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Background Plants in nature or crops in the field interact with a multitude of beneficial or parasitic organisms, including bacteria, fungi and viruses. Viruses are highly specialized to infect a limited range of host plants, leading in extreme cases to the full invasion of the host and a diseased phenotype. Resistance to viruses can be mediated by various passive or active mechanisms, including the RNA-silencing machinery and the innate immune system. Main text RNA-silencing mechanisms may inhibit viral replication, while viral components can elicit the innate immune system. Viruses that successfully enter the plant cell can elicit pattern-triggered immunity (PTI), albeit by yet unknown mechanisms. As a counter defense, viruses suppress PTI. Furthermore, viral Avirulence proteins (Avr) may be detected by intracellular immune receptors (Resistance proteins) to elicit effector-triggered immunity (ETI). ETI often culminates in a localized programmed cell death reaction, the hypersensitive response (HR), and is accompanied by a potent systemic defense response. In a dichotomous view, RNA silencing and innate immunity are seen as two separate mechanisms of resistance. Here, we review the intricate connections and similarities between these two regulatory systems, which are collectively required to ensure plant fitness and resilience. Conclusions The detailed understanding of immune regulation at the transcriptional level provides novel opportunities for enhancing plant resistance to viruses by RNA-based technologies. However, extensive use of RNA technologies requires a thorough understanding of the molecular mechanisms of RNA gene regulation. We describe the main examples of host RNA-mediated regulation of virus resistance.

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25–30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.

Summary Microrchidia (MORC) proteins comprise a family of proteins that have been identified in prokaryotes and eukaryotes. They are defined by two hallmark domains: a GHKL‐type ATPase and an S5‐fold. In plants, MORC proteins were first discovered in a genetic screen for Arabidopsis thaliana mutants compromised for resistance to a viral pathogen. Subsequent studies expanded their role in plant immunity and revealed their involvement in gene silencing and genome stabilization. Little is known about the role of MORC proteins of cereals, especially because knockout (KO) mutants were not available and assessment of loss of function relied only on RNAi strategies, which were arguable, given that MORC proteins in itself are influencing gene silencing. Here, we used a Streptococcus pyogenes Cas9 (SpCas9)‐mediated KO strategy to functionally study HvMORC1, one of the current seven MORC members of barley. Using a novel barley RNA Pol III‐dependent U3 small nuclear RNA (snRNA) promoter to drive expression of the synthetic single guide RNA (sgRNA), we achieved a very high mutation frequency in HvMORC1. High frequencies of mutations were detectable by target sequencing in the callus, the T0 generation (77%) and T1 generation (70%–100%), which constitutes an important improvement of the gene‐editing technology in cereals. Corroborating and extending earlier findings, SpCas9‐edited hvmorc1‐KO barley, in clear contrast to Arabidopsis atmorc1 mutants, had a distinct phenotype of increased disease resistance to fungal pathogens, while morc1 mutants of either plant showed de‐repressed expression of transposable elements (TEs), substantiating that plant MORC proteins contribute to genome stabilization in monocotyledonous and dicotyledonous plants.

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a \"peripheral infrastructure\" around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies.

Host defenses can have broader ecological roles, but how they shape natural microbiome recruitment is poorly understood. Aliphatic glucosinolates (GLSs) are secondary defense metabolites in Brassicaceae plant leaves. Their genetically defined structure shapes interactions with pests in Arabidopsis thaliana leaves, and here we find that it also shapes bacterial recruitment. In model genotype Col-0, GLSs (mostly 4-methylsulfinylbutyl-GLS) have no clear effect on natural leaf bacterial recruitment. In a genotype from a wild population, however, GLSs (mostly allyl-GLS) enrich specific taxa, mostly Comamonadaceae and Oxalobacteraceae. Consistently, Comamonadaceae are also enriched in wild A. thaliana, and Oxalobacteraceae are enriched from wild plants on allyl-GLS as carbon source, but not on 4-methylsulfinylbutyl-GLS. Recruitment differences between GLS structures most likely arise from bacterial myrosinase specificity. Community recruitment is then defined by metabolic cross-feeding among bacteria. The link of genetically defined metabolites to recruitment could lead to new strategies to shape plant microbiome balance.

Background In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes ( Sp Cas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated. Results We designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions. Conclusions Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects.

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. Cell-surface receptors form the front line of plant immunity. Here, the authors show that the RLP co-receptors SOBIR1 and BAK1 directly phosphorylate each other, leading to activation of the immune receptor complex in which RLCKs are differentially required for production of reactive oxygen species that play a role in resistance against Phytophthora palmivora .

Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators.

Most Gram-negative plant pathogenic bacteria translocate effector proteins (T3Es) directly into plant cells via a conserved type III secretion system, which is essential for pathogenicity in susceptible plants. In resistant plants, recognition of some T3Es is mediated by corresponding resistance ( ) genes or R proteins and induces effector triggered immunity (ETI) that often results in programmed cell death reactions. The identification of genes and understanding their evolution/distribution bears great potential for the generation of resistant crop plants. We focus on T3Es from pv. ( ), the causal agent of bacterial spot disease on pepper and tomato plants. Here, 86 lines mainly of the genus were screened for phenotypical reactions after -mediated transient expression of 21 different effectors to (i) identify new plant lines for T3E characterization, (ii) analyze conservation/evolution of putative genes and (iii) identify promising plant lines as repertoire for gene isolation. The effectors provoked different reactions on closely related plant lines indicative of a high variability and evolution rate of potential genes. In some cases, putative genes were conserved within a plant species but not within superordinate phylogenetical units. Interestingly, the effector XopQ was recognized by several spp. lines, and infection assays revealed that XopQ is a host range determinant in many species. Non-host resistance against and XopQ recognition in required , strongly suggesting the presence of a TIR domain-containing XopQ-specific R protein in these plant lines. XopQ is a conserved effector among most xanthomonads, pointing out the XopQ-recognizing R as candidate for targeted crop improvement.