The unequal functional redundancy of Arabidopsis INCURVATA11 and CUPULIFORMIS2 is not dependent on genetic background.

The paralogous genes INCURVATA11 (ICU11) and CUPULIFORMIS2 (CP2) encode components of the epigenetic machinery in Arabidopsis and belong to the 2-oxoglutarate and Fe (II)-dependent dioxygenase superfamily. We previously inferred unequal functional redundancy between ICU11 and CP2 from a study of the synergistic phenotypes of the double mutant and sesquimutant combinations of icu11 and cp2 mutations, although they represented mixed genetic backgrounds. To avoid potential confounding effects arising from different genetic backgrounds, we generated the icu11-5 and icu11-6 mutants via CRISPR/Cas genome editing in the Col-0 background and crossed them to cp2 mutants in Col-0. The resulting mutants exhibited a postembryonic-lethal phenotype reminiscent of strong embryonic flower (emf) mutants. Double mutants involving icu11-5 and mutations affecting epigenetic machinery components displayed synergistic phenotypes, whereas cp2-3 did not besides icu11-5. Our results confirmed the unequal functional redundancy between ICU11 and CP2 and demonstrated that it is not allele or genetic background specific. An increase in sucrose content in the culture medium partially rescued the post-germinative lethality of icu11 cp2 double mutants and sesquimutants, facilitating the study of their morphological phenotypes throughout their life cycle, which include floral organ homeotic transformations. We thus established that the ICU11-CP2 module is required for proper flower organ identity.

The m6A RNA Demethylase ALKBH9B Plays a Critical Role for Vascular Movement of Alfalfa Mosaic Virus in Arabidopsis.

The N 6-methyladenosine (m6A) pathway has been widely described as a viral regulatory mechanism in animals. We previously reported that the capsid protein (CP) of alfalfa mosaic virus (AMV) interacts with the Arabidopsis m6A demethylase ALKBH9B regulating m6A abundance on viral RNAs (vRNAs) and systemic invasion of floral stems. Here, we analyze the involvement of other ALKBH9 proteins in AMV infection and we carry out a detailed evaluation of the infection restraint observed in alkbh9b mutant plants. Thus, via viral titer quantification experiments and in situ hybridization assays, we define the viral cycle steps that are altered by the absence of the m6A demethylase ALKBH9B in Arabidopsis. We found that ALKBH9A and ALKBH9C do not regulate the AMV cycle, so ALKBH9B activity seems to be highly specific. We also define that not only systemic movement is affected by the absence of the demethylase, but also early stages of viral infection. Moreover, our findings suggest that viral upload into the phloem could be blocked in alkbh9b plants. Overall, our results point to ALKBH9B as a possible new component of phloem transport, at least for AMV, and as a potential target to obtain virus resistance crops.

INCURVATA11 and CUPULIFORMIS2 Are Redundant Genes That Encode Epigenetic Machinery Components in Arabidopsis.

All critical developmental and physiological events in a plant's life cycle depend on the proper activation and repression of specific gene sets, and this often involves epigenetic mechanisms. Some Arabidopsis thaliana mutants with disorders of the epigenetic machinery exhibit pleiotropic defects, including incurved leaves and early flowering, due to the ectopic and heterochronic derepression of developmental regulators. Here, we studied one such mutant class, the incurvata11 (icu11) loss-of-function mutants. We have identified ICU11 as the founding member of a small gene family that we have named CUPULIFORMIS (CP). This family is part of the 2-oxoglutarate/Fe(II)-dependent dioxygenase superfamily. ICU11 and its closest paralog, CP2, have unequally redundant functions: although cp2 mutants are phenotypically wild type, icu11 cp2 double mutants skip vegetative development and flower upon germination. This phenotype is reminiscent of loss-of-function mutants of the Polycomb-group genes EMBRYONIC FLOWER1 (EMF1) and EMF2 Double mutants harboring icu11 alleles and loss-of-function alleles of genes encoding components of the epigenetic machinery exhibit synergistic, severe phenotypes, and some are similar to those of emf mutants. Hundreds of genes are misexpressed in icu11 plants, including SEPALLATA3 (SEP3), and derepression of SEP3 causes the leaf phenotype of icu11 ICU11 and CP2 are nucleoplasmic proteins that act as epigenetic repressors through an unknown mechanism involving histone modification, but not DNA methylation.

Patterns of CRISPR/Cas9 activity in plants, animals and microbes.

The CRISPR/Cas9 system and related RNA-guided endonucleases can introduce double-strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on-target mutations), the targeting accuracy (likelihood of off-target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species-dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome-editing tools.