Mining the Plant-Herbivore Interface with a Leafmining Drosophila of Arabidopsis

Experimental infections of Arabidopsis thaliana (Arabidopsis) with genomically characterized plant pathogens such as Pseudomonas syringae have facilitated the dissection of canonical eukaryotic defence pathways and parasite virulence factors. Plants are also attacked by herbivorous insects, and the development of an ecologically relevant genetic model herbivore that feeds on Arabidopsis will enable the parallel dissection of host defence and reciprocal resistance pathways such as those involved in xenobiotic metabolism. An ideal candidate is Scaptomyza flava, a drosophilid fly whose leafmining larvae are true herbivores that can be found in nature feeding on Arabidopsis and other crucifers. Here, we describe the life cycle of S. flava on Arabidopsis and use multiple approaches to characterize the response of Arabidopsis to S. flava attack. Oviposition choice tests and growth performance assays on different Arabidopsis ecotypes, defence-related mutants, and hormone and chitin-treated plants revealed significant differences in host preference and variation in larval performance across Arabidopsis accessions. The jasmonate and glucosinolate pathways in Arabidopsis are important in mediating quantitative resistance against S. flava, and priming with jasmonate or chitin resulted in increased resistance. Expression of xenobiotic detoxification genes was reduced in S. flava larvae reared on Arabidopsis jasmonate signalling mutants and increased in plants pretreated with chitin. These results and future research directions are discussed in the context of developing a genetic model system to analyse insect–plant interactions.Organismic and Evolutionary Biolog

Activation of Arabidopsis thalianad defense response pathways by oligogalacturonides and Flagellin

Arabidopsis thaliana perçoit au travers de “pathogen- or microbe- associated molecular patterns” (PAMPs/MAMPs) l’attaque d’organismes pathogènes. De nombreuses études ont révélé des éléments communs dans les réponses de défense induites par les éliciteurs, mais les similarités et différences entre les voies de signalisation qu’ils induisent restent encore mal connues. Les oligogalacturonates (OGs) sont des éliciteurs dérivant de la paroi des plantes qui induisent une large variété de réponse de défenses. Le transcriptome de plantules traitées par les OGs révèle une réponse transitoire de nombreux gènes, notamment une réponse transitoire et précoce de deux cytochromes P450 codant pour CYP81F2 et CYP82C3, Une analyse de l’expression de ces gènes dans divers mutants de défense suggère que leur réponse rapide à cet éliciteur est indépendante des voies de signalisation du salicylate (SA), du jasmonate (JA) ou de l’éthylène (Et). Ces gènes utilisés comme rapporteurs de la réponse au OGs, sont aussi induits par d’autre MAMPs, notamment les lipopolysaccharides, la flagelline (Flg22), et la chitine. Une analyse additionnelle du transcriptome a été menée avec des plantules traitées par deux éliciteurs très différents, les OGs, polysaccharides provenant de l’hôte, et Flg22, un peptide de synthèse dérivé de la flagelline bactérienne. Ces deux éliciteurs déclanchent des réponses similaires, rapides et transitoires. Elles sont caractérisées par l’activation précoce de voies de signalisation multiples, associées au JA en particulier. Cependant l’activation par Flg22 est plus forte, il induit un plus grand nombre de gènes. L’amplitude de l’activation est dose-dépendante dans les deux cas, mais, même aux plus fortes concentrations, les OGs n’induisent pas autant de gènes que Flg22. De plus, une activation des processus de sénescence, des voies de sécrétion SA-dépendante et de l’expression la protéine PR1 ne sont observés qu’en réponse à Flg22. CYP81F2 participe à la réponse induite précoce aux eliciteurs (OGs and Flg22). Son expression est indépendante de SA, de JA et de l’éthylène. CYP81F2 semble coder pour une indole glucosinolate 4-hydroxylase requises pour la formation de callose en réponse à Flg22.Pathogen attacks are perceived in Arabidopsis thaliana through recognition of pathogen- or microbe- associated molecular patterns (PAMPs/MAMPs). Although the study of various elicitors has revealed significant overlaps in defense response, the degrees of similarity/difference between MAMPs are not well defined. Oligogalacturonides (OGs), plant cell wall-derived elicitors, induce a wide range of defenses responses. Transcript profiling of Arabidopsis seedlings treated with OGs indicates that the response to OGs involves a transient response in the regulation of many genes. Among the genes, two cytochrome P450s, CYP81F2 and CYP82C3, are significantly induced shortly after OGs treatment. Monitoring the expression of these genes in a variety of defense-related mutants suggests that their rapid induction, mediated by OGs is independent of SA, JA, or Et signaling pathways. These reporter genes are also highly expressed in response to other MAMPs, including lipopolysaccharide (LPS), flagellin (Flg22), or chitin. Additional transcriptional analysis was carried out with OGs and pathogensynthesized flagellin (Flg22), two very different elicitors. Both triggered a fast and transient response that are similar. This response is characterized by activation of the early stages of multiple defense signaling pathways, particularly JA-associated processes. However, the response to Flg22 is stronger in the number of genes differentially expressed and the amplitude of change. The magnitude of genes induction was in both cases dose-dependent, but, even at very high concentrations, OGs did not induce as many genes as Flg22. Moreover, activation of senescence processes, SA-dependent secretory pathway genes, and PR1 expression was only observed with Flg22 elicitation. These results suggest a lower threshold for activation of early responses than for sustained late innate immune defenses. Induction of the Arabidopsis CYP81F2 gene is part of the early induced response to elicitors (OGs and Flg22). CYP81F2 gene expression is independent of the SA-, JAand Et-signaling pathways. CYP81F2 seems to catalyze the 4 methoxylation of indolic glucosinolates, which is required for callose formation in response to Flg22

Activation des voies de défense chez Arabidopsis thaliana par les oligogalacturonides et la flagelline

Arabidopsis thaliana perçoit au travers de pathogen- or microbe- associated molecular patterns (PAMPs/MAMPs) l'attaque d'organismes pathogènes. De nombreuses études ont révélé des éléments communs dans les réponses de défense induites par les éliciteurs, mais les similarités et différences entre les voies de signalisation qu ils induisent restent encore mal connues. Les oligogalacturonates (OGs) sont des éliciteurs dérivant de la paroi des plantes qui induisent une large variété de réponse de défenses. Le transcriptome de plantules traitées par les OGs révèle une réponse transitoire de nombreux gènes, notamment une réponse transitoire et précoce de deux cytochromes P450 codant pour CYP81F2 et CYP82C3, Une analyse de l'expression de ces gènes dans divers mutants de défense suggère que leur réponse rapide à cet éliciteur est indépendante des voies de signalisation du salicylate (SA), du jasmonate (JA) ou de l'éthylène (Et). Ces gènes utilisés comme rapporteurs de la réponse au OGs, sont aussi induits par d'autre MAMPs, notamment les lipopolysaccharides, la flagelline (Flg22), et la chitine. Une analyse additionnelle du transcriptome a été menée avec des plantules traitées par deux éliciteurs très différents, les OGs, polysaccharides provenant de l'hôte, et Flg22, un peptide de synthèse dérivé de la flagelline bactérienne. Ces deux éliciteurs déclanchent des réponses similaires, rapides et transitoires. Elles sont caractérisées par l'activation précoce de voies de signalisation multiples, associées au JA en particulier. Cependant l'activation par Flg22 est plus forte, il induit un plus grand nombre de gènes. L'amplitude de l'activation est dose-dépendante dans les deux cas, mais, même aux plus fortes concentrations, les OGs n induisent pas autant de gènes que Flg22. De plus, une activation des processus de sénescence, des voies de sécrétion SA-dépendante et de l'expression la protéine PR1 ne sont observés qu en réponse à Flg22. CYP81F2 participe à la réponse induite précoce aux eliciteurs (OGs and Flg22). Son expression est indépendante de SA, de JA et de l'éthylène. CYP81F2 semble coder pour une indole glucosinolate 4-hydroxylase requises pour la formation de callose en réponse à Flg22.Pathogen attacks are perceived in Arabidopsis thaliana through recognition of pathogen- or microbe- associated molecular patterns (PAMPs/MAMPs). Although the study of various elicitors has revealed significant overlaps in defense response, the degrees of similarity/difference between MAMPs are not well defined. Oligogalacturonides (OGs), plant cell wall-derived elicitors, induce a wide range of defenses responses. Transcript profiling of Arabidopsis seedlings treated with OGs indicates that the response to OGs involves a transient response in the regulation of many genes. Among the genes, two cytochrome P450s, CYP81F2 and CYP82C3, are significantly induced shortly after OGs treatment. Monitoring the expression of these genes in a variety of defense-related mutants suggests that their rapid induction, mediated by OGs is independent of SA, JA, or Et signaling pathways. These reporter genes are also highly expressed in response to other MAMPs, including lipopolysaccharide (LPS), flagellin (Flg22), or chitin. Additional transcriptional analysis was carried out with OGs and pathogensynthesized flagellin (Flg22), two very different elicitors. Both triggered a fast and transient response that are similar. This response is characterized by activation of the early stages of multiple defense signaling pathways, particularly JA-associated processes. However, the response to Flg22 is stronger in the number of genes differentially expressed and the amplitude of change. The magnitude of genes induction was in both cases dose-dependent, but, even at very high concentrations, OGs did not induce as many genes as Flg22. Moreover, activation of senescence processes, SA-dependent secretory pathway genes, and PR1 expression was only observed with Flg22 elicitation. These results suggest a lower threshold for activation of early responses than for sustained late innate immune defenses. Induction of the Arabidopsis CYP81F2 gene is part of the early induced response to elicitors (OGs and Flg22). CYP81F2 gene expression is independent of the SA-, JAand Et-signaling pathways. CYP81F2 seems to catalyze the 4 methoxylation of indolic glucosinolates, which is required for callose formation in response to Flg22

Activation des voies de défense chez Arabidopsis thaliana par les oligogalacturonides et la flagelline

Arabidopsis thaliana perçoit au travers de pathogen- or microbe- associated molecular patterns (PAMPs/MAMPs) l'attaque d'organismes pathogènes. De nombreuses études ont révélé des éléments communs dans les réponses de défense induites par les éliciteursPathogen attacks are perceived in Arabidopsis thaliana through recognition of pathogen- or microbe- associated molecular patterns (PAMPs/MAMPs). Although the study of various elicitors has revealed significant overlaps in defense response, the degrees o

Resistance to Botrytis cinerea Induced in Arabidopsis by Elicitors Is Independent of Salicylic Acid, Ethylene, or Jasmonate Signaling But Requires PHYTOALEXIN DEFICIENT31[W]

Oligogalacturonides (OGs) released from plant cell walls by pathogen polygalacturonases induce a variety of host defense responses. Here we show that in Arabidopsis (Arabidopsis thaliana), OGs increase resistance to the necrotrophic fungal pathogen Botrytis cinerea independently of jasmonate (JA)-, salicylic acid (SA)-, and ethylene (ET)-mediated signaling. Microarray analysis showed that about 50% of the genes regulated by OGs, including genes encoding enzymes involved in secondary metabolism, show a similar change of expression during B. cinerea infection. In particular, expression of PHYTOALEXIN DEFICIENT3 (PAD3) is strongly up-regulated by both OGs and infection independently of SA, JA, and ET. OG treatments do not enhance resistance to B. cinerea in the pad3 mutant or in underinducer after pathogen and stress1, a mutant with severely impaired PAD3 expression in response to OGs. Similarly to OGs, the bacterial flagellin peptide elicitor flg22 also enhanced resistance to B. cinerea in a PAD3-dependent manner, independently of SA, JA, and ET. This work suggests, therefore, that elicitors released from the cell wall during pathogen infection contribute to basal resistance against fungal pathogens through a signaling pathway also activated by pathogen-associated molecular pattern molecules

The AtrbohD-Mediated Oxidative Burst Elicited by Oligogalacturonides in Arabidopsis Is Dispensable for the Activation of Defense Responses Effective against Botrytis cinerea1[W][OA]

Oligogalacturonides (OGs) are endogenous elicitors of defense responses released after partial degradation of pectin in the plant cell wall. We have previously shown that, in Arabidopsis (Arabidopsis thaliana), OGs induce the expression of PHYTOALEXIN DEFICIENT3 (PAD3) and increase resistance to the necrotrophic fungal pathogen Botrytis cinerea independently of signaling pathways mediated by jasmonate, salicylic acid, and ethylene. Here, we illustrate that the rapid induction of the expression of a variety of genes by OGs is also independent of salicylic acid, ethylene, and jasmonate. OGs elicit a robust extracellular oxidative burst that is generated by the NADPH oxidase AtrbohD. This burst is not required for the expression of OG-responsive genes or for OG-induced resistance to B. cinerea, whereas callose accumulation requires a functional AtrbohD. OG-induced resistance to B. cinerea is also unaffected in powdery mildew resistant4, despite the fact that callose accumulation was almost abolished in this mutant. These results indicate that the OG-induced oxidative burst is not required for the activation of defense responses effective against B. cinerea, leaving open the question of the role of reactive oxygen species in elicitor-mediated defense

A High-Performance, Small-Scale Microarray for Expression Profiling of Many Samples in Arabidopsis-Pathogen Studies

Studies of the behavior of biological systems often require monitoring of the expression of many genes in a large number of samples. While whole-genome arrays provide high-quality gene-expression profiles, their high cost generally limits the number of samples that can be studied. Although inexpensive small-scale arrays representing genes of interest could be used for many applications, it is challenging to obtain accurate measurements with conventional small-scale microarrays. We have developed a small-scale microarray system that yields highly accurate and reproducible expression measurements. This was achieved by implementing a stable gene-based quantile normalization method for array-to-array normalization, and a probe-printing design that allows use of a statistical model to correct for effects of print tips and uneven hybridization. The array measures expression values in a single sample, rather than ratios between two samples. This allows accurate comparisons among many samples. The array typically yielded correlation coefficients higher than 0.99 between technically duplicated samples. Accuracy was demonstrated by a correlation coefficient of 0.88 between expression ratios determined from this array and an Affymetrix GeneChip, by quantitative RT-PCR, and by spiking known amounts of specific RNAs into the RNA samples used for profiling. The array was used to compare the responses of wild-type, rps2 and ndr1 mutant plants to infection by a Pseudomonas syringae strain expressing avrRpt2. The results suggest that ndr1 affects a defense-signaling pathway(s) in addition to the RPS2-dependent pathway, and indicate that the microarray is a powerful tool for systems analyses of the Arabidopsis disease-signaling network