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

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.