Polymorphic residues in rice NLRs expand binding and response to effectors of the blast pathogen

Accelerated adaptive evolution is a hallmark of plant-pathogen interactions. Plant intracellular immune receptors (NLRs) often occur as allelic series with differential pathogen specificities. The determinants of this specificity remain largely unknown. Here, we unravelled the biophysical and structural basis of expanded specificity in the allelic rice NLR Pik, which responds to the effector AVR-Pik from the rice blast pathogen Magnaporthe oryzae. Rice plants expressing the Pikm allele resist infection by blast strains expressing any of three AVR-Pik effector variants, whereas those expressing Pikp only respond to one. Unlike Pikp, the integrated heavy metal-associated (HMA) domain of Pikm binds with high affinity to each of the three recognized effector variants, and variation at binding interfaces between effectors and Pikp-HMA or Pikm-HMA domains encodes specificity. By understanding how co-evolution has shaped the response profile of an allelic NLR, we highlight how natural selection drove the emergence of new receptor specificities. This work has implications for the engineering of NLRs with improved utility in agriculture

Natural Variation in Partial Resistance to Pseudomonas syringae Is Controlled by Two Major QTLs in Arabidopsis thaliana

BACKGROUND: Low-level, partial resistance is pre-eminent in natural populations, however, the mechanisms underlying this form of resistance are still poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we used the model pathosystem Pseudomonas syringae pv. tomato DC3000 (Pst) - Arabidopsis thaliana to study the genetic basis of this form of resistance. Phenotypic analysis of a set of Arabidopsis accessions, based on evaluation of in planta pathogen growth revealed extensive quantitative variation for partial resistance to Pst. It allowed choosing a recombinant inbred line (RIL) population derived from a cross between the accessions Bayreuth and Shahdara for quantitative genetic analysis. Experiments performed under two different environmental conditions led to the detection of two major and two minor quantitative trait loci (QTLs) governing partial resistance to Pst and called PRP-Ps1 to PRP-Ps4. The two major QTLs, PRP-Ps1 and PRP-Ps2, were confirmed in near isogenic lines (NILs), following the heterogeneous inbred families (HIFs) strategy. Analysis of marker gene expression using these HIFs indicated a negative correlation between the induced amount of transcripts of SA-dependent genes PR1, ICS and PR5, and the in planta bacterial growth in the HIF segregating at PRP-Ps2 locus, suggesting an implication of PRP-Ps2 in the activation of SA dependent responses. CONCLUSIONS/SIGNIFICANCE: These results show that variation in partial resistance to Pst in Arabidopsis is governed by relatively few loci, and the validation of two major loci opens the way for their fine mapping and their cloning, which will improve our understanding of the molecular mechanisms underlying partial resistance

Gene coexpression clusters and putative regulatory elements underlying seed storage reserve accumulation in Arabidopsis

Abstract Background In Arabidopsis, a large number of genes involved in the accumulation of seed storage reserves during seed development have been characterized, but the relationship of gene expression and regulation underlying this physiological process remains poorly understood. A more holistic view of this molecular interplay will help in the further study of the regulatory mechanisms controlling seed storage compound accumulation. Results We identified gene coexpression networks in the transcriptome of developing Arabidopsis (Arabidopsis thaliana) seeds from the globular to mature embryo stages by analyzing publicly accessible microarray datasets. Genes encoding the known enzymes in the fatty acid biosynthesis pathway were found in one coexpression subnetwork (or cluster), while genes encoding oleosins and seed storage proteins were identified in another subnetwork with a distinct expression profile. In the triacylglycerol assembly pathway, only the genes encoding diacylglycerol acyltransferase 1 (DGAT1) and a putative cytosolic "type 3" DGAT exhibited a similar expression pattern with genes encoding oleosins. We also detected a large number of putative cis-acting regulatory elements in the promoter regions of these genes, and promoter motifs for LEC1 (LEAFY COTYLEDON 1), DOF (DNA-binding-with-One-Finger), GATA, and MYB transcription factors (TF), as well as SORLIP5 (Sequences Over-Represented in Light-Induced Promoters 5), are overrepresented in the promoter regions of fatty acid biosynthetic genes. The conserved CCAAT motifs for B3-domain TFs and binding sites for bZIP (basic-leucine zipper) TFs are enriched in the promoters of genes encoding oleosins and seed storage proteins. Conclusions Genes involved in the accumulation of seed storage reserves are expressed in distinct patterns and regulated by different TFs. The gene coexpression clusters and putative regulatory elements presented here provide a useful resource for further experimental characterization of protein interactions and regulatory networks in this process.</p

The rice NLR pair Pikp-1/Pikp-2 initiates cell death through receptor cooperation rather than negative regulation

Plant NLR immune receptors are multidomain proteins that can function as specialized sensor/helper pairs. Paired NLR immune receptors are generally thought to function via negative regulation, where one NLR represses the activity of the second and detection of pathogen effectors relieves this repression to initiate immunity. However, whether this mechanism is common to all NLR pairs is not known. Here, we show that the rice NLR pair Pikp-1/Pikp-2, which confers resistance to strains of the blast pathogen Magnaporthe oryzae (syn. Pyricularia oryzae) expressing the AVR-PikD effector, functions via receptor cooperation, with effector-triggered activation requiring both NLRs to trigger the immune response. To investigate the mechanism of Pikp-1/Pikp-2 activation, we expressed truncated variants of these proteins, and made mutations in previously identified NLR sequence motifs. We found that any domain truncation, in either Pikp-1 or Pikp-2, prevented cell death in the presence of AVR-PikD, revealing that all domains are required for activity. Further, expression of individual Pikp-1 or Pikp-2 domains did not result in cell death. Mutations in the conserved P-loop and MHD sequence motifs in both Pikp-1 and Pikp-2 prevented cell death activation, demonstrating that these motifs are required for the function of the two partner NLRs. Finally, we showed that Pikp-1 and Pikp-2 associate to form homo- and hetero-complexes in planta in the absence of AVR-PikD; on co-expression the effector binds to Pikp-1 generating a tri-partite complex. Taken together, we provide evidence that Pikp-1 and Pikp-2 form a fine-tuned system that is activated by AVR-PikD via receptor cooperation rather than negative regulation

bZIP transcription factors in Arabidopsis

Jakoby M, Weisshaar B, Dröge-Laser W, et al. bZIP transcription factors in Arabidopsis. Trends in Plant Science. 2002;7(3):106-111.In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate processes including pathogen defence, light and stress signalling, seed maturation and flower development. The Arabidopsis genome sequence contains 75 distinct members of the bZIP family, of which approximately 50 are not described in the literature. Using common domains, the AtbZIP family can be subdivided into ten groups. Here, we review the available data on bZIP functions in the context of subgroup membership and discuss the interacting proteins. This integration is essential for a complete functional characterization of bZIP transcription factors in plants, and to identify functional redundancies among AtbZIP factors

The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance

BGPI : Equipe 4International audiencePlant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo-and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor

AKRP and EMB506 are two ankyrin repeat proteins essential for plastid differentiation and plant development in Arabidopsis.

International audienceEMB506 is a chloroplast protein essential for embryo development, the function of which is unknown. A two-hybrid interaction screen was performed to provide insight into the role of EMB506. A single interacting partner, AKRP, was identified among a cDNA library from immature siliques. The AKR gene (Zhang et al., 1992, Plant Cell 4, 1575-1588) encodes a protein containing five ankyrin repeats, very similar to EMB506. Protein truncation series demonstrated that both proteins interact through their ankyrin domains. Using reverse genetics, we showed that loss of akr function resulted in an embryo-defective (emb) phenotype indistinguishable from the emb506 phenotype. Transient expression of the signal peptide of AKRP fused to green fluorescent protein demonstrated the chloroplast localization of AKRP. The ABI3 promoter was used to express AKR in a seed-specific manner in order to analyse the post-embryonic effect of AKR loss of function in akr/akr seedlings. Homozygous fertile and viable akr/akr plants were obtained. These plants exhibited mild to severe defects in chloroplast and leaf cellular organization. We conclude that EMB506 and AKRP are involved in crucial and tightly controlled events in plastid differentiation linked to cell differentiation, morphogenesis and organogenesis during the plant life cycle