The results provide new insights into the evolution of cell walls and support the notion that the CGA were pre-adapted to life on land by virtue of the their cell wall biosynthetic capacity. These findings are highly significant for understanding plant cell wall evolution as they imply that some features of land plant cell walls evolved prior to the transition to land, rather than having evolved as a result of selection pressures inherent in this transition.

The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor. When activated, it splices the bZIP60 mRNA, producing a truncated transcription factor that upregulates genes involved in the unfolded protein response. Bax inhibitor 1 (BI-1) is another ER stress sensor that regulates cell death in response to environmental assaults. The potyvirus 6K2 and potexvirus TGB3 proteins are known to reside in the ER, serving, respectively, as anchors for the viral replicase and movement protein complex. This study used green fluorescent protein (GFP)-tagged Turnip mosaic virus (TuMV), Plantago asiatica mosaic virus (PlAMV), Potato virus Y (PVY), and Potato virus X (PVX) to determine that the IRE1/bZIP60 pathway and BI-1 machinery are induced early in virus infection in Arabidopsis thaliana, Nicotiana benthamiana, and Solanum tuberosum. Agrodelivery of only the potyvirus 6K2 or TGB3 genes into plant cells activated bZIP60 and BI-1 expression in Arabidopsis thaliana, N. benthamiana, and S. tuberosum. Homozygous ire1a-2, ire1b-4, and ire1a-2/ire1b-4 mutant Arabidopsis plants were inoculated with TuMV-GFP or PlAMV-GFP. PlAMV accumulates to a higher level in ire1a-2 or ire1a-2/ire1b-4 mutant plants than in ire1b-4 or wild-type plants. TuMV-GFP accumulates to a higher level in ire1a-2, ire1b-4, or ire1a-2/ire1b-4 compared with wild-type plants, suggesting that both isoforms contribute to TuMV-GFP infection. Gene silencing was used to knock down bZIP60 and BI-1 expression in N. benthamiana. PVX-GFP and PVY-GFP accumulation was significantly elevated in these silenced plants compared with control plants. This study demonstrates that two ER stress pathways, namely IRE1/bZIP60 and the BI-1 pathway, limit systemic accumulation of potyvirus and potexvirus infection. Silencing BI-1 expression also resulted in systemic necrosis. These data suggest that ER stress-activated pathways, led by IRE1 and BI-1, respond to invading potyvirus and potexviruses to restrict virus infection and enable physiological changes enabling plants to tolerate virus assault.

CRISPR/Cas9 editing efficacies in tetraploid potato were highly improved through the use of endogenous potato U6 promoters. Highly increased editing efficiencies in the Granular Bound Starch Synthase gene at the protoplast level were obtained by replacement of the Arabidopsis U6 promotor, driving expression of the CRISPR component, with endogenous potato U6 promotors. This translated at the ex-plant level into 35% full allelic gene editing. Indel Detection Amplicon Analysis was established as an efficient tool for fast assessment of gene editing in complex genomes, such as potato. Together, this warrants significant reduction of laborious cell culturing, ex-plant regeneration and screening procedures of plants with high complexity genomes.

Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

The charophycean green algae (CGA or basal streptophytes) are of particular evolutionary significance because their ancestors gave rise to land plants. One outstanding feature of these algae is that their cell walls exhibit remarkable similarities to those of land plants. Xyloglucan (XyG) is a major structural component of the cell walls of most land plants and was originally thought to be absent in CGA. This study presents evidence that XyG evolved in the CGA. This is based on a) the identification of orthologs of the genetic machinery to produce XyG, b) the identification of XyG in a range of CGA and, c) the structural elucidation of XyG, including uronic acid-containing XyG, in selected CGA. Most notably, XyG fucosylation, a feature considered as a late evolutionary elaboration of the basic XyG structure and orthologs to the corresponding biosynthetic enzymes are shown to be present in Mesotaenium caldariorum.