Abstract
Pinus monticola antimicrobial peptide (PmAMP1) inhibits growth of Cronartium ribicola and other fungal pathogens. C. ribicola causes white pine blister rust and has resulted in a dramatic reduction of native white pines across North America. Quantitative disease resistance (QDR) is a highly desirable trait screened in breeding programs for durable resistance against C. ribicola. Along with phenotyping on a collection of germplasms, we analyzed PmAMP1 transcript and protein expression and re-sequenced the full-length gene including its promoter region. A mixed linear model was used to identify the association of single nucleotide polymorphisms (SNPs) with accumulated protein and stem QDR levels. Among 16 PmAMP1 SNPs identified in the present study, we found an association of protein levels with 6 SNPs (P < 0.05), including 2 in the 5′-untranslated region (UTR), 3 in the open reading frame (ORF) region with 2 nonsynonymous SNPs, and 1 SNP in the 3′-UTR. Another set of six SNPs was associated with stem QDR levels (P < 0.05), with one localized in the promoter region and the other five in the ORF region with four nonsynonymous changes, suggesting that multiple isoforms may have antifungal activity to differing degrees. Of three common PmAMP1 haplotypes, the trees with haplotype 2 showed high QDR levels with moderate protein abundance while those trees with haplotype 3 exhibited low QDR levels in the susceptible range and the lowest level of protein accumulation. Thus, an association of gene variations with protein abundance and resistance-related traits may facilitate elucidation of physiological contribution of PmAMP1 to host resistance.
Similar content being viewed by others
Abbreviations
- AMP:
-
Anti-microbial peptide
- ORF:
-
Open reading frame
- PR:
-
Pathogenesis-related
- QDR:
-
Quantitative disease resistance
- qRT-PCR:
-
Quantitative reverse transcription-polymerase chain reaction
- SNP:
-
Single nucleotide polymorphism
- UTR:
-
Untranslated region
- WPBR:
-
White pine blister rust
References
Bowles DJ (1990) Defense-related proteins in higher plants. Annu Rev Biochem 59:873–907
Bradbury PJ, Zhang Z, Kroon DE, Casstevens TM, Ramdoss Y, Buckler ES (2007) TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23:2633–2635
Canales J, Avila C, Cánovas FM (2011) A maritime pine antimicrobial peptide involved in ammonium nutrition: pine AMP and ammonium nutrition. Plant Cell Environ 34:1443–1453
Clop A, Marcq F, Takeda H, Pirottin D, Tordoir X (2006) A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity. Nat Genet 38:813–818
Dracatos PM, Cogan NOI, Dobrowolski MP, Sawbridge TI, Spangenberg GC, Smith KF, Forster JW (2008) Discovery and genetic mapping of single nucleotide polymorphisms in candidate genes for pathogen defence response in perennial ryegrass (Lolium perenne L.). Theor Appl Genet 117:203–219
Ekramoddoullah AK, Liu J-J, Zamani A (2006) Cloning and characterization of a putative antifungal peptide gene (PmAMP1) in Pinus monticola. Phytopathology 96:164–170
Elfstrand M, Fossdal CG, Swedjemark G, Clapham D, Olsson O, Sitbon F, Sharma P, Lonneborg A, von Arnold S (2001) Identification of candidate genes for use in molecular breeding—a case study with the Norway spruce defensin-like gene, Spi1. Silvae Genet 50:75–81
Farnir F, Coppieters W, Arranz JJ, Berzi P, Cambisano N, Grisart B, Karim L, Marcq F, Moreau L, Mni M, Nezer C, Simon P, Vanmanshoven P, Wagenaar D, Georges M (2000) Extensive genome-wide linkage disequilibrium in cattle. Genome Res 10:220–227
Fins L, Byler J, Ferguson D, Harvey A, Mahalovich MF, McDonald G, Miller D, Schwandt J, Zack A (2002) Return of the giants—restoring western white pine to the inland Northwest. J For 100:20–26
Fritig B, Heitz T, Legrand M (1998) Antimicrobial proteins in induced plant defense. Curr Opin Immunol 10:16–22
Gao AG, Hakimi SM, Mittanck CA, Wu Y, Woerner BM, Stark DM, Shah DM, Liang J, Rommens CM (2000) Fungal pathogen protection in potato by expression of a plant defensin peptide. Nat Biotechnol 18:1307–1310
Hammami R, Ben Hamida J, Vergoten G, Flis I (2009) PhytAMP: a database dedicated to antimicrobial plant peptides. Nucleic Acids Res 37:D963–D968
Herrmann D, Barre P, Santoni S, Julier B (2010) Association of a CONSTANS-LIKE gene to flowering and height in autotetraploid alfalfa. Theor Appl Genet 121:865–876
Hua XJ, van de Cotte B, Van Montagu M, Verbruggen N (2001) The 5′-untranslated region of At-P5R gene is involved in both transcriptional and post-transcriptional regulation. Plant J 26:157–169
Hughes TA (2006) Regulation of gene expression by alternative untranslated regions. Trends Genet 22:119–122
Kanada S, Nakano N, Potaczek DP, Maeda K, Shimokawa N, Niwa Y, Fukai T, Sanak M, Szczeklik A, Yagita H, Okumura K, Ogawa H, Nishiyama C (2008) Two different transcription factors discriminate the -315C>T-polymorphism of the FcεRI α-chain gene: binding of Sp1 to -315C and of a high mobility group-related molecule to -315 T. J Immunol 180:8204–8210
King JN, David A, Noshad D, Smith J (2010) A review of genetic approaches to the management of blister rust in white pines. For Pathol 40:292–313
Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163
Kwan T, Benovoy D, Dias C, Gurd S, Provencher C, Beaulieu P, Hudson TJ, Sladek R, Majewski J (2008) Genome-wide analysis of transcript isoform variation in humans. Nat Genet 40:225–231
Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. doi:10.1093/bioinformatics/btp187
Liu J-J, Ekramoddoullah AK (2006) The family 10 of plant pathogenesis-related proteins: their structure, regulation, and function in response to biotic and abiotic stresses. Physiol Mol Plant Pathol 68:3–13
Liu J-J, Ekramoddoullah AK (2011) Genomic organization, induced expression and promoter activity of a resistance gene analog (PmTNL1) in western white pine (Pinus monticola). Planta 233:1041–1053
Liu J-J, Hunt R, Ekramoddoullah AK (2004a) Recent insights into genetic resistance of western white pine to white pine blister rust. Recent Res Dev Biotech Bioeng 6:65–76
Liu B, Zhang S, Zhu X, Yang Q, Wu S, Mei M, Mauleon R, Leach J, Mew T, Leung H (2004b) Candidate defense genes as predictors of quantitative blast resistance in rice. Mol Plant Microbe Interact 17:1146–1152
Liu J-J, Ekramoddoullah AK, Zamani A (2005) A class IV chitinase is up-regulated upon fungal infection and abiotic stresses and associated with slow-canker-growth resistance to Cronartium ribicola in western white pine (Pinus monticola, Dougl. Ex D. Don). Phytopathology 95:284–291
Liu J-J, Ekramoddoullah AK, Hunt RS, Zamani A (2006) Identification and characterization of random amplified polymorphic DNA markers linked to a major gene (Cr2) for resistance to Cronartium ribicola in Pinus monticola. Phytopathology 96:395–399
Liu J-J, Sturrock R, Ekramoddoullah AK (2010a) The superfamily of thaumatin-like proteins: its origin, evolution, and expression towards biological function. Plant Cell Rep 29:419–436
Liu J-J, Zamani A, Ekramoddoullah AK (2010b) Expression profiling of a complex thaumatin-like protein family in western white pine. Planta 231:637–651
Liu J-J, Sniezko RA, Ekramoddoullah AK (2011) Association of a novel Pinus monticola chitinase gene (PmCh4B) with quantitative resistance to Cronartium ribicola. Phytopathology 101:904–911
Manners JM (2009) Primitive defence: the MiAMP1 antimicrobial peptide family. Plant Mol Biol Rep 27:237–242
Marcus JP, Goulter KC, Green JL, Harrison SJ, Manners JM (1997) Purification, characterisation and cDNA cloning of an antimicrobial peptide from Macadamia integrifolia. Eur J Biochem 244:743–749
McManus AM, Nielsen KJ, Marcus JP, Harrison SJ, Green JL, Manners JM, Craik DJ (1999) MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins. J Mol Biol 293:629–638
Montesinos E (2007) Antimicrobial peptides and plant disease control. FEMS Microbiol Lett 270:1–11
Nei M, Li W-H (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 76:5269–5273
Padovan L, Scocchi M, Tossi A (2010) Structural aspects of plant antimicrobial peptides. Curr Protein Pept Sci 11:210–219
Quesada T, Gopal V, Cumbie WP, Eckert AJ, Wegrzyn JL, Neale DB, Goldfarb B, Huber DA, Casella G, Davis JM (2010) Association mapping of quantitative disease resistance in a natural population of loblolly pine (Pinus taeda L.). Genetics 186:677–686
Richardson BA, Ekramoddoulah AK, Liu J–J, Kim M-S, Klopfenstein NB (2010) Current and future molecular approaches to investigate the white pine blister rust pathosystem. For Pathol 40:314–331
Schwandt JW, Lockman IB, Kliejunas JT, Muir JA (2010) Current health issues and management strategies for white pines in the western United States and Canada. For Pathol 40:226–250
Sniezko RA, Kegley AJ, Danchok R (2008) White pine blister rust resistance in North America, Asian and European species—results from artificial inoculation trials in Oregon. Ann For Res 51:53–66
Sooriyaarachchi S, Jaber E, Covarrubias AS, Ubhayasekera W, Asiegbu FO, Mowbray SL (2011) Expression and β-glucan binding properties of Scots pine (Pinus sylvestris L.) antimicrobial protein (Sp-AMP). Plant Mol Biol 77:33–45
Van Loon LC, Rep M, Pieterse CMJ (2006) Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 44:135–162
Verma SS, Yajima WR, Rahman MH, Shah S, Liu J-J, Ekramoddoullah AKM, Kav NNV (2012) A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus). Plant Mol Biol 79:61–74
Watterson GA (1975) On the number of segregating sites in genetical models without recombination. Theor Popul Biol 7:256–276
Weir BS (1996) Genetic data analysis II. Sinauer Associates, Sunderland
Yu J, Pressoir G, Briggs WH, Vroh Bi I, Yamasaki M, Doebley JF, McMullen MD, Gaut BS, Nielsen DM, Holland JB, Kresovich S, Buckler ES (2006) A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. Nat Genet 38:203–208
Zamany A, Liu J-J, Ekramoddoullah AK, Sniezko R (2011) Antifungal activity of a Pinus monticola antimicrobial peptide 1 (PmAMP1) and its accumulation in western white pine infected with Cronartium ribicola. Can J Microbiol 57:667–679
Zarei A, Körbes AP, Younessi P, Montiel G, Champion A, Memelink J (2011) Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis. Plant Mol Biol 75:321–331
Acknowledgments
This research was supported in part by the Canadian Forest Service (CFS) and the CFS-Genomics R&D Initiative Fund awarded to J.-J.L. We thank J. Hutchinson and L. Baerg at CFS for support on DNA sequencing; J. Hill and A. Kegley at Dorena Genetic Resource Center (DGRC) for support on tree resistance assessment.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
425_2012_1747_MOESM1_ESM.pdf
Supplementary Fig. S1 Nucleotide sequence comparison of the PmAMP1 genomic sequences. Multiple sequence alignment analysis was performed using an on-line ClustalW2 program provided by the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Genomic DNA fragment was amplified using gene-specific primers from each of 150 individual trees for direct DNA sequencing. Amplified fragments showed only one unique sequence for each of 128 trees as an indicator of homozygous genotypes. Genomic DNA sequences derived from other 22 trees showed SNPs at multiple sites, suggesting a heterozygous genotype for the PmAMP1 locus in each of these trees (PDF 160 kb)
425_2012_1747_MOESM2_ESM.tif
Supplementary Fig. S2 Phylogenetic analysis of the PmAMP1 genomic sequences. Based on PmAMP1 nucleotide sequence alignment result as shown in Suppl. Fig. 1, a representative of phylogenetic trees was generated by MEGA 4.0 software using the neighbor-joining (NJ) method (TIFF 1,662 kb)
425_2012_1747_MOESM3_ESM.bmp
Supplementary Fig. S3 Linkage disequilibrium (LD) analysis of the PmAMP1 single nucleotide polymorphism (SNP) sites. Plot of intra-locus linkage disequilibrium between 16 polymorphic DNA variation sites in the PmAMP1 gene estimated using the TASSEL software package. Graphic r 2 is presented in the upper right and P-values are indicated in the lower left (BMP 1,040 kb)
425_2012_1747_MOESM4_ESM.ppt
Supplementary Fig. S4 The LD scatter-plots of squared correlation coefficient (r 2) as a function of DNA distance in base-pairs between informative polymorphic sites (f > 0.1 and P < 0.01) in the PmAMP1 gene (PPT 58 kb)
Rights and permissions
About this article
Cite this article
Liu, JJ., Zamany, A. & Sniezko, R.A. Anti-microbial peptide (AMP): nucleotide variation, gene expression, and host resistance in the white pine blister rust (WPBR) pathosystem. Planta 237, 43–54 (2013). https://doi.org/10.1007/s00425-012-1747-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-012-1747-2