Aphids secrete diverse repertoires of effectors into their hosts to promote the infestation process. While ‘omics’ approaches facilitated the identification and comparison of effector repertoires from a number of aphid species, the functional characterization of these proteins has been limited to dicot (model) plants. The bird cherry-oat aphid Rhopalosiphum padi is a pest of cereal crops, including barley. Here, we extend efforts to characterize aphid effectors with regard to their role in promoting susceptibility to the R. padi–barley interaction. We selected three R. padi effectors based on sequence similarity to previously characterized Myzus persicae effectors and assessed their subcellular localization, expression, and role in promoting plant susceptibility. Expression of R. padi effectors RpC002 and Rp1 in transgenic barley lines enhanced plant susceptibility to R. padi but not M. persicae, for which barley is a poor host. Characterization of Rp1 transgenic barley lines revealed reduced gene expression of plant hormone signalling genes relevant to plant–aphid interactions, indicating that this effector enhances susceptibility by suppressing plant defences in barley. Our data suggest that some aphid effectors specifically function when expressed in host species, and feature activities that benefit their corresponding aphid species.

1 The bird cherry aphid Rhopalosiphum padi and the green peach aphid Myzus persicae are 2 devastating insect pests on agriculturally important crops. While the host range of R. padi 3 consists mainly of cereal plants, M. persicae can infest thousands of plant species from 4 across different plant families. However, despite a broad host range, which includes 5 Nicotiana benthamiana, M. persicae performs poorly on barley. To gain insights into the 6 role of aphid effectors in determining aphid host range, we undertook a comparative 7 functional characterization approach for 3 predicted orthologous effector pairs from M. 8 persicae and R. padi. We compared subcellular localisation and protein stability of these 9 effectors in N. benthamiana and determined their contribution to host susceptibility. While 10 subcellular localization of members of each effector pair was similar, we observed 11 differences in proteins stability, especially in the case of RpC002 (R. padi C002)/MpC002 12 (M. persicae C002). For example, RpC002 was less expressed and/or stable than 13 MpC002 in N. benthamiana and, in contrast to MpC002, did not enhance susceptibility in 14 this plant species. However, expression of RpC002, as well as R. padi effector Rp1, in 15 transgenic barley lines enhanced plant susceptibility to R. padi but not M. persicae. Our 16 data suggests that some aphid effectors specifically function when expressed in host 17 species, and feature activities that benefit their corresponding aphid species. 18 Characterization of Rp1 transgenic barley lines revealed reduced gene expression of plant 19 hormone signalling genes relevant to plant-aphid interactions, suggesting this effector 20 enhances susceptibility by suppressing plant defences in barley. 21 22 42persicae, expression of ApC002 from A. pisum in these same plant species has no visible 43 impact on the host interaction with M. persicae (Pitino and Hogenhout, 2013). The 44 difference in effector activity was attributed to a motif sequence (NDQGEE) in the N-45 terminal region of MpC002, which is lacking in ApC002 (Pitino and Hogenhout, 2013). 46 Whether the presence/absence of this motif affects protein stability rather than activity 47 across plant species remains to be determined. In addition, several effectors from the 48 broad host range pest M. persicae have been implicated in promoting host susceptibility, 49 including Mp1 and Mp58 (Pitino and Hogenhout, 2013;Elzinga et al., 2014; Rodriguez et 50 al., 2017). However, the underlying mechanisms by which these effectors impact 51 susceptibility remain largely unknown. We previously described that Mp1 associates with 52 2 2 the host trafficking protein VPS52 (Vacuolar Sorting Associated Protein 52) to promote 53 plant susceptibility . Using different combinations of Mp1 and 54 VPS52 variants from different plant and aphid species, respectively, we showed that the 55 Mp1-VPS52 association is highly specific to the broad host range pest M. persicae and its 56 hosts, and likely shaped by plant-aphid co-evoluti...