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Sunflower polygalacturonase-inhibiting proteins (HaPGIP) are genetically conserved in cultivated sunflower (Helianthus annuus L.) but diverse in wild species

Molecular Breeding volume 36, Article number: 17 (2016) Cite this article

Abstract

Cultivation of sunflower is often associated with substantial pathogen-related yield losses. Natural defense mechanisms of plants might be improved by advantageous combination of genes encoding proteins that suppress important pathogenicity factors. Endopolygalacturonases (endoPGs) as cell-wall-degrading enzymes contribute to the virulence of fungi, bacterial pathogens, and oomycetes. However, plants have evolved families of polygalacturonase-inhibiting proteins (PGIPs) that specifically recognize and inactivate these polygalacturonases (PGs). Here, we describe the identification of four sunflower pgip genes. The genes were cloned, genetically mapped, in silico characterized, and subjected to expression analyses in response to pathogen infection. Three Helianthus annuus pgip genes (Hapgip2–4) are located on linkage group (LG) 10 and phylogenetically closely related to other dicot pgips. In contrast, Hapgip1 was mapped on LG1 and formed a separate Asteraceae branch, suggesting that HaPGIPs may represent structurally and functionally distinct proteins. All HaPGIPs displayed characteristic sequence structures such as the tandem repetition of leucine-rich domains. WRKY transcription factor-binding sites identified through in silico promoter analyses differed between Hapgips in number and architecture. A comprehensive diversity analysis indicated that HaPGIP1–HaPGIP4 are rather conserved in current breeding material but genetically diverse in wild species. Based on homologous positions determined as amino acids with known influence on PvPGIP–PG interaction, non-synonymous substitutions in HaPGIPs were identified in H. maximiliani, H. ciliaris, H. paradoxus, H. tuberosis, and H. petiolaris. Furthermore, relative real-time quantitative PCR analyses revealed enhanced Hapgip1–Hapgip4 expression upon Botrytis cinerea inoculation and an increased expression level of Hapgip3 in response to the oomycete Plasmopara halstedii.

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Acknowledgments

We thank Loren Rieseberg and the entire research consortium for the opportunity to use the sunflower reference genome (https://www.sunflowergenome.org/) for comparative sequence analyses and for mapping information. We are grateful to KWS SAAT SE, Einbeck, Germany (S. Wieckhorst), and University of Hohenheim, State Plant Breeding Institute, Germany (Volker Hahn), for providing seeds of sunflower accessions used for Hapgip diversity analysis. Seeds that originate from the USDA can be requested via http://www.ars-grin.gov/npgs/, French inbred lines at INRA Toulouse, France (Patrick Vincourt), respectively. We would like to thank A. Fiedler for the excellent technical assistance, O. Spring for providing Plasmopara halstedii race 730, and Ralph Hückelhoven for his generous gift of the Botrytis cinerea strain.

Authors’ contribution

M.L. designed the experiments, carried out bioinformatics analyses, and performed phytopathology experiments, SNP validation, genetic mapping and diversity analysis. S.S. designed and conducted the expression analyses. M.L., S.S, and C.C.S. drafted and revised the manuscript. All authors read and approved the final version of the manuscript.

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  1. Department of Plant Sciences, Plant Breeding, Technische Universität München, Liesel-Beckmann-Straße 2, 85354, Freising, Germany

    Maren Livaja, Sebastian Steinemann & Chris-Carolin Schön

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  1. Maren Livaja
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Correspondence to Maren Livaja.

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Figure S1

Phylogenetic relationship of sunflower PGIP proteins. Phylogenetic tree showing the evolutionary relationship of HaPGIP proteins and 49 deduced plant PGIP proteins by maximum likelihood method. The numbers at the branch nodes indicate the statistical support as obtained by 1000 bootstrap replicates, branch length corresponds to sequence distances. The bar indicates amino acid substitutions per site. (TIFF 25663 kb)

Figure S2

Distribution of W boxes within Hapgip promoters. Schematic view on putative binding sites for WRKY transcription factors in promoter regions of sunflower pgips. Numbers indicate the position in the 5′ region upstream of the transcriptional start site. The W boxes shown in rectangles represent the minimal consensus necessary for specific DNA binding. Circled are nucleotide residues directly 5′ adjacent to each element. (TIFF 1036 kb)

Text S1

Supplementary tables S1, S3–S6. The document contains an overview on amino acid variation in HaPGIP1–HaPGIP4 (Table S1), a list of primers designed for PCR and Sanger sequencing (Table S3), accession numbers of plant PGIPs used for phylogenetic analysis (Table S4) as well as information on oligonucleotides used for quantitative real-time PCR (Table S5). Further, putative cis-regulatory elements that possibly control the transcriptional activity of Hapgip gene promoters are summarized (Table S6). (DOCX 42 kb)

Supplementary material 4 (XLSX 20 kb)

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Livaja, M., Steinemann, S. & Schön, CC. Sunflower polygalacturonase-inhibiting proteins (HaPGIP) are genetically conserved in cultivated sunflower (Helianthus annuus L.) but diverse in wild species. Mol Breeding 36, 17 (2016). https://doi.org/10.1007/s11032-016-0444-4

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Keywords

  • Helianthus annuus
  • Polygalacturonase-inhibiting proteins
  • Leucine-rich repeat proteins
  • Gene diversity
  • Botrytis cinerea
  • Plasmopara halstedii