Silencing of the Phytoene desaturase gene mitigates oxidative stress through the accumulation of free amino acids

Phytoene desaturase (PDS) is a rate-limiting enzyme involved in the biosynthesis of carotenoids, which converts phytoene to zeta-carotene in a two-step desaturation reaction. Transiently blocked carotenogenesis by silencing the PDS gene in Nicotiana benthamiana (NbPDS) using the virus-induced gene silencing (VIGS) technique was used. Silencing of NbPDS induced dwarfism and an albino-type leaf trait in N. benthamiana. The NbPDS-silenced leaves accumulated free amino acids in amounts 9.5-folds greater than those of the GFP-silenced control leaves, but contained only 59.6% of total soluble proteins. When treatment with 10 and 100 μM paraquat was carried out to induce oxidative stress, NbPDS-silenced N. benthamiana demonstrated more resistance at both concentrations compared to the control plants. These data strongly suggest that high concentrations of free amino acids occur because they are inadequately incorporated into proteins of the NbPDS-silenced plants, but reduce injury inflicted by oxidative stress even without the assistance of important antioxidants like carotenoids.

Biolistic transformation of plants using CRISPR/Cas9 technology

Experiments on CRISPR/Cas9 mediated knock-in approaches in the PDS gene of various genes of interest were planned. Biolistic bombardment mediated delivery of target vectors to plant explants was suggested.

Efficient CRISPR/Cas9-Mediated Knockout of an Endogenous PHYTOENE DESATURASE Gene in T1 Progeny of Apomictic Hieracium Enables New Strategies for Apomixis Gene Identification

Most Hieracium subgenus Pilosella species are self-incompatible. Some undergo facultative apomixis where most seeds form asexually with a maternal genotype. Most embryo sacs develop by mitosis, without meiosis and seeds form without fertilization. Apomixis is controlled by dominant loci where recombination is suppressed. Loci deletion by γ-irradiation results in reversion to sexual reproduction. Targeted mutagenesis of genes at identified loci would facilitate causal gene identification. In this study, the efficacy of CRISPR/Cas9 editing was examined in apomictic Hieracium by targeting mutations in the endogenous PHYTOENE DESATURASE (PDS) gene using Agrobacterium-mediated leaf disk transformation. In three experiments, the expected albino dwarf-lethal phenotype, characteristic of PDS knockout, was evident in 11% of T0 plants, 31.4% were sectorial albino chimeras, and the remainder were green. The chimeric plants flowered. Germinated T1 seeds derived from apomictic reproduction in two chimeric plants were phenotyped and sequenced to identify PDS gene edits. Up to 86% of seeds produced albino seedlings with complete PDS knockout. This was attributed to continuing Cas9-mediated editing in chimeric plants during apomictic seed formation preventing Cas9 segregation from the PDS target. This successful demonstration of efficient CRISPR/Cas9 gene editing in apomictic Hieracium, enabled development of the discussed strategies for future identification of causal apomixis genes.

Modification of Tobacco rattle virus RNA1 to Serve as a VIGS Vector Reveals That the 29K Movement Protein Is an RNA Silencing Suppressor of the Virus

Tobacco rattle virus (TRV) has a bipartite, positive-sense single-stranded RNA genome and is widely used for virus-induced gene silencing (VIGS) in plants. RNA1 of TRV that lacks the gene for the cysteine-rich 16K silencing-suppression protein infects plants systemically in the absence of RNA2. Here, we attempted to engineer RNA1 for use as a VIGS vector by inserting heterologous gene fragments to replace 16K. The RNA1 vector systemically silenced the phytoene desaturase (PDS) gene, although less efficiently than when the original VIGS vector system was used, which consists of wild-type RNA1 and engineered RNA2 carrying the heterologous gene. Infectious RNA1 mutants with a dysfunctional 16K suppressed silencing and enhanced transgene expression in green fluorescent protein-transgenic Nicotiana benthamiana following inoculation by agroinfiltration, unlike mutants that also lacked 29K, a movement protein (MP) gene. The 30K MP gene of Tobacco mosaic virus complemented in cis the movement defect but not the silencing suppression functions of TRV 29K. Silencing suppression by 29K occurred in the context of RNA1 replication but not in an agroinfiltration assay which tested 29K alone for suppression of sense-mediated silencing. Both 29K and 16K were needed to avoid necrotic symptoms in RNA1-infected N. benthamiana. The results shed new light on virulence factors of TRV.