A conserved extracellular ribonuclease with broad-spectrum cytotoxic activity enables smut fungi to compete with host-associated bacteria

Abstract

SummaryRibotoxins are secreted ribonucleases that specifically target and cleave the universally conserved sarcin-ricin loop sequence of rRNA, which leads to inhibition of protein biosynthesis and subsequently to cell death. We have identified and characterized a secreted Ribo1 protein of plant pathogenic smut fungi. Heterologous expression in different model systems showed that smut Ribo1 has cytotoxic activity against bacteria, yeast, host and non-host plants. Recombinant expression of Ribo1 inNicotiana benthamianainduced plant cell death; however, an active site mutant induced cell death only when expressed as a secreted protein. In the maize smutUstilago maydis, transcription ofRibo1is specifically induced in early infection stages. While a knock-out mutant revealed that Ribo1 is dispensable forU. maydisvirulence, the overexpression of Ribo1in-plantahad a strong dominant negative effect on virulence and induced host defense responses including cell death. This suggests a function of Ribo1 during the epiphytic development rather than for invasive colonization of the host. Accordingly, in presence of the biocontrol bacteriaPantoeasp., which were isolated from maize leaves, theribo1knock-out mutant was significantly impaired in virulence. Together, we conclude that Ribo1 enables smut fungi to compete with host-associated bacteria during epiphytic development.