The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

Since the discovery that nucleases of the bacterial CRISPR(clustered regularly interspaced palindromic repeat)-associated(Cas) system can be used as easily programmable tools for genome engineering,their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double-strand break induction,resulting in mutations by non-homologous recombinatr e-tion. Strategies for performing such experiments à from Rthe design of guide RNA to the use of different transformation technologies à are evaluated. Furtherweive-more, we sum up recent developments regarding the use of nuclease-deficient Cas9/12 proteins, as DNAbinding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.

Development and molecular identification of pollen-somatic hybrid plants in Brassica spp.

As an important branch of sexual plant cell manipulation studies, gametosomatic hybridization has made great progress in the last decade. Fusion of protoplasts isolated from microspore tetrads, young and mature pollen grains with somatic protoplast achieved successes. However,. the studies so far mainly concentrated on the plant species in Solanaceae, and there has been no clear report on the fertility of hybrid plants. In the previous reports by Li et al. (1994), three plantlets were regenerated from young pollen-somatic protoplast fusion in Brassica and were preliminarily identified to be one allotriploid and two allotetraploids by means of chromosome counts and esterase

Transient transformation of pollen protoplasts via electroporation and temporal expression of Zml3-260-GUS-NOS chimeric gene in Brasska campestris var.purpurea

Using pollen as a vector of foreign genes for plant transformation is an active research area. An important link in this transformation system is the delivery of foreign genes into pollen. However, DNA transfer into pollen is difficult because of the existence of a thick pollen wall, whereas pollen protoplast deprived of pollen wall should be advantageous

Polarized Defense Against Fungal Pathogens Is Mediated by the Jacalin-Related Lectin Domain of Modular Poaceae-Specific Proteins

Modular proteins are an evolutionary answer to optimize performance of proteins that physically interact with each other for functionality. Using a combination of genetic and biochemical experiments, we charac-terized the rice protein OsJAC1, which consists of a jacalin-related lectin （JRL） domain predicted to bind mannose-containing oligosaccharides, and a dirigent domain which might function in stereoselective coupling of monolignols. Transgenic overexpression of OsJAC1 in rice resulted in quantitative broad- spectrum resistance against different pathogens including bacteria, oomycetes, and fungi. Overexpression of this gene or its wheat ortholog TAJA1 in barley enhanced resistance against the powdery mildew fungus. Both protein domains of OsJAC1 are required to establish resistance as indicated by single or combined transient expression of individual domains. Expression of artificially separated and fluorescence-tagged protein domains showed that the JRL domain is sufficient for targeting the powdery mildew penetration site. Nevertheless, co-localization of the lectin and the dirigent domain occurred. Phylogenetic analyses re- vealed orthologs of OsJAC1 exclusively within the Poaceae plant family. Dicots, by contrast, only contain proteins with either JRL or dirigent domain（s）. Altogether, our results identify OsJAC1 as a representative of a novel type of resistance protein derived from a plant lineage-specific gene fusion event for better function in local pathogen defense.

Identification of Early Nuclear Target Genes of Plastidial Redox Signals that Trigger the Long- Term Response of Arabidopsis to Light Quality Sh∽ts

Natural illumination conditions are highly variable and because of their sessile life style, plants are forced to acclimate to them at the cellular and molecular level. Changes in light intensity or quality induce changes in the reduction/oxidation （redox） state of the photosynthetic electron chain that acts as a trigger for compen- satory acclimation responses comprising functional and structural adjustments of photosynthesis and metabolism. Such responses include redox-controlled changes in plant gene expression in the nucleus and organelles. Here we describe a strategy for the identification of early redox-regulated genes （ERGs） in the nucleus of the model organism Arabidopsis thaliana that respond significantly 30 or 60 min after the generation of a reduction signal in the photosynthetic electron transport chain. By comparing the response of wild-type plants with that of the acclimation mutant stn7, we could specifically identify ERGs. The results reveal a significant impact of chloroplast redox signals on distinct nuclear gene groups including genes for the mitochondrial electron transport chain, tetrapyrrole biosynthesis, carbohydrate metabolism, and signaling lipid synthesis. These expression profiles are clearly different from those observed in response to the reduction of photosynthetic electron transport by high light treatments. Thus, the ERGs identified are unique to redox imbalances in photosynthetic electron transport and were then used for analyzing potential redox-responsive cis-elements, trans-factors, and chromosomal regulatory hot spots. The data identify a novel redox-responsive element and indicate extensive redox control at transcriptional and chromosomal levels that point to an unprecedented impact of redox signals on epigenetic processes.