Abstract
RNA-binding proteins play a central role in post-transcriptional mechanisms that control gene expression. Identification of novel RNA-binding proteins in fungi is essential to unravel post-transcriptional networks and cellular processes that confer identity to the fungal kingdom. Here, we carried out the functional characterisation of the filamentous fungus-specific RNA-binding protein RBP35 required for full virulence and development in the rice blast fungus. RBP35 contains an N-terminal RNA recognition motif (RRM) and six Arg-Gly-Gly tripeptide repeats. Immunoblots identified two RBP35 protein isoforms that show a steady-state nuclear localisation and bind RNA in vitro. RBP35 coimmunoprecipitates in vivo with Cleavage Factor I (CFI) 25 kDa, a highly conserved protein involved in polyA site recognition and cleavage of pre-mRNAs. Several targets of RBP35 have been identified using transcriptomics including 14-3-3 pre-mRNA, an important integrator of environmental signals. In Magnaporthe oryzae, RBP35 is not essential for viability but regulates the length of 3′UTRs of transcripts with developmental and virulence-associated functions. The Δrbp35 mutant is affected in the TOR (target of rapamycin) signaling pathway showing significant changes in nitrogen metabolism and protein secretion. The lack of clear RBP35 orthologues in yeast, plants and animals indicates that RBP35 is a novel auxiliary protein of the polyadenylation machinery of filamentous fungi. Our data demonstrate that RBP35 is the fungal equivalent of metazoan CFI 68 kDa and suggest the existence of 3′end processing mechanisms exclusive to the fungal kingdom.
Highlights
The rice blast fungus Magnaporthe oryzae causes significant economic yield losses in rice and wheat [1,2,3]
We prove that the Drbp35 mutant is affected in the TOR signaling pathway showing significant changes in nitrogen metabolism and protein secretion
Nothing it is known about pre-messenger RNA (mRNA) 39 end processing in filamentous fungi and our study suggest that their polyadenylation machinery differs from yeast and higher organisms
Summary
The rice blast fungus Magnaporthe oryzae causes significant economic yield losses in rice and wheat [1,2,3]. Early infection is initiated by the adhesion of three-celled conidia to the surface and the development within a few hours of a short germ tube that differentiates into a penetration structure known as an appressorium. A hyphal peg produced in the base of the appressorium breaches the leaf cuticle and an invasive hypha (IH) initiates the colonisation of epidermal cells. This IH is coated by a plant-derived layer called extra-invasive hyphal membrane [4], and fungal effectors which facilitate infection and/or induce host immune responses are transferred to the plant cytoplasm across this membrane [5,6]. Silencing pathways and fungal-specific small RNAs have been identified in M. oryzae [13,14,15], very little is known of the post-transcriptional regulatory network that control M. oryzae infection ability
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