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Anti-microbial peptide (AMP): nucleotide variation, gene expression, and host resistance in the white pine blister rust (WPBR) pathosystem

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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.

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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

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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.

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Authors and Affiliations

  1. Natural Resources Canada, Pacific Forestry Centre, Canadian Forest Service, 506 West Burnside Road, Victoria, BC, V8Z 1M5, Canada

    Jun-Jun Liu & Arezoo Zamany

  2. Dorena Genetic Resource Center, USDA Forest Service, 34963 Shoreview Road, Cottage Grove, OR, 97424, USA

    Richard A. Sniezko

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  1. Jun-Jun Liu
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Correspondence to Jun-Jun Liu.

Electronic supplementary material

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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)

Supplementary material 5 (DOC 31 kb)

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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

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