Identification and functional analysis of the type III effector AopBF1 in Acidovorax citrulli

Abstract:

[Background] Bacterial fruit blotch is a prevalent quarantine disease caused by Acidovorax citrulli in cucurbit crops. A. citrulli secretes the key pathogenic factor, known as type III effectors (T3Es), into plant cells via the type III secretion system (T3SS). However, the pathogenesis of T3Es remains elusive. In previous studies, we have identified several candidate T3Es in A. citrulli FC440. [Objective] To reveal the sequence features, transport characteristics, and the role in the pathogenic process of the candidate T3E AopBF1 from A. citrulli FC440 strain, so as to lay a theoretical foundation for dissecting the mechanism of pathogenesis. [Methods] Bioinformatic tools were used to predict and analyze the T3E sequence characteristics of AopBF1. Then, RT-qPCR and an avirulent protein reporter system analysis were conducted to reveal the regulation and transport characteristics of AopBF1. The pathogenicity of aopBF1 mutant (insertion mutation) and overexpressing strain was investigated to unveil the role of AopBF1 in pathogenicity. [Results] AopBF1 had the typical T3E characteristics, possessing a protein kinase domain and no other conserved domain. The expression of AopBF1 was significantly down-regulated in the mutants of the T3SS core regulatory genes hrpX and hrpG. The co-expression of aopBF1 and the AvrBs1 functional region (59-445 aa) in the avrBs1 mutant induced hypersensitive reactions (HRs) in the leaves of ECW-10R pepper containing the Bs1 protein. The pathogenicity of the aopBF1 mutant in cucumber was significantly reduced, while the accumulation of hydrogen peroxide, superoxide anion radical, and callose significantly increased. The overexpression of aopBF1 significantly enhanced the pathogenicity and delayed HRs in Nicotiana benthamiana. The AopBF1 transiently expressed in N. benthamiana was localized in the cytoplasm, nucleus, and cell membrane, and then induced HRs and up-regulated the expression of PTI and hormone pathway-related genes. [Conclusion] AopBF1 is demonstrated to be a T3E with a protein kinase domain in A. citrulli. It can inhibit PTI immune responses such as host reactive oxygen species and callose accumulation to enhance the pathogenicity of A. citrulli and can trigger PTI and hormone-related immune responses in N. benthamiana containing R proteins.

Key words:Acidovorax citrulli;AopBF1;type III effectors;virulent protein;protein kinase