Citrus canker bacterium can be an ally in biorenewables manufacturing

A study led by Brazilian scientists revealed the biological process used by Xanthomonas to weaken the defenses of plants and discovered a novel class of enzyme that can be used to obtain advanced sugars from agroindustrial waste.

By Luciana Constantino  |  Agência FAPESP – Xanthomonas citri, the bacterium that causes citrus canker, is a notorious “villain” among citrus growers but can be an ally in the manufacturing of biorenewables such as ethanol, dyes, plastics, and other chemicals currently derived from petroleum.

A study published in June in the journal Nature Communications by researchers at the Brazilian Center for Research in Energy and Materials (CNPEM) in Campinas, São Paulo state, reveals the biological processes used by the bacterium to weaken plants’ defense systems and the discovery of a new class of enzymes called CE20 that can assist infection.

According to the study, which was supported by FAPESP, the bacterium mobilizes these enzymes to destroy the plant’s cell walls, which function as a barrier against invasion of pathogens.

When the bacterium invades cells, it triggers the production of proteins that lead to an increase in virulence factors, including the type II secretion system, a sort of “molecular needle” inserted into the plant’s degraded cell walls to inject the proteins.

The study detailed the orchestrated atomic-level action of multiple enzyme components to break down xyloglucan, one of the complex carbohydrates that make plants’ primary cell walls resistant to pathogen invasion. The discovery reveals a novel signaling pathway that offers the bacterium a way in.

“We demonstrated the existence of conserved molecular machinery in this pathogen, specializing in xyloglucan. We elucidated at the biochemical and structural level the action of the machinery’s enzymatic components. Our discovery of the mechanisms for deconstructing this carbohydrate points to possible applications in the bioeconomy and biorefining. We can obtain novel combinations of enzyme cocktails that are more effective in breaking down plant biomass to produce ethanol, aviation fuel and other chemicals, for example,” said Mário Tyago Murakami, principal investigator for the study. Murakami is Scientific Director of CNPEM’s Biorenewables Laboratory (LNBR).

CNPEM is already developing other microbial platforms for biorefining, such as an enzyme cocktail produced by a fungus (RUT-C30). The platform, for which a patent application has been filed, is customized for Brazilian conditions and has been tested in an industrial setting. It will be used in biorefineries to obtain advanced sugars from agroindustrial waste, with a reduced environmental impact and as a substitute for fossil fuels.

According to Murakami, the discovery of novel components that potentiate plant infection also contributes to the development of strategies to combat citrus canker, including the production of inhibitors for this group of bacteria.

“Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans”, the article explains. “Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.”

Xanthomonas attacks other crops besides citruses, such as rice, cotton and banana. Considered one of the worst threats to citrus worldwide, citrus canker causes dark brown raised lesions on leaves and fruit, which drop prematurely, reducing the productivity of the affected plants. The blighted fruit is too unsightly to be sold, and once established, the disease can be controlled only by destroying all susceptible trees.

Citrus canker expanded 15% in 2020 compared with the previous year in the states of São Paulo and Minas Gerais, where most of Brazil’s oranges are grown, and affected some 34 million plants, according to Fundecitrus, a research organization maintained by the industry. It blamed São Paulo’s decision not to eradicate symptomatic trees as one of the reasons for the expansion. It said 1.27 million 40 kg boxes of oranges were lost to citrus canker in the 2020-21 crop year.

Applying science

The approach used by the researchers in the study was multidisciplinary, involving phylogenetics, transcriptomics, gene deletion, molecular cloning and analysis of mutagenesis, as well as X-ray scattering and diffraction at the Brazilian Synchrotron Light Laboratory (LNLS-CNPEM). Diffraction data was acquired from the MX2 macromolecular crystallography beamline. The researchers also performed genetic engineering and in vivo experiments on plants at the Brazilian Biosciences National Laboratory (LNBio).

The study took some five years and involved 23 researchers, including professors at the University of São Paulo (USP) and the University of Campinas (UNICAMP).

“It’s not often in Brazil that we have a set of PhD theses and postdoctoral projects looking systematically at the same problem. This kind of teamwork and collective sharing has made a great difference to our research, enhancing our capacity to deliver results and increasing the impact of our discoveries,” Murakami told Agência FAPESP. 

Another study by the same team involving the enzyme cocktail used by Xanthomonas to break down cell walls was published in January, also in Nature Communications. It used quantum mechanical and molecular mechanical methods, as well as high-resolution experiments, to show that enzymes of significant industrial importance (such as glycoside hydrolase) can function via alternative catalytic itineraries that are thermodynamically viable.

The study broke with the paradigm that there is a single catalysis pathway for each enzyme-substrate pair. A catalysis pathway or itinerary is a set of chemical and structural changes undergone by a given substrate owing to the action of an enzyme.

This line of research has continued on the Manacá macromolecular crystallography beamline at Sirius, LNLS’s new synchrotron light source. Manacá was the first experiment station to be opened to the scientific community and companies interested in investigating the composition and structure of matter in its various forms and with applications in a range of knowledge areas. 

Sirius is operated by LNLS and is part of CNPEM. It is a state-of-the-art electron accelerator designed to be one of the world’s most advanced synchrotron light sources in its category. “At Sirius we’ve left behind images and statistics to analyze dynamic events in videos of catalytic processes involving the enzymes discovered in this study,” Murakami said.

The research on Xanthomonas has been supported by FAPESP via two Thematic Projects (15/26982-0 and 15/13684-0), four postdoctoral fellowships (16/06509-0, 17/14253-9, 16/19995-0 and 19/13936-0), and two PhD scholarships (17/00203-0 and 18/03724-3). 

The article “Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors” is at: www.nature.com/articles/s41467-021-24277-4.