Trehalose Phosphate Synthase Complex-Mediated Regulation of Trehalose 6-Phosphate Homeostasis Is Critical for Development and Pathogenesis in Magnaporthe oryzae

ABSTRACT Trehalose biosynthesis pathway is a potential target for antifungal drug development, and trehalose 6-phosphate (T6P) accumulation is widely known to have toxic effects on cells. However, how organisms maintain a safe T6P level and cope with its cytotoxicity effects when accumulated have not been reported. Herein, we unveil the mechanism by which the rice blast fungus Magnaporthe oryzae avoids T6P accumulation and the genetic and physiological adjustments it undergoes to self-adjust the metabolite level when it is unavoidably accumulated. We found that T6P accumulation leads to defects in fugal development and pathogenicity. The accumulated T6P impairs cell wall assembly by disrupting actin organization. The disorganization of actin impairs the distribution of chitin synthases, thereby disrupting cell wall polymer distribution. Additionally, accumulation of T6P compromise energy metabolism. M. oryzae was able to overcome the effects of T6P accumulation by self-mutation of its MoTPS3 gene at two different mutation sites. We further show that mutation of MoTPS3 suppresses MoTps1 activity to reduce the intracellular level of T6P and partially restore ΔMotps2 defects. Overall, our results provide insights into the cytotoxicity effects of T6P accumulation and uncover a spontaneous mutation strategy to rebalance accumulated T6P in M. oryzae. IMPORTANCE M. oryzae, the causative agent of the rice blast disease, threatens rice production worldwide. Our results revealed that T6P accumulation, caused by the disruption of MoTPS2, has toxic effects on fugal development and pathogenesis in M. oryzae. The accumulated T6P impairs the distribution of cell wall polymers via actin organization and therefore disrupts cell wall structure. M. oryzae uses a spontaneous mutation to restore T6P cytotoxicity. Seven spontaneous mutation sites were found, and a mutation in MoTPS3 was further identified. The spontaneous mutation in MoTPS3 can partially rescue ΔMotps2 defects by suppressing MoTps1 activity to alleviate T6P cytotoxicity. This study provides clear evidence for better understanding of T6P cytotoxicity and how the fungus protects itself from T6P’s toxic effects when it has accumulated to severely high levels.

The reviewers suggested a number of additions and changes that will further strengthen the manuscript. In particular, one of the reviewers indicated that additional evidence may be necessary to demonstrate the T6P is induced during fungus development. We invite the submission of a revised manuscript that addresses the reviewers comments.
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The manuscript describes the balance of T6P accumulation in the rice blast disease which caused the defects in the fungal invasion structure and virulence. The author found a self-mutation of its MoTPS3 caused the recovery on the deletion of MoTps2. The author design various methods to verify the hypothesis that the organisms maintain a safe T6P level and cope with its cytotoxicity effects by the self-mutation. The manuscript was well-written, however, in my point, the ms lack some key evidences for the conclusion which led to the misleading to the readers.
The most important tissue is when the T6P enhanced? In line 86-87, When trehalose is no more required (such as in the absence of 86 stress)? However, the author showed no evidence here that during the development of the fungus the T6P was induced. As this evidence missing, the author lacks the results that the Tps3 mutation in vivo. I do not agree to the conclusion in the MS that the self-mutation of TPs3 is required for the virulence.
Another major gap is that, how the tps3 mutation happened? NO evidence here. Are these sites active at the basic level even not in the deletion of MoTps2? The mutation of Tps3 is only dependent to Tps2 or once immature of the appressorium? After reading carefully throughout the Ms, I still can not follow that why the mutation occurred. I think the author should provided more evidence here.
Reviewer #2 (Comments for the Author): Chen et al. investigated the mechanism by which the physiologic level of trehalose-6-phosphate (T6P) is regulated in the rice blast fungus M. oryzae.Despite excellent presentation, few grammatical errors need to be fixed in the manuscript. L128-132: You rather observed (not characterized) a spontaneous mutation that partially restores the defects. Also, this sentence is too long, making it difficult to grab the message. I suggest you break it into two simpler sentences. L142: define aa (as it appears here for the very first time  Fig. 1 A. L595: Delete one 'Afterwards'. The materials and methods section does not explain how the various forms of mutation that exist in the spontaneous strain were identified.

Response to Reviewer #1
The manuscript describes the balance of T6P accumulation in the rice blast disease which caused the defects in the fungal invasion structure and virulence. The author found a self-mutation of its MoTPS3 caused the recovery on the deletion of MoTps2. The author design various methods to verify the hypothesis that the organisms maintain a safe T6P level and cope with its cytotoxicity effects by the self-mutation. The manuscript was well-written, however, in my point, the ms lack some key evidences for the conclusion which led to the misleading to the readers.

Response
We thank the reviewer for the constructive criticism and time spent to analyze this manuscript. Our responses and explanations in relation to the comments are provided below. We hope that the changes we have made resolve all your concerns about the article. We are more than happy to make any further changes that will improve the paper and/or facilitate successful publication.
The most important tissue is when the T6P enhanced? In line 86-87, When trehalose is no more required (such as in the absence of 86 stress)? However, the author showed no evidence here that during the development of the fungus the T6P was induced. As this evidence missing, the author lacks the results that the Tps3 mutation in vivo. I do not agree to the conclusion in the MS that the self-mutation of TPs3 is required for the virulence.

Response
Thanks for your positive comments. Coupled with previous studies and our results, the accumulated T6P can only be found in the ΔMotps2 mutant (~2000-fold) from the fungal mycelia. Both ΔMotps1 and ΔMotps3 mutants are impaired of T6P production. In Fig. 4 e and Fig.6 a, our results also indicated that the intracellular T6P maintains a relatively low level in the mycelium. The ΔMotps2 mutant has severe defects in conidiation and appressorium formation. We could not obtain enough conidia and appressorial tissues for further GC-MS analysis. In Fusarium graminearum, loss of FgTps2 also leads to highly accumulated T6P level in both mycelia and conidia. It is possible that the conidia and appressoria of the ΔMotps2 mutant also accumulated high level of T6P and thus leads to the observed severe defects. MoTps1 is involved in the biosynthesis of T6P. To increase the intracellular level of T6P, we generated ΔMotps1-OE strains by overexpressing the trehalose 6-phosphate synthase MoTPS1 in the wild type ( Fig.R1 A). Although over-expression of MoTPS1 leads to defects in growth, conidiation and virulence, the ΔMotps1-OE strain has no significant effect on T6P production ( Fig.R1 B). Since trehalose biosynthesis is induced when exposed to environmental stresses, we further monitored the expression level of the TPS complex subunits through qRT-PCR assays. We noticed that the expression levels of MoTPS1 and MoTPS3 increased by ~1.8-fold under oxidative stress ( Fig.R1 C). In the Δ Motps1-OE strains, the expression level of the MoTPS1 was up-regulated by ~10-fold, while there was only little increase in the T6P level. These results also pushed us to investigate how M. oryzae physiologically overcomes the stress due to T6P accumulation. Interestingly, we noticed that the ΔMotps2 mutant undergoes spontaneous mutation in its MoTPS3 gene to take care of the accumulated T6P. Further results indicated that the spontaneous mutants have low level of intracellular T6P. This finding indicates an important role of the MoTps3 in modulating T6P level. Based on these results, we suggested that M. oryzae maintains a low level of T6P under normal condition, and the T6P level could increase under stress condition. Trehalose is a non-reducing disaccharide that protects proteins and cellular membranes from inactivation or denaturation caused by a variety of stress conditions. In response to specific environmental stresses, activities of the TPS complex subunits are increased and thus induce trehalose biosynthesis. With the recovery of stress, trehalose biosynthesis is no more required and trehalose needs to be degraded. Degradation of trehalose by trehalases provides the energy necessary for stress recovery. We are sorry that the sentence in L86-87 makes you confused and this sentence has been revised as "Under stress recovery condition, trehalose biosynthesis is no more required" in the revised manuscript.
In 2007 significantly smaller than those caused by the wild type (Fig.4, Table 2). Based on these results, we concluded that self-mutation in MoTPS3 down-lowers T6P level and thus partially rescues Δ Another major gap is that, how the tps3 mutation happened? NO evidence here.

Response
This is actually the very first work that unveils the occurrence of this mutation due to persistent T6P accumulation in M. oryzae. As such, we decided to dissect in details how this spontaneous mutation occurs and the mechanism by which T6P induces the mutation in our future studies. However, we discussed some hypotheses that could possibly help to uncover this mechanism to aid further work. Spontaneous mutation is ultimate source of genetic variation and a fundamental component of evolution. Spontaneous mutation rate tends to rise when they are under antibiotic treatment, starvation, or other stresses. These genetic variations in turn provide beneficial evolutionary changes for adaption (Flynn, Chain et al. 2017, Liu and Zhang 2019, Ho, Macrae et al. 2020. Genes encoding the proteins involved in trehalose biosynthesis are mechanistically linked to metabolism, cell wall homeostasis, stress responses, and virulence (Thammahong, Puttikamonkul et al. 2017). Our results indicated that the ΔMotps2 mutant displayed high sensitivity to cell wall stress (Fig.3) and rapamycin (unpublished data). Moreover, the accumulated T6P in ΔMotps2 mutant disrupt metabolite homeostasis (Fig.6). These results suggested that the accumulated T6P in ΔMotps2 mutant disrupts cellular homeostasis and could serve as a stress agent that induces the genetic variation. In M. oryzae, MoTps1 is responsible for the biosynthesis of T6P, and MoTps3 physically interacts with MoTps1 (Fig.5b). It is possible that the accumulated T6P in ΔMotps2 mutant can be sensed by MoTps1-MoTps3 and therefore induces the mutation in MoTps3.
In 2014, Song et al. indicated in Fusarium graminearum that the trehalose 6-phospahte phosphatase FgTps2 is required for fungal development, virulence and mycotoxin production (Song, Li et al. 2014). Loss of FgTps2 also leads to severe intracellular accumulation of T6P. To better understand how the accumulated T6P induces spontaneous mutation (and to satisfactorily address the reviewer's concern), we generated ΔFgtps2 mutant in F. graminearum. As shown in Fig. R2 (Table R1). In addition, both trehalose and T6P production were abolished in the ΔFgtps1-ΔFgtps2 mutant. These results indicated that the accumulated T6P could also act as a spontaneous mutation inducer in response to intracellular T6P accumulation and FgTPS1 or FgTPS3 could act as the potential mutation targets in F. graminearum. Are these sites active at the basic level even not in the deletion of MoTps2? The mutation of Tps3 is only dependent to Tps2 or once immature of the appressorium? After reading carefully throughout the Ms, I still cannot follow that why the mutation occurred. I think the author should provide more evidence here.

Response
As shown in Fig.R3, the spontaneous mutation sites identified in MoTPS3 are well conserved in different species. Our results also indicated that the spontaneous mutation strains ΔMotps2-m mutant has similar phenotypes when compared to the ΔMotps2 -ΔMotps3 mutant (Fig.4). It is possible that these mutations sites could be induced under other stress conditions. This is also a topic of further studies. As for the existing evidences, the spontaneous mutation is observed when MoTPS2 gene is deleted and this partially ameliorates the defects of the tps2 mutant. This therefore simply suggests that using MoTps2 as a drug target to combat the rice blast infection could be compromised by this spontaneous mutation of MoTPS3. Whether there are other conditions that can lead to this mutation apart from the deletion of MoTPS2 will make part of an interesting subject of our next research as this will shade more light on the feasibility of targeting the TPS complex for antifungal drug discovery.

Fig.R3
Amino acids alignment of Tps3 homologues in different species. The base deletion mutation site was marked by red cycle and the base insertion site was marked by blue arrow.
We would like to thank the editorial board and the reviewers for their constructive comments concerning this article (control no. mSystems00462-21 R0). These comments are all valuable and helpful for improving our article. All the authors have seriously discussed about all these comments. We therefore modified the manuscript accordingly to accommodate the reviewer's observations and meet the requirements of the journal. Point-by-point responses to the reviewer's comments are listed below.
L128-132: You rather observed (not characterized) a spontaneous mutation that partially restores the defects. Also, this sentence is too long, making it difficult to grab the message. I suggest you break it into two simpler sentences.

Response
Thanks a lot for your valuable suggestion to improve the quality of our manuscript. The sentence has been revised as "In addition, the TPS complex possesses a spontaneous 'correction' way to modulate T6P homeostasis by spontaneous mutation of the regulatory subunit MoTps3. This spontaneous correction function further suppresses the MoTps1 activity to down-regulate T6P, resulting in partial restoration of the ΔMotps2 mutant defects in growth, conidiation and pathogenicity." L142: define aa (as it appears here for the very first time)

Response
Thanks for your careful checks. We have defined "aa" in L142 in the revised manuscript.

Response
We are sorry for this grammatical error. The sentence has been revised as "The wild type (70-15), mutant (ΔMotps2) and complemented strain (ΔMotps2-com) were then cultured on CM, MM and RBM culture media for 7 days to compare their growth rates." The revision is shown in L149 -151.

Response
Thanks for this important comment; we have inserted the related references in L236. The sentence was revised as "Since the appropriate localization of CHS genes is essential for cell wall homeostasis (29,39)

Response
As shown in the Fig.S7, the ΔMotps2 mutant produced spontaneous suppressor strains when we revived ΔMotps2 mutant from filter papers on SYM medium. Based on our experience, the ΔMotps2 mutant produced spontaneous suppressor strains randomly. This happened when we performed the growth assays, cell wall stress assays, osmotic assays, etc. In our manuscript, the first part of our results section is mainly about the biological function of the MoTps2 (Fig.1, Fig.2 and Fig.3), while Fig.4, Fig.5 and Fig.6 focus on the identification of the mutation sites in the spontaneous suppressor strain and how the mutations of MoTPS3 restore the ΔMotps2 mutant's defects.

Response
Thanks for your correction; we have deleted one "afterwards" in L595. We are sorry for this mistake.
The materials and methods section does not explain how the various forms of mutation that exist in the spontaneous strain were identified.

Response
Thanks for your valuable suggestion. How various forms of mutation in the spontaneous strain were identified was added in the materials and methods part. This can be seen in L553-560 in the revised manuscript. 1st Revision -Editorial Decision Congratulations! Your manuscript has been accepted for publication in mSystems. Thank you for your careful consideration of the reviewers' comments.
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