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Functions of a novel chitinase-like effector family unique to aphids

Reference	BB/X002322/1
Principal Investigator / Supervisor	Dr Po-Yuan Shih
Co-Investigators / Co-Supervisors
Institution	Imperial College London
Department	Life Sciences
Funding type	Research
Value (£)	653,665
Status	Current
Type	Research Grant
Start date	28/03/2023
End date	27/03/2026
Duration	36 months

Abstract

Compared with studies on plant-pathogen interactions, we know relatively little of the molecular interplay between aphids and their hosts. This knowledge gap is a pressing problem in light of reduced availability or effectiveness of pesticides and breakdown of host resistance. Akin to pathogen-plant interaction models, aphids secrete protein effectors via saliva into their hosts, leading to either compatible or incompatible interactions. Although there are several published salivary proteomes, and some effectors are known to impact on host responses or aphid performance, only a few examples are known of how aphid effectors target host immunity. By comparing transcriptomes and salivary proteomes of aphids and F1 progeny that segregate for virulence, we discovered a single effector candidate in avirulent aphids. This protein has weak homology to chitinases but lacks the catalytic residues, and is a member of a novel aphid-specific chitinase-like (CHL) family that is intriguingly absent from even their closest relatives. We propose that CHLs evolved virulence functions by instead sequestering chitin, preventing activation of host chitin receptors. A secondary opposing function appears to be in triggering immunity via R-gene mechanisms through surface RLK/RLP or cytoplasmic NLR receptors. Here, we will seek the biochemical and biological functions of CHL effectors. In vitro biochemical approaches such as pull-downs will reveal whether they bind chitin and/or other proteins, thus revealing potential host targets. Expression in planta will test for activation or suppression of host immunity in presence or absence the RAP1 R gene. Genetic leverage on the plant side comes from hosts differing in R genes and mutants lacking chitin receptors; and we will create CRISPR KO aphids using the platform at INRAE, France. Finally, we will explore the mechanisms of these components of host immunity that we have found to be easily suppressed by both biotic and abiotic stresses.

Summary

Aphids are intrinsically fascinating insects, but they are also damaging pests in global agriculture, causing losses through feeding on plant sap and through spreading many viruses that cause plant diseases. Aphid populations can increase extremely fast due to their clonal reproduction and their unusual "Russian Doll" telescoped generations. Although chemical pesticides are widely used, problems often arise due to the aphids evolving resistance and no longer being able to be controlled. In addition, there may be environmental or health issues associated with certain pesticides that have led to some being withdrawn or restricted. There are a few crops such as tomato, melon, soybean and rice, with known genetic resistance to some aphids and related species. The genes involved are called R-genes (for resistance) and commonly code for receptor proteins that recognise invasion by the pest insect. However, insects in turn can evolve new means to overcome the plant's R-gene defences. Overall there is a pressing need to find new robust and sustainable ways to manage pests in agriculture. In this context, we compared molecular differences between aphids that thrived or died depending on which plant type they were attacking, and recently discovered a new class of proteins in aphid saliva that distantly resemble chitinase enzymes. Chitinases are present in all insects, being essential for maintaining the chitin polymer in their exoskeleton and mouthparts. However, these salivary proteins appear unable to act as chitinase enzymes, so we need to find another function to explain how they benefit the aphids. Intriguingly, this protein group is unique to aphids, being absent even from other closely related species of sap-sucking insects. Because aphids evolved as a distinct group of species around 300 million years ago, we think that the chitinase-like proteins have ancient origins that may underpin the enormous success of these organisms. Our starting idea is that chitinase-likeproteins may still have the ability to stick to chitin, in effect mopping up fragments of these molecules. This would prevent the host plant from detecting the presence of chitin, allowing the aphids to go under the radar of the plant's immune system. Other work on fungi shows that plants carry chitin-detecting receptor proteins on their cell surface, and there is no reason to think that these receptors couldn't also detect aphid chitin. Our second idea comes from finding one particular aphid chitinase-like protein that is strongly associated with exactly the opposite process: triggering plant immunity through an R-gene. Here we will directly test both these concepts. To uncover how chitinase-like proteins work, we will take a wide range of research approaches. We want to work out the common rules for the whole family, and also to discover the specific properties of the one member that leads to plant immunity and aphid death. First, we will make purified forms of the proteins to enable laboratory scale experiments. In particular, we will test whether the proteins really bind to chitin, or whether they have some other unexpected functions. We will also hunt for proteins or other components of plant cells that bind to the incoming chitinase-like salivary proteins. Both immune-suppressing and immune-activating modes are commonly known to result from protein-to-protein interactions in responses to fungal and bacterial diseases, so we will look for parallels in our aphid system. Working with colleagues in France, we will apply a genome editing technology called CRISPR to specifically knock-out the gene that codes for the immune-activating salivary protein. This will give us new aphids to see if they do better or worse than aphids that still have this gene. Finally, we discovered that plant immunity is suppressed under stressful environments, including drought that are increasingly prevalent due to climate, so we will explore mechanisms of immune robustness.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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