Geschmack ist vorhersagbar: Mit FlavorMiner. FlavorMiner heißt das Tool, das IPB-Chemiker und Partner aus Kolumbien jüngst entwickelt haben. Das Programm kann, basierend auf maschinellem Lernen (KI), anhand der…
Seit Februar 2021 bietet Wolfgang Brandt, ehemaliger Leiter der Arbeitsgruppe Computerchemie am IPB, sein Citizen Science-Projekt zur Pilzbestimmung an. Dafür hat er in regelmäßigen Abständen öffentliche Vorträge zur Vielfalt…
Ordon, J.; Martin, P.; Lee Erickson, J.; Ferik, F.; Balcke, G.; Bonas, U.; Stuttmann, J.;Disentangling cause and consequence: genetic dissection of the
DANGEROUS MIX2
risk locus, and activation of the DM2h NLR in autoimmunityPlant J.1061008-1023(2021)DOI: 10.1111/tpj.15215
Nucleotide-binding domain–leucine-rich repeat-type immune receptors
(NLRs) protect plants against pathogenic microbes through intracellular
detection of effector proteins. However, this comes at a cost, as NLRs
can also induce detrimental autoimmunity in genetic interactions with
foreign alleles. This may occur when independently evolved genomes are
combined in inter- or intraspecific crosses, or when foreign alleles are
introduced by mutagenesis or transgenesis. Most autoimmunity-inducing
NLRs are encoded within highly variable NLR gene clusters with no
known immune functions, which were termed autoimmune risk loci. Whether
risk NLRs differ from sensor NLRs operating in natural pathogen
resistance and how risk NLRs are activated in autoimmunity is unknown.
Here, we analyzed the DANGEROUS MIX2 risk locus, a major autoimmunity hotspot in Arabidopsis thaliana. By gene editing and heterologous expression, we show that a single gene, DM2h, is necessary and sufficient for autoimmune induction in three independent cases of autoimmunity in accession Landsberg erecta. We focus on autoimmunity provoked by an EDS1-yellow fluorescent protein (YFP)NLS fusion protein to characterize DM2h functionally and determine features of EDS1-YFPNLS
activating the immune receptor. Our data suggest that risk NLRs
function in a manner reminiscent of sensor NLRs, while
autoimmunity-inducing properties of EDS1-YFPNLS in this
context are unrelated to the protein\'s functions as an immune regulator.
We propose that autoimmunity, at least in some cases, may be caused by
spurious, stochastic interactions of foreign alleles with coincidentally
matching risk NLRs.
Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene-free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on the production of toxic 5-fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T1, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher-order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.