PREreview of Four cell wall-degrading enzymes ofXanthomonas campestrispv.campestrisdetermine bacterial escape from hydathodes to the leaf vasculature

Overview

This paper by Paauw et al. focuses on the role of the Type II Secretion System (T2SS) in the pathogenicity of Xanthomonas campestris pv. campestris (Xcc), particularly during the transition from hydathodes to xylem vasculature during natural infection. Both the Type III Secretion System (T3SS) and T2SS are critical for Xcc virulence but serve distinct roles. The T3SS injects effectors into plant cells to subvert immune responses, while the T2SS releases cell walls degrading enzyme (CDWEs) facilitating nutrient acquisition and tissue colonization.

Different X. campestris pathovars exhibit distinct infection strategies. Some colonize vascular tissues via hydathodes (e.g., X. campestris pv. campestris or Xcc), while others inhabit the leaf apoplast via stomata (e.g., X. campestris pv. raphani or Xcr). This specialization is underpinned by enzymes like CbsA, a cell wall-degrading enzyme crucial for vascular pathogenicity. However, the complexity of pathogenicity is evident; introducing the CWDE CbsA alone into non-vascular strains does not confer vascular colonization ability, indicating the involvement of additional, unidentified factors.

The authors bridge these knowledge gaps through innovative approaches, including generating T2SS and T3SS mutants in bioluminescent Xcc strains to track infection progression at the hydathodes. Their findings reveal critical roles for T2SS in accessing and spreading within plant vasculature, contrasting with T3SS functions. A comparative pangenomic analysis further identifies four CWDEs vital for vascular pathogenesis, suggesting niche-specific adaptations that merit further investigation.

General Comments

We really enjoyed reading this paper as it is a logical progression from our current understanding of the bacterial factors required for vascular colonization during hydathode infection. The results are robust with clear phenotypes. However, we think that complementation of some of these important genes like xpsD (T2SS mutant) would further strengthen their conclusions. Additionally, verification that the putative CWDEs do indeed have degrading properties would be nice to include in this study. We expand upon these limitations further in the results section.

Introduction:

The introduction is well written, comprehensive and includes all the information needed to understand this study.

Results:

Overall, the results reported are of high quality and logically structured. Below we have a few comments/suggestions based on the figures.

Line 119 – refer to Fig S1A not just Fig S1

Inclusion of a graphical model showing the movement of the bacteria between the hydathode, xylem endings, lateral veins, etc. would be helpful to visualize the bacterial tissue-to-tissue movement.

Fig 1 & 2 –

·      Figs. 1 and 2 demonstrate that xps-T2SS is required for movement between hydathode and midvein and then into lateral veins. A complementation of xpsD in the Xcc DxpsD mutant would further validate these claims.

·      Please refer to Fig S1A in the figure legend of Fig 1.

·      Fig S1B is not references at all in the manuscript. It could be added in line 149.

Fig S2 –

·      It is difficult to ascertain whether the xylem pores in Fig. S2 are actually blocked by biofilm. Also, more methodological information about how these samples were infected would help support their claims, given this is a different host.

·      We are not sure what this experiment adds to the overall manuscript as all they can state is that the xpsD mutant is able to colonize the xylem in cabbage.

Fig 3 –

·      There is a missed opportunity on Fig. 3A to show more statistics on orthogroups found, size of pangenome vs. core genome, etc. Please fill in this information

·      Highlighting the pathovars used in the experiments in Fig. 3B would be nice. In line 208 they state that the pathovars cluster together but do not demonstrate this in the figure.

·      Are the gene models in Fig. 3E to scale? A scale bar would be helpful.

·      Drawing a box around the orthologs used in this study in Fig 3C and D aka. The cutoff used to identify the unique orthologs would facilitate the readers understanding of how they identified the CWDEs.

·      Please provide a list of the genomes used for the pangenome analysis. Additionally, for the sake of transparency, at the time of publication if the authors could include all orthologs identified through their pangenome analysis, this would be a wonderful resource for the community.

Fig 4 –

·      Confirming the specific enzymatic activity and the expression of each CWDE would provide further context for the mutant infection phenotypes. Are the Xcc CWDEs functional? Plate assays have been conducted before with T2SS mutants by the authors previously (Pfeilmeier et al. 2024, PMID: 38172620). More specifically, these mutants are unable to generate halos in agar plates containing, milk, PGA, CMC, Azo-xyloglucan, and RBB-xylan. Testing the Xcc mutants against these same substrates will verify the enzymatic activity of the CWDEs

·      Xcr has two homologs of these CWDEs, does complementing this strain with their Xcc counterparts make it into a vascular colonizer? We know the cbsA alone does not do this (Dubrow et al. 2022, PMID: 35536128).

·      The cbsA mutant, quadruple mutants, and complemented lines should be infected via the cutting assay to demonstrate movement or lack of movement between the tissues, as the key message is the that these enzymes facilitate hydathode to vasculature movement.

Fig S4 –

·      This figure is a call back from an observation made in their previous work (Pfeilmeier et al. 2024, PMID: 38172620). However, no context or explanation for why this experiment was done is included in the results section.

Fig S5 –

·      The growth rates in Fig. S5 do appear different so their statement in line 262 is incorrect. More specifically, the quadruple CWDE mutant has a slower growth rate in most medias which may help explain some of the cutting assay movement phenotypes ie. the delay in vasculature migration.

Tables S1 &S2 –

·      Elongation factor Tu in Xc-specific orthogroups is found to be unique which warrants further investigation. This suggests they have diversified under host pressure. This would be a very interesting research project to follow up with.

Discussion:

The discussion is well written, and the authors propose several follow up experiments which we agree would be worthwhile to explore. This includes using SEM at the hydathodes and investigating the integrity of the xylem pits during infection. The authors suggest that the CWDEs of different pathovars may have different substrate specificity which could restrict host range suggesting that the xylem composition could be a host factor for vascular pathogenicity. Our proposed experiment of complementing CWDEs from Xcc into Xcr could provide an opportunity to test this hypothesis.

Our Conclusion

We thoroughly enjoyed reading this paper. The author’s findings expand our knowledge on the role of the T2SS and CWDEs in tissue-specific colonization of Xcc.

Competing interests

The authors declare that they have no competing interests.