The Irish potato famine pathogen subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface

Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved effector proteins to counteract antimicrobial autophagy. How and why adapted pathogens co-opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phythophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway, while antagonizing antimicrobial autophagy. Here we show that PexRD54 induces autophagosome formation by bridging small GTPase Rab8a-decorated vesicles with autophagic compartments labelled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing that specific trafficking pathways underpin selective autophagy. We discovered that Rab8a contributes to basal immunity against P. infestans, but PexRD54 diverts a sub-population of Rab8a vesicles to lipid droplets that associate with autophagosomes. These are then diverted towards pathogen feeding structures that are accommodated within the host cells. We propose that PexRD54 mimics starvation-induced autophagy by channeling host endomembrane trafficking towards the pathogen interface possibly to acquire nutrients. This work reveals that effectors can interconnect independent host compartments to stimulate complex cellular processes that benefit the pathogen. Graphical abstract


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We first validated PexRD54-Rab8a association through co-immunoprecipitation assays by co-214 expressing the potato Rab8a (herein Rab8a) with PexRD54 in planta. Notably, the AIM mutant of binding activity. Consistent with this, the AIMp failed to associate with Rab8a in pull down assays,

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although it still strongly interacted with ATG8CL ( Figure 2B). These results suggest that PexRD54's 219 N-terminal region preceding the C-terminal AIM mediates Rab8a association.

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To gain insights into association of PexRD54 with Rab8a, we investigated their subcellular distribution

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We next examined the extent to which Rab8a mutants colocalize with PexRD54. When co-expressed 254 with BFP:PexRD54, both GFP:Rab8a and GFP:Rab8a S29N consistently produced sharp fluorescence 255 signals that overlap with the typical ring-like autophagosomes marked by PexRD54 ( Figure S9A-B).

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However, GFP:Rab8a Q74L showed a similar localization pattern to the GFP control, and mostly did not 257 produce fluorescence signals that peak at BFP:PexRD54-puncta ( Figure S9C-D). We quantified these 258 observations in multiple independent experiments where GFP:Rab8a and GFP:Rab8a S29N frequently 259 (68%, N = 23) labeled BFP:PexRD54-puncta, whereas GFP:Rab8a Q74L only did so much less often are consistent with the results obtained in Figure 2B and 1A-B which revealed that PexRD54's AIM 264 peptide fails to associate with Rab8a and suppresses autophagosome formation. Finally, we tested 265 the affinity of PexRD54 to Rab8a and its mutants. Rab8a S29N pulled-down PexRD54 more than wild 266 type GFP:Rab8a or GFP:Rab8a Q74L in planta ( Figure 2D). This suggests that PexRD54 preferentially 267 associates with the GDP (S29N)

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Rab8 family contributes to immunity against P. infestans.

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We next investigated the potential role of Rab8a in immunity against P. infestans. First, we tested 289 whether silencing of Rab8a gene expression interferes with the pathogen growth. In the N.   Rab8a members led to a consistent increase in disease symptoms caused by P. infestans ( Figure   302 3A), supporting the view that the Rab8a family contributes to immunity. To determine the role of the in western blots, unlike the wild type Rab8a that was undetectable upon co-delivery of the infestans ( Figure 3B). Finally to gain supporting evidence of the positive role of Rab8a in immunity,

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we used a dominant negative form of potato Rab8a (Rab8a N128I ) to assay for immune phenotypes.

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Consistent with prior experiments, overexpression of GFP:Rab8a N128I , but not a GFP control, 313 enhanced plant susceptibility to P. infestans, supporting that the Rab8a family contributes to plant 314 immunity ( Figure 3C). Taken together, these results show that Rab8a members redundantly 315 contribute to plant immunity.

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To further ascertain the functional relationship between PexRD54 and Rab8a, we investigated the 364 degree to which Rab8a associates with autophagy machinery. We monitored the co-localization of puncta are frequently accompanied by RFP:Rab8a labelled vesicles. However, we detected an to wild type GFP:Rab8a ( Figure S20).

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As we previously describe that PexRD54 has varying binding affinity for Rab8a and its mutants (

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To better characterize the autophagy stimulated by PexRD54, we decided to further investigate the

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suggesting that PexRD54 mediated autophagy can override or mask starvation induced autophagy 503 ( Figure 6A-B). Furthermore, in plants stably expressing GFP:Rab8a that are exposed to 24 hours 504 dark period, we detected an increased degree of colocalization between RFP:ATG8CL and 505 GFP:Rab8a ( Figure 6C-D, S23), in a similar fashion to enhanced ATG8CL-Rab8a association 506 mediated by PexRD54 ( Figure 4A-D). Furthermore, similar to PexRD54-mediated decrease in Rab8a 507 levels ( Figure 5F-G), we noted a reduction in GFP:Rab8a levels but not free GFP protein levels 508 following 24 hour dark treatment of stable transgenic 35s::GFP:Rab8a plants ( Figure 6E). Collectively, 509 these data suggest that PexRD54 mimics carbon starvation induced autophagy.  Induction of autophagosomes by PexRD54 that are targeted to the perihaustorial interface prompted 611 the hypothesis that P. infestans could benefit by co-opting host autophagy to support its own growth.

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Thus, we explored the impact of autophagy activation by PexRD54 on P. infestans host colonization.

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We decided to transiently interfere with pathogen induced autophagy by expressing the AIMp. Our   Collectively, these results suggest that P. infestans relies on host autophagy function to support its host autophagy pathways while subverting defense-related autophagy, instead of just the AIM

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Here, we expanded our knowledge of PexRD54 triggered autophagy and discovered an effector 675 derived peptide that can specifically block autophagy. Chemical inhibitors of autophagy are often used 676 to measure autophagy flux and to overcome the limitations of standard genetic approaches. However, 677 these inhibitors are mostly inefficient and lack the required specificity. We discovered that PexRD54's 678 AIM peptide is a strong autophagy suppressor effective against all potato ATG8 isoforms.

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Conceivably, the AIM peptide competitively inhibits autophagosome biogenesis by occupying the W

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We propose that upon carbon deprivation, the plant uses LDs as an alternative membrane source to 718 accommodate for an increase in autophagosome biogenesis, and PexRD54 exploits this process to 719 stimulate autophagy. However, Rab8a does not appear to be engaged in all autophagy routes, as it 720 is dispensable for Joka2 mediated autophagy ( Figure 6). This is consistent with the finding that

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The N-terminal split YFP half was used via a linker peptide RPACKIPNDLKQKVMNH and the C-