Characterization of an antimicrobial and phytotoxic ribonuclease secreted by the fungal wheat pathogen Zymoseptoria tritici

Abstract

Summary The fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB) disease of wheat leaves. Zymoseptoria tritici secretes many functionally uncharacterized effector proteins during infection. Here, we characterized a secreted ribonuclease (Zt6) with an unusual biphasic expression pattern.Transient expression systems were used to characterize Zt6, and mutants thereof, in both host and non‐host plants. Cell‐free protein expression systems monitored the impact of Zt6 protein on functional ribosomes, and in vitro assays of cells treated with recombinant Zt6 determined toxicity against bacteria, yeasts and filamentous fungi.We demonstrated that Zt6 is a functional ribonuclease and that phytotoxicity is dependent on both the presence of a 22‐amino‐acid N‐terminal ‘loop’ region and its catalytic activity. Zt6 selectively cleaves both plant and animal rRNA species, and is toxic to wheat, tobacco, bacterial and yeast cells, but not to Z. tritici itself.Zt6 is the first Z. tritici effector demonstrated to have a likely dual functionality. The expression pattern of Zt6 and potent toxicity towards microorganisms suggest that, although it may contribute to the execution of wheat cell death, it is also likely to have an important secondary function in antimicrobial competition and niche protection.

A review of the known unknowns in the early stages of septoria tritici blotch disease of wheat

“…Studies investigating fungal proteins secreted during both the compatible (Kettles et al , ) and the incompatible (M’Barek et al ., ) interaction have made progress in answering the question of whether or not Z. tritici is a hemibiotroph. At present, and based on previously characterized proteins and current protein prediction models, it would appear that there is little evidence that feeding is taking place at a substantial level during the latent phase (Palma‐Guerrero et al ., ).…”

“…More recently, in 2017, the Z. tritici ribonuclease effector Zt6, toxic to bacteria, yeasts and tobacco but not Z. tritici itself, was shown to elicit cell death in wheat as well as serving a secondary role in antimicrobial competition (Kettles et al , ). Not only does Zt6 have dual functionality, it also displays a double peak in expression, first at 1 dpi, followed by a second peak at 14 dpi, coinciding, as do the effectors discovered by Rudd et al (), with the switch to necrotrophy.…”

“…Not only does Zt6 have dual functionality, it also displays a double peak in expression, first at 1 dpi, followed by a second peak at 14 dpi, coinciding, as do the effectors discovered by Rudd et al (), with the switch to necrotrophy. However, unlike the NbBAK1 and NbSOBIR1 effectors, the ability of Zt6 to induce cell death is independent of host receptor‐like kinase resistance genes (Kettles et al , ). Although this evidence points to a role in pathogenicity or virulence of the fungus, Δ Zt6 mutants had no loss of virulence when inoculated onto wheat leaves (Kettles et al , ).…”

“…However, unlike the NbBAK1 and NbSOBIR1 effectors, the ability of Zt6 to induce cell death is independent of host receptor‐like kinase resistance genes (Kettles et al , ). Although this evidence points to a role in pathogenicity or virulence of the fungus, Δ Zt6 mutants had no loss of virulence when inoculated onto wheat leaves (Kettles et al , ). This suggests, therefore, that perhaps the primary role of Zt6 is not toxicity towards the plant, but rather to competing microorganisms.…”

The inconspicuous gatekeeper: endophytic Serendipita vermifera acts as extended plant protection barrier in the rhizosphere

“…This has led to the hypothesis that microbe−microbe competition and co‐operation are crucial processes affecting plant–microbe interactions in the rhizosphere, and are possibly driven by microbial effector proteins (Snelders et al ., ). Recently, the Zymoseptoria tritici ribonuclease effector Zt6 was shown to display both phytotoxic as well as antimicrobial activity (Kettles et al ., ), providing first experimental evidence for a class of effector proteins required for plant colonisation as well as microbe−microbe interactions. Similarly, the gene tvhydii1 of Trichoderma virens , encoding a class II hydrophobin, is expressed during Arabidopsis thaliana root colonisation as well as confrontation with the phytopathogen Rhizoctonia solani (Guzman‐Guzman et al ., ).…”