Disentangling cause and consequence: genetic dissection of the DANGEROUS MIX2 risk locus, and activation of the DM2h NLR in autoimmunity.

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.

Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants.

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.

Genome-wide association links candidate genes to fruit firmness, fruit flesh color, flowering time, and soluble solid content in apricot (Prunus armeniaca L.).

BACKGROUND: Apricots originated from China, Central Asia and the Near East and arrived in Anatolia, and particularly in their second homeland of Malatya province in Turkey. Apricots are outstanding summer fruits, with their beautiful attractive color, delicious sweet taste, aroma and high vitamin and mineral content. METHODS AND RESULTS: In the current study, a total of 259 apricots genotypes from different geographical origins in Turkey were used. Significant variations were detected in fruit firmness (FF), fruit flesh color (FFC), flowering time (FT), and soluble solid content (SSC). A total of 11,532 SNPs based on DArT were developed and used in the analyses of population structure and association mapping (AM). According to the STRUCTURE (v.2.2) analysis, the apricot genotypes were divided into three groups. The mixed linear model with Q and K matrixes were used to detect the associations between the SNPs and four traits. A total of 131 SNPs were associated with FF, FFC and SSC. No SNP marker was detected associated with FT. CONCLUSION: The results demonstrated that AM had high potential of revealing the markers associated with economically important traits in apricot. The SNPs identified in the study can be used in future breeding programs for marker-assisted selection in apricot.

Receptor-like cytoplasmic kinases of different subfamilies differentially regulate SOBIR1/BAK1-mediated immune responses in Nicotiana benthamiana.

Cell-surface receptors form the front line of plant immunity. The leucine-rich repeat (LRR)-receptor-like kinases SOBIR1 and BAK1 are required for the functionality of the tomato LRR-receptor-like protein Cf-4, which detects the secreted effector Avr4 of the pathogenic fungus Fulvia fulva. Here, we show that the kinase domains of SOBIR1 and BAK1 directly phosphorylate each other and that residues Thr522 and Tyr469 of the kinase domain of Nicotiana benthamiana SOBIR1 are required for its kinase activity and for interacting with signalling partners, respectively. By knocking out multiple genes belonging to different receptor-like cytoplasmic kinase (RLCK)-VII subfamilies in N. benthamiana:Cf-4, we show that members of RLCK-VII-6, -7, and -8 differentially regulate the Avr4/Cf-4-triggered biphasic burst of reactive oxygen species. In addition, members of RLCK-VII-7 play an essential role in resistance against the oomycete pathogen Phytophthora palmivora. Our study provides molecular evidence for the specific roles of RLCKs downstream of SOBIR1/BAK1-containing immune complexes.