Functional Analysis of the CSLD Genes in Physcomitrella Patens by CRISPR/Cas9 Gene Knockout
Plant cellulose synthase catalytic subunits (CESAs) are known to synthesize the β-1,4-glucan chains that comprise cellulose microfibrils. These CESA subunits associate to form plasma membrane localized Cellulose Synthesis Complexes (CSCs) that facilitate microfibril assembly in concert with β-1,4-glucan polymerization. Cellulose synthase-like D proteins have also been suggested to be involved in cellulose biosynthesis. However, it is not known whether CSLDs form CSCs or produce cellulose in microfibrillar form. This has been difficult to evaluate due to interfering CESA activity being present in the same cells. Whereas CESA null mutations are lethal in vascular plants, some evidence suggests that CESAs are not essential for viability in the moss Physcomitrella patens. In order to study whether CSLDs form CSCs and produce microfibrillar cellulose, production of a P. patens genetic background in which all seven PpCESAs are rendered non-functional was attempted through the use of CRISPR/Cas9 mutagenesis. Knocking out the PpCESA4 and PpCESA10 in the ppcesa3/8/6/7 knockout (KO) background produced viable sextuple ppcesa KO lines and resulted in a new phenotype characterized by abnormal gametophore morphology. Subsequent attempts to knockout PpCESA5 in the sextuple KO generated lines with the expected ‘no gametophore’ ppcesa5 KO phenotype. Although these lines remain to be genotyped, these results suggest that all CESAs can be eliminated in wild type P. patens with no consequence to viability. A role for CSLDs in the development of moss tissues has been demonstrated previously through global knockdown of the P. patens CSLD family by RNA interference. In order to study the roles of specific CSLDs in P. patens development, double KO mutants for PpCSLD1/7 were generated. No obvious rhizoid growth and cellulose content differences were observed between wild type P. patens and ppcsld1/7 mutants, possibly due to gene redundancy and/or background CESA interference. These results also confirmed CRISPR/Cas9 as an efficient gene editing tool in P. patens.
"Functional Analysis of the CSLD Genes in Physcomitrella Patens by CRISPR/Cas9 Gene Knockout"
Dissertations and Master's Theses (Campus Access).