Comparative evaluation of pKSE401 and pHSE401 gRNA constructs for CRISPR/Cas9-Mediated resistance to cotton leaf curl virus

Farwa Yaqub, Sidra Ashraf, Ahmed Al-Harrasi, Bushra Akhtar, Aftab Ahmad, Nayla Munawar

Abstract


Cotton leaf curl disease (CLCuD), caused by begomoviruses and their betasatellites, is a major threat to cotton production, especially in South Asia, where periodic viral outbreaks continue to affect cotton yields, quality, and livelihoods. The advent of CRISPR/Cas9 gene-editing technology has transformed plant biotechnology, offering efficient, accurate, and programmable methods for combating viral pathogens at the genetic level. Here, the antiviral efficacy of two most widely used CRISPR/Cas9 binary plant expression vectors, pKSE401 and pHSE401, was tested in Nicotiana benthamiana against Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan betasatellite (CLCuMuB). The guide-RNAs (gRNAs) were designed to target viral genes that play significant roles in pathogenicity and replication, with pHSE401 encoding a single gRNA and pKSE401 a multiplex of two gRNAs. Agrobacterium-mediated transient transformation and viral inoculation experiments revealed that both CRISPR/Cas9 vectors effectively delayed symptom onset and reduced virus titers relative to infected controls. Remarkably, the multiplex pKSE401 system was more effective at suppressing viral infection, achieving about a 90% reduction in viral accumulation compared with a 75% reduction by the single gRNA pHSE401 construct. pKSE401-treated plants showed delayed symptom development, reduced severity, and partial recovery, demonstrating the improved efficiency of multiplex genome editing. The results demonstrate the cutting-edge potential of CRISPR/Cas9 multiplex approaches as next-generation methods for designing sustainable resistance to multifaceted plant virus diseases. This research not only contributes to our understanding of CRISPR-based antiviral response mechanisms but also provides a promising avenue for designing broad-spectrum, sustainable resistance against viral epidemics in cotton and other commercially valuable crops.


Keywords


CRISPR/Cas9 genome editing, Cotton leaf curl virus (CLCuV), pKSE401, pHSE401, Virus resistance

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References


Abbas, S., 2020. Climate change and cotton production: an empirical investigation of Pakistan. Environmental Science and Pollution Research 27, 29580-29588.

Ahmed, N., Mahmood, M.A., Amin, I., Mansoor, S., 2021. Geminiviruses also encode small proteins with specific functions. Trends in Microbiology 29, 1052-1054.

Ali, Z., Abul-Faraj, A., Li, L., Ghosh, N., Piatek, M., Mahjoub, A., Aouida, M., Piatek, A., Baltes, N.J., Voytas, D.F., Dinesh-Kumar, S., Mahfouz, M.M., 2015. Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Molecular Plant 8, 1288-1291.

Ali, Z., Ali, S., Tashkandi, M., Zaidi, S.S.E.A., Mahfouz, M.M., 2016. CRISPR/Cas9-mediated immunity to geminiviruses: differential interference and evasion. Scientific Reports 6, 26912-26944.

Aragão, F.J.L., Faria, J.C., 2009. First transgenic geminivirus-resistant plant in the field. Nature Biotechnology 27, 1086-1088.

Ashraf, S., Ahmad, A., Khan, S.H., Jamil, A., Sadia, B., Brown, J.K., 2023. LbCas12a mediated suppression of Cotton leaf curl Multan virus. Frontiers in Plant Science 14, 1233295.

Binyameen, B., Khan, Z., Khan, S.H., Ahmad, A., Munawar, N., Mubarik, M.S., Riaz, H., Ali, Z., Khan, A.A., Qusmani, A.T., Abd-Elsalam, K.A., Qari, S.H., 2021. Using multiplexed CRISPR/Cas9 for suppression of Cotton leaf curl virus. International Journal of Molecular Sciences 22, 12543-12558.

Briddon, R.W., Markham, P.G., 2001. Complementation of bipartite begomovirus movement functions by topocuviruses and curtoviruses. Archives of Virology 146, 1811-1819.

Broghammer, A., Krusell, L., Blaise, M., Sauer, J., Sullivan, J.T., Maolanon, N., Vinther, M., Lorentzen, A., Madsen, E.B., Jensen, K.J., Roepstorff, P., 2012. Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding. Proceedings of the National Academy of Sciences 109, 13859-13864.

Chen, K., Wang, Y., Zhang, R., Zhang, H., Gao, C., 2019. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annual Review of Plant Biology 70, 667-697.

Chen, W., Qian, Y., Wu, X., Sun, Y., Wu, X., Cheng, X., 2014. Inhibiting replication of Begomoviruses using artificial zinc finger nucleases that target viral-conserved nucleotide motif. Virus Genes 48, 494-501.

Cheng, X., Li, F., Cai, J., Chen, W., Zhao, N., Sun, Y., Guo, Y., Yang, X., Wu, X., 2015. Artificial TALE as a convenient protein platform for engineering broad-spectrum resistance to Begomoviruses. Viruses 7, 4772-4782.

El-Mounadi, K., Morales-Floriano, M.L., Garcia-Ruiz, H., 2020. Principles, applications, and biosafety of plant genome editing using CRISPR-Cas9. Frontiers in Plant Science 11, 1-16.

Farooq, J., Farooq, A., Riaz, M., Shahid, M.R., Saeed, F., Iqbal, M.S., Hussain, T., Batool, A., Mahmood, A., 2014. Cotton leaf curl virus disease: a principal cause of decline in cotton productivity in Pakistan: a mini review. Canadian Journal of Plant Protection 2, 9-16.

Healey, A., Furtado, A., Cooper, T., Henry, R.J., 2014. Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species. Plant Methods 10, 21.

Heyraud, F., Matzeit, V., Schaefer, S., Schell, J., Gronenborn, B., 1993. The conserved nonanucleotide motif of the geminivirus stem-loop sequence promotes replicational release of virus molecules from redundant copies. Biochimie 75, 605-615.

Hussain, T., Ali, M., 1975. A review of cotton diseases in Pakistan. Pakistan Cottons 19, 71-86.

Iqbal, Z., Sattar, M.N., Shafiq, M., 2016. CRISPR/Cas9: a tool to circumscribe cotton leaf curl disease. Frontiers in Plant Science 7, 1-11.

Ji, X., Zhang, H., Zhang, Y., Wang, Y., Gao, C., 2015. Establishing a CRISPR-Cas-like immune system conferring DNA virus resistance in plants. Nature Plants 1, 1-4.

Khan, S., Mahmood, M.S., Ur Rahman, S., Rizvi, F., Ahmad, A., 2020. Evaluation of the CRISPR/Cas9 system for the development of resistance against Cotton leaf curl virus in model plants. Plant Protection Science 56, 154-162.

Khan, Z., Khan, S.H., Ahmad, A., Aslam, S., Mubarik, M.S., Khan, S., 2018. CRISPR/dCas9-mediated inhibition of replication of Begomoviruses. International Journal of Agriculture and Biology 21, 711-718.

Mahmood, M., Naqvi, R., Irfan, N., Amin, I., Mansoor, S., 2022. First report of Cotton leaf curl Multan virus infecting Millettia pinnata in Pakistan. New Disease Reports 46, e12116.

Monga, D., Sain, S.K., 2021. Incidence and severity of cotton leaf curl virus disease on different BG II hybrids and its effect on the yield and quality of cotton crop. Journal of Environmental Biology 42, 90-98.

Mubarik, M.S., Wang, X., Khan, S.H., Ahmad, A., Khan, Z., Amjid, M.W., Razzaq, M.K., Ali, Z., Azhar, M.T., 2021. Engineering broad-spectrum resistance to cotton leaf curl disease by CRISPR-Cas9 based multiplex editing in plants. GM Crops & Food 12, 647-658.

Puchta, H., 2017. Applying CRISPR/Cas for genome engineering in plants: the best is yet to come. Current Opinion in Plant Biology 36, 1-8.

Rojas, M.R., Hagen, C., Lucas, W.J., Gilbertson, R.L., 2005. Exploiting chinks in the plant's armor: evolution and emergence of geminiviruses. Annual Review of Phytopathology 43, 361-394.

Rybicki, E., Fouquet, C., 1998. Geminiviridae classification: current concepts and demarcation criteria. 2nd International Workshop on Bemisia and Geminiviral Diseases, June 7-12, 1998, San Juan, Puerto Rico 65, 55-68.

Sattar, M.N., Kvarnheden, A., Saeed, M., Briddon, R.W., 2013. Cotton leaf curl disease—an emerging threat to cotton production worldwide. Journal of General Virology 94, 695-710.

Sera, T., 2005. Inhibition of virus DNA replication by artificial zinc finger proteins. Journal of Virology 79, 2614-2619.

Tang, L., Mao, B., Li, Y., Lv, Q., Zhang, L., Chen, C., He, H., Wang, W., Zeng, X., Shao, Y., Pan, Y., 2017. Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield. Scientific Reports 7, 14438-14445.

Zaidi, S.S.A., Mansoor, S., Ali, Z., Tashkandi, M., Mahfouz, M.M., 2016. Engineering plants for geminivirus resistance with CRISPR/Cas9 system. Trends in Plant Science 21, 279-281.




DOI: https://doi.org/10.33804/pp.010.04.6181

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