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Virus-induced gene silencing (VIGS) is a reverse genetic tool used to identify the function of individual genes by reducing their expression. Here VIGS was applied to identify the role of four genes in G. hirsutum; Polyphenol Oxidase (GhPPO), Phosphatidylserine Synthase (GhPSS), Anthocyanidin synthase (GhANS), and Anthocyanidin Reductase (GhANR) in verticillium wilt disease occurring in cotton. Silencing of GhPPO and GhPSS showed abnormal leaf growth such as leaf thinning, reduced leaf size, and leaf curling phenotype which indicates their importance in normal leaf development. RT-PCR and qPCR results showed reduced transcript levels of GhPPO, GhPSS, GhANS, and GhANR in TRV inoculated plants. Moreover silencing of four genes in cotton cultivar FM9160, partially resistant to Verticillium wilt disease compromised resistance by infecting with “King isolate” which is a more aggressive defoliating strain of Verticillium dahliae. The reduced mRNA level of GhPPO and GhPSS makes the cotton plants more susceptible approximately 80% and 76% of leaf wilting were observed while in GhANS and GhANR 66% and 62% leaf wilting were shown. In conclusion, this is the first report showing that the cotton gene GhPPO, GhPSS involved in resistance to Verticillium dahliae and have an important role in normal leaf development in G. hirsutum.

Clustered regularly interspaced palindromic repeats (CRISPR) and associated Cas9 nuclease (CRISPR-Cas9) systems provide adaptive immunity to prokaryotes against infectious phage particles that can be engineered as a genome-editing tool. Guided by an RNA strand, the class II type II CRISPR-Cas9 system can be employed to provide resistance against plant DNA viruses. Here we describe an efficient CRISPR-Cas9 genome editing system based on simultaneous targeting of the highly conserved intergenic region (IR) of the virus that can provide resistance against Cotton leaf curl Kokhran virus-Burewala strain (CLCuKoV-Bur) in Nicotiana benthamiana plants. The data revealed that upon infection, the transgenic plants harboring CRISPR-Cas9 and two gRNAs showed complete resistance against CLCuKoV-Bur/Cotton leaf curl Multan betasatellite (CLCuMB). All efforts failed to find the intact virus in CLCuKoV-Bur/CLCuMB challenged transgenic (OX:Cas9NB:IR) plants using either gene specific PCR primers or CLCuKoV-Bur as a probe in southern blot hybridization. Thus, our results have demonstrated an efficient CRISPR-Cas9 approach to engineer durable resistance against CLCuKoV-Bur in a model system. The implications of these findings are discussed.

Geminiviridae is one of the most important families in plant viruses with a single-stranded DNA genome comprises one or two genomes (DNA-A and DNA-B) of 2.5–5.2Kb. These viruses infect economically important food and fiber crops worldwide. Till to date nine genera (Mastrevirus, Curtovirus, Becurtovirus, Turncurtovirus, Begomovirus, Capulavirus, Topocuvirus, Eragrovirus, and Grablovirus) have been reported which are transmitted by four insect vectors (whiteflies, leafhoppers, aphids, and treehoppers). DNA-A encodes replication-associated protein, coat protein, replication enhancer protein, transcriptional activator protein, AV2, AC4, and AC5 while DNA-B encodes nuclear shuttle protein and movement protein. Satellite molecules have also been reported to be associated with the family Geminiviridae that includes alphasatellite, betasatellite which encode alpha rep and βC1 gene respectively. A non-coding deltasatellite has also been reported to be associated with geminivirus infections. Geminivirus encoded proteins interact with host proteins to perform a variety of functions which include reprogramming of cell cycle, inhibition of cell death pathways, transcriptional control of multiple genes, interference with cell signaling pathways and overcoming host defense response. Our current knowledge regarding interactions of geminivirus encoded proteins with the host has been described in the following chapter.

Spinach is a common vegetable crop and very little data is available about its virus infection. Symptomatic leaves of spinach were collected during field survey. Circular DNA molecules were amplified from symptomatic samples using rolling circle amplification (RCA). After restriction analysis, presumed bands of virus and satellites were cloned, sequenced and analyzed. Analysis of sequenced RCA product revealed the presence of chickpea chlorotic dwarf virus (CpCDV; Mastrevirus). Further analyses of the cloned virus showed that strain "C" of CpCDV was present in symptomatic samples of spinach collected from field associated with vein darkening, curling and enations on leaves. Amplification of alpha- and betasatellites with universal primers was performed. CpCDV showed association with cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellites (CLCuMA). Infectivity analysis of CpCDV and CpCDV/CLCuMB were done in N. benthamiana using particle bombardment method and the results showed that CpCDV was able to transreplicates CLCuMB in this host. To our knowledge, this is the first report of a dicot infecting mastrevirus (CpCDV) along with CLCuMB and CLCuMA associated with leaf curl disease of spinach in Pakistan. The significance of the results is discussed.

Begomoviruses interfere with host plant machinery to evade host defense mechanism by interacting with plant proteins. In the old world, this group of viruses are usually associated with betasatellite that induces severe disease symptoms by encoding a protein, βC1, which is a pathogenicity determinant. Here, we show that βC1 encoded by Cotton leaf curl Multan betasatellite (CLCuMB) requires Gossypium hirsutum calmodulin-like protein 11 (Gh-CML11) to infect cotton. First, we used the in silico approach to predict the interaction of CLCuMB-βC1 with Gh-CML11. A number of sequence- and structure-based in-silico interaction prediction techniques suggested a strong putative binding of CLCuMB-βC1 with Gh-CML11 in a Ca+2-dependent manner. In-silico interaction prediction was then confirmed by three different experimental approaches: The Gh-CML11 interaction was confirmed using CLCuMB-βC1 in a yeast two hybrid system and pull down assay. These results were further validated using bimolecular fluorescence complementation system showing the interaction in cytoplasmic veins of Nicotiana benthamiana. Bioinformatics and molecular studies suggested that CLCuMB-βC1 induces the overexpression of Gh-CML11 protein and ultimately provides calcium as a nutrient source for virus movement and transmission. This is the first comprehensive study on the interaction between CLCuMB-βC1 and Gh-CML11 proteins which provided insights into our understating of the role of βC1 in cotton leaf curl disease.

A Tobacco rattle virus (TRV)-based virus-induced gene silencing assay was employed as a reverse genetic approach to study gene function in cotton (Gossypium hirsutum). This approach was used to investigate the function of the Enoyl-CoA reductase (GhECR) gene in pathogen defense. Amino acid sequence alignment of Arabidopsis ECR with homologous sequence from G. hirsutum, G. arboreum, G. herbaceum and G. barbadense showed that ECRs are highly conserved among these species. TRV-based silencing of GhECR gene in G. hirsutum induced a cell death/necrotic lesion-like phenotype. Reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR showed reduced GhECR mRNA levels in TRV inoculated plants. Three isolates of Verticillium dahliae (V. dahliae) and Fusarium oxysporum f. sp. vasinfectum (FOV) were used to infect GhECR-silenced plants. Out of 6 races of 2 pathogens, down regulation of GhECR gene resulted in reduced resistance. This is the first report showing that cotton GhECR gene is involved in resistance to different strains of V. dahliae and FOV.