Filamentous fungal and oomycete plant pathogens that invade by direct penetration through the leaf epidermal cell wall cause devastating plant diseases. Plant pre-invasive immunity towards non-adapted filamentous pathogens is highly effective and durable. Pre- and post-invasive immunity correlates with the formation of evolutionarily conserved and cell-autonomous cell wall structures, named papillae and encasements, respectively. Yet, it is still unresolved how papillae/encasements are formed and whether these defense structures prevent pathogen ingress. Here we show that in Arabidopsis, the two closely related members of the SYP12 clade of syntaxins (PEN1 and SYP122) are indispensable for the formation of papillae and encasements. Moreover, loss-of-function mutants were hampered in pre-invasive immunity towards a range of phylogenetically distant non-adapted filamentous pathogens, underlining the versatility and efficacy of this defense. Complementation studies using SYP12s from the early diverging land plant, Marchantia polymorpha, showed that the SYP12 clade immunity function has survived 470 My of independent evolution. These results suggest that ancestral land plants evolved the SYP12 clade to provide a broad and durable pre-invasive immunity to facilitate their life on land, and pave the way to a better understanding of how adapted pathogens overcome this ubiquitous plant defense strategy.

27Many filamentous fungal and oomycete plant pathogens invade by direct penetration through 28 the leaf epidermal cell wall and cause devastating plant diseases. In response to attack, plants 29 form evolutionarily conserved cell autonomous defense structures, named papillae and 30 encasements, that are thought to block pathogen ingress. Previously, the syntaxin PEN1 in 31 Arabidopsis, like its orthologue ROR2 in barley, was found to mediate pre-invasive immunity 32 towards powdery mildew fungi, where it assures the timely formation of papilla defense 33 structures. However, this powdery mildew-specific function of PEN1 in papilla timing, thought 34 to take place at the trans-Golgi network, does not explain how plants generally ward off other 35 filamentous pathogens. In the present study, we found that PEN1 has a second function, shared 36 with its closest homologue SYP122, in the formation of papillae, as well as encasements. This 37 second function provides pre-invasive immunity towards highly diverse non-adapted 38 filamentous pathogens, underlining the versatility and efficacy of these defense structures. 39 PEN1 and SYP122 belong to the broadly conserved land plant syntaxin clade SYP12, suggested 40 to function in specialized forms of polarized secretion. In support of this, complementation 41 studies using SYP12s from the basal plant, Marchantia polymorpha, showed that the SYP12 42 clade immunity function has survived 450 My of independent evolution. As saprophytic 43 filamentous land fungi predate plant terrestrialization, we suggest ancestral land plants evolved 44 the SYP12 clade to provide a durable immunity to facilitate their life on land. 45 46Introduction: 47 49 S1) (1). These conserved defense structures are thought to provide effective and durable resistance 50 against diverse filamentous pathogens, and fossil evidence suggests they appeared very early in the 51 evolution of land plants (2)(3)(4). The discovery that the secretory syntaxin PEN1 is required for the 52 timely formation of papillae to defend against non-adapted powdery mildew fungi, highlighted the 53 key role played by membrane trafficking in plant immunity (5-7). PEN1 (also referred to as syntaxin 54 of plants 121 or SYP121) is primarily located at the plasma membrane. On the other hand, the ARF-55 GEF, GNOM, regulates recycling of PEN1 between the plasma membrane and the trans-Golgi 56 network, where it is needed for a fast papilla response (6). Accordingly, PEN1 is thought to mediate 57 3 membrane fusion both at the plasma membrane and the trans-Golgi network. The ROR2 syntaxin in 58 barley is orthologous to PEN1 (5), suggesting that the pre-invasive immunity dependent on 59 PEN1/ROR2 was present in early angiosperms before the divergence of monocots and dicots. Loss 60 of PEN1/ROR2 delays, but does not prevent, the papilla response after attack by the barley powdery 61 mildew Blumeria graminis f.sp. hordei (Bgh) (8, 9), while haustorial encasements are unaffected (10, 62 11). Moreover, although many filamentous pa...

Plant innate immunity toward cell-wall penetrating filamentous pathogens relies on the conserved SYP12 clade of secretory syntaxins. In Arabidopsis , the two closely related SYP12 clade members, PEN1 and SYP122, play an overlapping role in this general immunity, which can be complemented by two SYP12 clade members from Marchantia (MpSYP12A and MpSYP12B). However, in addition to the conserved SYP12 clade function, PEN1 alone mediates pre-invasive immunity toward powdery mildew fungi, which likely reflects a specialization of its functionality. Here, we show that the PEN1-specific specialization in immunity correlates with a continuous BFA-sensitive recycling and the ability to accumulate strongly at the growing cell plate. This contrasts with the behavior of SYP122, MpSYP12A, and MpSYP12B, all being more stable at the plasma membrane. We suggest that the highly mobile SYP12 specialization observed for PEN1 is required for a fast pre-invasive immune response to resist attack from powdery mildew fungi.