silencing
- Coccinia grandis
- Mechanical inoculation