Elsevier

Methods

Volume 32, Issue 3, March 2004, Pages 340-345
Methods

Knocking out expression of plant allergen genes

https://doi.org/10.1016/j.ymeth.2003.08.011Get rights and content

Abstract

Pollen of many grasses, trees, and weeds are the source of inhalant allergic proteins while various other plant products are allergenic only upon their ingestion as a food source. Allergenic proteins of pollen are exposed to human immune system after their rapid release from pollen upon coming in contact with moist surface of nasal mucosa. The advent of molecular cloning and ability to genetically transform plants now offer unprecedented opportunities to produce hypoallergenic plants by targeted switching off allergen production. Gene silencing strategies that operate at post-transcriptional level are highly suitable for blocking allergen production. We have demonstrated the concept of allergen gene silencing through antisense approach by producing ryegrass plants that do not produce major allergen in its pollen. Our results show the potential of antisense approach in reducing the allergenic potential of plants. Such a strategy can have a general applicability for production of transgenic plants depleted of both inhaled and ingested allergens. In addition, such an approach could also help in elucidating the in vivo function of allergen(s) in plants and contribution of an allergen to overall allergenic potential of an allergen source.

Introduction

Allergens are defined by their ability to cause the induction of hypersensitivity response when encountered by an immune system of sensitive individuals. Inhalation or ingestion of potential allergens leads to production of allergen-specific IgE antibodies. The incidence and severity of allergic disorders is steadily increasing worldwide. Exposure to common environmental antigens is the cause of allergic conditions such as hay fever, allergic asthma, and eczema affecting up to 25% of the population in developed countries.

Pollen grains of various weeds, trees, and grasses are significant source of inhalant allergens. In various regions of the world, the pollen of grass genera is the causative factor of respiratory allergy in more individuals than any other flowering plant family. At least 40% of type 1 allergic patients are sensitized against grass pollen allergens thus making grass pollen the most frequent elicitor of allergic symptoms [1]. Ryegrass and closely related grasses of subfamily Pooideae are distributed in cool temperate regions in North America, Europe, and Southern parts of Australia. In warm temperate and sub-tropical environment, pollen of Bermuda grass (subfamily, Chloridoideae) is an important source of allergens. Atopic individuals can also suffer from ingested food-based allergens. Food allergy affects 8% of children under 3 years of age and approximately 2% of the adult population. The perceived risks of allergies associated with foods containing biotechnology-based modifications have also highlighted the allergy problem. Most of the inhalant or food allergens of plant origin are proteins ranging from 10 to 50 kDa.

The advent of molecular biology era has led to isolation and cloning of genes encoding these allergens and potential applications of recombinant allergens in allergy diagnosis and therapy [2], [3], [4], [5], [6]. Besides, there is the exciting possibility of blocking allergen production by knocking out genes encoding these proteins. Any strategy that focuses on knocking out single genes, for example, the genetic selection of mutagenized plant populations is unlikely to lead to significant reduction in allergen production because most of the allergen proteins are present as isoforms encoded by genes belonging to multigene families. Therefore, post-transcriptional gene silencing such as use of antisense or RNAi based constructs are only feasible approaches to remove allergen production. Sequence micro-heterogeneity between different isoforms is a commonly observed feature of allergens. However, most of the genes encoding isoforms have conserved stretches of identical nucleotide sequences. It is possible to silence all the isoforms of a target allergen by using an interfering construct that will have significant homology to common nucleotide sequences shared by all the isoforms.

In grasses, two different proteins designated as belonging to groups 1 and 5 have been described as the major allergens [2]. More than 95% of grass pollen allergic patients show sera IgE antibodies that react with these allergens [3], [4]. Group 1 allergens, 30–35 kDa proteins, are present in the cytoplasm of the pollen grain but are released rapidly upon hydration [3]. The cDNAs encoding group 1 allergens from various grass genera have been cloned [2]. Group 5 allergens are highly IgE reactive proteins of molecular mass 28–32 kDa and were initially identified as IgE reactive fusion proteins in grass pollen cDNA expression libraries [4]. Group 1 and group 5 together account for most of the IgE binding capacity of crude pollen extracts. Both group 1 and 5 allergens exist as multiple isoforms encoded by multigene families [2], [7].

In our laboratory, we have demonstrated the concept of allergen gene silencing through antisense approach by producing ryegrass plants that do not produce major allergen, Lol p 5, in its pollen [8]. The mechanism of antisense action is based on hybridization of antisense sequence to complementary sequence in mRNA molecule coding for a specific protein. When produced in cell, antisense RNA behaves in a dominant manner, i.e., if the sequences have high levels of nucleotide identity, antisense RNA produced by one introduced sequence can ablate the function of all the related genes. This is particularly relevant for group 5 grass pollen allergens. For example, two Lol p 5 isoforms, Lol p 5a and Lol p 5b, show only 70% identity at the nucleotide level but both their sequences share long stretches of identical sequences, implying that the antisense DNA construct based on any one of these two isoforms should result in depletion of both isoforms. In our experiments an antisense construct based on Lol p 5A regulated by a pollen-specific promoter was used to genetically engineer grass plants. The transgenic pollen showed remarkably reduced allergenicity as reflected by the low IgE binding capacity of pollen extract as compared to control pollen [8]. Our results thus provided proof of the principle for the concept of allergen removal through gene silencing. This principle is potentially applicable for removing inhalant or food allergens in plants amenable to genetic transformation.

In the following sections, we will briefly describe the design of antisense construct for the silencing of Lol p 5 and the methodologies for the generation of hypoallergenic ryegrass plants lacking Lol p 5 production in pollen (Fig. 1).

Section snippets

Generation of antisense construct

Since grass allergens (such as Lol p 1 and 5) show pollen-specific expression, pre-requisite for the preparation of antisense construct is the use of a strong pollen-specific gene promoter so that production of endogenous and antisense RNA is well coordinated during pollen development to deplete the allergen in pollen. Accordingly, we used antisense construct comprising Lol p 5A and Ory s1 promoter sequences. Ory s1 promoter, originally isolated from rice, has been shown to be pollen specific

Concluding remarks

Recent advances in recombinant DNA technology and plant biotechnology have provided an opportunity to address some of the fundamental questions underpinning pollen allergy. Plant genetic engineering has made the introduction of foreign or synthetic genes possible in plants including grasses. The most successful and therefore favoured gene delivery system for monocots involves direct introduction of DNA constructs into intact cells using high velocity microprojectile bombardment while

Acknowledgements

The financial support from ARC and Melbourne Research Grant Scheme of The University of Melbourne is gratefully acknowledged.

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