Plants have highly effective immune systems which allow them to thrive in our environment today. However, these systems lack the adaptability we need for reliable sources of food.
Devastating wheat disease outbreaks in Africa and Asia have highlighted the need for rapid responsiveness in global agriculture. There are major concerns about the increasing threat of plant diseases on global food security which is why scientists at The Sainsbury Laboratory continue to advocate for global plant pathogen surveillance systems.
“Increased awareness of emerging plant pathogens is necessary for scientists and farmers to rapidly respond and mitigate disease outbreaks." says Clémence Marchal, postdoctoral scientist in Sophien Kamoun's lab at The Sainsbury Laboratory, "This information is critical to have disease resistant crop varieties ready before disaster strikes.”
Plant immune systems rely on the innate genetic information contained in each seed to protect themselves against the specific pathogens they will encounter in their lifetimes. To improve disease resistance in our crops, humans have relied on plant breeding methods since the dawn of agriculture.
Recent advances in genetic technologies have allowed us to speed up the process of crop improvement even further by engineering immune receptors within plant cells that can recognize the presence of pathogens and trigger an immune response. However, this requires researchers to sift through vast amounts of genetic data to find these particular resistance genes, making the discovery pipeline akin to ‘finding a needle in a haystack’.
The advantages of having a shorter and simpler approach in finding new genetic sources of disease resistance would be manifold.
Plant immunity often relies on intracellular nucleotide-binding, leucine-rich repeat receptors (NLR) to detect specific pathogens. Whilst singleton NLR proteins can recognise effectors (virulence proteins from the pathogen) and signal the immune response, research has also revealed paired NLRs in which each partner plays distinct roles in pathogen effector recognition or immune signalling.
Pik-1 and Pik-2 are such an NLR receptor pair from rice. The integrated heavy metal associated (HMA) integrated domain of Pik-1, which is responsible for recognizing pathogen presence, can be mutated to confer new pathogen effector specificities as shown by De la Concepcion and colleagues in 2019.
However, modifications restricted by the plant's own genetic repertoire for pathogen recognition will limit the potential for recognising a wider range of pathogens and pests.
Jiorgos Kourelis, who is also a postdoctoral scientist in the Kamoun lab, was intrigued by the hypothesis that Pik-1 could potentially recognize completely novel pathogen effectors via swapping its HMA domain with different domains.
If new domains could be generated against specific pathogens on demand, could the Pik immune receptor complex be the ultimate scaffold to engineer made-to-order plant immune receptors?
Jiorgos proposed using animal adaptive immunity as a potential source of on-demand domains, given that it has the capacity to generate antibodies against virtually any antigen it is exposed to. Together with Clémence, they tested the concept by focusing on the minimal antigen-binding fragment of single-domain heavy chain antibodies of camelid mammals, called nanobodies.
The researchers used published sequences of camelid nanobodies that target specific fluorescent proteins. They fused these nanobodies with the Pik immune receptor complex to create Pikobodies (Pik + nanobodies). To test the specificity of Pikobodies, two different fluorescent proteins were used, GFP and mCherry, which also allowed imaging techniques to be used for a more streamlined immune assay approach.
With the help of Andres Posbeyikian, a predoctoral intern in the Kamoun lab, Clémence conducted immune assay work which showed that these Pikobodies not only recognize their specific targets within the plant cell environment but also produce a functional immune response. This was tested by modifying Potato Virus X to produce the target fluorescent proteins and infect a stable transgenic model plant, Nicotiana benthamiana, containing the genes to generate Pikobodies. Reduced viral load showed that pathogen recognition translated into resistance within the plant to levels comparable to that of Rx, a natural resistance gene that recognises Potato Virus X.
Jiorgos believes that generating nanobodies in a non-plant system reduces the risk of off-target binding with plant-derived proteins within the plant cell. He also highlights that it would be possible to use bioengineered libraries, instead of mammalian systems, to generate new nanobodies for different pathogens.
“We’re very excited about the potential applications of this technology.” says Jiorgos, “The short and simple pipeline could allow for so much more gene discovery and increase the possibility of developing robust disease resistance that pathogens are less likely to overcome over time.”
There are widespread benefits to a shorter and more affordable, and therefore more accessible, discovery pipeline. Researchers in the Kamoun lab believe that the translation of their discoveries should be accessible and beneficial to communities all over the globe, especially those who have less options available to them to protect their crops.
The results of this proof-of-principle study show exciting potential for made-to-order resistance genes against any pathogen or pest that delivers effectors inside host plant cells. This is being followed up by proof-of-application studies.
Group leader, Sophien Kamoun, says, “Pikobodies are a promising new technology that could revolutionize the way we engineer disease resistance by providing a pseudo-adaptive immune system to plants. We will now endeavor to apply the Pikobodies technology to crops and bring it to farmers throughout the world.”
0/ Excited to share the final version of our #Pikobodies paper first seen @biorxiv : bioengineered plant immune receptors-nanobody fusions! https://t.co/Wdii6MIWXh— Jiorgos Kourelis (@JiorgosKourelis) March 2, 2023
Thanks to co-first author @ClemMarchal, and @adnroide @Adeline_Harant @KamounLab for their invaluable contributions
Watch this "aBIOTECH Virtual Seminar" to hear more about pikobodies from Professor Sophien Kamoun. You'll need to click the 'unmute' button on the player.
This is a series of academic activities sponsored by the editorial department of aBIOTECH & "Biotechnology Bulletin". Moderator: Chief editor of aBIOTECH, researcher Huang Sanwen, Chinese Academy of Agricultural Sciences; editorial board member of aBIOTECH, professor Dong Shameng of Nanjing Agricultural University.