New insights into natural rubber biosynthesis from rubber-deficient lettuce mutants expressing goldenrod or guayule cis-prenyltransferase.

Lettuce produces natural rubber (NR) with an average Mw of > 1 million Da in laticifers, similar to NR from rubber trees. As lettuce is an annual, self-pollinating, and easily transformable plant, it is an excellent model for molecular genetic studies of NR biosynthesis. CRISPR/Cas9 mutagenesis was optimized using lettuce hairy roots, and NR-deficient lettuce was generated via bi-allelic mutations in cis-prenyltransferase (CPT). This is the first null mutant of NR deficiency in plants. In the CPT mutant, orthologous CPT counterparts from guayule (Parthenium argentatum) and goldenrod (Solidago canadensis) were expressed under a laticifer-specific promoter to examine how the average Mw of NR is affected. No developmental defects were observed in the NR-deficient mutants. The lettuce mutants expressing guayule and goldenrod CPT produced 1.8 and 14.5 times longer NR, respectively, than the plants of their origin. This suggests that, although goldenrod cannot synthesize a sufficiently lengthy NR, goldenrod CPT has the catalytic competence to produce high-quality NR in the cellular context of lettuce laticifers. Thus, CPT alone does not determine the length of NR. Other factors, such as substrate concentration, additional proteins, and/or the nature of protein complexes including CPT-binding proteins, influence CPT activity in determining NR length.

CRISPR/Cas9 mediated targeted mutagenesis of LIGULELESS-1 in sorghum provides a rapidly scorable phenotype by altering leaf inclination angle.

Sorghum (Sorghum bicolor L. Moench) is one of the world's most cultivated cereal crops. Biotechnology approaches have great potential to complement traditional crop improvement. Earlier studies in rice and maize revealed that LIGULELESS-1 (LG1) is responsible for formation of the ligule and auricle, which determine the leaf inclination angle. However, generation and analysis of lg1 mutants in sorghum has so far not been described. Here, we describe CRISPR/Cas9 mediated targeted mutagenesis of LG1 in sorghum and phenotypic changes in mono- and bi-allelic lg1 mutants. Genome editing reagents were co-delivered to sorghum (var. Tx430) with the nptII selectable marker via particle bombardment of immature embryos followed by regeneration of transgenic plants. Sanger sequencing confirmed a single nucleotide insertion in the sgRNA LG1 target site. Monoallelic edited plantlets displayed more upright leaves in tissue culture and after transfer to soil when compared to wild type. T1 progeny plants with biallelic lg1 mutation lacked ligules entirely and displayed a more severe reduction in leaf inclination angle than monoallelic mutants. Transgene-free lg1 mutants devoid of the genome editing vector were also recovered in the segregating T1 generation. Targeted mutagenesis of LG1 provides a rapidly scorable phenotype in tissue culture and will facilitate optimization of genome editing protocols. Altering leaf inclination angle also has the potential to elevate yield in high-density plantings.