An ancient endosymbiosis involving early eukaryotic cells and cyanobacteriaresulted in the modern chloroplast organelle of the Archaeplastida clade, including all land plants. Throughout chloroplast evolution, many genes from the chloroplast genome were transferred to the nuclear genome, including the genetic pathway for synthesis of the bacterial polymer peptidoglycan (PG). In the ~1 billion years since the acquisition of the chloroplast, seed plants can now be grouped into species that retain ten PG genes (“full-PG”) or only four of the ten (“4-PG”). In Arabidopsis thaliana, a 4-PG flowering plant, knockout mutants for the PG gene murE show a proplastid phenotype, where chloroplast development is completely halted. In this report, A. thaliana mutants for the remaining three PG genes (mraY, murG, and ddl) were phenotyped to test for their functions. Two T-DNA insertion lines for each gene were selected from the Arabidopsis Biological Resource Center, and either confirmed as homozygotes or selfed to produce homozygous lines. These lines were then grown for at least two generations to confirm homozygous status and the location of the T-DNA insertion was identified using Sanger sequencing. All mutant lines had an insertion in either an exon or 5’ UTR of target genes. All lines were then grown on soil in conjunction with wild type plants for comparison of germination, morphology, and transcriptomics. No differences were observed in time to germination or morphology compared to the wild type control. Transcriptomics showed that target PG genes were differentially expressed in each line, but usually at a non-significant value. Genes related to flowering and reproduction were significantly differentially expressed in the mraY lines. Protoplasts were produced to image chloroplasts and identify any morphological or developmental effects. Chloroplast morphology was comparable to wild type; additionally, measurements of photosynthetic efficiency were not found to be significantly different than wild type. Together these data show that A. thaliana mutants for three PG genes do not display a phenotype like those for mutants of the fourth gene, murE. The results suggest that PG genes in A. thaliana are no longer linked to PG production and have diverged from the ancestral bacterial pathway.

Planococcus citri mealybugs are an incredibly unique species due to their nested endosymbiosis, in which Moranella endobia resides within Tremblaya princeps. These endosymbionts work together with their host to provide nutritional support throughout its life cycle and onto future offspring. Though what makes these endosymbionts even more interesting is that when viewed form a cell biological perspective, it becomes evident that they should have been exocytosed out of the host millions of years ago. One of the three membranes that surrounds Tremblaya, particularly the outermost vacuolar membrane, acts as the endosomal compartment around the bacteriome. In a traditional case of the endocytic cycle, the contents within the vacuole would be marked by the GTPase proteins Rab5 and Rab7 respectively until the fate of lysosomal digestion occurred. Though what is unique about the vacuolar membrane that surrounds Tremblaya is two things: first is that that membrane is lost and regained upon maternal transmission and secondly the endosymbionts within the membrane are not reaching their lysosomal fate, rather they are being passed down onto future generations. How these endosymbionts can redirect the endocytic pathway can possibly be explained by one of these four mechanisms: 1. Rabex-5 fails to recruit to the membrane of the early endosome, 2. Interruption of Rab7 activation by inhibiting membrane translocation of Ccz1, 3. Failure of the HOPS complex to bind to the late endosome, 4. Inhibition of translocation of ORP1L to the late endosome. Though the four mechanisms outlined above are very clearly regarding a cell biological process, they are not easily testable in a real-world setting. Thus, to adjust and account for this, the early and late endosomal marker proteins (Rab5 and Rab7) were used as the proteins of interest throughout immunofluorescence and western blot experimentation. These experiments revealed significant difficulties in working with commercially made antibodies but more importantly provided insight as to how is best to go forth with this research. In addition to this, qPCR experimentation and Rab7 epitope analysis did reveal that Rab5 and Rab7 are in fact key players in understanding how these endosymbionts are able to evade the endocytic cycle.