Identification and functional analysis of putative effector proteins from Diaphorina citri and Candidatus Liberibacter asiaticus

Robert Schuurink^1*Sarmina Dangol¹Márcio Fernandes Alves Leite²Mônica Neli Alves³marc galland⁴Paula J.M. Van Kleeff¹Gertjan Kramer²Josiane Cecília Darolt³Laudecir Raiol-Junior³Rafael Brandao Garcia³Leandro Peña⁵Nelson Arno Wulff³Marcelo Pedreira Miranda³Silvio Lopes³Harro Bouwmeester²

• ¹Plant Physiology, University of Amsterdam, Amsterdam, Netherlands
• ²Universiteit van Amsterdam, Amsterdam, Netherlands
• ³Fundo de Defesa da Citricultura, Araraquara, Brazil
• ⁴Institut de Genetique Environnement et Protection des Plantes, Le Rheu, France
• ⁵Universitat Politecnica de Valencia, Valencia, Spain

Phloem feeders, such as the psyllid Diaphorina citri (D. citri), feed on plants through inserting their stylet bundle followed by probing the apoplast before reaching the phloem. The psyllids secrete watery saliva containing various proteins into the phloem, that can act as effectors to facilitate their feeding or modulate host defense responses. Concomitantly, feeding is the main mode of transmitting the Candidatus Liberibacter asiaticus (CLas) bacteria to the phloem. CLas produces several effectors that has been hypothesized to contribute to Huanglongbing (HLB) virulence. Here, we aimed to identify putative effector proteins from both the D. citri and CLas. To achieve this, we used different omics techniques on different tissues and organs from both plants and insects. More specifically, we performed transcriptomics on heads of healthy and CLas-infected D. citri, proteomics of artificial diet and of phloem of four different plant species fed on by healthy and CLas-infected D. citri. Subsequently we used various criteria and bioinformatics tools to predict putative effectors. This resulted in the identification of four proteins from D. citri (Ferritin, prisilkin, CG31997-PA and Pterin-4-alpha-carbinolamine dehydratase-like Protein (PCBD)) and two Sec-dependent effectors from CLas, CLIBASIA_04560 and CLIBASIA_05320, that were used for further functional studies. The expression of these six proteins in Nicotiana benthamiana modified the ROS burst triggered by flagellin, indicating that they can indeed function as effector proteins in planta. In addition, expression of the psyllid effectors in planta significantly reduced the growth of Pseudomonas syringae pv. tabaci (Pta).