A novel chitinase-like family of candidate effectors unique to aphids

Molecular interactions between aphids and plants include delivery of salivary effector proteins into host cells, acting as virulence factors to suppress host immunity, or as avirulence functions triggering immune activation. However, understanding of virulence and avirulence mechanisms in aphid-plant systems is currently limited. Here, we report discovery of an effector candidate family that is unique to aphids. Using functional genomics data on divergent pea aphid (Acyrthosiphon pisum) genotypes and their F1 progeny, we filtered for differentially expressed saliva proteins that co-segregated with virulence or avirulence phenotypes. LOC100575698 (ACPISUM_029930), annotated as an uncharacterized protein, was the sole candidate effector for which RNA-Seq and saliva proteomics data showed significantly different expression both between avirulent and virulent parents and between their segregating F1 progeny, with this gene upregulated in avirulent genotypes. BLASTP searches revealed multiple divergent homologs only in genomes of the Aphidomorpha infra-order, suggesting a hitherto undefined ancient aphid-specific gene family. AlphaFold models indicate strong structural similarities but weak sequence homology to chitinases. Because the aphid-specific clade all lack canonical DxxDxDxE motifs for catalytic activity, we designate the proteins as a novel CHitinase-Like (CHL) family. Association of ACPISUM_029930 (ApCHL1) with avirulence was further supported by co-segregating SNPs and a genotype-specific alternatively spliced isoform. We hypothesise that CHL proteins may function similarly to phylogenetically unrelated chitin-binding fungal effectors that sequester chitin, also present in aphid stylets, potentially preventing defence activation through plant chitin receptors and/or blocking chitin degradation by host-secreted chitinases.