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11.21.16 - Protein Discovery: First Of Its Family To Ever To Be Sequenced

Drosera capensis, commonly known as the cape sundew


Hundreds of brutal, efficient killers crowd Carter Butts’ home, bringing grim death to any unwise enough to venture into range. But fear not. These are magnificent bug-eating plants that populate nearly every continent.

Drosera capensis, commonly known as the cape sundew, also replicate human digestive processes under extreme circumstances. So Butts, a UC Irvine sociology and statistics professor, and his research colleague Rachel Martin, a UCI associate professor of chemistry, molecular biology and biochemistry – and a fellow carnivorous plant enthusiast – wondered: why not pull one into their lab and see what it could teach humans?

The result: An 18-month project to sequence and study the genome of Drosera capensis, the first of its family and the third carnivorous plant ever to be sequenced.

They chose the cape sundew, in part because it drew Charles Darwin’s special attention in his 1875 book on insect-devouring plants. The 194 species of sundew use a sweet sticky secretion to snag insects on long tentacles. Other tentacles move to trap bugs and sessile glands digest them.
Butts and Martin overcame hurdles in extracting DNA, analyzed genomic sequences with tools typically used in sociology and explored novel enzymes that break down proteins – and which could provide significant medical benefits to humans.
“Carnivorous plants are neat,” Martin says. “Nobody knows exactly what proteins and enzymes they possess. They started out trying to kill insects to protect themselves from being eaten. This has evolved six separate times into mechanisms for trapping insects and using them for nutrients.”

Their discoveries, reported in two recent peer-reviewed journals, show the magnificent killers may be equally extraordinary in what they can do for humans – erasing biofilms from medical instruments and safely battling stubborn fungal infections, for example.
Environmental challenges faced by the plants suggest they have creative ways of surviving, according to Butts.
“It’s a lot harder for a carnivorous plant to eat things than it is for us,” he says. “You can’t heat up the environment to make the enzymes go faster and you have to compete with bacteria and fungus and other creatures that are also trying to eat your food. For them to survive, they need to have really effective and interesting properties.”

Butts and Martin, already collaborators on a National Science Foundation project focusing on proteins, created their project under the aegis of Calit2 – where Butts has his lab.

The institute provides for multidisciplinary research – in this case, pairing Butts’ methodology training and Calit2’s computational resources with Martin’s keen understanding of biochemistry.

“My work has mostly been on studying social networks, simulation methods and statistical methods,” Butts says. “This whole arena was an entirely new direction for me. We adapted a technique that I previously used to study individual life histories, believe it or not, to look at proteins.”

The team quickly ran into challenges. First, Butts and Martin needed to extract usable DNA – a challenge because plants excrete substances that mask their DNA when chopped up.

“A lot of the genome-sequencing techniques and the techniques for collecting the DNA are optimized for human studies because the driving force for a lot of this is medicine,” Martin says. “I mixed and matched techniques till I came up with one that actually worked.  UCI’s Genomics High Throughput Facility was very helpful in checking whether the genome was good enough to sequence.”
The effort took about three months, followed quickly by creation of the sequencing data – where technical developments have shortened the process to about a week.

“We took advantage of next-generation sequencing technology – which has reduced the cost and time quite a lot – to get usable reads,” Butts says. “That takes the extracted DNA, breaks it into little tiny pieces, and then uses a machine to read out the DNA sequences of these little pieces. You get a puzzle that has hundreds of millions of pieces and you have to put it together, which is a computational challenge.”

CalIt2 provided key resources during this step.
“It requires access to high-performance computing, which was something I have in my lab and I was able to bring to bear on the problem,” he says.

The team reviewed gigabytes of data to figure out how to organize the puzzle pieces into readable sections of genome.
“There’s no universal strategy that always works. You experiment with modifying the techniques and try different strategies. We went through quite a number of assembly algorithms and strategies for processing and cleaning the data until we found something that finally worked,” Butts says.

“We believe we have about 90 percent of what’s there. That’s pretty good for an initial assembly, and that means what we have is good enough that we can start to find proteins and do other kinds of studies of the organism with fairly high confidence,” he adds.
The data yielded a rich trove of information.

“It really is exciting to have this huge basket of stuff, and realize there are treasures in there. You just have to figure out how to get them out,” says Butts.

Martin quickly identified 44 protein sequences, far too many to economically produce. Using molecular modeling, they helped target the most novel and exciting sequences. She focused on proteins that may work as anti-fungal agents, producing three in her lab. Two are called chitinases because they generate enzymes that digest the chitin in bug exoskeletons.

“This is very valuable agent to use as an antifungal because you can use it to poison fungus and not humans, since humans don’t have chitin,” she explains.

Martin also has created a protein-specific insert, a sub-sequence of a protein that cleaves off after the protein is made. It is itself an enzyme whose functions may include defense against pathogens and cleaning cell walls.

“This is something the plant seems to be using as an antifungal or anti-bacterial agent. We’re really excited about catching that in the lab,” she says.

The duo has more genomes in the pipeline, too. And Martin and Butts are excited to explore what they may discover from other carnivorous plants.

“We found all this great stuff from only the first carnivorous plant that we’ve looked at and there are numerous kinds of plants and lineages. So we’re really excited about comparing what we found here to what’s going on in other carnivorous plants,” Martin says. “We expect to see a lot of similarities, because things evolve in similar directions to get the same function, but there is no reason it has to be exactly the same.”

Butts wants to continue developing the methodology that allowed the duo to go from genomic source code to the model of proteins and enzymes that has guided their experiments.

“This is a really valuable innovation,” he says. “We are trying to model more kinds of proteins, to model proteins under different conditions and to be able to predict more things about their structure and function.

“If we’re going to harness the fruits of the genomic revolution, we need to be able to dramatically improve our ability to speed that source-code-to-final-product process. A lot of people have put a lot of work into pieces of that, and we’re building on that work. We think the time is right to be able to put all those pieces together.”

-- William DiepenbrockSave