Engineered disease resistance in cotton using RNA-interference to knock down cotton leaf curl kokhran virus-Burewala and cotton leaf curl Multan betasatellite

Authors: AHMAD, AFTAB - University Of The Punjab; UR REHMAN M, ZIA - University Of The Punjab; HAMEED, USMAN - University Of The Punjab; RAO, ABDUL - University Of The Punjab; AHAD, AMMARA - University Of The Punjab; YASMEEN, ANEELA - University Of The Punjab; AKRAM, FAHEEM - University Of The Punjab; BAJWA, KAMRAN - University Of The Punjab; Scheffler, Jodi; NASIR, IDREES - University Of The Punjab; SHAHID, AHMAD - University Of The Punjab; IQBAL, JAVED - University Of The Punjab; HUSNAIN, TAYYAB - University Of The Punjab; HAIDER, MUHAMMAD - University Of The Punjab; BROWN, JUDITH - University Of Arizona

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/30/2017
Publication Date: 9/14/2017
Citation: Ahmad, A., Ur Rehman M, Z., Hameed, U., Rao, A.Q., Ahad, A., Yasmeen, A., Akram, F., Bajwa, K.S., Scheffler, J.A., Nasir, I.A., Shahid, A., Iqbal, J., Husnain, T., Haider, M.S., Brown, J.K. 2017. Engineered disease resistance in cotton using RNA-interference to knock down cotton leaf curl kokhran virus-Burewala and cotton leaf curl Multan betasatellite. Viruses. https://doi.org/10.3390/v9090257.
DOI: https://doi.org/10.3390/v9090257

Interpretive Summary: Cotton leaf curl Disease (CLCuD) is a devastating disease in Pakistan and is spreading to other countries. Millions of bales of cotton are lost each year due to this virus. It may be possible to decrease the spread of the virus using RNA interference (RNAi), an emerging technique that can target virus genes that are important for replication, transcription or infection and inactivate them, halting the virus spread. This technology can also cause a type of immune response so that after the original inoculation, the plant continues to recognize the virus and inactivate it if it attacks the plant. This study showed that cotton plants can be successfully inoculated, continue to be resistant to CLCuD attack and pass the trait on to their progeny.

Technical Abstract: Cotton Leaf Curl virus Disease (CLCuD) has caused enormous losses in cotton (Gossypium hirsutum) production in Pakistan. RNA interference (RNAi) is an emerging technique that could knock out CLCuD by targeting different regions of the pathogen genome that are important for replication, transcription and the infection cycle of the virus complex. In the current study, an amplicon based small interfering RNA (siRNA) construct (VB) was designed against AC1 of Cotton leaf curl Burewala virus (CLCuBuV), and the ßC1 and SCR genes of Cotton leaf curl Multan beta satellite (CLCuMB). The construct was verified in silico and transformed into cotton variety “VH-289” to test its ability to silence the virus proteins. The amplification of a 301bp fragment from the AC1 gene, using gene specific primers, confirmed successful insertion of the VB construct into VH-289. Confirmed transgenic cotton plants were subjected to virus pressure by inoculating each transgenic cotton plant and a non-transformed control plant with ten viruliferous whiteflies and observing the development of disease symptoms on the plants. The appearance of CLCuD symptoms on non-transgenic cotton plants as well as a high level of virus titer compared to asymptomatic transgenic cotton plants with low virus titer, indicated the effectiveness of the construct. The transgenic cotton plants and the non-transgenic controls were evaluated after three weeks for virus titer by using a real-time PCR quantification method for both the T0 and T1 generations. The virus titer values ranged from144 molecules/µl in a T0 generation transgenic plant to 15,590 molecules/µl in a T1 transgenic plant, in comparison to the control plants which were calculated to be 2,367,884 molecules/µl in T0 and 4,015,249 molecules/µl in T1 generation. The lack of symptoms and the difference in virus titer indicated that CLCuD could be successfully controlled through siRNA targeting of key genes in the viral genome. The most effective transgenic cotton plant was analyzed using florescent in situ hybridization (FISH) and karyotyping in the T1 generation and results showed a single copy transgene insert located on chromosome 6.