Small, but mitey: investigating the molecular genetic basis for mite domatia development and intraspecific variation in Vitis riparia using transcriptomics

Introduction

Mutualisms between plants and arthropods have evolved repeatedly across evolutionary time (Blattner et al., 2001; Bronstein et al., 2006), promoting the evolution of unique, heritable structures in plants that attract, reward, or protect mutualists (Romero & Benson, 2005; Bronstein et al., 2006). Investigating the genetic basis of mutualistic structures provides critical insights into how mutualisms evolved. Mite domatia (hereafter ‘domatia’) are tiny plant structures produced by many woody plant species on the underside of leaves that provide shelter for beneficial mites. Domatia facilitate a bodyguard mutualism between plants and mites: Mites benefit from the refuge provided by the domatia, which protects them from predators (Grostal & O'Dowd, 1994; Norton et al., 2001; Faraji et al., 2002a,b; Romero & Benson, 2005), and in return, plants receive protection from pathogenic fungi and/or herbivory via fungivorous and/or predacious mites (Agrawal & Karban, 1997; Norton et al., 2000; Romero & Benson, 2004). Domatia are common defenses in natural systems: They are present in over 5000 plant species and make up a large proportion of woody plant species in temperate deciduous forests (e.g. c. 50% of woody plant species in forests in Korea (O'Dowd & Pemberton, 1998) and Eastern North America (Willson, 1991)). They are present in several crop plants and have been studied as a pest control strategy in agriculture (Romero & Benson, 2005; Barba et al., 2019). Yet, despite their agricultural and ecological importance, we know relatively little about the genetic underpinnings of mite domatia in plants.

The genus Vitis is a powerful group for studying the genetics of domatia due to their heritable variation in domatia presence and size (English-Loeb et al., 2002; Graham et al., 2023) and the genetic and germplasm resources available. In Vitis, domatia are constitutive structures comprised of small, dense tufts of trichomes covering a depression/pit in the leaf surface in the abaxial vein axils, termed ‘tuft’ domatia. Norton et al. (2000) demonstrated that domatia in Vitis riparia, a wild grapevine species with relatively large domatia, led to a 48% reduction in powdery mildew in comparison with V. riparia plants with blocked domatia, which were inaccessible to mites. Given how effective domatia are as biological control agents in this system, there is interest in understanding domatia in domesticated grapevine (Vitis vinifera) and related species. The species V. riparia has been utilized for studies investigating domatia in Vitis due to the large variation of domatia size present in V. riparia (English-Loeb & Norton, 2006). Vitis riparia leaves have also been shown to harbor both beneficial fungivorous mites (Orthotydeus lambi) (English-Loeb et al., 1999) and predatory mites (Typhlodromus caudiglans and Typhlodromus pyri) (English-Loeb et al., 2002).

Two previous studies investigated the genetic basis of domatia in Vitis (Barba et al., 2019; LaPlante et al., 2021). Barba et al. (2019) measured mite abundance, domatia, and general trichome traits in the segregating F₁ family of a complex Vitis hybrid cross. They identified multiple quantitative trait loci (QTLs) influencing domatia-related traits, including a major QTL on chromosome 1. They also found additional support for a relationship between overall leaf and leaf trichome development, previously demonstrated in Vitis (Chitwood et al., 2014). LaPlante et al. (2021) investigated the genetic basis of trichome and domatia traits in a genome-wide association study (GWAS) using a common garden of V. vinifera cultivars. They identified a single nucleotide polymorphism (SNP) associated with domatia hair density near several candidate genes on chromosome 5. Only one gene was identified that was shared in both studies: Glabrous Inflorescence Stems 2 (VIT_205s0077g01390), which is thought to encode a zinc finger protein (ZFP) that regulates trichome development (LaPlante et al., 2021). The minimal overlap between the two studies is likely due to differences in the scale of genetic diversity investigated in QTL mapping and GWAS. As a result, the various molecular pathways involved in domatia development remain relatively unknown.

While little is known about the development of tuft domatia specifically, work in related structures in other species may provide clues regarding the genes involved in domatia development. Substantial work has characterized the genes involved in the development of trichomes, which are an essential component of tuft domatia. However, the molecular pathways involved in trichome development have yet to be elucidated in Vitis, though they have been characterized in other angiosperms. Further, increased trichome density is not the only component of domatia; the pit that forms in the lamina underneath the trichomes is also a key element with uncharacterized developmental pathways. The molecular mechanisms regulating leaf pit or laminar invagination formation in other systems, such as leaf pit galls, are also unresolved. Previous work has characterized the genes involved in another form of domatia that house ants, called tuber domatia. Tuber domatia are functionally like tuft domatia in providing shelter for mutualistic arthropods in return for defense but are tubers formed from stem tissue. The genes involved in domatia development may overlap with those previously implicated in trichome and tuber domatia development, providing additional hypotheses to investigate regarding domatia development in V. riparia.

Here, we investigate the molecular genetic mechanisms of development and intraspecific variation in domatia of V. riparia, the riverbank grape. We hypothesized that genes differentially expressed in V. riparia domatia (1) share similarities with pathways previously identified in trichome development, including transcription factors (TFs) and cell wall modification pathways (Dong et al., 2022; Han et al., 2022), (2) are involved in responses to biotic organisms as has been previously identified with functionally similar tuber domatia (Pu et al., 2021), and (3) involve auxin-signaling due to its role in both trichome (Han et al., 2022) and tuber domatia development (Pu et al., 2021). We also hypothesized that intraspecific variation in domatia size in V. riparia may be driven by differences in overall leaf morphology, as previous work has demonstrated a link between leaf morphology and trichomes in Vitis (Chitwood et al., 2014; Barba et al., 2019). We sequenced the transcriptomes of domatia in two V. riparia genotypes that differ in their investment in domatia alongside control leaf tissue to identify key pathways involved in domatium development in Vitis. We also landmarked leaves from these two genotypes and compared leaf shapes to identify possible morphological differences that may impact domatia traits.