Effector XopQ-induced stromule formation inNicotiana benthamianadepends on ETI signaling components ADR1 and NRG1

Abstract

In Nicotiana benthamiana, expression of the Xanthomonas effector XANTHOMONAS OUTER PROTEIN Q (XopQ) triggers RECOGNITION OF XOPQ1 (ROQ1)-dependent effector triggered immunity (ETI) responses accompanied by accumulation of plastids around the nucleus and the formation of stromules. Both plastid clustering and stromules were proposed to contribute to ETI-related hypersensitive cell death and thereby to plant immunity. Whether these reactions are directly connected to ETI signaling events has not been tested. Here we utilized transient expression experiments to determine whether XopQ-triggered plastid reactions are a result of XopQ perception by the immune receptor ROQ1 or a consequence of XopQ virulence activity. We found that N. benthamiana mutants lacking ROQ1, ENHANCED DISEASE SUSCEOTIBILITY 1 (EDS1) or the helper NUCLEOTIDE-BINDING LEUCIN-RICH REPEAT IMMUNE RECEPTORS (NLRs) N-REQUIRED GENE 1 (NRG1) and ACTIVATED DISEASE RESISTANCE GENE 1 (ADR1), fail to elicit XopQ-dependent host cell death and stromule formation. Mutants lacking only NRG1 lost XopQ-dependent cell death but retained some stromule induction that was abolished in the nrg1_adr1 double mutant. This analysis aligns XopQ-triggered stromules with the ETI signaling cascade but not to host programmed cell death. Furthermore, data reveal that XopQ-triggered plastid clustering is not strictly linked to stromule formation during ETI. Our data suggest that stromule formation, in contrast to chloroplast perinuclear dynamics, is an integral part of the N. benthamiana ETI response and that both NRG1 and ADR1 hNLRs play a role in this ETI response.

Stromule geometry allows optimal spatial regulation of organelle interactions in the quasi-2D cytoplasm

“…However, at least in the context of our work, inoculation with GV3101 (pMP90) (Fig. 3A and B), and triggering of ETI by the expression of XopQ results in only a subset of plastids relocating to the nucleus (Erickson et al, 2014;Prautsch et al, 2023), while the rest remain evenly spread (Fig. 3B and Supplementary Fig.…”

“…It is readily accepted that when plants are confronted with stress stimuli, interactions and communication between organelles, cytosol and plasma membrane increases in importance (Medina-Puche and Lozano-Duran, 2022). Recent attention has been paid to instances of plastid repositioning to the nucleus, which readily occurs upon confrontation with biotic stress, such as exposure to pathogen-derived effectors and peptides that trigger immune responses (Erickson et al, 2014(Erickson et al, , 2018Caplan et al, 2008Caplan et al, , 2015Ding et al, 2019;Prautsch et al, 2023). In this context, plastid proximity to the nucleus is cited as important for the efficient transfer of retrograde signals such as H2O2 and proteins from plastids to nuclei (Caplan et al, 2008(Caplan et al, , 2015Park et al, 2018;Ding et al, 2019).…”

Pepper mild mottle virus coat protein interacts with pepper chloroplast outer envelope membrane protein OMP24 to inhibit antiviral immunity in plants

“…In plants, PCC is a general response of plants under biotic and abiotic stresses [ 16 , 17 , 42 ]. In N. benthamiana , expression of the Xanthomonas effector XANTHOMONAS OUTER PROTEIN Q (XopQ) or TMV p50 activates ETI immunity accompanied by chloroplast relocation around the nucleus [ 49 ]. Stromules are associated with PCC and are thought to guide chloroplasts to the nucleus and to transfer the retrograde signaling such as ROS from chloroplasts to the nucleus to regulate resistance gene expression [ 13 , 16 , 26 , 27 ].…”

Small family, big impact: RNL helper NLRs and their importance in plant innate immunity

“…Arabidopsis NRG1.1 and NRG1.2 serve as redundant signaling components specifically required for TNL-induced immunity and contribute to basal resistance in the absence of ADR1s [ 8 , 9 ]. Interestingly, functional redundancy has not only been reported within the RNL subfamilies, but also between both subfamilies [ 11 , 13 ]. It is remarkable, however, that in Arabidopsis and Nicotiana benthamiana ADR1s and NRG1s contribute to some sensor NLR-triggered immune responses in an unequally redundant manner.…”

“…In contrast, in N . benthamiana NRG1 is sufficient to mediate resistance against tobacco mosaic virus (trigger of the TNL N) or Pseudomonas syringeae (trigger of the TNL Roq1) infections, since knocking out NbADR1 does not affect these resistance responses [ 13 – 16 ]. This may indicate that during evolution, the RNL subfamilies have subfunctionalized in different species or that the preference of the upstream TNLs (see below) for one or the other subfamily has changed.…”