Membrane Contact Sites Between Chloroplasts and Pathogen Interface Underpin Plant Focal Immune Responses

Abstract

When a plant detects a pathogen, chloroplasts terminate photosynthetic activity and uptake vital roles in the immune system to help stave off infection, including the production of defense hormone precursors and antimicrobial reactive oxygen species. Additionally, chloroplasts associate with the nucleus and produce greater numbers of tubular extensions called stromules during immune challenge. We previously showed that during infection by the potato blight pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen haustoria, hyphal extensions that are accommodated within the host cell. However, the extent to which chloroplast positioning around haustoria, or at the nucleus, contributes to immunity during infection remains unknown. Here we show a striking increase in the susceptibility to P. infestans of Nicotiana benthamiana CRISPR knock-out lines lacking the chloroplast movement and anchoring gene, CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1). However, the positioning of chloroplasts around the haustorium or nucleus is not impaired in the absence of CHUP1. Further, loss of CHUP1 leads to an extreme clustering of chloroplasts around the nucleus in the presence and absence of infection, showing that greater chloroplast-nucleus association does not necessarily equate to more robust immunity. While plants lacking CHUP1 have reduced basal stromules, they are still able to induce stromules following immune stimulation, indicating that multiple populations of stromules exist. Lastly, we found that CHUP1 is required for proper deposition of callose - a cell wall material implicated in pathogen penetration resistance - around P. infestans haustorium, but not for other core immune processes. Our results implicate chloroplasts in plant focal immunity and point to a key role of CHUP1 in facilitating the deposition of defense material at the pathogen interface.

Emerging Roles of Motile Epidermal Chloroplasts in Plant Immunity

“…The role of epidermal chloroplasts in response to the oomycete pathogen P. infestans has also been studied in the model solanaceous plant N. benthamiana [ 95 , 96 , 97 ]. In this section, I mention recent research on the dynamics of epidermal chloroplast movements and the involvement of CHUP1 in antioomycete immunity, although there is ambiguity regarding the adaptive or nonadaptive characteristics of P. infestans to N. benthamiana , which shows an ambiguous age-related resistance to P. infestans ; mature plants are resistant, whereas young plants are susceptible [ 98 ].…”

“…Given that epidermal chloroplasts of N. benthamiana are usually detected at the surface area of pavement cells, regardless of pathogen inoculation, the working mechanisms of epidermal chloroplasts in response to pathogen attacks were not entirely the same between A. thaliana and N. benthamiana . Furthermore, it is possible that the CHUP1-related ECR and focal accumulation of epidermal chloroplasts to the fungal penetration sites are independent events in A. thaliana because the pathogen interface-specific accumulation of epidermal chloroplasts in N. benthamiana is CHUP1-independent ( Figure 5 ) [ 97 ]. In this context, it remains to be elucidated whether a part of the epidermal chloroplasts of A. thaliana , which focally accumulate at the fungal penetration sites, has a distinct role(s) from ECR-associated epidermal chloroplasts, as well as that in N. benthamiana .…”

“…Savage et al reported that the positioning of epidermal chloroplasts at the interface between N. benthamiana and the haustoria of P. infestans occurred independent of CHUP1 [ 97 ], which was slightly different from Arabidopsis ECR [ 48 ]. However, they demonstrated that CHUP1 is required for penetration resistance in epidermal cells of N. benthamiana against P. infestans [ 97 ].…”

“…Savage et al reported that the positioning of epidermal chloroplasts at the interface between N. benthamiana and the haustoria of P. infestans occurred independent of CHUP1 [ 97 ], which was slightly different from Arabidopsis ECR [ 48 ]. However, they demonstrated that CHUP1 is required for penetration resistance in epidermal cells of N. benthamiana against P. infestans [ 97 ]. Silencing or knockout of two CHUP1 alleles in N. benthamiana clearly enhances susceptibility to P. infestans , whereas the accumulation of epidermal chloroplasts around haustoria is not impaired in the CHUP1 knockout mutant ( chup1 ) [ 97 ].…”

“…However, they demonstrated that CHUP1 is required for penetration resistance in epidermal cells of N. benthamiana against P. infestans [ 97 ]. Silencing or knockout of two CHUP1 alleles in N. benthamiana clearly enhances susceptibility to P. infestans , whereas the accumulation of epidermal chloroplasts around haustoria is not impaired in the CHUP1 knockout mutant ( chup1 ) [ 97 ]. The Arabidopsis chup1 mutant also showed decreased preinvasive NHR against MAE of nonadapted fungal pathogens [ 48 ], suggesting the importance of the chloroplast-associated protein CHUP1 in epidermal plant immunity against both oomycete and fungal pathogens.…”