The plasma membrane (PM) is composed of heterogeneous subdomains, characterized by differences in protein and lipid composition. PM receptors can be dynamically sorted into membrane domains to underpin signaling in response to extracellular stimuli. In plants, the plasmodesmal PM is a discrete microdomain that hosts specific receptors and responses. We exploited the independence of this PM domain to investigate how membrane domains can independently integrate a signal that triggers responses across the cell. Focusing on chitin signaling, we found that responses in the plasmodesmal PM require the LysM receptor kinases LYK4 and LYK5 in addition to LYM2. Chitin induces dynamic changes in the localization, association, or mobility of these receptors, but only LYM2 and LYK4 are detected in the plasmodesmal PM. We further uncovered that chitin-induced production of reactive oxygen species and callose depends on specific signaling events that lead to plasmodesmata closure. Our results demonstrate that distinct membrane domains can integrate a common signal with specific machinery that initiates discrete signaling cascades to produce a localized response.

Plasmodesmata are cytosolic bridges, lined by the plasma membrane and traversed by endoplasmic reticulum; plasmodesmata connect cells and tissues, and are critical for many aspects of plant biology. While plasmodesmata are notoriously difficult to extract, tissue fractionation and proteomic analyses can yield valuable knowledge of their composition. Here we have generated two novel proteomes to expand tissue and taxonomic representation of plasmodesmata: one from mature Arabidopsis leaves and one from the moss Physcomitrium patens and leveraged these and existing data to perform a comparative analysis to identify evolutionarily conserved protein families that are associated with plasmodesmata. Thus, we identified β-1,3-glucanases, C2 lipid-binding proteins and tetraspanins as core plasmodesmal components that likely serve as essential structural or functional components. Our approach has not only identified elements of a conserved plasmodesmal proteome, but also demonstrated the added power offered by comparative analysis for recalcitrant samples. Conserved plasmodesmal proteins establish a basis upon which ancient plasmodesmal function can be further investigated to determine the essential roles these structures play in multicellular organism physiology in the green lineages.

14The plasma membrane (PM) that lines plasmodesmata has a distinct protein and lipid 15 composition, underpinning specific regulation of these connections between cells. The 16 plasmodesmal PM can integrate extracellular signals differently from the cellular PM, but it 17 is not known how this specificity is established or how a single stimulus can trigger 18 independent signalling cascades in neighbouring membrane domains. Here we have used the 19 fungal elicitor chitin to investigate signal integration and responses at the plasmodesmal PM. 20 We found that the plasmodesmal PM employs a receptor complex composed of the LysM 21 receptors LYM2 and LYK4 which respectively change their location and interactions in 22 response to chitin. Downstream, signalling is transmitted via a specific phosphorylation 23 signature of an NADPH oxidase and localised callose synthesis that causes plasmodesmata 24 closure. This demonstrates the plasmodesmal PM deploys both plasmodesmata-specific 25 components and differential activation of PM-common components to independently 26 integrate an immune signal. 27 28 93that plasmodesmata are regulated independently of other immune responses, suggesting that 94 there is a critical requirement for a cell to finely tune connectivity to its neighbours. 95 4 Results 96Chitin-triggered plasmodesmata closure is dependent on LYK4 and LYK5 97 We previously identified that LYM2 is a GPI-anchored, LysM receptor protein that is 98 resident in the plasmodesmal PM (Faulkner et al., 2013). As LYM2 has no intracellular 99 domains we reasoned that it must interact with other proteins to initiate downstream signals 100 that result in plasmodesmal responses. Ligand perception by LysM RKs and RPs often 101 involves multiple members of the LysM protein family: chitin perception in rice involves 102 both the RP CHITIN ELICITOR BINDING PROTEIN (OsCEBiP) and the RK CHITIN 103 ELICITOR RECEPTOR KINASE 1 (OsCERK1) (Kaku et al., 2006; Hayafune et al., 2014); 104 peptidoglycan perception in Arabidopsis involves the RK CERK1, and RPs LYM1 and 105 LYM3 (Willmann et al., 2011); and PM chitin perception in Arabidopsis involves CERK1 106 (also called LYK1) and the RKs LYK4 and LYK5 (Cao et al., 2014). Thus, we hypothesised 107 that LYM2 might partner with a LysM RK for signalling. The Arabidopsis LysM RK family 108 consists of 5 members: CERK1/LYK1, LYK2, LYK3, LYK4 and LYK5. To narrow down 109 plasmodesmata signalling candidates we screened publicly available data sets for LYK gene 110 expression. Comparing data sets from seedlings (GSE74955, Yamada et al., 2016; 111 GSE78735, Hillmer et al., 2017) and mature leaves (eFP browser, Winter et al. 2007) we 112 identified variable expression patterns for the LYK family members (Fig. S1). Thus, we 113 performed RT-PCR to identify members of the family expressed in mature Arabidopsis 114 leaves where we assay for and detect LYM2 function. Only transcripts from CERK1, LYK3, 115LYK4 and LYK5 were detected in mature leaves grown in our conditions, eliminating LYK2 116 f...

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Plasmodesmata connect neighbouring plant cells across the cell wall. They are cytosolic bridges, lined by the plasma membrane and traversed by endoplasmic reticulum to connect these cell components between cells and tissues. While plasmodesmata are notoriously difficult to extract, tissue fractionation and proteomic analyses have yielded valuable knowledge of their composition. Most proteomic profiles originate from cell suspension cultures in which simple plasmodesmata dominate and have been exclusively generated from dicotyledonous plant species. Here we have generated two novel proteomes to expand tissue and taxonomic representation of plasmodesmata: one from mature Arabidopsis leaves and one from the moss Physcomitrium patens. We have leveraged these and existing data to perform a comparative analysis that, owing to comparing proteomes from an expanded taxonomic tree, allowed us to identify conserved protein families that are associated with plasmodesmata that likely serve as core structural or functional components. Thus, we identified β-1,3-glucanases, C2 lipid-binding proteins and tetraspanins as core plasmodesmal components, with proteins from P. patens and Arabidopsis maintaining plasmodesmal association across diverse species. Our approach has not only identified elements of a conserved, core plasmodesmal proteome, but also demonstrated the added power offered by comparative analysis. Conserved plasmodesmal proteins establish a basis upon which ancient plasmodesmal function can be further investigated to determine the essential roles these structures play in multicellular organism physiology in the green lineages.