Cotton leaf curl is a serious disease of cotton and several other malvaceous plant species that is transmitted by the whitefly Bemisia tabaci . The disease is, at this time, endemic throughout Pakistan and western India [2, 3]. Affected cotton plants exhibit a range of symptoms that include leaf curling, stunted growth and a poor yield of cotton fibre [1, 4]. Additionally, affected plants may develop leaf-like outgrowths from the veins on the underside of leaves. The disease is caused by a geminivirus complex that involves several distinct begomoviruses (genus Begomovirus, family Geminiviridae) that interact with a disease-specific DNA satellite, Cotton leaf curl Multan betasatellite (CLCuMB) [3, 5–7].
The geminiviruses are a rapidly emerging group of plant viruses, which can be attributed to various factors, including increased insect vector populations, the presence of alternate hosts and recombination among viruses [8, 9]. Geminiviruses are plant-infecting viruses with circular single-stranded (ss)DNA genomes of 2.5-5.6 kb . Whitefly-transmitted geminiviruses are classified in the genus Begomovirus which encompasses many of the agriculturally most destructive geminiviruses. All begomoviruses native to the New World have bipartite genomes, with components known as DNA A and DNA B. In the Old World, although a few bipartite begomoviruses have been identified, the majority of begomoviruses have genomes consisting of a single component, homologous to the DNA A component of the bipartite viruses [11, 12]. A small number of truly monopartite begomoviruses have been characterized, such as Tomato leaf curl virus and Tomato yellow leaf curl Sardinia virus (TYLCSV) [13, 14]. However, the majority of monopartite begomoviruses are associated with addition ssDNA components, known as betasatellites and alphasaltellites . The betasatellites are symptom modulating satellites which, in some cases, are required by their helper begomoviruses to systemically infect the plants from which they were isolated; this is the case for the begomoviruses that cause CLCuD, such as Cotton leaf curl Multan virus (CLCuMV), which require CLCuMB to efficiently infect cotton and induce bona fide disease symptoms [7, 16]. The betasatellites encode a single protein which is a pathogenicity (symptom) determinant and a suppressor of RNA interference (RNAi)-based host defenses [17–19]. The alphasatellites are associated with the majority of begomovirus-betasatellite complexes, including that which causes CLCuD [20–22]. They are not essential but recent results suggest that they may be involved in overcoming host defenses .
The genomes of monopartite begomoviruses encode six proteins with genes in the virion and complementary-sense separated by a non-coding intergenic (IR) region that contains control sequences as well as the virion-sense origin of replication . The genes in the virion-sense encode the V2 protein, which is involved in virus movement and is a suppressor of RNAi, and the coat protein, the only structural protein of geminiviruses that is required to form the characteristic geminate particles, for movement in plants and interacts with the whitefly vector for transmission plant-to-plant. In the complementary-sense the genes encode the replication associated protein (Rep; the only virus encoded protein required for viral DNA replication, which is a rolling-circle replication-initiator protein), the transcriptional activator protein (TrAP; involved in the up-regulation of late (virion-sense) genes, modulating host gene expression and may be a suppressor of RNAi), the replication-enhancer protein (REn; interacts with and enhances Rep activity) and the C4 protein (a suppressor of RNAi that may be involved in virus movement).
The control of CLCuD, as is the case for most geminivirus diseases, is mainly based on control of the vector using insecticides and the cultivation of resistant crop varieties . Resistant cotton cultivars were introduced in the mid to late 1990 s that were developed by conventional breeding/selection. After initially showing promise in the control of CLCuD, the virus complex ultimately overcame the resistance , although the precise changes in the complex responsible remain unclear. Both the virus and the betasatellite associated with resistance breaking have been shown to be recombinant [3, 5] and efforts continue to identify the precise molecular basis for their ability to overcome host resistance in cotton. Thus, alternate strategies are required to prevent losses due to the disease. Among the possible strategies, those based on RNAi have shown some promise .
RNAi is a ubiquitous phenomenon in eukaryotic organisms that is triggered by double-stranded RNA (dsRNA) that plays important roles in diverse biological processes. The key features of RNA silencing include the production of 21-25 nucleotide small interfering RNAs (siRNA) by enzymes known as Dicers and the formation of RNA-induced silencing complexes (RISCs) which contain Argonaute proteins that directly carry out gene silencing at the transcriptional or posttranscriptional levels . RNA silencing can be activated by introducing transgenes, RNA viruses or DNA sequences that are homologous to expressed genes. This phenomenon has been utilized as a tool to study various molecular processes in the cell, chromosome organization, functions of genes and for obtaining resistance to viruses in plants and animals. As a counter defense viruses have evolved "suppressor" proteins, which are able to prevent or counter RNAi .
Several studies investigating the use of RNAi for obtaining resistance against geminiviruses have been reported, with different levels of success (reviewed by [29, 30]). However, there are only two reports investigating the use of RNAi against a begomovirus-betasatellite complex [31, 32]. Here RNAi has been investigated as a means of obtaining resistance against one of the viruses causing CLCuD.