Codiaeum variegatum in Pakistan harbours pedilanthus leaf curl virus and papaya leaf curl virus as well as a newly identified betasatellite.

Codiaeum variegatum (common name, garden croton) is an ornamental plant grown for its bright yellow variegated leaf morphology. Two C. variegatum plants with upward leaf curling and vein swelling symptoms were collected in Faisalabad, Pakistan. Sequencing of clones obtained by PCR amplification with specific primers showed one plant infected with the monopartite begomoviruses pedilanthus leaf curl virus (PeLCV) and papaya leaf curl virus (PaLCuV) and the other to be infected with only PeLCV. Both plants also harboured a betasatellite that was distinct from all previously identified betasatellites, for which the name "codiaeum leaf curl betasatellite" (CoLCuB) is proposed. This is the first identification of a begomovirus and an associated betasatellite infecting C. variegatum in Pakistan. Both PeLCV and PaLCuV cause problems in a number of crop plants, and C. variegatum may act as a reservoir for these agriculturally important viruses. The precise impact and geographical distribution of the newly identified CoLCuB will be investigated.

Viral vectors as carriers of genome-editing reagents.

The presence of a transgene in the genome of plants is a regulatory challenge. Recently, Liu et al. reported an engineered tomato spotted wilt virus (TSWV) that can carry large clustered regularly interspaced palindromic repeats (CRISPR)/Cas reagents for targeted genome editing in various crops without the integration of the transgene into the genome.

Establishment of Transcriptional Gene Silencing Targeting the Promoter Regions of GFP, PDS, and PSY Genes in Cotton using Virus-Induced Gene Silencing.

Virus-induced gene silencing (VIGS) by deploying viral-based vectors such as tobacco rattle virus (TRV) is a homology-based gene silencing technique in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) to validate the function of particular genes. The study presented here showed the induction of DNA methylation in the promoter regions of three phenotypic marker genes in different cotton accessions, including two endogenous genes such as phytoene desaturase (PDS) and phytoene synthase (PSY), and an exogenous gene, such as green fluorescent protein (GFP). First, DNA methylation was established in transgenic GFP cotton where methylation persisted up to S3 generation. Afterward, the promoter of PSY was targeted following the same conditions. Significant silencing of PSY was observed and methylation of the promoter was found up to S2 generation in red leaf cotton as detected in GFP cotton. Silencing of PDS resulted in a photobleaching phenotype; interestingly, the strength of this phenotype was diverse within the plants and was not observed in the next generation. Bisulfite sequencing results showed methylation percentage of the cytosine residues was high at CG and CHG sites of the targeted promoter sequences in the silenced plants. The findings of this paper suggest that TRV-based vector system can be used to monitor DNA methylation for both exogenous and endogenous gene levels in cotton and offer a very useful tool for plant epigenetic modification.

Cotton Mi-1.2-like Gene: A potential source of whitefly resistance.

Whitefly (Bemisia tabaci) inflicts tremendous yield losses to cotton crops in many parts of the world by sap-sucking and transmitting viral diseases. The tomato-associated Mi-1.2 gene has been successfully deployed in tomato cultivars to attain whitefly resistance. In the current study, putative Mi-1.2-like orthologs were identified in five whitefly hosts and functionally validated through virus-inducing gene silencing (VIGS) in cotton plants. The expression profiling and qPCR results depicted differential regulation of the Mi-1.2-like gene in various tissue types and under different biotic and abiotic stresses, especially in whitefly susceptible and resistant cotton plants. The upregulation of the Mi-1.2-like gene (Gadrp RPP-13 Like gene) was observed at 24 h and 48 h post-whitefly exposure (PWFE) in whitefly resistant (FDH-228) and tolerant (Mac7) cotton plants as compared to susceptible plants of Coker-312. However, delayed expression was recorded at 72 h of PWFE in Coker-312 plants. In TRV based gene silencing experiment, silencing of the Mi-1.2-like gene, significantly enhanced the whitefly infestation on both whitefly-resistant and susceptible cotton genotypes. Based on these results, we conducted the evolutionary analysis of Mi-1.2-like orthologs among cotton, cassava, tomato, papaya, and cucumber hosts. This indicated that cotton associated Mi-1.2 like gene has a close relation with cassava and tomato. These results suggested that Mi-1.2-like R genes could be the potential candidate for deriving whitefly resistance response in cotton plants.

G5, a Phage Single-Stranded DNA-Binding Protein, Fused with a Nuclear Localization Signal, Attenuates Symptoms and Reduces Begomovirus-Betasatellite Accumulation in Transgenic Plants.

Cotton leaf curl disease is caused by several monopartite begomoviruses and is the major threat to cotton production in the Indian subcontinent. The disease has been shown to be associated with four distinct species, including Cotton leaf curl Kokhran virus (CLCuKoV), and a specific betasatellite-Cotton leaf curl Multan betasatellite (CLCuMuB). Transgenic Nicotiana benthamiana plants were produced which constitutively express the Escherichia coli phage M13 encoded, sequence nonspecific single-stranded (ss) DNA-binding protein, G5 alone and fused with the maize opaque-2 nuclear localization signal (NLS), to evaluate resistance against CLCuKoV-CLCuMuB. Transgenic plants expressing only G5 performed poorly exhibiting symptoms of infection and high virus DNA levels upon inoculation with CLCuKoV and CLCuKoV with CLCuMuB. In contrast, plants transformed with G5 fused to the NLS developed mild symptoms and showed a reduction in virus and betasatellite DNA levels in comparison to nontransformed plants. The results show that G5 may be useful in developing broad-spectrum resistance against ssDNA viruses.