Characterization of Transcription Factors Following Expression Profiling of Medicago truncatula-Botrytis spp. Interactions

Medicago truncatula may be used as a model plant to study the pathosystem Vicia faba/Botrytis spp. This study aims to investigate the profiling of the transcription factors (TFs) of M. truncatula involved in the interaction with Botrytis cinerea and Botrytis fabae. The response of two genotypes of M. truncatula to the pathogens was first established, defining genotype A17 as partially resistant and genotype Esp162 as susceptible. We found that B. fabae is more aggressive as a pathogen of M. truncatula than B. cinerea. The profiling of the TFs involved in the interaction was subsequently carried out using an available genome guide qPCR-based platform for the quantitative measurement of 1,084 M. truncatula TFs. Microscopic studies showed that the infection process had started 24 h after inoculation. A total of 126 TFs showed significant differences in their expression after inoculation irrespective of the genotype, while 37 TFs were differentially expressed between the resistant and the susceptible genotypes and 70 TFs showed different levels of expression between the genotypes unaltered by infection. This has allowed characterization at the transcriptional regulation level of the differential response of two genotypes of M. truncatula to two very similar pathogens differing in their aggressiveness. These results will be valuable to increase the functional knowledge of the M. truncatula genome and to help in breeding programmes of V. faba for resistance to Botrytis spp.Peer Reviewe

Transcription factor profiling leading to the identification of putative transcription factors involved in the Medicago truncatula-Uromyces striatus interaction

Understanding the host response to Uromyces sp., the causal agent of rust in many crop species, is crucial in elucidating the specific biology of rust resistance. In an attempt to unravel the Medicago truncatula-U. striatus interaction, we performed a global analysis of transcription factor (TF) expression in resistant and susceptible accessions of the model plant M. truncatula during infection with U. striatus. For this purpose, an established qPCR platform was applied, consisting of specific primer pairs for more than 1,000 predicted TF genes. A total of 107 putative TF genes out of the 1,084 studied were differentially expressed. Thirteen of the TFs that were differentially expressed between resistant and susceptible genotypes are known to be relevant in cellular defense. These data suggest that resistance could be mediated both by genes that are constitutively expressed and by genes, which are activated/repressed when plants are inoculated. These defense related TFs sequences were amplified in chickpea DNA with the aim of determining the location of these genes on the genetic map of this crop and identifying possible DNA regions involved in resistance mechanisms. © 2010 Springer-Verlag.Financial support by FP6-2002-FOOD-1-506223 European Union Integrated Project and by Spanish projects AGL2005-07497-CO2-01/AGR and AGL2008-01239 is acknowledged.Peer Reviewe

Single-Cell Analysis of Multiple Steps of Dynamic NF-κB Regulation in Interleukin-1α-Triggered Tumor Cells Using Proximity Ligation Assays

The frequently occurring heterogeneity of cancer cells and their functional interaction with immune cells in the tumor microenvironment raises the need to study signaling pathways at the single cell level with high precision, sensitivity, and spatial resolution. As aberrant NF-κB activity has been implicated in almost all steps of cancer development, we analyzed the dynamic regulation and activation status of the canonical NF-κB pathway in control and IL-1α-stimulated individual cells using proximity ligation assays (PLAs). These systematic experiments allowed the visualization of the dynamic dissociation and re-formation of endogenous p65/IκBα complexes and the nuclear translocation of NF-κB p50/p65 dimers. PLA combined with immunostaining for p65 or with NFKBIA single molecule mRNA-FISH facilitated the analysis of (i) further levels of the NF-κB pathway, (i) its functionality for downstream gene expression, and (iii) the heterogeneity of the NF-κB response in individual cells. PLA also revealed the interaction between NF-κB p65 and the P-body component DCP1a, a new p65 interactor that contributes to efficient p65 NF-κB nuclear translocation. In summary, these data show that PLA technology faithfully mirrored all aspects of dynamic NF-κB regulation, thus allowing molecular diagnostics of this key pathway at the single cell level which will be required for future precision medicine

Developmental specificity of auxin response by pairs of ARF and Aux/IAA transcriptional regulators

The plant hormone auxin elicits many specific context-dependent developmental responses. Auxin promotes degradation of Aux/IAA proteins that prevent transcription factors of the auxin response factor (ARF) family from regulating auxin-responsive target genes. Aux/IAAs and ARFs are represented by large gene families in Arabidopsis. Here we show that stabilization of BDL/IAA12 or its sister protein IAA13 prevents MP/ARF5-dependent embryonic root formation whereas stabilized SHY2/IAA3 interferes with seedling growth. Although both bdl and shy2-2 proteins inhibited MP/ARF5-dependent reporter gene activation, shy2-2 was much less efficient than bdl to interfere with embryonic root initiation when expressed from the BDL promoter. Similarly, MP was much more efficient than ARF16 in this process. When expressed from the SHY2 promoter, both shy2-2 and bdl inhibited cell elongation and auxin-induced gene expression in the seedling hypocotyl. By contrast, gravitropism and auxin-induced gene expression in the root, which were promoted by functionally redundant NPH4/ARF7 and ARF19 proteins, were inhibited by shy2-2, but not by bdl protein. Our results suggest that auxin signals are converted into specific responses by matching pairs of coexpressed ARF and Aux/IAA proteins