| The moss Physcomitrella patens is an attractive model organism, due to its small genome, dominant haploid phase, and high rate of homologous recombination. P. patens has eight genes that form a family known as the Cellulose Synthase-Like Ds (CSLDs). They may be involved in the biosynthesis of crystalline or non-crystalline cellulose and polarized tip growth, but their direct role is still unknown. In order to study the functions of CSLDs, RT-qPCR was preformed, and knockout (KO) mutants for PpCSLD1, PpCSLD2, and PpCSLD8 were developed, but showed no obvious phenotype. As a result, PpCSLD4, PpCSLD5, and PpCSLD6 KO vectors were created to produce double KO mutants. For global silencing of the entire CSLD family, an RNAi vector (pUD8D8i) was developed containing both sequences that are highly similar among all P. patens CSLDs and sequences containing beta-glucuronidase. This construct has been placed in a P. patens line that expresses a nuclear localization signal (NLS) for green fluorescent protein fused to beta-glucuronidase, in order to monitor active silencing. The P. patens colonies silenced by pUD8D8i had a significantly smaller area and greater circularity and solidity (P < 0.001) than P. patens silenced by the control vector pUGi. Preliminary Carbohydrate Binding Module (CBM) labeling of crystalline cellulose in csld1 KO mutants and wild-type moss showed an unexpected increase in crystalline cellulose deposition in cells grown on mannitol. As a result, a comprehensive polysaccharide labeling study was performed with various monoclonal antibodies and CBMs. We have identified six cell wall carbohydrates that exhibit increased deposition and one that exhibit decreased deposition in cells grown on mannitol... |
