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

Ribotoxins 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 in Nicotiana benthamiana induced plant cell death; however, an active site mutant induced cell death only when expressed as a secreted protein. In the maize smut Ustilago maydis , transcription of Ribo1 is specifically induced in early infection stages. While a knock-out mutant revealed that Ribo1 is dispensable for U. maydis virulence, the overexpression of Ribo1 in-planta had 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 bacteria Pantoea sp., which were isolated from maize leaves, the ribo1 knock-out mutant was significantly impaired in virulence. Together, we conclude that Ribo1 enables smut fungi to compete with host-associated bacteria during epiphytic development. ### Competing Interest Statement The authors have declared no competing interest.