The plant immune system is fundamental to plant survival in natural ecosystems and productivity in crop fields. Substantial evidence supports the prevailing notion that plants possess a two-tiered innate immune system, called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is triggered by microbial patterns via cell surface-localized pattern-recognition receptors (PRRs), whereas ETI is activated by pathogen effector proteins via mostly intracellularly-localized receptors called nucleotide-binding, leucine-rich repeat proteins . CC-BY-NC-ND 4.

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23The plant immune system is fundamental to plant survival in natural ecosystems and productivity 24 in crop fields. Substantial evidence supports the prevailing notion that plants possess a two-tiered 25 innate immune system, called pattern-triggered immunity (PTI) and effector-triggered immunity 26 (ETI). PTI is triggered by microbial patterns via cell surface-localized pattern-recognition 27 receptors (PRRs), whereas ETI is activated by pathogen effector proteins via mostly 28 intracellularly-localized receptors called nucleotide-binding, leucine-rich repeat proteins 29 50 from both pathogenic and nonpathogenic microbes. NLRs, on the other hand, are intracellular 51 proteins that sense pathogen-derived effector proteins inside the plant cell and can be further 52 classified into the coiled coil (CC)-type, Toll/interleukin-1 receptor (TIR)-type, or RPW8 (CCR)-53 type, depending on their N-terminal domain 7 . However, PRR-and NLR-mediated signaling 54 pathways result in many similar downstream immune outputs, including defense gene 55 expression, production of ROS and callose deposition at the plant cell wall 8,9 . The underlying 56 reason is not clear and mechanistic relationship between the two immune pathways remains 57 largely enigmatic. Notably, while many PRR signaling components have been identified and 58 anti-pathogen mechanisms described, the downstream signaling events in ETI and how ETI halts 59 pathogen growth still remain poorly understood, despite recent breakthroughs in the 60 understanding of NLR protein structures and activities 10-13 . 61 62 Requirement of PRR/co-receptors for ETI 63 Using Arabidopsis thaliana-Pseudomonas syringae pathosystem, we accidentally discovered a 64 striking and unexpected role of PRR/co-receptors in ETI. Specifically, an "avirulent", ETI-65 eliciting bacterial strain, P. syringae pv. tomato (Pst) DC3000(avrRpt2), which activates RPS2 66 (Resistance to P. syringae 2)-dependent ETI in wild-type Col-0 plants 14,15 , failed to elicit 67 effective ETI in two separate PRR/co-receptor Arabidopsis mutants, fls2/efr/cerk1 (fec) and 68 bak1/bkk1/cerk1 (bbc) mutants, which are mutated in major PRR/co-receptors recognizing 69 bacteria-associated molecular patterns 16 . As shown in Fig. 1a, the fec and bbc mutants did not 70 mount an effective ETI against Pst DC3000(avrRpt2). To determine whether a requirement of 71 PRR/co-receptors for ETI is specific to Pst DC3000(avrRpt2) or is a more general phenomenon, 72 we tested two other ETI-triggering "avirulent" effectors, AvrPphB and AvrRps4, which are 73 recognized by RPS5 17 and RPS4 18 , respectively, in Arabidopsis Col-0 accession. We found that 74 the compromised ETI phenotype in fec and bbc mutants held true for both AvrPphB and 75 AvrRps4 (Extended Data Fig. 1), suggesting a potentially broad role of PRR/co-receptors in ETI 76 pathways. We subsequently focused on AvrRpt2-triggered ETI for in-depth characterization. 77 Hypersensitive response (HR), manifested by fast cell death under high bacterial inoculum, is a...

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Biphasic production of reactive oxygen species (ROS) has been observed in plants treated with avirulent bacterial strains. The first transient peak corresponds to PTI (pattern-triggered immunity)-ROS, whereas the second long-lasting peak corresponds to ETI (effector-triggered immunity)-ROS. PTI-ROS are produced in the apoplast by plasma membrane-localized NADPH oxidases, and the recognition of an avirulent effector increases the PTI-ROS regulatory module, leading to ETI-ROS accumulation in the apoplast. However, how apoplastic ETI-ROS signaling is relayed to the cytosol is still unknown. Here, we found that in the absence of cytosolic ascorbate peroxidase 1 (APX1), the second phase of ETI-ROS accumulation was undetectable in Arabidopsis (Arabidopsis thaliana) using luminol-based assays. In addition to being a scavenger of cytosolic H2O2, we discovered that APX1 served as a catalyst in this chemiluminescence ROS assay by employing luminol as an electron donor. A horseradish peroxidase (HRP)-mimicking APX1 mutation (APX1W41F) further enhanced its catalytic activity toward luminol, whereas an HRP-dead APX1 mutation (APX1R38H) reduced its luminol oxidation activity. The cytosolic localization of APX1 implies that ETI-ROS might accumulate in the cytosol. When ROS were detected using a fluorescent dye, green fluorescence was observed in the cytosol 6 h after infiltration with an avirulent bacterial strain. Collectively, these results indicate that ETI-ROS eventually accumulate in the cytosol, and cytosolic APX1 catalyzes luminol oxidation and allows monitoring of the kinetics of ETI-ROS in the cytosol. Our study provides important insights into the spatial dynamics of ROS accumulation in plant immunity.

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