The prevalent occurrence of herbicide resistant weeds increases the necessity for new site of action herbicides for effective control as well as to relax selection pressure on the known sites of action. As a consequence, interest increased in the unexploited molecule cinmethylin as a new solution for the control of weedy grasses in cereals. Therefore, the mechanism of action of cinmethylin was reevaluated. We applied the chemoproteomic approach cellular Target Profiling™ from Evotec to identify the cinmethylin target in Lemna paucicostata protein extracts. We found three potential targets belonging to the same protein family of fatty acid thioesterases (FAT) to bind to cinmethylin with high affinity. Binding of cinmethylin to FAT proteins from Lemna and Arabidopsis was confirmed by fluorescence-based thermal shift assay. The plastid localized enzyme FAT plays a crucial role in plant lipid biosynthesis, by mediating the release of fatty acids (FA) from its acyl carrier protein (ACP) which is necessary for FA export to the endoplasmic reticulum. GC-MS analysis of free FA composition in Lemna extracts revealed strong reduction of unsaturated C18 as well as saturated C14, and C16 FAs upon treatment with cinmethylin, indicating that FA release for subsequent lipid biosynthesis is the primary target of cinmethylin. Lipid biosynthesis is a prominent target of different herbicide classes. To assess whether FAT inhibition constitutes a new mechanism of action within this complex pathway, we compared physiological effects of cinmethylin to different ACCase and VLCFA synthesis inhibitors and identified characteristic differences in plant symptomology and free FA composition upon treatment with the three herbicide classes. Also, principal component analysis of total metabolic profiling of treated Lemna plants showed strong differences in overall metabolic changes after cinmethylin, ACCase or VLCFA inhibitor treatments. Our results identified and confirmed FAT as the cinmethylin target and validate FAT inhibition as a new site of action different from other lipid biosynthesis inhibitor classes.

Phakopsora pachyrhizi is a biotrophic fungus provoking SBR disease. SBR poses a major threat to global soybean production. Though several R genes provided soybean immunity to certain P. pachyrhizi races, the pathogen swiftly overcame this resistance. Therefore, fungicides are the only current means to control SBR. However, insensitivity to fungicides is soaring in P. pachyrhizi and, therefore, alternative measures are needed for SBR control. In this article, we discuss the different approaches for fighting SBR and their potential, disadvantages, and advantages over other measures. These encompass conventional breeding for SBR resistance, transgenic approaches, exploitation of transcription factors, secondary metabolites, and antimicrobial peptides, RNAi/HIGS, and biocontrol strategies. It seems that an integrating approach exploiting different measures is likely to provide the best possible means for the effective control of SBR.

Amaranthus tuberculatus, Amaranthus hybridus, and Amaranthus palmeri are agronomically important weed species. Here, we present the most contiguous draft assemblies of these three species to date. We utilized a combination of Pacific Biosciences long-read sequencing and chromatin contact mapping information to assemble and order sequences of A. palmeri to near-chromosome-level resolution, with scaffold N50 of 20.1 Mb. To resolve the issues of heterozygosity and co-assembly of alleles in diploid species, we adapted the trio binning approach to produce haplotype assemblies of A. tuberculatus and A. hybridus. This approach resulted in an improved assembly of A. tuberculatus, and the first genome assembly for A. hybridus, with contig N50s of 2.58 and 2.26 Mb, respectively. Species-specific transcriptomes and information from related species were used to predict transcripts within each assembly. Syntenic comparisons of these species and Amaranthus hypochondriacus identified sites of genomic rearrangement, including duplication and translocation, while genetic map construction within A. tuberculatus highlighted the need for further ordering of the A. hybridus and A. tuberculatus contigs. These multiple reference genomes will accelerate genomic studies in these species to further our understanding of weedy evolution within Amaranthus.

SummaryNonhost resistance (NHR) of plants to fungal pathogens comprises different defense layers. Epidermal penetration resistance of Arabidopsis to Phakopsora pachyrhizi requires functional PEN1, PEN2 and PEN3 genes, whereas post-invasion resistance in the mesophyll depends on the combined functionality of PEN2, PAD4 and SAG101. Other genetic components of Arabidopsis post-invasion mesophyll resistance remain elusive.We performed comparative transcriptional profiling of wild-type, pen2 and pen2 pad4 sag101 mutants after inoculation with P. pachyrhizi to identify a novel trait for mesophyll NHR. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis and microscopic analysis confirmed the essential role of the candidate gene in mesophyll NHR.UDP-glucosyltransferase UGT84A2/bright trichomes 1 (BRT1) is a novel component of Arabidopsis mesophyll NHR to P. pachyrhizi. BRT1 is a putative cytoplasmic enzyme in phenylpropanoid metabolism. BRT1 is specifically induced in pen2 with post-invasion resistance to P. pachyrhizi. Silencing or mutation of BRT1 increased haustoria formation in pen2 mesophyll. Yet, the brt1 mutation did not affect NHR to P. pachyrhizi in wild-type plants.We assign a novel function to BRT1, which is important for post-invasion NHR of Arabidopsis to P. pachyrhizi. BRT1 might serve to confer durable resistance against P. pachyrhizi to soybean.

SummaryNonhost resistance (NHR) is the most prevalent form of plant immunity. In Arabidopsis, NHR requires membrane-localized ATP-binding cassette (ABC) transporter PENETRATION (PEN) 3. Upon perception of pathogen-associated molecular patterns, PEN3 becomes phosphorylated, suggestive of PEN3 regulation by post-translational modification. Here, we investigated the PEN3 protein interaction network.We probed the Arabidopsis protein microarray AtPMA-5000 with the N-terminal cytoplasmic domain of PEN3. Several of the proteins identified to interact with PEN3 in vitro represent cellular Ca 2+ sensors, including calmodulin (CaM) 3, CaM7 and several CaM-like proteins, pointing to the importance of Ca 2+ sensing to PEN3-mediated NHR. We demonstrated co-localization of PEN3 and CaM7, and we confirmed PEN3-CaM interaction in vitro and in vivo by PEN3 pull-down with CaM Sepharose, CaM overlay assay and bimolecular fluorescence complementation. We also show that just like in pen3, NHR to the nonadapted fungal pathogens Phakopsora pachyrhizi and Blumeria graminis f.sp. hordei is compromised in the Arabidopsis cam7 and pen3 cam7 mutants.Our study discloses CaM7 as a PEN3-interacting protein crucial to Arabidopsis NHR and emphasizes the importance of Ca 2+ sensing to plant immunity.