PREreview of Breaching the cell-envelope barriers of gram-positive and fungal microbes by a type VI secretion system in Acidovorax citrulli

In this short manuscript by Pei et al., authors describe that a plant pathogen, Acidovorax citrulli, uses a T6SS to compete with Gram-positive bacterial and fungal species. A brief characterization of a new effector (named RhsB) is described, which belongs to the nuclease superfamily PD-(D/E)XK. The article touches upon a current topic that regards the ability of Gram-negatives to attack a Gram-positive species via T6SSs. This manuscript follows another paper on the same topic which is also on bioRxiv (https://doi.org/10.1101/2021.03.04.433973).

Major comments:

1)    It is always important to complement the ΔtssM mutant in all competition experiments (Fig 1). It is possible that the number of prey survival (which is also the number of prey recovered) might represent growth or metabolic differences between the two attacker strains (WT and ΔtssM) that are due to some non-specific acquired mutation in another locus.

2)    In my opinion this article has the same problem as the second one available on bioRxiv in regard of the T6SS activity against Gram-positives. It is important to include other techniques/experiments to show that the attacker (Gram-negative) is actually killing the prey (Gram-positive). The CFU (colony forming units) assays used in the paper only shows the number of viable cells after a certain time. It is not clear whether the prey was unable to grow or whether it was actually killed.

3)    I think it is very important to repeat the prey survival experiments (Fig. 1) in liquid media to determine whether the T6SS effect depends on contact between cells.

4)    The sentence in line 1 page 7 is overstated. The effector could be released in the extracellular medium and later internalized by a transporter, similarly to what was reported by Song et al. 2021 (PMID: 33462232).

Minor comment:

5)    The DNAse activity shown in Fig 2i does not seem to be fully reversed by the KD-AA mutation, which seems to have some degree of activity left. Maybe it would be best to co-incubate the native protein with its immunity in this assay.