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Study details the functioning of a key protein in the development of citrus canker

Discoveries allow us to understand how bacteria modulate cell growth in plants, paving the way for the creation of control methods

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Researchers from the National Biosciences Laboratory (LNBio) revealed details of the structure and functioning of a key protein for the development of citrus canker – a disease that affects the main commercially important citrus species.

caused by bacteria Xanthomonas citri, the disease is characterized by the uncontrolled growth of plant cells, which generates tumors on the surface of leaves, fruits and branches. Through these lesions, the bacteria spreads and, depending on the degree of infection, the disease can cause premature fruit and leaf fall, compromising plant productivity.

“For these lesions to develop, the pathogen needs to neutralize a host cell protein called MAF1, responsible for controlling cell growth. In this work, we explored the regulatory pathways of citrus MAF1 and also the regions of its molecular structure that are important for antitumor function”, said Celso Benedetti, coordinator of the supported research from FAPESP and professor at the Biology Institute (IB) at Unicamp. The results were published in the journal Structure.

Benedetti's group has been investigating for more than 10 years at LNBio – which is part of the National Center for Research in Energy and Materials (CNPEM), in Campinas – the mechanisms by which Xanthomonas citri causes disease in plants of the genus Citrus.

Researchers have mainly studied “pathogenicity factors”, that is, molecules released by bacteria to “circumvent” the host cell's defense system and favor infection.

“In a previous study, we demonstrated that the bacteria inject proteins known as 'TAL effectors' (transcription activator-like effectors, its acronym in English), which reprogram cell growth in the plant. Our results suggest that this reprogramming occurs largely due to the inactivation of the citrus MAF1 protein,” said Benedetti.

MAF family proteins have been widely studied in yeast, drosophila and mammals – humans and mice mainly – as they are involved in diseases such as cancer and obesity.

Evidence from the scientific literature indicates that MAF1 controls gene transcription and cell growth by binding to the RNA polymerase 3 protein complex, responsible for manufacturing the “ingredients” necessary for protein synthesis.

“To grow, the cell needs proteins and, to do so, it activates RNA polymerase 3 to manufacture ribosomes and tRNAs [transport RNA] necessary for protein synthesis. MAF1 is a regulator of RNA polymerase 3 activity and, when it stops working, cell growth and division are induced”, explained Benedetti.

Until the publication of the article in Structure, the scientific literature only had a description of the structure of human MAF1 – obtained using the X-ray diffraction crystallography technique (which consists of crystallizing proteins and observing how this crystal diffracts the radiation emitted on it).

The crystallographic structure of the citrus MAF1 protein now obtained by the LNBio group reveals structural elements absent in the structure of human MAF1. Furthermore, it maps regions of the molecule that are important for its interaction with RNA polymerase 3.

“MAF1 activity depends on a process known as phosphorylation [addition of phosphate groups to the polypeptide chain]. When MAF1 is phosphorylated [receives phosphate adornments], it 'turns off' RNA polymerase 3, which then becomes active. Our data indicate that the phosphorylation of citrus MAF1 is mediated by the TOR kinase, one of the most important kinases,” said Benedetti.

Proteins whose main function is to phosphorylate other proteins and, thus, regulate their activity are known as kinases. According to Benedetti, the cellular signaling pathway mediated by the TOR kinase had already been described for human MAF1, but it was not yet known that the process in plants was similar to that in humans.

The new findings allowed the LNBio group to elucidate the signaling cascade associated with cell proliferation in plants, which is ultimately controlled by a hormone known since the time of the English naturalist Charles Darwin (1809-1882): auxin.

“It has long been known that auxin controls cell growth in plants, but it was not understood exactly how. Our data are suggesting that auxin regulates the activity of the TOR kinase, which, in turn, regulates the repressive activity of MAF1 on RNA polymerase 3. This knowledge could have major implications for research aimed at producing biomass for energy generation.” , said Benedetti.

Applied knowledge

In order to prove the central role of TOR in regulating cell growth in plants, the group carried out an experiment with orange leaves (Citrus), who were infected with X. citri and exposed to a solution containing a compound capable of inhibiting the activity of this kinase.

“Treatment with the TOR inhibitor almost completely prevented the formation of citrus canker, while in infected leaves treated with a solution containing only placebo the lesion developed as expected,” said Benedetti.

Currently, the LNBio group is investigating the possibility of using this inhibitor – as well as other compounds that act on the TOR/MAF1 regulation pathway – to control the disease.

“However, any substance that presents an inhibitory effect on the TOR kinase in citrus must be harmless to the plant, humans, animals and the environment. Furthermore, to be economically viable, the compound would need to have a low production cost, or it would considerably increase the final price of the product, making its application in orchards unfeasible,” he explained.

Currently, there are no completely effective methods for eliminating citrus canker. Control of the disease is carried out by the citrus growers themselves, through the risk mitigation system, according to rules from the Ministry of Agriculture, Livestock and Supply (MAPA) that came into force in March 2017.

Measures such as spraying the orchard with copper-based products, replacing infected plants with healthy seedlings and more resistant varieties, using windbreaks, in addition to controlling the citrus leafminer, an insect that facilitates the spread of canker, are recommended. citric.

Although the new legislation allows the presence of plants with citrus canker in orchards – their eradication is no longer mandatory –, citrus growers are prohibited from selling fruits with lesions or symptoms of the disease. “In this new scenario of living with the disease, new control measures are important,” said Benedetti.

The article Crystal Structure and Regulation of the Citrus Pol III Repressor MAF1 by Auxin and Phosphorylation, by Adriana Santos Soprano, Celso Eduardo Benedetti and others, can be read at:www.sciencedirect.com/science/article/pii/S0969212617302198#!.
 

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Orange grove in the São Paulo city of Olímpia: citrus canker causes early drop of fruits and leaves

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