Characterization of the interaction between the bacterium TR3 and Magnaporthe oryzae

Xue Zhang, Purdue University

Abstract

Rice blast disease caused by the filamentous ascomycete Magnaporthe oryzae, is one of the most destructive diseases that affect global rice production and food security. Bacteria have been explored and developed as biological control agents (Walsh, Morrissey et al. 2001). Pseudomonas syringae pv. tomato (Pst) DC3000 delivers effector proteins into plant cells by the bacterial type III secretion system (T3SS). Some of these P. syringae effectors target fundamental cellular processes that are conserved among eukaryotes. Heterologous expression of bacterial type III effectors (T3Es) HopAI manipulates the conserved cell pathway, MAPK signaling pathway in M. oryzae, which indicates T3E HopAI maintained its targets and the mode of action in evolutionary distant organisms. Moreover, M. oryzae may be used to characterize the T3Es function. HopAI is a phosphothreonine lyase that possesses the same in vitro catalytic activity as OspF and SpvC on Erk2 MAP kinases. In this study, we expressed two T3SS effectors HopAI and HopI1 from DC3000 in the rice blast fungus M. oryzae. Expression of HopAI resulted in defects in conidiation and hyphae growth similar to those of the ∆mps1 mutant. The phosphorylation level of Mps1 also was reduced in the HopAI transformants. Co- immunoprecipitation assays confirmed the physical interaction of Mps1 with HopAI, and Pmk1 also weakly interacted with HopAI. To understand the role of Mps1 during invasive growth, we expressed HopAI under the promoter of the MIR1 or BAS1 gene that specifically expressed at the infectious stage. In rice leaf sheath penetration assays, the PBAS1- or PMIR1-HopAI transformants were defective in invasive growth, indicating that MAPK signaling is important for cell-to-cell movement of infectious hyphae. The Bas1-GFP and Bas4-GFP fusion proteins in the PMIR1-HopAI transformants were not detected, which indicates that the expression of HopAI in invasive hyphae may play a role in fungal effectors localization or expression. Attachment with host cells is critical for T3SS activation of secretion (Notti and Stebbins 2016). To further characterize the interaction between the bacterium and M. oryzae, seven bacteria were screened for its inhibitory effect against M. oryzae required contact-based interaction. In this study, Pseudomonas fluorescens strain TR3 was isolated from rice leaves infected by M. oryzae and identified by sequence analysis. When co-cultured on Complete Medium (CM) plates, strain TR3 caused fungal cell death in a contact-based manner. Separation by a dialysis tube with the cutoff molecular weight of 14 kDa eliminated the inhibitory or lysis effects of TR3 on M. oryzae, excluding the possible involvement of small antifungal metabolites. Treatment with TR3 affected the phosphorylation level of Pmk1 and reduced the rate of appressorium formation and virulence in M. oryzae. The type 3 secretion system (T3SS) was proven to play a role in bacterial and fungal interaction (BFI) using T3SS inducing medium (M9 medium). Disruption of hrcC, an essential T3SS gene of T3SS, blocked flagella development in TR3 and reduced growth inhibition activities to M. oryzae. These results indicate that strain TR3 may deliver effector proteins into fungal cells by T3SS and TR3-M. oryzae can be used for studying bacterial-fungal interactions at the molecular level.

Degree

Ph.D.

Advisors

Xu, Purdue University.

Subject Area

Botany|Agriculture|Plant Pathology

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