Microbial enzymes are key to the digestion of pectin in leaf nails

In a new study, a team of researchers at the Max Planck Institute for Chemical Ecology in Jena, Germany, shows how leaf beetles have successfully exploited new and previously indigestible food sources
during evolution.
These insects obtain enzymes from microbes through horizontal gene transfer, enabling them to degrade pectin, a solid component of plant cell walls
.
Because the degradation products produced by pectin digestion are not inherently critical to the growth and development of beetles, the researchers concluded that beetles destroy the cell walls to obtain protein-rich cytoplasm from plant cells, which is the nutrients
they need.

Fossilized discoveries of insects preying on destroyed plants prove that insects have been using plants as a food source for more than 400 million years
.
Researchers led by Roy Kirsch and Yannick Pauchet of the Insect Symbiosis Department are investigating how herbivorous insects first break down hard-to-digest components of
plant food.
In their earlier work, they have shown that pectin-degrading enzymes are
widespread in herbivorous beetle species.
They were also able to show that the enzymes of these beetles always came from microorganisms
.
Therefore, the question of the current study is how important these enzymes are to the nutrition and health of insects, in this case, the mustard leaf beetle Phaedon cochleariae
.

"Our goal is to better understand how herbivorous insects handle plant cell walls, which are their primary food
.
Pectin is the matrix of embedded cellulose and hemicellulose fibers within the cell wall of plants, and is the main component
of the intermediate layer that connects cells.
Therefore, pectin must first be digested so that cellulase and hemicellulase can come into contact with their substrates, and plant cells are eventually liberated from their protective cell walls
.
In this case, the role of pectinase, the pectin-degrading enzyme, is key to efficient digestion of leaf beetle food," said
first author Roy Kirsch.

To study the role of pectinase, the researchers generated beetle strains
that these enzymes did not exist.
At first, this was much
more difficult than expected.
"Even a drastic reduction in pectinase activity by knockout RNAi experiments in leaf beetle larvae is not enough to effectively inhibit pectin digestion
.
" We can only completely knock out pectinase-encoded genes in leaf beetles by using CRISPR/Cas9 genome editing," said study leader Yannick Pauchet, who underscored the importance of "gene scissors," Emmanuelle Charpentier and Jennifer Doudna who won the 2020 Nobel Prize
in Chemistry for the technique.

The larvae of the "pectinase deletion mutant" produced by this method have very low
viability.
A further question now is whether oral administration of pectin-digested degradation products to beetle larvae improves survival of
these beetles.
However, feeding experiments have shown that this is not the case
.
"On the one hand, the result was a bit of a surprise
for us.
But on the other hand, this is an important indicator, and leaf beetles do not digest pectin, using their decomposition products for their own metabolism, but instead destroy pectin and other plant cell wall polysaccharides to access the protein-rich cytoplasm of plant cells," Roy Kirsch concludes the results
.

In recent years, studies including the Yannick Pauchet project team have shown that obtaining microbial enzymes through horizontal gene transfer contributes to the abundance of leaf beetle species, but also contributes to representatives of other insect families, such as weevils, bark beetles and slub worms
.
Getting enzymes from other microbes is like an evolutionary shortcut; In any case, it creates the conditions
for insects to turn on pectin-rich plants as a food source.
"Perhaps somewhat ironically, pectinase may have evolved in plants first to dynamically adjust pectin structures to adapt to the changing needs
of plants.
" These enzymes are then acquired by plant pathogenic microorganisms, which in turn donate them to herbivorous insects
.
Plant adaptation strategies obviously always involve risks and side effects," Roy Kirsch said
.

However, there are also some leaf beetle species whose symbiotic bacteria produce pectinase for their hosts
.
In so-called "knock-in" experiments, using CRISPR-Cas9 to introduce pectinase genes into the beetle genome, it is now planned to show that this new beetle trait affects not only interactions between plants and insects, but also between insects and their bacterial chaperones
.
Future research will also focus on other enzymes
that do not degrade pectin but rather other plant cell wall components.
The work of Roy Kirsch, Yannick Pauchet, and their team suggests that insects are critical to surviving on host plants not just adaptations to plant defenses
, such as toxins, feeding inhibitors, and other secondary metabolites.
Scientists are more concerned with primary metabolism, which has so far been overlooked in the study of plant-insect interactions
.
"The ability of insects to digest these primary metabolites is equally important for the evolutionary success of herbivorous insects," Yannick Pauchet said
.

Journal Reference:

1. Roy Kirsch, Yu Okamura, Wiebke Haeger, Heiko Vogel, Grit Kunert, Yannick Pauchet.
   Metabolic novelty originating from horizontal gene transfer is essential for leaf beetle survival.
   Proceedings of the National Academy of Sciences, 2022; 119 (40) DOI: 10.
   1073/pnas.
   2205857119