Functional Analysis of Effector Hasp83 in the Pathogenicity of Puccinia striiformis f. sp. tritici

Wheat stripe rust is a serious disease on wheat, which is caused by Puccinia striiformis f. sp. tritici (Pst). Pst is an obligate biotrophic fungus, which can form haustorium during infection and absorb nutrients from the host plants via haustorium. Moreover, Pst secretes effectors through haustorium to regulate host immunity and promotes the infection process.

The objective of this study is to clarify the function and mechanism of Pst effectors, and to reveal the pathogenicity mechanism of Pst.

By comparing the transcriptome of Pst urediospore, germinated tube and haustorium, Hasp83 encoding secreted protein was identified to be significantly induced in haustorium, and whether it could inhibit the cell death caused by BAX on Nicotiana benthamiana leaves was observed through Agrobacterium-mediated transient expression. qRT-PCR was used to detect the expression level of Hasp83 during different Pst infection stages in wheat. The type Ⅲ secretion system (T3SS) of Pseudomonas fluorescens EtHan and host induced gene silencing (HIGS) were carried out to investigate the function of Hasp83 during Pst infection. The yeast two-hybrid (Y2H) system was used to screen the proteins interacting with Hasp83 in wheat, and co-inmunoprecipitation (Co-IP) assay was used to further verify the interaction by co-expressing Hasp83 and its candidate target proteins in N. benthamiana cells.

The open reading frame (ORF) of Hasp83 is 522 bp, encoding 173 amino acids. Hasp83 contains no conserved domain, and the N-terminal 1-29 amino acids encode a signal peptide, which could inhibit the cell death caused by BAX on N. benthamiana leaves through Agrobacterium-mediated transient expression. qRT-PCR analysis revealed that Hasp83 was up-regulated during Pst infection in wheat. Transient expression of Hasp83 in wheat via T3SS could inhibit callose accumulation caused by P. fluorescens, and lead to 19.35%-38.62% decrease of reactive oxygen species (ROS) accumulation area and necrotic cell area caused by the avirulent Pst race CYR23 in wheat. Silencing of Hasp83 by HIGS in Suwon11 wheat leaves infected with the virulent Pst race CYR31 significantly reduced pathogenicity of Pst compared to controls, resulting in less urediospore sporulation, shorter infection hyphal length, smaller infection area, and decreased haustorium number. Y2H result showed that the effector Hasp83 interacted with wheat hypersensitive-induced reaction (HIR) protein, Tahir1. The interaction between Hasp83 and Tahir1 was further confirmed by Co-IP assay in N. benthamiana.

Pst effector Hasp83 can suppress wheat immunity caused by the non-pathogenic bacteria and avirulent Pst, and enhance the pathogenicity of Pst.