Natural genetic variation and negative density effects in plant-nematode interactions.

Arabidopsis thaliana is a suitable host for phytoparasitic nematodes of the genus Meloidogyne. Successful nematode infection leads to the formation of root galls. We tested for natural genetic variation and inoculation density effects on nematode reproductive success in the interaction between A. thaliana and Meloidogyne javanica. We inoculated different Arabidopsis genotypes with two sources of nematodes at two different doses, using a mild protocol for inoculum preparation. We counted root galls and egg masses 2 months after inoculation. We obtained a high number of successful nematode infections. Infection success differed among Arabidopsis genotypes in interaction with the nematode source. Overall, infection success and reproductive success of nematodes were lower at a higher inoculum dose of nematodes. Our results indicate that natural genetic variation in both host plants and nematodes, as well as short- and long-term negative density effects, shape nematode reproductive success.

Identifying and Isolating Meiotic Mutants in a Polyploid Brassica Crop.

This chapter provides a detailed description of TILLING and CRISPR-Cas9 approaches for the purpose of studying genes/factors involved in meiotic recombination in the polyploid species B. napus. The TILLING approach involves the screening and identification of EMS-mutagenized M2 B. napus plants. The strategy for high-throughput plant pooling, the set up for microfluidic PCR and sequencing is provided and the parameters for the analysis of sequence results and the detection of mutants are explained. The CRISPR-Cas system relies on the optimal design of guide RNAs and their efficient expression. The procedure for the generation and detection of knockout mutants is described with the aims to simultaneously target homologous genes.

Methyl Transfer in Glucosinolate Biosynthesis Mediated by Indole Glucosinolate O-Methyltransferase 5.

Indole glucosinolates (IGs) are plant secondary metabolites that are derived from the amino acid tryptophan. The product of Arabidopsis (Arabidopsis thaliana) IG core biosynthesis, indol-3-ylmethyl glucosinolate (I3M), can be modified by hydroxylation and subsequent methoxylation of the indole ring in position 1 (1-IG modification) or 4 (4-IG modification). Products of the 4-IG modification pathway mediate plant-enemy interactions and are particularly important for Arabidopsis innate immunity. While CYP81Fs encoding cytochrome P450 monooxygenases and IGMTs encoding indole glucosinolate O-methyltransferases have been identified as key genes for IG modification, our knowledge about the IG modification pathways is not complete. In particular, it is unknown which enzyme is responsible for methyl transfer in the 1-IG modification pathway and whether this pathway plays a role in defense, similar to 4-IG modification. Here, we analyze two Arabidopsis transfer DNA insertion lines with targeted metabolomics. We show that biosynthesis of 1-methoxyindol-3-ylmethyl glucosinolate (1MOI3M) from I3M involves the predicted unstable intermediate 1-hydroxyindol-3-ylmethyl glucosinolate (1OHI3M) and that IGMT5, a gene with moderate similarity to previously characterized IGMTs, encodes the methyltransferase that is responsible for the conversion of 1OHI3M to 1MOI3M. Disruption of IGMT5 function increases resistance against the root-knot nematode Meloidogyne javanica and suggests a potential role for the 1-IG modification pathway in Arabidopsis belowground defense.

The N-end rule pathway regulates pathogen responses in plants.

To efficiently counteract pathogens, plants rely on a complex set of immune responses that are tightly regulated to allow the timely activation, appropriate duration and adequate amplitude of defense programs. The coordination of the plant immune response is known to require the activity of the ubiquitin/proteasome system, which controls the stability of proteins in eukaryotes. Here, we demonstrate that the N-end rule pathway, a subset of the ubiquitin/proteasome system, regulates the defense against a wide range of bacterial and fungal pathogens in the model plant Arabidopsis thaliana. We show that this pathway positively regulates the biosynthesis of plant-defense metabolites such as glucosinolates, as well as the biosynthesis and response to the phytohormone jasmonic acid, which plays a key role in plant immunity. Our results also suggest that the arginylation branch of the N-end rule pathway regulates the timing and amplitude of the defense program against the model pathogen Pseudomonas syringae AvrRpm1.

Metabolic engineering in Nicotiana benthamiana reveals key enzyme functions in Arabidopsis indole glucosinolate modification.

Indole glucosinolates, derived from the amino acid Trp, are plant secondary metabolites that mediate numerous biological interactions between cruciferous plants and their natural enemies, such as herbivorous insects, pathogens, and other pests. While the genes and enzymes involved in the Arabidopsis thaliana core biosynthetic pathway, leading to indol-3-yl-methyl glucosinolate (I3M), have been identified and characterized, the genes and gene products responsible for modification reactions of the indole ring are largely unknown. Here, we combine the analysis of Arabidopsis mutant lines with a bioengineering approach to clarify which genes are involved in the remaining biosynthetic steps in indole glucosinolate modification. We engineered the indole glucosinolate biosynthesis pathway into Nicotiana benthamiana, showing that it is possible to produce indole glucosinolates in a noncruciferous plant. Building upon this setup, we demonstrate that all members of a small gene subfamily of cytochrome P450 monooxygenases, CYP81Fs, are capable of carrying out hydroxylation reactions of the glucosinolate indole ring, leading from I3M to 4-hydroxy-indol-3-yl-methyl and/or 1-hydroxy-indol-3-yl-methyl glucosinolate intermediates, and that these hydroxy intermediates are converted to 4-methoxy-indol-3-yl-methyl and 1-methoxy-indol-3-yl-methyl glucosinolates by either of two family 2 O-methyltransferases, termed indole glucosinolate methyltransferase 1 (IGMT1) and IGMT2.

The gene controlling the indole glucosinolate modifier1 quantitative trait locus alters indole glucosinolate structures and aphid resistance in Arabidopsis.

Glucosinolates are defensive secondary compounds that display large structural diversity in Arabidopsis thaliana and related plants. Much attention has been paid to variation in the biosynthesis of Met-derived aliphatic glucosinolates and its ecological consequences, but little is known about the genes that cause qualitative and quantitative differences in Trp-derived indole glucosinolates. We use a combination of quantitative trait locus (QTL) fine-mapping and microarray-based transcript profiling to identify CYP81F2 (At5g57220), encoding a cytochrome P450 monooxygenase, as the gene underlying Indole Glucosinolate Modifier1 (IGM1), a metabolic QTL for the accumulation of two modified indole glucosinolates, 4-hydroxy-indole-3-yl-methyl and 4-methoxy-indole-3-yl-methyl glucosinolate. We verify CYP81F2 function with two SALK T-DNA insertion lines and show that CYP81F2 catalyzes the conversion of indole-3-yl-methyl to 4-hydroxy-indole-3-yl-methyl glucosinolate. We further show that the IGM1 QTL is largely caused by differences in CYP81F2 expression, which results from a combination of cis- and trans-acting expression QTL different from known regulators of indole glucosinolate biosynthesis. Finally, we elucidate a potential function of CYP81F2 in plant-insect interactions and find that CYP81F2 contributes to defense against the green peach aphid (Myzus persicae) but not to resistance against herbivory by larvae from four lepidopteran species.

Teaching (and learning from) metabolomics: the 2006 PlantMetaNet ETNA Metabolomics Research School.

Under the auspices of the European Training and Networking Activity programme of the European Union, a 'Metabolic Profiling and Data Analysis' Plant Genomics and Bioinformatics Summer School was hosted in Potsdam, Germany between 20 and 29 September 2006. Sixteen early career researchers were invited from the European Union partner nations and the so-called developing nations (Appendix). Lectures from invited leading European researchers provided an overview of the state of the art of these fields and seeded discussion regarding major challenges for their future advancement. Hands-on experience was provided by an example experiment - that of defining the metabolic response of Arabidopsis to treatment of a commercial herbicide of defined mode of action. This experiment was performed throughout the duration of the course in order to teach the concepts underlying extraction and machine handling as well as to provide a rich data set with which the required computation and statistical skills could be illustrated. Here we review the state of the field by describing both key lectures given at and practical aspects taught at the summer school. In addition, we disclose results that were obtained using the four distinct technical platforms at the different participating institutes. While the effects of the chosen herbicide are well documented, this study looks at a broader number of metabolites than in previous investigations. This allowed, on the one hand, not only to characterise further effects of the herbicide than previously observed but also to detect molecules other than the herbicide that were obviously present in the commercial formulation. These data and the workshop in general are all discussed in the context of the teaching of metabolomics.

Mapping of QTL for resistance against the crucifer specialist herbivore Pieris brassicae in a new Arabidopsis inbred line population, Da(1)-12 x Ei-2.

BACKGROUND: In Arabidopsis thaliana and other crucifers, the glucosinolate-myrosinase system contributes to resistance against herbivory by generalist insects. As yet, it is unclear how crucifers defend themselves against crucifer-specialist insect herbivores. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed natural variation for resistance against two crucifer specialist lepidopteran herbivores, Pieris brassicae and Plutella xylostella, among Arabidopsis thaliana accessions and in a new Arabidopsis recombinant inbred line (RIL) population generated from the parental accessions Da(1)-12 and Ei-2. This RIL population consists of 201 individual F(8) lines genotyped with 84 PCR-based markers. We identified six QTL for resistance against Pieris herbivory, but found only one weak QTL for Plutella resistance. To elucidate potential factors causing these resistance QTL, we investigated leaf hair (trichome) density, glucosinolates and myrosinase activity, traits known to influence herbivory by generalist insects. We identified several previously unknown QTL for these traits, some of which display a complex pattern of epistatic interactions. CONCLUSIONS/SIGNIFICANCE: Although some trichome, glucosinolate or myrosinase QTL co-localize with Pieris QTL, none of these traits explained the resistance QTL convincingly, indicating that resistance against specialist insect herbivores is influenced by other traits than resistance against generalists.