Don’t Cry for Me: Evolutionary and Functional Analysis of Two Rice Pollen Allergens, Ory s 1 and Ory s 12

Abstract

Hay fever is a globally relevant disease that is characterised by sneezing, itchiness of eyes and general discomfort. Some proteins in pollen react with human Immunoglobulin E (IgE) to trigger an immune response, and these proteins are called allergens. Allergens are abundant in grass pollen and, due to grass being wind pollinated, is difficult to avoid. Despite the high prevalence of hay fever and grass pollen allergy, little is known about how these allergen genes evolved, what the specific function of these genes are and how they pertain to pollen development, and whether there is potential for the development of a hypoallergenic derivative in grasses. To investigate how allergen genes evolved in grasses, I conducted a phylogenetic and structural analysis of allergens in the model grass, rice (Oryza sativa). Two pollen allergens and allergen families were identified; Ory s 1 belonged to the group 1 grass pollen allergen family homologous to β-expansins, and Ory s 12 belonged to the group 12 allergen family homologous to profilins. Group 1 genes were found only in monocots and appeared to have evolved recently and rapidly following speciation, and most grass species contained 3-4 copied of these genes. However, group 12 genes arose from an ancient duplication that resulted in reproductively expressed and vegetatively expressed copies that were found in most monocots and dicots. No correlation was found between the evolutionary patterns of Ory s 1 and Ory s 12, suggesting that these two gene families evolved allergenicity independently of each other. To understand the function of allergen genes, 3 mutant Ory s 1 and 3 mutant Ory s 12 lines were generated using the CRISPR-Cas9 system. These mutant lines were assessed for pollen fertility, pollen tube germination and growth rates, and seed fertility. All mutant lines had normal pollen fertility, but showed low pollen tube germination rates and slow pollen tube development, leading to lowered seed setting. To understand this further, wild type protein was transiently over-expressed with GFP (green fluorescent protein) in pollen grains. Ory s 1 caused the pollen tubes to grow rapidly compared to the wild type, suggesting Ory s 1 is involved in efficient pollen tube growth to promote rapid fertilisation of the ovule. Overexpression of Ory s 12 lead to aborted pollen tubes, similar to the mutant phenotype, suggesting that this profilin may be involved in preventing over-polymerization or under-polymerization of actin filaments, both of which may prevent efficient pollen tube growth. Interestingly, these two allergens have highly specific, but different, roles in pollen tube growth, suggesting allergenicity is not a result of shared function but of shared abundance in the mature pollen grain. To investigate the potential for a hypoallergenic derivative, the mutant lines were assessed for seed fertility and assayed against a pool of pollen allergy patients. Ory s 12 mutants showed low seed setting, and are unlikely to be suitable as a semi-viable plant. However, homozygous mutant Ory s 1 plants showed higher seed setting rates under some environmental conditions and maintained some, if not all, viability. The homozygous mutant pollen proteins were assayed against human allergen patients from Queensland, and showed almost no binding to human sera, unlike pollen proteins from the wild type and mutant Ory s 12 lines, suggesting the Ory s 1 mutant is both a viable and hypoallergenic rice derivative. In summary, Ory s 1 and Ory s 12 have evolved independently of each other, share similar localisation in the mature pollen grain and have highly specific roles in pollen tube development. However, a homozygous Ory s 1 mutant showed decreased binding to human allergy patient sera, suggesting that the construction of viable hypoallergenic grass derivatives is possible.