Journal of Plant Research

, Volume 130, Issue 2, pp 239–253 | Cite as

Mining the Cicer arietinum genome for the mildew locus O (Mlo) gene family and comparative evolutionary analysis of the Mlo genes from Medicago truncatula and some other plant species

  • Reena Deshmukh
  • V. K. Singh
  • Brahma Deo Singh
Regular Paper


The mildew locus O (Mlo) gene family is ubiquitous in land plants. Some members of this gene family are involved in negative regulation of powdery mildew resistance, while others are involved in several other biological functions. Mlo proteins have characteristic seven transmembrane domains and a calmodulin-binding domain at their C-termini, and are associated with plasma membrane. The Mlo gene family has been studied in several economically important cereals, but little information is available on this gene family in the important legumes, Medicago truncatula Gaertn. and Cicer arietinum L. We carried out a comprehensive and comparative investigation of the Mlo gene family in these two species using the genome sequences available at the M. truncatula genome database (Mt v4.0) and NCBI (C. arietinum). A genome-wide homology-based search using Arabidopsis Mlo proteins as query identified 16 MtMlo (M. truncatula Mlo) and 14 CarMlo (C. arietinum Mlo) genes. The MtMlo and CarMlo genes had comparable gene structure, protein sequence and topology. Their chromosomal locations indicated the occurrence of extensive reorganization in the genomes of the two species after their divergence from the common ancestor. A multiple sequence alignment of 53 Mlo proteins from these two and several other species showed a highly conserved sequence block of seven amino acids, viz., L-ETPTW, towards their N-termini. The evolutionary phylogenetic analysis grouped the MtMlo and CarMlo members into four clusters, and most of the MtMlo and CarMlo members formed one-to-one ortholog pairs. The ka/ks analyses indicated that the MtMlo and CarMlo genes are subjected to intense purifying selection.


Cicer arietinum Gene duplication Medicago truncatula Mlo genes Phylogeny 



The work was supported by the University Grant Commission, New Delhi research fellowship to RD. The authors are thankful to the Department of BioTechnology, New Delhi funded SUB-DIC Bioinformatics Centre at School of Biotechnology, Banaras Hindu University for software and technical help. The authors have no conflict of interest.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

Supplementary material

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Supplementary material 1 (PDF 10520 kb)
10265_2016_868_MOESM2_ESM.xlsx (11 kb)
Supplementary material 2 (XLSX 10 kb)


  1. Alberts B, Johnson A, Lewis J, et al (2002) Membrane proteins. In: Molecular biology of the cell. 4th edn. Garland Science, New YorkGoogle Scholar
  2. Altenhoff AM, Studer RA, Robinson-Rechavi M, Dessimoz C (2012) Resolving the ortholog conjecture: orthologs tend to be weakly, but significantly, more similar in function than paralogs. PLoS Comput Biol 8:e1002514CrossRefPubMedPubMedCentralGoogle Scholar
  3. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefPubMedGoogle Scholar
  4. Bai Y, Pavan S, Zheng Z, Zappel NF, Reinstädler A, Lotti C, De Giovanni C, Ricciardi L, Lindhout P, Visser R, Theres K, Panstruga A (2008) Naturally occurring broad-spectrum powdery mildew resistance in a Central American tomato accession is caused by loss of mlo function. Mol Plant Microbe Interact 21:30–39CrossRefPubMedGoogle Scholar
  5. Bailey TL, Williams N, Misleh C, Li WW (2006) MEME: discovering and analyzing DNA and protein sequence motifs. Nucl Acids Res 34:W369–W373CrossRefPubMedPubMedCentralGoogle Scholar
  6. Benedito VA, Torres-Jerez I, Murray JD et al (2008) A gene expression atlas of the model legume Medicago truncatula. Plant J 55:504–513CrossRefPubMedGoogle Scholar
  7. Büschges R, Hollricher K, Panstruga R et al (1997) The barley MLO gene: a novel control element of plant pathogen resistance. Cell 88:695–705CrossRefPubMedGoogle Scholar
  8. Chen Z, Hartmann HA, Wu MJ, Friedman EJ, Chen JG, Pulley M, Schulze-Lefert P, Panstruga R, Jones AM (2006) Expression analysis of the AtMLO gene family encoding plant-specific seven-transmembrane domain proteins. Plant Mol Biol 60:583–597CrossRefPubMedGoogle Scholar
  9. Cheng H, Kong W, Hou D, Lv J, Tao X (2013) Isolation, characterization, and expression analysis of CmMLO2 in muskmelon. Mol Biol Rep 40:2609–2615CrossRefPubMedGoogle Scholar
  10. Choi HK, Mun JH, Kim DJ et al (2004) Estimating genome conservation between crop and model legume species. Proc Natl Acad Sci USA 101:15289–15294CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cooper DN (1999) Contractions and expansions in gene size and number. In: Human gene evolution, 1st edn. Elsevier, pp 329–335Google Scholar
  12. Devoto A, Hartmann HA, Piffanelli P et al (2003) Molecular phylogeny and evolution of the plant-specific seven-transmembrane MLO family. J Mol Evol 56:77–88CrossRefPubMedGoogle Scholar
  13. Devoto A, Piffanelli P, Nilsson I, Wallin E, Panstruga R, von Heijne G, Schulze-Lefert P (1999) Topology, subcellular localization, and sequence diversity of the Mlo family in plants. J Biol Chem 274:34993–35004CrossRefPubMedGoogle Scholar
  14. Duarte JM, Cui L, Wall PK, Zhang Q, Zhang X, Leebens-Mack J, Ma H, Altman N, dePamphilis CW (2006) Expression pattern shifts following duplication indicative of subfunctionalization and neofunctionalization in regulatory genes of Arabidopsis. Mol Biol Evol 23:469–478CrossRefPubMedGoogle Scholar
  15. Elliott C, Zhou F, Spielmeyer W, Panstruga R, Schulze-Lefert P (2002) Functional conservation of wheat and rice Mlo orthologs in defense modulation to the powdery mildew fungus. Mol Plant Microbe Interact 15:1069–1077CrossRefPubMedGoogle Scholar
  16. Elliott C, Müller J, Miklis M, Bhat RA, Schulze-Lefert P, Panstruga R (2005) Conserved extracellular cysteine residues and cytoplasmic loop–loop interplay are required for functionality of the heptahelical MLO protein. Biochem J 385:243–254CrossRefPubMedGoogle Scholar
  17. Fan C, Chen Y, Long M (2008) Recurrent tandem gene duplication gave rise to functionally divergent genes in Drosophila. Mol Biol Evol 25:1451–1458CrossRefPubMedPubMedCentralGoogle Scholar
  18. Feechan A, Jermakow AM, Dry IB (2009) Grapevine MLO candidates required for powdery mildew pathogenicity? Plant Signal Behav 4:522–523CrossRefPubMedPubMedCentralGoogle Scholar
  19. Finn RD, Bateman A, Clements J et al (2014) Pfam: the protein families database. Nucl Acids Response 42:D222–D230CrossRefGoogle Scholar
  20. Fitch W (1970) Distinguishing homologous from analogous proteins. Syst Zool 19:99–106CrossRefPubMedGoogle Scholar
  21. Franceschini A, Szklarczyk D, Frankild S et al (2013) STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucl Acids Res 41:808–815CrossRefGoogle Scholar
  22. Fukasawa Y, Leung RK, Tsui SK, Horton P (2014) Plus ça change—evolutionary sequence divergence predicts protein subcellular localization signals. BMC Genom 15:46CrossRefGoogle Scholar
  23. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98Google Scholar
  24. Hatsuzawa K, Tagaya M, Mizushima S (1997) The hydrophobic region of signal peptides is a determinant for SRP recognition and protein translocation across the ER membrane. J Biochem 121:270–277CrossRefPubMedGoogle Scholar
  25. He J, Benedito VA, Wang M et al (2009) The Medicago truncatula gene expression atlas web server. BMC Bioinform 10:441–449CrossRefGoogle Scholar
  26. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucl Acids Res 27:297–300CrossRefPubMedPubMedCentralGoogle Scholar
  27. Hu B, Jin J, Guo A, Zhang H, Luo J, Gao G (2015) GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics 31:1296–1297CrossRefPubMedGoogle Scholar
  28. Humphry M, Reinstädler A, Ivanov S, Bisseling T, Panstruga R (2011) Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1. Mol Plant Pathol 12:866–878CrossRefPubMedGoogle Scholar
  29. Jain M, Misra G, Patel RK et al (2013) A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.). Plant J 4:715–729CrossRefGoogle Scholar
  30. Jørgensen IH (1992) Discovery, characterization and exploitation of Mlo powdery mildew resistance in barley. Euphytica 63:141–152CrossRefGoogle Scholar
  31. Kaufmann H, Qiu X, Wehmeyer J, Debener T (2012) Isolation, molecular characterization, and mapping of four rose MLO orthologs. Front Plant Sci 3:244–257CrossRefPubMedPubMedCentralGoogle Scholar
  32. Kim DS, Hwang BK (2012) The pepper MLO gene, CarMlo2, is involved in the susceptibility cell-death response and bacterial and oomycete proliferation. Plant J 72:843–855CrossRefPubMedGoogle Scholar
  33. Kim MC, Lee SH, Kim JK et al (2002a) MLO, a modulator of plant defense and cell death, is a novel calmodulin-binding protein. J Biol Chem 277:19304–19314CrossRefPubMedGoogle Scholar
  34. Kim MC, Panstruga R, Elliott C, Müller J, Devoto A, Yoon HW, Park HC, Cho MJ, Schulze-Lefert P (2002b) Calmodulin interacts with MLO protein to regulate defence against mildew in barley. Nature 416:447–450CrossRefPubMedGoogle Scholar
  35. Konishi S, Sasakuma T, Sasanuma T (2010) Identification of novel Mlo family members in wheat and their genetic characterization. Genes Genet Syst 85:167–175CrossRefPubMedGoogle Scholar
  36. Kumar J, Hückelhoven R, Beckhove U, Nagarajan S, Kogel KH (2011) A compromised Mlo pathway affects the response of barley to the necrotrophic fungus Bipolaris sorokiniana (Teleomorph: Cochliobolus sativus) and its toxins. Phytopathology 91:127–133CrossRefGoogle Scholar
  37. Lecourieux D, Ranjeva R, Pugin A (2006) Calcium in plant defense- signaling pathways. New Phytol 171:249–269CrossRefPubMedGoogle Scholar
  38. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucl Acid Res 30:325–327CrossRefGoogle Scholar
  39. Li YY, Yu H, Guo ZM et al (2006) Systematic analysis of head-to-head gene organization: evolutionary conservation and potential biological relevance. PLoS Comput Biol 2:e74CrossRefPubMedPubMedCentralGoogle Scholar
  40. Liu Q, Zhu H (2008) Molecular evolution of the Mlo gene family in Oryza sativa and their functional divergence. Gene 409:1–10CrossRefPubMedGoogle Scholar
  41. Lyngkjær MF, Newton AC, Atzema JL, Baker SJ (2000) The barley mlo-gene: an important powdery mildew resistance source. Agronomie 20:745–756CrossRefGoogle Scholar
  42. Miyata T, Yasunaga T (1980) Molecular evolution of mRNA: a method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its application. J Mol Evol 16:23–36CrossRefPubMedGoogle Scholar
  43. Ohta T (1989) Role of gene duplication in evolution. Genome 31:304–310CrossRefPubMedGoogle Scholar
  44. Panstruga R (2005) Serpentine plant MLO proteins as entry portals for powdery mildew fungi. Biochem Soc Trans 33:389–392CrossRefPubMedGoogle Scholar
  45. Pessina S, Pavan S, Catalano D, Gallotta A, Visser RG, Bai Y, Malnoy M, Schouten HJ (2014) Characterization of the MLO gene family in Rosaceae and gene expression analysis in Malus domestica. BMC Genom 15:618–629CrossRefGoogle Scholar
  46. Qin B, Zheng F, Zhang Y (2015) Molecular cloning and characterization of a Mlo gene in rubber tree (Hevea brasiliensis). J Plant Physiol 175:78–85CrossRefPubMedGoogle Scholar
  47. Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R (2005) InterProScan: protein domains identifier. Nucl Acids Res 33:116–120CrossRefGoogle Scholar
  48. Reddy VS, Ali GS, Reddy AS (2003) Characterization of a pathogen-induced calmodulin-binding protein: mapping of four Ca2+-dependent calmodulin-binding domains. Plant Mol Biol 52:143–159CrossRefPubMedGoogle Scholar
  49. Reinstädler A, Müller J, Czembor JH, Piffanelli P, Panstruga R (2010) Novel induced mlo mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein. BMC Plant Biol 10:31. doi: 10.1186/1471-2229-10-31 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Singh VK, Singh AK, Chand R, Singh BD (2012) Genome wide analysis of disease resistance mlo gene family in sorghum [Sorghum bicolor (L.) Moench]. J Plant Genom 2:18–27Google Scholar
  51. Shen Q, Zhao J, Du C et al (2012) Genome-scale identification of MLO domain-containing genes in soybean (Glycine max L. Merr.). Genes Genet Syst 87:89–98CrossRefPubMedGoogle Scholar
  52. Swarbreck D, Wilks C, Lamesch P et al (2008) The Arabidopsis Information Resource (TAIR): gene structure and function annotation. Nucl Acids Res 36:D1009–D1014CrossRefPubMedGoogle Scholar
  53. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefPubMedPubMedCentralGoogle Scholar
  54. Tang H, Krishnakumar V, Bidwell S, Rosen B, Chan A, Zhou S, Gentzbittel L, Childs KL, Yandell M, Gundlach H, Mayer KF, Schwartz DC, Town CD (2014) An improved genome release (version Mt4.0) for the model legume Medicago truncatula. BMC Genom 15:312–326CrossRefGoogle Scholar
  55. Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22:4673–4680CrossRefPubMedPubMedCentralGoogle Scholar
  56. Tiuryn J, Randomski JP, Slonimski PP (2000) A formal model of genomic DNA multiplication and amplification. In: Nadeau JH (ed) Comparative genomics: empirical and analytical approaches to gene order dynamics, map alignment and the evolution of gene families, 1st edn. Springer, Netherlands, pp 503–514CrossRefGoogle Scholar
  57. Treangen T, Messeguer X (2006) M-GCAT: interactively and efficiently constructing large-scale multiple genome comparison frameworks in closely related species. BMC Bioinformatics 7:433–448CrossRefPubMedPubMedCentralGoogle Scholar
  58. Tusnády GE, Simon I (2001) The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850CrossRefPubMedGoogle Scholar
  59. Varshney RK, Song C, Saxena RK et al (2013) Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement. Nat Biotechnol 31:240–246CrossRefPubMedGoogle Scholar
  60. Young ND, Debellé F, Oldroyd GE et al (2011) The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 480:520–524CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan 2016

Authors and Affiliations

  • Reena Deshmukh
    • 1
  • V. K. Singh
    • 1
  • Brahma Deo Singh
    • 1
  1. 1.Centre for Bioinformatics, School of Biotechnology, Faculty of ScienceBanaras Hindu UniversityVaranasiIndia

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