Research details functioning of key protein for development of citrus canker

Brazilian scientists’ discovery of ways in which bacterium Xanthomonas citri modulates plant cell growth paves way for novel methods to control the disease.

By Karina Toledo  |  Agência FAPESP – Researchers at the National Bioscience Laboratory (LNBio), in Brazil, have revealed details of the structure and function of a key protein for the development of citrus canker, a disease that affects the commercially most important species of citrus fruit.

Caused by the bacterium Xanthomonas citri, this disease is characterized by runaway plant cell growth that produces lesions on leaf, fruit and branch surfaces. The bacteria spread via these lesions, and when the infection is severe enough, the disease can cause leaves and fruit to drop prematurely, decreasing crop yield.

“For the lesions to develop, the pathogen must neutralize a host cell protein called MAF1, which is responsible for controlling cell growth. Our research explored the MAF1 regulation pathways in citrus as well as the regions of the plant’s molecular structure that are important for anti-tumor function,” said Celso Benedetti, principal investigator for the study, which was supported by FAPESP. The results were published in the journal Structure.

Benedetti’s group has investigated the mechanisms by which X. citri causes disease in citrus plants for over ten years at LNBio, which is attached to the National Energy and Materials Research Center (CNPEM) in Campinas, São Paulo State.

Above all, the researchers have studied the “factors of pathogenicity”, molecules released by bacteria to circumvent the host cells’ defense system and favor infection. “In a previous study, we showed that bacteria inject host cells with proteins known as TAL (transcription activator-like) effectors, which reprogram cell growth in the plant. Our results for citrus suggest this reprogramming is driven largely by inactivation of MAF1,” Benedetti said.

The MAF1 protein family has been widely studied in yeast, fruit flies and mammals, especially humans and mice, because of its involvement in diseases such as cancer and obesity.

Evidence in the scientific literature suggests MAF1 controls gene transcription and cell growth by binding to RNA polymerase 3 (also known as Pol III), a protein complex that produces the “ingredients” needed for protein synthesis. 

“Cells need proteins in order to grow and use RNA polymerase 3 to make ribosomes and tRNAs, transfer RNAs, for protein synthesis,” Benedetti explained. “MAF1 is a regulator of RNA polymerase 3 activity, and cell growth and division increase when it stops working.”

Until publication of the article in Structure, the scientific literature offered only a description of the structure of human MAF1, obtained by X-ray diffraction crystallography (a technique that consists of crystallizing proteins and observing how the crystal scatters a beam of incident X-rays).

The crystal structure of citrus MAF1 now obtained by the group at LNBio has structural elements not found in human MAF1. In addition, this structure maps regions of the molecule that are important for its interaction with Pol III.

“The activity of MAF1 depends on a process known as phosphorylation [addition of phosphate groups to polypeptide chains]. When MAF1 is phosphorylated [receives phosphate adornments], it ‘uncouples’ from RNA polymerase 3, which therefore becomes active. Our findings show that citrus MAF1 phosphorylation is mediated by TOR, one of the most important kinases,” Benedetti said. 

Kinases are proteins whose main function is to phosphorylate other proteins and thereby regulate their activity. According to Benedetti, the cell signaling pathway mediated by TOR had been described for human MAF1, but it was not known whether the process in plants was similar to the process in humans.

The new findings enabled the LNBio group to elucidate the signaling cascade associated with plant cell proliferation, which is ultimately controlled by auxin, a hormone known since the pioneering experiments performed with plants by English naturalist Charles Darwin (1809-1882).

“Auxin has long been known to control plant cell growth, but exactly how it does so has been poorly understood,” Benedetti said. “Our findings suggest auxin regulates the activity of the kinase TOR, which in turn regulates MAF1-mediated repression of RNA polymerase 3 transcription. This knowledge has major implications for research on biomass energy production.”

Knowledge applied

To demonstrate the central role of TOR in plant cell growth regulation, the group performed an experiment with leaves of the orange tree (Citrus sinensis), which were infected with X. citri and exposed to a solution containing a compound that inhibited the activity of this kinase.

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

The LNBio group are currently investigating the possibility of using the inhibitor – as well as other compounds that act on the TOR/MAF1 regulation pathway – to control citrus canker.

“However, any substance that may have an inhibitory effect on TOR in citrus must be shown to be innocuous to the plant and also to humans, animals and the environment,” Benedetti said. “Moreover, to be economically feasible, the compound’s production cost must be low. Otherwise, it would considerably raise the final price of the product, making its application to citrus groves unviable.”

Today, there are no totally effective ways to eliminate citrus canker, which is managed by growers by using a risk mitigation system in accordance with Ministry of Agriculture rules in force since March 2017.

The rules require copper spraying of citrus groves, replacement of infected plants with healthy saplings and more resistant varieties, windbreaks, and control of the citrus leafminer (Phyllocnistis citrella), an insect that facilitates the spread of citrus canker.

While the new legislation allows growers to leave plants with citrus canker standing and does not require eradication, it prohibits the sale of fruit with lesions or other symptoms of the disease. “New control measures are important in this new scenario of living with the disease,” Benedetti said.

The article “Crystal structure and regulation of the citrus Pol III repressor MAF1 by auxin and phosphorylation” (https://doi.org/10.1016/j.str.2017.07.004) by Adriana Santos Soprano, Celso Eduardo Benedetti et al. can be retrieved from: sciencedirect.com/science/article/pii/S0969212617302198!.