A SNP associated with alternative splicing of RPT5b causes unequal redundancy between RPT5a and RPT5b among Arabidopsis thaliana natural variation

<p>Abstract</p> <p>Background</p> <p>The proteasome subunit RPT5, which is essential for gametophyte development, is encoded by two genes in <it>Arabidopsis thaliana</it>; <it>RPT5a </it>and <it>RPT5b</it>. We showed previously that <it>RPT5a </it>and <it>RPT5b </it>are fully redundant in the Columbia (Col-0) accession, whereas in the Wassilewskia accession (Ws-4), <it>RPT5b </it>does not complement the effect of a strong <it>rpt5a </it>mutation in the male gametophyte, and only partially complements <it>rpt5a </it>mutation in the sporophyte. <it>RPT5b^Col-0</it>and <it>RPT5b^Ws-4</it>differ by only two SNPs, one located in the promoter and the other in the seventh intron of the gene.</p> <p>Results</p> <p>By exploiting natural variation at <it>RPT5b </it>we determined that the SNP located in <it>RPT5b </it>intron seven, rather than the promoter SNP, is the sole basis of this lack of redundancy. In Ws-4 this SNP is predicted to create a new splicing branchpoint sequence that induces a partial mis-splicing of the pre-mRNA, leading to the introduction of a Premature Termination Codon. We characterized 5 accessions carrying this A-to-T substitution in intron seven and observed a complete correlation between this SNP and both a 10 to 20% level of the <it>RPT5b </it>pre-mRNA mis-splicing and the lack of ability to complement an <it>rpt5a </it>mutant phenotype.</p> <p>Conclusion</p> <p>The accession-dependent unequal redundancy between <it>RPT5a </it>and <it>RPT5b </it>genes illustrates an example of evolutionary drifting between duplicated genes through alternative splicing.</p

The XPF-ERCC1 Complex Is Essential for Genome Stability and Is Involved in the Mechanism of Gene Targeting in Physcomitrella patens

The XPF-ERCC1 complex, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair, and homologous recombination. XPF-ERCC1 incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here, we have examined the role of the XPF-ERCC1 complex in the model bryophyte Physcomitrella patens which exhibits uniquely high gene targeting frequencies. We undertook targeted knockout of the Physcomitrella ERCC1 and XPF genes. Mutant analysis shows that the endonuclease complex is essential for resistance to UV-B and to the alkylating agent MMS, and contributes to the maintenance of genome integrity but is also involved in gene targeting in this model plant. Using different constructs we determine whether the function of the XPF-ERCC1 endonuclease complex in gene targeting was removal of 3′ non-homologous termini, similar to SSA, or processing of looped-out heteroduplex intermediates. Interestingly, our data suggest a role of the endonuclease in both pathways and have implications for the mechanism of targeted gene replacement in plants and its specificities compared to yeast and mammalian cells

A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens

CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing

Mécanismes moléculaires impliqués dans le ciblage génique chez Physcomitrella patens

Le ciblage génique est l’intégration d’un fragment d’ADN exogène partageant deshomologies de séquences avec un génome receveur, au locus génomique correspondant. Onparle alors d’intégration ciblée de façon opposée à l’intégration aléatoire qui se fait de façonnon homologue. Le mécanisme moléculaire impliqué dans le ciblage génique est la voie deréparation de l’ADN par recombinaison homologue, basée sur l’action de la protéine RAD51qui permet l’échange entre séquences homologues et sur l’action d’endonucléases et derésolvases qui permettent de rétablir la conformation de la double hélice d’ADN. Chez laplupart des eucaryotes, la transformation génétique avec un fragment d’ADN exogène se faitmajoritairement par intégration aléatoire. Il existe cependant quelques exceptions comme lalevure Saccharomyces cerevisiae ainsi que la mousse Physcomitrella patens, dans lesquellesles intégrations se font préférentiellement de manière ciblée. Alors que chez la levure, toutesles intégrations ciblées résultent de deux évènements de recombinaison homologueconduisant au remplacement du gène, chez la mousse les intégrations ciblées résultentégalement d’insertions ciblées ou correspondant à un seul évènement de recombinaisonhomologue accompagné d’une intégration qui apparait comme non homologue. Afin d’étudierles mécanismes moléculaires impliqués dans le ciblage génique chez la mousse, lors desremplacements de gène mais également lors de ces insertions ciblées, j’ai réalisé une analysefonctionnelle des protéines PpRAD51, en utilisant une approche comparative entre une espècevégétale très peu compétente pour le ciblage génique, Arabidopsis thaliana, et une espècevégétale très compétente pour le ciblage génique, Physcomitrella patens. Cette étude a mis enévidence une disjonction de fonction des protéines PpRAD51 : en effet, la moitié N-terminalede la protéine est fonctionnelle pour les divisions mitotiques mais ne l’est pas pour le ciblagegénique. Parallèlement, j’ai mesuré l’effet de l’absence de l’endonucléase RAD1-RAD10 surle ciblage génique en étudiant les mutants de délétion Δrad1, Δrad10 et Δrad1Δrad10. Cetteétude a permis de montrer le rôle important de ce complexe endonucléase dans le ciblagegénique chez la mousse, et plus particulièrement dans les insertions ciblées

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

Beyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR–induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in

Simple and efficient targeting of multiple genes through CRISPR-Cas9 in Physcomitrella patens

Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene knock-out-technology in a variety of species. However, in a number of situations knocking out or modifying a single gene is not sufficient, this is particularly true for genes belonging to a common family or for genes showing redundant functions. Like many plants the model organism Physcomitrella patens has experienced multiple events of polyploidization during evolution that resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the co-delivery of a CAS9 expressing cassette, multiple sgRNA vectors and a cassette for transient transformation selection for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9 mediated targeting of five different genes allows the selection of a quintuple mutant and all possible sub-combinations of mutants in one experiment with no mutations detected in potential off target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to alternative End Joining pathway for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families in Physcomitrella is substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants

Pollen tube growth mutations in Arabidopsis

Pollen tube growth mutations in Arabidopsis. 17. International Botanical Congres

The Arabidopsis Proteasome RPT5 Subunits Are Essential for Gametophyte Development and Show Accession-Dependent Redundancy[W]

We investigated the role of the ubiquitin proteasome system (UPS), which allows proteins to be selectively degraded, during gametophyte development in Arabidopsis thaliana. Three mutant alleles altering the UPS were isolated in the Wassilewskija (Ws) accession: they affect the Regulatory Particle 5a (RPT5a) gene, which (along with RPT5b) encodes one of the six AAA-ATPases of the proteasome regulatory particle. In the heterozygous state, all three mutant alleles displayed 50% pollen lethality, suggesting that RPT5a is essential for male gametophyte development. However, a fourth mutant in the Columbia (Col) accession did not display such a phenotype because the RPT5b Col allele complements the rpt5a defect in the male gametophyte, whereas the RPT5b Ws allele does not. Double rpt5a rpt5b mutants showed a complete male and female gametophyte lethal phenotype in a Col background, indicating that RPT5 subunits are essential for both gametophytic phases. Mitotic divisions were affected in double mutant gametophytes correlating with an absence of the proteasome-dependent cyclinA3 degradation. Finally, we show that RPT5b expression is highly increased when proteasome functioning is defective, allowing complementation of the rpt5a mutation. In conclusion, RPT5 subunits are not only essential for both male and female gametophyte development but also display accession-dependent redundancy and are crucial in cell cycle progression