ABOVE: Scientists brought plant disease to a halt with a drug that blocks bacterial proteins. Kinya Nomura.

Using a microscopic syringe fashioned out of protein, bacteria inject an arsenal of virulent proteins into plant cells, inflicting disease and devastating crops.1 Plant microbiologists at Duke University study these proteins with the hope of discovering drugs that could shield plants from their offensives. In a study in Nature, Sheng Yang He from Duke University and his colleagues uncovered the structure and function of a large family of virulent proteins that eluded scientists for a quarter of a century.2 This information guided the researchers to develop a novel treatment for crop disease.

Among the slew of virulent proteins that bacteria inject into plant cells are Avirulence protein E (AvrE) and its relatives.3 “This protein has been known for 25 years, and people have tried to work with it, but it’s very large, and its size has made it very difficult to produce protein to study,” noted Jeff Dangl, a plant immunologist at the University of North Carolina who was not involved with the work. What’s more, He noted, “It’s actually toxic to plants, yeast, animal cells—any cell it touches.” This hampers experiments to study protein function. 

These proteins are highly conserved in structure across bacterial species, making them ideal targets for new drugs, so He and his colleagues were determined to unravel their functions.4 First, the team deciphered the structure of AvrE and its relatives. Structural analysis of large proteins is difficult with experimental techniques alone, so they turned to the machine learning tool AlphaFold2.5,6 

The team found that four related proteins from different bacterial species all adopted a similar shape, each carrying a hollow barrel, implying that they may behave as transport channels if inserted in the cell membrane. “One of the key breakthroughs in this story was the use of AlphaFold to predict a structure, and that allowed the authors to immediately come up with a hypothesis because the predicted structure suggested a clear function,” Dangl noted.

See also: “Lasker Award for Revolutionizing Protein Structure Predictions”

To explore whether these channels transport ions, He and his team injected frog eggs with mRNA coding for two of the virulent proteins. Frog eggs are a useful model for studying the electric currents induced by ion transport across cell membranes because they naturally possess fewer of their own ion channels than most cell types and thus produce less background signal.7 

The team found that the virulence proteins induced electric currents across the membrane when a voltage was applied, suggesting that the channels lodge themselves within the membrane and allow ions to cross freely through their hollow barrels. Coincidentally, they also noticed swelling in the eggs that carried the proteins and hypothesized that the channels could also transport water. To test this, they moved the eggs into a simple water solution with a lower ion concentration than inside the frog eggs. This imbalance resulted in water rushing into eggs expressing the channels, causing them to swell and burst. Thus, the team confirmed that the channels transport water as well as ions across the cell membrane.

From their structural analysis, the researchers predicted that the channels have an opening 15 to 20 ångströms (Å) wide—approximately one-millionth of the width of a paper straw. So, they next tested whether the channels could also transport complex compounds. Large compounds like fluorescent proteins measuring 30 Å in width did not traffic into the frog eggs, but smaller compounds like the 7 Å-wide fluorescein entered easily.

The big question is how these channels benefit the bacteria. “Bacterial pathogens live in the spongey air space inside a leaf, and that space is pretty dry,” Dangl said. Bacteria need moisture to grow but they can’t breach the cell wall to directly access the plant’s resources. Therefore, they drain surrounding cells in a process called water soaking.8 The team speculated that these channels might be key players for sourcing water and nutrients from the cells. In the future, the research team plans to capture these channels in action within the leaf by measuring the flow of fluorescently labelled compounds.

Finally, the team searched for compounds that block the channel and limit disease. For this, they turned to polyamidoamine G1, a polymer measuring 22 Å that is wider than the channel diameter. This drug showed promise in frog eggs: It blocked the ion currents, swelling and bursting, and uptake of fluorescein. 

See also: “DeepMind AI Speeds Up the Time to Determine Proteins’ Structures”

Polyamidoamine G1 successfully prevented disease in plants, too. The drug stopped two common crop pathogens, Erwinia amylovora and Pseudomonas syringae, from producing disease symptoms in plants, including dark boils known as fire blight on pears and brown spots (dead cells) on the leaves of the model plant Arabidposis thaliana.9,10 In future work, He plans to resolve the structure of the drug bound to one of the proteins to determine how it blocks the channel.

The drug did not inhibit bacterial growth in the lab even though it staved off disease in plants, suggesting that it doesn’t kill bacteria like an antibiotic but instead limits their virulence. Alternatively, it’s possible that the plant immune system cleared the infection when stimulated by the drug; however, the team did not detect activation of plant immune proteins, suggesting that the protective effect came down to channel inhibition.

“This will be applicable to all bacterial pathogens that contain this family of proteins. There are many of them, and they are major pathogens,” He said. Aside from preventing fire blight in apple and pear trees, the drug may combat a pandemic strain of P. syringae that ravages kiwifruit across Asia, Europe, and New Zealand, he suggested.

“I’m really excited about this research, which really employed cutting-edge structural biology technologies to solve a long sought after mechanism of an important virulent protein produced by bacterial plant pathogens,” said Wenbo Ma, a microbiologist at the Sainsbury Laboratory who was not involved with the work but who has collaborated with one of the authors in the past.

“This is a very unique target that’s not produced by eukaryotes,” Ma said. Drugs that target bacterial proteins bearing little similarity to host proteins are ideal as they may be less likely to cause side effects in the host. “But whether this particular compound can affect other unrelated proteins or functions, we don’t know, and that needs to be tested,” she added.

References

  1. Büttner D. Protein export according to schedule: Architecture, assembly, and regulation of yype III secretion systems from plant- and animal-pathogenic bacteria. Microbiol. Mol. Biol. 2012;76(2):262-310.
  2. Nomura K, et al. Bacterial pathogens deliver water- and solute-permeable channels to plant cells. Nature. 2023; 621(7979):586-591.
  3. Lorang JM, et al. avrA and avrE in Pseudomonas syringae pv. tomato PT23 play a role in virulence on tomato plants. MPMI. 1994;7(4):508-515. 
  4. Baltrus DA, et al. Dynamic evolution of pathogenicity revealed by sequencing and comparative genomics of 19 Pseudomonas syringae isolates. Plos Pathog. 2011;7(7):e1002132.
  5. Cheng Y. Membrane protein structural biology in the era of single particle cryo-EM. COSB. 2018;52:58-63.
  6. Jumper J, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7867):583-589.
  7. Dascal N. The Use of Xenopus oocytes for the study of ion channel. Crit. Rev. Biochem. 1987;22(4):317-387.
  8. Schwartz AR, et al. TALE-induced bHLH transcription factors that activate a pectate lyase contribute to water soaking in bacterial spot of tomato. PNAS. 2017;114(5):E897-E903.
  9. Sharifazizi M, et al. Evaluation of biological control of Erwinia amylovora, causal agent of fire blight disease of pear by antagonistic bacteria. Biocontrol. 2017;104:28-34.
  10. Donati I, et al. New insights on the bacterial canker of kiwifruit (Pseudomonas syringae pv. actinidiae). J. Berry Res. 2014;4(2):53-67.