Evolution and functional characterization of CAZymes belonging to subfamily 10 of glycoside hydrolase family 5 (GH5_10) in two species of phytophagous beetles

Abstract

Hemicelluloses, such as xyloglucan, xylan and mannans, consist of a heterogeneous array of plant-derived polysaccharides that form the plant cell wall. These polysaccharides differ from each other in their structure and physiochemical properties, but they share a β-(1,4)-linked sugar backbone. Hemicelluloses can be hydrolyzed by plant-cell-wall-degrading enzymes (PCWDEs), which are widely distributed in phytopathogenic microbes. Recently, it has become apparent that phytophagous beetles also produce their own PCWDEs. Our previous work identified genes encoding putative mannanases belonging to the subfamily 10 of glycoside hydrolase (GH) family 5 (GH5_10) in the genomes of the leaf beetle, Gastrophysa viridula (Chrysomelidae, Chrysomelinae; one gene), and of the bean beetle, Callosobruchus maculatus (Chrysomelidae, Bruchinae; four genes). In contrast to proteins from other GH5 subfamilies, GH5_10 proteins are patchily distributed within the tree of life and have so far hardly been investigated. We addressed the following questions: Are beetle-derived GH5_10s active PCWDEs? How did they evolve? What is their physiological function? Using heterologous protein expression and enzymatic assays, we show that the G. viridula GH5_10 protein is an endo-β-1,4-mannanase. We also demonstrate that only one out of four C. maculatus GH5_10 proteins is an endo-β-1,4-mannanase, which has additional activity on carboxymethyl cellulose. Unexpectedly, another C. maculatus GH5_10 protein has evolved to use xylan instead of mannans as a substrate. RNAi experiments in G. viridula indicate (i) that the sole GH5_10 protein is responsible for breaking down mannans in the gut and (ii) that this breakdown may rather be accessory and may facilitate access to plant cell content, which is rich in nitrogen and simple sugars. Phylogenetic analyses indicate that coleopteran-derived GH5_10 proteins cluster together with Chelicerata-derived ones. Interestingly, other insect-derived GH5_10 proteins cluster elsewhere, suggesting insects have several independent evolutionary origins.

Cellulose degradation in Gastrophysa viridula (Coleoptera: Chrysomelidae): functional characterization of two CAZymes belonging to glycoside hydrolase family 45 reveals a novel enzymatic activity

“…The patterns of esterified pectin and beet pectin show the least degradation, most likely due to substitutions of the pectin polymer with either methyl ester (esterified pectin) or acetyl groups (beet pectin). These results are in agreement with our previous work confirming both mannanase (Busch et al, ) and pectinase activity (Kirsch et al, ) in G. viridula .…”

“…If this logic is true, and taking into account that protein‐bound nitrogen is the limiting factor required for growth in phytophagous insects (Kainulainen et al, ; Rossi et al, ; Kerslake et al, ), reduced access to plant cells should result in striking phenotype differences compared to control animals. However, and as stated elsewhere (Busch et al, ), reduced xyloglucanase activity in GH45‐1‐silenced insects may be compensated for by the action of other glycoside hydrolase families present in G. viridula, such as GH5 mannanases (Busch et al, ) or GH28 pectinases (Kirsch et al, ). It is also possible that the gut microbiota may contribute to the degradation of the plant cell wall in phytophagous beetles and may compensate for the reduction of the xyloglucanase and cellulase activity due to the silencing of the GH45 genes.…”

“…As a consequence, the animal may take advantage of the released sugar monomers and, more important, improve its access to the nitrogen‐rich plant cell content. Based on the present work and our previous studies, focusing on GH28 pectinases and GH5 mannanases (Kirsch et al, ; Busch et al, ), G. viridula possesses the ability to break down almost every component of the plant primary cell wall, i.e. pectins, mannans, amorphous cellulose and xyloglucan.…”

“…Although co‐incubation experiments with both G. viridula GH45s in our lab did not support this hypothesis, it is likely that other GH families encoded by G. viridula may contribute to the proposed synergism, e.g. GH5 and GH28s (Kirsch et al, ; Busch et al, ).…”

“…A similar lack of striking changes in phenotype was observed in GH45‐1 silenced insects compared to controls, suggesting that the breakdown products of the individual cell wall polysaccharides may play a less important role in nutrient acquisition than anticipated. A lack of striking changes in the phenotype of RNAi‐treated insects may have occurred because both GH45 enzymes may function as secondary enzymes by allowing the insect to access the plant cell content rather than being responsible for the exploitation of the plant cell wall polysaccharides for metabolic purposes, as has been suggested in our previous work (Busch et al, ). Consequently, the insect would not need to break down the recalcitrant polysaccharide network down to sugar monomers, but would gain access to many simple sugars and proteins from the plant cells.…”