Identification and functional analysis of the type Ⅲ effector AopAI in Acidovorax citrulli

Abstract:

[Background] Bacterial fruit blotch caused by Acidovorax citrulli is a prevalent disease in cucurbitaceous crops, and the type III effectors (T3Es) secreted by the type III system into plants are the major pathogenic factors of A. citrulli. However, the knowledge about the T3Es in A. citrulli is limited. [Objective] To identify a novel T3E, Acidovorax outer protein AI (AopAI), in A. citrulli, and investigate its impact on the bacterial pathogenicity and interference with plant immunity. [Methods] Bioinformatics tools were used to analyze the sequence characteristics of AopAI, and then the AvrBs1 as an avirulent reporter was used to verify the transport function of AopAI. Fluorescence quantitative polymerase chain reaction (PCR) was carried out to determine the expression of aopAI and the effect of AopAI on the expression of marker genes of the PAMP-triggered immunity (PTI) signaling pathway in plants. Furthermore, the gene insertion mutation and functional complementation methods were employed to examine the pathogenicity of the pathogen and the accumulation of hydrogen peroxide and callose in plants. Finally, the transient expression method was used to predict the subcellular localization and the ability of AopAI to inhibit elicitor-induced cell death. [Results] AopAI had a conserved domain of adenosine diphosphate (ADP) ribosyltransferase and no transmembrane helix region or signal peptide. The expression of aopAI was significantly down-regulated in the mutants of hrpX and hrpG (core regulatory genes of T3SS). The co-expression of aopAI and the AvrBs1 functional region (59-445 aa) induced the hypersensitive reaction in the leaves of 'ECW-10R' pepper, which indicated that AopAI was a transporter protein. The aopAI mutant presented declined pathogenicity to cucumber cotyledons, while it increased the accumulation of hydrogen peroxide and callose. After transient expression of AopAI in Nicotiana benthamiana, the protein was localized in the cell membrane and nucleus. Additionally, AopAI inhibited the elicitor NIP-induced leaf cell death and down-regulated the expression of the PTI marker genes GRAS2 and ACRE31. [Conclusion] We identified a typical T3E, AopAI in A. citrulli, which was localized in the nucleus and cell membrane and had an ADP ribosyltransferase domain. AopAI functioned as a virulent protein inhibiting NIP-induced cell death and reduced hydrogen peroxide and callose by inhibiting the ACRE31-regulated immune pathway, thus suppressing the PTI defense mechanism in plants.