Nitrosothiols in the immune system: Signaling and protection

Antioxidants and Redox Signaling 18.3 (2013): 288-308Significance: In the immune system, nitric oxide (NO) has been mainly associated with antibacterial defenses exerted through oxidative, nitrosative, and nitrative stress and signal transduction through cyclic GMP-dependent mechanisms. However, S-nitrosylation is emerging as a post-translational modification (PTM) involved in NO-mediated cell signaling. Recent Advances: Precise roles for S-nitrosylation in signaling pathways have been described both for innate and adaptive immunity. Denitrosylation may protect macrophages from their own S-nitrosylation, while maintaining nitrosative stress compartmentalized in the phagosomes. Nitrosothiols have also been shown to be beneficial in experimental models of autoimmune diseases, mainly through their role in modulating T-cell differentiation and function. Critical Issues: Relationship between S-nitrosylation, other thiol redox PTMs, and other NO-signaling pathways has not been always taken into account, particularly in the context of immune responses. Methods for assaying S-nitrosylation in individual proteins and proteomic approaches to study the S-nitrosoproteome are constantly being improved, which helps to move this field forward. Future Directions: Integrated studies of signaling pathways in the immune system should consider whether S-nitrosylation/denitrosylation processes are among the PTMs influencing the activity of key signaling and adaptor proteins. Studies in pathophysiological scenarios will also be of interest to put these mechanisms into broader contexts. Interventions modulating nitrosothiol levels in autoimmune disease could be investigated with a view to developing new therapiesFinanced by the Spanish Government grants CSD2007-00020 (RosasNet, Consolider-Ingenio 2010 programme), CP07/00143 (Miguel Servet programme), and PS09/00101; and PI10/0213

Progastrin Represses the Alternative Activation of Human Macrophages and Modulates Their Influence on Colon Cancer Epithelial Cells

Macrophage infiltration is a negative prognostic factor for most cancers but gastrointestinal tumors seem to be an exception. The effect of macrophages on cancer progression depends on their phenotype, which may vary between M1 (pro-inflammatory, defensive) to M2 (tolerogenic, pro-tumoral). Gastrointestinal cancers often become an ectopic source of gastrins and macrophages present receptors for these peptides. The aim of the present study is to analyze whether gastrins can affect the pattern of macrophage infiltration in colorectal tumors. We have evaluated the relationship between gastrin expression and the pattern of macrophage infiltration in samples from colorectal cancer and the influence of these peptides on the phenotype of macrophages differentiated from human peripheral monocytes in vitro. The total number of macrophages (CD68+ cells) was similar in tumoral and normal surrounding tissue, but the number of M2 macrophages (CD206+ cells) was significantly higher in the tumor. However, the number of these tumor-associated M2 macrophages correlated negatively with the immunoreactivity for gastrin peptides in tumor epithelial cells. Macrophages differentiated from human peripheral monocytes in the presence of progastrin showed lower levels of M2-markers (CD206, IL10) with normal amounts of M1-markers (CD86, IL12). Progastrin induced similar effects in mature macrophages treated with IL4 to obtain a M2-phenotype or with LPS plus IFNγ to generate M1-macrophages. Macrophages differentiated in the presence of progastrin presented a reduced expression of Wnt ligands and decreased the number and increased cell death of co-cultured colorectal cancer epithelial cells. Our results suggest that progastrin inhibits the acquisition of a M2-phenotype in human macrophages. This effect exerted on tumor associated macrophages may modulate cancer progression and should be taken into account when analyzing the therapeutic value of gastrin immunoneutralization

Differential regulation of nuclear and mitochondrial Bcl-2 in T cell apoptosis

Activated T cells require anti-apoptotic cytokines for their survival. The anti-apoptotic effects of these factors are mediated by their influence on the balance of expression and localisation of pro- and anti-apoptotic members of the Bcl-2 family. Among the anti-apoptotic Bcl-2 family members, the expression level of Bcl-2 itself and its interaction with the pro-apoptotic protein Bim are now regarded as crucial for the regulation of survival in activated T cells. We studied the changes in Bcl-2 levels and its subcellular distribution in relation to mitochondrial depolarisation and caspase activation in survival factor deprived T cells. Intriguingly, the total Bcl-2 level appeared to remain stable, even after caspase 3 activation indicated entry into the execution phase of apoptosis. However, cell fractionation experiments showed that while the dominant nuclear pool of Bcl-2 remained stable during apoptosis, the level of the smaller mitochondrial pool was rapidly downregulated. Signals induced by anti-apoptotic cytokines continuously replenish the mitochondrial pool, but nuclear Bcl-2 is independent of such signals. Mitochondrial Bcl-2 is lost rapidly by a caspase independent mechanism in the absence of survival factors, in contrast only a small proportion of the nuclear pool of Bcl-2 is lost during the execution phase and this loss is a caspase dependent process. We conclude that these two intracellular pools of Bcl-2 are regulated through different mechanisms and only the cytokine-mediated regulation of the mitochondrial pool is relevant to the control of the initiation of apoptosis

Ectopic Pregnancy as a Model to Identify Endometrial Genes and Signaling Pathways Important in Decidualization and Regulated by Local Trophoblast

The endometrium in early pregnancy undergoes decidualization and functional changes induced by local trophoblast, which are not fully understood. We hypothesized that endometrium from tubal ectopic pregnancy (EP) could be interrogated to identify novel genes and pathways involved in these processes. Gestation-matched endometrium was collected from women with EP (n = 11) and intrauterine pregnancies (IUP) (n = 13). RNA was extracted from the tissue. In addition, tissues were prepared for histological analysis for degree of decidualization. We compared a) the samples from EP that were decidualized (n = 6) with non-decidualized samples (n = 5), and b) the decidualized EP (n = 6) with decidualization-matched IUP (n = 6) samples using an Affymetrix gene array platform, with Ingenuity Pathway Analysis, combined with quantitative RT-PCR. Expression of PRL and IGFBP1 was used to confirm the degree of decidualization in each group. There were no differences in PRL or IGFBP1 expression in the decidualization-matched samples but a marked reduction (P<0.001) in the non-decidualized samples. Decidualization was associated with increased expression of 428 genes including SCARA5 (181-fold), DKK1 (71-fold) and PROK1 (32-fold), and decreased expression of 230 genes including MMP-7 (35-fold) and SFRP4 (21-fold). The top canonical pathways associated with these differentially expressed genes were Natural Killer Cell and Wnt/b-Catenin signaling. Local trophoblast was associated with much less alteration of endometrial gene expression with an increase in 56 genes, including CSH1 (8-fold), and a reduction in 29 genes including CRISP3 (8-fold). The top associated canonical pathway was Antigen Presentation. The study of endometrium from tubal EP may promote novel insights into genes involved in decidualization and those influenced by factors from neighboring trophoblast. This has afforded unique information not highlighted by previous studies and adds to our understanding of the endometrium in early pregnancy

EcoTILLING in Capsicum species: searching for new virus resistances

<p>Abstract</p> <p>Background</p> <p>The EcoTILLING technique allows polymorphisms in target genes of natural populations to be quickly analysed or identified and facilitates the screening of genebank collections for desired traits. We have developed an EcoTILLING platform to exploit <it>Capsicum </it>genetic resources. A perfect example of the utility of this EcoTILLING platform is its application in searching for new virus-resistant alleles in <it>Capsicum </it>genus. Mutations in translation initiation factors (eIF4E, eIF(iso)4E, eIF4G and eIF(iso)4G) break the cycle of several RNA viruses without affecting the plant life cycle, which makes these genes potential targets to screen for resistant germplasm.</p> <p>Results</p> <p>We developed and assayed a cDNA-based EcoTILLING platform with 233 cultivated accessions of the genus <it>Capsicum</it>. High variability in the coding sequences of the <it>eIF4E </it>and <it>eIF(iso)4E </it>genes was detected using the cDNA platform. After sequencing, 36 nucleotide changes were detected in the CDS of <it>eIF4E </it>and 26 in <it>eIF(iso)4E</it>. A total of 21 <it>eIF4E </it>haplotypes and 15 <it>eIF(iso)4E </it>haplotypes were identified. To evaluate the functional relevance of this variability, 31 possible eIF4E/eIF(iso)4E combinations were tested against <it>Potato virus Y</it>. The results showed that five new <it>eIF4E </it>variants (<it>pvr2¹⁰</it>, <it>pvr2¹¹</it>, <it>pvr2¹²</it>, <it>pvr2¹³</it>and <it>pvr2¹⁴</it>) were related to PVY-resistance responses.</p> <p>Conclusions</p> <p>EcoTILLING was optimised in different <it>Capsicum </it>species to detect allelic variants of target genes. This work is the first to use cDNA instead of genomic DNA in EcoTILLING. This approach avoids intronic sequence problems and reduces the number of reactions. A high level of polymorphism has been identified for initiation factors, showing the high genetic variability present in our collection and its potential use for other traits, such as genes related to biotic or abiotic stresses, quality or production. Moreover, the new <it>eIF4E </it>and <it>eIF(iso)4E </it>alleles are an excellent collection for searching for new resistance against other RNA viruses.</p

A mutation in the melon Vacuolar Protein Sorting 41prevents systemic infection of Cucumber mosaic virus

[EN] In the melon exotic accession PI 161375, the gene cmv1, confers recessive resistance to Cucumber mosaic virus (CMV) strains of subgroup II. cmv1 prevents the systemic infection by restricting the virus to the bundle sheath cells and impeding viral loading to the phloem. Here we report the fine mapping and cloning of cmv1. Screening of an F2 population reduced the cmv1 region to a 132 Kb interval that includes a Vacuolar Protein Sorting 41 gene. CmVPS41 is conserved among plants, animals and yeast and is required for post-Golgi vesicle trafficking towards the vacuole. We have validated CmVPS41 as the gene responsible for the resistance, both by generating CMV susceptible transgenic melon plants, expressing the susceptible allele in the resistant cultivar and by characterizing CmVPS41 TILLING mutants with reduced susceptibility to CMV. Finally, a core collection of 52 melon accessions allowed us to identify a single amino acid substitution (L348R) as the only polymorphism associated with the resistant phenotype. CmVPS41 is the first natural recessive resistance gene found to be involved in viral transport and its cellular function suggests that CMV might use CmVPS41 for its own transport towards the phloem.The TILLING platform is supported by the Program Saclay Plant Sciences (SPS, ANR-10-LABX-40) and the European Research Council (ERC-SEXYPARTH). This work was supported by grants AGL2009-12698-C02-01 and AGL2012-40130-C02-01 from the Spanish Ministry of Science and Innovation, the Spanish Ministry of Econom and Competitiveness, through the "Severo Ochoa Programme for Centres of Excellence in R&D" 2016-2019 (SEV-2015-0533)" and the CERCA Programme/Generalitat de Catalunya.Giner, A.; Pascual, L.; Bourgeois, M.; Gyetvai, G.; Rios, P.; Picó Sirvent, MB.; Troadec, C.... (2017). A mutation in the melon Vacuolar Protein Sorting 41prevents systemic infection of Cucumber mosaic virus. Scientific Reports. 7:1-12. https://doi.org/10.1038/s41598-017-10783-3S1127Anderson, P. K. et al. Emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers. Trends in Ecology & Evolution 19, 535–544, doi: 10.1016/j.tree.2004.07.021 (2004).Nicaise, V. Crop immunity against viruses: outcomes and future challenges. Frontiers in Plant Science 5, 660, doi: 10.3389/fpls.2014.00660 (2014).Kang, B.-C., Yeam, I. & Jahn, M. Genetics of plant virus resistance. Annual Review of Phytopathology 43, 581–621, doi: 10.1146/annurev.phyto.43.011205.141140 (2005).Fraser, R. S. S. The genetics of plant-virus interactions: implications for plant breeding. Euphytica 63, 175–185, doi: 10.1007/bf00023922 (1992).Truniger, V. & Aranda, M. Recessive resistance to plant viruses. Advances in virus research 119–159 (2009).Sanfaçon, H. Plant translationfactors and virus resistance. Viruses 7, 3392–3419, doi: 10.3390/v7072778 (2015).Yang, P. et al. PROTEIN DISULFIDE ISOMERASE LIKE 5-1 is a susceptibility factor to plant viruses. Proceedings of the National Academy of Sciences of the United States of America 111, 2104–2109, doi: 10.1073/pnas.1320362111 (2014).Ouibrahim, L. et al. Cloning of the Arabidopsis rwm1 gene for resistance to Watermelon mosaic virus points to a new function for natural virus resistance genes. The Plant Journal 79, 705–716, doi: 10.1111/tpj.12586 (2014).Rubio, M., Nicolaï, M., Caranta, C. & Palloix, A. Allele mining in the pepper gene pool provided new complementation effects between pvr2-eIF4E and pvr6-eIF(iso)4E alleles for resistance to pepper veinal mottle virus. Journal of General Virology 90, 2808–2814, doi: 10.1099/vir.0.013151-0 (2009).Ibiza, V. P., Cañizares, J. & Nuez, F. EcoTILLING in Capsicum species: searching for new virus resistances. BMC Genomics 11, 631–631, doi: 10.1186/1471-2164-11-631 (2010).Chandrasekaran, J. et al. Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology. Molecular Plant Pathology. doi: 10.1111/mpp.12375 (2016).Rodríguez-Hernández, A. et al. Melon RNA interference (RNAi) lines silenced for Cm-eIF4E show broad virus resistance. Molecular plant pathology 13, 755–763, doi: 10.1111/j.1364-3703.2012.00785.x (2012).Edwardson, J. R. & Christie, R. G. In CRC Handbook of Viruses Infecting Legumes (eds J.R. Edwardson & R.G. Christie) 293–319 (CRC Press, 1991).Roossinck, M. J. Cucumber mosaic virus, a model for RNA virus evolution. Molecular Plant Pathology 2, 59–63 (2001).Caranta, C. et al. QTLs involved in the restriction of Cucumber mosaic virus (CMV) long-distance movement in pepper. Theor Appl Genet 104, 586–591 (2002).Valkonen, J. P. & Watanabe, K. N. Autonomous cell death, temperature sensitivity and the genetic control associated with resistance to Cucumber mosaic virus (CMV) in diploid potatoes (Solanum spp). Theor Appl Genet 99, 996–1005 (1999).Ohnishi, S. et al. Multigenic system controlling viral systemic infection determined by the interactions between Cucumber mosaic virus genes and quantitative trait loci of soybean cultivars. Phytopathology 101, 575–582, doi: 10.1094/phyto-06-10-0154 (2011).Kobori, T., Satoshi, T. & Osaki, T. Movement of Cucumber mosaic virus is restricted at the interface between mesophyll and phloem pathway in Cucumis figarei. Journal of General Plant Pathology 66, 159–166, doi: 10.1007/pl00012939 (2000).Argyris, J. M., Pujol, M., Martín-Hernández, A. M. & Garcia-Mas, J. Combined use of genetic and genomics resources to understand virus resistance and fruit quality traits in melon. Physiologia Plantarum 155, 4–11, doi: 10.1111/ppl.12323 (2015).Diaz, J. A. et al. Potential sources of resistance for melon to nonpersistently aphid-borne viruses. Plant Disease 87, 960–964 (2003).Karchi, Z., Cohen, S. & Govers, A. Inheritance of resistance to Cucumber Mosaic virus in melons. Phytopathology 65, 479–481 (1975).Risser, G., Pitrat, M. & Rode, J. C. Etude de la résistance du melon (Cucumis melo L.) au virus de la mosaïque du concombre. Ann Amél Plant 27, 509–522 (1977).Dogimont, C. et al. Identification of QTLs contributing to resistance to different strains of Cucumber mosaic cucumovirus in melon. Acta Hortic 510, 391–398 (2000).Essafi, A. et al. Dissection of the oligogenic resistance to Cucumber mosaic virus in the melon accession PI 161375. Theoretical and Applied Genetics 118, 275–284 (2009).Guiu-Aragonés, C., Díaz-Pendón, J. A. & Martín-Hernández, A. M. Four sequence positions of the movement protein of Cucumber mosaic virus determine the virulence against cmv1-mediated resistance in melon. Molecular Plant Pathology 16, 675–684, doi: 10.1111/mpp.12225 (2015).Guiu-Aragonés, C. et al. The complex resistance to Cucumber mosaic cucumovirus (CMV) in the melon accession PI 161375 is governed by one gene and at least two quantitative trait loci. Molecular Breeding 34, 351–362, doi: 10.1007/s11032-014-0038-y (2014).Guiu-Aragonés, C. et al. cmv1 is a gate for Cucumber mosaic virus transport from bundle sheath cells to phloem in melon. Mol. Plant Pathology 17, 973–984 (2016).Sanseverino, W. et al. Transposon Insertions, Structural Variations, and SNPs Contribute to the Evolution of the Melon Genome. Molecular Biology and Evolution 32, 2760–2774, doi: 10.1093/molbev/msv152 (2015).Pols, M. S., ten Brink, C., Gosavi, P., Oorschot, V. & Klumperman, J. The HOPS Proteins hVps41 and hVps39 Are Required for Homotypic and Heterotypic Late Endosome Fusion. Traffic 14, 219–232, doi: 10.1111/tra.12027 (2013).Asensio, C. S. et al. Self-assembly of VPS41 promotes sorting required for biogenesis of the regulated secretory pathway. Developmental cell 27, 425–437, doi: 10.1016/j.devcel.2013.10.007 (2013).Kolesnikova, L. et al. Vacuolar protein sorting pathway contributes to the release of Marburg virus. Journal of Virology8 3, 2327–2337, doi: 10.1128/JVI.02184-08 (2009).Nuñez-Palenius, H. G. et al. Melon Fruits: Genetic Diversity, Physiology, and Biotechnology Features. Critical Reviews in Biotechnology 28, 13–55, doi: 10.1080/07388550801891111 (2008).Castelblanque, L. et al. Improving the genetic transformation efficiency of Cucumis melo subsp. melo “Piel de Sapo” via Agrobacterium. Procedings of the IXth UECARPIA Meeting on Genetics and Breeding of Cucurbitaceae, 21-24th May, Avignon,, 627–631 (2008).Dahmani-Mardas, F. et al. Engineering melon plants with improved fruit shelf life using the TILLING approach. PLoS ONE 5, doi: 10.1371/journal.pone.0015776 (2010).Esteras, C. et al. SNP genotyping in melons: genetic variation, population structure, and linkage disequilibrium. Theoretical and Applied Genetics 126, 1285–1303, doi: 10.1007/s00122-013-2053-5 (2013).Leida, C. et al. Variability of candidate genes, genetic structure and association with sugar accumulation and climacteric behavior in a broad germplasm collection of melon (Cucumis melo L.). BMC Genetics 16, 28, doi: 10.1186/s12863-015-0183-2 (2015).Balderhaar, H. Jk & Ungermann, C. CORVET and HOPS tethering complexes – coordinators of endosome and lysosome fusion. Journal of Cell Science 126, 1307–1316, doi: 10.1242/jcs.107805 (2013).Darsow, T., Katzmann, D. J., Cowles, C. R. & Emr, S. D. Vps41p Function in the Alkaline Phosphatase Pathway Requires Homo-oligomerization and Interaction with AP-3 through Two Distinct Domains. Molecular Biology of the Cell 12, 37–51 (2001).Ostrowicz, C. W. et al. Defined subunit arrangement and Rab interactions are required for functionality of the HOPS tethering complex. Traffic 11, 1334–1346, doi: 10.1111/j.1600-0854.2010.01097.x (2010).Solinger, J. A. & Spang, A. Tethering complexes in the endocytic pathway: CORVET and HOPS. FEBS Journal 280, 2743–2757, doi: 10.1111/febs.12151 (2013).Rehling, P., Dawson, T., Katzmann, D. J. & Emr, S. D. Formation of AP-3 transport intermediates requires Vps41 function. Nature Cell Biology 1, 346–353 (1999).Niihama, M., Takemoto, N., Hashiguchi, Y., Tasaka, M. & Morita, M. T. ZIP genes encode proteininvolved inmembrane trafficking of the TGN–PVC/Vacuoles. Plant and Cell Physiology 50, 2057–2068, doi: 10.1093/pcp/pcp137 (2009).Wang, A. & Krishnaswamy, S. Eukaryotic translation initiation factor 4E-mediated recessive resistance to plant viruses and its utility in crop improvement. Molecular Plant Pathology 13, 795–803, doi: 10.1111/j.1364-3703.2012.00791.x (2012).Ruffel, S. et al. A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E). The Plant Journal 32, 1067–1075, doi: 10.1046/j.1365-313X.2002.01499.x (2002).Morosky, S., Lennemann, N. J. & Coyne, C. B. BPIFB6 regulatessecretory pathway trafficking and Enterovirus replication. Journal of Virology 90, 5098–5107, doi: 10.1128/jvi.00170-16 (2016).Serra-Soriano, M., Pallás, V. & Navarro, J. A. A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements. The Plant Journal 77, 863–879, doi: 10.1111/tpj.12435 (2014).Vale-Costa, S. & Amorim, M. J. Recycling Endosomes and Viral Infection. Viruses 8, 64, doi: 10.3390/v8030064 (2016).Carette, J. E. et al. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 477, 340–343, doi: 10.1038/nature10348 (2011).Barajas, D., Martín, I. Fd. C., Pogany, J., Risco, C. & Nagy, P. D. Noncanonical role for the host Vps4 AAA + ATPase ESCRT protein in the formation of Tomato bushy stunt virus replicase. PLoS Pathog 10, e1004087, doi: 10.1371/journal.ppat.1004087 (2014).Richardson, L. G. L. et al. A unique N-Tterminal sequence in the Carnation Italian ringspot virus p36 replicase-associated protein interacts with the host cell ESCRT-I component Vps23. Journal of Virology 88, 6329–6344, doi: 10.1128/JVI.03840-13 (2014).Jiang, J., Patarroyo, C., Garcia Cabanillas, D., Zheng, H. & Laliberté, J.-F. The vesicle-forming 6K2 protein of Turnip mosaic virus interacts with the COPII coatomer Sec. 24a for viral systemic infection. Journal of Virology 89, 6695–6710, doi: 10.1128/jvi.00503-15 (2015).Genovés, A., Navarro, J. A. & Pallás, V. Theintra- and intercellular movement of Melon necrotic spot virus (MNSV) depends on an active secretory pathway. Molecular Plant-Microbe Interactions 23, 263–272, doi: 10.1094/MPMI-23-3-0263 (2010).Amano, M. et al. High-resolution mapping of zym, a recessive gene for Zucchini yellow mosaic virus resistance in cucumber. Theoretical and Applied Genetics 126, 2983–2993, doi: 10.1007/s00122-013-2187-5 (2013).Lewis, J. D. & Lazarowitz, S. G. Arabidopsis synaptotagmin SYTA regulates endocytosis and virus movement protein cell-to-cell transport. Proceedings of the National Academy of Sciences of the United States of America 107, 2491–2496, doi: 10.1073/pnas.0909080107 (2010).Rizzo, T. & Palukaitis, P. Construction of full-length cDNA clones of Cucumber mosaic virus RNAs 1, 2 and 3: Generation of infectious RNA transcripts. Molecular and General Genetics 122, 249–256 (1990).Zhang, L., Handa, K. & Palukaitis, P. Mapping local and systemic symptom determinants of Cucumber mosaic cucumovirus in tobacco. The Journal of General Virology 75, 3185–3191 (1994).Fukino, N., Sakata, Y., Kunihisa, M. & S., M. Characterisation of novel simple sequence repeat (SSR) markers for melon (Cucumis melo L.) and their use for genotype identification. Journal of Horticultural Science & Biotechnology 82, 330–334 (2007).Garcia-Mas, J. et al. The genome of melon (Cucumis melo L.). Proceedings of the National Academy of Sciences 109, 11872–11877, doi: 10.1073/pnas.1205415109 (2012).Untergasser, A. et al. Primer3—new capabilities and interfaces. Nucleic Acids Research 40, e115–e115, doi: 10.1093/nar/gks596 (2012).Gonzalo, M. J. et al. Simple-sequence repeat markers used in merging linkage maps of melon (Cucumis melo L.). Theor Appl Genet 110, 802–811 (2005).Doyle, J. J. & Doyle, J. L. Isolation of plant DNA from fresh tissue. Focus1 2, 13–15 (1990).Argyris, J. M. et al. Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly. BMC Genomics 16, 4, doi: 10.1186/s12864-014-1196-3 (2015).Proost, S. et al. PLAZA 3.0: an access point for plant comparative genomics. Nucleic Acids Research 43, D974–D981, doi: 10.1093/nar/gku986 (2015).Choi, Y., Sims, G. E., Murphy, S., Miller, J. R. & Chan, A. P. Predicting the Functional Effect of Amino Acid Substitutions and Indels. PLoS ONE 7, e46688, doi: 10.1371/journal.pone.0046688 (2012).Li, B. et al. Automated inference of molecular mechanisms of disease from amino acid substitutions. Bioinformatics 25, 2744–2750, doi: 10.1093/bioinformatics/btp528 (2009).Earley, K. W. et al. Gateway-compatible vectors for plant functional genomics and proteomics. The Plant Journal 45, 616–629, doi: 10.1111/j.1365-313X.2005.02617.x (2006).Gonzalez-Ibeas, D. et al. MELOGEN: an EST database for melon functional genomics. BMC Genomics 8, 306 (2007).Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Research 29, 2002–2007 (2001).Dalmais, M. et al. UTILLdb, a Pisum sativum in silico forward and reverse genetics tool. Genome Biology 9, R43–R43, doi: 10.1186/gb-2008-9-2-r43 (2008).Saladié, M. et al. Comparative transcriptional profiling analysis of developing melon (Cucumis melo L.) fruit from climacteric and non-climacteric varieties. BMC Genomics 16, 440, doi: 10.1186/s12864-015-1649-3 (2015)

Geographical gradient of the <em>eIF4E</em> alleles conferring resistance to potyviruses in pea (<em>Pisum</em>) germplasm

<div><p>Background</p><p>The eukaryotic translation initiation factor 4E was shown to be involved in resistance against several potyviruses in plants, including pea. We combined our knowledge of pea germplasm diversity with that of the <i>eIF4E</i> gene to identify novel genetic diversity.</p><p>Methodology/Principal findings</p><p>Germplasm of 2803 pea accessions was screened for <i>eIF4E</i> intron 3 length polymorphism, resulting in the detection of four <i>eIF4E^A-B-C-S</i> variants, whose distribution was geographically structured. The <i>eIF4E^A</i> variant conferring resistance to the P1 PSbMV pathotype was found in 53 accessions (1.9%), of which 15 were landraces from India, Afghanistan, Nepal, and 7 were from Ethiopia. A newly discovered variant, <i>eIF4E^B</i>, was present in 328 accessions (11.7%) from Ethiopia (29%), Afghanistan (23%), India (20%), Israel (25%) and China (39%). The <i>eIF4E^C</i> variant was detected in 91 accessions (3.2% of total) from India (20%), Afghanistan (33%), the Iberian Peninsula (22%) and the Balkans (9.3%). The <i>eIF4E^S</i> variant for susceptibility predominated as the wild type. Sequencing of 73 samples, identified 34 alleles at the whole gene, 26 at cDNA and 19 protein variants, respectively. Fifteen alleles were virologically tested and 9 alleles (<i>eIF4E^{A-1-2-3-4-5-6-7}</i>, <i>eIF4E^B-1</i>, <i>eIF4E^C-2</i>) conferred resistance to the P1 PSbMV pathotype.</p><p>Conclusions/Significance</p><p>This work identified novel <i>eIF4E</i> alleles within geographically structured pea germplasm and indicated their independent evolution from the susceptible <i>eIF4E^S1</i> allele. Despite high variation present in wild <i>Pisum</i> accessions, none of them possessed resistance alleles, supporting a hypothesis of distinct mode of evolution of resistance in wild as opposed to crop species. The Highlands of Central Asia, the northern regions of the Indian subcontinent, Eastern Africa and China were identified as important centers of pea diversity that correspond with the diversity of the pathogen. The series of alleles identified in this study provides the basis to study the co-evolution of potyviruses and the pea host.</p></div

TILLING - a shortcut in functional genomics

Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING