Host autophagy machinery is diverted to the pathogen interface to mediate focal defense responses against the Irish potato famine pathogen

[Editors’ note: the author responses to the first round of peer review follow.]

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

There was general agreement that the study is interesting. However, the study falls short of providing enough new mechanistic insights to be suitable for a research advance. Specifically, there are important issues that are not fully substantiated, which raised questions:

-Do the events described in the manuscript happen during infection? (That is, what is the biological relevance during natural infection?)

-Are autophagosomes actually directed toward the pathogen interface under native conditions? Additional microscopy (e.g. electron microscopy) can help address this important issue.

We are very pleased to submit the manuscript “Host autophagosomes are diverted to the pathogen interface to mediate focal defense responses against the Irish potato famine pathogen” for publication in eLife as a Research Advance. We recently showed that the Irish potato famine pathogen P. infestans subverts host autophagy related defences by secreting an effector protein (Dagdas et al., eLife, 2016, 5:e10856; Maqbool et al., JBC, 2016, 291:20270). In this manuscript, we provide an exciting cellular biology dimension to our previous model and show that defense-related selective autophagy pathway is diverted to the plant-microbe interface in order to limit the pathogen growth, instead of taking the default vacuolar degradation route. In turn, the pathogen antagonizes the antimicrobial-autophagy launched to destroy itself by deploying an effector protein that disarms the defense-related autophagosomes across the perimicrobial host membrane. Interestingly, the autophagosome biogenesis machinery, marked by the conserved early autophagosome protein ATG9, is also diverted to hostmicrobe interface. Furthermore, we assign a previously unknown antimicrobial function to ATG9 protein, as its overexpression significantly improved plant immunity to pathogen infection.

The extent to which autophagy contributes to immunity and/or serves adapted pathogens is currently under debate. In Dagdas et al. (eLife, 2016), we showed that the effector protein PexRD54 of P. infestans outcompetes host autophagy cargo receptor Joka2 in binding the core autophagy adaptor ATG8. This results in suppression of Joka2-mediated immunity. Interestingly, the effector also stimulates the formation of the autophagosomes, which suggests that the pathogen could subvert host autophagy for its own benefit. In this study, we demonstrate that autophagosomes are diverted towards haustoria, specialized hyphal extensions that grow inwards host cells. This reveals a new pathway that is re-routed towards the pathogen interface and provides evidence for extensive remodelling of the host endomembrane system by plant pathogens.

In addition to showing importance of focal autophagic defense responses, our work has wider implications. It sheds light on membrane biogenesis in plant cells that accommodate microbial structures and explains the differences in protein composition between plant plasma membrane and perimicrobial

membranes. This work also points to a possible source for the enigmatic extracellular vesicles that have been reported in host-microbe interfaces of symbiotic and parasitic interactions.

Although the essence of this story is similar to our previous submission that was rejected, this is almost a new manuscript, not a cosmetically re-decorated version of the previous one. We have added several new composite figures and modified the text accordingly. A summary of the new data and figures are as follows:

1) We provide RNAi based genetic evidence demonstrating autophagosome targeting to haustorial interface is dependent on the core autophagy components ATG4 and ATG9. This is now presented in Figure 2.

2) We have performed high resolution imaging of ATG8CL/Joka2/PexRD54 autophagosomes to show that they associate with the perimicrobial host membrane. These data are presented in Figure 1—figure supplement 1, Figure 3—figure supplement 1, and Figure 4—figure supplement 2.

3) We have performed domain-function analyses of Joka2 to show that domains that mediate oligomerization and protein-protein interaction are important for haustorial accumulation of Joka2. Furthermore, we did pathogenicity assays that shows focal accumulation of Joka2 is important for its positive role in antimicrobial immunity. These data are now presented in Figure 3, Figure 3—figure supplement 2, Figure 3—figure supplement 4.

4) We have performed high-resolution imaging and pathogenicity assays that showed perihaustorial niche is a hot spot for autophagosome biogenesis and overexpression of early autophagosome biogenesis marker ATG9 enhances plant immunity. These data are now presented in Figure 5, Figure 5—figure supplement 1.

[Editors' note: the author responses to the re-review follow.]

This manuscript has been improved significantly from previous submission. The work uncovers selective autophagy targeted to haustoria as a defense mechanism, represents one of the first papers to tackle plant cargo receptors, and is a significant contribution to our understanding of how autophagy may contribute immunity. We are happy to offer publication of the manuscript after the authors have addressed the following issues:

We are delighted to hear about the positive evaluation of our manuscript by the reviewers and the editor. We addressed all the raised issues as following:

1) It is helpful to explain in the Introduction differences of "selective autophagy", "antimicrobial-autophagy", and "xenophagy". This will help readers.

We now included new text in the Introduction to clarify different types of autophagy.

2) Where does ATG8CL localize without infection?

We now present images (Figure 1—figure supplement 1A-B) showing punctate and vacuolar localization of ATG8CL in uninfected cells.

3) The title is a bit misleading by stating "autophagosomes are diverted to the pathogen interface", since it implies initiation of autophagosomes elsewhere then move to haustoria. What the authors show is that autophagosomes are formed around huastoria.

We changed the title to “Host autophagy machinery is diverted to the pathogen interface to mediate focal defense responses against the Irish potato famine pathogen”.

4) Many of the figures show fluorescence of BFP protein fusions using a blue color. Although this is intuitive, it is very difficult to see the blue color against the black background. Since this is a false color, anyhow, I strongly suggest replacing the blue color with something brighter such as yellow or magenta.

We thank the editor for this suggestion. We updated all the figures and changed all blue channels to teal and green channels to yellow.

5) Figure 4—figure supplement 1 and Figure 4—figure supplement 2 present critical data to support the conclusion that PexRD54 and ATG8CL co-localize in puncta at the perihaustorial membrane. This should be moved to main figures.

We have revised the figure as suggested. These data are now presented in Figure 4.

6) In the Discussion section the authors allude to phylogenetic analyses that they have performed on the ATG8 family in plants. It would be helpful to explicitly state whether ATG8CL is conserved across flowering plants, or whether it is specific to the Solanaceae family. I gather that it is not broadly conserved. If it is not, do they speculate that other ATG8 members fulfill this role in other plant families?

We included following text in the Discussion “The expansion of plant ATG8 members seems to have occurred earlier in evolution, and each plant family carries a unique set of ATG8 isoforms that have been diversifying over millions of years. Thus, it is likely that in different plant families different ATG8 isoforms are specialized to carry out defense-related tasks.”

7) Images in Figure 3 were collected 3-4 days post infection. Based on Figure 3—figure supplement 4, protein levels differ significantly. Please comment whether this affect interpretation of the data.

We think this is unlikely because we excluded the cells that didn’t look healthy or showed no Joka2 expression. Additionally, we measured the frequency of haustoria that has Joka2 puncta rather than quantifying the abundance of Joka2 puncta/cell. Consistently, our image quantification does not show any significant differences in number of haustoria that accommodate Joka2:BFP puncta in images obtained three (32/35 of haustoria, 91.4%) vs four (14/15 of haustoria 93.3%) days post infection.