Review 2: "Viral Proteins Activate PARIS-Mediated tRNA Degradation and Viral tRNAs Rescue Infection"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review:

In this manuscript, the Isaev, Wiedenheft and Bikard groups have characterized the activity of the recently discovered PARIS bacterial immune system. They find that the Paris immune system encodes two proteins AriA and AriB, which form a large, oligomeric complex. This AriA6B3 complex is the inactive state, and recognition of the phage Ocr protein triggers AriB release and activation. AriB is a nuclease that degrades host tRNA to induce translational shutdown and abortive infection, but this defense can be overcome by phage-encoded mutant tRNA that resist AriB nuclease activity.

Overall, this is a very strong manuscript. The combination of cryo-EM structures with functional data provides compelling evidence for the Paris mechanism and will be of great interest to the expanding bacterial immunity field.

If the authors could address the following points, the manuscript would be strengthened further:

1. The conformational change in the AriB cis to trans is not very clear. Mode vectors would help with this visualization.

2. Interface between AriA and AriB is rather small, and AriB appears to be sub-stoichiometric (at least based on the weak density in the 2D and 3D classes). This is the case for the Corbett lab preprint. In that case, how is the toxic effect prevented?

3. The authors used local refinement to improve the density for an asymmetric unit of the AriAB map. Did the authors attempt symmetry expansion prior to focused 3D classification and local refinement? If not, why?

4. The description in the methods section of how 3DVA was performed and how the results were visualized is insufficient. How was this done? What resolution was the map low pass filtered to, what was the output mode? Showing different conformations from 3DVA as a supplementary figure would be insightful and useful for the reader.

5. Bootstrap scale of phylogenetic tree in Fig. 6 is unclear. It is hard to see the different shades of green.

6. The phylogenetics is very fascinating, particularly the fused Paris systems. Since the canonical Paris seems to function through the disassembly of the AriAB complex, how would this work if both proteins were a single fused polypeptide?

7. The Hi-C experiments in Fig. S8 are a great idea, but the data is not terribly convincing that the chromosome changes compactness.

8. Based on the AF2 model of the AriB dimer, can the authors speculate about what makes this nuclease specific for tRNA cleavage, and how the phage tRNA mutations prevent cleavage activity? Where would the AriB active sites map to the aligned tRNA and the positions of the phage tRNA mutations?