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Brief summary of the study

This study investigated plant pathogen fungi from the species Colletotrichum and Magnaporthe oryzae (and others), identifying two factors responsible for pathogenicity caused by penetration of plant cells. The study identified the genes PKS2 and PBG13 responsible for dihydroxyhexanoic acid (DHHA) biosynthesis, generating turgor in fungal cells. The authors show convincing evidence by bioinformatics (conservation analysis of the genes among fungal species), gene complementation analyses, biochemical assays, microscopy, and more. The study also presents an impressive amount of experiments and models in the supplementary data, like for example localisation of PKS2 and PBG13 proteins.

Major comments (validity or strength of the methodology, experiments and analyses, strength of the conclusions)

• The use of a combination of reverse genetics and biochemical analysis in the methodology strengthens the validity of the findings of this study. It was also shown that the loss of PKS2 and PBG13 genes disrupted the turgor generation and pathogenicity of fungi without affecting melanin production, however the use of varied environmental conditions or other fungi strains may further buttress the conclusions from this study.

Minor comments (clarifications to statements in the text, interpretation of the results, presentation of the data/figures)

• The introduction and  discussion section could have been more elaborate. Some key figures/data crop yield losses due to diseases caused by the investigated fungi could have contextualized and reinforced the significance of these findings.

• Readers might benefit from a definition on terminologies related to turgor pressure measurements and osmolyte interactions, such as "cytorrhysis"

Comments on reporting (information on the statistical analyses or availability of data)

• The report is complete and thorough in its reasoning, experimentation and interpretation

• Data analysis is thorough and corroborates the interpretation

• Perhaps some information/data/experiments shown in the supplementary material could be moved in the main text. This information is very valuable!

Suggestions for future studies

• As already mentioned by the authors, investigation of the potential interactions between DHHA polymers and other cell wall components can be done to provide further information on the role of DHHA polymers in maintaining cell wall integrity and function.

• Future exploration of the environmental factors influencing PKS2 and PBG13 expression could be related to how climate change may affect fungal pathogenicity

• It could be interesting to identify natural substances affecting DHHA for possible novel antifungal leads

• What could be resistance/coping mechanisms of fungal pathogens depleted of DHHA polymers?

Conflicts of interest of reviewers

• There are no conflicts of interest to declare.

Competing interests

The authors declare that they have no competing interests.