Peer review of: A fungal pathogen induces systemic susceptibility and systemic shifts in wheat metabolome (2019)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The manuscript by Seybold et al. carried out comparative analysis of the interaction of Z. tritici in 
susceptible and resistant wheat using coinfection assays, comparative metabolomics, and 
microbiome profiling. Z. tritici was found to suppress the production of immune-related 
metabolites in a susceptible cultivar. The fungus-induced immune suppression appears to spread 
within the leaf and to other leaves. Defense-related biosynthetic pathways are suppressed and 
induced in susceptible and resistant cultivars, respectively. In addition, infection by the fungus also 
affects the wheat leaf microbiome. 

Although the data presented are potentially interesting, they are almost exclusively descriptive and 
failed to provide mechanistic insights on how immunity to Z. tritici is regulated/achieved in wheat. 
The differences in P. syringae pv. oryzae growth between mock and Z. tritici treatment were 
marginal and the differences in systemic fungal growth between mock and Z. tritici treatment were 
even smaller, which make the biological meaning of these differences questionable. 

Reviewer #2 (Remarks to the Author): 

The field of plant pathology is changing, climate change, toxic pesticides and resistance challenge 
efforts. The authors studied the mode of action of the fungal wheat pathogens Zymoseptoria tritici. 
They found a new mechanism, which they term “systemic induced susceptibility”. This was 
supported by a defense-related biosynthetic pathway that are suppressed and induced in 
susceptible and resistant cultivars, and the finding that that Z. tritici suppresses the production of 
immune-related metabolites in a susceptible cultivar. This is an interesting finding, logic in my 
eyes and well known from other microbe-host interactions. 

The strength of the study is the comprehensive experimental design including coinfection assays, 
comparative metabolomics, and microbiome profiling to study the interaction of Z. tritici in 
susceptible and resistant wheat as well as the logic, hypothesis-driven experimental design. 

The weakness of the study is that this new plant-pathogen interaction was shown only this this 
artificial system for one pathogen, even for one strain of the pathogen. Here, it needs more 
evidence that it occurs in the field or works for other pathogens too. 

The pathogen induced strong shifts in wheat leaf microbiota. It is of value that these studies are 
integrated, and the extend of the shift shows how important this is. However, as presented these 
results are isolated, and should be more linked with the other results. Moreover, I would suggest, 
to look at the whole pathosystem from the microbiome perspective. The pathogen is a member of 
the microbiome as well. Qualitative data of the microbiota are missing! 

The definition of plant pathogens in these categories biotrophic, hemibiotrophic etc. is a really 
anthropocentric view, which needs revision. In this manuscript I would not place it so central that 
Zymoseptoria is hemibiotrophic. It’s the function of all fungi to degrade organic matter. 

The title has to be changed – it is to broad, only one pathosystem was studied. 

Discussion section would profit from shortening. 

Reviewer #3 (Remarks to the Author): 

The manuscript reports on the correlation between SAR and pathogenic fungi. 
The study shows very interesting results, a possibility for fungi to suppress plant immune system 
based on metabolomics data. 
I would like to recommend the publication of the manuscript. 

One comment: 
Line 123, 37,664 seems the number of MS peaks. Then, to say the number is for metabolites is 
rather doubtful. So, better to say it is the number of peaks or signals in the analytical system. 

Reviewer #4 (Remarks to the Author): 

The manuscript aims to investigate the interaction between the fungal pathogen Z. tritici and 
wheat. The work presents three related stories. In the first story, the authors showed that upon 
infection with the fungal pathogen Zymoseptoria tritici an induced susceptibility in adjacent regions 
of the leaf is triggered. Second, they showed that there are differences in the accumulation of 
metabolomes in two wheat cultivars, one susceptible and one resistant. The third story is based on 
a microbiome analysis of wheat leaves and demonstrates that in the resistant cultivar there is a 
reduction in the richness and changes in the structure of the bacterial community upon infection 
with the pathogen. The work presents a very original work and provides good quality data which is 
of significance to the field. However, the work lacks a general focus and sometimes it is difficult to 
understand the logic of the text. Although the three stories are novel and related, the connection 
between them is not clear in the manuscript. 
The authors have a very novel and interesting finding. They demonstrate that Z. tritici triggers 
induced susceptibility. They call this phenomenon “systemic induced susceptibility” (SIS). The 
main goal of the work is to characterize SIS, but the analysis performed does not provide 
evidences for the consequences of SIS in the microbiome or the metabolites involved in this 
phenomenon. To achieve this, first of all, the comparisons in the metabolome should be performed 
between mock and infected plants. In the results and figures, it seems (although it is not very 
clear) that this is not the main comparison performed. Comparisons between the two cultivars is 
valid, but would not provide information on what are the compounds involved in SIS (and SAR). 
This is mainly due to the complex genetic background of both wheat cultivars and that SIS (and 
SAR) are induced upon infection, as shown in figure 2. In addition, changes in microbiome were 
only observed in the resistant cultivar, which points more towards the direction that SIS does not 
lead to changes in the microbiome. If there are evidences for SIS changing the microbiome, it 
should be explained in the text and figures. I suggest that either the focus of the manuscript 
switches towards the characterization of induced resistance by Z. tritici in wheat or that the data 
providing mechanistic understanding of SIS is highlighted. 

More detailed comments can be found below: 
1- The representation of the metabolome is not very clear. In figure 4b, it is very difficult to 
understand which comparison was performed based on the provided nomenclature. Based on the 
text, it seems that the main comparison was performed between both cultivars. If the hypothesis 
is that changes in the content of some metabolites lead to the observed phenotype upon infection 
by Z. tritici, then the authors should compare the metabolomic profile of infected and uninfected 
leaves. Most frequently the comparisons are between cultivars, which is also relevant, but not 
conclusive for SIS and SAR. 
The authors had interesting results related to the content of BXs in resistant and susceptible 
cultivars. In the resistant cultivar they observed increased levels of the inactive forms in adjacent 
tissues of the infection by the fungus. However, this was not observed in the susceptible cultivar. 
The authors conclude from this data that the fungus might manipulate the host in order to prevent 
the induction of this compound. However, it could very well be that only in Chinese Spring these 
inactive forms are accumulated and not in Obelisk. Actually, this seems to be the case since in Fig. 
S9 there is no induction of the BXs, but CS has higher constitutive levels of them. Thus, their 

function in SIS remains unclear and Z. tritici does not seem to have capacity to suppress their 
accumulation. 
In addition, it is known that Chinese Spring basal resistance is not higher than in other cultivars, 
but this cultivar is specifically able to recognize an effector of some strains of Z. tritici and 
subsequently trigger an immune response that hinders the progression of the pathogen (by the 
way this is key information that should be described in the introduction). The accumulation of the 
metabolites investigated in this work could contribute to the resistance response, but if this is the 
case, there should be differences in CS between the Mock and Zt treatments in figure 3c,d and S9. 
Based on the provided data this does not seem the case. 
In summary, I think that the authors should focus on differentially accumulated compounds upon 
infection in order to have more conclusive data on SIS (and also on SAR). 
2- In the discussion, the authors indicate that the data showed that pathogens have an active role 
in manipulating the microbiome (lines 316-322) in a susceptible host. Although this is true for the 
co-infections performed with Pseudomonas, it is not the case for the global microbiome analysis 
performed, since there are no differences between mock and Zt treatments in cultivar Obelisk in 
the figure 5. 
The main goal is to characterize the effect of SIS in the microbiome and based on the data 
presented, there is an effect on the bacterial community upon a resistance response against Z. 
tritici, but not in the compatible interaction. If there were observed changes in the susceptible 
cultivar, they should be shown in the results. 
3- Finally, although in the results it is said that there are statistical differences in figure 6b and 6d 
at 16 dpi, it is not clear in the figure. Either the asterisks are missing or they are difficult to see. In 
case that there are no statistical differences, it cannot be concluded that induced susceptibility in 
systemic leaf tissues is a mechanism to promote further infections by Z. tritici and also the results 
section should be modified. 
4- I think that there are some sentences that are not accurate in the discussion/results. For 
instance, the authors mentioned that the role of fungal effector proteins in SIS is demonstrated in 
the experiment of T3SS. For making this statement, first the authors need to clarify if the hrcC 
mutant grows more in the presence of the fungus. Based on the figure S5, there are no statistical 
differences between mock “Pst hcC“ and “Zt Pst hrcC“. Therefore, the sentences “the increased 
growth of Pst hrcC in Z. tritici-infected Obelisks…” (line 254) and “Coinfection with Z. tritici also 
enabled growth of the T3SS-defective…” (line 110) are not correct. Even if this is the case, this 
experiment only highlights that effectors of Pst are not required, most probably because of a 
suppression of the immune response by Z. tritici, but it is not sugesting anything about the role of 
Z. tritici effectors in suppressing the immune response of wheat. 

Minor comments: 
- The results shown in figure 1a and 1b are already known in the literature, but it was not cited 
properly. The authors should highlight that this was already known and cite Brown et al. 2001. 
- The title of figure S5 is very difficult to understand. 
- In figure S7 there is a typo. It should say Obelisk. 
- In the metabolome section, it is not clear if the analysis was performed for the annotated or for 
the global metabolome. In line 125 it is mentioned that only annotated metabolites will be 
analyzed, but in line 126, it is mentioned that the complete metabolome dataset will be analysed. 
- In this analysis, it is striking to observe that there are differences in the mock between local and 
adjacent. Do the authors have any explanation for that? 
- Figure 3c,d would be clearer if it will also indicate which are the active and inactive forms. 
- In line 162, it is mentioned “We suspect that the absence of BXs in Obelisk is partly responsible 
for the increased susceptibility of this cultivar”. However, since we know that the resistance of CS 
is due to the immune response triggered upon recognition of an effector protein, it is most 
probably the other way round that the presence of BXs is responsible for the resistance of Chinese 
Spring. 
Related to this, it is mentioned that BXs are not accumulated in cv. Obelisk (line 161), but this is 
not what is shown in figure S9. 

In any case, in order to conclude about SIS and SAR, the authors should compare infected and 
non-infected tissues. Only upon infection, they observed differential colonization by the bacteria. 
Thus, if these metabolites are having a role, they should be differentially accumulated upon 
infection and not between cultivars. 
- The sentence written in lines 183 and 184 is not very accurate, since there are no enough 
evidences to highlight that Z. tritici manipulates immune-related biosynthetic pathways in 
susceptible wheat cultivars. 
- The statistical analysis was performed correctly, but I miss information on how many times were 
the experiments performed independently. Specially for the infection assays. 
- Line 409 in materials and methods. How were the seeds washed? 
- The results section has frequently information that should have been provided in the introduction 
section.