Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)–mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes.

BackgroundRNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.Methodology/Principal FindingsIn this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.Conclusions/SignificanceSimilar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.

Microbial pathogens and pests produce effectors to modulate host processes. Aphids are phloem-feeding insects, which introduce effectors via saliva into plant cells. However, it is not known if aphid effectors have adapted to modulate processes in specific plant species. Myzus persicae is a polyphagous insect that colonizes Arabidopsis thaliana and Nicotiana benthamiana, while the pea aphid Acyrthosiphon pisum specializes on colonizing plant species of the family Fabaceae. We found that M. persicae reproduction increased on transgenic Arabidopsis, producing the M. persicae effectors C002, PIntO1 (Mp1), and PIntO2 (Mp2), whereas reproduction of M. persicae did not increase on Arabidopsis producing the A. pisum orthologs of these three proteins. Plant-mediated RNA interference experiments showed that c002- and PIntO2-silenced M. persicae produce less progeny on Arabidopsis and N. benthamiana than nonsilenced aphids. Orthologs of c002, PIntO1, and PIntO2 were identified in multiple aphid species with dissimilar plant host ranges. We revealed high nonsynonymous versus synonymous nucleotide substitution rates within the effector orthologs, indicating that the effectors are fast evolving. Application of maximum likelihood methods identified specific sites with high probabilities of being under positive selection in PIntO1, whereas those of C002 and PIntO2 may be located in alignment gaps. In support of the latter, a M. persicae c002 mutant without the NDNQGEE repeat region, which overlaps with an alignment gap in C002, does not promote M. persicae colonization on Arabidopsis. Taken together, these results provide evidence that aphid effectors are under positive selection to promote aphid colonization on specific plant species.