Mass Spectrometric Analysis Reveals Remnants of Host–Pathogen Molecular Interactions at the Starch Granule Surface in Wheat Endosperm

Abstract

The starch granules of wheat seed are solar energy-driven deposits of fixed carbon and, as such, present themselves as targets of pathogen attack. The seed's array of antimicrobial proteins, peptides, and small molecules comprises a molecular defense against penetrating pathogens. In turn, pathogens exhibit an arsenal of enzymes to facilitate the degradation of the host's endosperm. In this context, the starch granule surface is a relatively unexplored domain in which unique molecular barriers may be deployed to defend against and inhibit the late stages of infection. Therefore, it was compelling to explore the starch granule surface in mature wheat seed, which revealed evidence of host-pathogen molecular interactions that may have occurred during grain development. In this study, starch granules from the soft wheat Triticum aestivum cv. AC Andrew and hard wheat T. turgidum durum were isolated and water washed 20 times, and their surface proteins were digested in situ with trypsin. The peptides liberated into the supernatant and the peptides remaining at the starch granule surface were separately examined. In this way, we demonstrated that the identified proteins have a strong affinity for the starch granule surface. Proteins with known antimicrobial activity were identified, as well as several proteins from the plant pathogens Agrobacterium tumefaciens, Pectobacterium carotovorum, Fusarium graminearum, Magnaporthe grisea, Xanthomonas axonopodis, and X. oryzae. Although most of these peptides corresponded to uncharacterized hypothetical proteins of fungal pathogens, several peptide fragments were identical to cytosolic and membrane proteins of specific microbial pathogens. During development and maturation, wheat seed appeared to have resisted infection and lysed the pathogens where, upon desiccation, the molecular evidence remained fixed at the starch granule surface.

The starch granule associated proteomes of commercially purified starch reference materials from rice and maize

“…The peptides associated with the starch granule surface were collected according to our modified protocol [17]. Briefly, the trypsin-treated granules were centrifuged at 18,000 ×g for 1 min.…”

“…Many of these proteins are enzymes involved in starch biosynthesis, and include adenosine 5′ diphosphate-glucose pyrophosphorylase, granule-bound starch synthase (GBSS), soluble starch synthase, starch branching enzyme, starch debranching enzyme, and plastidial starch phosphorylase [16]. Other proteins are involved in defense, transport, signaling, protein folding, and stress response [17,18].…”

“…It is for this reason that we initiated a tandem mass-spectrometry-based survey of commercially available starch reference materials to catalog their trace proteomes. We have previously investigated the SGAPs in wheat [17,27] using strict washing protocols at a laboratory scale. Here, we have investigated the proteomes of rice and maize starch granules following commercial starch purification.…”

Alpha-Lactalbumin in Human Milk Alters the Proteolytic Degradation of Soluble CD14 by Forming a Complex

“…Protein bands were excised from SDS PAGE gels and subjected to in-gel tryptic digestion as previously described (19,20). Tryptic digestions were performed for 8 h at 37°C with sequencing grade trypsin (Promega, Madison, WI).…”

Diversity and distribution of puroindoline-D1 genes in Aegilops tauschii

“…The tryptophan domain is WRWWKWWK and is located between the third and fourth Pina cysteine and is WPTKWWK for Pinb. The amino acid sequences of both puroindolines (Pina and Pinb) have up to 60 % similarity in bread wheat (Wall et al 2010). In bread wheat, the texture of the soft endosperm is associated with the wild-type allelic forms of Pina and Pinb, whereas in hard wheat the texture of the endosperm is associated with mutations in these two genes (Chebotar et al 2012;Xia et al 2008).…”