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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.

Plant receptor-like kinases (RLKs) are important players in response to pathogen infections. Verticillium and Fusarium wilts, caused by Verticillium dahliae (Vd) and Fusarium oxysporum f. sp vasinfectum (Fov), respectively, are among the most devastating diseases in cotton (Gossypium spp). To understand the cotton response to these soil-borne fungal pathogens, we performed a genome-wide in silico characterization and functional screen of diverse RLKs for their involvement in cotton wilt diseases. We identified Gossypium hirsutum GhWAK7A, a wall-associated kinase, that positively regulates cotton response to both Vd and Fov infections. Chitin, the major constituent of the fungal cell wall, is perceived by lysin-motif-containing RLKs (LYKs/CERK1), leading to the activation of plant defense against fungal pathogens. A conserved chitin sensing and signaling system is present in cotton, including chitin-induced GhLYK5-GhCERK1 dimerization and phosphorylation, and contributes to cotton defense against Vd and Fov. Importantly, GhWAK7A directly interacts with both GhLYK5 and GhCERK1 and promotes chitin-induced GhLYK5-GhCERK1 dimerization. GhWAK7A phosphorylates GhLYK5, which itself does not have kinase activity, but requires phosphorylation for its function. Consequently, GhWAK7A plays a crucial role in chitin-induced responses. Thus, our data reveal GhWAK7A as an important component in cotton response to fungal wilt pathogens by complexing with the chitin receptors.

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.