Chloroplasts alter their morphology and accumulate at the pathogen interface during infection byPhytophthora infestans

Abstract

Chloroplasts are light harvesting organelles that arose from ancient 20 endosymbiotic cyanobacteria. Upon immune activation, chloroplasts switch off 21 photosynthesis, produce anti-microbial compounds, and develop tubular 22 65 development relies on surface immune signalling, whereas the pathogen subverts this 66 remotely by switching off surface immune pulses. These results implicate chloroplast 67 photo-relocation machinery in plant focal immune responses and demonstrate that 68 chloroplasts play a direct role in antagonizing pathogen invasion.69 Results & Discussion: 70 To gain insights into defence-related chloroplast functions, we investigated chloroplast 71 dynamics during P. infestans infection of the solanaceous model plant Nicotiana 72 benthamiana. Confocal microscopy of infected leaf epidermal cells stably expressing 73 GFP in chloroplast stroma (CpGFP hereafter) revealed that chloroplasts accumulate at 74 58% of haustoria (n = 299 haustoria) (Fig. 1A-C). Notably, chloroplasts are mainly 75 positioned around infection sites, sieging haustoria in a highly dynamic fashion (Fig. 1C, 761E and Movie S1-3). Furthermore, compared to non-infected controls, we recorded a 5-77 fold increase in stromule formation in infected CpGFP cells ( Fig. 1A-B, 1D). Notably, 78 chloroplasts seized haustoria, tightly embracing the EHM through stromules that extend 79 and coil around the pathogen (Fig. 1A, 1C, Movie S3). Expression of the EHM marker 80 protein RFP:REM1.3 (Bozkurt et al., 2014) in infected CpGFP plants, allowed us to 81 collect further evidence that chloroplasts intimately associate with the EHM (Fig. S1, 82Movie S4). Remarkably, time lapse-microscopy showed that chloroplasts plunge towards 83 haustoria and embrace the EHM following haustorial penetration (Fig. 1E, Movie S5, Fig. 84 S2), suggesting that chloroplasts may participate in focal immunity. 85To determine the extent to which chloroplasts physically associate with the EHM, we 86 next employed optical tweezers in combination with Total Internal Fluorescence 87 Microscopy (TIRF) in infected CpGFP plants. Using optical tweezers, we successfully 88 trapped and moved 17% of chloroplasts (n = 29) in non-haustoriated cells, a distance 89 greater than the 10 μm threshold. In comparison, we were unable to trap and move any 90 chloroplasts (0%, n = 18) neighbouring haustoria past the threshold, indicating a strong 91 association between the chloroplasts and the EHM. Consistent with this, we recorded a 92 few instances where these chloroplasts were initially pulled away from the EHM, but 93 before they passed the distance threshold they escaped the trap and sprang back 94 towards their former position (22%, n = 18) ( Fig. 1G, Movie S6). Taken together, these 95 results demonstrate that chloroplasts mobilize towards haustoria and tightly embrace the 96 EHM through induction of stromules, possibly to increase the surface area of interaction, 97 in a manner similar to chloroplast-nucleus communication upon activation of antiviral 98 immunity (Kumar et al., 2018).

Host-interactor screens ofPhytophthora infestansRXLR proteins reveal vesicle trafficking as a major effector-targeted process

“…These findings, along with related cell biology and biochemical studies, indicate that P. infestans massively reprograms host membrane trafficking during infection (Du et al, 2015;Tomczynska et al, 2018). Membrane trafficking perturbations of haustoriated cells include the rerouting to the haustorial interface of components of the endocytic pathways (Lu et al, 2012;Bozkurt et al, 2015), autophagy machinery (Dagdas et al, 2018;Pandey et al, 2020) and chloroplasts (Toufexi et al, 2019).…”

Devastating intimacy: the cell biology of plant–Phytophthora interactions

“…However, in cells penetrated by multiple haustoria Toufexi et al . (2019) observed chloroplasts clustered around all of them. Given that there is only one nucleus per cell, it is more likely that chloroplasts move to haustoria independently of the nucleus.…”

“…Toufexi et al . (2019) describe clustering of chloroplasts around haustoria. The chloroplasts were observed to extend stromules and wrap haustoria in a chloroplast–stromule network.…”

The Complete Chloroplast Genomes of Punica granatum and a Comparison with Other Species in Lythraceae

“…A new study found that chloroplast retrograde signaling can regulate nuclear alternative splicing of a subset of Arabidopsis thaliana transcripts [14,15]. Interestingly, researchers have found that chloroplasts play diverse roles in plant defense, including contributing to the production of defense compounds [16]. Chloroplasts contain their own genome, the chloroplast DNA (cpDNA), which is highly conserved in genomic structure, gene content, and gene order.…”