Reviewer #1 (Public review):

Summary:

The central question of this manuscript is the role of RNase III in supporting Salmonella infection. The authors begin with an RNAseq analysis of a collection of food or clinical Salmonella isolates from China, identifying RNase III (encoded by rnc) as an upregulated gene in clinical ("high virulence") isolates. Based on follow-up studies with knockout and complemented strains, the authors propose that RNase III has two roles - one in the upregulation of sodA expression to counter host-derived ROS, and the other in general degradation of dsRNA to dampen host immune responses. Overall, the manuscript is logical and the authors make largely reasonable interpretations of their data. However, the depth of supporting evidence limits the breadth of the authors' conclusions in their current form. Thus, this manuscript will be useful to researchers in directly related fields of study, but more work is required to understand how these proposed mechanisms function during infection.

Strengths:

(1) The use of comparative RNAseq between different isolates to identify potential virulence mechanisms is a powerful approach to understanding what makes certain strains more likely to cause infection over others.

(2) The experiments identifying dsRNA as the factor contributing to increased innate immune induction in the rnc knockout strain are particularly thorough.

(3) The authors observed an in vivo mammalian infection defect for RNase III-deficient Salmonella, a novel finding for the field and strong evidence that this protein is required to support pathogen fitness.

Weaknesses:

(1) The strengths of the manuscript are in places obscured by a lack of clarity and justification in the manuscript about strain selection and rationale for using some backgrounds over others. Moreover, several aspects of the organization and flow of the manuscript could be improved, as data is described out of order and the text description of results does not always align with the data presented.

(2) The specific claim that the relatively modest increase in expression of RNase III in some isolates (Figure 1A) accounts for their "virulence" is not well-supported, since the only comparisons in the study are between total knockouts or wild-type (and not overexpression) and the actual protein levels of RNase III are not quantified.

(3) Although the experiments on dsRNA are strong, they would have benefited from measurements of cytokine production/immune responses during infection with the actual knockout strains instead of transfected RNA along with quantification of Salmonella burdens.

(4) The contribution of RNase III catalytic activity (i.e., through the use of a catalytically dead mutant) was not assessed, which means that a role for general RNA binding or protein-protein interactions cannot be ruled out from this study.

(5) The in vivo work was limited to survival analysis, so whether the proposed mechanisms account for the defects observed could not be resolved.

(6) Statistical analysis throughout the manuscript is inconsistently applied, making it hard in places to determine whether the differences seen in phenotypes are biologically significant.