RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome.

Transcription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance (R) genes. For example, the pepper Bs3 and rice Xa27 genes are hypersensitive reaction plant R genes that are transcriptionally activated by corresponding TALEs. Both R genes have a hallmark expression pattern in which their transcripts are detectable only in the presence and not the absence of the corresponding TALE. By transcriptome profiling using next-generation sequencing (RNA-seq), we tested whether we could avoid laborious positional cloning for the isolation of TALE-induced R genes. In a proof-of-principle experiment, RNA-seq was used to identify a candidate for Bs4C, an R gene from pepper that mediates recognition of the Xanthomonas TALE protein AvrBs4. We identified one major Bs4C candidate transcript by RNA-seq that was expressed exclusively in the presence of AvrBs4. Complementation studies confirmed that the candidate corresponds to the Bs4C gene and that an AvrBs4 binding site in the Bs4C promoter directs its transcriptional activation. Comparison of Bs4C with a nonfunctional allele that is unable to recognize AvrBs4 revealed a 2-bp polymorphism within the TALE binding site of the Bs4C promoter. Bs4C encodes a structurally unique R protein and Bs4C-like genes that are present in many solanaceous genomes seem to be as tightly regulated as pepper Bs4C. These findings demonstrate that TALE-specific R genes can be cloned from large-genome crops with a highly efficient RNA-seq approach.

Sequence-based SNP genotyping in durum wheat.

Marker development for marker-assisted selection in plant breeding is increasingly based on next-generation sequencing (NGS). However, marker development in crops with highly repetitive, complex genomes is still challenging. Here we applied sequence-based genotyping (SBG), which couples AFLP®-based complexity reduction to NGS, for de novo single nucleotide polymorphisms (SNP) marker discovery in and genotyping of a biparental durum wheat population. We identified 9983 putative SNPs in 6372 contigs between the two parents and used these SNPs for genotyping 91 recombinant inbred lines (RILs). Excluding redundant information from multiple SNPs per contig, 2606 (41%) markers were used for integration in a pre-existing framework map, resulting in the integration of 2365 markers over 2607 cM. Of the 2606 markers available for mapping, 91% were integrated in the pre-existing map, containing 708 SSRs, DArT markers, and SNPs from CRoPS technology, with a map-size increase of 492 cM (23%). These results demonstrate the high quality of the discovered SNP markers. With this methodology, it was possible to saturate the map at a final marker density of 0.8 cM/marker. Looking at the binned marker distribution (Figure 2), 63 of the 268 10-cM bins contained only SBG markers, showing that these markers are filling in gaps in the framework map. As to the markers that could not be used for mapping, the main reason was the low sequencing coverage used for genotyping. We conclude that SBG is a valuable tool for efficient, high-throughput and high-quality marker discovery and genotyping for complex genomes such as that of durum wheat.

Multidisciplinary approach to unravelling the relative contribution of different oxylipins in indirect defense of Arabidopsis thaliana.

The oxylipin pathway is commonly involved in induced plant defenses, and is the main signal-transduction pathway induced by insect folivory. Herbivory induces the production of several oxylipins, and consequently alters the so-called 'oxylipin signature' in the plant. Jasmonic acid (JA), as well as pathway intermediates are known to induce plant defenses. Indirect defense against herbivorous insects comprises the production of herbivore-induced plant volatiles (HIPVs). To unravel the precise oxylipin signal-transduction underlying the production of HIPVs in Arabidopsis thaliana and the resulting attraction of parasitoid wasps, we used a multidisciplinary approach that includes molecular genetics, metabolite analysis, and behavioral analysis. Mutant plants affected in the jasmonate pathway (18:0 and/or 16:0 -oxylipin routes; mutants dde2-2, fad5, opr3) were studied to assess the effects of JA and its oxylipin intermediates 12-oxo-phytodienoate (OPDA) and dinor-OPDA (dnOPDA) on HIPV emission and parasitoid (Diadegma semiclausum) attraction. Interference with the production of the oxylipins JA and OPDA altered the emission of HIPVs, in particular terpenoids and the phenylpropanoid methyl salicylate, which affected parasitoid attraction. Our data show that the herbivore-induced attraction of parasitoid wasps to Arabidopsis plants depends on HIPVs that are induced through the 18:0 oxylipin-derivative JA. Furthermore, our study shows that the 16:0-oxylipin route towards dnOPDA does not play a role in HIPV induction, and that the role of 18:0 derived oxylipin-intermediates, such as OPDA, is either absent or limited.

Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack.

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium (Pseudomonas syringae pv. tomato), a pathogenic leaf fungus (Alternaria brassicicola), tissue-chewing caterpillars (Pieris rapae), cell-content-feeding thrips (Frankliniella occidentalis), or phloem-feeding aphids (Myzus persicae). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. Analysis of global gene expression profiles demonstrated that the signal signature characteristic of each Arabidopsis-attacker combination is orchestrated into a surprisingly complex set of transcriptional alterations in which, in all cases, stress-related genes are overrepresented. Comparison of the transcript profiles revealed that consistent changes induced by pathogens and insects with very different modes of attack can show considerable overlap. Of all consistent changes induced by A. brassicicola, Pieris rapae, and E occidentalis, more than 50% also were induced consistently by P. syringae. Notably, although these four attackers all stimulated JA biosynthesis, the majority of the changes in JA-responsive gene expression were attacker specific. All together, our study shows that SA, JA, and ET play a primary role in the orchestration of the plant's defense response, but other regulatory mechanisms, such as pathway cross-talk or additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response.

Natural variation in RPS2-mediated resistance among Arabidopsis accessions: correlation between gene expression profiles and phenotypic responses.

Natural variation in gene expression (expression traits or e-traits) is increasingly used for the discovery of genes controlling traits. An important question is whether a particular e-trait is correlated with a phenotypic trait. Here, we examined the correlations between phenotypic traits and e-traits among 10 Arabidopsis thaliana accessions. We studied defense against Pseudomonas syringae pv tomato DC3000 (Pst), with a focus on resistance gene-mediated resistance triggered by the type III effector protein AvrRpt2. As phenotypic traits, we measured growth of the bacteria and extent of the hypersensitive response (HR) as measured by electrolyte leakage. Genetic variation among accessions affected growth of Pst both with (Pst avrRpt2) and without (Pst) the AvrRpt2 effector. Variation in HR was not correlated with variation in bacterial growth. We also collected gene expression profiles 6 h after mock and Pst avrRpt2 inoculation using a custom microarray. Clusters of genes whose expression levels are correlated with bacterial growth or electrolyte leakage were identified. Thus, we demonstrated that variation in gene expression profiles of Arabidopsis accessions collected at one time point under one experimental condition has the power to explain variation in phenotypic responses to pathogen attack.

Induced parasitoid attraction by Arabidopsis thaliana: involvement of the octadecanoid and the salicylic acid pathway.

Plants can use indirect defence mechanisms to protect themselves against herbivorous insects. An example of such an indirect defence mechanism is the emission of volatiles by plants induced by herbivore feeding. These volatiles can attract the natural enemies of these herbivores, for example, parasitoid wasps. Here, it is shown that the octadecanoid and the salicylic acid pathways are involved in the induced attraction of the parasitoid wasp Cotesia rubecula by Arabidopsis thaliana infested with the herbivore Pieris rapae. Besides exogenous application of jasmonic acid or salicylic acid, use is also made of transgenic Arabidopsis that do not show induced jasmonic acid levels after wounding (S-12) and transgenic Arabidopsis that do not accumulate salicylic acid (NahG). Treatment of Arabidopsis with jasmonic acid resulted in an increased attraction of parasitoid wasps compared with untreated plants, whereas treatment with salicylic acid did not. Transgenic plants impaired in the octadecanoid or the salicylic acid pathway were less attractive than wild-type plants.

A conserved transcript pattern in response to a specialist and a generalist herbivore.

Transcript patterns elicited in response to attack reveal, at the molecular level, how plants respond to aggressors. These patterns are fashioned both by inflicted physical damage as well as by biological components displayed or released by the attacker. Different types of attacking organisms might therefore be expected to elicit different transcription programs in the host. Using a large-scale DNA microarray, we characterized gene expression in damaged as well as in distal Arabidopsis thaliana leaves in response to the specialist insect, Pieris rapae. More than 100 insect-responsive genes potentially involved in defense were identified, including genes involved in pathogenesis, indole glucosinolate metabolism, detoxification and cell survival, and signal transduction. Of these 114 genes, 111 were induced in Pieris feeding, and only three were repressed. Expression patterns in distal leaves were markedly similar to those of local leaves. Analysis of wild-type and jasmonate mutant plants, coupled with jasmonate treatment, showed that between 67 and 84% of Pieris-regulated gene expression was controlled, totally or in part, by the jasmonate pathway. This was correlated with increased larval performance on the coronatine insensitive1 glabrous1 (coi1-1 gl1) mutant. Independent mutations in COI1 and GL1 led to a faster larval weight gain, but the gl1 mutation had relatively little effect on the expression of the insect-responsive genes examined. Finally, we compared transcript patterns in Arabidopis in response to larvae of the specialist P. rapae and to a generalist insect, Spodoptera littoralis. Surprisingly, given the complex nature of insect salivary components and reported differences between species, almost identical transcript profiles were observed. This study also provides a robustly characterized gene set for the further investigation of plant-insect interaction.