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Ferroptosis contributes to developmental cell death in rice blast

Qing Shen, Meiling Liang, Fan Yang, Yi Zhen Deng, Naweed I. Naqvi

Preprint posted on November 21, 2019 https://www.biorxiv.org/content/10.1101/850560v1

Article now published in New Phytologist at http://dx.doi.org/10.1111/nph.16636

Iron-regulated cell death is crucial for pathogenic development of the blast fungus as well as the host immune response that counters it.

Selected by Hiral Shah

Background

Ferroptosis is an iron-dependent regulated cell death (Dixon and Stockwell, 2018), which was first described in cultured mammalian cancer cells. It occurs through an oxidative mechanism that involves the formation of lipid peroxides leading to membrane damage. Small molecule screens have discovered several inducers of ferroptosis, like erastin and RSL3. On the other hand, iron chelators and lipid antioxidants can prevent or reduce the cell death induced by these chemicals. All these compounds have been an important part of understanding the mechanism of ferroptosis over the past decade. A large part of the interest in the field stems from the ability to induce cell death in cancer cells. However, recent years have shown the presence of this unique cell death process in other systems, including plants. In a key discovery, ferroptosis was observed in a resistant rice variety during the hypersensitive immune response to the rice blast fungus Magnaporthe oryzae (Dangol et al., 2019).

The blast fungus destroys millions of tons of rice each year and is a major issue for crop production and global food security. During pathogenic development, the fungus produces three-celled asexual spores called conidia (Fernandez and Orth, 2018). Once a conidium lands on the rice leaf surface, it germinates and forms a dome-shaped infection cell called the appressorium. The mature appressorium then helps rupture the leaf surface and forms a penetration peg to enter the host tissue. During appressorium maturation, storage material from the spore cells is transported to the appressorium as the conidium undergoes cell death. In this preprint, the authors investigated the possibility of iron-dependent ferroptotic cell death during pathogenic development of the blast fungus.

Major findings

First, the authors show that developmental cell death in spores is dependent on iron and can be prevented by addition of iron chelators. Since spore death was iron-dependent, the authors next used ferroptosis specific inhibitors, Lip-1 and Fer-1, which prevented spore death while inducers like ferric ion enhanced spore cell death.  The authors used collapsed nuclei (disappearance of the histone-GFP signal) and vital staining to distinguish live versus dead conidial cells and found that the three spore cells underwent a sequential cell death, starting from the terminal cell.

Having established the ferroptotic nature of cell death, the authors looked for the hallmarks of ferroptosis, presence of lipid peroxides. On probing germinating spores with C11-BODIPY, the authors observed an accumulation of lipid peroxides particularly in the terminal cell and subsequently to the other two cells of the conidium. They also found high levels of malondialdehyde, a common oxidative breakdown product of lipids in the rice blast fungus undergoing cell death. The accumulation of peroxides was prevented upon treatment with ferroptosis inhibitors, confirming that iron-dependent ferroptotic cell death occurs in the pathogenic fungus.

Interestingly, erastin and RSL3, common inducers of ferroptosis in cancer cells did not have an impact on spore cell death, once again raising the possibility of either target protein diversification or involvement of a different pathway.

Iron-dependent NADPH oxidase, Nox2, is a known source of lipid peroxides and is expressed in the conidium. Absence of Nox2 activity reduced cell death and enhanced conidium viability. Further, these defects due to the loss of Nox2 were restored upon addition of ferric ions but were further impaired by ferroptosis inhibitors, indicating that Nox2 is a key regulator of ferroptotic cell death during early pathogenic development.

 

Iron-dependent conidium cell death drives pathogenic development of the blast fungus

 

Conidium cell death is known to be autophagic and autophagy is known to regulate ferroptosis in mammalian cells. So, is there a link between the two forms of regulated cell death during appressorium formation? The absence of autophagy, i.e. in the atg8Δ mutant, led to low levels of iron and membrane-aasociated lipid peroxides and significantly improved viability in conidial cells. Interestingly, exogenous iron could induce cell death in the atg8Δ mutant. Thus, autophagy probably sensitizes the conidium to ferroptosis by altering cellular iron availability. On the other hand, ferroptosis inhibitors had no obvious effect on autophagy, thus implying that autophagy precedes ferroptosis during infection-related development in Magnaporthe.

Next, the authors turned to the role of ferroptosis during host-pathogen interaction. Most importantly, ferroptotic cell death was found to play a role in appressorium maturation, as treatment with ferroptosis inhibitors led to a decline in host penetration and delayed the process of infection (Fig. 5(b)), a phenotype shared with the nox2Δ mutant.

Recently, ferroptosis was reported in resistant rice cultivars in response to the blast fungus infection (Dangol et al., 2019). Moving forward from this work, the authors investigated whether ferroptosis altered the general host-pathogen interaction and prevented spread of the fungus by inducing host specific treatments (using erastin and RSL3 that did not induce ferroptosis in the fungus) at times when spore cell death had already occurred. Induction of ferroptosis in a susceptible rice cultivar led to a resistance response or incompatible host-pathogen outcome, such that the fungus penetrated the host tissue but failed to spread to the neighbouring cells.

 

Why I selected this preprint

This study is important because it reports the role of ferroptosis in the development of a fungal pathogen. It takes our understanding of appressorium development further, just when we thought it was the best studied stage in the infection process. Importantly, the preprint describes the role of ferroptosis during physiological processes in both the host and the pathogen of the rice-blast pathosystem. Interestingly, ferroptosis is involved in the immune response in host plants while in the fungus it is crucial for pathogenic development, two processes that have opposite effects on the host-pathogen interaction.

 

Questions for the authors

Ferroptosis occurs sequentially in a conidium. Is there a preference for which terminal cell goes first? Is it the germinating cell or the one opposite to it?

Does the process of nuclear degradation, used as an indicator of cell viability in this study, go through stages as well? Does the diffused histone signal start to appear with autophagy or becomes visible only after ferroptosis has occurred?

How might autophagy affect iron homeostasis? What is the other ways in which autophagy could prepare the cell for ferroptosis?

Is ferroptosis likely to occur at other stages of development in the rice blast fungus?

 

References

Fernandez J, Orth K. 2018. Rise of a Cereal Killer: The Biology of Magnaporthe oryzae Biotrophic Growth. Trends Microbiol 26(7): 582-597.

Dangol S, Chen Y, Hwang BK, Jwa N-S. 2019. Iron- and Reactive Oxygen Species-Dependent Ferroptotic Cell Death in Rice-Magnaporthe oryzae Interactions. The Plant Cell 31(1): 189-209.

Dixon, SJ, & Stockwell, BR. 2018. The Hallmarks of Ferroptosis. Annual Review of Cancer Biology 3(1). doi:10.1146/annurev-cancerbio-030518-055844

 

Tags: appressorium maturation, autophagy, fungal development, iron homeostasis, plant immune response, spore death

Posted on: 30th November 2019 , updated on: 2nd December 2019

doi: https://doi.org/10.1242/prelights.15449

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Authors' response

The author team shared

Ferroptosis occurs sequentially in a conidium. Is there a preference for which terminal cell goes first? Is it the germinating cell or the one opposite to it?

Based on the C11-BODIPY (lipid peroxidation), calcein-AM and vital staining, the terminal cell (at the apex/tip of the conidium) has a relatively higher frequency of undergoing cell death first, but the underlying mechanism is really not clear.
Both the terminal cells have the potential to geminate. Although, we frequently see instances of cessation of germ tube elongation from one of the terminal cells, especially upon appressorium initiation from the other. It is still unclear whether there is a link between conidial germination and the initiation of ferroptosis per se.

Does the process of nuclear degradation, used as an indicator of cell viability in this study, go through stages as well? Does the diffused histone signal start to appear with autophagy or becomes visible only after ferroptosis has occurred?

The process of nuclear degradation in theory goes through stages, but the overall process is very fast. As described in the preprint, autophagy is robust at 2-4 hpi, while the death of the conidial cells, as indicated by diffusion and then disappearance of Histone-GFP signal, is evident at 6 hpi and becomes more obvious at the later time points during development. This needs more investigation to establish the hierarchy, but we believe that nuclear degradation occurs concomitantly with conidial cell death, in which ferroptosis and autophagy are equally involved.

How might autophagy affect iron homeostasis? What is the other ways in which autophagy could prepare the cell for ferroptosis?

Our working hypothesis regarding the autophagy-ferroptosis relationship is that autophagy affects iron homeostasis by degradation of ferritin complex(es) responsible for intracellular iron storage, in this way controlling internal iron availability and at the same time preparing the cell for ferroptosis. Other possibility could be that autophagy directly regulates the intracellular oxidative status or redox homeostasis, but this hypothesis needs to be verified.

Is ferroptosis likely to occur at other stages of development in the rice blast fungus?

According to our unpublished data, yes. The in planta growth stage of the rice blast fungus is also affected upon chemical inhibition of ferroptosis.

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