The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae

Attack by the host powdery mildew Erysiphe cichoracearum usually results in successful penetration and rapid proliferation of the fungus on Arabidopsis. By contrast, the nonhost barley powdery mildew Blumeria graminis f. sp. hordei (Bgh) typically fails to penetrate Arabidopsis epidermal cells. In both instances the plant secretes cell wall appositions or papillae beneath the penetration peg of the fungus. Genetic screens for mutations that result in increased penetration of Bgh on Arabidopsis have recently identified the PEN1 syntaxin. Here we examine the role of PEN1 and of its closest homologue, SYP122, identified as a syntaxin whose expression is responsive to infection. pen1 syp122 double mutants are both dwarfed and necrotic, suggesting that the two syntaxins have overlapping functions. Although syp122-1 and the cell wall mur mutants have considerably more pronounced primary cell wall defects than pen1 mutants, these have relatively subtle or no effects on penetration resistance. Upon fungal attack, PEN1 appears to be actively recruited to papillae, and there is a 2-h delay in papillae formation in the pen1-1 mutant. We conclude that SYP122 may have a general function in secretion, including a role in cell wall deposition. By contrast, PEN1 appears to have a basal function in secretion and a specialized defense-related function, being required for the polarized secretion events that give rise to papilla formation.     

A SNARE-protein has opposing functions in penetration resistance and defence signalling pathways

Penetration resistance is often the first line of defence against fungal pathogens. Subsequently induced defences are mediated by the programmed cell death (PCD) reaction pathway and the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signalling pathways. We previously demonstrated that full penetration resistance in Arabidopsis against the non-host barley powdery mildew fungus (Blumeria graminis f.sp. hordei) requires the syntaxin SYP121 (PEN1). Here we report that SYP121, together with SYP122, functions as a negative regulator of subsequently induced defence pathways. The SA level in the syntaxin double mutant syp121-1 syp122-1 is dramatically elevated, resulting in necrosis and dwarfism. This phenotype is partially rescued by introducing the SA-signalling mutations eds1-2, eds5-3, sid2-1 and npr1-1 as well as the NahG transgene. These partially rescued triple mutants have an unknown defence to Pseudomonas syringae pv. tomato, and have increased HR-like responses to non-host and host powdery mildew fungi. The HR-like responses cause efficient resistance to the latter. These defence pathways are SA-independent. Furthermore, the JA/ET signalling marker, PDF1.2, is highly upregulated in the triple mutants. Thus SYP121 and SYP122 are negative regulators of PCD, SA, JA and ET pathways through a molecular function distinct from that of SYP121 in penetration resistance. Our data suggest that individual cells preferentially express either penetration resistance or the subsequently induced defences.     

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.     

Activity determinants and functional specialization of Arabidopsis PEN1 syntaxin in innate immunity

In eukaryotes, proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family are believed to have a general role for the fusion of intracellular transport vesicles with acceptor membranes. Arabidopsis thaliana PEN1 syntaxin resides in the plasma membrane and was previously shown to act together with its partner SNAREs, the adaptor protein SNAP33, and endomembrane-anchored VAMP721/722 in the execution of secretory immune responses against powdery mildew fungi. We conducted a structure-function analysis of PEN1 and show that N-terminal phospho-mimicking and non-phosphorylatable variants neither affected binary nor ternary SNARE complex formation with cognate partners in vitro. However, expression of these syntaxin variants at native protein levels in a pen1 mutant background suggests that phosphorylation is required for full resistance activity in planta. All tested site-directed substitutions of SNARE domain or "linker region" residues reduced PEN1 defense activity. Two of the variants failed to form ternary complexes with the partner SNAREs in vitro, possibly explaining their diminished in planta activity. However, impaired pathogen defense in plants expressing a linker region variant is likely because of PEN1 destabilization. Although Arabidopsis PEN1 and SYP122 syntaxins share overlapping functions in plant growth and development, PEN1 activity in disease resistance is apparently the result of a complete functional specialization. Our findings are consistent with the hypothesis that PEN1 acts in plant defense through the formation of ternary SNARE complexes and point to the existence of unknown regulatory factors. Our data indirectly support structural inferences that the four-helical coiled coil bundle in ternary SNARE complexes is formed in a sequential order from the N- to C-terminal direction.     

The ARP2/3 complex,acting cooperatively with Class I formins,modulates penetration resistance in Arabidopsis against powdery mildew invasion

The actin cytoskeleton regulates an array of diverse cellular activities that support the establishment of plant–microbe interactions and plays a critical role in the execution of plant immunity. However, molecular and cellular mechanisms regulating the assembly and rearrangement of actin filaments (AFs) at plant–pathogen interaction sites remain largely elusive. Here, using live-cell imaging, we show that one of the earliest cellular responses in Arabidopsis thaliana upon powdery mildew attack is the formation of patch-like AF structures beneath fungal invasion sites. The AFs constituting actin patches undergo rapid turnover, which is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR regulatory complex (W/SRC). The focal accumulation of phosphatidylinositol-4,5-bisphosphate at fungal penetration sites appears to be a crucial upstream modulator of the W/SRC–ARP2/3 pathway-mediated actin patch formation. Knockout of W/SRC–ARP2/3 pathway subunits partially compromised penetration resistance with impaired endocytic recycling of the defense-associated t-SNARE protein PEN1 and its deposition into apoplastic papillae. Simultaneously knocking out ARP3 and knocking down the Class I formin (AtFH1) abolished actin patch formation, severely impaired the deposition of cell wall appositions, and promoted powdery mildew entry into host cells. Our results demonstrate that the ARP2/3 complex and formins, two actin-nucleating systems, act cooperatively and contribute to Arabidopsis penetration resistance to fungal invasion.

ARP2/3 complex, acting cooperatively with Class I formins, modulates actin patch formation beneath fungal penetration sites, contributing to the penetration resistance of Arabidopsis against powdery mildew invasion.     

Arabidopsis Sec1/Munc18 Protein SEC11 Is a Competitive and Dynamic Modulator of SNARE Binding and SYP121-Dependent Vesicle Traffic

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.     

Nonhost resistance in Arabidopsis-Colletotrichum interactions acts at the cell periphery and requires actin filament function

Pathogenesis of nonadapted fungal pathogens is often terminated coincident with their attempted penetration into epidermal cells of nonhost plants. The genus Colletotrichum represents an economically important group of fungal plant pathogens that are amenable to molecular genetic analysis. Here, we investigated interactions between Arabidopsis and Colletotrichum to gain insights in plant and pathogen processes activating nonhost resistance responses. Three tested nonadapted Colletotrichum species differentiated melanized appressoria on Arabidopsis leaves but failed to form intracellular hyphae. Plant cells responded to Colletotrichum invasion attempts by the formation of PMR4/GSL5-dependent papillary callose. Appressorium differentiation and melanization were insufficient to trigger this localized plant cell response, but analysis of nonpathogenic C. lagenarium mutants implicates penetration-peg formation as the inductive cue. We show that Arabidopsis PEN1 syntaxin controls timely accumulation of papillary callose but is functionally dispensable for effective preinvasion (penetration) resistance in nonhost interactions. Consistent with this observation, green fluorescent protein-tagged PEN1 did not accumulate at sites of attempted penetration by either adapted or nonadapted Colletotrichum species, in contrast to the pronounced focal accumulations of PEN1 associated with entry of powdery mildews. We observed extensive reorganization of actin microfilaments leading to polar orientation of large actin bundles towards appressorial contact sites in interactions with the nonadapted Colletotrichum species. Pharmacological inhibition of actin filament function indicates a functional contribution of the actin cytoskeleton for both preinvasion resistance and papillary callose formation. Interestingly, the incidence of papilla formation at entry sites was greatly reduced in interactions with C. higginsianum isolates, indicating that this adapted pathogen may suppress preinvasion resistance at the cell periphery.     

Mechanisms of functional specificity among plasma-membrane syntaxins in Arabidopsis

Syntaxins and interacting SNARE proteins enable membrane fusion in diverse trafficking pathways. The Arabidopsis SYP1 family of plasma membrane-localized syntaxins comprises nine members, of which KNOLLE and PEN1 play specific roles in cytokinesis and innate immunity, respectively. To identify mechanisms conferring specificity of action, we examined one member of each subfamily-KNOLLE/SYP111, PEN1/SYP121 and SYP132-in regard to subcellular localization, dynamic behavior and complementation of knolle and pen1 mutants when expressed from the same promoters. Our results suggest that cytokinesis-specific syntaxin requires high-level accumulation during cell-plate formation, which necessitates de novo synthesis rather than endocytosis of pre-made protein from the plasma membrane. In contrast, syntaxin in innate immunity does not need upregulation of expression but instead requires pathogen-induced and endocytosis-dependent retargeting to the infection site. This feature of PEN1 is not afforded by SYP132. Additionally, PEN1 could not substitute for KNOLLE because of SNARE domain differences, as revealed by protein chimeras. In contrast, SYP132 was able to rescue knolle as did KNOLLE-SYP132 chimeras. Unlike KNOLLE and PEN1, which appear to have evolved to perform specialized functions, SYP132 stably localized at the plasma membrane and thus might play a role in constitutive membrane fusion.     

Immobilized Subpopulations of Leaf Epidermal Mitochondria Mediate PENETRATION2-Dependent Pathogen Entry Control in Arabidopsis

The atypical myrosinase PENETRATION2 (PEN2) is required for broad-spectrum invasion resistance to filamentous plant pathogens. Previous localization studies suggested PEN2-GFP association with peroxisomes. Here, we show that PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria and that PEN2 has the capacity to form homo-oligomer complexes. We demonstrate pathogen-induced recruitment and immobilization of mitochondrial subpopulations at sites of attempted fungal invasion and show that mitochondrial arrest is accompanied by peripheral accumulation of GFP-tagged PEN2. PEN2 substrate production by the cytochrome P450 monooxygenase CYP81F2 is localized to the surface of the endoplasmic reticulum, which focally reorganizes close to the immobilized mitochondria. Exclusive targeting of PEN2 to the outer membrane of mitochondria complements the pen2 mutant phenotype, corroborating the functional importance of the mitochondrial PEN2 protein subpool for controlled local production of PEN2 hydrolysis products at subcellular plant-microbe interaction domains. Moreover, live-cell imaging shows that mitochondria arrested at these domains exhibit a pathogen-induced redox imbalance, which may lead to the production of intracellular signals.     

Requirement of vesicle-associated membrane protein 721 and 722 for sustained growth during immune responses in Arabidopsis

Extracellular immune responses to ascomycete and oomycete pathogens in Arabidopsis are dependent on vesicle-associated secretion mediated by the SNARE proteins PEN1 syntaxin, SNAP33 and endomembrane-resident VAMP721/722. Continuous movement of functional GFP-VAMP722 to and from the plasma membrane in non-stimulated cells reflects the second proposed function of VAMP721/722 in constitutive secretion during plant growth and development. Application of the bacterium-derived elicitor flg22 stabilizes VAMP721/722 that are otherwise constitutively degraded via the 26S proteasome pathway. Depletion of VAMP721/722 levels by reducing VAMP721/722 gene dosage enhances flg22-induced seedling growth inhibition in spite of elevated VAMP721/722 abundance. We therefore propose that plants prioritize the deployment of the corresponding secretory pathway for defense over plant growth. Interstingly, VAMP721/722 specifically interact in vitro and in vivo with the plasma membrane syntaxin SYP132 that is required for plant growth and resistance to bacteria. This suggests that the plant growth/immunity-involved VAMP721/722 form SNARE complexes with multiple plasma membrane syntaxins to discharge cue-dependent cargo molecules.     

Synaptotagmin 5 Controls SYP132-VAMP721/722 Interaction for Arabidopsis Immunity to Pseudomonas syringae pv tomato DC3000

Vesicle-associated membrane proteins 721 and 722 (VAMP721/722) are secretory vesicle-localized arginine-conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) to drive exocytosis in plants. They are involved in diverse physiological processes in plants by interacting with distinct plasma membrane (PM) syntaxins. Here, we show that synaptotagmin 5 (SYT5) is involved in plant defense against Pseudomonas syringae pv tomato (Pst) DC3000 by regulating SYP132-VAMP721/722 interactions. Calcium-dependent stimulation of in vitro SYP132-VAMP722 interaction by SYT5 and reduced in vivo SYP132-VAMP721/722 interaction in syt5 plants suggest that SYT5 regulates the interaction between SYP132 and VAMP721/722. We interestingly found that disease resistance to Pst DC3000 bacterium but not to Erysiphe pisi fungus is compromised in syt5 plants. Since SYP132 plays an immune function to bacteria, elevated growth of surface-inoculated Pst DC3000 in VAMP721/722-deficient plants suggests that SYT5 contributes to plant immunity to Pst DC3000 by promoting the SYP132-VAMP721/722 immune secretory pathway.     

The physiological role of SYP4 in the salinity and osmotic stress tolerances

The trans-Golgi network (TGN) contains multiple sorting domains and acts as the compartment for cargo sorting. Recent evidence indicates that the TGN also functions as an early endosome, the first compartment in the endocytic pathway in plants. The SYP4 group, plant Qa-SNAREs localized on the TGN, regulates both secretory and vacuolar transport pathways. Consistent with a secretory role, SYP4 proteins are required for extracellular resistance to fungal pathogens. However, the physiological role of SYP4 in abiotic stress remains unknown. Here, we report the phenotypes of a syp4-mutant in regard to salinity and osmotic response, and describe the physiological roles of the SYP4 group in the abiotic stress response.     

A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition in Arabidopsis

A few membrane vesicle trafficking (SNARE) proteins in plants are associated with signaling and transmembrane ion transport, including control of plasma membrane ion channels. Vesicle traffic contributes to the population of ion channels at the plasma membrane. Nonetheless, it is unclear whether these SNAREs also interact directly to affect channel gating and, if so, what functional impact this might have on the plant. Here, we report that the Arabidopsis thaliana SNARE SYP121 binds to KC1, a regulatory K⁺ channel subunit that assembles with different inward-rectifying K⁺ channels to affect their activities. We demonstrate that SYP121 interacts preferentially with KC1 over other Kv-like K⁺ channel subunits and that KC1 interacts specifically with SYP121 but not with its closest structural and functional homolog SYP122 nor with another related SNARE SYP111. SYP121 promoted gating of the inward-rectifying K⁺ channel AKT1 but only when heterologously coexpressed with KC1. Mutation in any one of the three genes, SYP121, KC1, and AKT1, selectively suppressed the inward-rectifying K⁺ current in Arabidopsis root epidermal protoplasts as well as K⁺ acquisition and growth in seedlings when channel-mediated K⁺ uptake was limiting. That SYP121 should be important for gating of a K⁺ channel and its role in inorganic mineral nutrition demonstrates an unexpected role for SNARE–ion channel interactions, apparently divorced from signaling and vesicle traffic. Instead, it suggests a role in regulating K⁺ uptake coordinately with membrane expansion for cell growth.     

A novel motif essential for SNARE interaction with the K(+) channel KC1 and channel gating in Arabidopsis

The SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) protein SYP121 (=SYR1/PEN1) of Arabidopsis thaliana facilitates vesicle traffic, delivering ion channels and other cargo to the plasma membrane, and contributing to plant cell expansion and defense. Recently, we reported that SYP121 also interacts directly with the K(+) channel subunit KC1 and forms a tripartite complex with a second K(+) channel subunit, AKT1, to control channel gating and K(+) transport. Here, we report isolating a minimal sequence motif of SYP121 prerequisite for its interaction with KC1. We made use of yeast mating-based split-ubiquitin and in vivo bimolecular fluorescence complementation assays for protein-protein interaction and of expression and electrophysiological analysis. The results show that interaction of SYP121 with KC1 is associated with a novel FxRF motif uniquely situated within the first 12 residues of the SNARE sequence, that this motif is the minimal requirement for SNARE-dependent alterations in K(+) channel gating when heterologously expressed, and that rescue of KC1-associated K(+) current of the root epidermis in syp121 mutant Arabidopsis plants depends on expression of SNARE constructs incorporating this motif. These results establish the FxRF sequence as a previously unidentified motif required for SNARE-ion channel interactions and lead us to suggest a mechanistic framework for understanding the coordination of vesicle traffic with transmembrane ion transport.     

Arabidopsis nonhost resistance gene PSS1 confers immunity against an oomycete and a fungal pathogen but not a bacterial pathogen that cause diseases in soybean

ABSTRACT: BACKGROUND: Nonhost resistance (NHR) provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species. Three Arabidopsis thaliana PEN (penetration deficient) genes, PEN1, 2 and 3 have been shown to provide NHR against the barley pathogen Blumeria graminis f. sp. hordei at the prehaustorial level. Arabidopsis pen1-1 mutant lacking the PEN1 gene is penetrated by the hemibiotrophic oomycete pathogen Phytophthora sojae, the causal organism of the root and stem rot disease in soybean. We investigated if there is any novel nonhost resistance mechanism in Arabidopsis against the soybean pathogen, P. sojae. RESULTS: The P. sojae susceptible (pss) 1 mutant was identified by screening a mutant population created in the Arabidopsis pen1-1 mutant that lacks penetration resistance against the non adapted barley biotrophic fungal pathogen, Blumeria graminis f. sp. hordei. Segregation data suggested that PEN1 is not epistatic to PSS1. Responses of pss1 and pen1-1 to P. sojae invasion were distinct and suggest that PSS1 may act at both pre- and post-haustorial levels, while PEN1 acts at the pre-haustorial level against this soybean pathogen. Therefore, PSS1 encodes a new form of nonhost resistance. The pss1 mutant is also infected by the necrotrophic fungal pathogen, Fusarium virguliforme, which causes sudden death syndrome in soybean. Thus, a common NHR mechanism is operative in Arabidopsis against both hemibiotrophic oomycetes and necrotrophic fungal pathogens that are pathogenic to soybean. However, PSS1 does not play any role in immunity against the bacterial pathogen, Pseudomonas syringae pv. glycinea, that causes bacterial blight in soybean. We mapped PSS1 to a region very close to the southern telomere of chromosome 3 that carries no known disease resistance genes. CONCLUSIONS: The study revealed that Arabidopsis PSS1 is a novel nonhost resistance gene that confers a new form of nonhost resistance against both a hemibiotrophic oomycete pathogen, P. sojae and a necrotrophic fungal pathogen, F. virguliforme that cause diseases in soybean. However, this gene does not play any role in the immunity of Arabidopsis to the bacterial pathogen, P. syringae pv. glycinea, which causes bacterial blight in soybean. Identification and further characterization of the PSS1 gene would provide further insights into a new form of nonhost resistance in Arabidopsis, which could be utilized in improving resistance of soybean to two serious pathogens.     

Blocking ER export of the Golgi SNARE SYP31 affects plant growth

We recently identified a novel and transplantable di-acidic motif (EXXD) that facilitates ER export of the Golgi syntaxin SYP31 (type IV protein) and which may function also for type I and type II proteins in plants. By mutagenesis of Arabidopsis thaliana SYP31 and live cell imaging experiments in tobacco leaf epidermal cells, we determined that replacing the MELAD sequence of SYP31 with gagag retained SYP31 in the ER, which demonstrates that the di-acidic motif ELAD is critical for SYP31 ER export. To investigate whether blockage of a Golgi SNARE in the ER have consequences for plant growth, we produced tobacco plants stably overexpressing either the wild type MELAD or the mutant gagag form of SYP31. Whereas tobacco plants overexpressing the wild-type SYP31 developed to set seed, tobacco plants overexpressing the mutant form gagag rapidly became chlorotic, ceased their growth and invariably died after several weeks. This indicated that retention of overexpressed SYP31 in the ER is likely toxic for the secretory pathway and, therefore, plant development. Putative explanations for this observation are discussed taking into account SNARE properties and possible interactions.Key words: plant growth, ER-golgi interface, ER export, Golgi SNAREs, SYP31, SNARE interactions, di-acidic motifSNAREs (soluble N-ethyl-maleimide sensitive factor attachment receptor proteins) are components of the molecular machinery that facilitates vesicular transport in the secretory pathway of eukaryotic cells,^1,2 and are critical for numerous plant physiological functions.^1,3 SYP31 is a type IV syntaxin localized at the Golgi and it is required for anterograde traffic from the ER to the Golgi.^4,5 In the search for putative ER export signals in the sequence of SYP31, we identified a di-acidic motif (M) ELAD(G) of the type EXXD.⁶ This di-acidic motif was essential for ER export and Golgi targeting of SYP31, and we suggested an interaction of this motif with the COPII machinery (reviewed in ref. ⁶). To investigate whether blocking a Golgi SNARE in the ER may affect plant growth, we have produced transgenic tobacco plants overexpressing either the wild type MELAD or the mutant gagag form of A. thaliana SYP31.     

Glutathione and tryptophan metabolites are key players in Arabidopsis nonhost resistance against Colletotrichum gloeosporioides

The nonhost resistance of Arabidopsis against hemibiotrophic fungi in the genus Colletotrichum consists of pre- and post-invasive immune responses. Previously, we reported EDR1 and PEN2 as important components of Arabidopsis pre-invasive resistance toward non-adapted Colletotrichum gloeosporioides (Cg). However, despite their defect in entry control pen2 and edr1 mutants terminated further growth of this pathogen by activating the post-invasive hypersensitive response (HR) accompanied by plant cell death. In the present study, we showed that γ-glutamylcysteine synthetase (GSH1), which is required for glutathione biosynthesis, and tryptophan (Trp) metabolism contribute to pre- and post-invasive non-host resistance against Cg. We found GSH1 to be involved in the PEN2-dependent entry control of Cg. Opposite to pen2 and edr1, gsh1 mutants failed to restrict the invasive growth of the pathogen, which demonstrated the requirement for GSH1 during post-invasive non-host resistance. Based on the infection and metabolic phenotypes of Arabidopsis mutants defective in Trp metabolism, we showed that the biosynthesis of Trp-derived phytochemicals is also essential for resistance to Cg during the post-invasive HR. By contrast, GSH1 and these metabolites are dispensable for the induction of HR cell death, which is triggered in the non-invaded mesophyll cells adjacent to the Cg-invaded epidermal cells.