When it comes to growing niche products, any sort of harm to the plants could prove disastrous to the overall yield itself. This is especially so if that plant is only grown in certain regions due to soil and weather requirements. That is the case for the subject of chocolate, a billion dollar industry that is restricted primarily to West African countries that make up more than 60% of global production and the rest being split between South America and Indonesia.

But the cacao tree, Theobroma cacao, has its own problems that put the livelihood of the smallholder farms that grow it in jeopardy year after year. There are a wide variety of pathogens that love to feast upon the cacao bean pods and these generally wipe out nearly a third of the globally produced pods prior to harvest time every year. And the outbreaks are often localized so that, if they infect a farm, they will usually end up wiping out 100% of that farmer’s crop for that season.

Regulating Immune Defense

There have been numerous attempts over the years to improve tree resistance to the pathogens and to reduce the risk of infection happening in the first place. Genetic investigations of both the pathogens and the tree itself have taken place in the past, giving new insights into possibly tackling the issue once and for all.

One of the specific genes in the cacao tree was identified in a 2010 study, with an overexpression experiment in 2015 confirming its role in the plant immune system. That gene, named TcNPR1 (Non-expressor of Pathogenesis-Related 1), appears to be a master regulatory switch for the immune system as a whole and can prevent infection by some of the worst pathogens like Phythophthora, the “plant destroyer” water mold.

Several other connected genes have also been characterized over the intervening period, including what appears to be a negative regulator of the NPR1 gene that was named NPR3 and is salicylic acid based in its regulation. Knocking out NPR3 seems to improve immune resistance to pathogens as well, as it prevents the gene from reducing NPR1 activity in managing those systems. The method used involving microRNA (miRNA) knockout was not a permanent fix, however, and scientists at Penn State wanted to see if they could use CRISPR-Cas9 to make a more long-lasting cultivar with high infectivity resistance.

Leaves and Embryos

The major downside to all such genetic or breeding work with the cacao tree is that it is actually a rather slow-growing plant and any research with it generally takes a significant time gap before results can be recorded. To get around this, the researchers decided to try a transient expression test first with CRISPR. This was done with detached leaves taken from a cacao plant and which were then inoculated with an Agrobacterium vector housing the CRISPR knockout system. They were then left for 48 hours to allow the insertion of the CRISPR system and for the NPR3 gene knockout to take place in the leaf cells.

After that period, the leaves were then exposed to a strain of the Phytophthora tropicalis pathogen for 72 hours. Pictures were taken after this, along with tissue samples that were flash-frozen to prevent any further changes from occurring. Extracted DNA from the leaves were tested to confirm that the knockout had taken place. They also knew that Agrobacterium wasn’t known to be able to infect every cell in a leaf, so they tested the proportion of the leaves that were successfully transformed.

In total, they confirmed that around 27% of the leaf cells had their TcNPR3 gene knocked out. They saw that the pathogen-caused lesions were significantly smaller in size on the transformed leaves as compared to the control group, proving that the knockout had given the plant cells greater resistance to overall infection.

With that success confirmed, the scientists turned to the larger experiment: making transgenic knockout plants starting from an embryo so that all of the plant cells would have the transformation. They did so and confirmed a stable integration of the change, but it will probably be quite some time before a followup study can show the effects of the resistance in an adult cacao tree.

They did notice that the embryos with the change grew more slowly than the controls and theorize that metabolic drag from lacking the NPR3 gene may be responsible. Other work in Arabidopsis with the same gene knockout had shown some negative growth symptoms as a result. The scientists hope to further test any potential off-target effects as the plants grow, but have shown that all the off-target sites found thus far have not affected any form of gene expression in the embryos.

A Long Term Project

Overall, they feel that in the short term, transient leaf transformation may be the best option, as a 27% knockout rate appears to be enough to generally protect the trees from infection and will likely improve the ability of a farmer’s crop living until harvest. The long run may see more stable knockout cultivars be developed and spread worldwide to naturally have the plant’s immune system protect themselves from pathogens.

But, for now, we’re continuing to learn about how these plants function and what we can do to improve plant growth and plant health around the world.

Press Article Link

Study Link

Photo CCs: Cacao from Wikimedia Commons

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