Deconstructing the plant cell wall

Serving as the largest reservoir of organic carbon on Earth, plant cell walls are metabolically inaccessible to most animals (Rose 2003). Composed primarily of cellulose, hemicellulose, lignin, and pectin, these polysaccharides define the mechanical properties and endow the wall with its characteristic rigidity (Mohnen 2008; Burton, Gidley, and Fincher 20Ю). Instrumental towards ensuring the structural integrity of the plant cell, the polysaccharidic matrix also contributes towards adhesion and signal transduction (Burton, Gidley, and Fincher 20Ю). As the foremost barrier separating the nutritionally rich cytosol from the extracellular matrix, the plant cell wall safeguards against intracellular infection by pathogens and parasites (Underwood 2012). For folivores, the benefits of degrading the plant cell wall are thus twofold: (1) an upgraded carbon economy by tapping into a recalcitrant source of energetically valuable sugars, and (2) mediating an efficient extraction of limiting nitrogenous content and lipids from the cytosol for a more balanced diet.

Microbial contributions to insect folivory include the degradation of recalcitrant plant cell wall polysaccharides

FIGURE 13.1 Microbial contributions to insect folivory include the degradation of recalcitrant plant cell wall polysaccharides (e.g. pectin, cellulose, and hemicellulose), the avoidance of noxious secondary compounds, and the preservation of leaves as their ecological niche. Overcoming these obstacles allow folivores to exploit an otherwise imbalanced nutritional resource.

Towards deconstructing the plant cell wall, a conserved battery of enzymes (e.g. glucoside hydrolases, polysaccharide lyases, etc.) are necessary to transform representative polysaccharidic sequences into simple sugars that can be metabolized throughout the digestive tract (Kubicek, Starr, and Glass 2014). Complementary to a range of endogenous digestive enzymes (Calderon-Cortes et al. 2012; McKenna et al. 2019), functional descriptions across a myriad of herbivore gut microbiomes also revealed the importance of the resident community in mediating plant biomass degradation (Martens et al. 2011; Engel, Martinson, and Moran 2012; Pope et al. 2012; Patel et al. 2014). In ruminants, relatively stable gut microbiomes serve as bioreactors for plant cell wall degradation and fermentation (Dai et al. 2015). Koalas and wombats—generalist and specialist herbivores, respectively—harbor highly convergent microbial communities enriched for the production of cellulases and xylanases to process a leafy diet (Shiffman et al. 2017). While the consistent annotation of carbohydrate-active enzymes may highlight the adaptive potential of gut microbiomes to their herbivorous hosts, assigning specific functions to individual taxa within these communities remains challenging given their complexity. As highlighted earlier, these limitations are less pronounced in the partnerships insects form with their symbionts (Douglas 2015). Stabilized through millions of years of coevolution between host and microbe, the symbioses that foliage-feeding ants and beetles engage in with microorganisms serve as some of the most streamlined partnerships both compositionally and functionally (Figure 13.2).

Leaf-cutting ants (Hymenoptera: Formicidae: Attini) are among the most prolific defoliators in tropical forests (Mueller et al. 2001; Wirth et al. 2003). Conspicuous and widespread in the New World tropics, members of the Attini tribe form

Microbial symbionts of folivores mediate host plant use across a diversity of insect hosts,

FIGURE 13.2 Microbial symbionts of folivores mediate host plant use across a diversity of insect hosts, (a) Ca Stammera capleta (above) produces two pectinolytic enzymes that facilitate degradation of plant cell walls, making the cytosolic content available for its tortoise beetle hosts (below), (b) The fungal partner of leaf-cutting ants functions as an external digestive system, both degrading most types of recalcitrant sugars present in leaves, as well as detoxifying plant phenolics. (c) Bacteria in the oral secretions of the Colorado potato beetle deceive tomato plants into perceiving herbivore attack as microbial, diverting plant resources into mounting an incorrect defense strategy (credit Tavo Romann). (d) The velvet bean caterpillar seems to avoid plant protease inhibitors by associating with gut bacteria whose proteases are unaffected by plant inhibitors, (e) Gypsy moth caterpillars feeding on quaking aspen acquire a gut microbiome able to degrade their host plant salicinoids. (f) Wolbachia symbionts allow leaf miners to maintain a green island in yellowing leaves, preserving their food source despite a decaying environment (credit Dr. Susannah Lydon).

enormous colonies that can host millions of workers and feature nests that subsist for decades (Wirth et al. 2003). Displaying a division of labor characteristic of other social insects, colonies host reproductively active queens, and differentially sized morphological worker castes divided to fulfill an array of specialized tasks (Weber 1966; Currie and Stuart 2001). This includes defending the colony, clearing the nest of debris and waste, and foraging for freshly cut leaves (Currie and Stuart 2001). As individual colonies are capable of foraging hundreds of kilograms of leaves each year, leaf-cutting ants drastically alter forest ecosystems and drive nutrient cycling (Wirth et al. 2003). But at the center of the ants’ remarkable capacity to deconstruct massive amounts of plant biomass is a specialized, cultivated leucocoprineous fungus that the colony depends on for nutrition (Weber 1966; Mueller et al. 2001). A partnership dated at ~60 million years old, attini ants and their cultivars have diversified rapidly since the origin of the symbiosis, totaling 220 known species (Mueller et al. 2001). Provisioning their cultivars with fresh leaves, the ants manage their fungiculure in ways that parallel human agricultural practices (Mueller et al. 2005). Here, the ants inoculate, manipulate and transplant their cultivars to maximize growth. Carefully tended and continuously manured through fecal fluids, the fungus is cultivated for several weeks ahead of harvesting the mature mycelium and its associated gongylidia (specialized nodules) (Mueller et al. 2005). Rich in nutrients, the gongylidia serve as vacuolized bundles that nourish the colony. Larval development depends entirely on consuming the cultivated fungus (Quinlan and Cherrett 1978), while adult workers supplement their cultivar meals with sugary extracts from floral nectaries and other plant juices (Murakami and Higashi 1997). Given the elemental role of the cultivar, attine ants transmit the fungus vertically through trophophoresy (Wirth et al. 2003). Acquiring inocula from their natal gardens, reproductive females transfer the fungal cultivar via specialized pockets towards establishing a starter culture (Mueller et al. 2005).

As the main food source for the colony, the cultivar’s primary function is to convert plant biomass into nutrients for the ants (Suen et al. 2010). Serving as an external digestive system, the central metabolic features of the fungal gardens within the nest functionally resemble the gut microbiomes of bovines and other folivorous ruminants (Suen et al. 2010; Aylward et al. 2015). Structurally differentiated into distinct strata, fresh foliar material is introduced only to the top layer ahead of stepwise degradation process that lasts for six weeks and concludes with the removal and expulsion of degraded biomass by worker ants into refuse dumps (Wirth et al. 2003). Throughout this process, the quantification of plant cell wall polysaccharides revealed marked reduction in cellulose, hemicellulose and pectin. Most prominently, cellulose content decreased by 30% following passage through the fungal garden (Suen et al. 2010). Genome annotation of cultivars isolated from Atta cephalotes and Acromyrmex echinatior identified 145 predicted plant biomassdegrading enzymes, including 81 glycoside hydrolases, 6 polysaccharide lyases and nine carbohydrate esterases (Aylward et al. 2013). Complementary metaproteomic characterization predicted many of these enzymes hydrolyze the complete spectrum of polysaccharides that comprise the plant cell wall. Gene expression analyses of the cultivar revealed that genes encoding plant cell wall degrading enzymes reached their highest expression in the bottom section of the fungal garden (Grell et al. 2013). This is consistent with the observed physical transformation of plant substrates from green, leafy material on the top layer, to an amalgam of degraded biomass and mature fungal cells. Strikingly, many of these glycolytic enzymes are characterized in the fecal droplets the ants deposit to manure the garden throughout the cultivation process (Schiptt et al. 2010). This suggests that the proteins survive the ants’ digestive system ahead of reapplication to fresh plant material, possibly as a secondary adaptation to prolong the deconstructive efficacy of enzymes within the gardens (Schiptt et al. 2010).

While enormous colony sizes and a clear division of labor allows for leaf-cutting ants to engage in an ancient agricultural practice that culminates with outsourcing essential digestive processes to an external partner, other insects coopt microbial metabolic diversity by engaging in highly intimate nutritional symbioses. One clear example involves tortoise leaf beetles (Coleoptera; Chrysomelidae: Cassidinae) and their symbiont, Candidatus Stammera capleta (henceforth Stammera). With ~3000 described species arranged in 170 genera and 24 tribes, cassidines are an exceptionally diverse group of herbivorous beetles (Chaboo 2007). Despite their cosmopolitan distribution, cassidines nonetheless diversified most rapidly in the tropics where their biogeography closely aligns with their host plants (Windsor 1987; Windsor, Riley, and Stockwell 1992; Morrison and Windsor 2018). Towards acquiring essential digestive enzymes to process a strictly folivorous diet, tortoise leaf beetles engage in a highly streamlined symbiosis with Stammera.

Localized extracellularly in symbiotic organs connected to the foregut, Stammera populations are maintained as monocultures resulting in low strain diversity across host populations (Salem et al. 2017). Strikingly, given the microbe’s localization, Stammera possesses a drastically reduced genome (0.27 Mb) that is largely dedicated to informational processing (transcription, translation), replication, and the production essential digestive enzymes. Encoded w'ithin Stammera's limited metabolism are two pectinolytic enzymes, polygalacturonase and rhamnogalacturonan lyase (Salem et al. 2017). The former is an endo-active glycoside hydrolase (family 28) that cleaves homogalacturonan, nature’s most abundant pectic class; while the latter is a polysaccharide lyase (family 4) that hydrolyzes the heteropolymeric backbone of pectin through a beta-elimination reaction (Salem et al. 2017). Collectively, both symbiont-derived enzymes are responsible for the insect’s pectinolytic phenotype, an essential adaptation given the high abundance of pectin in foliage (Burton. Gidley, and Fincher 2010) and the recalcitrant complexity of the polysaccharide (Mohnen 2008). Consistent with the specialized role of Stammera, symbiont elimination diminishes the digestive capacity of cassidines, notably in relation to the two pectic classes (Salem et al. 2017). This corresponds with low larval survivorship and slow- developmental times relative to symbiotic insects. Given the mutualistic impact of Stammera, female leaf beetles ensure a stable continuum of the symbiosis through strict vertical transmission by packaging the microbe into caplet-like structures deposited individually over the anterior pole of each egg. Upon hatching, emerging larva consume the caplets, and in the process, acquire their starting inoculum of a pectinase-producing partner.

In line with the assessment that Stammera possesses the smallest know-n genome of any extracellular microbe (Salem et al. 2017), transcriptional profiling revealed a tightly regulated and metabolically integrated symbiosis, expanding our view of the minimal metabolism required to sustain life outside of a host cell (Bauer et al. 2020). In contrast to endosymbionts w-ith highly reduced genomes, Stammera does not utilize aerobic respiration for energy generation. Rather, energy production and the recovery of reducing equivalents are achieved through the oxidation of sugars to pyruvate via glycolysis, followed by the fermentation of pyruvate to lactate through the activity of lactate dehydrogenase (Bauer et al. 2020). Aerobic respiration is typically conserved in most nutritional endosymbionts since amino acid and vitamin biosynthesis relies on precursors produced through the citric acid cycle, tying respiratory energy generation with the mutualistic factors that underlie the host-symbiont partnership (McCutcheon and Moran 2012). But since Stammera’s mutualistic role does not lie in supplementing micronutrients, selection to maintain a complex respiratory apparatus is relaxed in favor of fermentative one. While less efficient in terms of ATP output per unit of glucose, the symbiont’s obligate reliance on fermentation for energy generation is unlikely to be costly given the host’s carbohydrate-rich diet. This is supported by the upregulation of sugar transport into the symbiotic organ to fuel the minimal fermentative metabolism of an essential digestive symbiont (Bauer et al. 2020).

Broadscale genome sequencing of representative Stammera strains revealed that the differential distribution of symbiont-encoded pectinolytic enzymes can drastically shape the digestive physiology of Cassidinae beetles with direct implications on host plant use (Salem et al. In press). Conserved across the Stammera pangenome is the ability to produce and supplement polygalacturonase, highlighting the homogalacturonan-targeting pectinase as a foundational enzyme for the stability of the symbiosis with cassidines. In contrast, the annotation of rhamnogalacturonan lyase is limited to a subset of Stammera strains, as is the ability to deconstruct the heteropolymeric sequence of pectin (Salem et al. In press). Consistent with in silico predictions, beetles harboring Stammera encoding polygalacturonase and rhamnogalacturonan lyase display a greater pectinolytic range relative to cassidines whose symbionts only supplement the former of the two digestive enzymes (Salem et al. In press). Matching an ability to metabolize a greater diversity of universal plant polysaccharides, cassidines deploying both pectinases have radiated to exploit a wider range of host plants. In reconciling detailed records of life history traits with comparative genomics, transcriptomics and biochemical assays, the symbiosis between tortoise leaf beetles and Stammera serves as an example of how small changes to a symbiont’s metabolic range can drastically impact the phenotypic complexity and the adaptive potential of its metazoan host.

< Prev   CONTENTS   Source   Next >