Promoter trapping in the rice blast fungus Magnaporthe grisea

Lei Li, Purdue University

Abstract

Rice blast disease, caused by Magnaporthe grisea, is the major disease that threatens the rice production throughout the world. Many studies have focused on the signaling and biology in the early stages of infection, and dozens of pathogenicity or virulence genes have been characterized. However, there is little knowledge about the molecular events occurring during later stages of the pathogen-host interactions. The goal of this study was to identify M. grisea genes that are specifically or highly expressed during rice infection, especially stages after penetration. Enhanced green fluorescent protein (EGFP)-based promoter trapping vectors were transformed into M. grisea to generate a collection of 4398 transformants by restriction enzyme-mediated insertion (REMI) or Agrobacterium tumefaciens -mediated transformation (ATMT). After screening 1727 transformants, a total of 20 transformants defective in infection-related morphogenesis or expressed EGFP in different infection structures were identified. The genomic sequences flanking the transforming vectors were recovered from four of these 20 transformants by plasmid rescue or thermal asymmetric interlaced PCR (TAIL-PCR). One of the transformants, L1320, was further characterized. The EGFP signal was detected in the nuclei of the infectious hyphae of L1320. A novel gene named MIR1 (for Magnaporthe infection-related gene 1, MIR1) that is unique to M. grisea was disrupted in L1320 by the transforming vector. The N-terminal 107 residues of Mir1 were fused in-frame with EGFP. The fusion gene was under the control of MIR1 promoter. However, no significant defect in colony morphology or pathogenicity was observed in either transformant L1320 or the knockout mutants of MIR1. The expression level of MIR1 in infected tissues was 165-fold higher than that in mycelia grown in complete medium. Deletion analyses of 1.5 kb upstream of the MIR1 gene indicated that the 458-bp region upstream of the start codon was sufficient to induce the expression of MIR1 in planta. Further analysis indicated that Mir1 is a low complexity protein and its interaction with other M. grisea proteins may be required for nuclear localization in infectious hyphae.

Degree

Ph.D.

Advisors

Xu, Purdue University.

Subject Area

Plant pathology

COinS