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

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

Monitoring systemic infection by cucumber mosaic virus using a small fluorescent protein iLOV in plants

Tracking virus infection in plants is useful to assess plant susceptibility or resistance. The recombinant cucumber mosaic virus (CMV) expressing a 2b-fused green fluorescent protein (GFP, 25 kDa) gradually loses (within weeks) the GFP gene in plants. Here, we constructed CMV expressing a flavin-based small fluorescent protein (iLOV, 10 kDa). CMV-iLOV still retains iLOV after 28 dpi in upper leaves of inoculated Nicotiana benthamiana and allows long-term monitoring of its distribution in plants. Using CMV-iLOV, we showed that pot1, a recessive allele for resistance to potyviruses, does not confer CMV resistance in tomato

RECHERCHES METHODOLOGIQUES EN VUE DE L'ISOLEMENT DE MUTANTS D'EXPRESSION DU GENE NUCLEAIRE RPL21

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Exploring New Routes for Genetic Resistances to Potyviruses: The Case of the Arabidopsis thaliana Phosphoglycerates Kinases (PGK) Metabolic Enzymes

International audienceThe development of recessive resistance by loss of susceptibility is a consistent strategy to combat and limit damages caused by plant viruses. Susceptibility genes can be turned into resistances, a feat that can either be selected among the plant’s natural diversity or engineered by biotechnology. Here, we summarize the current knowledge on the phosphoglycerate kinases (PGK), which have emerged as a new class of susceptibility factors to single-stranded positive RNA viruses, including potyviruses. PGKs are metabolic enzymes involved in glycolysis and the carbon reduction cycle, encoded by small multigene families in plants. To fulfil their role in the chloroplast and in the cytosol, PGKs genes encode differentially addressed proteins. Here, we assess the diversity and homology of chloroplastic and cytosolic PGKs sequences in several crops and review the current knowledge on their redundancies during plant development, taking Arabidopsis as a model. We also show how PGKs have been shown to be involved in susceptibility—and resistance—to viruses. Based on this knowledge, and drawing from the experience with the well-characterized translation initiation factors eIF4E, we discuss how PGKs genes, in light of their subcellular localization, function in metabolism, and susceptibility to viruses, could be turned into efficient genetic resistances using genome editing techniques

eIF4E-mediated resistance to potyviruses in plants: from natural alleles to edited genes

Resistance to viruses is an important aspect of plant breeding. One way to achieve it is to select genetic resistances based on the susceptibility factors hijacked by the virus to infect the plants. Here, we recount work done on genes encoding translation initiation factors eIF4E, some of the most successful targets for obtaining resistance to potyviruses, starting from their characterization 20 years ago. With examples from different plant species, pepper, tomato, tobacco and arabidopsis, we present the basis of this type of resistances and their characteristics, highlighting the role of gene redundancy among 4E factors, their specificity for the virus and the need for the plant of a trade-off between resistance and development. Finally, we show how the new genome editing techniques could be used in plant breeding to develop eIF4E-based resistances in crops, mimicking the functional alleles that have been selected during evolution in many crops

eIF4E Resistance: Natural Variation Should Guide Gene Editing

eIF4E translation initiation factors have emerged as major susceptibility factors for RNA viruses. Natural eIF4E-based resistance alleles are found in many species and are mostly variants that maintain the translation function of the protein. eIF4E genes represent major targets for engineering viral resistance, and gene-editing technologies can be used to make up for the lack of natural resistance alleles in some crops, often by knocking out eIF4E susceptibility factors. However, we report here how redundancy among eIF4E genes can restrict the efficient use of knockout alleles in breeding. We therefore discuss how gene-editing technologies can be used to design de novo functional alleles, using knowledge about the natural evolution of eIF4E genes in different species, to drive resistance to viruses without affecting plant physiology