PREreview de Salicylic acid-triggered apoplastic proteolysis releases cryptic phytocytokines with distinct immunogenic functions

Katzy et al. showed that the immune phytohormone salicylic acid (SA) triggers the release of cryptic phytocytokines in maize to regulate immunity. The authors found that SA increases serine hydrolase activity and remodels the maize apoplastic peptidome. Through peptidomic analysis and functional assays, they further showed that two peptides, PC13 and PC14, have opposing effects on resistance to Ustilago maydis. Overall, the experiments are carefully executed, and the manuscript is clearly written. This work advances our understanding of plant immunity by linking SA signaling to the release of cryptic phytocytokines, a connection that has not been extensively studied. The data provide solid support for SA-induced remodeling of the apoplastic peptidome, but the opposing effects of these peptides on infection require further validation.

Major comments

1. Although Figure 1D shows that trypsin treatment abolished the PR gene-inducing activity of the apoplastic fraction from SA-treated plants, I have some questions about the reinfiltration experiments in Figure 1. Could differences in residual SA levels between the mock- and SA-treated fractions contribute to the observed responses? I suggest measuring SA levels in the reinfiltrated fractions and testing whether the corresponding concentrations can inducePR gene expression. These experiments may help clarify the contribution of residual SA and strengthen the authors’ conclusions.

2. Are the opposing effects of PC13 and PC14 specific to U. maydis infection, or do they reflect broader effects on disease resistance? Given that both peptides induce PR gene expression, I suggest pretreating maize with the synthetic peptides and subsequently performing infection assays with U. maydis and additional pathogens. These experiments would help clarify the roles of these peptides in immunity and complement the transcriptomic analysis following peptide treatment.

3. The authors propose that PC13 activates SA signaling and promotes programmed cell death (PCD). However, SA signaling is known to promote cell survival, so induction of SA-associated genes alone does not establish a role in promoting PCD. In addition, the RNA-seq analysis does not clearly explain how PC14 promotes susceptibility, as the enriched GO terms do not provide clear support for its proposed role in preserving host cell viability. Measuring SA levels and assessing cell death, for example through ion leakage assays, would help substantiate these interpretations.

Minor points

1. In Figure 1C, the asterisk above PR10 in the SA-derived APF treatment at 12 h is questionable, as the expression level appears similar to that of the control. Checking the raw data and corresponding statistical analysis would help confirm whether the asterisk indicating statistical significance is appropriate.

2. The disease category labels are missing from the leftmost set of plots in Figure 2F. Adding the disease category labels to the leftmost set of plots in Figure 2F would help readers interpret the symptom scores and compare disease severity among the different fungal strains.

3. For Figure 3B, the authors may consider including a heatmap of gene-level expression changes rather than showing the number of genes to help readers compare responses across treatments and time points.

4. In Figure 5A, all four lanes are currently labeled “+”. It would be helpful to verify the “−” and “+” labels to ensure that the treatment conditions are correctly indicated.

5. To facilitate comparison between Figures 5A and 5B, it might be helpful to present the data consistently as either a percentage of the mock control or relative fluorescence intensity. This could make it easier for readers to follow the changes in apoplastic serine hydrolase activity over time after SA treatment.

I reviewed this preprint as part of the HHMI-Transparent and Accountable Peer Review training program. This report was written with the support of my research mentor.

Yixuan (Jack) Chen

Ph.D. student | Dong Lab | HHMI-Duke University

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they did not use generative AI to come up with new ideas for their review.