Biology subjects

Zeilmaker, T.

Publications and source records attributed to Zeilmaker, T..

4 recordsLinked to original sources

bioRxiv2022

Vitis vinifera plants edited in DMR6 genes show improved resistance to downy mildew.

The production and cultivation of vines (Vitis vinifera) tolerant or resistant to diseases such as downy mildew (DM) is a promising strategy to reduce fungicides and help viticulture sustainability. In many crops, generation of knock-out mutants in host genes controlling susceptibility to DM, such as Downy Mildew Resistant 6 (DMR6) is a strategy of proven success to obtain resistant plants, while the effect of mutations in DMR6 genes has yet to be demonstrated in grapevine. In addition, small mutations in genes governing important traits can be obtained by gene-editing while maintaining the genetic background of commercially important clones. Moreover, very recent advances in the technology of gene-editing allowed to produce non-transgenic grapevine mutants, by regeneration of protoplasts previously edited with the CRISPR-Cas9 ribonucleoprotein. This approach may revolutionize the production of new grapevine varieties and clones, but it requires knowledge on the targets, and an extensive evaluation of the impact of their mutation on plant phenotype and fitness. In this work we generated single and double knock-out mutants in DMR6 susceptibility (S) genes in multiple grapevine cultivars with improved resistance to DM.

plant biology↗

bioRxiv2021

Regeneration of Plants from DNA-free Edited Grapevine Protoplasts

CRISPR-Cas technology has widely extended the application fields of genome editing in plant breeding, making possible specific and minimal mutations within a genetic pool. With respect to standard genome editing technologies, CRISPR-Cas machinery can be introduced in the form of ribonucleoproteins (RNPs), thus avoiding the introduction of exogenous DNA into cells. The interest on the application of DNA-free delivery into plant cells is constantly increasing, especially in the case of valuable woody plants elite varieties where CRISPR-Cas9 technology would preserve their genotype, while still resulting into targeted genetic modifications. The use of single cells fits well the requirements of New Breeding Technologies, by ensuring both the CRISPR-Cas DNA-free delivery as RNPs and, since every plant will be regenerated from a single edited cell, the absence of chimerism. However, the use of protoplasts cell culture from woody plants is generally hampered by low editing efficiencies and an unsuccessful regenerative process. We here describe a successful DNA-free methodology to obtain fully edited grapevine plants, regenerated from protoplasts obtained from V. vinifera cv. Crimson seedless L. embryogenic callus. The transfected protoplasts were edited on the Downy Mildew susceptibility gene VvDMR6-2. The regenerated edited plants exhibited homozygous deletions of 1bp or 2bp, and homozygous insertion of 1bp.

plant biology↗

bioRxiv2020

Mining downy mildew susceptibility genes: a diversity study in grapevine.

Several pathogens continuously threaten viticulture worldwide. Until now, the investigation on resistance loci has been the main trend to understand the interaction between grapevine and mildew causal agents. Dominantly inherited gene-based resistance has shown to be race-specific in some cases, to confer partial immunity and to be potentially overcome within a few years since its introgression. Recently, on the footprint of research conducted on Arabidopsis, the putative hortologues of genes associated with downy mildew susceptibility in this species, have been discovered also in the grapevine genome. In this work, we deep-resequenced four putative susceptibility genes in 190 highly genetically diverse grapevine genotypes to discover new sources of broad-spectrum recessively inherited resistance. The scouted genes are VvDMR6-1, VvDMR6-2, VvDLO1, VvDLO2 and predicted to be involved in susceptibility to downy mildew. From all identified mutations, 56% were Single Nucleotide Polymorphisms (SNPs) in heterozygosity, while the remaining 44% were homozygous. Regarding the identified mutations with putative impact on gene function, we observed ~4% genotypes mutated in VvDMR6-1 and ~8% mutated in VvDMR6-2, only a handful of genotypes that were mutated in both genes. ~2% and ~7% genotypes showed mutations in VvDLO1 and VvDLO2 respectively, and again a few genotypes resulted mutated in both genes. In particular, 80% of impacting mutations were heterozygous while 20% were homozygous. The current results will inform grapevine genetics and corroborate genomic-assisted breeding programs for resistance to biotic stresses. Significance statementA survey on the genetic diversity of downy mildew susceptibility genes in grapevine varieties and wild species reveals a potential valuable for genomic-assisted breeding as well as tailored gene editing to induce disease resistance.

plant biology↗

bioRxiv2017

Arabidopsis JASMONATE-INDUCED OXYGENASES down-regulate plant immunity by hydroxylation and inactivation of the hormone jasmonic acid

The phytohormone jasmonic acid (JA) is vital in plant defense and development. Although biosynthesis of JA and activation of JA-responsive gene expression by the bioactive form JA-isoleucine (JA-Ile) have been well-studied, knowledge on JA metabolism is incomplete. In particular, the enzyme that hydroxylates JA to 12-OH-JA, an inactive form of JA that accumulates after wounding and pathogen attack, is unknown. Here, we report the identification of four paralogous 2-oxoglutarate/Fe(II)-dependent oxygenases in Arabidopsis thaliana as JA hydroxylases and show that they down-regulate JA-dependent responses. As they are induced by JA we named them JASMONATE-INDUCED OXYGENASEs (JOXs). Concurrent mutation of the four genes in a quadruple Arabidopsis mutant resulted in increased defense gene expression and increased resistance to the necrotrophic fungus Botrytis cinerea and the caterpillar Mamestra brassicae. In addition, root and shoot growth of the plants was inhibited. Metabolite analysis of leaves showed that loss of function of the four JOX enzymes resulted in over-accumulation of JA and in reduced turnover of JA into 12-OH-JA. Transformation of the quadruple mutant with each JOX gene strongly reduced JA levels, demonstrating that all four JOXs inactivate JA in plants. The in vitro catalysis of 12-OH-JA from JA by recombinant enzyme could be confirmed for three JOXs. The identification of the enzymes responsible for hydroxylation of JA reveals a missing step in JA metabolism, which is important for the inactivation of the hormone and subsequent down-regulation of JA-dependent defenses.\n\nSIGNIFICANCE STATEMENTIn plants, the hormone jasmonic acid (JA) is synthesized in response to attack by pathogens and herbivores, leading to activation of defense responses. Rapidly following JA accumulation, the hormone is metabolized, presumably to prevent inhibitive effects of high JA levels on growth and development. The enzymes that directly inactivate JA were so far unknown. Here, we identify four jasmonate-induced oxygenases (JOXs) in Arabidopsis that hydroxylate jasmonic acid to form inactive 12-OH-JA. A mutant that no longer produces the four enzymes hyperaccumulates JA, exhibits reduced growth, and is highly resistant to attackers that are sensitive to JA-dependent defense. The JOX enzymes thus play an important role in determining the amplitude and duration of JA responses to balance the growth-defense tradeoff.

plant biology↗