Nuclear processes associated with plant immunity and pathogen susceptibility

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...

Adaptive mutations play an important role in molecular evolution. However, the frequency and nature of these mutations at the intra-molecular level is poorly understood. To address this, we analysed the impact of protein architecture on the rate of adaptive substitutions, aiming to understand how protein biophysics influences fitness and adaptation. Using Drosophila melanogaster and Arabidopsis thaliana population genomics data, we fitted models of distribution of fitness effects and estimated the rate of adaptive amino-acid substitutions both at the protein and amino-acid residue level. We performed a comprehensive analysis covering genome, gene and protein structure, by exploring a multitude of factors with a plausible impact on the rate of adaptive evolution, such as intron number, protein length, secondary structure, relative solvent accessibility, intrinsic protein disorder, chaperone affinity, gene expression, protein function and protein-protein interactions. We found that the relative solvent accessibility is a major driver of adaptive evolution, with most adaptive mutations occurring at the surface of proteins. Moreover, we observe that the rate of adaptive substitutions differs between protein functional classes, with genes encoding for protein biosynthesis and degradation signalling exhibiting the fastest rates of protein adaptation. Overall, our results suggest that adaptive evolution in proteins is mainly driven by inter-molecular interactions, with host-pathogen coevolution likely playing a major role.

Modern omics platforms have made the determination of susceptible/resistance genes feasible in any species generating huge numbers of potential _targets for crop protection. However, the efforts to validate these _targets have been hampered by the lack of a fast, precise, and efficient gene _targeting system in plants. Now, the repurposing of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has solved this problem. CRISPR/Cas9 is the latest synthetic endonuclease that has revolutionized basic research by allowing facile genome editing in prokaryotes and eukaryotes. Gene knockout is now feasible at an unprecedented efficiency with the possibility of multiplexing several _targets and even genome-wide mutagenesis screening. In a short time, this powerful tool has been engineered for an array of applications beyond gene editing. Here, we briefly describe the CRISPR/Cas9 system, its recent improvements and applications in gene manipulation and single DNA/RNA molecule analysis. We summarize a few recent tests _targeting plant pathogens and discuss further potential applications in pest control and plant–pathogen interactions that will inform plant breeding for crop protection.