Cotton is a most important source of natural fiber for textile, hosiery, paper industries and, vegetable oil worldwide. India is the first largest cotton producer (5.75 million tonnes of lint) and second largest exporter of cotton (1.17 million tonnes) according to International Cotton Advisory Committee (ICAC) report for the month of March 2017. Most commercially cultivated cotton (~90%) in India belongs to different cultivars of Gossypium hirsutum species, whereas other species like G. arboreum, G. Barbedense and G. harbaceum collectively contributed about 10% of total cultivation. Cotton is mostly grown in Punjab, Haryana, Rajasthan, Gujarat, Maharashtra, Andhra Pradesh, and Karnataka states in India. The cotton fields of north western states like Punjab, Haryana, and Rajasthan are highly cotton leaf curl disease (CLCuD) prone because of cross- border movement of viruliferous whiteflies carrying recombinant begomoviruses. The most popular varieties of Gossypium hirsutum is HS6 in Haryana, F-846 in Punjab and RST-9 in Rajasthan which is highly susceptible to Cotton leaf curl virus (CLCuV). All the varieties of Gossypium hirsutum cultivated in Indian subcontinent are susceptibility to CLCuD causing distinct monopartite cotton leaf curl begomovirus species complex and its associated alpha and beta satellite molecules.
Presently, there are five CLCuV species belonging to closed circular single-stranded (ss) DNA viruses of genus Begomovirus, family Geminiviridae, transmitted exclusively by whitefly (Bemisia tabaci) in a circulative persistent manner. The closed circular ssDNA begomovirus may be either monopartite in nature or bipartite in nature. The monopartite begomovirus associated with CLCuD is a complex of DNA A and DNA satellite molecules, viz. DNA 1 and DNA ? (Briddon et al., 2003; Mansoor 2003b).
The CLCuD inflicts enormous losses to the cotton crops by reducing yield and compromising quality. The symptoms of CLCuD are initially characterized by swelling and darkening of the veins, downward curling of the leaf margins, enations on the underside of a leaf which develop into cup-shaped, leaf-like structures. Since last three decades, CLCuD is a major disaster to the cotton production in the Indian subcontinent. Apart from Cotton, crops such as tomato, tobacco, chillies, okra, papaya, vegetables and ornamental plants grown in these areas are affected by whitefly transmitted begomoviruses (Khan et al., 2009).
The small genome (~2751 nts) of CLCuV has six overlapping genes which utilize the bidirectional mode of transcription for effective processing of genes (Rojas et al., 2005). The two genes in virion-sense strand are V1 encoding coat protein (CP) and V2 encoding pre-coat/movement protein (MP). The four genes in complementary-sense strand are C1 encoding replication-associated protein (Rep), C2 encoding transcriptional activator protein (TrAP), C3 encoding replication enhancer protein (REn) and the C4 encoding a multifunctional protein. The C4 gene of monopartite geminiviruses encodes multifunctional protein which manifests several functions. It is involved in symptom determination, virus movement and suppressing RNA silencing synergistically with transcriptional activator protein (C2 transcript). The AC4 gene of bipartite geminivirus was first studied in Tomato golden mosaic virus (TGMV) which was later found to be a suppressor of gene silencing (Elmer et al., 1988). This gene is least conserve among geminiviruses, showing clear-cut functional divergence in bipartite and monopartite viruses but converge in functioning as a silencing suppressor (Krake et al., 1998). The intergenic region (IR) contains an origin of replication (Ori), bidirectional promoters and repeated upstream motifs (iterons) required for the control of gene expression and replication (Mullineaux et al., 1993; Hanley-Bowdoin et al., 1999 and Dry et al., 2000).
The betasatellite encodes a single protein (?C1) which is a pathogenicity determinant and suppressor of host defence (Saunders et al., 2004; Cui et al., 2005). The C4 and ?C1 proteins may affect the activity of the DICER-like proteins (DCL1/DCL2/DCL3/DCL4) in host plants that function in silencing suppression. These proteins could either downregulate transcription of a host protein that acts in the PTGS pathway in the cytoplasm or could activate transcription of a host PTGS inhibitor.
The conventional strategies to control CLCuD is time consuming, hazardous and environmentally unsafe due to excessive use of chemicals. Simultaneously, they completely fail due to emergence of recombinant viruses and satellite molecules. Different non-conventional strategies have been developed and applied to control CLCuD with various degree of success. Among them, RNA interference (RNAi) based technology has shown remarkable performance against begomovirus infection (Akmal et al., 2017, Wamiq and Khan 2018, Mansoor et a., 2006 and Hashmi et al., 2011). The small RNA (sRNA) molecules like, microRNAs (miRNAs) and small interfering RNAs (siRNAs) mediated gene silencing with sequences complementary to virus sequences have been successfully used to generate virus resistance in host plants. The siRNA-based resistance makes use of the plant post-transcriptional gene silencing (PTGS) mechanism to degrade viral RNAs.
In this study Gossypium hirsutum cv. HS6 was transformed with hairpin forming (hp) RNAi construct to scrutinize the function role of silencing suppressor gene (C4) in the process of gene silencing, level of transgene expression, level of gene specific anti-viral siRNA in transformed plants, assessment of resistance against CLCuV and finally transgene stability in the second generation transformed cotton plants were performed. The transformed cotton showing resistance against CLCuV were significantly kept under strong virus inoculation pressure in greenhouse conditions. Here we found that the transgenic cotton plants having high expression of pathogen-derived silencing suppressor gene or their counter anti-viral siRNA showed no symptoms of CLCuD with significantly low virus DNA titre in comparison to the nontransformed control plants as revealed by the RCA studies after 15, 30, 45 and 60 days post inoculation (DPI). This RNAi strategy is very effective and has the advantage of reducing possible bio-safety risks associated with other strategies. The PTGS based technique is environmental friendly for developing virus resistance in transformed crops.