Ananya Ghosal, MAKAUT (WB)
Agricultural sectors are being harmed by Begomoviruses which cause a huge loss to economically important crops. To prevent such losses, intricate studies of viral genomes and functions are needed. The genomes of viruses are directly exploited by infectious clone approaches and applications. The critical instrument for basic characterization of the notable and newly discovered viruses are infectious clones of DNA Viruses. Understanding the structure and composition of viruses plays a major role in the evolution of molecular plant pathology.
Constitution of Begomovirus
Infection clones achieve when cDNA was inserted into the plasmid vector. Begomoviruses have bipartite and monopartite genome which infects only dicot plants via whitefly as a vector. Bipartite Begomovirus consists of DNA-A and DNA-B. The function of DNA-A is to make proteins responsible for encapsidation, expression regulation, and replication. On the other hand, DNA-B makes protein responsible for the mechanism of viral DNA molecules.
DNA-A can replicate autonomously but systematic infections occur due to DNA-B. DNA-A and DNA-B consist of 200bp of conserved sequence. Begomovirus has six open reading frames AV1 and AV2 whereas the complementary strand has four encoded proteins such as AC-1, AC-2, AC-3, AC-4. AC-1 is called Replication association protein, which initiates viral DNA replication. Replication association protein (Rep) attaches to the plant homolog in Retinoblastoma protein which supports viral DNA replication.
AC-2 protein is called Transactivational activation protein which helps in gene silencing and transactivates the expression of viral sense gene expression. AC-4 helps in the determination of symptoms that further affects cell-cycle controls which counters the host responses for Rep expression. DNA-B has two proteins BV1 and BC1 protein, where BV1is nuclear-shuttle protein and BC1 is movement protein.
Construction strategy of infectious clones
Infectious clones can exploit the viral genome. The first step to construct an infectious clone is to isolate the genome. We can get the viral genome by extracting host symptomatic plant total DNA. The next step is to produce a full-length infectious clone by amplification of the viral genome. Polymerase Chain Reaction (PCR) and Rolling Circle Amplification (RCA) are the practices for amplifying Begomovirus. The product of amplification is long single-stranded DNA with repeating bases that complements the template.
Then cloning of the full genome is done due to the size of Begomovirus. To infect the host cell production of tandem repeats are required which consist of two origins of replication. Three methods are used for the transformation of Agrobacterium tumefaciens. They are:
- Triparental mating: It is the most effective method for cloning a mobilized plasmid into Agrobacterium tumefaciens.
- Electroporation: It is the most efficient way for inserting a gene of interest into Agrobacterium tumefaciens.
- Freeze/thaw method: It is an alternative, fast and straightforward method for Agrobacterium tumefaciens with foreign DNA.
The next step is the Agroinoculation of the host plant which allows transmission of Begomovirus infectious clones into host plants with the support of Agrobacterium tumefaciens. The infectious clones thus constructed, give way for the functional analysis at replication of the virus, its pathogenesis, and transmission. Inoculum is used for resistance screening which is required to avoid genetically inconsistent viral population. Infectious clone gives an alternative which is the ineffective procedure and the infection is transmitted via an insect vector. We can get Virus-induced gene silencing by alteration of infectious clones. Despite this, the uncontrolled use of infectious clones may create a risk to the environment.
Also read: Whitefly hijacks a plant gene- Let’s learn how is that possible?
Reference:
- Saad, M. F. M., Sau, A. R., Akbar, M. A., Baharum, S. N., Ramzi, A. B., Talip, N., & Bunawan, H. (2021). Construction of infectious clones of begomoviruses: Strategies, techniques and applications. Biology, 10(7), 604. https://doi.org/10.3390/biology10070604
- The in-text image of the genomic organization of begomoviruses has been extracted from: https://talk.ictvonline.org/ictv-reports/ictv_online_report/ssdna-viruses/w/geminiviridae/392/genus-begomovirus
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