Arabidopsis immune secretory pathways to powdery mildew fungi

ABSTRACT

Innate immune responses in host plants begin with the recognition of pathogen-specific nonself molecules and terminate with the secretion of immune molecules. In the dicotyledonous model plant, Arabidopsis thaliana, two distinct secretory pathways required for disease resistance to powdery mildew fungi have been identified so far. One is an exocytic pathway consisting of PEN1, SNAP33 and VAMP721/722 SNARE proteins, but the other is an efflux-mediated one composed of PEN2 atypical myrosinase and PEN3 ABC transporter. Based on the conservation of the mechanically same exocytic pathway in the monocotyledonous plant barely, the former is regarded as an ancient secretory pathway, whereas the latter is considered as a newly evolved one in the Brassicaceae family including Arabidopsis. We recently identified synaptotagmin 1 as an additional regulator of these two secretory pathways. With current results, we discuss how these two secretory pathways contribute to Arabidopsis immunity depending on fungal adaptedness to Arabidopsis.

Powdery mildew fungi are obligate biotrophic phytopathogens that require living host plants, which is thought to result from loss of genes encoding proteins functioning in metabolic, regulatory and transporting activities.^1,2 To gain nutrients from plants, a powdery mildew fungus should form a feeding structure called haustorium inside a plant cell. For this, a landed fungal spore first develops an appressorium to penetrate the hard plant cell wall. If successful in penetration, a fungus then invaginates the plant plasma membrane to develop a haustorium. Surely, a fungus should additionally block the host salicylic acid (SA)-related suicide event called the hypersensitive response (HR), to continuously uptake nutrients via the established haustorium from a plant cell until generating offspring spores (Fig. 1A).³

Figure 1.

Arabidopsis two distinct immune secretory pathways. (A) A scheme depicting interactions between powdery mildew fungal pathogenicity and plant resistance mechanisms. An initial attempt of leaf-landed fungal spores to form haustoria inside host plant cells is frustrated by pre-invasive resistance, which is contributed by the PEN1-SNAP33-VAMP721/722 exocytic and the PEN2/PEN3 secretory pathways (Failed entry, stained fungi with Coomassie). Some fungi can successfully form haustoria by using an effector to circumvent pre-invasive resistance (Successful entry, stained with aniline blue to label the haustorium-containing plant cell wall). The second defense mechanism is post-invasive resistance in host plant cells in which fungal haustoria are formed. Salicylic acid leads to the death of infected host cells (called hypersensitive response (HR)) to protect neighboring host cells from pathogen infection (Host cell death (HR), stained with aniline blue to mark deposited callose in the PM of a dead plant cell). However, some fungi can finally reproduce to generate progeny spores again by using an effector to bypass post-invasive resistance (Successful reproduction, stained fungal mycelia and conidiophores). (B) Two immune secretory pathways (SNARE-mediated exocytosis (blue) and PEN2/3-engaged secretion (red)) differentially contribute to Arabidopsis pre-invasive resistance depending on the adaptedness of powdery mildew fungi.

In Arabidopsis, the pathogenic powdery mildew Golovinomyces orontii fungus can finish its life cycle by producing progeny spores, whereas the barley powdery mildew Blumeria graminis and the pea powdery mildew Erysiphe pisi fungi cannot reproduce, which indicates that the latter two fungi are non-adapted to Arabidopsis. Interestingly, E. pisi can establish haustoria better than B. graminis in Arabidopsis cells.^3,4 Although both fungi fail to generate reproducing organs, this additionally indicates that E. pisi is slightly more adapted than B. graminis to Arabidopsis (Fig. 1B). Comparative genomic analysis revealed the non-redundant conservation of candidate effectors among these three powdery mildew fungi.² This suggests that the fungal adaptedness to a host plant species is determined by whether a fungus possesses an effector to block host immune responses (Fig. 1A).

Although it is poorly known how plants detect powdery mildew fungi, the last step of immune responses, secretion of immune molecules, has been identified by screening Arabidopsis mutants that allow more penetration of B. graminis. One is the SNARE-assisted exocytic secretion. Although externally released molecules are unknown yet, this exocytosis is driven by complex formation of the plasma membrane (PM) PEN1 syntaxin, the PM-localized SNAP33, and the vesicle-residing VAMP721/722.^5,6 The other is the PM-located PEN3 ABC transporter-mediated extrusion of yet-unidentified indole glucosinolate derivatives which are metabolized by the unusual peroxisomal PEN2 myrosinase.^3,7 Genetic analyses revealed that these two distinct secretory pathways additively work in inhibiting the early growth of powdery mildew fungi.³

In the monocotyledonous barley, the PM-localized MLO containing seven transmembrane motifs is critically required for susceptibility of B. graminis, because its deletion results in almost complete resistance in barley.⁸ Likewise, it was also found that the Arabidopsis homologs of barley MLO, AtMLO2, 6, and 12, act negatively in resistance to G. orontii.⁹ Compromised resistance to G. orontii by introducing a mutation of PEN1 or PEN2 in mlo mutant plants indicates that the MLO is a negative regulator of the above-mentioned two known immune secretory pathways,⁹ although its biochemical activity is not known yet.

Recently, we found that synaptotagmin 1 (SYT1) also acts negatively in immune responses to powdery mildew fungi in Arabidopsis, because fungal growth is severely inhibited in syt1 plants.⁴ Re-elevated fungal growth by inserting a mutation in PEN1 or PEN2 gene into syt1 plants indicates that SYT1 is an additional negative regulator of both immune secretory pathways.⁴ While genetically dissecting the relationship of SYT1 to PEN1 and PEN2, we interestingly observed differences in degree of contribution to early immune responses in Arabidopsis to powdery mildew fungi between the SNARE-driven exocytosis and the PEN2/PEN3 extrusion pathways. This is primarily due to a milder defense-boosted phenotype in syt1 plants than the atmlo2/6/12 triple mutant plants, in which powdery mildew fungi are almost completely unable to penetrate host plant cells. The PEN1 mutation in syt1 plants allows more penetration of the most non-adapted B. graminis fungus than the PEN2 mutation.⁴ However, the slightly more adapted E. pisi and the fully adapted G. orontii fungi penetrate better in pen2 syt1 plants than pen1 syt1 plants.⁴ These indicate that the immune activity of PEN1-SNAP33-VAMP721/722 exocytic pathway is more effective on a highly non-adapted fungus rather than on adapted fungi in Arabidopsis (Fig. 1B).

Previously, we also identified the same exocytosis-driving SNAREs, HvROR2, HvSNAP34 and HvVAMP721, in barley as in Arabidopsis.⁶ The high co-expression patterns of these barley and corresponding Arabidopsis SNARE genes¹⁰ suggest that the SNARE-driven exocytic pathway is an ancient immune secretory pathway conserved in both monocots and dicots. This additionally implies that the immune exocytosis might have been a long target to be overcome by pathogens, likely resulting in relatively easy neutralization of this exocytosis now by a pathogen-developed effector. In contrast to the above immune exocytosis, the PEN2/PEN3-involved extrusion mechanism is a relatively new secretory pathway and found in the Brassicaceae including Arabidopsis but not in barley.¹⁰ Therefore, it is likely that not so many pathogens may succeed in inventing an effector to disarm this PEN2/PEN3 secretory pathway. This may explain why the PEN2/PEN3 secretory pathway is currently more potent in defense against a broader range of powdery mildew fungi than the PEN1-SNAP33-VAMP721/722 exocytosis in Arabidopsis. Unlike the PEN2/PEN3-associated pathway, the PEN1-SNAP33-VAMP721/722 pathway is additionally required for plant growth, development and abiotic stress responses. Indeed, the amounts of VAMP721/722 determine sustained plant growth under biotic/abiotic stresses,^11,12 suggesting that plants cannot invest whole this exocytic pathway to the immune function. Therefore, it is also possible that the diverse biological functions of the PEN1-SNAP33-VAMP721/722 exocytic pathway may result in a weaker immune activity than the PEN2/PEN3 pathway in Arabidopsis.

The Arabidopsis SYT1 binds PEN1 and phospholipids in a Ca²⁺-dependent manner.^4,13 Since SYT1 controls the PM-endosome cycling¹⁴ and down-regulates PEN1 abundance,⁴ it seems that SYT1 stimulates PEN1 degradation via endocytosis of a part of PEN1-contained PM, likely to fine-tune the PEN1-SNAP33-VAMP721/722 exocytic activity. While the PEN1 protein level is elevated, interestingly, the PM-localized PEN3 level is not changed in syt1 plants.⁴ This indicates that SYT1 distinctly regulates the immune activity of PEN2/PEN3 secretory pathway. Therefore, it is of great interest to understand how SYT1 controls the PEN2/PEN3 pathway. It was recently reported that PEN2 is additionally localized to mitochondria and its substrate is produced at the endoplasmic reticulum (ER) surface, which are all re-oriented to fungal attack sites.¹⁵ Since SYT1 also contributes to reinforcing the ER-PM junction for resistance to mechanical stresses,¹⁶ one possibility is that SYT1 may affect the action or substrate accessibility of PEN2 probably via modulating the tightness of PM-ER contact sites.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by grants from National Research Foundation (2016R1D1A1B02007322 to C.K.) and from ReSEAT Program of Korea Institute of Science and Technology Information (to B.G.K.), Korea.