Authors
King, J., Rahnama, M.
Abstract
Functional genomic analysis of essential genes in haploid filamentous fungi, such as the rice blast pathogen Magnaporthe oryzae (syn. Pyricularia oryzae), requires conditional expression systems since gene deletions often cause lethality. Here, we present a reproducible protocol for Conditional Promoter Replacement (CPR) using Agrobacterium-mediated transformation and homologous recombination to substitute a native promoter with the nitrogen-responsive pMoNIA1promoter from the M. oryzae NIA1 gene. The system enables precise, nitrogen source-dependent gene regulation and is validated using the BUF1 melanin biosynthesis gene. Replacement of the BUF1 promoter with pMoNIA1 produces a clear phenotypic switch from wild-type dark gray/black pigmentation under inducing conditions (KNO) to buff/tan under repressing conditions (glutamate). Molecular confirmation verified correct cassette integration, and quantitative RT-PCR showed a 44-65-fold repression of BUF1 expression under repressing conditions, substantially stronger than previously reported pMoNIA1 systems. This approach provides a robust, visually tractable, and broadly applicable method for conditional gene control in M. oryzae, facilitating functional studies of essential, pleiotropic, or stage-specific genes in filamentous fungi.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 01 Nov 2025.
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